Application of an Endothelialized Modular Construct for ...€¦ · Rohini Gupta . Doctor of Philosophy . Chemical Engineering and Applied Chemistry University of Toronto, 2010

Application of an Endothelialized Modular Construct for Islet Transplantation

by

Rohini Gupta

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Chemical Engineering and Applied Chemistry and Institute of Biomaterials and Biomedical Engineering

University of Toronto

© Copyright by Rohini Gupta 2010

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Application of an Endothelialized Modular Construct for Islet

Transplantation Rohini Gupta

Doctor of Philosophy Chemical Engineering and Applied Chemistry

University of Toronto, 2010 Abstract

Successful survival of large volume engineered tissues depends on the development of a

vasculature to support the metabolic demands of donor tissue in vivo. Pancreatic islet

transplantation is a cell therapy procedure to treat Type 1 diabetes that can potentially benefit

from such a vascularization strategy. The treatment is limited as the majority of transplanted

islets (60%) fail to engraft due to insufficient revascularization in the host(1, 2). Modular tissue

engineering is a means of designing large volume functional tissues using micron sized tissues

with an intrinsic vascularization. In this thesis, we explored the potential of endothelialized

modules to drive vascularization in vivo and promote islet engraftment. Human endothelial cells

(EC) covered modules were transplanted in the omental pouch of athymic rats and human EC

formed vessels near implanted modules until 7 days when host macrophages were depleted. Rat

endothelial cells covered modules were similarly transplanted in the omental pouch of allogeneic

rats with and without immunosuppressants. When the drugs were administered, endothelialized

modules significantly increased the vessel density. Moreover, donor GFP labelled EC formed

vessels that integrated with the host vasculature and were perfusable until 60 days; this key result

demonstrate for the first time that unmodified primary endothelial cells form stable vessels in an

allograft model. Transplantation of islets in such endothelialized modules significantly improved

the vessel density around transplanted islets. Donor endothelial cells formed vessels near

transplanted islets in allogeneic immunesuppressed recipients. Meanwhile, there was an increase

in islet viability with transplantation of endothelialized modules in syngeneic recipients but this

difference was not significant. In summary, endothelialized modules were effective in

promoting stable vascularization and improving transplanted islet vascularisation. Future work

should promote faster maturity of donor vessels and modulate the host immune and

inflammatory responses to significantly improve transplanted islet engraftment.

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Acknowledgements

I thank Dr. Michael Sefton for his supervision and guidance over the past years. He has taught

me a great deal about pursuing research ideas, designing experiments, understanding what the

data really means and telling a story effectively. I am grateful for his continual encouragement

to follow my curiosity, ideas and passion. His mentorship has helped me to identify and define

my own scientific path. I also thank my committee members, Prof. Molly Shoichet, Prof. Mike

Wheeler and Prof. Phil Marsden for their support and guidance through the research project and

also their helpful edits and comments for the thesis.

I thank members of the Sefton lab, Chuen, Brendan, Dean, Omar, Lindsay and Mark, for

brainstorming sessions, discussing research ideas, and supporting experiments. Their friendship

and companionship has enriched my graduate life and made it enjoyable to work these past

years. I especially thank Chuen Lo for his invaluable expertise and laborious work with animal

surgery, blood collection, and perfusion studies. His enthusiasm and willingness allowed me to

explore and implement many research ideas.

I also thank all the collaborators in this project: Deb Dixon (Dr. Ray Rajotte lab) for isolating

and supplying islets, Chyan-Jang Lee (Dr. J. Medin lab) for transduction of RAEC with the

eGFP lentivirus, Lisa Yu (Dr. M. Henkelman lab) for microCT imaging, Melanie Melacast and

Kelvin So for optimizing several immunoshistochemical stains. I acknowledge the following

funding sources: University of Toronto Fellowships, Natural Sciences and Engineering Research

Council (PGS Scholarship), US National Institute of Health and Canadian Institute of Health

Research.

Finally, I thank my family, my parents and brother, for encouraging me at each step, guiding my

focus to the long term goals and helping me through many of life’s chores so I can focus on my

academic work. I thank my friends, Noniya, Rayna, Reema, Nadia, Bernadette, Raquel, Anna,

Dharmesh for listening to my ideas and providing much needed distractions from the lab. And I

thank my husband, Kunal, for shedding a different perspective on the research questions and his

continual patience and support in this journey.

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Table of Contents

Acknowledgements ................................................................................................................... iii

Table of Contents ...................................................................................................................... iv

List of Figures ........................................................................................................................... ix

List of Tables ............................................................................................................................ xi

Abbreviations ........................................................................................................................... xii

1 Research Introduction, Hypothesis and Experimental Plan ..................................................... 1

1.1 Research Overview ......................................................................................................... 2

1.2 Hypothesis ...................................................................................................................... 4

1.3 Research Objectives ........................................................................................................ 4

1.4 Experimental plan ........................................................................................................... 5

1.4.1 Transplant model: Surgically created Omental pouch ......................................... 5

1.4.2 Objective 1: HUVEC endothelialized modules in partially immunecompromised rats .................................................................................... 6

1.4.3 Objective 2: RAEC endothelialized modules in allogeneic rats ........................... 7

1.4.4 Objective 3: Islets embedded in RAEC endothelialized modules in allogeneic diabetic rats ......................................................................................................... 8

1.4.5 Objective 4: Islets embedded in RAEC endothelialized modules in syngeneic diabetic rats ......................................................................................................... 9

1.5 Thesis Organization ...................................................................................................... 10

2 Endothelial Cells and Tissue Engineering & Current Challenges with Islet Transplantation . 11

2.1 Introduction .................................................................................................................. 12

2.2 Endothelial cells and Tissue Engineering ...................................................................... 13

2.2.1 The role of EC in coagulation and inflammation ................................................ 13

2.2.2 EC as anti-coagulants ........................................................................................ 14

2.2.3 Mechanisms of angiogenesis ............................................................................. 15

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2.3 Transplantation of primary endothelial cells to promote vascularization ........................ 18

2.3.1 Endothelial cell sources ..................................................................................... 18

2.3.2 Biomaterials for EC culture ............................................................................... 19

2.3.3 EC transplantation models ................................................................................. 20

2.3.4 Donor EC modifications and co-transplantation with supporting cells ............... 22

2.3.5 Organization of EC prior to transplantation ....................................................... 24

2.3.6 EC transplantation to improve vascularization in engineered or ischemic tissues ............................................................................................................... 25

2.3.7 Summary of EC transplantation ......................................................................... 26

2.4 Biomaterials and angiogeneic growth factor delivery .................................................... 30

2.5 Current Challenges with Islet Transplantation ............................................................... 32

2.5.1 Type 1 Diabetes and Islet Transplantation ......................................................... 32

2.5.2 Transplanted islet engraftment ........................................................................... 34

2.5.3 Instant Blood Mediated Inflammatory Reaction ................................................. 34

2.5.4 IBMIR and coagulation ..................................................................................... 35

2.5.5 IBMIR and inflammation .................................................................................. 37

2.5.6 Immune Regulation and Immunosuppressant Toxicity ....................................... 38

2.5.7 Revascularization of transplanted islets ............................................................. 39

3 Fate of Endothelialized Modular Constructs Implanted in an Omental Pouch in Nude Rats .. 42

3.1 Abstract ........................................................................................................................ 43

3.2 Introduction .................................................................................................................. 44

3.3 Materials and Methods .................................................................................................. 46

3.3.1 Cells .................................................................................................................. 46

3.3.2 Module Fabrication ........................................................................................... 46

3.3.3 Module transplants ............................................................................................ 46

3.3.4 Histology and immunostaining .......................................................................... 47

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3.3.5 Histology quantification .................................................................................... 47

3.3.6 Statistical Analysis ............................................................................................ 48

3.4 Results .......................................................................................................................... 49

3.4.1 Remodeling of collagen gel modules ................................................................. 49

3.4.2 Transplanted HUVEC ....................................................................................... 51

3.4.3 Vessel Maturity ................................................................................................. 55

3.4.4 Apoptotic cells .................................................................................................. 57

3.4.5 Inflammatory cells............................................................................................. 58

3.5 Discussion .................................................................................................................... 60

3.6 Conclusions .................................................................................................................. 63

3.7 Acknowledgements ....................................................................................................... 63

4 Endothelialized modules drive stable chimeric vascularization in allogeneic rats. ................ 64

4.1 Abstract ........................................................................................................................ 65

4.2 Introduction .................................................................................................................. 66


4.3.1 Cells .................................................................................................................. 68


4.3.3 Module transplants ............................................................................................ 68

4.3.4 Perfusion studies ............................................................................................... 69



4.3.7 Statistical Analysis ............................................................................................ 70

4.4 Results .......................................................................................................................... 72

4.4.1 Remodeling of endothelialized modules and tissue response .............................. 72

4.4.2 Blood vessel formation around transplanted modules ........................................ 74

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4.4.3 Transplanted RAEC form vessels in immunosuppressant treated rats................. 77

4.4.4 Inflammatory cell response to endothelialized modules ..................................... 81

4.5 Discussion .................................................................................................................... 83

4.6 Conclusions .................................................................................................................. 85

4.7 Acknowledgements ....................................................................................................... 85

5 Application of an Endothelialized Modular Construct for Islet Transplantation .................... 86

5.1 Abstract ........................................................................................................................ 87

5.2 Introduction .................................................................................................................. 88


5.3.1 Cells .................................................................................................................. 90


5.3.3 In vitro characterization ..................................................................................... 91

5.3.4 Static glucose challenge assay ........................................................................... 91

5.3.5 Diabetic animals ................................................................................................ 92

5.3.6 Omental pouch transplants ................................................................................ 92

5.3.7 Metabolic follow up .......................................................................................... 92



5.4 Results and Discussion.................................................................................................. 95

5.4.1 In vitro Characterization of islets in endothelialized modules ............................ 95

5.4.2 Transplanted intra-islet vessel density ............................................................... 98

5.4.3 Peripheral vessel density of transplanted islets ..................................................100

5.4.4 RAEC isolated from Lewis rats ........................................................................103

5.4.5 Transplanted islet viability ...............................................................................104

5.4.6 Transplanted islet function ...............................................................................107

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5.4.7 Why does increased vessel density not correlate with improved islet viability or function? ......................................................................................................112

5.5 Conclusions .................................................................................................................114

5.6 Acknowledgements ......................................................................................................114

6 Conclusion and Future Work ..............................................................................................115

6.1 Conclusions .................................................................................................................116

6.2 Recommendations and Future work .............................................................................118

6.2.1 Characterize allogeneic endothelialized modules ..............................................119

6.2.2 Improve donor derived vessel maturation .........................................................120

6.2.3 Prevent host inflammation and immune responses ............................................121

6.2.4 Improve engraftment of islets in endothelialized modules .................................122

6.2.5 Syngeneic endothelialized modular constructs ..................................................123

6.2.6 Endothelialized modules and in vivo blood compatibility .................................124

7 Reference List .....................................................................................................................126

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List of Figures Figure 1-1: Overview of Experimental Plan ............................................................................... 5

Figure 2-1: Endothelial cells (EC) as a non-thrombogeneic surface. . ....................................... 15

Figure 2-2: Proposed schematic of donor EC derived vascularization.. .................................... 21

Figure 2-3: Summary of procedures in islet isolation and transplantation. ................................ 33

Figure 2-4: Simplified schematic of coagulation cascade. ...................................................... 35

Figure 3-1: Schematic representation of a modular construct. ................................................. 45

Figure 3-2: Trichrome staining of HUVEC endothelialized modules ....................................... 50

Figure 3-3: High magnification trichrome images of HUVEC endothelialized modules ........... 51

Figure 3-4: UEA-1 stained sections of HUVEC endothelialized modules ................................. 53

Figure 3-5: High magnification (400x) UEA-1 sections at days 3 and 7.. ................................. 54

Figure 3-6: Double staining with UEA-1 lectin and vWf.......................................................... 56

Figure 3-7: Double staining with UEA-1 lectin and rat smooth muscle α-actin………………..57

Figure 3-8: TUNEL staining of animals treated with clodronate liposome ............................... 58

Figure 3-9: ED2 stained sections of HUVEC endothelialized modules...................................... 59

Figure 4-1 Trichrome staining of RAEC modules with and without drug treatment.. ................ 73

Figure 4-2: Apoptotic (TUNEL), proliferating (Ki67) cells, and myofibroblasts (SMA). .......... 74

Figure 4-3: Rat microvessels (BS-1 lectin positive) around RAEC modules. ............................ 75

Figure 4-4: BS-1 positive microvessel density counts cells. ...................................................... 76

Figure 4-5: microCT images of whole omental pouch containing endothelialized modules. . ... 77

x

Figure 4-6: Transplanted eGFP-RAEC around endothelialized modules. .................................. 78

Figure 4-7: Average number of GFP positive vessels over time.. .............................................. 79

Figure 4-8: GFP-RAEC vessels with supporting cells at day 60.. ............................................. 80

Figure 4-9: Fluorescent beads in GFP positive vessels at day 60. ............................................. 81

Figure 4-10: Average counts of CD68 (ED1, macrophages) positive cells. ............................. 82

Figure 4-11: T cells around endothelialized modules. . ........................................................... 82

Figure 5-1: Live/Dead assay and insulin measurements of islets in vitro. . .............................. 96

Figure 5-2: Islets in endothelialized modules in culture............................................................. 98

Figure 5-3: Transplanted intra-islet microvascular density at day 21. . .................................... 99

Figure 5-4: Transplanted islet peripheral vessel density at day 21 ........................................... 101

Figure 5-5: GFP-RAEC around islets in endothelialized modules ........................................... 102

Figure 5-6: Lewis rat endothelialized modules. .................................................................... 104

Figure 5-7: Insulin positive islets in allogeneic and syngeneic recipients……………………. 106

Figure 5-8: Line plot of average blood glucose levels over time of diabetic recipients.. .......... 109

Figure 5-9: Line plot of blood glucose levels of individual syngeneic recipients. .................. 111

Figure 5-10: Plot of non-fasting serum insulin levels. . ......................................................... 111

Figure 6-1: Schematic of future areas of focus with allogeneic endothelialized modules ........ 118

Figure 6-2 Trichrome sections of RAEC modules via the portal vein ..................................... 124

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List of Tables Table 1-1: Summary of project plan detailing specific aims and objectives ................................. 4

Table 1-2: Summary of Experimental model.............................................................................. 6

Table 2-1: Summary of endothelial cell transplantation studies ................................................ 29

Table 3-1: List of monoclonal antibodies and their target antigens. .......................................... 48

Table 3-2: Macrophage accumulation around HUVEC endothelialized modules ..................... 59

Table 4-1: List of antibodies and their target antigens used for immunostaining ....................... 71

Table 5-1: List of antibodies and their target antigens used for immunostaining ....................... 94

Table 5-2: Average insulin positive area in diabetic recipients at 21 days .............................. 105

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Abbreviations

APC: Activated Protein C

β: Beta cell

BS-1: Bandeiraea simplicifolia

CD: Clustered Antibody

EC: Endothelial cells

ECM: Extracellular Matrix

ELISA: Enyzme linked immunosorbent assay

EPC: Endothelial Progenitor Cell

FBS: Fetal Bovine Serum

FGF: Fibroblast Growth Factor

GFP: Green Fluorescent Protein

H&E: Hematoxylin and Eosin

HUVEC: Human Umbilical Vein Endothelial Cells

IBMIR: Instant Blood Mediated Inflammatory Reaction

IEQ: Islet Equivalents

IL: Interleukin

LEW: Lewis strain

MHC: Major Histocompatibility Complex

MMP: Matrix MetalloProteinases

mm: Millimeter

mmol: Millimole

MSC: Mesenchymal Stem Cells

MVD: MicroVessel Density

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NK: Natural Killer

NO: Nitric Oxide

OGTT: Oral Glucose Tolerance Test

PAF: Platelet Activating Factor

PECAM: Platelet-endothelial adhesion molecule

PDGF: Platelet Derived Growth Factor

RAEC: Rat Aortic Endothelial Cell

SEM: Standard Error of the Mean

SMA: α-Smooth Muscle Actin

SCID: Severe Combined Immunodeficiency

SD: Sprague Dawley strain

STZ: Streptozotocin

t-PA: Tissue-type Plasminogen Activator

TAT: Thrombin-Antithrombin complex

TF: Tissue Factor

TFPI: Tissue Factor Pathway Initiator

TGF: Transforming Growth Factor

TNF: Tissue Necrosis Factor

TUNEL: Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling

UEA-1: Ulex Europaeus Agglutinin I

µm: micron

VEGF: Vascular Endothelial Growth Factor

VE: Vascular Endothelial

vWf: Von Willebrand Factor

1

1 Research Introduction, Hypothesis and Experimental

Plan

2

1.1 Research Overview

Engineering functional tissues is a means of replacing, restoring or repairing diseased organs and

tissues. Engineering thick (greater than 1mm), 3-D functional tissues is challenging as cells in

the interior of thick tissues do not receive adequate nutrients due to mass transfer limitations.

This leads to a hypoxic cell core in large volume tissue substitutes. Moreover, when engineered

tissues are transplanted, they are not immediately connected with the host blood supply.

Although, there is some in-growth of host blood vessels, vascularization is generally too slow

and inadequate to support the metabolic demands of the new tissue. This is in contrast to whole

organ transplantation where existing donor blood vessels are anastomosed directly to host blood

vessels and the donor tissue is well perfused. Successful survival of engineered tissues depends

on strategies to develop a new vasculature that will support the metabolic demands of donor

tissue. Strategies to induce vascularization have included the delivery of primary endothelial

cells to initiate vascularization. Endothelial cells alone and with supporting cells have been

transplanted in ischemic tissues and co-transplanted with functional cells to improve

vascularization in immunedeficient animals. In some of these cases, donor endothelial cells form

stable vessels that connect to the host vasculature(3, 4). Primary EC transplantation appears to

be a promising strategy for improving vascularization of engineered tissues. However, there is a

need to understand how normal inflammatory and immune response influence donor EC derived

vascularization and functional tissue survival.

Pancreatic islet transplantation is a cell therapy procedure that can benefit from a vascularization

strategy. Islet transplantation is a means of replacing insulin producing cells (islets) and

potentially maintaining normoglycemia in Type I diabetics. Currently, the treatment is limited,

however, as the majority of transplanted islets (60%) fail to engraft due to an immediate blood

mediated inflammatory reaction and lack of revascularization in the host(1, 2). During isolation,

much of the vascular endothelium is lost after transplantation and revascularization takes 7-14

days. In the early post-transplant period, a majority of the islets are hypoxic and undergo

apoptosis(5-7). Several groups have shown that delivery of pro-angiogeneic growth factors

increased vascular density around transplanted islets and in some cases improved islet

3

engraftment and functionality in diabetic recipients. Islet co-transplantation with mesenchymal

stem cells has also resulted in improved vascular density and engraftment. These studies suggest

that improving vessel density around transplanted islets can in fact enhance islet engraftment.

To date, islet co-transplantation with primary endothelial cells has not been attempted and may

be an effective way to enhance islet engraftment

Modular tissue engineering is a means of designing large volume functional tissue replacements

using micro-tissues with an inherent vascularization. Other features of modular issue

engineering include the capability of creating complex tissues with mixed cell populations,

scaleability and a non-thrombogeneic surface. Previous work with modular tissue engineering

has shown that the endothelialized surface provides a non-thrombogeneic surface that permits

blood flow in vitro. Moreover, embedded cells (HepG2) were viable at high densities within

modules indicating that the modules support functional cells without any mass transfer

limitations(8, 9). We became interested in exploring the potential of endothelialized modules to

drive vascularization in vivo. Moreover, we wanted to evaluate whether endothelialized modules

will promote vascularization and engraftment of transplanted islets. Our goal was to assess

whether endothelialized modular technology can a) be used to create a stable vascular network

and b) can the engineered vascular network support co-transplanted islets.

4

1.2 Hypothesis

1. Transplant of endothelialized modules will result in an increased vascular density in the

omentum.

2. Such vascularization will enhance engraftment of pancreatic islets embedded in

endothelialized modules as compared to free islets or islets in modules (no EC).

1.3 Research Objectives

As detailed in Table 1, the project has two aims: 1) characterize remodeling and vascularization

of endothelialized modules in vivo and 2) characterize islets in endothelialized modules in

diabetic recipients.

Table 1-1: Summary of project plan detailing specific aims and objectives

Specific Aim Objectives

Characterize remodeling, vascularization of endothelialized modules in vivo

1. Assess host response to endothelialized modules in a partially immunocompromised model

2. Develop and assess the remodeling and vascularization that occurs with transplantation of rat endothelialized modules in an allogeneic model

Characterize islets in endothelialized modules in diabetic recipients

3. Assess the host response and function of islets embedded in endothelialized modules transplanted in a diabetic allogeneic model

4. Evaluate function of islets embedded in endothelialized modules for treatment of diabetic rats in a syngeneic model

5

1.4 Experimental plan

The overall experimental plan for the project was to prepare and characterize endothelialized

modules with/without islets in vitro, transplant modules in vivo and evaluate host response and

vascularization via immunohistology as outlined in Fig. 1-1. For islet transplantation studies,

serum glucose and insulin measurements were used in addition to assess the viability and

function of transplanted islets. For each objective, an appropriate animal model was selected as

summarized in Table 1-2.

Figure 1-1: Overview of Experimental Plan

1.4.1 Transplant model: Surgically created Omental pouch

The omentum, a fatty tissue layer in the peritoneum, has been explored for a number of years as

a vascularized organ for regeneration and as a cell transplant site. In larger animals (humans,

dogs), the omentum consists of both a greater omentum tissue and lesser omentum sac; in

rodents, only the greater omentum exists as a fatty apron-like tissue attached to the gastric wall.

The omentum consists of adipose tissue and lymphoid tissue (milky spots). The milky spots are

areas with host immune and inflammatory cells that rapidly expand in the presence of an

inflammatory stimuli(10). The omentum is highly vascularized and is fed by both gastric and

splenic vessels. The right side of the omentum is fed by arteries originating from the gastric

system and the left side is fed by arteries from the spleen, both sides drain into the portal system.

While the right and left side of the omentum are well vascularized, the middle omental tissue is

fragile and avascular(11). Blood vessel density in the milky spots is 2-3x greater than around the

6

adipose tissue. The vascularized omentum has higher levels of VEGF expression compared to

other tissues and activation by inflammatory/hypoxic stimuli increases VEGF production, blood

vessel density, and progenitor cell recruiting factors in the omentum (12). The vast supply of

growth factors and high degree of vascularization make the omentum a rich environment for cell

transplantation. Tissues can be transplanted into the omentum via either the roll-up technique or

alternatively, the omentum can be sutured to create a pouch. Moreover, some studies have

shown that the omentum can be used for islet transplantation; islets have been transplanted in the

omental pouch of large animals such as dogs(13), non-human primates(14) and also rodents(15,

16). The omental pouch was selected as the site for all module transplantation studies as it

allows for delivery of a large volume of implants and can be easily retrieved for histological

analysis. The particular transplant model used for each objective is summarized in Table 1-2 and

discussed in depth below. Table 1-2: Summary of Experimental model

Objective Module Type Cell (EC and islet) source

Animal Model Drugs

1 Human umbilical vein endothelial cells (HUVEC) endothelialized modules

Pooled human umbilical veins

Omental pouch in Athymic (Nude) rats

+/- Macrophage depletion

2 Rat aortic endothelial cells (RAEC) endothelialized modules

Sprague Dawley rats

Omental pouch in Sprague Dawley rats

+/- Immunosuppressant and Atorvastatin

3 Islets embedded in RAEC endothelialized modules

Sprague Dawley rats

Omental pouch in diabetic Sprague Dawley rats

Immunosuppressant and Atorvasatin

4 Islets embedded in RAEC endothelialized modules

Lewis rats Omental pouch in diabetic inbred Lewis rats

None

1.4.2 Objective 1: HUVEC endothelialized modules in partially immunecompromised rats

The first objective was to assess and characterize the host response to endothelialized modules

without embedded cells. Previous work in the lab had demonstrated that HUVEC

7

endothelialized modules form a non-thrombogenic surface in vitro. Moreover, others have

demonstrated that HUVEC in immunecompromised mice (SCID) formed primitive vessels. We

evaluated HUVEC endothelialized modules in the omental pouch of athymic (nude) rats.

Athymic rats lack production of all mature T lymphocytes including CD4+ and CD8+ cells and

cannot initiate T cell mediated rejection of xeno- and allo-grafts. As nude rats age, there is some

repopulation of CD4+ T cells, but T lymphocytes always have decreased activity in comparison

with normal euthymic rats(17). All other leukocytes (monocytes/macrophages, B cells and

natural killer (NK) cells) are present in the nude rat and in fact nude rats are reported to have

higher levels of NK cells in comparison to euthymic rats(18). NK cells also harbor allo-

recognition antigens and can participate in the rejection of allogeneic cells, although this type of

rejection is severely limited when compared to T cell mediated rejection. With respect to

xenografts, NK cells in combination with macrophages and xenoreactive antibodies are known to

reject vascularized xenografts in the nude rat (18, 19). However, human EC transplant in the

nude rats has not been explored extensively. HUVEC endothelialized modules were transplanted

in the nude rat to prevent adverse T cell rejection of xenogeneic human EC. HUVEC

remodeling and survival under normal and with a reduced inflammatory response by

administration of clodronate liposomes was assessed.

1.4.3 Objective 2: RAEC endothelialized modules in allogeneic rats

Although there are several reports of human EC survival in immunocompromised animals, the

vascularization potential of donor EC has been only barely explored in immunecompetent

animals. Thus, the potential of endothelialized modules to drive vascularization in a clinically

relevant allograft model was investigated. Rat aortic endothelial cells isolated from outbred

Sprague Dawley (SD) rats were cultured on the surface of collagen modules and transplanted in

the omental pouch of outbred SD rats. Sprague Dawley is a common outbred rat strain with

genetic heterogeneity; the exact MHC variability between individual rats within the strain is

unfortunately unknown. Reports of SD donor cells transplanted into SD rats are rare as

transplantation studies either evaluate fully characterized MHC mismatch models or syngeneic

models. Some have reported that allogeneic SD pancreatic islets survive well in the

8

“immunepriviledged” retinal space without immunesuppressants(20). On the other hand, central

nervous (CNS) fetal grafts transplanted to outbred SD rats were rejected; in particular, T cells

were located near the endothelium of CNS grafts indicating that there is an immune reaction to

outbred endothelial cells (21). Consistent with this, adult kidneys from SD donor were rejected

when transplanted into the SD host(22). Although the degree of mismatch between outbred SD

recipients is unknown, the studies above confirm that the outbred strain rejects vascularized

tissues, making this a clinically relevant model for evaluation of allogeneic endothelial cell

transplantation. Allogeneic endothelialized modules were evaluated in the SD rat, and donor

RAEC survival and vessel formation was evaluated with and without the use of a common

transplant immunesuppressant (Tacrolimus) and an anti-inflammatory, EC protective agent

(Atorvastatin).

1.4.4 Objective 3: Islets embedded in RAEC endothelialized modules in allogeneic diabetic rats

The next objective was to evaluate whether endothelialized modules derived vascularization can

support islets in an allograft diabetic model. Building on the model in objective 2, islets isolated

from outbred SD rats embedded in RAEC endothelialized modules were compared with islets

alone in outbred diabetic SD rats with the same drugs as used in objective 2. Only a handful of

groups have evaluated transplantation of allogeneic islets in the presence of immunosuppressants

in a rodent model. Immunosuppressants are toxic to transplanted islets and a large number of

islets (~4000 islets/200g rat) are required for reversing diabetes(23). Here, a minimal islet mass

(~2000 islets/250g rat) model was used to assess whether endothelialized modules improve

transplanted islet vascularization, viability and/or function in comparison to free islets in the

omental pouch of diabetic rats. Rats were made diabetic by injection of streptozotocin (STZ), a

common method of inducing chemical diabetes. Streptozotocin is a toxic glucose analog that is

selectively uptaken by pancreatic ϐ cells via the GLUT2 glucose transporter. Streptozotocin

then promotes ϐ cell necrosis by causing DNA fragmentation, protein methylation and acting as

an intracellular NO donor. In rodents, 55-80 mg/kg doses of streptozotocin leads to ϐ cell death

and high blood glucose levels within 48hrs(24).

9

1.4.5 Objective 4: Islets embedded in RAEC endothelialized modules in syngeneic diabetic rats

Next, a syngeneic model of islet transplantation was used to evaluate transplanted islet function

without the toxic effects of immunesuppressants. Islets isolated from Lewis (Lew/Crl substrain)

embedded in either collagen only modules or endothelialized (RAEC isolated from Lew/MolTac

substrain) modules were transplanted in Lewis (Lew/Crl substrain) diabetic recipients. The

Lewis strain is a common inbred strain with several Lewis substrains including the Lew/Crl

substrain maintained at Charles River and the Lew/MolTac substrain maintained at Taconic. The

MHC haplotype of the inbred strain is RT1l and syngeneic donor cells are tolerated well without

the need for immunesuppressants. This strain is susceptible to streptozotocin induced diabetes

and is commonly used for islet transplantation studies. In the Lewis rat, ~2000 syngeneic islets

under the kidney capsule or in the omental pouch are known to reverse diabetes long term(15).

Here, islets (~2000) embedded in collagen only or endothelialized modules were transplanted

without immunosuppressants to assess the impact of donor endothelial cells in improving

transplanted islet vascularization, viability and function in the omental pouch of syngeneic

diabetic rats. Whereas free islets were used as a control group in the allogeneic model, in this

syngeneic model, islets in collagen only (without EC) were selected as the control group to

directly elucidate the effects of donor EC.

10

1.5 Thesis Organization

The thesis chapters are organized as follows:

Chapter 1: Research Introduction

Chapter 2: Literature Review: Endothelial cell transplant and islet transplantation

Chapter 3: Objective 1: Fate of Endothelialized modular construct in nude rats; Published

manuscript, Tissue Engineering, 2009

Chapter 4: Objective 2: Endothelialized modules drive stable chimeric vascularization in

allogeneic rats; Manuscript in Preparation

Chapter 5: Objective 3-4: Application of endothelialized modular construct for islet

transplantation; Manuscript in Preparation

Chapter 6: Conclusions and Future Work

11

2 Endothelial Cells and Tissue Engineering & Current

Challenges with Islet Transplantation

12

2.1 Introduction

Engineering functional tissues is a means of replacing, restoring or repairing diseased organs and

tissues. Combining functional cells with a variety of polymers and extracellular matrices (ECM)

has successfully created artificial skin and bladder. Engineering thick (greater than 1mm), 3-D

functional tissues is challenging as cells in the interior of thick tissues do not receive adequate

nutrients(25, 26). This leads to a hypoxic cell core in large volume tissue substitutes. Moreover,

when engineered tissues are transplanted, they are not immediately connected with the host

blood supply. Although, there is some in-growth of host blood vessels, vascularization is

generally too slow and inadequate to support the metabolic demands of the new tissue. This is in

contrast to whole organ transplantation where existing donor blood vessels are anastomosed

directly to host blood vessels and the donor tissue is well perfused. Successful survival of

engineered tissues depends on strategies to develop a new vasculature that will support the

metabolic demands of donor tissue.

Ischemic disorders (myocardial infarction, peripheral vascular disorder) are examples of

situations where new blood vessel generation is particularly useful. Much work has been done

with ischemic tissues and vascularization strategies have focused on creating new blood vessels

by delivering pro-angiogenic growth factors, transplanting primary endothelial cells, and

encouraging endothelial progenitor cells to the ischemic site (the latter is not discussed further in

this review). Angiogeneic strategies can also be readily adapted to create a vasculature for

transplanted tissues. The following review focuses on the role of endothelial cells (EC) in

physiological angiogenesis, and describes the current work on EC transplantation as a

vascularization strategy. Growth factor delivery as another vascularization strategy is briefly

presented and the application of growth factor delivery for improving pancreatic islet

engraftment is discussed.

13

2.2 Endothelial cells and Tissue Engineering

Endothelial cells (EC) line blood vessel lumens and form a selectively permeable membrane to

regulate the movement of molecules from the bloodstream to the underlying tissue. EC serve

more than just as a selective barrier, they modulate vascular tone, hemostasis, inflammatory and

immune responses in normal and pathological conditions. Particularly relevant to tissue

engineering is the role of EC in modulating blood coagulation, inflammatory, and angiogenesis

processes. The following describes these innate mechanisms and reviews their relevance for

tissue engineering.

2.2.1 The role of EC in coagulation and inflammation

Environmental conditions such as inflammatory stimuli, vessel injury and hypoxic stress can

activate endothelial cells to promote coagulation and inflammation. Procoagulant EC facilitate

the production of a blood clot which consists of aggregated platelets in a fibrin matrix. EC

secrete platelet-activating factor (PAF) and von Willebrand factor (vWf) to recruit platelets to

site of injury. Although vWF is constitutively expressed in the subendothelium matrix, upon

activation, vWf is rapidly mobilized to the luminal side and acts to both stabilize coagulation

factor VIII and bind platelets. EC lose expression of heparan sulfate and thrombomodulin and

secrete tissue factor to activate the coagulation pathway and initiate thrombin production. In

addition, EC secrete plasminogen activator inhibitors to prevent fibrinolysis. The combination of

platelet aggregration, thrombin generation and inhibition of fibrnolysis leads to the formation of

a clot and fibrin deposition(27-29).

EC secrete proinflammatory cytokines, interleukins and chemotactic molecules to initiate

inflammation and recruit leukocytes (neutrophils, monocytes and platelets) to the site of injury.

EC directly capture circulating leukocytes from the bloodstream by expressing three types of

adhesion molecules on their luminal surface: selectins, integrins and immunogluoblins. E-

selectin and P-selectin on the EC surface interact with their reciprocal selectins on the leukocyte

14

membrane to slow down and capture the rolling leukocytes. Then, surface integrins such as

intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion module-1 (VCAM-1)

promote firm adhesion of leukocytes(29, 30) to the EC membrane. Next, immunoglobulins such

as platelet-endothelial cell adhesion molecule-1 (PECAM-1) expressed on both the EC and

leukocyte surface allow the leukocytes to transverse through the EC intercellular junctions to the

subluminal tissue space. Depending on the injury, leukocytes also participate in coagulation to

create a thrombotic clot.

2.2.2 EC as anti-coagulants

An important function of the endothelium is to tightly regulate the coagulation and complement

systems. As summarized in Fig. 2-1, quiescent (non-activated) EC directly inhibit coagulation,

prevent platelet aggregation and initiate the fibrinolytic system(31). Coagulation is inhibited

primarily through the protein C/protein S pathway and the tissue factor pathway initiator (TFPI)

mechanism (27, 31, 32). EC expressed thrombomodulin binds plasma thrombin to activate

protein C and activated protein C (APC) complexes with EC secreted protein S to inactivate

factors Va and VIIIa. Also, surface TFPI binds tissue factor (TF) and forms a quaternary

structure with factor VIIa and Xa. Additionally, EC secrete prostacyclin (PGI2), and nitric oxide

(NO) to prevent platelet aggregation and activate tissue-type plasminogen activator (t-PA) to

degrade fibrin. The endothelium secretes several complement inhibitory molecules (C1-

inhibitor, vitronectin and clusterin) and contains surface molecules (CD55, CD46 and CD59) that

neutralize complement activation products(33). Together these mechanisms allow quiescent

endothelial cells to maintain a non-thrombogenic blood flow environment. For a number of

years, endothelial cells have been exploited as a non-thrombogenic lining for vascular grafts and

there is strong evidence that endothelialized small diameter vascular grafts in vivo prevent

coagulation and outlast their non-seeded counterparts(34, 35).

15

EC

PGI2 NO

APC PS

TM TF

Va VIIIa

VIIa Xa

TFPI T

tPA

Coagulation

Platelets

Fibrinolysis

PC

EC EC

Figure 2-1: Endothelial cells (EC) prevent platelet aggregation, coagulation and initiate fibrinolytic system to modulate hemostasis. Prostacyclin (PGI2) and nitric oxide (NO) inhibit platelet activation, shape change and adhesion to the EC surface. Thrombomodulin (TM) converts thrombin (T) to an anticoagulant form and this complex activates protein C (PC) to APC. EC secrete protein S (PS) which binds with APC to degrade Factors Va and VIIa, major cofactors of the coagulation cascade. Tissue factor pathway inhibitor (TFPI) expressed on EC surface binds tissue factor (TF) and factors Xa and VIIa to further inhibit coagulation. EC secrete tissue plasminogen activator (tPA) to initiate the fibrinolytic system and degrade fibrin clots.

2.2.3 Mechanisms of angiogenesis

Physiological angiogenesis in the adult occurs during ovulation, inflammatory and wound

healing responses. New blood vessels are created through either sprouting of existing vessels or

splitting of a vessel lumen (intussusceptive) mechanism(27). Angiogenesis is a complex process

driven primarily by endothelial cells (EC) and involves the cooperation of several growth factors,

basement membrane proteins, proteolytic enzymes (MMPs), and supporting cell types (pericytes,

smooth muscle cells, macrophages).

Sprouting angiogenesis is a well studied mechanism and occurs in 4 stages: existing blood

vessel disruption and EC migration, EC proliferation and formation of immature tubules, vessel

stabilization by supporting cells, and connection with existing vasculature to establish blood flow

16

in new vessels. In response to a hypoxic stress or tissue injury, cells (EC, wound macrophages)

secrete an array of angiogenic growth factors including vascular endothelial growth factor

(VEGF) and fibroblast growth factor (FGF). The growth factors target specific endothelial cells

known as the ‘tip cells’, located in small capillaries and venules, and change the targeted EC

from a quiescent state to an activated angiogenic state(36). As part of this angiogenic switch, EC

express several proteases such as MT1-MMP (membrane type 1 matrix metalloproteinase),

MMP-2, MMP-9 and u-PA on their membranes(37). Proteinases such as ADAM-15 dissociate

the strong EC-EC junctional contacts (VE-Cadherin) and MT1-MMPs degrade the underlying

EC basement membrane. Basement membrane proteins play an important signaling role in

angiogenesis; EC interact with the ECM through specific surface receptors known as integrins.

Degradation of the normal basement membrane (mostly laminin and collagen Type 1V) exposes

EC to Type 1 collagen and EC integrins α2ϐ1 and α1ϐ1 engagement with Type I collagen localizes

MMP-2 to the EC surface. MMP-2 further initiates ECM digestion to allow for EC migration

away from the parent vessel(38). MMP activity is highly regulated in this process and the

degradation of specific ECM component releases growth factors that in turn guide EC migration.

After EC migration, growth factors such as VEGF and FGF stimulate EC proliferation and the

formation of tubules. EC accumulate intracellular vacuoles that coalesce to form lumens. The

lumens fill with erythrocytes and further organize into tubules. Next, the immature tubules

undergo a number of vessel stabilization steps that include pericyte recruitment and basement

membrane changes. EC secrete growth factors such as PDGF-ϐ to recruit pericytes, smooth

muscle cells and fibroblasts to stabilize the neovessels(27, 31, 39, 40). Other growth factors such

as TGF-ϐ, Angiopoietin/Tie-2 (Tie-2 receptor on EC surface) help to stabilize the EC and

perictye interaction and prevent further EC proliferation(41). As part of the tubular

morphogenesis process, the provisional basement membrane that consists mainly of collagen

Type IV and laminin-8 changes to a mature membrane that includes the addition of laminin-10.

Interaction of EC integrins α5ϐ3 with laminin in the mature matrix stabilizes vessels by increasing

anti-apoptotic gene signaling in EC. Also, pericyte-EC interaction drives TIMP (tissue inhibitor

of metalloproteinases) inhibition of MMP’s to prevent further ECM degradation(37). Stabilized

vessels connect to existing capillaries and blood flow to the vessel continues to downregulate the

angiogenic signaling.

17

A lesser studied angiogenic mechanism is vessel growth through intussusceptive angiogenesis,

which is an efficient and rapid method of increasing the capillary network density and has been

implicated in many organogenesis, tissue repair and tumor angiogenesis models. In this

mechanism, existing capillaries split directly into two vessels. First, opposing capillary walls of

an existing capillary come into contact. Next, EC junctional proteins reorganize and allow EC to

stretch around a transluminal pillar (column of collagen fibers), the pillar increases in size and is

eventually supported by pericytes and fibroblasts to form two separate vessels (42, 43). The new

capillary bed is further organized to balance out the feeding (arteriorles) and emptying (venule)

vessels and new vessels will change morphology and form either vessels of the arterial or venule

side. Also, vessel branches are resized and unnecessary vessels are closed (vessel pruning) to

optimize the blood flow in the vascular bed. In contrast to sprouting angiogenesis, vessel

formation through intussusceptive mechanism occurs while flow is present in the native vessel

and moreover, does not require EC migration and proliferation. It is proposed that during vessel

development in the embryo and/or tumor growth, the sprouting mechanism initiates a primitive

capillary bed and the capillary bed is expanded and remodeled via intussuscpetive

angiogenesis(42). Although it has not been well studied in tissue engineering, intussusceptive

angiogenesis has shown to play a role in wound healing responses to biomaterials and in the res-

establishment of blood flow to transplanted fetal procine islet-like clusters in athymic mice

(44),(45).

18

2.3 Transplantation of primary endothelial cells to promote

vascularization

Endothelial cells alone and with supporting cells have been transplanted in ischemic tissues and

co-transplanted with functional cells to improve vascularization in vivo. Endothelial cell

transplantation was first investigated for improving small diameter vessel grafts(35) and clinical

studies to date indicate that a healthy endothelium lining can improve graft patency(46). Also,

complete blood vessels have been engineered by combining EC and supporting vascular cells in

a defined pattern(47, 48). Rather than engineering complete blood vessels, endothelial cell

transplantation has since been explored as a tool for inducing vascularization in vivo.

2.3.1 Endothelial cell sources

Endothelial cells can be derived from adult vessels, stem cells, bone marrow and cord blood

progenitor cells. Adult endothelial cells are isolated through enzymatic and mechanical

treatments from different vessels and expanded ex vivo. Animal (rat, sheep, canine, and bovine)

EC have been isolated from both large vessels such as aorta, pulmonary artery, jugular vein,

saphenous vein, and from microvessels in the heart, lung and fat tissues. Human endothelial

cells are primarily isolated from discarded umbilical veins and from saphenous veins. Although

it is well understood that EC from different anatomical sites in the vasculature have

heterogeneous structure, marker expression and function(49), EC in culture show evidence of

plasticity. For example large vessel endothelial cells can display microvascular phenotypes in

vitro(50). A possible explanation for this observed plasticity is that EC respond to their

microenvironment; thus EC isolation and subsequent culture in a different environment modifies

their natural phenotype and function. For tissue engineering applications, both macro and micro

vessel EC have been used for developing a microvasculature in vivo.

19

2.3.2 Biomaterials for EC culture

EC can be cultured on a variety of natural biopolymers and select synthetic polymers. The

underlying matrix modulates a number of EC properties in vitro including viability,

thrombogenecity and phenotype. The most common material employed for EC culture and

subsequent transplantation is Type 1 collagen. Other materials that have also been used for EC

transplantation are fibronectin, matrigel, and synthetic polymers (PLA, PLGA).

Collagen, the most abundant polymer synthesized in the body(51), is available in 20 different

forms. Type 1 collagen is most commonly used in tissue engineering applications including

vascular grafts(52). Type 1 collagen can be isolated and solubilized through proteolytic enzymes

and this soluble collagen is maintained at acidic conditions in low temperatures until ready to be

used(53). For cell culture applications, low density Type 1 collagen (0.3 - 3.0 mg/mL), is

polymerized under neutral conditions to form a soft porous gel (interfibril spacing of approx 1

μm)(53). Cells embedded in collagen hydrogels are viable in vitro and implanted hydrogels have

low immunogenicity and antigenicity in vivo. The mechanical strength of collagen gels can be

increased by covalent cross linkages through chemical agents such as glutaraldehyde.

Endothelial cells have been routinely cultured on collagen gels. Macro and micro vessel

endothelial cells form a monolayer when cultured on the surface of the collagen gels and the

confluent monolayer displays a non-thrombogeneic phenotype(8). Activation of the EC

monolayer with growth factors causes the EC to assume an angiogenic sprouting phenotype.

Similarly, when EC are embedded inside 3D collagen gels or collagen is overlaid on a confluent

EC layer, EC switch to an angiogenic state and sprout to form a capillary-like network in vitro.

EC have a strong interaction with collagen gels and can realign the collagen fibers to create a

compact gel. In turn, Type 1 collagen gel stiffness modulates EC formation of vascular networks

in vitro. Interestingly, while some groups have reported that human umbilical vein endothelial

cells (HUVEC) undergo apoptosis when cultured within 3D collagen gels(54, 55), others have

shown that HUVEC, bovine and rat endothelial cells survive well and form capillaries within

similar collagen gels(56, 57). A possible explanation in the discrepancy is that a lower EC

density was used within gels where EC survived well (1.0 x 106 vs. 2.0x106 cells/mL). Others

have explored the addition of other extracellular matrix proteins such as collagen Type IV to

collagen gels to promote vessel growth in culture(50).

20

Another ECM protein that has been used for EC culture is fibronectin. Fibronectin can be

isolated from plasma and added as either a surface coating or incorporated within hydrogels.

Fibronectin is a strong modulator of EC organization and is known to enhance EC adhesion,

spreading, proliferation and the initiation of angiogenesis(58). EC bind to the fibronectin matrix

with integrin α5β1 both in culture and in vivo and this integrin engagement mediates the

reorganization of the underlying matrix and tube formation. Also, fibronectin is a key ECM

protein that is deposited by EC during stimulated angiogenesis in Type 1 collagen gels in

vitro(59, 60). Fibronectin has been added to collagen gels for EC culture and found to modulate

EC behavior. For example, the addition of fibronectin promoted a greater EC migration into

collagen gels(61). Also, the addition of fibronectin to collagen gels elongated the microvessels

generated from aortic endothelial cells in culture compared to collagen only gels (50).

Other common materials for EC culture include Matrigel® and synthetic materials. Matrigel®,

secreted from Engelbroth-Holm Swarm mouse sarcoma cells, is composed of many basement

membrane proteins (collagen Type 1, fibronectin, laminin) and growth factors; however, the

exact composition of Matrigel® is largely unknown. EC cultured on top of Matrigel® layers

sprout and form capillary-like networks in vitro. A common approach for assessing

angiogenesis is through a Matrigel plug in which liquid Matrigel® is injected directly into a host

to create a plug that is conducive to host cell invasion and angiogenesis. Also, donor EC sprout

into a capillary network when injected directly into a Matrigel plug in vivo(62). Porous

biocompatible medical grade materials such as poly (l-lactic acid) (PLLA) and poly (d,l-lactic

acid-co-glycolic acid) (PLGA) are synthetic materials and have been traditionally employed for

tissue engineering and growth factor delivery. Blends of PLGA and PLLA can also be used for

EC culture and subsequent transplantation. The large porosity supports embedded cells in vitro

and cells invade into the material when implanted in vivo(63). Modification of material porosity

modulates EC growth characteristics in vitro.

2.3.3 EC transplantation models

Although a few types of EC have been transplanted to initiate vascularization in vivo, HUVEC

have mostly commonly been used for transplantation studies. HUVEC have been combined in a

21

number of scaffolds and transplanted in either ischemic areas or under the skin of severe

combined immunedeficient (SCID) mice. Majority of the transplanted EC are apoptotic after

transplantation and without intervention will disappear in a few weeks. However, when EC

apoptosis is prevented, donor EC initiate angiogenesis in the host and participate in the formation

of new blood vessels that integrate with the host vasculature and show normal blood flow

physiology characteristics(3, 64). The general vascularization process can be characterized in 4

major steps (Fig. 2-2): donor EC migration into host tissue, investment by host EC and/or

supporting vascular cells, establishment of blood flow to vessels and differentiation of vessels

into capillary-like, venule-like and arteriole-like vessels. The newly formed vasculature has also

shown to support functional cells and improve blood flow in ischemic tissues in

immunecompromised mice.

Figure 2-2: Proposed schematic of donor EC derived vascularization. Donor EC migrate into surrounding tissue to form vessels that are stabilized by vascular cells. New vessels insoculate with host vessels to form chimeric vessels and blood flow is established. Vascular remodeling continues with differentiation of vessels into arteriole-like, venule-like and capillary-like vessels.

22

The process of angiogenesis by EC transplantation is fragile and requires both anti-apoptotic

signaling in transplanted EC and stabilization of the donor primitive vessels, otherwise donor

vessels regress over time. For example, human dermal endothelial cells (HDMEC) seeded in

Matrigel® and implanted into porous PLLA scaffolds in SCID mice lead to human vessels short

term but the vessels started to regress at 14 days(65). Similarly, another group transplanted

HUVEC in a Matrigel plug in SCID mice and noted a decrease in HUVEC derived vessel density

over time. Although some HUVEC derived vessels survived until 100 days and included

erythrocytes in their lumens, there was no indication of functional perfusion within the HUVEC

derived vessels(66). Therefore, although transplantation of human EC alone in SCID animals

can result in the formation of primitive vessels, donor vessels are not functional long term.

2.3.4 Donor EC modifications and co-transplantation with supporting cells

In order to achieve stabilization of donor EC derived vessels, a pre-requisite of vessel function,

methods such as inducing anti-apoptotic gene signaling in transplanted EC and co-

transplantation with supporting vascular cells have been explored. A successful modification to

prolong donor derived vascularization has been to down regulate apoptotic signaling in donor

endothelial cells. Serum starved or growth factor deprived EC have been rescued by VEGF

stimulation of EC through upregulation of an anti-apoptotic gene, Bcl-2, suggesting that Bcl-2

overexpression can prolong EC survival in vitro(67). Based on this, HUVEC were transfected

with Bcl-2 (Bcl-2 HUVEC), suspended in collagen-fibronectin gels and transplanted in the

abdominal wall of SCID mice. Bcl-2 HUVEC formed mature vascular networks that were

supported by host cells while unmodified HUVEC only formed immature vessels at 30 days(55).

The Bcl-2 HUVEC derived vessels developed into a complete microvascular bed capable of

assuming all arterial, venous and capillary EC phenotypes at 60 days after transplant (64). Host

smooth muscle cells and endothelial cells integrated within the donor derived vessels to form

chimeric vessels. Moreover, the chimeric vessels were perfusable by injected dextran and

maintained a physiological selective barrier.

Another successful modification to stabilize donor vessels has been to incorporate supporting

vascular cells directly with donor EC prior to transplantation. It has been well established that

23

during angiogenesis, vascular cells (fibroblasts, smooth muscle cells and pericytes) migrate to

support developing neovessels. In vitro, pericyte co-cultures stabilized EC derived vessels in 3D

collagen gels(68) and fibroblasts promoted EC formation of a tubular network(69). Also, EC

promote the differentiation of perivascular cell precursors to supporting vascular cells in

vitro(70). Building on this, both 10T1/2 (perivascular precursors) and embryonic fibroblasts

have been co-cultured with HUVEC in porous PLGA/PLL scaffolds(3, 4). Transplantation of

10T1/2 and HUVEC in PLGA/PLL scaffolds led to functional and perfusable donor vessels that

lasted at least 1 year after transplantation in SCID mice. Similarly embryonic fibroblasts

stabilized the HUVEC derived vessels until 60 days in SCID mice. Both the 10T1/2 cells and

embryonic fibroblasts differentiate into supporting smooth muscle cells for EC derived vessels

which likely promoted the long term survival of donor vessels in vivo. Also, mature (aortic)

smooth muscle cells added to the Bcl-2-HUVEC culture prior to transplantation further improved

the maturation of HUVEC derived vessels in SCID mice(71). Thus, prevention of donor EC

apoptosis and incorporating supporting vascular cells with donor EC can result in formation of a

functional donor EC derived vascular network long term in immunecompromised recipients.

Other ways to support developing vessels is to add growth factors that stimulate angiogenesis

and prevent EC apoptosis in vivo. In fact, human microvascular endothelial cells (HMVEC) co-

transplanted with VEGF releasing PLG scaffolds in SCID mice showed a significant increase in

vessel density at 14 days(63). Interestingly, delivery of VEGF from alginate microparticles

combined with Bcl-2-HUVEC in collagen-fibronectin scaffolds increased the donor derived

vessel density in ischemic limbs of SCID mice at 14 days but not at 28 days(72). Similarly

transplantation of HUVEC overexpressing PDGF-BB led to increased inflammation and

destabilization of donor vessels over time(73). These limited studies suggest that whereas it may

be possible to achieve synergistic effects of growth factor delivery with EC transplantation, the

correct growth factor cocktail and temporal distribution must first be optimized to enable long

term vessel stabilization.

24

2.3.5 Organization of EC prior to transplantation

Rather than transplanting single endothelial cells, a pre-formed capillary bed can also be

transplanted. When EC are embedded in porous gels, they sprout into a capillary-like network in

vitro. Since donor EC form a similar primitive network after transplantation, some groups have

explored whether transplantation of pre-formed capillary-like structure advances the vascular

development in vivo. HUVEC and lung fibroblasts in fibrin gels were allowed to form capillary-

like structures for one week in vitro. Transplantation of these capillary-like structures induced a

greater vessel density than HUVEC and fibroblasts alone (no capillary pre-formation) in Rag-/-

mice at 14 days(74). On the other hand, both freshly explanted and one week cultured rat

microvessel fragments (human or rat) formed a perfusable vascular network until 28 days in

SCID mice(75). Donor vessels remained in the middle of the implanted construct while host

vessels were observed at the boundary suggesting that host insoculation occurred with donor

microvessels rather than the formation of the chimeric vessels observed with transplantation of

individual endothelial cells. Another manipulation of EC has been to culture EC without any

biomaterials. Recently, HUVEC have been cultured as spheroids (100 cells in each spheroid)

and these spheroids show outgrowth of capillary-like structures when embedded in different

matrices(76). The intercellular EC-EC contact within the spheroid configuration is thought to

provide anti-apoptotic signaling for HUVEC. Moreover, the EC spheroids embedded in

Matrigel-fibrin matrix in combination with VEGF and FGF produced stable human vessels in

SCID mice for 20 days. However, addition of a single growth factor produced immature vessels

(76) and transplantation of EC spheroids alone without growth factors was not attempted. The

EC spheroid technique was combined with osteoblasts in processed bone cancellous bone

scaffolds (no growth factors) and human vessel with supporting smooth muscle cells were found

at 21 days(77). Since evaluation of donor vessel formation was limited to 3 weeks, the long term

remodeling of either pre-formed capillary-like structures or spheroid aggregation has not been

studied.

25

2.3.6 EC transplantation to improve vascularization in engineered or ischemic tissues

Considerable attention has been provided to skin tissue engineering where reduced

vascularization has been attributed to failure of both traditional full thickness skin grafts and

tissue engineered skin grafts (78). Tissue engineered skin grafts (split thickness grafts) consist of

both the dermis and epidermis layers. Although tissue engineered skin grafts provide an

alternate source of skin grafting, they do not consist of an internal vasculature and rely on the

migration of host vessels into the graft which can take up to 14 days. Attempts to improve skin

graft revascularization have included the addition of human EC alone and human EC derived

capillary-like structures. HUVEC and human fibroblasts formed a capillary network on a

reconstructed dermal equivalent (human keratinocytes on a collagen/chitosan polymer) after 31

days of culture in vitro(79). Transplantation of the endothelialized dermal equivalent on a skin

wound in nude mice resulted in human EC derived vessels that were perfused with mouse blood

as early as 4 days and persisted until 14 days(69). More importantly, it was noted that

endothelialized dermal equivalents had faster insoculation with mouse capillaries (4 days as

opposed to 14 days) than normal dermal equivalents. Similarly, vascularization of skin grafts

with human dermal microvascular EC (HDMEC) resulted in human EC derived vessels in

athymic mice that persisted until 4 weeks, however the authors also noted a poorer epidermal

organization in vitro with the addition of EC to dermal implants(80). Also, Bcl-2 HUVEC added

to dermal equivalents 24 hrs prior to implantation improved the vascular density of the dermal

grafts in SCID mice as compared to non vascularized controls(81). HUVEC derived vessels

persisted primarily in the centre of the graft (until 6 weeks) and the donor (mouse) vessels

invaded from the graft periphery. Also, transplantation of a whole endothelial cell culture sheet

with syngeneic skin grafts increased vessel density in the skin graft after transplantation,

however transplanted EC were unlabelled and so there is no data on the actual survival of

syngeneic EC(82). Although the addition of endothelial cells has improved vascular density in

the dermal grafts in the studies above, it is not clear whether the vascularization also improved

engraftment or longevity of the implant.

26

Another application of vascularization derived by EC transplantation has been to rescue ischemic

tissues formed by peripheral artery occlusions and myocardial infarctions. Transplantation of

Bcl-2 HUVEC in collagen-fibronectin scaffolds with/without VEGF has shown to improve blood

flow to ischemic limbs(55, 72). In fact, just the addition of endothelial cells alone is sufficient to

promote vascularization in myocardial infarction as evidenced by increased vascular density at 6

weeks by injection of allogeneic rat aortic endothelial cells (RAEC) (83) and improved left

ventricular function after injection of HUVEC(84). Similarly, transplantation of microvessel

grafts in myocardial infarctions of SCID mice improved vascular density and improved cardiac

function at 14 days(85). A more elegant treatment of myocardial scars is to introduce

cardiomyocytes and restore some degree of cardiac function; however transplanted cell survival

is dependent on adequate perfusion. Transplantation of co-cultured rat heart microvessel EC and

neonatal cardiomyocytes sheets improved cardiac function, decreased fibrosis and increased

vascular densities in a myocardial infarct in nude rats(86). Also, HUVEC and embryonic

fibroblasts combined with hESC derived cardiomyocytes or myoblasts in porous PLGA/PLLA

scaffolds increased vessel density in both normal cardiac muscle and skeletal muscle implants as

compared to non-vascularized implants. Although promising, the HUVEC and fibroblast

approach have yet to be tested in ischemic tissues to rigorously assess whether the increased

vessel density improves muscle functionality(4, 87).

2.3.7 Summary of EC transplantation

A number of studies with human endothelial cells transplanted in immunedeficient animals show

a general trend for EC derived vascularization in vivo as summarized in Table 2-1. As outlined

in Fig. 2-2, the general trend after transplantation of primary endothelial cells in SCID mice is

that donor EC form lumens as early as day 3, lumens accumulate erythrocytes by 7 days,

supporting vascular cells (either of donor or host origin) are invested at 14 days and mature

donor vessels are formed by 21 days. In general, mature donor vessels past this time point are

chimeric (i.e. supported by host endothelial and/or vascular smooth muscle cell). Even when

donor EC derived vessels regress, just the initial presence of EC seems to be sufficient to signal

27

host endothelial cells to initiate angiogenesis and increase vascular density at the desired

location(65).

Factors that influence stabilization of donor EC derived vessels include: transplant model (EC

source, transplant matrix and transplant site), EC organization prior to transplantation and anti-

apoptotic manipulations to enhance donor EC survival. Various types of adult EC (HUVEC,

HDMEC) have shown to successfully initiate vascularization after transplantation and modified

adult EC (BCl-2-HUVEC) were equivalent in their vascularization potential as cord-blood, adult

blood or hES derived EC in SCID mice(88). A few combinations of EC type and underlying

matrix have shown to be successful in promoting EC vessel development in immunedeficient

animals, but there is little information about how these parameters influence EC remodeling in

vivo. The transplant site can dictate vessel remodeling of donor EC as noted by variations in

donor vessel density and sizes by microvessel fragments transplanted in a myocardial infarct vs.

the subcutaneous space(75, 85). This correlates with the view that cues in the microenvironment

govern endothelial cell heterogeneity in different vascular sites(49). Pre-formation of capillary-

like structures in vitro prior to transplant appears to hasten the insoculation of donor vessels with

host vessels, albeit the long term impact of pre-formed capillary formation remains to be

understood. Certainly, preventing EC apoptosis after transplantation is a key factor in promoting

donor EC survival in vivo. Genetically enhancing anti-apoptotic mechanisms in donor EC or co-

transplantation with supporting vascular cells has shown significant improvements in the

development of a stable vasculature in vivo.

Some donor EC derived vessels appear to be directly perfused as measured by the injection of

india ink, fluorescent dextran or lectin at 21 days(4, 73, 75). However, complete donor vessel

functionality including perfusion of all donor vessels (rather than a few), permselectivity, and

response to exogenous stimuli has only been demonstrated at 60 days (64). Thus, it is not known

whether donor vessels observed prior to 60 days actually improve the functional vascular density

in the transplant site. An indirect measurement of vessel function is to assess whether the vessel

development improves blood flow and/or functional cell engraftment. EC transplantation has

improved blood flow in ischemic tissues at 21 days, albeit the observed blood flow could have

occurred in host vessels (since host vessel density also increases with EC transplantation) rather

than donor derived vessels(64). Also, although EC co-transplantation increases vessel density

28

around functional cells, there is no strong evidence of improved viability or engraftment of

functional cells.

Although much progress has been made to achieve stable vascularization by donor EC, the exact

mechanism of this process is largely unknown. It is presumed that donor EC initiate an

angiogenic response to form a neo vasculature. Since EC transplantation to date has mainly been

evaluated in immunedeficient animals, it remains to be seen how normal inflammatory and

immune responses influence the donor EC remodeling. Two groups have transplanted

allogeneic or syngeneic endothelial cells in animal models and reported on survival and

contribution of these cells to host vessel density. For example, 12% of rat jugular vein

endothelial cells injected intra-arterially into the hindlimb of syngeneic recipients were detected

within the capillary bed of the muscle 1 month after transplantation(89). Still there is a need to

better understand the long term remodeling and response of allogeneic endothelial cell

transplantation and critically evaluate whether this strategy can be used for improving

vascularization in ischemic tissues or tissue engineered implants.

29

Table 2-1: Summary of endothelial cell transplantation studies

Cells Scaffold Animal model

Functional cell/Ischemia

Study (days) Key result Ref

HDMEC

PLLA + Matrigel® SCID mouse None 28 Human vessels regress at day 14 and were replaced by mouse vessels

(65)

HUVEC Matrigel plugs Rag 2-/- None 100 Limited HUVEC vessel survival long term (66) HMVEC VEGF releasing PLG SCID mouse none 14 Significant increase in vascular density with

VEGF delivery for 14 days (63)

HUVEC + lung fibroblasts

Fibrin gels SCID mice Dermal equivalents

14 Prevascularized implants had faster (5 days) insoculation than single cell delivery

(74)

HUVEC spheroids Matrigel- fibrin + VEGF + FGF

SCID mouse Osteoblasts 21 Formation of human derived vessels that were supported by host smooth muscle cells

(76, 77)

HUVEC transfected with Bcl-2

Collagen-fibronectin +/- VEGF releasing PLG

SCID mouse Skin grafts, Ischemic limbs

60 Human vessels were stable, functional and differentiate into complete vascular bed; VEGF increased donor density short term only

(55, 64, 72, 81)

HUVEC + Embryonic fibroblasts

PLGA/PLL scaffolds SCID mouse Skeletal muscle

14 Increased vessel density around muscle implants with EC + fibroblasts

(4)

HUVEC + perivascular precursors +/- PDGF-BB

Collagen - fibronectin SCID mouse None 360 Stable, perfusable vessels formed with HUVEC + precursors implant. Overexpression of PDGF-BB results in destability of vessels

(3, 73)

Rat fat microvessel fragments

Collagen Type 1 SCID mice Myocardial infarction

28 Perfusion into microvascular bed at 7 days; improved LV function

(75, 85)

RAEC None SD rat with CsA

Myocardial Cyroinjury

45 Improved vascular density and blood flow, labeled cells detected only at 2 weeks

(83)

Rat jugular vein endothelial cells

None Syngeneic Wistar

None 28 EC injected into the hindlimb incorporated into vessels – 12% remaining by end of study

(90)

30

2.4 Biomaterials and angiogeneic growth factor delivery

Therapeutic angiogenesis strategies have exploited the delivery of growth factors to improve

vascularization in ischemic tissues. Isoforms of VEGF, FGF, and PDGF have been delivered as

either whole proteins or as genes encoding for target growth factors. Although, pre-clinical

studies with growth factor delivery were promising and showed improved perfusion in ischemic

model, majority of clinical trials with growth factor delivery have failed to show real

improvements in tissue perfusion. Intramuscular injection of naked plasmid DNA encoding for

FGF reduced ischemic pain and improved ulcer healing for peripheral arterial occlusive disease

in Phase I trials(91); however the same trials failed in Phase II studies(92). Ongoing issues with

growth factor delivery include non-targeted delivery, short half-life of growth factors in vivo,

generation of immature vessels and vessel regression over time. The following focuses on

approaches to improve the therapeutic effect of growth factor delivery by incorporating

biomaterials to target the angiogenic delivery site.

A variety of natural and synthetic biomaterials have been employed to control spatial and

temporal delivery of growth factors and promote stable vascularization in animal models. A

common approach has been to incorporate growth factor proteins directly into biomaterials and

modulate the release rates by controlling biomaterial properties. Incorporation of VEGF to

collagen matrices prolonged the release time and angiogenic effect as compared to a VEGF bolus

solution subcutaneously in mice(93). Also, sustained release of bFGF from gelatin scaffolds

improved tissue vascular density and functional performance in a rabbit hindlimb ischemic

model(94), a rat myocardial infarction model(95) and a sternum regeneration model(96). bFGF

loaded gelatin microspheres have also shown to improve blood flow and relieve symptoms of

limb ischemia in one Phase I clinical trial(97). Synthetic materials such as poly(lactide-

coglycolide) (PLG) have been useful in controlling VEGF release to local tissue sites while

minimizing systemic VEGF distribution in mice(98). VEGF delivered in a biodegradable PLG

scaffolds improved blood flow and vessel density in a limb ischemia model(99). Interestingly,

spatial control of VEGF delivery in layered PLG microspheres improved vascular density and

functionality as compared to a uniform delivery of VEGF(92) in PLG scaffolds. While most

studies have simply compared angiogenic effects of growth factor loaded biomaterial to sham

31

implants, one study evaluated biomaterials as a growth factor delivery tool. FGF-2 delivery from

gelatin combined with PLL or PLG scaffolds improved tissue perfusion and capillary density as

compared to a bolus FGF-2 injection in an ischemic limb model for 8 weeks(100).

Since physiological angiogenesis is regulated by multiple growth factors in controlled stages, it

is not surprising that single delivery of growth factors has shown limited success. Strategies to

improve angiogenesis in ischemic models have included the delivery of multiple growth factors

via biomaterials. Nillesen et al. showed that a combination of FGF and VEGF in collagen gels

increased blood vessel density at day 7 compared to collagen with VEGF alone but no significant

differences were noted at day 21. However, when FGF-2 was bound in heparin modified

collagen gels, co-delivery with VEGF improved blood vessel density and maturity (number of

SMA positive cells) at 21 days(101). Similarly, VEGF and PDGF delivery in PLG microspheres

led to a significant increase in vessel density and maturity at 4 weeks relative to delivery of

either single growth factor alone or bolus injections of both growth factors. Spatial control of

VEGF and PDGF delivery from PLG scaffold layers also showed improved vessel maturity as

compared to VEGF release alone(102). In these studies, sustained delivery of multiple growth

factors is considered crucial for vessel stabilization(103).

The ability of growth factor induced vascularization to improve functional cell survival has also

been tested for transplanted islets (discussed in section 5.7) and hepatocytes. VEGF releasing

alginate scaffolds were used to prevascularize liver lobes for 7 days. Implantation of primary

syngeneic hepatocytes into the pre-vascularized lobes improved cell survival compared to

implantation into control (non-VEGF) scaffolds at 12 days(104). Similarly, bFGF loaded poly-l-

lactic discs improved vascularization and engraftment of hepatocytes in syngeneic rats at 14

days, however it was noted however that majority of bFGF was released from the material by 72

hrs(105). In summary, although growth factor delivery is an attractive vascularization strategy,

delivery of systemic single growth factors is generally not successful. A promising approach is

to deliver multiple growth factors with spatial and temporal control. A variety of biomaterials

can be used to achieve controlled delivery. However, it remains to be seen which combination of

controlled growth factors will eventually lead to functional cell engraftment and improved

ischemic tissue perfusion.

32

2.5 Current Challenges with Islet Transplantation

2.5.1 Type 1 Diabetes and Islet Transplantation

In Canada, over 2 million people suffer from diabetes and 10% are diagnosed with the severe

Type 1 condition(106). Also referred to as Juvenile diabetes, this autoimmune disease is

characterized by immune mediated destruction of insulin producing (β cells) in the pancreas

which results in absolute insulin deficiency(107). Without insulin, glucose cannot be

metabolized by the body and it accumulates in the blood (hyperglycemia). Daily insulin

injections are lifesaving but they provide inadequate regulation of glucose levels and patients

typically develop serious retinal, renal and cardiovascular complications(108). Insulin therapy is

also linked with periods of hypoglycemia (low blood sugar levels) and over time patients lose

awareness of hypoglycemic onset which can be fatal(109). Only pancreas transplantation is

capable of restoring innate insulin production, reversing the hyperglycemic state and preventing

vascular complications(108). However, it is a high-risk procedure and generally only performed

in combination with kidney transplants(1, 108). Recently, islet transplantation has emerged as an

alternative to replace pancreatic tissue and restore euglycemia(1, 2, 108, 110).

Islets of Langerhans, cell clusters ranging from 50-500 μm in size, make up 1-2% of pancreatic

tissue(111). Islets have a β cell (insulin producing) core surrounded by endocrine cells

(glucagon, somatostatin and pancreatic polypeptide producing), neurons, inflammatory cells

(macrophages) and a densely fenestrated capillary bed(111). Islets are isolated from a donor

pancreas using enzymatic and mechanical disruption and are purified with Ficoll acid gradients

to remove exogenous endocrine tissue(1). Purified islets in animal models have been implanted

in a number of sites including the renal subcapsular space, immunepriviliged tissue (eyes, testes)

and under the skin. Clinically, islets are injected into the portal vein, a large blood vessel leading

into the liver, where they engraft and deliver insulin similar to native pancreas. Access into the

human portal vein is typically achieved through percutaneous transhepatic catheterization under

fluorescence guidance(1). In 2000, Shapiro et al. used the Edmonton protocol (summarized in

Fig. 2-3) to demonstrate that transplanted islets can achieve insulin independence in severe Type

33

1 diabetics(2, 110). Protocol success was attributed to optimal isolation techniques, the use of a

large dose of transplanted islets (10,000 islet equivalents/kg) and steroid free

immunosuppressants. To date, islet transplantation has treated 500 patients worldwide and on

average (60%) patients remain insulin independent for one year(1). By the 5 year mark, only

10% of patients are still insulin independent. However, even after transplanted islets fail to

correct hyperglycemia, patients have lower exogenous insulin requirements and the majority

(65%) of patients show sufficient C-peptide secretion to prevent hypoglycemia(2). Also, islet

transplantation may improve diabetes related complications such as microvascular angiogpathy,

nephropathy, and overall patient survival(112). Thus, islet transplantation has the potential to

restore endogenous insulin production and improve the diabetic condition.

Figure 2-3: Summary of procedures in islet isolation and transplantation. Donor pancreas is continuously immersed in enzyme solution and digested in a semi-automatic Ricordi chamber where solution is circulated through a 450 um mesh to prevent released islets from further digestion. Digested islet suspension is purified by computerized centrifugation in Ficoll gradients. Islets are slowly infused via a closed bag system (suspended in 200 mL media) within the portal vein while monitoring portal venous pressure. Immunosuppressive therapy consists of dacluzimab, sirolimus and tacrolium. [Adapted from Merani S, Clinical Science 2006]

34

2.5.2 Transplanted islet engraftment

A number of limitations persist with islet transplantation including the need for multiple donors

(2 to 3 pancreata) for successful treatment and the loss of transplanted islet functionality over

time. During the early post-transplant period, islets are apoptotic and a majority of the

transplanted islets (>60%) fail to engraft; the islets that do successfully engraft continue to lose

functionality over time(1, 2, 108). Although the exact cause of islet dysfunction is unknown, a

number of factors have been implicated. Isolated islets are fragile as their underlying ECM and

vasculature have been disrupted and are prone to apoptosis even during routine culture.

Transplantation of isolated islets presents a further stressful environment including host

inflammation, immune and coagulation responses, immunosuppressant toxicity, and a lack of

adequate vascularization; taken together these factors contribute to transplanted islet

dsynfunction.

2.5.3 Instant Blood Mediated Inflammatory Reaction

The earliest initiator of islet graft apoptosis is a severe inflammatory reaction, Instant Blood

Mediated Inflammatory Reaction (IBMIR) which ensues immediately (within 15 mins) after islet

exposure to portal vein blood. IBMIR is characterized by activation of the coagulation and

complement systems, rapid platelet activation and adhesion to islets, leukocyte infiltration and

eventually islet apoptosis(113-115). Transplanted islets are known to express a number of pro-

coagulation markers that initiate IBMIR. In particular, the endocrine cells (mainly the α cells) of

isolated islets display an alternatively spliced form of tissue factor (as-TF) and have procoaulant

activity in culture. After transplantation, islet expressed TF triggers the extrinsic (FVII/VIIa)

coagulation pathway (Fig. 2-4) to activate thrombin generation, platelet binding and fibrin

deposition around the islets(116, 117). Also, islets express over 50 inflammation associated

genes including monocyte chemotactic protein-1 (MCP-1), and interleukin-8 which induce

inflammation and activate the complement system(115).

35

Intrinsic Extrinsic Tissue Factor

VIIa VII

X Xa Xa

PT Thrombin

XIII XIIIa

PT, Platelets, Va

Fibrinogen Fibrin Clot

Ca++, PP

Ca++

XIIa XII

XIa XI

IX IXa

Ca++, PP VIIa

Antithrombin

Fibrin

Figure 2-4: Simplified schematic of coagulation cascade. Activation of tissue factor leads to thrombin generation and fibrin clot formation. Antithrombin inhibits thrombin and this complex (TAT) is detected in plasma as an indicator of thrombin generation. (Abbreviations: PT – prothrombin, PP- platelet phospholipids, VII – Factor VII etc.)

2.5.4 IBMIR and coagulation

The occurrence of IBMIR was initially observed in vitro when human islets were exposed to

ABO compatible whole blood within heparnized PVC tubing; in this model the coagulation

system was rapidly activated and islets were entrapped within large clots in 15 minutes.

Clinically, high thrombin-antithrombin (TAT) complex levels have been noted in the

bloodstream 15 minutes after islet infusion (into the liver) indicating the presence of

IBMIR(117). Moreover, high TAT levels immediately after transplantation correlated with low

C-peptide levels after 7 days in patients suggesting that IBMIR reduced islet graft functionality

(116). It is presumed that IBMIR results in both an immediate destruction of islets and also that

clot formation around islets prevents islets from travelling to smaller branches in the liver; the

latter has not been directly observed in vivo. Also, islets exposed to blood in vitro bind a number

36

of complement proteins and immunoglobulins including the C3b/iC3b complex indicating that

complement activation may be another mechanism by which islets are immediately

destroyed(118).

Strategies to avoid IBMIR have included islet modification to allow the surface to be more blood

compatible and administering soluble drugs in vivo to block the generation of thrombin and other

coagulation products. Heparin (known to inhibit thrombin generation) binding to the islet

surface was successful in reducing platelet binding in an in vitro blood loop model and lowered

serum TAT levels in pigs after intraportal transplantation at 15 mins(119). Similarly, human

aortic endothelial cells (HAEC) coated directly on human islet surface lowered activation of the

coagulation and complement systems in vitro; however, the improvement dependent largely on

the extent of EC coverage(120). The long term effect of heparin binding and EC coating to islet

surface was not evaluated. In another approach, tissue factor activity on the islet surface was

blocked by the addition of a human monoclonal anti-TF antibody (CNTO859) to monkey islets

in culture. Although blocking TF in monkeys with a human antibody was only partially

successful in reducing IBMIR in vivo (no significant difference in TAT levels within an hour

after transplantation), minimal mass (5000 IEQ/kg) transplantation in allogeneic

immunosuppressed monkeys led to improved graft function (not statistically significant) at 2

months post-transplant (121).

The majority of strategies used to circumvent IMBIR have evaluated the use of soluble drugs to

prevent coagulation activation in vivo. Initial studies with heparin in combination with soluble

complement receptor 1 (sCR1) demonstrated that blocking both coagulation and complement

activation prevented platelet adhesion, clot formation and leukocyte infiltration into islets in an

in vitro blood flow loop but neither heparin or sCRI alone were effective in inhibiting

IBMIR(122). Similarly, the addition of Melagatran (thrombin inhibitor) and low molecular

weight dextran sulfate (LMW-DS, tissue factor inhibitor) to blood exposed to islets reduced TAT

generation, platelet consumption and inhibited macroscopic clotting in an in vitro blood loop

model(122, 123); interestingly the coagulation markers also prevented complement activation

suggesting that the complement system may only be activated as secondary to the coagulation

pathway. Perhaps the most successful technique to modulate IBMIR has been through

administration of recombinant activated protein C (APC), a physiological anti-coagulant released

37

by EC that inhibits thrombin production and regulates the inflammatory process. Murine APC

given prior to syngeneic intrahepatic islet transplantation improved islet graft survival, reduced

fibrin deposition and leukocytes infiltration in transplanted islets(124), the decrease in

inflammation was a secondary benefit to the coagulation benefit. Although these approaches

highlight the multiple benefits of reducing thrombin generation, they pose clinical challenges as

the drugs impair coagulation homeostasis and may induce bleeding.

2.5.5 IBMIR and inflammation

A secondary result of the coagulation mechanism is activation of the inflammation response.

Transplanted islets can activate liver macrophages (Kupffer cells) which in turn secrete a host of

inflammatory cytokines that cause islet damage. Also, endothelial cells of the hepatic sinusoids

are activated in response to transplanted islets and recruit macrophages and leukocytes(124).

Inflammatory cytokines such as interleukin-1(Il-1ϐ), tissue necrosis factor α (TNF-α), and

interferon-γ (IFN-γ) are particularly toxic to transplanted islets and have been noted shortly after

clinical islet transplantation(113). Il-1ϐ decreases insulin biosynthesis in islets and can also

cause islet death; while Il-1ϐ alone simulated islet death in rodent models, Il-1ϐ in combination

with TNF-α or IFN-γ was required for human islet toxicity. Il-1ϐ damage is mediated mainly by

Il-1ϐ activation of the inducible nitric oxide synthase (iNOS) pathway on islets which in turn

releases the free radical nitric oxide (NO). NO production has been implicated in decreasing

insulin synthesis from ϐ cells, breaking DNA strands to induce apoptosis and mediating necrosis

of both rodent and human islets(125).

Kupffer cells, sinuosdial endothelial cells and transplanted islets are all known to produce NO

resulting in cytokine mediated islet damage(126), thus strategies to prevent inflammation have

looked at modifying all three types of cells. Depletion of macrophages was associated with

lower levels of plasma Il-1ϐ, and NO after islet transplantation which resulted in longer graft

survival in allogeneic rats(127). Meanwhile reduction of EC activation (as measured by plasma

levels of adhesion molecules) by APC administration correlated with preservation of

transplanted islet function and decreased islet apoptosis in syngeneic mice(124). Also, directly

interfering with islet production of cytokines by either suppressing iNOS activity(128), blocking

38

Il-1ϐ function or blocking the p38 pathway (known to prevent Il-1ϐ, TNF-α and Il-6 secretion by

macrophages) in human islets has led to improved graft function in athymic recipients. When

the p38 pathway was blocked, LPS induced islet production of pro-inflammatory mediators

(TNF-α, iNOS, COX-2) and islet apoptosis was prevented in culture suggesting that these

strategies are directly mitigating the inflammatory stresses(129). Similarly, treating islet cultures

with lisofylline (LSF), a synthetic anti-inflammatory compound known to reduce the

inflammation response to stimuli, prevented inflammatory mediated human islet apoptosis and

fewer LSF-treated islets were needed for reversing diabetes in SCID mice(130). The strategies

to prevent inflammation by manipulating islets in vitro appear promising however it remains to

be seen how these strategies will translate to an immunocompetent model.

2.5.6 Immune Regulation and Immunosuppressant Toxicity

Islet transplantation in a clinical allogeneic setting faces an additional stress of immune reactivity

and immunosuppressant toxicity. In spite of immunosuppression, there are still reports of T cell

reactivity to transplanted islets due to HLA mismatch and recurrence of T cell autoimmunity

which can lead to islet graft damage. In fact, clinical reports indicate that a lower cellular allo-

and auto-reactivity in patients correlate with improved insulin production after islet

transplantation(131, 132). Immunosuppressant therapy allows for acceptance of allogeneic grafts

by interfering with the normal immune cells (mainly T cells) recognition and response. The

Edmonton protocol immunosuppressant regimen consists of Dacluzimab (anti-CD5 antibody),

Sirolimus (mTOR inhibition) and Tacrolimus (calcerniun inhibitor)(2). Both Sirolimus and

Tacrolimus have shown to be directly toxic to islets in vitro, can cause diabetes in normal rats

and are associated with insulin resistance of transplanted islets in allogeneic rats. Moreover,

continual immunosuppression is affiliated with lack of islet function over time(133). These

studies highlight that modulation of the immune response while minimizing islet toxicity is a key

consideration for long term islet graft survival in the clinical setting.

39

2.5.7 Revascularization of transplanted islets

A significant contributor to islet graft apoptosis is inadequate islet revascularization both during

the early transplant period and long term which places transplanted islets under continual

hypoxic stress. Hypoxic stress induces islet apoptosis, decreases glucose stimulated insulin

release and may in turn lower the angiogenic capacity of intraislet EC(134). Native islets are

highly vascularized, receive greater blood flow than exogenous pancreatic tissue and have high

demands for oxygen consumption. They contain a dense network of fenestrated capillaries and

are located adjacent to major blood vessels to efficiently secrete insulin(111). Although islets

only comprise 1-2% of the pancreas, they receive 5-15% of pancreatic blood flow. During

isolation, islets lose much of the internal endothelium and rely on host revascularization for

blood supply which can take upto two weeks and eventual revascularization is still insufficient to

support the metabolic demands of the islet tissue.

The majority of vessel development around transplanted islets is via in-growth of blood vessels

from the host. Generally, the time for re-vascularization is lengthy irrespective of transplant site

and while blood vessels appear within islet grafts 3-5 days after transplantation, blood flow

establishment to islets can take up to 14 days(7). During this revascularization process, a

majority of the transplanted islets are hypoxic and secrete VEGF which likely stimulates

angiogenesis. A detailed view of islets transplanted under a kidney capsule showed that islets

assemble close to pre-existing host vessels and transform these vessels to an angiogenic state at

day 4, host vessels infiltrate the periphery of islet grafts by day 7 and functional vessels

connecting intraislet capillaries to host vasculature are finally established by 14 days(135).

Recently it was discovered that intraislet EC also contribute to revascularization. Generally,

isolated islets tend to lose intraislet endothelial cells rapidly, however, when intraislet EC were

preserved they migrated to form chimeric functional vessels in the periphery of islet grafts at

least until 3 weeks transplantation (136, 137). Although donor intraislet EC contribution to

vascularization has only been demonstrated in mice, human intraislet EC are similarly

angiogenic in vitro and may similarly contribute to revascularization(136). However, even after

revascularization has been complete, long term angiogenesis around the islets is insufficient.

Transplanted islets (mouse or human) transplanted under the kidney capsule or intrahepaticaly

40

exhibit lower intraislet and peripheral islet vascular density, receive decreased blood flow and

experience decreased oxygen tension in comparison to endogenous islets(7, 138).

To improve vascularization around transplanted islets, growth factor delivery, in particular

VEGF has been explored (134, 139-141). VEGF (VEGF-A isoform in particular) has been

implicated as a key factor in establishing islet vasculature both in development and after

transplantation(142). Some methods of simply adding VEGF protein to islet culture prior to

transplantation have not shown to be successful in improving islet revascularization. Attempts

to pre-treat mouse islets with VEGF or FGF-2 in vitro did not improve transplanted islet vascular

density or functionality in diabetic mice(141). Similarly, encapsulation of VEGF with islets in a

collagen matrix increased blood vessel density around transplanted islets after 14 days, but there

was no difference in vessel density after 1 month between control and VEGF encapsulated

islets(143). On the other hand, controlled overexpression of VEGF (under rat insulin promoter)

was successful in improving short term (14 days) vascular density, blood flow and islet

mass(134) in SCID mice.

Improving islet vascularization should translate to improved islet engraftment and a few studies

have demonstrated this success. Transplantation of islets with multiple growth factors (VEGF,

and heparin bound FGF) loaded scaffolds improved vascular density and islet engraftment as

noted by a larger number of diabetic animals cured, albeit the average time to reverse

hyperglacemia was quite long (27 days)(144). Also, exogenous VEGF delivery to transplanted

islets improved oxygen tension at the transplant site (kidney capsule) and prolonged euglycaemia

in syngeneic aniamls and delayed rejection in allogeneic recipients(139). Similarly,

transplantation of islets with elevated VEGF production increased vessel density and improved

glucose control for 16 days when compared to mock transduced control islets(140); non-

transduced islets were not compared here so it is still not clear how this strategy compares to

islets alone. In general, attempts to improve vascularization by VEGF delivery look promising

in improving the short term vessel density and some islet functionality. But as discussed earlier,

VEGF growth factor delivery leads to leaky mature vessels and since long term vascularization

has not been evaluated to date, it is unclear whether VEGF delivery will enable a clinical islet

transplantation strategy.

41

Other ways to improve islet revascularization have included the addition of supporting cells that

are known to enhance vascularization. Mesenchymal stem cells (MSC) are one such cell type

that have angiogenic properties in vivo. Bone marrow derived mesenchymal stem cells (MSC)

co-transplanted with islets under the kidney capsule improved glycemic control and increased

vessel density in syngeneic rats until 39 days(145). Similarly, mobilization of bone marrow

derived angioblasts to islets transplanted intrahepatically improved vascularization and glucose

control(146). Although co-transplantation of islets with endothelial cells has been explored as a

strategy to prevent adverse inflammatory reactions, the use of primary endothelial cells for islet

vascularization has not been attempted.

Combined, these approaches highlight the short term impact of angiogenic strategies in

preserving functional islet mass post transplantation. None of the approaches to date address the

immediate post-transplant period in which the majority of islets are hypoxic, and it is unclear

whether growth factor delivery can lead to an accelerated vascularization. More importantly,

islet vascularization strategies have not been tested under the normal clinical stress associated

with intrahepatic transplantation combined with immunosuppressant toxicity. As mentioned,

immunosuppressants are known to be anti-angiogenic in vivo(147). Thus, it remains to be seen

whether improved vascularization can enable long term vascularization and islet engraftment in

a clinical setting.

The scope of this thesis is to evaluate the delivery of endothelial cell covered micron-sized

tissues as a vascularization strategy to support islet engraftment in diabetic recipients. Building

upon the work by others in the EC transplantation literature, preliminary studies will look at

human EC transplantation in immunocomprimised recipients. Next, EC transplantation will be

explored in a clinically applicable, allogeneic immunosuppressed model. Finally, EC

transplantation in combination with islets will be tested in both an allogeneic immunosuppressed

and a syngeneic diabetic model.

42

3 Fate of Endothelialized Modular Constructs Implanted

in an Omental Pouch in Nude Rats

Published in: Tissue Eng Part A. 2009; Oct 15(10):2875-87.

Authors: Rohini Gupta, Nico Van Rooijen , and Michael V. Sefton.

Contributions: R.G., M.V.S. designed research. R.G. performed research, analyzed data. R.G.,

N.V.R and M.V.S contributed new reagents/analytic tools. R.G. and M.V.S. wrote the

manuscript.

43

3.1 Abstract

Modular tissue engineering is a novel micro scale approach that aims to assemble tissue

constructs with inherent vascularization. We transplanted endothelialized modules (sub-mm

sized collagen gel cylinders covered with HUVEC on the outside surface) in the omental pouch

of nude rats to characterize remodeling of the collagen gels and the fate of the transplanted

HUVEC. Endothelialized modules randomly assembled in vivo to form channels among

individual modules that persisted for at least 14 days. Transplanted HUVEC migrated and

formed primitive vessels in these channels, however host inflammation limited HUVEC survival

beyond 3 days. Temporary depletion of peritoneal macrophages (by treatment with clodronate

liposomes) prolonged the survival of HUVEC derived vessels to 7 days and some vessels

appeared to be perfused with host erythrocytes and invested with host vascular cells (either rat

von Willebrand Factor or smooth muscle α-actin positive cells). Despite treatment, HUVEC

were presumed to be still subject to immune rejection. The presence of primitive HUVEC

derived vessels is encouraging in this first in vivo study of the modular approach, in this partially

immune-compromised animal model. It suggests that with appropriate attention to the host

response to transplanted endothelial cells and improved vessel survival, cells that would be

embedded in modules could be adequately perfused.

44

3.2 Introduction

A critical challenge for engineering large scale tissue replacements is to ensure that the

transplanted tissue grafts are well vascularized within the host. Without an internal vascular

supply, tissue survival is limited by diffusion of essential nutrients and oxygen. Hence, only

cells within a diffusion distance of 150-200 μm of the surrounding blood vessels are viable, and

there can be significant cell loss at the core of transplanted tissues without vascularization(25,

26). This phenomenon has been documented for several tissue engineering applications(141,

148).

New blood vessel formation in the adult occurs typically through angiogenesis and there has

been much effort to promote therapeutic angiogenesis through delivery of growth factors (e.g.

VEGF, bFGF) but multiple growth factors are likely necessary to produce a mature

vasculature(149). Several groups have shown that transplantation of primary endothelial cells,

particularly human umbilical vein endothelial cell (HUVEC) in immunocompromised rodents,

can induce mature vessels. For example, HUVEC transfected with an antiapoptotic gene Bcl-2,

suspended in a collagen-fibronectin gel, and transplanted into the abdominal wall of SCID mice

developed into a complete microvascular bed capable of assuming arterial, venous and capillary

EC phenotype(55, 64). Increased vessel density was shown to increase perfusion in an ischemic

hindlimb model(64) and also to effectively vascularize skin substitutes(88). Others have

transplanted a mixture of HUVEC with mesenchymal precursor cells to form functional blood

vessels that integrated with the host vasculature and were stable for 1 year(3). In another

example, Levenberg et al. demonstrated that HUVEC derived vascularization of 3D skeletal

muscle tissue increases perfusion and survival of muscle constructs in vivo(4). Combined, these

studies suggest that primary EC transplantation can drive vascularization in vivo.

Modular tissue engineering is a means of assembling constructs with uniform cell density,

scalability, mixed cell populations and vascularization(8). Functional cells are embedded in sub-

millimetre sized collagen rods, and the outside surface is covered with endothelial cells (EC).

We refer to the collagen rod with embedded cells and EC composite unit as a module. Many

modules are used to fill a tissue cavity creating interconnected channels by virtue of the spaces

among the randomly packed modules (Fig. 3-1). HUVEC grow to confluence on the surface of

45

the collagen modules and remain non-thrombogenic when exposed to blood flow in vitro(8). By

varying the type of embedded cells, it is expected that modular tissue engineering can be used for

a number of applications; HepG2 cells(9) and smooth muscle cells(150) have been the subject of

reports to date.

Figure 3-1: Schematic representation of a modular construct. Individual collagen gel cylinders (modules) normally with embedded cells are covered with endothelial cells on the outside surface. Modules randomly pack in situ and tissue spaces between individual modules form channels that allow for blood flow. In this manner, embedded cells are expected to be perfused with blood within the modular construct.

Here, we report on the fate of HUVEC covered modules implanted in a surgically created

omental pouch. No cells were embedded within the modules in the studies reported here. We

chose the omentum pouch as it is well vascularized, permits a large number of implants and can

be easily retrieved for histological analysis(151). In pilot studies, we noted an early loss of

transplanted HUVEC (in the nude rats) and significant inflammatory cell infiltration around the

module implants. In order to prolong HUVEC survival, we explored the use of clodronate

liposomes to temporarily deplete peritoneal macrophages. Clodronate (dichloromethylene-

bisphosphonate) is encapsulated in liposomes and when ingested by phagocytotic cells causes

them to be apoptotic(152). Repeated injections (2 or 3 days apart) of clodronate liopsomes have

shown to successfully deplete (temporarily) peritoneal and omental macrophages in rats(153).

For both untreated and treated rats our aim was to assess i) remodelling of the collagen modules

ii) HUVEC fate in vivo and iii) host inflammatory response to implanted modules.

46

3.3 Materials and Methods

3.3.1 Cells

Pooled human umbilical vein endothelial cells were purchased from Cambrex Corporation (East

Rutherford, NJ) and maintained in endothelial cell growth media with 2% FBS (EGM-2,

Cambrex) at 37°C and 5% CO2. Cells were used between passages 3-5.

3.3.2 Module Fabrication

Modules were prepared without embedded cells as before using bovine Type 1 collagen (3.1

mg/mL, Cohesion Technologies, Palo Alto CA), gelled (37°C, 60 minutes) in sterile 0.71 mm ID

polyethylene tubing (PE60, Intramedic - BD Canada, Oakville, ON)(9). Tubing was cut into

small pieces (~2mm long x 0.6mm diameter) using a custom automatic cutter and collected in

HUVEC medium. Collagen modules were separated from the outer tubing with gentle vortexing.

HUVEC (2.0x 106) were seeded dynamically onto the surface of 1 mL of modules (produced

using 3 m of tubing) for 45 min on a low speed shaker and incubated for 7 days prior to implant

(HUVEC contracted collagen modules to ~1mm long x 0.5 mm diameter). Collagen only

modules were prepared as above but without endothelial cell seeding.

3.3.3 Module transplants

Adult male (5 weeks of age) nude (athymic) rats (200-250g) were purchased from Charles River

Laboratories (Wilmington, MA). They were individually housed in sterile cages and fed ad

libitum. The study was approved by the University of Toronto animal care committee. Animals

were divided into 2 groups: untreated and clodronate liposome-treated. Treated animals

received two injections of 1 mL clodronate liposomes into the peritoneal cavities, 4 days and 1

day prior to surgery with a 23G needle(153). Clodronate liposomes were prepared as previously

described(153) and clodronate was a kind gift from Roche Diagnostics GMbH (Mannheim,

Germany). Untreated animals received no injections. For module implants, animals were

anesthetized and an upper midline incision exposed the abdomen. The greater omentum was

spread out and 7-0 silk suture was run along the edges and the top to create a pouch with a small

opening. Modules (0.5 mL, with or without HUVEC) suspended in PBS were gently delivered

47

by a sterilized micropipette (1 mL pipette tip) and allowed to settle. The pouch was folded over

and sutured to secure the modules inside. The muscle layer was closed with 6-0 Vicryl sutures

and the skin layer sutured with 4-0 nylon sutures. Animals were given a 0.3 mL injection of

0.25 mg/mL Buphrenophrine solution for recovery. For both untreated and clodronate liposome

treated groups, animals were transplanted with endothelialized modules for 3 (n=4), 7 (n=5), and

14 (n=5) days. Collagen only modules were transplanted for 3 or 7 days in clodronate liposome

treated animal (n=1) and untreated rats (n=3).

3.3.4 Histology and immunostaining

At explant, animals were sacrificed and the omental pouch was excised in 4% neutral buffered

formalin (Sigma Aldrich, Oakville, ON) and fixed for 48 hrs. Tissue samples were embedded in

paraffin wax and five 4 µm sections were cut at 3 levels (each level was 100µm apart) within the

block. Sections were processed and stained for hematoxylin and eosin (H&E, Fisher), Masson

trichrome (Fisher), and various antibodies as outlined in Table 3-1. Apoptotic cells were

identified by TUNEL staining. For immunostaining, endogenous peroxide and biotin activity

was blocked with 3% hydrogen peroxide (Fisher) for 15 minutes and avidin/biotin blocking kit

(SP-2001, Vector) and sections were developed with 30 minutes each of a biotinylated-linking

reagent (ID labs, London ON) and horseradish peroxidase-conjugated ultrastreptavidin labeling

reagent (ID labs). After washing well in PBS, color development was done with freshly prepared

NovaRed solution (SK-4800, Vector). Finally, sections were counterstained lightly with Mayer’s

hematoxylin, dehydrated in alcohol, cleared in xylene and mounted in Permount (Fisher).

Fluorescent sections (SMA and vWf) were followed by a streptavidin -FITC reagent (Vector:

SA-5001) for 30 minutes and cover-slipped with Vectashield (Vector: H-1000) mounting

medium for fluorescence. All sections were viewed with a Zeiss Axiovert light and fluorescent

microscope equipped with a CCD camera.

3.3.5 Histology quantification

UEA-1 stained positive cells at Day 3, 7 and 14 were counted using a Chalkley method(154)

adapted from the tumour angiogenesis literature. At low magnification (100x), 3 ‘hotspots’ were

identified as areas containing the most positively stained cells in each section. At each hot spot,

a 25-point Chalkley eyepiece graticule was applied (Chalkley grid area = 0.196 mm2) at 200x

48

magnification and the eyepiece was rotated to align the maximum number of dots with UEA-1

positive structures (structures could be vessels or single cells). The dots that overlaid UEA-1

positive structures were counted and the mean of the three counts was used for statistical

analysis. The microvessel density (MVD) method was applied to count UEA-1 positive vessels

(with a defined lumen). For each animal, the number of vessels in the 3 hot spots (same as

identified in the Chalkley method) was counted at 400x magnification. Average of the three

MVD counts was used for statistical analysis.

Macrophages (ED1 or ED2 positive) were counted in five representative omentum sections at

400x magnification and the average number for each of four animals in each treatment group

was recorded. The degree of inflammation was then described as either: mild (0-50

macrophages), moderate (50-100 macrophages) or severe (100-150 macrophages).

Table 3-1: List of monoclonal antibodies and their target antigens.

Antibody Supplier Conditions Target Antigen Ulex Europaeus Agglutinin I lectin (UEA-1)

Vector Laboratories, Burlingame CA

Heat induced temperature retrieval, 1:300 for 1 hr

Human endothelial cells (does not stain rat endothelial cells)

ED1 (MCA341) and ED2 (MCA342)

AbD Serotec, Raleigh, NC

1% pepsin pre-treatment, 1:600 for 1 hr

Rat macrophages and monocytes (does not stain human cells)

Von Willebrand factor (CL20176A-R, vWf)

Cedarlane, Burlington ON

After UEA-1 application, 1:5000 overnight

Rat endothelial cells and platelets (does not stain human cells)

α-Smooth muscle actin (AB5694, SMA)

Abcam, Cambridge, MA

After UEA-1 application, 1:1000 overnight

Rat smooth muscle cells (does not stain rat endothelial cells)

TUNEL assay (DNA Polymerase 1 Large (Klenow)

Promega 1% pepsin pre-treatment, Cocktail for 1 hr at 37°C

Apoptotic cells (stains fragmented DNA of both rat and human cells)

3.3.6 Statistical Analysis

A one way Anova with Tukey post hoc analysis was applied to compare means between

multiple groups. Data was considered statistically significant at p <0.05. All analysis was done

with Statistica Version 5.1 software (Statsoft, USA).

49

3.4 Results

3.4.1 Remodeling of collagen gel modules

Implanted modules were identified with Masson trichrome stain (collagen stained green).

Endothelialized modules retained their cylindrical shape and randomly assembled such that

there were channels (albeit often containing cells) among individual modules that persisted for

at least 14 days in both untreated (Fig. 3-2a-c) and clodronate liposome treated (Fig. 3-2d-f) rats.

Low magnification pictures for both untreated and clodronate liposome treated animals show

that channels were formed among the majority of the implanted modules. In some implants a

large number of modules assembled in one location (Fig. 3-2b) while in others, modules were

more dispersed (Fig. 3-2c). While ~400 modules were implanted, only about 100-200

modules/animal could be found by histology; many modules were likely lost on filling the

pouch or during tissue processing. Module assembly, distribution and channel spacing was not

affected by treatment with clodronate liposomes (Fig. 3-2d-f). Meanwhile, collagen alone (no

EC) modules remodeled significantly and began to degrade as early as Day 3 (Fig. 3-2g).

Similar remodeling and degradation occurred with collagen only module implants in 7 week old

nude rats (without clodronate treatment – results not shown) at days 3, 7 and 14 and in all cases,

collagen modules lost their cylindrical shapes and did not form channels as seen with

endothelialized modules.

A mild inflammatory cell infiltration was observed with collagen only implants. A similar

response was observed in sham (no implant) operated animals (results not shown) suggesting

that some of the observed inflammatory response is largely due to the trauma of suturing the

omentum. With all endothelialized module implants, some level of cellular infiltration in the

module derived channels could be seen at all time points (Figs. 3-2,3). Many of the cells

appeared to be erythrocytes (Fig 3-3), but there were also several inflammatory cells. With

untreated rats, there was a heavier cell infiltration at day 3 (Fig. 3-3a) which was reduced by day

7 (Fig. 3-3b) and few cells were detected by day 14 (Fig. 3-3c). Meanwhile, with clodronate

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liposome treatment, mild cellular infiltration was detected around the modules at day 3 (Fig. 3-

3d) which was greater by day 7 (Fig. 3-3e) but reduced again by day 14 (Fig. 3-3f).

Figure 3-2: Trichrome staining showed that in both untreated (a-c) and clodronate liposome treated rats (d-f), endothelialized modules randomly assembled to form channels (indicated by white arrows) in situ. Channels were the spaces, often cell-filled, between individual modules and could be seen as long as 14 days in both groups. Scale bar = 500 μm. In comparison collagen only modules without EC (indicated by black arrows) in clodronate treated rats degraded as early as 3 days (g) and continued to remodel until day 7(h); channels were not apparent without EC. Scale bar = 200 μm

51

Figure 3-3: High magnification trichrome sections show that in untreated rats, cells (mainly erythrocytes and some inflammatory cells, white arrows) infiltrated module derived channels at day 3(a). There was only moderate infiltration at day 7(b) which decreased by day 14(c). In clodronate liposome treated rats, there was little cellular infiltration at day 3(d), more at day 7(e) and minimal at day 14(f). Scale bar = 25 μm

3.4.2 Transplanted HUVEC

Transplanted HUVEC were identified with UEA-1 lectin (Fig. 3-4,5), a human EC specific

marker. Collagen only implants did not stain with UEA-1, confirming that the lectin did not

stain rodent cells (Fig. 3-4g-h). In untreated rats, HUVEC formed primitive microvessels (i.e.,

UEA-1 positive structures with a defined lumen) in module derived channels at 3 days (Fig. 3-

4a). A majority of these vessels disappeared after 7 days (Fig. 3-4b) and by day 14, there were

few UEA-1 positive cells observed (Fig. 3-4c). These vessels are seen in higher magnification in

Figure 5 where some of the vessels included erythrocytes in their lumens (Fig. 5a). In clodronate

52

liposome treated rats, HUVECs were apparent on the surface of the modules at day 3 (Fig. 3-4d)

and by day 7, HUVECs migrated into module derived channels to form primitive vessels (Fig. 3-

4e). These UEA-1 positive vessels of varying sizes, a majority of which contained erythrocytes,

are shown in Fig. 3-5d. Similar to control rats, HUVEC derived vessels receded by day 14 and

few UEA-1 positive cells were observed (Fig. 3-4f). In all cases, HUVEC derived vessels were

located in close proximity to the modules and there were no UEA-1 positive cells detected

elsewhere in the omentum. There was no apparent effect of location within the implant on EC

survival, at least for the small 0.5 mL implants used here.

The Chalkley method is an established method of quantifying angiogenesis and was used to

count UEA-1 positive structures (vessels and single cells). Consistent with the images, the UEA-

1 cell population was high at day 3 and declined significantly (p<0.05) after 14 days in both

groups (Fig. 3-4i), presumably due to a macrophage response (see below). At day 7, there were

generally more UEA-1 positive cells in clodronate liposome treated animals, however due to

large variations within the group, none of the differences were statistically significant. On the

other hand, the vessel structures at the early time points were markedly different between the

treated and control groups as discussed below. The microvessel density (MVD, Fig. 3-5e)

method was to quantify UEA-1 positive vessels (i.e. those structures that also contained lumens).

There were significantly (p<0.05) fewer vessels on day 7 than on day 3 in the untreated group.

An opposite trend was observed for clodronate liposome treated animals as the average MVD

count increased (but not statistically significantly) from day 3 to 7.

53

Figure 3-4: UEA-1 stained sections of endothelialized modules from untreated rats show vessel formation at a) day 3 b), loss of a majority of vessels by day 7 (c) and few UEA-1 cells at day 14. Sections from clodronate treated animals show UEA-1 cells on the surface of modules at d) 3 days e) a number of HUVEC derived vessels at day 7 (f) and few UEA-1 positive cells by 14 days. g-h)Collagen only implants confirm that UEA-1 lectin was HUVEC specific . Arrows show examples of UEA-1 positive cells, scale bar = 100 μm. (i) Mean of Chalkley grid counts show that UEA-1 counts were significantly lower (p <0.05) at day 7 and 14 for untreated animals and at day 14 in the clodronate treated group (relative to day 3). Data is mean of each group +/- SE.

54

Figure 3-5: High magnification (400x) UEA-1 sections at days 3 and 7. In untreated rats, numerous vessels (arrows) were located at a) day 3 (b), while few vessels were found by day 7. In clodronate liposome treated rats, few vessels were detected at c) day 3 d) while numerous vessels of varying sizes were detected at day 7 with erythrocytes present in their lumen. Scale bar = 25 μm. (e) Microvessel density (MVD) counts were significantly lower in the untreated group at day 7 relative to day 3 (p<0.05). For clodronate liposome treated animals, MVD counts increased (not significant) from day 3 to day 7. The mean MVD count for clodronate liposome animals at day 7 was higher (but not significantly, p =0.17) than the corresponding untreated group. Data is represented as mean of MVD count (n=4 for day 3 and n = 5 for day 7) +/- SE.

55

3.4.3 Vessel Maturity

Sections were double stained with UEA-1 lectin (human endothelial cells) and either rat

FVIII/vWf (rat endothelial cells - Fig. 3-6) or smooth muscle α-actin (rat smooth muscle cells –

Fig. 3-7). At day 3 in both untreated and clodronate liposome treated rats, there were no host

(rat) vWF positive or SMA positive cells associated with UEA-1 vessels and single cells. At day

7, while the majority of UEA-1 stained vessels were not associated with rat cells, some UEA-1

vessels (i.e., with human EC) also contained rat endothelial cells (Fig. 3-6a,c) and were

surrounded by rat smooth muscle cells (Fig. 3-7a,c) in both untreated and treated animals. As

noted earlier, there were generally more UEA-1 vessels with clodronate liposome treatment at

day 7; however the extent of association of host cells was not dependent on treatment. By day

14 in both groups, although only few UEA-1 positive cells and vessels were detected, these few

UEA-1 cells were generally in close proximity to rat endothelial cells (Fig. 3-6b,d). In a few

instances, rat smooth muscle cells (Fig. 3-7b,d) were also seen around UEA-1 positive vessels.

Also, a number of rat vWf or SMA positive vessels (without UEA-1 cells) were seen near

endothelialized modules at day 14.

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Figure 3-6: Double staining with UEA-1 lectin (red) and rat von Willebrand factor (vWf, green) of endothelialized modules in untreated (a-b) and clodronate liposome treated (c-d) animals at days 7 and 14. Images show UEA-1 positive structures (red) invested with rat vWF positive cells (green) at day 7. At day 14, there were few, isolated UEA-1 positive cells, but these were associated with vWf positive rat cells. vWf stained vessels (without UEA-1 staining) are particularly noticeable in panel (d) – clodronate liposome treatment, day 14. Dashed lines show vessels, with and without UEA-1 staining. Scale bar = 25 µm.

57

Figure 3-7: Double staining with UEA-1 lectin (red) and rat smooth muscle α-actin (SMA, green) of endothelialized modules in untreated (a-b) and clodronate liposome treated (c-d) animals at days 7 and 14. Images show UEA-1 positive structures (red) invested with SMA positive cells (green). At day 7 in both untreated (a) and treated (c) animals some UEA-1 vessels were surrounded by SMA positive cells (as outlined); these structures were larger in the clodronate liposome treated animals. By day 14 (b, d) there was a close proximity between the two cells but the SMA positive cells did not surround the UEA-1 cells (arrows) as at day 7. Fig 7d also shows many SMA positive cells, not associated with the UEA-1 cells. Dashed lines show vessels, with and without UEA-1 staining. Scale bar = 25 µm

3.4.4 Apoptotic cells

Apoptotic cells were identified with a TUNEL stain. There was no TUNEL staining detected

around endothelialized modules in untreated rats at days 3 and 7. With clodronate liposome

treatment, there were no apoptotic cells seen at day 3; however, there were a few apoptotic cells

near endothelialized modules at day 7 (Fig. 3-8). TUNEL staining was not seen elsewhere in the

omentum.

58

Figure 3-8: TUNEL staining (brown nuclei) of animals treated with clodronate liposome show occasional apoptotic cells around endothelialized modules at day 7. Scale bar = 25 µm

3.4.5 Inflammatory cells

Inflammation around endothelialized module implants (Fig. 3-9) was characterized by rat

macrophage markers, ED1 (expressed in non-activated macrophages and dendritic cells) and

ED2 (expressed in activated macrophages)(155). Results shown are with the ED2 marker and

similar trends were seen with ED1 staining. A large number of macrophages were detected in

untreated rats at all time points (Fig. 3-9a-c). In clodronate liposome treated rats (treated 4 days

and 1 day before module implantation), there were few macrophages identified at day 3 with

greater numbers at day 7 and finally full macrophage repopulation by Day 14 (Fig. 3-9d-f

respectively). Similar results were observed with collagen only implants in clodronate liposome

treated rats (results not shown): there was minimal macrophage presence at day 3 and

macrophage repopulation was detected at day 7. Five representative ED2 sections for 4 animals

in both untreated and clodronate liposome treated groups (Table 3-2) were counted and there was

a significant reduction in macrophage numbers with clodronate liposome treatment at day 3. In

both untreated and clodronate liposome treated rats, the number of macrophages peaked at day 7.

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Figure 3-9: ED2 stained sections of endothelialized modules show high macrophage infiltration in untreated rats at day 3, 7 and 14 (a-c). For clodronate liposome treated rats, macrophages were minimal at day 3 (d), but began to repopulate at day 7 (e) and tissue is fully repopulated by day 14 (f). Scale bar = 100 μm

Table 3-2: Macrophage accumulation around endothelialized modules in clodronate liposome treated and control rats at day 3, 7, 14. When compared to untreated animals, there were fewer (statistically significant) ED1 and ED2 positive macrophages with clodronate treatment at day 3, (** = p<0.01). At the later time points, although there were generally fewer macrophages with clodronate treatment, these differences were not statistically significant (day 7 p = 0.07, day 14 p = 0.12). Data reported is average of each group +/-SEM.

Day Clodronate liposome Treated Untreated

ED1 ED2 ED1 ED2

3 65.9±7.9** 30.4±5.9** 107.4±7.2 82.0±11.0

7 81.5±5.0 78.0±10.8 102.3±11.2 99.0±18.1

14 68.0±8.1 47.9±10.1 85.8±6.0 72.9±9.3

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3.5 Discussion

Fabrication of viable 3D tissue is dependent on sufficient vascularization in vivo. We have been

exploring a modular tissue engineering strategy which incorporates intrinsically an internal

“vasculature” and which can be used with a variety of functional cells. These studies were the

first exploration of the strategy in vivo. There was substantial remodeling and agglomeration of

the collagen modules without endothelial cells. This was not apparent with the endothelialized

modules (Fig. 3-2). Collagen modules are larger and softer than those with endothelial cells,

since the HUVEC contract the gel and this likely contributed to the observed remodeling. More

importantly, there were channels separating the HUVEC bearing modules, suggesting that if the

channels were connected to the host vasculature, blood could reach all implanted modules.

Transplanted HUVEC also appeared to have migrated from the surface of the modules to form

primitive vessels within the module derived channels (Fig. 3-5). This type of remodeling was

encouraging since this suggests that individual modules (and eventually embedded cells) will be

close to a vascular supply.

With UEA-1 staining, we were able to distinguish and enumerate transplanted HUVEC. At day

3, HUVEC derived microvessels (i.e UEA-1 positive structures with a lumen) appeared to

consist completely of human endothelial cells in both untreated (Fig. 3-5a) and clodronate

liposome treated rats (Fig. 3-5c); UEA-1 does not stain rat endothelial cells and vessels were not

positive for rat vWf or SMA. At day 7, some HUVEC derived vessels were also associated with

host (rat) smooth muscle (Fig. 3-7) or endothelial cells (Fig. 3-6) but by day 14 the majority of

UEA-1 vessels had receded and very few UEA-1 positive cells were observed Also, some

vessels were invested with erythrocytes suggesting that the vessels had anastomosed with host

vasculature (Fig. 3-5d). Interestingly in both groups at day 14, the few detectable UEA-1

positive cells were almost always seen in close proximity to rat endothelial or smooth muscle

cells suggesting that transplanted cells may need to be supported by host cells for prolonged

survival (Fig. 3-6d, 3-7d). Also at day 14, a number of host vessels (rat SMA and FVIII positive

cells, not necessarily with UEA-1 positive cells) were seen inside the module derived channels

suggesting that vascularization of the modules with host cells was occurring (even though the

majority of HUVEC did not persist).

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The poor survival of HUVEC vessels in our study was surprising since others have implanted

human dermal microvascular endothelial cells in PLGA scaffolds in SCID mice and showed that

the human cells begin to form vessels at day 3, anastomose (accumulate erythrocytes) with host

vasculature at day 7 and were invested with supporting host cells that persisted until 28

days(156). On the other hand, others have shown that it is necessary to co-transplant HUVEC

with supporting cells (mesencyhmal precursors(3) or embryonic fibroblasts(4) or to transfect the

cells with an anti-apoptotic gene, Bcl-2(157) in order to form chimeric HUVEC/murine vessels

in SCID mice for extended period of time. We checked for apoptosis with TUNEL staining in

clodronate liposome treated rats (Fig. 3-8) and noticed some TUNEL staining around the

modules at day 7 suggesting that at least some of the HUVEC were undergoing apoptosis at that

time point. This may account for the loss of HUVEC by day 14.

Omental transplants are more invasive than dermal implants so that there may have been

increased surgery related inflammation relative to other studies(158). In addition SCID mice do

not have B cells and have fewer macrophages than the T cell deficient nude rats used here. We

inferred from the beneficial, (albeit temporary) effect of clodronate that macrophage responses

directed at the xenogeneic endothelial cells were responsible for the low HUVEC survival.

Although nude rats are T cell deficient, they maintain normal or slightly higher levels of other

leukocytes (macrophages, B cells and natural killer cells) than euthymic rats and are known to

reject xenografts(17, 19, 159). Thus, anti-HUVEC antibodies produced by the still-active B

cells may have played a significant role in the rejection. Also, in xenogeneic vascularized organ

transplantation, host complement activation can directly cause donor endothelium damage(160).

For example, complement depletion in nude rats receiving guinea pig cardiac transplants has

shown to extend xenograft survival24.

Macrophages in the nude rat are known to produce cytokines that are directly toxic to donor

endothelial cells(18). When nude rats treated with lelunomide and anti-asialoGM-1 to deplete

xenoreactive antibodies and natural killer cells received hamster hearts, infiltrating macrophages

alone were capable of rejecting the vascularized xenografts23. In our studies, macrophages

clearly infiltrated endothelialized modules and it is likely that rejection mediated by these cells

contributed to low HUVEC survival (Fig. 3-9). It is also conceivable that some isolated UEA-1

62

cells (those not in vascular structures) were actually a remnant of cells engulfed by macrophages;

this possibility cannot be excluded since macrophages were generally present in the vicinity of

modules.

To temporarily suppress the macrophage response, we treated the nude rats with clodronate

liposomes. This initial macrophage depletion prolonged HUVEC viability until 7 days (Fig. 3-

5e) which suggests that macrophage suppression could benefit HUVEC survival in the nude rat.

However, macrophages were only completely depleted until day 3 and began to repopulate by

day 7 (Fig. 3-9e) correlating to the HUVEC loss by day 14. Others have shown that with

clodronate liposome treatment, macrophages are depleted for approximately 4 days but with the

addition of Freunds adjuvant (to elicit an inflammatory response), macrophage repopulation is

accelerated(153). Similarly, the presence of a xenograft likely accelerated the inflammatory

response in spite of clodronate liposome treatment. Also, the omental tissue contains a number

of macrophage precursors(10) which were not depleted with clodronate liposomes (precursors

are not phagocytotic) and at the onset of inflammation, these cells likely matured and invaded

host tissue to participate in xenograft rejection(161).

Interestingly, with clodronate liposome treatment and subsequent macrophage depletion, vessel

formation was delayed until 7 days (Fig. 3-5d). Macrophages secrete growth factors that induce

HUVEC migration and vessel formation, as seen for example in a report of endothelial

progenitor cell transplantation for wound healing(162). This also highlights the need to

investigate such transplants in the presence of an inflammatory response. Ideally, we would like

to induce HUVEC vessel formation without an adverse host response such that the donor derived

vessels stabilize and integrate with host vasculature. Some human cell transplants have been

explored in nude rats. For example, intravenous injection of large numbers of human endothelial

progenitor cells (EPC – CD34+) in nude rats improves vascularization in myocardial infarctions

and ischemic limbs (163-166). These progenitor cells embed in various vessels and can be

detected up to 1 month after transplant26. Interestingly, only high doses of CD34+ cells are

capable of inducing a therapeutic benefit and only in the presence of ischemic tissue(167). Not

only are the models different, but also progenitor cells lack expression of mature human EC

markers (unlike HUVEC) and hence may induce less of an xenogeneic response(168). Our

observations hint at the possibility that HUVEC in nude rats are capable of driving

63

vascularization; however they presumably elicit a host (immune) response which limits this

capacity. For this reason we are currently pursuing allogeneic and syngeneic endothelial

transplant scenarios.

3.6 Conclusions

Transplantation of endothelialized modules (with HUVEC) in nude rats resulted in what appears

to be primitive vessels in channels among modules at early times. With clodronate liposome

induced macrophage depletion, HUVEC derived vessels persisted longer (until day 7) and some

were invested with host vascular cells even at 14 days. However, inflammatory cell infiltration

and a presumed immune response (in this xenogenic, partially immune compromised animal

model) lead to HUVEC cell loss and vessel regression by day 14 in both untreated and

macrophage depleted animals. Although the detection of primitive HUVEC vessels is

encouraging, better means of modulating the host response are needed to preserve the long term

viability and stability of HUVEC derived vessels.

3.7 Acknowledgements

The authors acknowledge the financial support of the US National Institutes of Health

(EB001013), and the technical expertise of Chuen Lo.

64

4 Endothelialized modules drive stable chimeric

vascularization in allogeneic rats.

Manuscript in Preparation

Authors: Rohini Gupta*, M. Dean Chamberlain*, and Michael V. Sefton.

*Both authors contributed equally to this work. R.G., M.D.C. and M.V.S. designed research

R.G., M.D.C performed research; R.G., M.D.C. contributed new reagents/analytic tools; R.G.,

M.D.C analyzed data; and R.G., M.D.C. and M.V.S. wrote the paper.

65

4.1 Abstract

Successful survival of engineered tissues depends on strategies to develop a new vasculature to

support the metabolic demands of donor tissue. This is particularly relevant for large volume

tissues in which oxygen diffusion limitations mean that cells further than 150-200μm of the

surrounding host blood vessels will not survive. Modular tissue engineering is a means of

designing large volume functional tissues using micron sized constructs with an intrinsic

vascularization. We have previously demonstrated that human endothelial cell covered modules

formed primitive vessels in the channel spacing amongst modules in the nude rat short term

(Chapter 3). Here, we evaluated the potential of rat endothelial cell covered modules to form a

vascular network in outbred, allogeneic rats. RAEC covered modules were transplanted in the

omental pouch of Sprague Dawley untreated rats and rats treated with an immunosuppressant

(Tacrolimus) and a mild anti-inflammatory and EC protective drug (Atorvastatin) for 3 to 60

days. Transplantation of endothelialized modules resulted in a significant increase in vessel

density at all time points in the drug treated rats as compared to untreated rats. In some cases,

donor RAEC were transduced with stable GFP expression prior to transplantation. GFP positive

cells migrated from the surface of the modules and formed primitive vessels at day 7; in

untreated rats, GFP positive vessels were not seen past day 7. Meanwhile, in drug treated rats,

GFP positive vessels matured over time, accumulated erythrocytes, were supported by host

smooth muscle cells and formed chimeric vessels until day 60. Transplantation of

endothelialized modules resulted in the formation of a densely vascularised and perfusable

network in drug treated rats as compared to untreated rats at day 60. Moreover, donor GFP

derived vessels were directly perfusable as indicated by injection of fluorescent beads. To our

knowledge, this is the first study that demonstrates that primary unmodified endothelial cells

without the addition of supporting cells form a chimeric and stable vascular bed in allogeneic

recipients.

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4.2 Introduction

A significant challenge in tissue engineering is the development of an adequate blood supply for

the survival of an engineered tissue construct. Without an internal vascular system, tissue

survival is limited by diffusion of essential nutrients and oxygen. Hence, only cells within a

diffusion distance of 150-200 μm of the surrounding host blood vessels are adequately perfused

and there can be significant cell loss at the core of implanted engineered tissues without an

adequate vascular network(25, 26).

Modular tissue engineering is a means of assembling constructs with benefits that include

uniform cell density, scalability, mixed cell populations and vascularisation (8). Functional cells

are embedded in sub-millimetre sized collagen rods and the outside surface is coated with

endothelial cells (endothelialized). These units are referred to as modules. By varying the type

of embedded cells in the individual modules, it is expected that complex modular tissue

engineered constructs can be produced for different applications; HepG2 cells (9), smooth

muscle cells (150) and others (pancreatic islets, cardiomyocytes, adipose and bone marrow

derived stem cells) have been incorporated into modular constructs to date. The modules are

used to fill a tissue cavity and the randomly packed modules create channels by the virtue of the

spaces among the modules. HUVEC endothelialized modules implanted in the nude rat formed

primitive vessels within the spaces between modules until 7 days with macrophage

depletion(169). While the channels were blood perfuseable in vitro(8) the results presented here

suggest at least some of the resulting vasculature is due to infiltration and reorganization of host

cells. Here, we explored the use of such modules in an allograft model with a view to establish

stable vascularisation, which would ultimately benefit the survival of embedded cells.

Several groups have shown that transplantation of primary endothelial cells, particularly

HUVEC, in immunocompromised rodents can produce mature vessels under specific

circumstances. Transplanted HUVEC transfected with an anti-apoptotic gene, Bcl-2, (55, 64) or

HUVEC mixed with mesenchymal precursor cells (3) were shown to form stable blood vessels in

SCID mice. Also, HUVEC derived vessels were shown to increase overall tissue perfusion in an

ischemic hindlimb model (3) and to effectively vascularize tissues including skin substitutes (88)

67

and 3D skeletal muscle constructs (4). Combined, these studies suggest that primary EC

transplantation can drive vascularization in vivo at least in immunodeficient animals. However,

long term HUVEC survival and vessel formation was only realized after either the HUVEC were

transfected with anti-apoptotic genes (Bcl-2) or co-transplanted with supporting cells

(mesenchymal stem cells, embryonic fibroblasts or perivascular cell precursors) as HUVEC

alone did not achieve stable vascularisation in vivo(3, 55, 64, 88). Since we are ultimately

interested in testing tissue engineering strategies in clinically relevant, immune competent animal

models, we have explored endothelial transplantation in an allograft model. Reports of

allogeneic endothelial cell transplantation are scarce but promising. Sprague Dawley (SD) rat

aortic endothelial cells (RAEC) were transplanted in a myocardial scar in SD rats (an allograft)

with cyclosporine and were shown to increase vascular density in the infarct region (83).

We used Tacrolimus and Atorvastatin to improve endothelial cell survival after transplantation in

an omental pouch allograft model. Tacrolimus is an immunosuppressant that decreases host

inflammation and immune response. Tacrolimus inhibits calcineurin, blocking the normal

function of T cells and the production of IL-2, a stimulator of T cell proliferation (170, 171).

Atorvastatin is in the class of 3-hydroxyl-3-methyl coenzyme A [HMG-CoA] reductase

inhibitors anti-cholesterol drugs. Statins have also recently been shown to have anti-

inflammatory, anti-coagulant and immunomodulatory effects (172). Statins decrease several

pro-inflammatory mediators including IL-2, IL-12, IFNγ and TNFα (173, 174). They also

decrease the expression of several surface immune and inflammatory markers including P-

selectin, chemokine receptors (CCR1, CCR2, CCR4 and CCR5) and adhesion molecules such as

ICAM1(175, 176). Atorvastatin may also directly decrease the immune response to endothelial

cells as demonstrated by decreased proliferation of T cells exposed to xenogeneic endothelial

cells in vitro(177). Statins also improve endothelial survival (178), increase NO production

(179, 180) and increase the number of circulating endothelial progenitor cells (181). Here, we

expected Atorvastatin to similarly reduce inflammatory response and promote donor endothelial

cell survival. In this paper, modules coated with rat aortic endothelial cells (RAEC) isolated

from outbred SD rats were transplanted into an omental pouch formed in both untreated

allogeneic rats and rats treated with Tacrolimus and Atrovastatin to study the formation and

survival of blood vessels.

68


4.3.1 Cells

Sprague-Dawley rat aortic endothelial cells (RAEC) were purchased from VEC technologies

(Rensselaer, NY) and maintained on 0.2% gelatin coated flasks in MCDB-131 complete medium

with 10% FBS at 37°C and 5% CO2. Cells were used between passages 3-5. In some cases,

RAEC were stably transduced with HIV-1 based recombinant lentivirus (LV) encoded for

enhanced green fluorescent protein (eGFP, kindly provided by Dr. Medin, Ontario Cancer

Institute). Briefly, vesicular stomatitis virus glycoprotein-pseudotyped (VSV-g) LV including

the pHR’-cPPT-EF-GW-SIN plasmid (containing eGFP) were generated by transient transfection

of 293T cells as described before (182, 183). For cell transduction, RAEC were infected with

recombinant LV-eGFP at a multiple of infection (M.O.I.) of 10 in the presence of 8 µg/ml

protamine sulphate. Sixteen to 18 hours post-infection, the supernatant was removed and eGFP

transduced RAEC (eGFP-RAEC) were cultured with fresh medium for at least two days prior to

use.


Modules were prepared as before by gelling bovine Type 1 collagen (3.1 mg/mL, Cohesion

Technologies, Palo Alto CA) at 37°C for 60 minutes in sterile 0.71 mm ID polyethylene tubing

(PE60, Intramedic – BD, Oakville, ON)(9, 169). Tubing was cut into small pieces (~ 2mm long

x 0.6mm diameter) using a custom automatic cutter and collected in MCDB-131 medium.

Collagen modules were separated from the outer tubing by gentle vortexing. Either untransduced

RAEC or eGFP transduced RAEC (2.5 x 106) were seeded dynamically onto the surface of 1 mL

of modules (produced using 2.5 m of tubing) for 45 min on a low speed shaker and incubated for

7 days prior to implantation (RAEC contracted collagen modules to ~1mm long x 0.5mm

diameter). Collagen only modules were prepared as above but without endothelial cell seeding.

4.3.3 Module transplants

Adult female Sprague-Dawley rats (7 weeks of age, 250-300 g; Charles River Laboratories, QC)

were individually housed and fed ad libitum. Animals were divided into 2 groups: untreated and

69

drug (Tacrolimus and Atorvastatin) treated. Tacrolimus (Astellas, Markham, ON) was

administered intramuscularly daily (days 1-6: 0.3 mg/kg, days 7-60: 0.2 mg/kg) in a saline

solution and Atorvastatin (Pfizer, Kirkland, QC) was administered daily for 21 days via oral

gavage at a dose of 0.5 mg/kg in sterile water. Modules were implanted in an omental pouch

created as described before(184). Modules suspended in 0.5 mL of PBS were gently delivered

by a sterilized 1mL micropipette into the pouch and allowed to settle. The pouch was folded

over and sutured to secure the modules inside. For both untreated and drug treated groups,

animals were transplanted with endothelialized modules for 3, 7, 14, 21 and 60 days (n=5).

Collagen only modules (without RAEC) were also transplanted in untreated and treated animals

for 3 or 7 days (n=2). The study was approved by the University of Toronto animal care

committee.

4.3.4 Perfusion studies

Animals were heparinzied (500 units, LEO Pharma Inc.) 5 min. prior to the procedure by s.c.

injection. Following a published protocol(185), the descending aorta was cannulated and

heparinized PBS (5 U/mL) was perfused at a constant pressure of 100 mmHg until the blood was

flushed from the vascular system. Animals were then perfused with either 25 mL of Microfil®

solution (MV-122, Flow-tech, Carver, MA). The Microfil solution was allowed to polymerize

for at least 90 minutes and then the omental tissue was excised into 4% formalin (Sigma

Aldrich), embedded in 1% agar solution and images were obtained with a General Electric

Medical Systems MS8 microCT. In some studies, animals were perfused with 10mL (1%

solution) of dark red fluorescent microspheres (F8807, Molecular Probes), tissue was excised

into 4% formalin and all tissue processing was done in the dark.


Animals were sacrificed and the omental pouch was excised into 4% neutral buffered formalin

and fixed for 48 hrs. Tissue samples were embedded in paraffin and 4 µm sections were cut at 3

levels that were 100µm apart. Sections were processed and stained for hematoxylin and eosin

(H&E, Fisher), Masson’s trichrome (Fisher), and various antibodies as outlined in Table 4-1 as

before(169). Sections were viewed with a Zeiss Axiovert light microscope equipped with a CCD

camera; fluorescent images were taken with an Olympus Upright microscope equipped with a

70

Photometrics CoolSNAP camera.


BS-1, a microvascular EC marker (186), or vWF positive cells were counted using Chalkley and

microvessel density (MVD) methods by a blinded observer as described before (184). Briefly, a

25-point Chalkley eyepiece graticule was applied and the eyepiece was rotated to align the

maximum number of dots with BS-1 positive cells in three representative areas at 200x

magnification. The dots that overlaid BS-1 positive structures were counted and the mean of the

three counts were represented semi-quantitatively where a mean of eight or greater was counted

as high (+) and a mean of less than eight was counted as low (-); a separate score (+/-) was

reported for each animal. The MVD method was applied to count BS-1 or vWF positive vessels

at 400x magnification in representative areas (3 areas counted for BS-1 and 5 areas counted for

vWf) and the average of these counts was used for statistical analysis. Macrophages (CD68

positive) were counted in five representative sections at 400x magnification and the average of

these counts was used for statistical analysis. GFP positive vessels were counted in one section

of the whole omentum tissue. The whole microscope slide (stained with GFP) was digitized

using the Aperio ScanScope XT and cross-sectional diameter of each GFP vessel was measured

manually in the Aperio ImageScope software. Vessels were then binned according to size ranges

as either: capillaries (1-9 µm), small arterioles/venules (9-15 µm), large arterioles/venules (15-75

µm) or abnormal (>75 µm).

4.3.7 Statistical Analysis

A one way ANOVA with Tukey post hoc analysis was applied to compare means between

multiple groups. Data was considered statistically significant at p <0.05. All analysis was done

with Statistica Version 5.1 software (Statsoft, USA).

71

Table 4-1: List of antibodies and their target antigens used for immunostaining

Antibody Supplier Conditions Target Antigen Bandeiraea Simplicifolia Lectin I (BS-1)


Heat induced temperature retrieval, 1:300 for 1 hr

Rat microvessel endothelial cells

GFP (6556) Abcam, Cambridge, MA

Heat induced temperature retrieval and citrate buffer, 1:8000 dilution for 1 hr

Donor endothelial cells

CD68 (ED1, MCA341) AbD Serotec, Raleigh, NC


Macrophages and monocytes

TCR (R73, αβ T cell Receptor)

AbD Serotec 1% pepsin pre-treatment, 1:600 for 1 hr

Rat T cells



1% pepsin pre-treatment 1:3000 for 1 hr

Endothelial cells and platelets

Smooth muscle α-actin (AB5694, SMA)

Abcam 1% pepsin pre-treatment 1:1000 overnight

Smooth muscle cells

TUNEL assay

Promega 1% pepsin pre-treatment, Cocktail applied for 1 hr at 37°C

Apoptotic cells (stains fragmented DNA)

72

4.4 Results

4.4.1 Remodeling of endothelialized modules and tissue response

The host response remodelled transplanted modules and surrounding tissue over time as shown

in the Masson trichrome images (Fig. 4-1). By day 7, the collagen only modules were largely

resorbed into the surrounding tissue (Fig. 4-1a, b). Endothelialized modules elicit a more

complex host response. At day 3, the endothelialized modules were surrounded by a hematoma

in both the drug treated and untreated animals (Fig. 4-1c, h). As time progressed, the hematoma

resolved and was largely reabsorbed by day 21 (Fig. 4-1f, k). The hematoma was not apparent in

sham operated animals or in animals with collagen only modules (no EC). Assemblies of

modules were seen both in the interior and exterior of the hematoma and intact modules were

seen throughout the 60 days (e.g., Fig. 4-1g, l). A few modules were also seen among the fat

cells in the omentum.

At day 7, an influx of cells was seen surrounding the modules at the exterior of the hematoma

(Fig. 4-1d, i). A number of these cells surrounding the modules were stained positive for Ki67

indicating that they are a proliferating cell population (Fig. 4-2b, d). Some of these proliferating

cells were likely the transplanted RAEC cells (indicated by histology showing cells that the cells

were both GFP and Ki67 positive, not shown) while other were of host origin. Some of the

infiltrating host cells were monocytes but a large number of the cells stained positive for smooth-

muscle α-actin (SMA) and were presumably myofibroblasts or perhaps pericytes (Fig. 4-2e). At

the same time, there was also a collagen-rich region that formed around these modules; this

collagen rich area continued to increase in size as the hematoma resolved (Fig. 4-1d-g,i-l). This

deposit was morphologically different from the transplanted endothelialized modules, which

retained their roughly cylindrical shape even after the EC migrated off their surface, and might

reflect degraded modules or collagen produced by the infiltrating cells. Additionally, blood

vessels began to appear in the collagen deposit region surrounding the modules at day 7. The

number of these blood vessels increased over time in both the treated and untreated groups.

73

Figure 4-1 Trichrome staining of transplanted modules (thin arrows) with and without drug treatment. Collagen only modules collapsed together and degraded over 7 days (a-b). At day 3, a hematoma (RBC) formed in the untreated (c) and treated (h) rats transplanted with endothelialized modules but not with collagen only modules (a). At day 7, host cells infiltrated around endothelialized modules in both treatment conditions (d, i). In the untreated group, infiltrating cells persisted until day 60 (g) but not in the treated group (l). There was a formation of a collagen deposit (CD) around the modules (d-g and i-l). This collagen deposit contained a number of blood vessels (thick arrows), which increased with time, in both the treated (j-l) and untreated (e-g) animals. Magnification is 50x.

74

TUNEL Ki67 SMA U

ntre

ated

Trea

ted

Figure 4-2: Apoptotic (TUNEL), proliferating (Ki67) cells, and myofibroblasts (SMA) around endothelialized modules in both untreated and drug treated animals at day 7. a) A few apoptotic cells (arrows) were detected on the surface of the modules in untreated and c) drug treated rats. b) Several proliferating cells were seen in the tissue surrounding the modules in untreated and d) drug treated rats. e) Some of the proliferating cells were positive for SMA in treated rats. Modules are outlined by dashed lines. Scale bar = 100 µm.

4.4.2 Blood vessel formation around transplanted modules

At day 3, there were single EC cells as determined by staining with BS-1 lectin, a microvascular

EC marker (186), near the modules (Fig. 4-3a,e) and over time these cells were found to form

blood vessels (structures with a lumen) particularly in the treated animals (Fig. 4-3 f-h). The

number of BS-1 staining cells, as measured by Chalkley counts, were higher with drug treatment

at all time points except at day 21 (Fig. 4-4a). By day 21, most of the individual BS-1 positive

cells were found in vessels giving rise to a low Chalkley count but a higher microvessel density

count (MVD)(Fig. 4-4b). BS-1 MVD counts were higher with drug treatment at all time points

after day 7 (Fig. 4-4b). Total vessels as determined by vWf staining were counted at days 21 and

60 and similar to BS-1 results, vWf positive vessel density was significantly higher with drug

treatment (Fig. 4-4c).

b

dd c

ee

a

75

Figure 4-3: BS-1 lectin was used to identify rat microvessel endothelial cells. a,e) There were isolated BS-1 positive cells (arrows) around endothelialized modules (dashed line) at day 3 in both groups; in some cases, BS-1 positive cells were on the surface of the modules . f) At day 7, there were a combination of isolated BS-1 positive cells and a few vessels surrounding endothelialized modules in drug treated animals. c,g) By day 14, there were many BS-1 vessels near modules and the vascular density was more pronounced with drug treatment; d,h) this trend for increased vascular density continued until day 21. Scale bar = 100μm.

a)

b)

Day 3 Day 7 Day 14 Day 21 Untreated +---- +---- +++-- +++-- Treated +++-- +++++ +++++ +----

76

c) Figure 4-4: BS-1 positive cells counted by a) Chalkley method and b) MVD method. a) Drug treated animals had more BS-1 positive cells as determined by the average chalkley counts than the untreated animals at each time point except for day 21. A mean score of eight or higher is positive (+) and less than eight is negative (-) b) Drug treated animals had significantly more BS-1 positive microvessels than untreated animals after day 7. c) Similarly, drug treated animals had significantly number of total blood vessels than untreated animals at day 21 and 60. Data reported is average of each group +/- SEM, *at (p<0.05).

The vascularised bed matured over time as confirmed by microCT imaging (Fig. 4-5). Whole

tissue microCT imaging at day 21 showed a higher overall vessel density around implanted

modules in the drug treated animals (Fig. 4-5a,c). These newly formed vessels integrated with

the host vasculature but were presumable leaky at day 21 as shown by the leaking of the Microfil

from the vessels. By 60 days, there was a decrease in the leakiness in the vascular bed and an

increase in the perfusable vascularization of the tissue implanted with modules (Fig. 4-5d).

Day 21 Day 60

Unt

reat

ed

b

a

77

Trea

ted

Figure 4-5: microCT images of whole omental pouch containing endothelialized modules. Drug treated animals had a greater overall vessel density when compared to non-treated animals. At day 21, there was a large leaky core detected (arrows) in both a) untreated (c) and treated animals. d) At day 60 there was still a greater vessel density in the treated animal and the leaky core (arrows) had decreased in size.

4.4.3 Transplanted RAEC form vessels in immunosuppressant treated rats

RAEC transduced with eGFP (eGFP-RAEC) were used to track the transplanted endothelial cells

and were identified through fluorescent imaging or with an anti-GFP antibody and chromogenic

staining (Fig. 4-6). eGFP-RAEC remained mostly on the surface of the modules in both

untreated and treated animals at day 3 (Fig. 4-6a, f, k, p) but by day 7, some GFP positive vessels

(with lumens) were seen near the modules (Fig. 4-6b, g, l, q). In untreated rats, no GFP positive

cells were seen after day 7 (Fig. 4-6c-e, h-j). There were some apoptotic cells, presumably

transplanted RAEC, at day 7 (Fig. 4-2a,c) in both untreated and treated animals. Although day 7

is the latest time at which eGFP-RAEC cells were seen in the untreated animal, there appeared to

be no difference in the number of apoptotic cells between the two groups. Meanwhile, with drug

treatment, eGFP-RAEC were seen in vessels of varying sizes near the modules at days 14, 21

and 60 (Fig. 4-6m-o,r-t). Most of the eGFP-RAEC derived vessels contained erythrocytes in

their lumens. The number of GFP positive blood vessels decreased from 14 days to 60 days

(Fig. 4-7a). This decrease in vessel density is expected after the initial burst of angiogenesis as

unneeded vessels are pruned(187). Over time, there was a stablization in the number and

maturity of blood vessels. The ratio of large arterioles and venules to capillaries (based on vessel

diameter) increased from 14 to 60 days suggesting that the capillaries were maturing as part of

the angiogenic process (Fig. 4-7b).

dd c

78

Day 3 Day 7 Day 14 Day 21 Day 60Un

treat

edTr

eate

dUn

treat

edTr

eate

d

a c d e

f g h i

k m n

p r s t

b

j

l o

q

Figure 4-6: Transplanted eGFP-RAEC around endothelialized modules (dashed outline) were detected either (a-e, k-o) fluorescently (arrows show example of eGFP-RAEC, green; images are overlaid with DAPI (blue)) or (f-j, p-t) stained with anti-GFP (arrows show example of eGFP-RAEC, brown). (a, f, k ,p) At day 3, eGFP-RAEC were primarily on the surface of modules and at day 7, eGFP-RAEC migrated off the modules to form tubes in both the (b, g) untreated and (l, q) treated animals. After day 14, eGFP-RAEC were not detected in (c-e, h-j) untreated animals but a number of eGFP-RAEC were incorporated into vessels in (m-o, r-t) drug treated animals. Scale bar = 100 µm.

79

a)

Figure 4-7: a) The average number of vessels that contain at least one GFP positive cell. There were significantly more GFP positive vessels at day 14 than at 60 days (p<0.05). Data reported is average value of each group +/- SEM. b) At each time point, the percent of GFP vessels with diameter in the range of capillaries, small arterioles/venules, large arterioles/venules or abnormal. From day 14 to 60, there was a decrease in the numbers of capillaries and an increase in the numbers of large arterioles/venules.

At day 14, there were many GFP positive vessels but little incorporation of SMA positive cells in

the walls of these vessels. The vessels matured over time and by day 60, three types of eGFP-

RAEC derived vessels could be distinguished: small diameter vessels without SMA (capillary-

like vessels; Fig. 4-8a,d,g), larger diameter vessels with a thin layer of SMA (venule-like vessels;

Fig. 4-8b,e,h), or large diameter vessels surrounded by a thicker ordered ring of host SMA

positive cells (arteriole-like vessels; Fig. 4-8c,f,i). eGFP-RAEC were commonly seen in vessels

at 60 days and generally also included host endothelial cells (vWf positive; GFP negative) to

form chimeric vessels. Also, at 60 days, some of the eGFP-RAEC derived vessels were

perfusable as indicated by fluorescent beads into the interior of vessel lumen (Fig. 4-9).

Size range (μm) Day 7 Day 14 Day 21 Day 60

Capillaries 9.0-1.0 71.6% 55.0% 54.8% 51.1% Small Arteriole or Venule 15.0-9.0 19.4% 29.0% 24.4% 24.2% Large Arteriole or Venule 75.0-15.0 9.0% 15.8% 20.6% 24.5% Other (Abnormal) >75.0 0.0% 0.1% 0.2% 0.2%

80

Day 60_capillary-like Day 60_vein-like Day 60_artery like

α-GFP

SMA

vWf

Figure 4-8: Serial sections of a-c) eGFP-RAEC by anti-GFP antibody in vessels around endothelialized modules, d-f) corresponding SMA positive cells (g-i) and vWF positive cells at days 60. Transplanted cells formed chimeric vessels that were either a) capillaries (d) with little no supporting SMA positive cells, b) venules (e) with a small layer of SMA positive cells or c) arterioles (f) with a thick layer of SMA positive cells. g-i) In all cases, GFP vessels were positive for rat vWF. Scale bar = 25 µm.

a

g h

b c

d e f

i

81

Day 60

Figure 4-9: Fluorescent images of endothelialized modules transplanted in allogeneic immunosuppressed animals at 60 days perfused with fluorescent beads. Images show examples of fluorescent beads (red, arrows) in the lumen of GFP positive (green) vessels.

4.4.4 Inflammatory cell response to endothelialized modules

Inflammatory cell numbers (CD68 positive cells, monocytes and macrophages) increased from

day 3 to 7 in untreated animals but not in treated animals (Fig. 4-10; p < 0.05). In untreated

animals, CD68 positive cells remained high until day 21 and were significantly higher than at

day 3 (Fig. 4-10; p < 0.05). With drug treatment, inflammatory cells peaked at day 14 and were

reduced by day 21 almost to the initial levels at day 3 (Fig. 4-10; p < 0.05). When compared to

untreated animals, there were significantly fewer cells at days 7 and 21 with drug treatment (Fig.

4-10; p < 0.05). T cells were seen around modules in untreated animals after 7 days with higher

numbers at day 14 (Fig. 4-11). In treated animals, no T cells were observed at any time point as

expected suggesting that Tacrolimus was effective in suppressing the T cell response (Fig. 4-11).

82

0

20

40

60

80

100

120

140

160

Day 3 Day 7 Day 14 Day 21

CD

68+

Cel

ls

untreated Treated

* ***

Figure 4-10: Average counts of CD68 (ED1, macrophages) positive cells. In untreated animals, the number of CD68 positive cells significantly increased from day 3 to 7 (p < 0.05) and remained high until 21 days. With drug treatment, cells peaked at day 14 and were significantly reduced by 21 days. When compared to control animals, there were significantly fewer inflammatory cells at days 7 and 21. Data shown is the mean of each group (5 sections/animal x 5 animals/group) +/- SEM, * at p < 0.05.

Day Untreated Treated 14

Figure 4-11: T cells around endothelialized modules. T cells (R73, αβ T cell Receptor, brown stain) were seen near endothelialized modules (dashed line) at day 14 (a) in untreated animals but with b) immunosuppressant treatment, no T cells were seen. Scale bar = 100 µm.

a b

83

4.5 Discussion

Upon implantation of the modules, remodelling of both modules and the surrounding tissue

occurred. There appeared to be four stages in this remodelling process: formation of a

hematoma, inflammatory cell infiltration, neovascularisation/proliferation and vascularised

granulation tissue formation. The latter three steps are similar to the stages of wound healing.

The hematoma around endothelialized modules formed as early as 3 days and may reflect the

formation of leaky blood vessels (via sprouting) due to the secretion of growth factors from the

implanted RAEC. Such a hematoma did not form without endothelialized modules or in sham

surgeries suggesting that the transplanted endothelial cells were a causative agent.

An inflammatory response characterized by the influx of monocytes and macrophages around

endothelialized modules was seen at day 3. The number of inflammatory cells peaked at day 7

without treatment and day 14 with drug treatment (Fig. 4-10). Similar to the initial stage of

wound healing, the infiltrating macrophages likely stimulated the migration of fibroblasts and

host endothelial cells into the wound bed to lay down new extracellular matrix (the collagen

deposit seen in Fig. 4-1e,j). The proliferative stage began at day 7 and there was an increase in

infiltrating cells around endothelialized modules (Fig. 4-1d,i) in both untreated and

immunosuppressed animals. Many of the infiltrating cells were proliferative (positive for Ki67;

Fig. 4-2) and likely myofibroblastic in nature (positive for SMA; Fig. 4-2e). These infiltrating

cells were presumably responsible for the observed collagen formation which enables the

neovascularisation around endothelialized modules (Fig. 4-1e,j). Remodelling continued with

the formation of a vascularized collagen bed with embedded modules (Fig. 4-1f-g, k-l).

We noted that host derived neovacularization, with BS-1 positive host vessels, in

immunosuppressed animals started at day 7 and was higher than in untreated animals at all time

points as evidenced by MVD counts (Fig. 4-4b) and vWf vessel counts (Fig. 4-4c). Few

transplanted eGFP-RAEC were seen in untreated animals after 7 days suggesting that the

contribution to neovascularisation from this source was limited without drug treatment. We

presumed that in immunosuppressed rats, the transplanted RAEC contributed both directly and

indirectly to vessel formation: RAEC migrated from the modules to participate in vessel

formation (Fig. 4-6) and secreted factors that recruited host cells. eGFP-RAEC derived vessels

84

persisted for at least 60 days and several of the eGFP-RAEC vessels were chimeric, i.e. invested

with host endothelial cells. eGFP-RAEC vessel maturation began at 21 days, at which time

vessels were integrated with host vessels but were leaky (Fig. 4-5). Over time there was a

stablization in the number and maturity of blood vessels with the emergence of vessels with

venule-like or arteriole-like quality with larger diameters and layers of SMA positive cells of

varying thickness (Fig. 4-8). Moreover, the vascular bed was more mature at day 60 as

evidenced by a decrease in the leakiness and an increase in the perfusable vascularization of the

tissue implanted with modules (Fig. 4-5).

That transplanted RAEC begin to form vessels 7 days after transplantation in immunosupressed

allogeneic recipients is consistent with other studies of rat endothelial cell transplants.

Syngeneic RAEC (BrdU labelled) injected into a myocardial scar (83) and rat microvascular

endothelial cells injected into hindlimbs of Lewis rats were incorporated into capillaries as early

as day 7 and persisted for at least 1 month (188). Allogeneic RAEC transplanted in Sprague

Dawley rats treated with cyclosporine lead to increased blood vessel density in the myocardial

scars as compared to non-EC controls 6 weeks after transplantation (83). Schechner et al., have

reported that HUVEC suspended in collagen-fibronectin gels formed thin walled capillaries in

immunodeficient mice and when cells were transduced with Bcl-2, the resulting vessels formed

arterioles-, venule- and capillary- like structures that increased blood flow in ischemic tissues 60

days after transplantation (55). In our studies, eGFP-RAEC began to form capillary like

structures at day 7, these capillaries were seen to have erythrocytes in their lumens at day 14 and

matured over time to form capillary-, arterioles- or venule- like structures (Fig. 4-8). Here

unmodified (i.e. without addition of anti-apoptotic genes) primary rat aortic endothelial cells

alone, without supporting cells were capable of generating multiple vessel types in

immunosuppressed allogeneic rats.

We hypothesize that in the allograft model presented here, infiltrating cells (macrophages,

myofibroblasts) supported newly formed RAEC derived vessels. In wound healing,

macrophages are known to secrete several pro-angiogeneic growth factors to induce

angiogenesis as well as regulate the migration of infiltrating fibroblasts and smooth muscle

cells(189). We noted an increase in inflammatory cells (Fig. 4-10) and SMA positive cells (Fig.

4-8e) surrounding eGFP-RAEC derived vessels in drug treated animals at day 14. Thus, it is

85

likely that transplantation of allogeneic EC in immunosuppressed animals initiated a wound

healing-like angiogenic response and newly developed vessels were supported by host

macrophages or fibroblasts. This might explain why donor derived vessels persist long term

without the need of supporting cells as required with human EC transplantation in immune

compromised animals. It may be interesting to explore whether the addition of supporting cell

types or cytoprotective genes further enhances the neovascularisation capacity of this model.

Studies with supporting mesenchymal stem cells are underway. We are also interested in

characterizing whether these vessels can support functional cell types in vivo and studies that

include rat islets in the modules are also underway.

4.6 Conclusions

With immunosuppressant therapy, allogeneic rat aortic endothelial cells (without functional

modification or accessory cells) embedded in collagen gel modules and implanted into an

omental pouch formed capillary like structures as early as 7 days after transplantation. These

vessels recruited host endothelial cells and began to form chimeric vessels with a mixture of

donor and host cells at day 21. By day 60, donor EC derived vessels formed capillary-,

arterioles- or venule- like structures. To our knowledge, this is the first report that unmodified

endothelial cells develop into mature blood vessels in an allogeneic transplant model. This

model of vascularization can be applied to support functional cells under normal inflammatory

and immune responses relevant to clinical transplantation.



(EB001013), the Canadian Institutes of Health Research (MOP-89864) and Natural Sciences and

Engineering Research council of Canada (Post-graduate scholarship). We are grateful to Chuen

Lo and his technical expertise in animal surgeries. Also, we thank Chyan-Jang Lee (Dr. J

Medin) for generation of the eGFP-RAEC, Lisa Yu (Dr. R. M. Henkelman) for microCT

imaging and Toronto General Hospital’s Pathology research group for immunostaining services.

86

5 Application of an Endothelialized Modular Construct

for Islet Transplantation

Manuscript in Preparation.

Authors: Rohini Gupta, Michael V. Sefton.

Contributions: R.G., M.V.S. designed research. R.G. performed research, contributed new

reagents/analytic tools, analyzed data; and R.G. and M.V.S. wrote the manuscript.

87

5.1 Abstract

Modular tissue engineering is a novel approach to assemble micro-tissues with an intrinsic

vascularization; transplantation of endothelialized modules leads to the formation of a stable

vascular network in allogeneic recipients (Chapter 4). We evaluated whether the endothelialized

modular approach promotes islet engraftment by improving vascular density and islet survival in

diabetic immunosuppressed allogeneic and syngeneic recipients. Transplantation of islets in

endothelialized modules significantly increased the vessel density around transplanted islets. In

allogeneic immunosuppressed recipients, donor endothelial cells (GFP positive) formed vessels

near co-transplanted islets; donor vessels were seen until 21 days, included erythrocytes in their

lumens and were supported by host smooth muscle cells. Insulin positive islets were detected

immunohistochemically for at least 21 days. In syngeneic recipients, islets transplanted in

endothelialized modules had on average a higher number (not statistically significant) of insulin

positive cells than islets transplanted in collagen only (no EC) modules at 21 days.

Transplantation of 2000 islets in allogeneic immunosuppressed animals lowered glucose levels

immediately but there was graft failure within one week. Graft failure rate as indicated by blood

glucose levels was the same with transplantation of free islets and islets in endothelialized

modules, however, serum insulin levels were slightly higher with the latter group at day 21.

Meanwhile, 2000 islets reversed diabetes in 40% of syngeneic recipients until 60 days; there was

no difference in blood glucose and serum insulin levels between recipients with islets in

endothelialized modules and those with islets in collagen only modules (no EC). This study was

the first exploration of primary EC co-transplanted with islets in allogeneic immunosuppressed

and syngeneic diabetic recipients. While the endothelialized modular approach increased vessel

density around transplanted islets, it is expected that further modulation of the host immune and

inflammatory reactions is needed to improve islet engraftment.

88

5.2 Introduction

With the Edmonton protocol, islet transplantation has emerged as a minimally invasive

procedure to replace insulin producing cells (islets) and reverse Type I diabetes. To date, islet

transplantation has treated 500 patients worldwide and on average patients remain insulin

independent for one year(1). However a number of limitations persist with islet transplantation

including the need to transplant a large islet mass (isolated from 2-3 pancreata) for successful

treatment. Overtime, transplanted islets continue to fail as only 10% of patients remain insulin

independent after 5 years. In fact, a majority of transplanted islets (>60%) fail to engraft shortly

after transplantation(1, 2, 108). Early apoptotic initiators include incompatibility of blood with

the islet surface and insufficient oxygen and nutrient supply to engrafted islets(113).

A major contributor to islet graft apoptosis is inadequate islet revascularization in the host.

Native islets are highly vascularized, contain a dense network of capillaries and are located

adjacent to major blood vessels to efficiently secrete insulin(111). During isolation, much of the

vascular endothelium is lost and after transplantation revascularization takes 7-14 days. Hence,

in the early post-transplant period, a majority of the islets are hypoxic and undergo apoptosis(5-

7). The islets that do persist continue to have significantly lower levels of vascular density,

receive decreased blood flow and experience a hypoxic environment in comparison to

endogenous islets as long term revascularization is not sufficient(7, 138). Several groups have

shown that increasing vascular density around transplanted islets by delivery of vascular

endothelial growth factor improved islet survival (detailed in section 5.4.7); this highlights that

revascularization may be a key factor in islet engraftment.

Another way to drive neo-vascularization is through transplantation of primary endothelial cells.

Donor endothelial cells drive new blood vessel formation that can support functional cells. For

example, HUVEC co-transplanted with mesenchymal precursor cells integrated with the murine

host vasculature to create a stable vascular network that promoted survival of co-transplanted

skeletal and myocardial tissue(75, 84). Similar studies with primary rat endothelial cells are

quite limited; for example allogeneic heart microvascular EC (BrDu labeled) incorporated into

new vessels in a myocardial scar after 6 weeks resulting in improved perfusion in ischemic

89

myocardium(83). The use of primary endothelial cell driven vascularization to support

transplanted islets has not been explored.

Endothelialized modular tissue engineering is a means of assembling constructs with a mixed

cell population and an inherent vascularisation(8). Functional cells are embedded in sub-

millimetre sized collagen rods and the outside surface is coated with endothelial cells (EC).

These units are referred to as endothelialized modules. By varying the type of embedded cells in

the individual modules, it is expected that complex modular tissue engineered constructs can be

applied for different applications; HepG2 cells(9), smooth muscle cells(150) have been

incorporated into modular constructs to date. Endothelial cells on the surface of the modules

enable a non-thrombogeneic, vascularised construct. We have shown that when human

umbilical vein endothelial cells (HUVEC) endothelialized modules were packed in situ, the

modules randomly assembled to create channels (spaces among packed modules) that permitted

blood without EC activation(8). Also, implantation of individual HUVEC endothelialized

modules in nude rats resulted in formation of HUVEC derived primitive vessels modules short

term(169). Furthermore, transplantation of rat aortic endothelial cell (RAEC) covered modules

in the omental pouch resulted in the formation of a stable, perfusable vascular construct in

immunosuppressed allogeneic recipient (Chapter 4). Here, we explored the use of this RAEC

endothelialized modular construct for supporting transplanted islets in both diabetic

immunosuppressed allogeneic and syngeneic recipients.

90


5.3.1 Cells

Rat aortic endothelial cells (RAEC) from Sprague Dawley and Lewis rats were purchased from

VEC technologies (Rensselaer, NY) and maintained on 0.2% gelatin coated flasks in MCDB-131

complete medium with 10% FBS at 37°C and 5% CO2. Cells were used between passages 3-5.

RAEC were transduced with HIV-1 based recombinant lentivirus vectors to engineer the stable

expression of enhanced green fluorescent protein (eGFP) (kindly provided by Dr. Medin,

University Health Network, Toronto). Briefly, vesicular stomatitis virus glycoprotein-

pseudotyped (VSV-g) LV including the pHR’-cPPT-EF-GW-SIN plasmid (containing eGFP)

was generated by transient transfection of 293T cells as described before (182, 183). For cell

transduction, RAEC were infected with recombinant LV-eGFP at an M.O.I. of 10 in the presence

of 8 µg/ml protamine sulphate. 16~18 hours post-infection, the supernatant was removed and

eGFP-RAEC were cultured with fresh medium for at least two days prior to use (RAEC

transduction was conducted by Dr. Chyan-Jang Lee in Dr. Medin laboratory). Islets were

isolated from male Sprague Dawley or Lewis rats as previously described (15) at the University

of Alberta (isolations done by Ms. Dixon at Dr. Ray Rajotte lab) and were shipped overnight in

RPMI-1640 medium (Invitrogen, Burlington, ON) with 10% FCS (50 mL tubes maintained at

approximately 37°C). Islets were received at the University of Toronto and the shipment

medium was replaced with fresh RPMI-1640 medium; islets were maintained overnight prior to

any further studies.


Modules were prepared as before (8, 9, 150) using bovine Type 1 collagen (3.1 mg/ mL) with

slight modifications. Islets were suspended in collagen solution (2000 islets/ 1mL collagen

solution) and the mixture was gelled (37°C, 45 minutes) in sterile 0.71 mm ID polyethylene

tubing (PE60, Intramedic - BD Canada, Oakville, ON). Tubing was cut into small pieces (~ 1.5

mm long x 0.6 mm diameter) using a custom automatic cutter and collected in RPMI-1640

medium. With this concentration, there were roughly 3 islets/module; modules were separated

from the outer tubing by gentle vortexing and maintained in RPMI-1640 medium. In some cases,

91

RAEC (untransduced or eGFP-RAEC) (2.5 x 106/mL settled modules) were seeded dynamically

(on a low speed shaker) onto the surface of modules for 60 min in MCDB-131 complete (VEC

technologies, RAEC compatible) medium and cultured overnight in a co-culture medium (50%

RPMI-1640 and 50% MCDB-131). Islets were then switched to RPMI-1640 medium for culture

and maintained for 2-7 days in culture. For transplantation studies, free islets, islets in modules

(no EC) or islets in endothelialized modules were transplanted after two days in culture.

5.3.3 In vitro characterization

For VE-cadherin staining, RAEC endothelialized modules were fixed in 4% paraformaldehye for

10 mins and permeabilized with 0.1% Triton-X for 4 minutes. Modules were incubated with

anti-rat VE Cadherin (SC-6458, Santa Cruz biotechnology, Delaware, CA) at 1:50 dilution for 1

hr, followed by a secondary antibody Alexa Fluor 488 (A-11059, Invitrogen) at 1:300 dilution

for 30 minutes and counterstained with 1µg/ml of Hoechst 33258 (H-3570, Invitrogen) for 5

minutes at room temperature. For Live/Dead staining, free islets or islets embedded in RAEC

covered modules were incubated with 1 uL of Calcein AM and 2 uL of Ethidium homodimer-1

(L-3224, Live/Dead kit, Invitrogen) in 1mL of PBS for 20 minutes at 37°C. Modules were

washed with 3x5 min PBS and immediately visualized with Zeiss LSM510 confocal microscope.

5.3.4 Static glucose challenge assay

Four samples of either 50 free islets or 50 islets in endothelialized modules were incubated in

low glucose (4.2 mM) Krebs-Ringer bicarbonate (KRB, K4002, Sigma-Aldrich, Oakville ON)

buffer with 0.25% bovine serum albumin (BSA, A7409, Sigma) and then in high glucose (16.7

mM) KRB buffer with 0.25% BSA for 1 hr at 37°C. 100 µL aliquots were taken after low

glucose and high glucose incubation and stored at -20°C. Samples were analyzed for rat insulin

using an ELISA kit per manufacturer instructions (Mercodia, Sweden) by Mount Sinai Services

(Toronto, ON). The stimulation index was calculated as the insulin concentration in high

glucose solution divided by low glucose solution for each sample and the average value for the

group was reported +/- SEM.

92

5.3.5 Diabetic animals

Adult male Sprague Dawley and Lewis rats (250-300 g) were purchased from Charles River

Laboratories (Wilmington, MA). They were individually housed and fed ad libitum. Animals

were rendered diabetic with 50 mg/kg of streptozotocin (S0130, Sigma-Aldrich) freshly prepared

in pH 4.5 citrate buffer (82585, Sigma-Aldrich). Blood was sampled through the tail vein and

measured with a glucometer (Onetouch Ultrasmart, LifeScan, Milpitas, CA). Animals with non-

fasting blood glucose levels over 16 mM for 2 consecutive readings (bi-weekly) were considered

diabetic. Sprague Dawley animals were immunosuppressed as follows: Tacrolimus (Astellas,

Markham, ON) was administered intramuscularly daily (day 1-6: 0.3 mg/kg, day 7-21: 0.2

mg/kg) in a saline solution and Atorvastatin (Pfizer, Kirkland, QC) was administered daily until

21 days via oral gavage at a dose of 0.5 mg/kg dissolved in sterile water. Lewis animals received

no additional drug treatments. The study was approved by University of Toronto animal care

committee.

5.3.6 Omental pouch transplants

For transplant, the greater omentum was spread out and 7-0 silk suture was run along the edges

and the top to create a pouch with a small opening. 2000 free islets, islets in modules (no EC) or

islets in endothelialized modules (0.5 mL volume) suspended in PBS were gently delivered by a

sterilized 1mL micropipette into the pouch and allowed to settle. The pouch was folded over and

sutured to secure the modules inside. For allogeneic transplants, animals were transplanted with

free islets or islets in endothelialized modules for 14-30 days. For syngeneic transplants, animals

were transplanted with islets in modules (no EC) or endothelialized modules for 14-60 days.

5.3.7 Metabolic follow up

For blood glucose monitoring, blood was sampled bi-weekly for days 0-14 and weekly for days

14-60 through the tail vein and measured with a glucometer. Animals that were not

hyperglycemic (blood glucose < 11.1mM for 5 consecutive days) were subjected to an oral

glucose tolerance test (OGTT) at day 60. After 4 hrs of fasting, animals were gavaged with a 2

mg/kg glucose solution and blood glucose was measured at 0-120 minutes. For insulin

measurements, blood was collected weekly from the tail vein in microtubes (CB 300Z, Sarstedt,

93

Germany), centrifuged (10,000 rpm for 5 mins) and serum was stored at -20°C. Serum insulin

measurements were made with a rat insulin ELISA kit as above.


At explantation, animals were sacrificed and the omental pouch was excised into 4% neutral

buffered formalin (Sigma Aldrich) and fixed for 48 hrs. All histological processing was done at

the Pathology Research Program Laboratories (University Health Network, Toronto, ON).

Tissue samples were embedded in paraffin wax blocks and 4 µm serial sections were cut at 3

levels (each level was 100µm apart) within the block. Sections were processed and stained as

before (184) for hematoxylin and eosin (H&E, Fisher Scientific, Ottawa ON), Masson trichrome

(Fisher), and various antibodies as outlined in Table 5-1. All sections were viewed with a Zeiss

Axiovert light microscope or an Olympus BX50 fluorescent microscope with a CCD camera.


BS-1 positive intra-islet vessels were counted in 5 representative islets at 400x magnification.

Individual islet diameter was manually measured in the ImagePro software and the vessel density

was converted to #BS-1 vessels/150 µm diameter islet. vWf positive islet peripheral vessels

(vessels counted outside of islets) were counted in 5 representative sections at 200x (field of

view area of 0.785 mm2) and the vessel density was converted to # vWf vessels/ mm2. Insulin

positive clusters were counted in the whole omentum tissue in 3 sections (5µm thick, each level

was 100 µm apart). The whole microscope slide (stained with anti-insulin) was digitized using

the Aperio ScanScope XT and the area of positively stained cells was measured using the

positive pixel algorithm in the Aperio ImageScope software. Data is reported as the average

value for each group +/- SEM.

94

Table 5-1: List of antibodies and their target antigens used for immunostaining

Antibody Supplier Conditions Target Antigen Bandeiraea Simplicifolia Lectin I (BS-1)


Heat induced temperature retrieval, 1:300 dilution for 1 hr

Microvascular (host, intra-islet) endothelial cells

Insulin (18-0067) Invitrogen 1% pepsin pre-treatment 1:100 for 1 hr

Insulin

GFP (Ab6556) Abcam, Cambridge, MA

Citrate pre-treatment, 1:8000 for 1 hr

GFP molecule – donor endothelial cells

CD68 (ED1, MCA341) AbD Serotec, Raleigh, NC


Macrophages and monocytes

TCR (R73, αβ T cell Receptor)

AbD Serotec, Raleigh, NC


T cells



1:5000 overnight All endothelial cells and platelets

95

5.4 Results and Discussion

5.4.1 In vitro Characterization of islets in endothelialized modules

Islets were characterized in vitro to assess viability and function after embedding in

endothelialized modules. With a live/dead assay, after 2 days in culture, free islets in culture and

those embedded in modules with endothelial cells (endothelialized) were generally viable in

RPMI-1640 medium (Fig. 5-1a,c). After 7 days, there were some dead cells associated with both

free islets (Fig. 5-1b) and islets embedded in endothelialized modules (Fig. 5-1d); although no

quantitative analysis was done, there appeared to be a similar amount of dead cells in both

conditions. To evaluate islet function, islets were exposed to a static glucose challenge and

insulin measurements were made with a rat insulin ELISA kit. Islets embedded in

endothelialized modules had slightly higher insulin secretion than free islets both after 2, and 7

days in culture (Fig. 5-1e). However, after 7 days in culture, insulin secretion in both groups was

reduced; this is consistent with reports from other groups that isolated rat and human islets lose

responsiveness to glucose simulation over time(190).

Day 2 Day 7

Free

isle

ts

Isle

ts in

End

othe

lializ

ed

mod

ules

c

b a

d

96

e)

0

1

2

3

4

5

6

Day 2 Day 7

Insu

lin st

imul

atio

n in

dex

Islets alone Islets in endothelialized modules

Figure 5-1: Free islets and islets in endothelialized modules in culture (RPMI-1640 medium) were characterized by: (a-d) Live/Dead assay and e) insulin measurements after a static glucose challenge. a) Free islets c) and islets in endothelialized modules were generally viable (green) after 2 days in culture. b) A few dead (red) cells were seen in free islets and with d) islets in endothelialized modules after 7 days in culture. e) Islets in endothelialized modules had higher insulin secretion than free islets in culture. Insulin secretion was reduced at day 7 in both free islets and islets in endothelialized modules. Data presented is average of the group +/- SEM; n=4.

A critical element of this study was the co-culture of islets with endothelial cells and

considerable effort was used in defining suitable co- culture conditions. The effect of co-culture

on Sprague Dawley strain rat aortic endothelial cells (RAEC) in islet embedded modules was

evaluated by VE-cadherin expression. VE-cadherin is a key endothelial cell junction marker

and its presence determines endothelial cell contact integrity(191, 192). Recent studies suggest

that while VE-cadherin mediates cell-contact derived growth inhibition, it may also mediate anti-

apoptotic signaling in endothelial cells(191). We have previously shown that RAEC grown to

confluence (7 days) on the surface of collagen modules (without embedded cells) have strong VE

cadherin expression (Chapter 4). When RAEC were seeded on islet embedded modules in

RMPI-1640 medium, RAEC showed sparse coverage on the surface of the modules after 7 days

(Fig. 5-2c). To improve RAEC coverage, RAEC were seeded on islet embedded modules in

native EC medium (MCDB-131 complete), cultured overnight in co-culture (50% MCDB-131

and 50% RPMI-1640) medium and then switched to islet (RPMI-1640) medium; this protocol is

97

referred to as a co-culture condition from here on. With this protocol, RAEC completely

covered the surface after 7 days in culture with strong VE cadherin expression (Fig. 5-2d). Islets

in embedded endothelialized modules following the co-culture protocol were viable at day 2

(Fig. 5-2b) similar to when islets were only cultured in RPMI-1640 (Fig. 5-2a); no further studies

were done to measure islet function.

Based on the in vitro characterization studies, it was clear that islets (free or in endothelialized

modules) were viable and functional for only short term in culture (2 days); this is consistent

with reports from other groups where islets loser viability and function in culture over time

(193). For transplantation studies, free islets (cultured in RPMI-1640 alone) and islets in

endothelialized modules (co-culture conditions) were transplanted after 2 days in culture to

preserve islet viability and functionality. Although after 2 days in culture, endothelial cells were

sub-confluent on the surface of the modules; this was presumed to be a less crucial parameter

than embedded islet viability. Since previous studies with endothelialized modules in allogeneic

rats (Chapter 4) demonstrated that donor EC migrate from the surface of modules to form

vessels, it was possible that sub-confluent donor EC on the surface of endothelialized modules

may similarly migrate to form vessels in surrounding tissue.

98

RPMI-only Co-culture conditions D

ay 2

Day

7

a

c d

Figure 5-2: Islets cultured in a) RPMI-1640 medium only or b) in co-culture conditions were characterized by Live/Dead assay at day 2. Both culture conditions resulted in generally viable islets (green). c-d) Rat aortic endothelial cells on the surface of islet embedded modules were characterized by VE cadherin staining. c) RAEC were sparse on the surface of the modules with culture in RPMI only but d) with co-culture conditions, RAEC were confluent at day 7.

5.4.2 Transplanted intra-islet vessel density

The intra-islet vessel density was quantified with BS-1 lectin as it was reported to be a strong

marker for intra-islet EC(194). Revascularization of islets is known to take up to two weeks,

thus vessels were counted at 21 days after transplantation to measure complete vascular density.

Several BS-1 positive intra-islet vessels of various sizes were seen 21 days after transplantation

(Fig. 5-3a-d). In both the allogeneic and syngeneic model, the intra-islet vessel density was

slightly lower in the control group (islets for the allogeneic model and islets in modules (no EC)

for syngeneic model) but not significantly different (~14 vessels/150µm islet) from islets

transplanted in endothelialized modules (Fig. 5-3e). This was not surprising as endothelial cells

were transplanted on the surface of the modules and were not expected to impact the intra-islet

vessel density.

99

Allogeneic Syngeneic

Free

Isle

ts/Is

lets

in

mod

ules

(no

EC)

Isle

ts in

End

othe

lializ

ed

mod

ules

e) Figure 5-3: BS-1 lectin was used to identify intra-islet microvascular endothelial cell at day 21. Images show examples of islets (dashed outline) with intra-islet BS-1 positive microvessels (arrows). In allogeneic rats, some intra-islet microvessels were seen with both a) both islets alone and c) islets transplanted in endothelialized modules. Similarly, in syngeneic recipients, intra-islet BS-1 vessels were found in both b) islets in modules (without EC) and d) in endothelialized modules. Scale bar = 100 µm. e) Average intra-islet BS-1 vessel density (#vessels/150 µm diameter islet) was slightly higher (not significant) in islets transplanted in endothelialized modules at day 21 in both immunosuppressed allogeneic and syngeneic groups. Data presented is average value of 25 representative islets (5 islets/animal x 5 animals/group) +/- SEM; (p < 0.05).

a

c d

b

100

5.4.3 Peripheral vessel density of transplanted islets

vWf positive vessels of various sizes formed near (peripheral) transplanted islets at day 21 (Fig.

5-4a-d). More vessels were detected around islets when they were transplanted in

endothelialized modules (Fig. 5-4c-d). The peripheral vessel density was counted and there was

a significant increase, approximately a 25% increase in vessel density, when islets were

transplanted in endothelialized modules compared to islets alone in the allogeneic model at day

21 (Fig. 5-4e). Similarly, compared to islets in modules (no EC), there was a 20% increase in

vessel density in the syngeneic model (Fig. 5-4e). Interestingly, vessel density was also higher

with the endothelialized modules in the syngeneic group than the allogeneic group.

Allogeneic Syngeneic

Free

isle

ts/Is

lets

in

mod

ules

(no

EC)

Isle

ts in

End

othe

lializ

ed

mod

ules

a b

c d

101

e)

Figure 5-4: vWF antibody was used to identify all EC at day 21. Images show examples of islets (dashed outline) surrounded by vWF positive vessels (arrows). b-d) Implanted modules could also be identified by a weak brown staining. There was a greater vessel density surrounding islets (peripheral vessels) transplanted in endothelialized modules in both the c) allogeneic and d) syngeneic recipients. e) Average peripheral vWF positive vessel density (# vessels/mm2) was significantly higher around islets transplanted in endothelialized modules than free islets in allogeneic immunosuppressed recipients and also compared to islets in modules (no EC) in syngeneic recipients at day 21. Data presented is average value of 25 representative islets (5 islets/animal x 5 animals/group) +/- SEM; (p < 0.05).

The contribution of donor EC to islet peripheral vessel density was observed by double staining

with anti-GFP and insulin antibodies. In allogeneic immunosuppressed recipients, GFP positive

vessels were prominent around the islets as early as day 7 (Fig. 5-5a-b), included erythrocytes by

day 14 and continued to increase in size until 21 days (Fig. 5-5c-d). GFP positive vessels were

only detected on the periphery of islets and never in the intra-islet space; this observation is

consistent with the report that while intra-islet vessel density remained the same, peripheral islet

vessel density significantly increased with transplantation of islets in endothelialized modules

(Fig. 5-3e, 4e). The remodeling of GFP vessels was similar to that noted with previous studies

with endothelialized modules alone in allogeneic recipients (Chapter 4). However, in this model,

sub-confluent endothelialized modules after only 2 days in culture were capable of forming

vessels in vivo that appeared to integrate with the host vasculature (accumulated erythrocytes)

and were supported by SMA actin cells at day 21 (Fig. 5-5e). Moreover, some GFP positive

vessels formed in close (50-100 µm) proximity to islets suggesting that donor derived vessels

were directly feeding transplanted islets. It is likely that angiogenic signaling by co-transplanted

islets also promoted donor EC derived vessel formation. Others have reported that intra-islet EC

102

migrate out to form chimeric vessels with host EC(136, 137, 194). Since intra-islet EC were not

labeled in our study, we cannot determine whether they also contributed to revascularization

here. The incorporation of GFP cells into vessels also explains the observed increase in vessel

density as donor EC likely secreted growth factors to initiate angiogenesis and were available for

integration into new vessels.

Day 7 – Islets Day 7 – GFP vessels

Day 14 Day 21

Figure 5-5: GFP-RAEC around islets in endothelialized modules transplanted in allogeneic immunosuppressed rats. At day 7, serial sections show a) islets (insulin positive, dashed outline) surrounded

c d

e

a b

103

by b) donor EC (GFP positive, arrows). Scale bar = 100 µm c-d) Sections were double stained with an anti-insulin antibody (purple) to identify transplanted islets and an anti-GFP antibody (brown) to identified transplanted EC. GFP positive vessels (arrows) were near transplanted islets (dashed outline) at a) day 14 b) and day 21. Scale bar = 50 µm. c) By day 21, GFP vessels (green, arrows) formed vessels of various sizes, accumulated erythrocytes and were supported by smooth muscle actin positive cells (orange).

5.4.4 RAEC isolated from Lewis rats

GFP positive cells were not seen with transplantation of islets in endothelialized modules in

syngeneic recipients after 14 days. This surprising finding can be attributed to two observations.

Syngeneic EC (Lewis strain donor) were much slower in proliferation and did not grow to

confluence on the surface of the modules (as determined by VE cadherin staining) even after 7

days in culture (Fig. 5-6a); this was starkly differently from previous work with RAEC isolated

from the Sprague Dawley strain. Moreover, transplantation of syngeneic endothelialized

modules appeared to initiate an immune response (T cells) in pilot studies (Fig. 5-6d-e) and GFP

positive donor syngeneic EC were seen at day 7 but not at day 21 (Fig. 5-6b-c). Current studies

are further investigating whether a variation in the substrain between donor and recipient may

have initiated this immune response. Donor RAEC were isolated from a Lewis/MolTac

substrain and the transplant recipients were of a Lewis/Crl substrain. Although the MHC

variability between the two substrain has not been clearly identified, there were reported genetic

variation between the two substrain (195, 196). Regardless, in the syngeneic model used here,

even though donor EC did not persist, their addition to the surface of the modules resulted in a

higher vessel density around transplanted islets at 21 days. The donor EC likely released growth

factors at the early time points to initiate a host angiogenic response which resulted in this

increased vessel density.

a)

104

Day 7 Day 21

GFP

(don

or E

C)

TCR

(T c

ells)

Figure 5-6: Lewis rat endothelialized modules. a) VE-cadherin staining of endothelialized modules (no islets) indicated that RAEC (Lewis strain) were sparse on the surface of the modules after 7 days in culture. b-c) Anti-GFP antibody was used to identify donor Lewis EC and d-e) TCR-α,ϐ antibody was used to identify host T cells with transplantation of endothelialized modules in syngeneic rats. b) Donor EC were seen at day 7 (arrows) around modules (dashed outline) c) but not at day 21. d-e). d) There was a large T cell response near transplanted modules at day 7 e) and by day 21, fewer T cells were seen. Scale bar = 100 µm.

5.4.5 Transplanted islet viability

Insulin staining was used to detect viable ϐ cells in transplanted islets. Rodent islets contain

roughly 2000-4000 cells, 70-80% of which are ϐ cells (197). Each ϐ cell contains roughly

10,000 insulin granules. Anti-insulin antibody recognition of insulin granules can be used as an

indication of viable ϐ cells which in turn is used to estimate a viable islet mass. Some insulin

positive islets were found until day 21 after transplantation of free islets (Fig. 5-7a-b) and islets

in endothelialized modules (Fig. 5-7c-d) in allogeneic immunosuppressed rats. Similarly,

transplanted islets were insulin positive in all syngeneic rats until 21 days (regardless of

treatment, Fig. 5-7e,g) and in treated (normoglycaemic) rats, insulin positive islets were detected

until 60 days both in collagen modules (without EC, Fig. 5-7f) and in endothelialized modules

b c

d e

105

(Fig. 5-7g). Free islets were found distributed randomly among the fat tissue in the omentum

and tended to agglomerate by 21 days (Fig. 5-7a-b). Meanwhile, islets in modules (collagen only

or endothelialized) remained either inside the modules (Fig. 5-7e,h) or were seen in the tissue

spaces between individual modules (Fig. 5-7d,f,g). The presence of viable islets in

endothelialized modules is encouraging as others have reported that islets embedded in collagen

gels were not viable in SCID mice at 28 days(198).

To quantify this observation, the area of all insulin positive cells in 3 sections (5µm depth each)

was measured at 21 days after transplantation of 2000 islets (Table 5-2). In allogeneic

immunosuppressed animals, the area of insulin positive cells was the same whether islets were

transplanted alone or in endothelialized modules. In syngeneic rats, the average area of insulin

positive cells was higher when islets were transplanted in endothelialized modules rather than in

collagen modules (no EC) but due to the large variation within the group, the difference was not

statistically significant (p=0.07). Interestingly, the insulin positive area in the syngeneic group

with endothelialized modules was significantly higher than free islets or islets in endothelialized

modules in the allogeneic rats. The lower islet viability in allogeneic rats is presumed to be a

result of the toxic effects of the daily administered immunosuppressant(23, 199).

Table 5-2: Average insulin positive area for diabetic animals transplanted with 2000 islets 21 days after transplantation. In allogeneic animals, the average insulin positive cells area was the same (0.15 mm2) for animals transplanted with free islets or islets in endothelialized modules. In syngeneic rats, animals transplanted with islets in endothelialized modules had a higher (not statistically significant) insulin positive cell area (0.85 mm2) than islets in modules without EC (0.48mm2).

Group Average insulin positive area (mm2) SEM N

Islets alone allogeneic 0.148 0.036 5 Islets in endothelialized modules allogeneic 0.149 0.008 5 Islets in modules syngeneic 0.477 0.137 3 Islets in endothelialized modules syngeneic 0.854 0.258 3

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Figure 5-7: Anti-insulin antibody was used to identify transplanted islets in (a-d) immunosuppressed allogeneic and (e-h) syngeneic recipients; arrows show examples of insulin positive islets and modules are highlighted by dashed outline. Viable islets were seen with transplanted (a-b) free islets and (c-d) islets in endothelialized modules until 21 days. Similarly, insulin positive clusters were seen in syngeneic recipients with (e-f) islets in modules and (g-h) islets in endothelialized modules until 60 days. Scale bar = 100 µm.

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5.4.6 Transplanted islet function

To evaluate transplanted islet function, non-fasting blood glucose and insulin levels were

measured in diabetic rats. In allogeneic immunosuppressed rats, there was an immediate

decrease in blood glucose with transplantation of 2000 free islets or islets in endothelialized

modules (Fig. 5-8a). However, blood glucose continued to rise over time and within a week,

glucose levels in both groups were similar to the levels prior to transplantation indicating that

transplanted islets were rejected during this period (Fig. 5-8a). This level of graft failure is

expected as the mass required for successful reversal in allogeneic immunosuppressed rats is

15,000 IEQ/kg as compared to our minimal mass implant of 8000 IEQ/kg(23). There was no

difference in the graft failure rate between transplanted free islets or islets in endothelialized

modules. Islet function was also assessed by comparing non fasting serum insulin levels and

measurements at 21 days indicated that all animals transplanted with 2000 islets (free or in

endothelialized modules) had higher serum insulin levels as compared to sham animals but not as

high as non-diabetic controls (Fig. 5-10a). The higher serum levels correlated with the

observation that at least some islets were viable in transplanted animals at day 21 and were likely

secreting insulin. However, the low mass of surviving islets meant that insulin secretion was not

sufficient to reverse hyperglycaemia.

In the syngeneic rats, there was no immediate decrease in blood glucose after the transplantation

of 2000 islets in collagen modules or in endothelialized modules when the results were averaged

together, but there were large variations within each group (Fig. 5-8b). In fact, 40% (2 out of 5)

of the animals transplanted with 2000 islets in modules (no EC) and in endothelialized modules

had a significant decrease in blood glucose levels and were treated until the end of the study (60

days) (Fig. 5-9b). Although blood glucose level decreased immediately after transplantation,

normal (< 11.1 mmol) blood glucose levels were generally achieved after 21 days. This trend

was similar to pilot studies in which transplantation of 2000 free islets in syngeneic rats resulted

in treatment of 1 out of 3 recipients (Fig. 5-9a). Also, average serum insulin levels were

significantly (p<0.05) higher when animals were transplanted with 2000 islets in endothelialized

modules compared to sham controls and were equivalent to control non-diabetics. There was a

large variation in serum insulin levels within the group as treated animals had higher serum

insulin levels than the non-treated animal (Fig. 5-10b). Treated animals were further subjected to

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an oral glucose tolerance test at 60 days after transplantation where glucose measurements were

made after introduction of a glucose challenge. Compared to non-diabetic control animals, all

treated animals displayed slower glucose clearance, but by the end of the test all animals returned

back to normoglycaemic levels (Fig. 5-9c). The slower glucose clearance in treated animals has

been noted in other studies with transplantation into the omentum(15) and it is suggested that

islets are not innervated as well in the omentum which may explain the slow response to an

increase in serum glucose(200).

The finding that transplantation of 2000 islets (free islets, islets in modules (no EC) or in

endothelialized modules) in the syngeneic rats was only successful in treating 40% of

transplanted recipients was inconsistent with reports from another group where 2000 islets in the

omentum reversed diabetes in 100% of syngeneic rats until 6 weeks(15). The omental pouch as

a transplant site has not been explored extensively and there are some indications that compared

to the subcapsular kidney space, more islets are required for treatment of diabetes in the

omentum(200). Consistent with this, one group reported that the same number of islets (250) in

the omentum only reversed diabetes in 20% of mice as compared to 100% of mice treated with

transplantation under the kidney capsule or intrahepatically(144). It is not clear why the

omentum requires a larger mass of islets to reverse hyperglycemia since it is a well vascularized

tissue space that allows for portal (same as native pancreas) delivery of insulin(11). On the other

hand, it is possible that there is a large islet loss during transplantation as the omental pouch is

technically challenging. Also, the omentum harbors milky spots with leukocytes which may get

recruited to the transplant site(10). A thorough comparison of the omental pouch site with other

transplant sites is required to fully characterize the impact of this transplant site.

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a)

b) Figure 5-8: Line plot of average blood glucose levels over time of diabetic a) immunosuppressed allogeneic and b) syngeneic recipients that underwent sham surgery, or were transplanted with 2000 free islets, islets in modules (no EC) or islets in endothelialized modules. a) In allogeneic recipients, there was a sharp decrease in blood gluose levels one day after transplantation but all animals became hyperglcaemic again within one week; there was no difference between animals transplanted with islets alone or islets in endothelialized modules. b) In syngeneic recipients, the average glucose levels did not decrease after transplantation but there were large variations within the group. Data presented is average values of blood glucose levels +/- SEM, sham (n=2), allogeneic and syngeneic groups (n=5).

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a)

b)

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c) Figure 5-9: a) Line plot of blood glucose levels of individual syngeneic recipients that were transplanted with 2000 islets; 1 out of 3 recipients became normoglycaemic. b) Line plot of blood glucose levels of individual syngeneic recipients treated until 60 days with islets in modules (no EC) or in endothelialized modules. b) Line plot of blood glucose levels over time after an oral gluocse challenge of non-diabetic control and syngeneic treated (normal blood glucose levels > 5 days) recipients 60 days after transplantation. The transplanted animals had increased blood gluocose levels 30 minutes after glucose administration as compared to non-diabetic controls. By the end of the test, 120 minutes after glucose challenge, all animals returned to normoglycaemic levels. Blood glucose levels lower than 11.1 mM were defined as normoglycemic.

a) b)

Figure 5-10: Plot of non-fasting serum insulin levels. a) immunosuppressed allogeneic (n=2) and (b) syngeneic recipients (n=5) that were transplanted with 2000 free islets, islets in endothelialized modules or islets in modules (no EC) for 21 days. For each group, serum levels for individual animal are marked by (▲)

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and the group average is represented as (-). a) Allogeneic immunosuppressed recipients that were transplanted with 2000 islets (alone or in endothelialized modules) had higher serum insulin levels than the diabetic sham group but not as high as the non-diabetic control group. Animals that received islets in endothelialized modules had slightly higher serum insulin levels than animals with islets alone. b) Transplantation of 2000 islets in endothelialized modules significantly(p<0.05) increased the serum insulin levels at day 21 as compared to diabetic shams and serum levels were equivalent to non-diabetic controls.

5.4.7 Why does increased vessel density not correlate with improved islet viability or function?

Overall there was little difference in islet function and viability when islets were transplanted in

endothelialized modules compared to controls in both immunosuppressed allogeneic and

syngeneic recipients. This was somewhat surprising as we noted an increased vessel density

with endothelialized modules and presumed it would improve islet engraftment. A few groups

have reported improved vessel density by VEGF delivery which related to improved function of

transplanted islets. For example, there have been short term improvements in engraftment of

islets with overexpression of VEGF transplanted under the kidney capsule. Elevated VEGF

production in islets increased vessel density and improved glucose control for 16 days in SCID

mice(140). Similarly, controlled VEGF production in islets (isolated from transgenic mice that

secrete VEGF under the rat insulin promoter) restored normoglycaemia in a greater percentage

of syngeneic mice, increased vascular density and blood flow 12 days after transplantation(134).

Rather than direct transduction of islets, delivery of VEGF and FGF through biomaterials also

improved glucose control in the omentum of syngeneic mice, although vessel density was not

measured in this study. The long term benefit of VEGF therapy was also demonstrated as

exogenous VEGF delivery to transplanted islets improved oxygen tension at the transplant site

(kidney capsule) and prolonged euglycaemia in syngeneic animals until 6 months and delayed

rejection in allogeneic recipients (12 days as compared to 9 days without

immunosuppressant(139). Interestingly, the authors also reported other benefits of VEGF

therapy including anti-apoptotic signaling and prevention of central core necrosis in vitro which

likely also supported islet engraftment in vivo. Yet another method to improve vessel density for

transplanted islets has been by co-transplantation with bone marrow derived mesenchymal stem

cells (MSC); islet engraftment and vessel density were improved with co-transplantation of MSC

in syngeneic rats until 39 days; however, oddly 2000 free islets alone under the kidney capsule

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were not sufficient to reverse diabetes in their hands(145). Also, syngeneic (but not allogeneic)

MSC improved long term survival of a suboptimal number (1200) of allogeneic islets and

reversed diabetes in allogeneic rats; the role of MSC in modulating the immune and

inflammation response was shown to be key factor in promoting islet engraftment(16). The

above studies suggest that increased vascular density can improve transplanted islet engraftment.

However, rather than just increase vessel density for transplanted islets, the therapies likely

benefit transplanted islets through multiple mechanisms. For example, VEGF delivery also

prevented islet apoptosis through anti-apoptotic signaling and transplantation of MSC modulated

the host inflammation/immune response. Therefore, while an increase in vessel density alone

may promote islet viability, it is likely that a combination of interventions are required to

promote eventual islet engraftment.

There may be several reasons for the lack of improvement in islet engraftment with

transplantation in endothelialized modules. In allogeneic rats, although donor EC certainly

improved vessel density at day 21, our previous studies indicate that donor derived vessel

maturity and perfusion takes 2-3 weeks. This lag time in increasing functional vessel density

likely did not support islets in the early transplant period during which the majority of

transplanted isles were lost. It is also possible that survival of the remaining islets in the

endothelialized modules at a later time point, when donor vessels were functional, would be

improved over free islets; thus it might be interesting in future studies to evaluate islet survival

after 3 weeks. Additionally, the administration of immunosuppressant to allogeneic rats

increased islet toxicity which may have resulted in a large loss of islet mass regardless of

vascularization; previous studies with VEGF delivery and MSC co-transplantation have not

evaluated islet function in the presence of immunosuppressants. In syngeneic rats, although

there was an increased vessel density at 21 days, there were no donor EC remaining which meant

that the vessels were all host derived. It is not clear whether the new host derived vessels also

matured slowly and whether they supported transplanted islets in the early post-transplant period.

Also, the activated immune system to donor EC may have also harmed co-transplanted islets;

which explains why there was only a slight increase in transplanted islet viability (insulin

positive cells). Still, an increase in the number of insulin positive stained cells with

transplantation in endothelialized modules was encouraging as it hints that with sufficient

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modulation of the host immune system, an improvement in vessel density may improve islet

viability.

5.5 Conclusions

We have demonstrated that transplantation of islets in endothelialized modules increased the islet

peripheral vessel density in both allogeneic immunosuppressed and syngeneic rats. In allogeneic

rats, donor EC contributed directly to the vessels seen near co-transplanted islets and included

host vascular supporting cells by day 21. At least some islets were viable and functional until 21

days in both models. While 2000 islets were not sufficient to reverse diabetes in allogeneic

immunosuppressed rats, 2000 islets reversed diabetes in 40% of syngeneic recipients. In the

allogeneic immunosuppresed rats, there was some improvement in islet function as measured by

serum insulin levels with transplantation of islets in endothelialized modules as compared to free

islets at day 21. Meanwhile, in the syngeneic model, there was an increase in islet viability as

measured by insulin positive cells with islets in endothelialized modules compared to islets in

modules (no EC) at day 21. This study was the first exploration of primary islet transplantation

with endothelialized modules and suggests that with sufficient modulation of the immune

systems, endothelialized modules may improve islet engraftment.



(EB001013), the Canadian Institutes of Health Research (MOP-89864) and Natural Sciences and

Engineering Research council of Canada (Post-graduate scholarship). We are grateful to Chuen

Lo and his technical expertise in animal surgeries. Also, we thank Chyan-Jang Lee (Dr. J

Medin) for generation of the eGFP-RAEC, Deb Dixon (Dr. Rajotte lab) for islet isolations,

Mount Sinai Services for Insulin ELISA assay and Toronto General Hospital’s Pathology

research group for all histology and immunostaining services.

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6 Conclusion and Future Work

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6.1 Conclusions

In this thesis, we explored endothelialized modular constructs as a means of promoting

vascularization and supporting the engraftment of islets in diabetic recipients. Allogeneic

endothelialized modular constructs generated a perfusable vascular network in

immunesuppressed recipients. This result is the first demonstration that primary unmodified

donor endothelial cells can form stable vessels in a clinically relevant allograft model.

Transplantation of endothelialized modules resulted in a wound healing response with

recruitment of inflammatory cells and the formation of a vascular network. Donor endothelial

cells, HUVEC and RAEC, contributed directly to the angiogeneic response by forming vessels

around transplanted modules in athymic and allogeneic recipients respectively. The extent of

vessel formation and survival of donor derived vessels depended on the cell type and transplant

model. In the nude rats, xenogeneic HUVEC formed vessels only until day 3 but with temporary

depletion of macrophages HUVEC derived vessels survived until day 7. Similarly, RAEC

derived vessels were rejected by day 7 in allogeneic recipients but minimization of immune and

inflammation mediated (by the use of Tacrolimus and Atorvastatin) rejection led to donor EC

survival for at least 60 days. In allogeneic immunesuppressed recipients, donor EC formed all

three types of vessels: capillary-like, venule-like and arteriole-like vessels. Donor derived

vessels were at least partially functional at 60 days as evidenced by fluorescent beads and

microfil perfusion. Transplantation of endothelialized modules resulted in a significant increase

in vessel density which included new host vessels, donor EC derived vessels and chimeric (host

+ donor EC) vessels. Thus, endothelialized modules were an effective method of vascularising

tissues when immune and inflammatory responses were modulated appropriately.

Primary islets co-cultured in endothelialized modules were viable and functional in vitro.

Transplantation of islets in endothelialized modules significantly improved peripheral islet vessel

density in syngeneic and immunesuppressed allogeneic recipients compared to islets in modules

(no EC) and free islets respectively. For treatment of diabetes, 2000 islets in the omental pouch

were not sufficient to restore nomoglycaemia in allogeneic immunesuppressed recipients but

reversed diabetes in 40% of syngeneic recipients. Unfortunately, the enhanced endothelialized

modules associated vessel density did not improve transplanted islet function as measured by

serum glucose and insulin levels in comparison to free islets or islets in collagen modules. On

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the other hand, there was a trend towards increased islet viability with transplantation in

endothelialized modules in syngeneic recipients. It is expected that further modulation of host

immune and inflammatory responses is required to successfully improve islet engraftment.

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6.2 Recommendations and Future work

Engineering complex tissues with adequate vascularization is crucial for engineering viable

tissues. The current thesis demonstrated that endothelialized modules offer a way of assembling

micro-tissues with an inherent vascularization that integrates with the host vasculature. While

some studies with primary EC transplantation have successfully induced new vessel growth in

immunecompromised animals, this study demonstrated that unmodified (i.e. not transfected with

anti-apoptotic genes or transplanted with accessory cells) endothelialized modules form a stable

vasculature in immunesuppressed allogeneic recipients. Moreover, this study demonstrated that

endothelialized modules improved vessel density for transplanted islets. Clearly, the failure to

restore normoglycaemia with transplantation of islets in endothelialized modules requires further

work. The following highlights key focus areas for future work and recommends a few

strategies that might be explored within these areas. Future work with allogeneic endothelialized

modular constructs as shown in Fig. 6-1 includes characterization of the cellular and molecular

events and donor derived vessel functionality which will in turn guide strategies to improve

donor derived vessel maturation and prevent activation of host immune and inflammatory

responses to improve engraftment of co-transplanted islets. Additionally, other areas for future

work are optimization of the syngeneic endothelialized modular construct and exploring

endothelialized modules as a non-thrombogeneic surface in vivo.

Allogeneicendothelialized

modular constructs

Characterize molecular and cellular events

(0-7 days post transplant)

Characterize donor derived vessel

functionality (7-21 days)

Improve vessel maturation

Reduce host inflammation and

immune activation

Improve islet engraftment in

endothelialized modules

Figure 6-1: Schematic of future areas of focus with allogeneic endothelialized modules

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6.2.1 Characterize allogeneic endothelialized modules

Allogeneic RAEC endothelialized modules transplanted in immunosuppressed allogeneic

recipients drove the formation of a stable and functional vasculature long term. Vascularization

by endothelialized modules was measured by histological quantification of vessel density at

several time points. Qualitative microCT imaging also confirmed increased vascularization and

to some extent, the functionality of the new vascular bed. These results serve as a starting point

for further studies to fully characterize the response of endothelialized modules. Some areas that

remain to be fully understood in this model are the host cellular and molecular responses to

donor EC that lead to the observed vascularization, the extent of vessel ‘leakiness’ at the early

time period, and complete functionality of stable donor EC derived vessels.

Although it is clear that donor EC initiate a large angiogeneic response, the cellular and

molecular events surrounding the donor EC driven vascularization is not known. In a normal

sprouting angiogeneic response, tip cells of blood vessels migrate from parent vessels to the site

of growth factor release. With transplantation of endothelialized modules, by day 3, there were a

number of BS-1 positive (not GFP positive) cells around transplanted modules indicating that

host EC migrated into the transplant site within 3 day after transplantation. At day 7, GFP

positive primitive vessels were observed and host EC likely recruited donor EC to migrate from

underlying the collagen matrix to participate in vessel formation. This two-way interaction of

donor and host EC is an important phenomenon and the molecules involved in this interaction

have yet to be identified. Angiogeneic growth factors such as VEGF, FGF, PDGF, and Tie-2

likely play an important role in these interactions. In addition, donor EC may drive the

angiogeneic response by hypoxic signaling and release of pro-inflammatory cytokines.

Molecular analysis for angiogeneic growth factors, hypoxic markers and inflammatory cytokines

between days 1-7 after transplantation can be done by measurement of serum molecules via flow

cytometery (if the systemic concentrations are high enough), and analysis of whole tissue or

microdisssected gene arrays (microarrays followed by RT-PCR) (201, 202). Understanding the

molecular mechanisms involved will help to develop strategies that further improve

vascularization by endothelialized modules.

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The function of the newly developed vessels 1-3 weeks after transplantation remains to be

understood. Analysis of the whole omentum tissue with endothelialized modules by microCT

imaging indicated that the vascular bed was leaky at day 21; however, it was not clear how many

of the newly developed vessels were leaky and whether the leaky vessels were of donor, host or

chimeric origin. Building on the current model, further perfusion studies can be carried out with

fluorescent dextrans or beads followed by whole tissue imaging techniques such as two-photon

confocal imaging or optical projected tomography techniques to fully characterize perfusion in

the vascular bed. Fluorescent whole tissue imaging will permit identification of donor derived

(GFP positive) vessels from host vessels. Additionally, advanced angiogenesis imaging models

such as dorsal skin-fold chambers and cranial windows from tumor literature could perhaps be

adapted to understand endothelialized modular derived vessel formation(203).

Finally, it will be important to further characterize the function of mature donor derived vessels.

In the current work, assuming vascular remodeling has been complete by day 60, a combination

of immunohistological evaluation of supporting cells and microCT imaging to assess perfusion

concluded that donor derived EC contribute to a complete pefusable vascular bed consisting of

capillaries, venule-like and arteriole-like vessels. The next stage of characterization would be to

further delineate the function of donor EC derived vessels. Ex-vivo whole tissue flow

measurements via Doppler-flow imaging modality can assess whether flow through the vascular

bed is normal. Moreover, complete function of donor derived vessels specifically can be

assessed by measuring permselectivity to assess EC barrier properties and introducing an

inflammatory stimuli to assess regulation of cytokines, interleukins and adhesion molecules as

previously described(64). Further characterization of the current model will enable a complete

picture on the remodeling and mechanisms involved for endothelialized module derived

vascularization.

6.2.2 Improve donor derived vessel maturation

A drawback of the current method of vascularization by endothelialized modules is the long time

required for formation of functional vessels. Although primitive donor derived vessels were seen

at day 7, perfusion into the implant site likely occurred much later. Forming a perfusable

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vascular network faster would be advantageous for supporting functional cells. Some groups

have shown the benefit of co-transplanting supporting vascular cells (adult smooth muscle cells,

embryonic fibroblasts, mesenchymal precursors) to endothelial cells in immunodeficient

recipients(3, 4, 71). In the allogeneic model, transplanted RAEC recruited supporting cells by

day 14, but it is possible that co-transplantation with supporting cells will hasten maturation of

donor EC derived vessels. Another way to achieve vessel maturation is by pre-forming

capillary–like structures prior to transplant. A few groups have transplanted microvascular

fragments (with pre-formed capillary-like structures) and have reported that that host

insoculation and flow into the capillaries began as early as day 4(74, 75). Perhaps, instead of

delivering modules with EC on the outside surface, modules with a capillary-like network can be

implanted. One way to form a capillary network is by embedding EC inside of individual

modules (rather than on the outside surface) to initiate EC sprouting (56, 57). Alternatively, EC

covered modules can be embedded in a provisional matrix (laminin, collagen IV blend) to further

promote sprouting and capillary-like formation outside of modules. The capillary networks can

be further augmented by incorporating supporting vascular cells (smooth muscles, embryonic

fibroblasts, mesenchymal cells) to form a mature vessel network prior to implant.

6.2.3 Prevent host inflammation and immune responses

Transplantation of endothelialized modules resulted in a large inflammatory and immune host

response in all models tested. Another area of improvement with the current model is to reduce

or minimize these responses. In some respect, the observed response is expected as vascularized

organ allo-transplantation has revealed that allogeneic EC directly activate immune and

inflammation responses(204). Prior to transplantation, EC form a quiescent layer on the surface

of the module but it is likely that after transplantation, donor EC become activated.

Characterization of molecular events during the early post-transplant period will identify the

main factors implicated in the resulting inflammatory response. Then, tools to prevent EC

activation can include genetic modification of donor EC to downregulate production of

inflammatory cytokines. For example, EC overexpression of Bcl-2 and Bcl-XL, anti-apoptotic

genes, inhibited NF-κϐ gene transcription and subsequent expression of adhesion molecules,

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chemokines and procoagulant factors on the EC surface(205). Moreover, Bcl-2 expression on

the EC surface has been correlated with accommodation of vascularized hamster xenografts

(206) and human allografts (207). Genetic manipulation of EC can lead to unwarranted side

effects however and other ways to achieve EC quiescence may be to modify the underlying

matrix. For example, it has been reported that embedding EC in Gelfoam matrices prevents

allogeneic T cell recognition and immune response in vivo (208). Thus genetic modification of

donor EC and/or substrate modifications may allow for allogeneic endothelialized module

transplant with minimal host inflammation and immune responses.

6.2.4 Improve engraftment of islets in endothelialized modules

Endothelialized modules offer a versatile tool for vascularization of functional cells and tissues.

A lack of adequate vascularization has been suggested as the basis for hypoxia mediated islet

death so an increase in islet vascularization is expected to improve islet viability. However, in

this study, although there was a significant improvement in vascularization of islets when

transplanted in endothelialized modules, there were only slight improvements in islet viability as

measured by insulin positive staining at day 21 in syngeneic recipients. In addition, islet

function as measured by serum glucose and insulin levels in diabetic recipients was similar with

transplantation of islets in endothelialized modules as compared to both free islets and islets in

modules (no EC). One explanation for this is that donor EC derived vessels were not mature and

functional during the early post-transplant period during which majority of the islets were lost.

Future work should further evaluate the vessel development by characterizing oxygen tension for

transplanted islets and measuring blood flow to transplanted islets as previously done with islet

transplantation under the kidney capsule(141). Improvements in maturing donor vessels faster as

discussed earlier may benefit co-transplanted islets and prevent islet loss. Alternatively, islets

can be introduced into a pre-vascularized space where a vascular network has already been

established. For example, others have reported that islets transplanted in pre-vascularized fat

chambers or splenic arteries can treat diabetic recipients(209). Similarly, collagen gels with

microvessels have been used to deliver islets(198). However, these models still need to be

rigorously tested to understand whether they offer improved engraftment over free islets in a

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translational model. Another possible reason for islet loss may be the strong inflammation and

immune responses to donor EC and prevention of these responses as discussed earlier will also

benefit co-transplanted islets. Future experiments can combine either prevention of these

responses or add anti-inflammatory interventions to promote islet engraftment.

6.2.5 Syngeneic endothelialized modular constructs

Complete prevention of the activation of immune system with endothelialized allogeneic

transplant may be difficult. An alternative would be to utilize a syngeneic model for functional

cell transplantation and avoid the use of immunesuppressants and their related toxicity. In this

thesis, transplantation of islets in endothelialized modules in a syngeneic model led to increased

vessel density when compared to collagen modules alone. However, the use of syngeneic EC

(Lewis rat strain) provided two surprising results: a) RAEC isolated from Lewis donor did not

proliferate in vitro and grew poorly on the surface of modules and b) donor EC only survived

until 14 days and in fact initiated an immune response (Fig. 2). The poor culture of EC in vitro

may be a Lewis strain specific phenomenon as limited studies indicate heterogeneity in the

growth and angiogeneic potential of endothelial cells from different mouse strains (210, 211). In

that case, it will be of interest to investigate other anatomical sites for EC harvest to yield more

robust EC culture or explore EC harvest from other syngeneic rats (i.e. Fischer strain). In

addition, the collagen matrix can be modified with other ECM components (laminin, fibronectin,

collagen type IV) to promote EC adhesion and spreading. In vivo activation of the immune

system may be a result of the difference between substrains of donor EC (Lew/MolTac) and

recipient (Lew/Crl) and pilot studies are currently investigating this theory. In the future, in vivo

immune activation can be avoided by matching the sub-strain of donor EC and recipient

substrain. If fact, a few studies have shown that syngeneic EC do not activate the immune

system (T lymphocytes) in comparison to allogeneic EC(212, 213), therefore it is expected that a

syngeneic EC transplant model can be developed. It will be of interest to characterize syngeneic

donor EC vessel development and maturation as with the allogeneic model. Once the syngeneic

model has established the benefit of vascularization for functional cells, future studies will be

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needed to incorporate innovative ways to modulate the immune system to allow for clinically

relevant allograft transplantation.

6.2.6 Endothelialized modules and in vivo blood compatibility

The confluent endothelial cell layer on the surface of modules has previously shown to be a non-

thrombogeneic, blood compatible surface in vitro(8). Although this property was not explored in

the current thesis, there are many applications of a non-thrombogeneic surface. One such

application is the use of endothelialized modules for delivering islets via the portal vein. The

portal vein is the clinical model for islet transplantation; however blood contact with an activated

islet surface is known to initiate immediate coagulation and inflammatory reaction(115, 116). A

confluent and quiescent endothelial lining might prevent these reactions(120). Pilot studies

showed that modules can be transplanted via the portal vein and lodge into the liver lobes (Fig.

6-2). A full study with transplantation of endothelialized modules via the portal vein and

measurement of serum levels for common coagulation markers such as TAT, FVIII-AT complex

within one hour and histological evaluation of leukocyte infiltration to graft site can be carried

out. Islets can be incorporated within endothelialized modules to assess whether the endothelial

surface prevents the instant blood mediated inflammatory reaction mediated islet apoptosis.

a) b) Figure 6-2 a-b) Trichrome sections of RAEC covered modules via the portal vein and embedded in the liver

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Finally, endothelialized modular tissue engineering is a platform technology that can be applied

for vascularization of ischemic tissues such as myocardial infarction, peripheral limb occlusion,

engineering functional tissues such as skin, muscle grafts, metabolic tissues and even developed

as a model for tumor angiogenesis to study the impact of anti-angiogeneic therapies. This thesis

provided a framework for endothelialized module remodeling in four different models:

immunecompromised, allogeneic, immunesuppressed allogeneic and syngeneic rats. Moreover,

the impact of the vascular development on viability and function of co-transplanted islets was

characterized. The results from this work can be applied to design many other applications of

the endothelialized microtissues.

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Application of an Endothelialized Modular Construct for ...€¦ · Rohini Gupta . Doctor of Philosophy . Chemical Engineering and Applied Chemistry University of Toronto, 2010