Sodium Ascorbate as a Potent Stimulator of Elastic Fiber ... · Sodium Ascorbate as a Potent Stimulator of Elastic Fiber Production Master of Science (2011), Hyunjun Kim (Jonathan),

Sodium Ascorbate as a Potent

Stimulator of Elastic Fiber Production

By

Hyunjun Kim (Jonathan)

A thesis submitted in conformity with the requirements for

the degree of Masters of Science

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

Copyright by Hyunjun Kim (2011)

Sodium Ascorbate as a Potent Stimulator of Elastic Fiber Production

Master of Science (2011), Hyunjun Kim (Jonathan),

Department of Laboratory Medicine and Pathobiology, the University of Toronto

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ABSTRACT

The complicated problem of efficient stimulation of elastic fiber production in already

developed human tissues has not yet been solved. The present study introduces sodium

ascorbate (SA) as a stimulator of elastogenesis in cultures of different cell types including

fibroblasts isolated from patients with elastopathy genetic diseases. We then elucidated

mechanisms of elastogenic action of SA. SA exercises its net elastogenic effect only after being

actively transported into the cell interior through two separate mechanisms. These are the ―fast

effect,‖ which reflects the greater stability of intracellular tropoelastin, and the ―late effect,‖

which reflects the true enhancement of the elastin gene expression occurring after SA-induced

activation of c-src tyrosine kinase and the consecutive phosphorylation of IGF-1 receptor, which

triggers the downstream signals leading to activation of the elastin gene expression. In

conclusion, for the first time we have established that SA is a potent stimulator of elastic fiber

production.

Sodium Ascorbate and Elastogenesis Hyunjun Kim (Jonathan)

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ACKNOWLEDGEMENTS

I would like to express my greatest acknowledgement to my father Choonkwon and my

mother Chosim, who provide unconditional caring, love and sacrifice so their children might

have a better education and lifestyle.

This thesis would not have been possible without my brother Hyunsuk, who not only

influenced me to be where I am today, but also helped me write the thesis while my arm was

broken. I am also grateful to my dear friend Heeyeon Park, as she spent a lot of time with me

while I was writing this thesis, providing me with laughter. She also proofread a portion of the

thesis. I would also like to thank my band members and my cell group members in Dongshin

Church and Agape Impact, who were considerate and understood my busy schedule while

writing the thesis.

I owe my deepest gratitude to my supervisor, Dr. Aleksander Hinek, who continuously

provided me encouragement, guidance and support. His passion in research has inspired me to

pursue a research-based master’s degree. He did not just teach me about science; he helped me

mature and provided me advice that will guide me in my future career and the rest of my life. I

would like to acknowledge the friendship and assistance from fellow members of the Dr. Hinek

Laboratory: Yangting Wang, Andrew Wang, Junyan Shi and Sanjana Sen. Without their

insightful data analysis and technical help, I would have not finished this thesis.

I am also thankful to my committee members, whose suggestions provided exceptional

insight into this work: Dr. Maurice Ringuette and Dr. David Chitayat.

Finally, I would like to acknowledge the Canadian Institute of Health Research (Dr.

Hinek, Grant PG 13920) for supporting the research presented in this thesis. I also would like to

acknowledge student’s scholarship from the University of Toronto.


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TABLE OF CONTENTS

INTRODUCTION……………………………………………….….…........1

Regenerative Medicine……….………………………………………………….…….....1

The Extracellular Matrix…….………………………………………………….…….....3

Collagen Fibers…………………………………………………………………….4

Synthesis…………………………………………………………………...4

Ascorbic Acid as a Stimulator of Collagen Synthesis………………………..5

The Role of Collagen in Fibrosis……………………………………………….6

Elastic Fibers….……………...……..…………………………………...…...……6

Elastin Gene Regulation…………………………………………………….7

Tropoelastin Synthesis and Secretion ………………………..…………...8

Assembly of Elastic Fibers ………………..……………………………....9

Diseases and Conditions Affecting Elastic Fibers: Elastinopathies…….………...12

Primary Elastinopathies…………………………………………………….…13

Secondary Elastinopathies…………………………………………………….14

Role of Elastin in Development of Common Vascular Diseases………………...16

Role of Elastin in Maintaining of Normal Structure of Myocardium

and During Post-infarct Cardiac Remodeling…………………………………....17

Ascorbic Acid and Its Derivatives in Elastogenesis………………………..………….18

Sodium Dependent Vitamin C Transporters……………………………………..19

Sodium Ascorbate………………………………………………………………..20

RATIONALE AND HYPOTHESES………………………………..……22

MATERIALS AND METHODS….……………………………………...24

Materials…………………………………………………………………………24

Cell Isolation………………...………………………………………….……….25

Isolation of Skin-Derived Fibroblasts……………………………………….25

Isolation of Fat-Derived Fibroblasts……………………………………25

Isolation of Smooth Muscle Cells…………………………………………….26

Isolation of Human Cardiac Fibroblasts………………………………..26

Cell Cultures………………………………..…………………………………...26

Immunostaining………………………………………………………………….27

Quantitative assays of Insoluble Elastin…………………………………………28

Histological Assessment…………………………………………………………28

One-Step RT-PCR Analysis……………………………...……………………...29

Western Blotting…………………………………….…………………………...29

Immunoprecipitation………………………..……………………………………30

Data Analysis…………………………………………………………………….31


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RESULTS…………………………………………………………….……..32

SA induces the deposition of elastic fibers in monolayer cultures of human

skin-derived fibroblasts…………………………………………………………...32

SA also penetrates to the full thickness skin explants kept in organ cultures

and stimulates deposition of new elastic fibers…………….……………………..32

The elastogenic effect of SA can be also observed in cultures of fat

tissue-derived fibroblasts; the magnitude of SA-induced elastogenic

stimulation exceeds effects observed in parallel cultures treated with an

elastogenic growth factor, IGF-1.…………..……………………………….........33

SA induces both elastogenesis and collagenogenesis in cultures of aortic

smooth muscle cells and cardiac fibroblasts, but the addition of spironolactone

to SA-treated cultures exclusively reduces formation of new collagen fibers…...34

In contrast to 100 µM SA, identical concentrations of sodium ions

applied in the forms of NaCl or ascorbic acid do not upregulate deposition of

elastic fibers in cultures of skin-derived fibroblasts..………………………….... 35

SA exercises its elastogenic effect only after being transported into the cell

interior…………………………………………………………………………….36

Cultures of skin-derived fibroblasts maintained in the presence of 2% FBS

revealed two peaks of transient upregulation in the levels of intracellular

tropoelastin protein occurring between 3-6 hours and 18-24 hours after

the addition of SA………………………………………………………………...36

SA-treated fibroblasts demonstrate a heightened level of intracellular

tropoelastin even after their translation machinery has been inhibited with

cycloheximide. This elastogenic effect occurs only after SA is transported

into the cell interior……………………………………………………………….36

Cultures of skin-derived fibroblasts maintained in the presence of 2% FBS

demonstrate a transient upregulation in the net level of tropoelastin mRNA

occurring 18 hours after SA addition……………………………………………..37

The ―late effect‖ of SA leading to upregulation of tropoelastin mRNA

levels is executed through the enhancement of the primary elastogenic

signals triggered by the IGF-1 receptor.………………………………………….38

Treatment with SA also stimulates the net deposition of immuno-detectable

elastic fibers and enhances levels of insoluble elastin in cultures of skin

fibroblasts isolated from patients with Loeys-Dietz Syndrome……...…………..40


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Addition of SA to cultures of skin fibroblast isolated from Williams-

Beuren Syndrome patients upregulates the level of tropoelastin mRNA

transcripts and the ultimate deposition of elastic fibers.………………………….40

FIGURES……………………….………………………………………….42

Figure 1…………………………………………………………………………..42

Figure 2…………………………………………………………………………..44

Figure 3…………………………………………………………………………..45

Figure 4…………………………………………………………………………..47

Figure 5…………………………………………………………………………..48

Figure 6…………………………………………………………………………..50

Figure 7…………………………………………………………………………..52

Figure 8…………………………………………………………………………..54

Figure 9…………………………………………………………………………..55

Figure 10..………………………………………………………………………..57

Figure 11..………………………………………………………………………..59

Figure 12…..……………………………………………………………………..61

Figure 13..………………………………………………………………………..63

Figure 14..………………………………………………………………………..65

Figure 15..………………………………………………………………………..67

Figure 16…………………………………………………………………………69

Figure 17..………………………………………………………………………..70

Figure 18…..……………………………………………………………………..71

Figure 19..………………………………………………………………………..73

Figure 20..………………………………………………………………………..75

CONCLUSIONS……………….………………………………………….77

DISCUSSION……………………………………………………………...78

REFERENCES…………………………………………………………….86


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LIST OF ABBREVIATIONS

AA - Ascorbic acid

ADSC - Adipose-derived stromal/stem cell

AoSMC – Aortic smooth muscle cell

ADCL - Autosomal dominant cutis laxa

APP - Ascorbyl 2-phosphate 6-palmitate

ARCL - Autosomal recessive cutis laxa

ASC - Adult stem cells

b-FGF - basic fibroblast growth factor

β-Gal – Beta-galactosidase

BMSC - Bone marrow stromal/stem cell

CF- Human fetal cardiac fibroblasts

CL - Cutis laxa

DTT - Dithiothreitol

DIDS - 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid

DMEM – Dulbecco’s modified eagle medium

EBP – Elastin-binding protein

ECM – Extracellular matrix

EDTA - Ethylenediaminetetraacetic acid

EGTA - Ethylene glycol tetraacetic acid

ELN - Elastin

EMILIN - Elastin microfibril interface-located protein

FBS – Fetal Bovine Serum


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FDF - Fat-derived fibroblast

GAG – Glycosaminoglycan

GAPDH - Glyceraldehyde 3-phosphate dehydrogenase

GLUT - Glucose transporter

HRP - Horseradish peroxidase

IGF-1 - Insulin-like growth factor-1

IGF-1R - Insulin-like growth factor-1 receptor

IL - Interleukin

IP – Immunoprecipitation

LDS1 – Loeys-Dietz syndrome -1

LDS2 – Loeys-Dietz syndrome -2

LOX - Lysyl oxidase

MAGP – Microfibril-associated glycoprotein

MRP - Multidrug resistance-associated protein

NF-B - Nuclear factor-kappa B

MMP - Matrix metalloproteinase

Neu-1 - Neuraminidase-1

PBS - Phosphate buffered saline

PMSF - Phenylmethanesulfonylfluoride

PPCA - Protective protein/cathepsin A

PPP - Cyclolignan PPP

ROS - Reactive oxygen species

SA – Sodium ascorbate


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SITS - 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic

SDF - Skin-derived fibroblast

SDS-PAGE – Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SKP - Skin-derived precursors

SMC – Smooth muscle cells

SVAS - Supravalvular aortic stenosis

SVCT - Sodium-dependent vitamin C transporter

TGFβ-1 – Transforming growth factor beta -1

TGFβR – Transforming growth factor beta receptor

TNF- - Tumor necrosis factor-alpha

WBS- Williams-Breuren syndrome


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INTRODUCTION

Regenerative Medicine

Adult stem cell therapy is an emerging and rapidly evolving field of research and

medicine. It has aimed to repair, replace or regenerate cells or tissues to restore impaired

function in organs that are incapable of self-regeneration after damage to the areas such as the

heart, bones, skin, lungs, kidneys and spinal cord.1 Despite the successful experimental

employment of embryonic stem cells (ESCs) for regeneration of selected human tissues,

concerns regarding the still-limited availability of these cells and unresolved issues concerning

the safety and outcome over time of therapeutic ESC transplantation stimulate research on the

stem cells derived from adult individuals (ASCs). Indeed, ASC transplantation has been

successfully used in regenerative medicine.

The initial interest for the source of adult stem cells was in bone marrow stromal/stem

cells (BMSCs), which can differentiate into several mesodermal lineages, such as bone, muscle,

cartilage, fat, epithelium, and neural progenitors.2 Many studies have proven that BMSC

transplantation actually increases the functional activity of various organs after injury.2 However,

BMSC incidence is 1~10 per 105 mesenchymal mononuclear cells in bone marrow.

3 BMSCs

cannot be easily isolated and expanded, and obtaining BMSCs requires invasive procedures.4

In recent years, the general focus has turned to the discovery of adipose-derived stromal/stems

cells (ADSCs) for three reasons: They are available in greater quantities in adipose stromal

tissues, they are less invasive and less expensive to obtain, and they are easier to isolate, expand

and manipulate.5, 6

Similarly to BMSCs, ADSCs possess the potential to differentiate into


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mesenchymal lineages, including bone muscles cartilage, fat, heart and neuronal progenitors; for

this reason, ADSC transplantation on different organs has been attempted.7 The definitive

objective of stem cell therapy is the hope that stem cells can permanently differentiate into

functional organ-specific cells, because some organs have limited capacities to self-regenerate

after damage.8 As an example, ADSC transplantation has shown improvement on bone defects

by osteogenic differentiation.9

However, transplantation of ADSCs shows only poor engrafting in post-infarct

myocardium and only few ADSCs transdifferentiated into the cardiomyocytes.4, 10-12

In addition,

ADSC transplantation would improve cardiac function after infarction through paracrine

stimulation of new angiogenesis and reducing ischemic size.4, 10-13

Interestingly, functional

recovery of the heart after ADSC transplantation exceeded that seen after BMSC engrafting.12, 13

Furthermore, the increase in new vasculogenesis observed after ADSC transplantation cannot

solely explain the beneficial healing of the infarcted heart; although engrafting fibroblasts to the

infarcted heart also induced angiogenesis, it did not improve heart contractility in contrast to

ADSCs.14, 15

Additionally, ADSCs have shown their collective ability to produce collagen type

I/III and fibronectin in skin grafts and in skin defects.16-20

Another source of multipotent cells that could be utilized in regenerative medicine is the

skin. The skin possesses more regenerative potentials than internal organs and contains a

number of different residential stem cell populations, referred to as skin-derived precursors

(SKPs).21

The SKP population contains both neural and mesodermal precursors that can also

replicate and differentiate into major dermal cell types such as keratinocytes, melanocytes,

fibroblasts and other cell types.21-23

It also has been shown that a certain fraction of SKPs can be

experimentally stimulated to differentiate into insulin-producing cells or into endothelium.24, 25


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However, the regenerative potentials of ASCs that reside in different tissues have not

been fully elucidated. The regeneration of connective tissue frameworks present in the heart,

lungs and skin after numerous types of injury is often not well-balanced and is complicated with

fibrosis. Thus, there is a need for a better understanding of the regeneration process, especially

for the development of drugs that would stimulate well-balanced production of major

components of ECM.

It is particularly obvious that the proper remodeling of the injured heart, lungs and skin

should include production of new elastic fibers that would ensure recovery of natural elasticity

and resilience of these organs.

The Extracellular Matrix

The extracellular matrix (ECM) is a complex network of proteins and carbohydrates that

provide the framework and physical support for structural organization of practically all tissues

and organs. The major components of the ECM are structural fibrous proteins (e.g., collagen and

elastin). While collagen fibers, the most abundant ECM molecules, provide tissues with

mechanical strength, the elastic fibers provide extensibility, elastic recoil, and resiliency.

The other group of ECM components comprises adhesive fibrous glycoproteins (e.g.,

fibronectin and laminin) and polysaccharide chains of hyaluronic acid (HA) that associate with

numerous glycosaminoglycans (GAG) and proteoglycans.26, 27

There is complex cross-talk between cell and ECM components. Relative amounts of

ECM components vary in different tissues and organs, modulated by cells as they respond to

numerous paracrine and endocrine factors, including various cytokines, growth factors and


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hormones.28

On the other hand, ECM components regulate the development, differentiation,

growth, and functional properties of numerous cell types in the building of particular tissues.29,

30 In order to exercise such modulatory effects, particular ECM components have to be engaged

first in complex interactions with other ECM- or serum-derived factors, and then with the

specific cell surface-exposed cellular moieties and receptors. For example, the hydrophilic

molecules GAG and HA can attract water, thus facilitating the diffusion of minerals, hormones,

and nutrients. They also interact with cells and other ECM macromolecules in regulating the

activity and stability of signaling molecules secreted by cells.31, 32

Collagen Fibers

Synthesis

Twenty-seven different types of collagen, each encoded by a specific gene, have been

described to date.33

Collagen type I is the most abundant and is the major component of the

ECM. It is encoded by two genes, COL1A1 and COL1A2, located in chromosomes 17 and 7,

respectively, in humans.

Production of collagen type I fibers involves several stages. The mRNA transcribed from

the COL1A1 and COL1A2 genes undergoes processing, and the mature mRNA then attaches to

the site of actual protein synthesis on rough endoplasmic reticulum, producing the pro-α1(I) and

pro-α2(I) polypeptides into the lumen of rough endoplasmic reticulum.33

In rough endoplasmic

reticulum, these procollagen molecules undergo extensive posttranslational modifications by a

number of molecular chaperones and enzymes assisting its folding and trimerization.33

For

example, hydroxylation of proline and lysine residues, which are processed by prolyl-4-

hydroxylase and lysyl hydroxylases, respectively, is required for proper folding and


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trimerization of procollagen molecules.33-36

These properly folded and trimerized procollagen

molecules are carried to the Golgi complex, in which N-linked carbohydrate groups are further

processed. After all modification occurs, the molecules are finally transported to the cell

membrane via secretory vesicles and secreted by exocytosis to the extracellular space.34

After being secreted into the extracellular space, the procollagen is converted into

collagen by the removal of C and N propeptides; this occurs when procollagen N and C-

proteinases create non-triple helical terminal telopeptides at each end, which triggers self-

assembly of collagen into fibrils.35

Finally, lysyl oxidase oxidizes selected lysine residues within

the N- and C-terminal telopeptides to insolubilize and stabilize the collagen molecules within

the fibers.35

Ascorbic Acid as a Stimulator of Collagen Synthesis

Ascorbic acid (AA) is one of the cofactors for the enzymatic activity of prolyl

hydroxylase and lysyl hydroxylase that hydroxylate prolyl and lysyl residues, respectively, in

procollagen, elastin, and other proteins with collagenous domains.36-41

AA is required for

collagen synthesis because one-third of prolyl residues in collagen need to be hydroxylated in

order to obtain the triple-helical conformation that stabilizes collagen molecules to be further

modified.40

In addition to its function as a cofactor in hydroxylation, AA has been found to

independently augment collagen mRNA levels, secretion rate, collagen processing enzymes

activities, and the inhibition of collagenases in different cell types; hence, it increases deposition

of collagen.42-47


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The Role of Collagen in Fibrosis

Under normal conditions the ECM is stable, but it can be steadily dismounted during the

normal aging process or rapidly damaged by numerous pathological processes.48, 49

The

consecutive regenerative and healing processes of the injured tissues include remodeling of the

ECM, which is often complicated by the accelerated degradation of selected ECM components

and/or the production of a new matrix lacking a particular component. Abnormalities in any step

of the synthesis and/or degradation of ECM components may result in various pathological

states,50

such as excessive collagenous fibrotic diseases,32

and often associate with the explicit

deficiency of elastic fibers.51

Elastic Fibers

Elastic fibers are a major component of the ECM that provides extensibility and elastic

recoil to dynamic connective tissues.52-54

Their ability to stretch repetitively and reversibly is

important for sustaining repeated cycles of extension and recoil in blood vessels and the heart,

as well as for the proper functioning of the bladder, skin, lungs, and cartilage.52

Many tissue-

specific properties and functionalities of elastic fibers depend on their organization and

architecture.55

Elastic fibers are complex structures composed of cross-linked, insoluble elastin, which

makes up most of the amorphous core, and the microfibrillar scaffold consisting of fibrillin,

fibulins, microfibril-associated glycoproteins, and numerous accessory and regulatory proteins

and GAGs building the interface between elastin and microfibrils or the cell surfaces.56-58

This

group consists of such molecules as elastin microfibril interface-located proteins (EMILINs)59

,

fibulins60, 61

, and chondroitin sulfate proteoglycans like versican, biglycan, and decorin.62


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Elastic fibers are produced by several types of cells including smooth muscle cells

(SMCs), endothelial cells, fibroblasts, keratinocytes and chondrocytes.63

The intracellular and

extracellular biosynthesis of elastic fibers involves a complex process.

The pericellular assembly of microfibrillar proteins is required at an early stage of

elastogenesis.64

The precursor proteins, called tropoelastin, are secreted to the already assembled

microfibril scaffold, where they are properly processed, assembled and covalently cross-linked

with each other to form a resilient and extensible polymer, insoluble elastin.64

The polymeric

elastin is metabolically inert, shows little turnover and constitutes the most durable ECM

component that may last throughout the entire human lifetime, under optimal conditions.49

Elastin Gene Regulation

Several factors that regulate transcription of the elastin gene have been reported. In

addition to several endogenous factors (glucocorticoids65

, IGF-166

, TGFβ-167

, and aldosterone68

),

synthetic glucocorticoid, dexamethasone69

, retinoids70

, ferric ions71

, and nitric oxide72

have also

been introduced as potent stimulators of elastin gene expression. In contrast, tumor necrosis

factor-α (TNF-),73

interleukin (IL)-174

, basic fibroblast growth factor (b-FGF)75

, Vitamin

D376

, and phorbol esters (TPA)77

have been shown to downregulate elastin gene expression.

The signaling pathways triggered by numerous endogenous factors that could affect

elastogenesis have been intensively investigated. For example, it has been established that the

IGF-1 dependent induction of elastin gene expression occurs only after removal of the negative

transcriptional regulator, Sp1/Sp3, from the retinoblastoma control element on the proximal

promoter of the elastin gene.66

Additionally, recently published work from our laboratory

showed that the elastogenic effect of aldosterone depends on the cross-activation of IGF-1


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receptor, which occurs after the aldosterone-triggered phosphorylation of Gα13, the activation of

C-src kinase and the additional phosphorylation of Tyr1316

on the IGF-I R.68, 78-80

This induces

the permissive conformation of IGF-1R, which allows the binding of phosphatidylinositol 3-

kinase (PI3K) and subsequent activation of PI3K and AKT, leading to elastogenesis in cardiac

and skin fibroblasts.68, 79, 81, 82

Tropoelastin Synthesis and Secretion

In humans, tropoelastin is encoded by the single elastin gene, which is located on

chromosome 7; with a size of 45 kb, it contains 36 small exons separated by larger introns.83, 84

The elastin gene codes for an mRNA of ~3.5 kb after extensive alternative splicing of the elastin

gene that translates into tropoelastin isoforms varying in size from 68 to 74 kDa.84, 85

Deletion or

inclusion of a particular exon often occurs in a cassette-like fashion during transcript splicing of

the elastin gene.54

Exon 36, which codes for C-terminal end, contains a large 3'-untranslated

region that regulates the stability of tropoelastin mRNA.86

Synthesis of tropoelastin occurs in the rough endoplasmic reticulum, but unlike other

ECM proteins, tropoelastin undergoes little post-translational modification and is not

glycosylated.87

Two major domains are found in tropoelastin: 1) hydrophobic domains rich in

non-polar residues (Gly, Val, Pro, and Ala), which are putatively responsible for the elastic

properties; and 2) hydrophilic domains typically rich in Lys and Ala, which are involved in

cross-linking.54

Because the signal peptide is cleaved after translation and is highly hydrophobic,

tropoelastin requires a protective chaperone to be carried to the cell surface.

An elastin-specific molecular chaperone, the 67-kDa elastin-binding protein (EBP) has

been identified as the enzymatically inactive splice variant of β-galactosidase (β-Gal).88

It binds


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directly to tropoelastin, but it also forms the molecular complex with two enzymatically active

proteins: neuraminidase-1 (Neu-1), and carboxypeptidase-protective protein/cathepsin A

(PPCA). After binding to the intracellular tropoelastin, the entire complex is translocated to the

cell surface where it facilitates extracellular assembly of the transported tropoelastin into the

growing elastic fibers.89

Assembly of Elastic Fibers

It has been established that Neu-1, also known as lysosomal sialidase, has to be

proteolytically activated by PPCA; then it catalyzes the removal of terminal sialic acids from the

carbohydrate chains of microfibrillar glycoproteins. This, in turn, allows for the exposure of

their penultimate galactosugars that can interact with the galectin domain of EBP, thereby

inducing the release of transported tropoelastin molecules.90, 91

In order to build up the extracellular elastic fibers, the newly synthesized tropoelastin,

attached to its transporting chaperone complex, must be delivered to the microfibrillar

framework that must be first assembled close to the cell surface.92

Microfibrils are complex structures composed of several types of glycoproteins that

appear as repeating globules on filamentous linear arrays.54

Microfibrils not only act to direct

elastin deposition, but can also contribute to the orientation of formed elastic fibers that guide

load bearing.93

Fibrillins are the principal structural components that provide the framework of

the microfibrils.52, 94

Fibrillin-1 contains two high-affinity binding sites for tropoelastin.95

Tropoelastin can bind to a specific fibrillin-1 sequence in this site through a transglutaminase

cross-linkage and thus stabilize the newly deposited tropoelastin. Microfibril-associated

glycoprotein-1 (MAGP-1), which provides structural integrity to the microfibrils, binds strongly


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in a calcium-dependent manner to the N-terminal sequence of fibrillin-1 and facilitates the

deposition of tropoelastin, since it contains multiple tropoelastin-interacting sites.52, 92, 96

Fibulins seem to stabilize the primary association of tropoelastin to the microfibrilar scaffolds,

since fibulins bind tropoelastin. The stabilizing interaction of fibulin-5 with both cell-surface

integrins and elastic fibers was postulated to be a mechanism facilitating elastic fiber and

cellular association.57

Upon the delivery of the initial tropoelastin molecules and its assembly on the

microfibrillar scaffold, the consecutively-delivered tropoelastin molecules can also aggregate

with each other (coacervation).54

Once coacervated, tropoelastin becomes rapidly cross-linked

without any further modifications or proteolytic processing.97

Lysyl residues of the tropoelastin

molecule initially undergo oxidative deamination by the copper-requiring enzyme lysyl

oxidase.98, 99

The cross-links are then spontaneously formed by the condensation of these

allysine molecules with themselves or with unmodified lysine.100

Formation of these cross-links

(desmosines and isodesmosines) connecting adjacent tropoelastin molecules is a prerequisite for

the growth of the mature polymer of ―insoluble‖ elastin that constitutes the core of elastic

fibers.100, 101


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The following diagram illustrates the secretion and assembly of tropoelastin.


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Diseases and Conditions Affecting Elastic Fibers: Elastinopathies

Elastic fibers are expressed and assembled mainly from the second half of development

up to the early postnatal period.102, 103

However, production of elastin fibers decreases through

adulthood.104

In normal physiological circumstances, elastic fibers do not generally undergo

extensive turnover and are supposed to last for one’s lifetime.54

However, in certain pathological

conditions, various enzymes such as matrix metalloproteinases (MMPs) and serine proteases or

physical/chemical damages can cause extensive degradation of elastic fibers. Elastic fibers

cannot be repaired, and once damaged, they have to be replaced by new ones.105

Loss of

elasticity due to degradation of elastic fibers also contributes to the aging of connective tissues

in various organs, resulting in skin wrinkles, aortic aneurysm and lung emphysema.55, 106-108

Furthermore, the insufficient production of elastic fibers during tissue regeneration in scar tissue

causes stiffness along with excess collagen production, which leads to decreased contractile

properties in the myocardium and arteries, less resiliency in lung and skin tissues, and, in turn,

maladaptive fibrosis.58, 109-112

Environmental factors are not the only causes of impaired elastic fibers; there are several

genetic diseases that compromise elastogenesis, affecting either the production of tropoelastin or

the assembly of elastic fibers. Alterations in various proteins that are involved in the complex,

multi-step processes of elastic fiber formation can severely reduce the total amount of deposited

elastic fibers. Genetic disorders of the elastic fiber are grouped into two categories according to

the specific molecular component altered by the underlying mutations.


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Primary Elastinopathies

Primary elastinopathies are caused by mutations of the elastin gene that are characterized

by inadequate deposition of elastic fibers.113

Clinical examples of primary elastinopathies are

cutis laxa (CL), supravalvular aortic stenosis (SVAS), and Williams-Beuren syndrome (WBS).

CL is characterized by an abnormal structure of dermal and vascular elastic fibers

resulting in loose, redundant, sagging and inelastic skin, as well as cardiovascular diseases

found in a heterogeneous group of genetic disorders.114-117

There are more than three autosomal

recessive heritable forms of cutis laxa (ARCL) in which the elastin gene is affected.115, 118

ARCL is a severe form of cutis laxa accompanied by cardiovascular complications and

pulmonary emphysema leading to death in childhood.113

On the other hand, autosomal dominant

cutis laxa (ADCL) is usually considered a milder disorder with possible additional

manifestations including hernias and genital prolapse, gastrointestinal diverticula, aortic and

arterial dilatation and tortuosity, and pulmonary artery stenosis.113, 117, 119

Mutations in the

elastin gene, mostly single nucleotide deletions occurring in exons 30 and 32 in the 3′-end of the

coding region, have been shown to cause ADCL.113, 117

It has recently been reported that CL can

be caused by a mutation in the gene encoding Fibulin-4 or Fibulin-5.120, 121

Genetic haploinsufficiency of the elastin gene, which also causes cardiovascular

complications in early childhood, has been clinically found. As an example, supravalvular aortic

stenosis (SVAS) is inherited in an autosomal dominant isolated manner or as part of the

complex developmental disorder Williams-Beuren syndrome (WBS), which is caused by a

deletion of up to 27 additional genes from the long arm of chromosome 7q, resulting in a more

complex developmental delay coupled with neurobehavioral, facial and metabolic disorders.122-

125 The clinical manifestations of SVAS are identical in both cases.

52 Deletion of the elastin gene


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or heterozygous mutations leading to premature termination codons and unstable tropoelastin

mRNA production is the underlying cause of obstructive vascular disease in SVAS.126

This

haploinsufficiency of elastin decreases deposition of elastin associated with increased vascular

cell proliferation and an increased volume of the medial layer leading to stenosis, which is also

accentuated by fibrous intimal thickening.113

Hence, patients with severe SVAS may have

narrowing of the ascending aorta with thinner and disorganized elastic fiber lamellae, smooth

muscle cell hypertrophy in the medial layer, and a fibrous ring above the aortic valves with

medial and intima thickening.52, 113

In addition to the aorta, other major arteries may also exhibit

narrowing and wall thickening including the pulmonary, coronary, carotid and renal arteries.

This may lead to stroke, left ventricle hypertrophy, and congestive heart failure in childhood.127-

129 Furthermore, patients with haploinsufficiency of elastin have the symptoms of premature

skin aging resulting from inadequate production of elastic fibers.130, 131

Secondary Elastinopathies

Not only does mutation in the elastin gene result in the severe pathological outcome of

elastogenic defects, but alterations of the genes that encode proteins involved in elastogenesis

can cause severe diseases by affecting production of elastic fibers. This group of diseases is

known as secondary elastinopathies.

One example of secondary elastinopathies is Marfan syndrome, which is a relatively

common disorder.132

It is an autosomal dominant hereditary disorder of connective tissue, with

major cardiovascular, skeletal and ocular defects.133

It is caused by mutations in the gene

encoding the major microfibrillar protein, fibrillin-1.133

Similarly to primary elastinopathies,

cardiovascular complications following elastic fiber degeneration such as progressive dilatation

of the ascending aorta and acute aortic dissection cause premature death in patients with Marfan


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syndrome.132

The normal fibrillin-1 protein sequesters TGF-β, but the mutant fibrillin-1 protein

cannot, causing excess TGF-β signaling.134

Secondary to the disarray of elastic fibers due to

mutated fibrillin-1, this excess TGF-β signaling in patients with Marfan syndrome results in

developmental defects in the cardiovascular, pulmonary, ocular, and skeletal systems.134, 135

Loeys-Dietz syndrome (LDS types 1A, 1B, 2A, and 2B) has recently been described as a

rare, autosomal dominant connective tissue disorder caused by mutations in the transforming

growth factor β receptor 1 and 2 (TGFβR1 or TGFβR2) genes.136

LDS has phenotypic overlap

with Marfan syndrome, but LDS patients present more severe pathological manifestations than

patients with Marfan syndrome.137

Therefore, life expectancy of LDS patients is significantly

shortened compared to patients with Marfan syndrome. Despite the fact that both disorders are

caused by mutations in different genes, the considerable overlap presented in LDS and Marfan

syndrome, including abnormal fragmentation and disarray of elastic fibers, suggests that the

pathological mechanism may be similar in both conditions.

Since aberrant transforming growth factor β (TGFβ) signaling contributes to the

connective tissue pathology in Marfan syndrome due to the inability of fibrillin-1 to sequester

TGFβ,134

it has been suggested that mutations in TGFβR-1/2-encoding genes would lead to

persistent and increased TGFβ-induced signals in LDS patients.136, 138

However, this does not

correlate well with the fact that TGFβ is normally a potent stimulator of elastic and collagen

fiber formation.67

Hence, the molecular mechanism leading to impaired elastogenesis in LDS is

required to be elucidated.

There are several other connective tissue disorders that can be considered as secondary

elastinopathies. Such disorders include Ehlers-Danlos syndrome, in which impaired function of

the cross-linking enzyme, lysyl oxidase, leads to impaired elastic fiber assembly;139

Costello140


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and Hurler141

syndromes, in which excessive, extracellular accumulation of chondroitin sulfate

and dermatan sulfate, respectively, disrupts proper assembly of elastic fibers; GM1-

gangliosidosis and Morquio type B, in which deficiency in the EBP (the 67-kD splice-variant of

β-galactosidase) leads to impaired elastic fiber assembly;142

and sialidosis and galactosialidosis,

which are characterized by deficiencies of Neu1 and PPCA, respectively, resulting in improper

assembly of elastic fibers.91, 143

Role of Elastin in Development of Common Vascular Diseases

Vascular smooth muscle cells (SMCs) are the main cell types residing in the tunica

media of arteries and veins. During embryonic development, they are responsible for producing

and organizing components of elastic fibers and lamellae that are mostly responsible for the

resiliency of vascular walls and for carrying the pulsatile flow of blood through the aorta and

large arteries.54

SMCs also play a major role in the pathogenesis of atherosclerosis, which is

responsible for the development of atherosclerotic plaques and pathologic intima — the

thickening of the injured arteries.

Stimulated by numerous exogenous and endogenous factors, SMCs contribute to both

the degradation of ECM components, including elastic fibers and lamellae, and to the often

overzealous processes leading to pathological thickening.144

During the initial phase, they would

release numerous ECM-degrading enzymes, including matrix metalloproteinases that break

down elastic fibers and lamellae.145

In the repair stage, they respond to numerous signals,

including those initiated by the degradation products of the ECM that stimulate SMC

proliferation, migration and deposition of the often imbalanced new ECM. This newly produced

ECM contains a disproportional amount of collagen and scarce and disorganized elastic fibers


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that cause further thickening and stiffening of injured arteries.146

The steady degradation of

elastic fibers in veins is also connected to the formation of varices.147

The Role of Elastin in Maintaining of Normal Structure of Myocardium and During Post-

Infarct Cardiac Remodeling

Cardiac fibroblasts (CF) are the most abundant cell type of the myocardium responsible

for regulating levels of various ECM proteins.148

However, in the injured hearts these stromal

cells usually respond to numerous hormones and cytokines with the overzealous production of

collagen that leads to the formation of rigid post-infarct scars and myocardial fibrosis. Recently,

it has been reported that the degradation of elastic fibers is evident in myocardiac infarction,149

and inhibiting the proteolytic degradation of existing elastic fibers during a cardiac infarction

reduces inflammatory infiltration and cardiac dilation.150, 151

It has also been suggested that

elastic fibers degraded during myocardiac infarction followed remodeling can be replaced by

collagen, resulting in fibrosis.152

Additionally, Mizuno et al has shown that transplanting transfected fibroblasts that over-

express tropoelastin into infarcted myocardium modifies the scar content, increasing cardiac

elasticity, ventricular function and cardiac outputs.153, 154

It has been recently shown that that the

blockade of the mineralocorticoid receptors with eplerenone or spironolactone leads to the

exclusive propagation of the elastogenic effect of aldosterone that counterbalances the

production of collagen fibers after a cardiac infarction.68, 78, 155, 156

Together, these studies

suggest that increasing the elastogenesis of CF in a post-infarct myocardium may improve

cardiac function by counterbalancing collagen fiber stiffness and increase elasticity of cardiac

tissues.


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Ascorbic Acid and Its Derivatives in Elastogenesis

In contrast to the well-documented stimulatory effects of ascorbic acid (AA) on collagen

production, this vitamin inhibits the deposition of new elastin.45, 157-159

The mechanism of this

effect is still controversial and not well understood. One group suggested that AA suppresses

tropoelastin expression by destabilizing tropoelastin mRNA,45

while other groups suggested that

the administration of AA does not affect the level of tropoelastin mRNA,160, 161

but hypothesized

that since AA is a co-factor of the hydroxylation reaction, AA causes the accumulation of

tropoelastin inside cells and reduces the assembly of insoluble elastic fibers due to

overhydroxylation on prolyl/lysyl residues of tropoelastin.157, 160-162

However, another group

showed that the secretion and assembly of tropoelastin into insoluble fibers were irrelevant to

levels of hydroxylation on tropoelastin.163

It has been recently reported that certain AA derivatives in addition to stimulation of

collagenogenesis can also affect elastin. It has been shown that Ascorbyl 2-phosphate 6-

palmitate, an amphipathic AA derivative, induced elastogenesis in the atrophic skin of

copper/zinc superoxide dismutase (CuZn-SOD)-deficient mice.164

The molecular mechanism

has not been elucidated. Additionally, a basic derivative of AA, sodium ascorbate (SA), lowered

material stiffness and increased the elasticity and tensile strength of vascular smooth muscle

cells (SMCs) embedded in hyaluronan-based vascular constructs using another unknown

mechanism.165

It has also been reported that combined topical treatment of AA and

madecassoside on the photoaged skin of female patients has also shown to induce elastogenesis

with significant improvement of skin elasticity by unknown mechanisms.166


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Sodium-Dependent Vitamin C Transporters

AA is slowly absorbed into cells via sodium-dependent Vitamin C transporters (SVCTs)

and it is easy for it to be oxidized to dehydroascorbate in the extracellular space.167

However,

once oxidized to dehydroascorbate, it enters cells via sodium-independent glucose transporters

(GLUTs) at a much faster rate than ascorbate via SVCTs.168

In humans, there are two types of SVCTs: SVCT1 and SVCT2; they do not have

redundant functions.169

SVCT transports stoichiometry of two sodium cations for each ascorbate

anion transportation.167

The energy required for these co-transport activities is supplied by

systems through the concentration gradient of sodium ion across the plasma membrane.170

SVCT1 and SVCT2 are encoded by the SLC23A1 and SLC23A2 genes, and they share 65%

sequence identity with a 12-transmembrane-domain structure and cytoplasmic C and N-terminal

domains.171, 172

SVCT1 is a low-affinity/high-capacity transporter, while SVCT2 is a high-

affinity/low-capacity carrier that becomes inactive in the absence of divalent metal ions such as

calcium and magnesium.169, 173

SVCT1 is mainly expressed in epithelial cells in the liver,

kidneys, and intestines, and in a few endocrine tissues. SVCT2 expression, on the other hand, is

widely distributed.167

SVCTs can be inhibited by anion transporter inhibitors such as

sulfinpyrazone, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), 4-acetamido-4′-

isothiocyanostilbene-2,2′-disulfonic (SITS) acid and probenecid.174, 175

Among these inhibitors, probenecid is one of most efficient inhibitors; it is relatively

harmless in clinical usage. Probenecid has been primarily used to increase uric acid excretion by

competitively inhibiting the organic anion transporter. This inhibitor also has been shown to

inhibit the tubular secretion of antibiotics such as penicillins or cephalosporins.176, 177

Probenecid also has been documented to block uptake of ascorbate by blocking the SVCT.175


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Additionally, probenecid is widely used as a standard inhibitor of multidrug transporters, such

as multidrug resistance-associated protein (MRP).178, 179

Sodium Ascorbate

There are several reports showing that the molecular effect of AA is significantly less

than its derivatives, which are modified to enter cells more efficiently.166, 180, 181

This may occur

because the majority of AA enters cells as a molecularly inactive, oxidized form of ascorbate

called dehydoascorbate, although it may be rapidly followed by its intracellular reduction to

ascorbate by several enzymes in certain cell types.167

In particular, SA exhibits a more potent

molecular effect than AA because it has been reported that a millimolar concentration of SA

reduces cell proliferation and viability significantly more than the same concentration of AA.181

Additionally, it has been shown that SA produces a cytotoxic effect in an array of malignant cell

lines by inducing apoptosis.182-184

SA is also effective in reducing reperfusion injury in skeletal

muscle.185

Additionally, it has been suggested that because of the proximity of sodium ions and

ascorbate that potentiate the SVCTs to transport SA at their maximum kinetics, SA possesses

more potent cytotoxic action than AA does.174, 181

There is a quickly growing need for basic research to introduce safe compounds that

allow for pharmacological stimulation of numerous cell types present in underdeveloped,

injured or metabolically damaged human tissues, to resume production of selected ECM

components (including elastin) that would contribute to their regeneration. The selective

stimulation of elastic fiber production seems to be particularly needed for the production of


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artificial constructs of arteries, heart valves, bladders and skin substitutes made of human cells

placed on biodegradable polymers.

Thus, the presented study introducing SA as a potent stimulator of new elastogenesis and

the offered explanation of the respective cellular action of this AA derivative contributes to the

bulk of the still-incomplete knowledge on the modulation of the elastic fiber formation and

encourages a new SA-based pharmacological intervention in this process.


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RATIONALE

This paper is concerned with the constantly growing need for the replacement of

damaged human connective tissues, particularly skin and production of bio-prostheses of the

blood vessels, and bladder-stimulated intensive research on efficient in vivo regeneration and in

vitro production of these organs. While only limited number of cells can be obtained from

human tissues, there is a need for effective propagation of cells in cultures and stimulation of

their capabilities to produce the extracellular matrix containing components representative to the

original targeted tissues. Among the numerous natural and synthetic compounds used for the

stimulation of ECM production, multiple vitamins were also tested. AA has already been

identified as a potent stimulator of collagen production and an inhibitor of effective

elastogenesis. However, the ECM-related actions of AA derivatives have not yet been

adequately tested. Because recent studies from our laboratory indicated that an elevation of

sodium levels could associate with upregulation in elastic fiber production, we decided to test

whether sodium ascorbate would stimulate both collagen and elastic fiber deposition.

HYPOTHESIS

1. The vitamin C derivative sodium ascorbate would be a more comprehensive stimulator

of ECM production than vitamin C itself.

2. Sodium ascorbate, in addition to collagen production, would also stimulate deposition of

elastic fibers by cells derived from the frameworks of skin, fat, arteries and heart.


P a g e | 23

3. Sodium ascorbate would also enhance elastic fiber formation by cells derived from

patients with genetic diseases such as Loeys-Dietz Syndrome and Williams-Beuren

Syndrome, characterized by inadequate elastogenesis.


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MATERIALS AND METHODS

Materials

All chemical-grade reagents, ascorbic acid (AA), sodium chloride, sodium ascorbate

(SA), proteinase inhibitors, recombinant human insulin-like growth factor-I (IGF-I), insulin-like

growth factor receptor-I (IGF-IR) inhibitor the cyclolignan PPP (PPP), protein synthesis

inhibitor cycloheximide, and the mineralocorticoid receptor antagonist spironolactone were

obtained from Sigma (St. Louis, MO). C-src inhibitor PP2 was purchased from Calbiochem

(San Diego, CA). Probenecid was purchased from ICN Biomedicals Inc. (Aurora, OH).

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), 0.2% trypsine, 0.02%

EDTA, and other cell culture products were acquired from GIBCO Life Technologies

(Burlington, ON). Polyclonal antibody to tropoelastin was purchased from Elastin Products

(Owensville, MI). Polyclonal collagen type I antibody and monoclonal fibrillin-1 antibody were

purchased from Chemicon (Temecula, CA). Monoclonal antibody against β-actin was purchased

from Cell Signaling Technology, Inc. (Danvers, MA). Monoclonal antibody

against

phosphotyrosine (PY99) and polyclonal antibody against IGF-IR were obtained from Santa

Cruz

Biotechnology (Santa Cruz, CA). Secondary antibody fluorescein-conjugated goat anti-rabbit

(GAR-FITC) and fluorescein-conjugated goat anti-mouse (GAM-FITC) were purchased from

Sigma, and secondary antibody fluorescein-conjugated rabbit anti-goat (RAG-FITC) was

purchased from Chemicon. Secondary antibody HRP-conjugated goat anti-rabbit (GAM-HRP)

and HRP-conjugated goat anti-mouse (GAM-HRP) were purchased from Santa

Cruz

Biotechnology. The radiolabeled reagent [3H]-valine was purchased from Amersham

Biosciences Canada Ltd. (Oakville, ON). Pre-cast 4-12% tris-glycine gel and Dynabeads Protein


P a g e | 25

G were purchased from Invitrogen Canada Inc. (Burlington, ON). A DNeasy Tissue system for

DNA assay, RNeasy Mini Kit for isolating total RNA and One-Step RT-PCR Kit were

purchased from Qiagen (Mississauga, ON).

Cell Isolation

Isolation of skin-derived fibroblasts

Skin-derived fibroblasts (SDF) were propagated from six biopsies of normal skin (gift of

Dr. T Mitts from the Department of Plastic Surgery, University of California) and from skin

biopsies derived from four patients with Loeys-Dietz syndrome (LDS) bearing different

mutations of TGFβR1 (S241L, R487Q) and TGFβR2 (A355P, R528H) and three patients with

Williams-Beuren Syndrome that were obtained from the Hospital for Sick Children in Toronto

after permission of the Institutional Ethics Committee. All SDF grew from the explants of the

full thickness skin biopsies, and we have confirmed that they exclusively migrate out of the

basal layers located between epidermis and dermis of those skin explants.

Isolation of fat-derived fibroblasts

Human subcutaneous adipose tissue samples were obtained from Thermogenesis

(Rancho Cordova, CA). They were purified from the adipose tissue of three female patients

following routine liposuction procedure, performed with the permission of the Institutional

Ethics Committee. The aspirated fat was digested in collagenase type I solution (Worthington

Biochemical) under gentle agitation for 1 h at 37°C, filtered with 500-μm and 250-μm Nitex

filters, and centrifuged at 200g for 5 min to separate the stromal cell fraction (pellet) from

adipocytes. The fraction was centrifuged at 300 g for 5 min. The supernatant was discarded, and


P a g e | 26

the cell pellet was resuspended in endothelial growth medium-2 MV (EGM-2MV, Cambrex),

which consists of endothelial basal medium-2 (EBM-2), 5% fetal bovine serum (FBS), and the

supplemental growth factors vascular endothelial growth factor (VEGF), basic fibroblast growth

factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1). The

cultured cells were characterized as newly differentiated fibroblasts using monoclonal antibody

specific to vimentin.

Isolation of Smooth muscle cells

Smooth muscle cells were grown from small aortic fragments collected during the

autopsy of a patient who died in a traffic accident in the media as described before.186

The

cultured cells were characterized as smooth muscle cells using a monoclonal antibody specific

to smooth muscle actin.

Isolation of Human Cardiac Fibroblasts

Cardiac fibroblasts (CFs) isolated from human fetal hearts at 20-22 weeks gestation (a

generous gift from Dr. John Coles obtained in accordance with an institutional (the Hospital for

Sick Children) review board-approved protocol) were propagated as previously described.78, 187

Cell Cultures

Cells were routinely passaged by trypsinization and maintained in Dulbecco’s modified

eagle’s medium (DMEM|) supplemented with 1% antibiotics/antimycotics, and 10% FBS. In all

described experiments passages 2–8 were used. In experiments aimed at assessing ECM

production, fibroblasts were initially plated (100,000 cells/dish) and maintained in normal

medium until confluency at which point they produce abundant ECM. Confluent cultures were


P a g e | 27

then treated once (for all experiments except immunostaining) or twice at day 0 and 3 (only for

immunostaining with or without treatment, which were 100 μM NaCl, 200µM ascorbic acid

(AA), 10 to 400µM sodium ascorbate (SA), 50 ng/ml IGF-1, or 1ng/ml TGFβ-1. The inhibitors

of protein synthesis (10 μg/ml cycloheximide), organic anion transporters (400μM

Probenecid)175

IGF-1 receptor (0.5M the cyclolignan PPP)188

, and the mineralocorticoid

receptor (2μM spironolactone)189, 190

were added 1 hour before SA treatment.

Immunostaining

At day 6 after day 0 and day 3 treatment with the indicated treatment, confluent cultures

were either fixed in cold 100% methanol at -20°C (for elastin, fibrillin-1, vimentin, and CD90

staining) or in 4% paraformaldehyde at room temperature (for collagen staining) for 30 minutes

and blocked with 1% normal goat serum for 1 hour at room temperature. The cultures were then

incubated for 1 hour with 10 g/ml of polyclonal antibody to tropoelastin, 10 g/ml of

monoclonal antibody to fibrillin-1, vimentin, or CD90 staining or with 10 µg/ml of polyclonal

antibody to collagen type I. All cultures were then incubated for an additional hour with

fluorescein-conjugated goat anti-rabbit (GAR-FITC), goat anti-mouse (GAM-FITC) or with

rabbit anti-goat (RAG-FITC) secondary antibodies to detect elastin, (monoclonal staining

fibrillin-1, vimentin, and CD90) and collagen type I staining respectively. Nuclei were

counterstained with propidium iodide. Secondary antibody alone was used as a control. All of

the cultures were then mounted in elvanol and examined with a Nikon Eclipse E1000

microscope attached to a cooled CCD camera (QImaging, Retiga EX) and a computer-generated

video analysis system (Image-Pro Plus software, Media Cybernetics, Silver Springs, MD).


P a g e | 28

Quantitative Assays of Insoluble Elastin

Fetal human cardiac fibroblasts were grown to confluency in 35-mm culture dishes

(100,000 cells/dish) in quadruplicate. Then 2 µCi of [3H]-valine/ml of fresh media were added

to each dish along with or without the indicated treatment at day 0. Cultures were incubated for

3 days, and the insoluble elastin was assessed separately in each dish. The cells were

extensively washed with PBS, and cultures containing cell remnants and deposited insoluble

extracellular matrix were scraped and boiled in 500 l of 0.1 N NaOH for 30 minutes to

solubilise all matrix components except elastin. The resulting pellets containing the insoluble

elastin were then solubilised by boiling in 200 l of 5.7 N HCl for 1 hour, and the aliquots were

mixed in scintillation fluid and counted.191

Aliquots taken from each culture were also used for

DNA determination, according to Rodems et al.192

using the DNeasy Tissue System from

Qiagen. Final results reflecting amounts of metabolically labelled insoluble elastin in individual

cultures were normalized per their DNA content and expressed as CPM/1 µg DNA.

Histological Assessment

Four skin biopsy explants from two female patients with or without scar (obtained from

Dr. Thomas Mitts, University of California) were cultured in DMEM media containing 5% FBS,

in the presence or absence of 200 µg/ ml of SA or iron ascorbate (added every 24 hours). The

7 day-old cultures of dermal explants were fixed in 4% paraformaldehyde and embedded in

paraffin. All the paraffin-embedded tissues were then cut into 4-µm thick sections that were

subjected to histochemistry with the pentachrome Movat staining.193


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One-Step RT-PCR Analysis

Confluent SDF cultures were treated with or without the specified treatment for different

periods of time (3 hours to 24 hours) or for 18 hours as indicated in the figure legend. Total

RNA was extracted using the RNeasy Mini Kit according to the manufacturer’s instructions, 1

µg of total RNA was added to each one-step RT-PCR (Qiagen One-Step RT-PCR Kit), and

reactions were set up according to the manufacturer’s instructions in a total volume of 25 µl.

The reverse transcription step was performed for elastin and GAPDH reactions at 50 ˚C for 30

minutes, followed by 15 minutes at 95 ˚C. The elastin PCR reaction (sense primer: 5'-

GGTGCGGTGGTTCCTCAGCCTGG-3', antisense primer: 5'-GGGCCTTGAGATACCCCA-

GTG-3'; designed to produce a 255 bp product) was performed under the following conditions:

25 cycles at 94 ˚C denaturation for 20 seconds, 63 ˚C annealing for 20 seconds, 72 ˚C extension

for 1 minute, and 1 cycle at 72 ˚C final extension for 10 minutes. The GAPDH PCR reaction

(sense primer: 5'-TCCACCACCCTGTTGCTGTAG-3', antisense primer: 5'-

GACCACAGTCCATGCCATCACT-3'; designed to produce a 450 bp product) was performed

under the following conditions: 21 cycles at 94 ˚C denaturation for 20 seconds, 58 ˚C annealing

for 30 seconds, 72 ˚C extension for 1 minute, and 1 cycle at 72 ˚C final extension for 10 minutes.

5 µl samples of the elastin, collagen type I, Gα13, and GAPDH PCR products from each

reaction were run on a 2% agarose gel and post-stained with ethidium bromide. The amount of

tropoelastin mRNA was standardized relative to the amount of GAPDH mRNA.

Western Blotting

Confluent SDF cultures were exposed for different periods of time (3 hours to 24 hours

or 2 hours to 8 hours) or for 3 hours as indicated to 100 μM SA in the presence or absence of

one-hour pretreated 400 μM probenecid or 10 μg/ml cycloheximide. At the end of each


P a g e | 30

experiment cells were lysed using an NP-40 buffer [(in mM: 20 Tris · HCl, pH 7.5, 150 NaCl,

1 EDTA, 1 EGTA, 1% NP-40) containing a cocktail of antiproteases (20 µg/ml leupeptin,

10 µg/ml aprotinin, 0.1 mM PMSF, 1 mM DTT) and antiphosphases (2.5 mM Na4O7P2, 1 mM

β-Glycerolphosphate, 1mM Na3VO4)], and 30-50 µg of protein extract was resuspended in

sample buffer (0.5 M Tris · HCl,

pH 6.8; 10% SDS; 10% glycerol; 4% 2-β-mercaptoethanol; and

0.05% bromophenol blue), and the mixture was boiled for 5 minutes. Protein lysates were

resolved by pre-cast SDS-PAGE gel (4–12% gradient), transferred to nitrocellulose membranes,

blocked for an hour and then immunoblotted with polyclonal anti-tropoelastin antibody at 4 ˚C

overnight. All blots were then incubated with the goat-anti-rabbit HRP-conjugated secondary

antibodies for an hour and examined using the enhanced chemiluminescence detection system.

Blots were stripped and re-probed using monoclonal anti-β-actin antibodies to standardize

relative to the amount of β-actin. The degree of expression was measured by densitometry.

Immunoprecipitation

To evaluate the level of IGF-IR-β phosphorylation in SDF cultures were incubated for

15 minutes in the presence or absence of 100 μM SA or 50 ng/ml IGF-1 with or without 30-

minute pretreated 0.5 μM PPP or 10 μM PP2 in 2 % containing DMEM as specified in the

figure legends. Parallel cultures were incubated in serum-free conditions in the presence or

absence of 100 μM SA or 50 nM IGF-1 with or without 30-minute pretreated 0.5 μM PPP. At

the end of each experiment the cells were lysed as specified above.

Prior to cell lysis, G-protein coupled to superparamagnetic Dynabeads was washed and

incubated with monoclonal anti-p-Tyr antibody (PY99) for 1 hour at 4 ˚C followed by addition


P a g e | 31

of 400 µg of protein extract from the cell lysates for 1 hour at 4 ˚C, as described in the

Invitrogen protocols. The resulting protein-antibody conjugate was washed four times with

PBS/0.01% Tween-20. The final conjugate was re-suspended in sample buffer and the proteins

were resolved at SDS-PAGE, transferred to the immobilon membrane and subjected to the

Western blotting with antibody recognizing the β subunit of the IGF-IR.

For all immunoprecipitation experiments, cell extracts were also incubated with normal

goat IgG and the IP products served as negative controls. They did not produce any unspecific

products that would be recognized by the respective Western blotting (data not shown).

Data Analysis

In all biochemical studies, quadruplicate samples in each experimental group were

assayed in three separate experiments. Mean and standard deviations (SD) were calculated for

each experimental group, and statistical analyses were carried out by ANOVA, followed by

Bonferroni's test comparing selected groups, or by t test, as appropriate. P value of less than

0.05 was considered significant.


P a g e | 32

P a g e | 32

RESULTS

SA induces the deposition of elastic fibers in monolayer cultures of human skin-derived

fibroblasts.

As the ascorbic acid has been established as a potent stimulator of collagen fiber

production,45

it has been also recognized as a factor that negatively interferes with deposition of

elastic fibers.161

We therefore decided to investigate whether and how AA derivatives would

affect production of both fibrotic components of the ECM.

Results of the initial experiments clearly indicated that treatment with 100 μM SA

remarkably upregulated deposition of the elastic fibers that could be detected with antibodies

recognizing tropoelastin (Fig 1), and the major component of microfibrillar scaffold, fibrillin-1

(Fig 2). Treatment with 100 µM SA also stimulated deposition of collagen fibers in a more

potent manner than treatment with the comparable concentration of AA (Fig 1). As predicted,

cultures treated with 100 µm AA did not produce elastic fibers (Fig.1).

We have further established that the elastogenic effect could be observed in cultures

treated with SA concentrations ranging from 25µM to 200 µM, and that further increases in SA

concentration abolished deposition of the immuno-detectable elastic fiber (Fig 3 A). The

quantitative assessment of metabolically labeled insoluble elastin confirmed results obtained

with immunohistochemistry (Fig 3B).

SA also penetrates to the full thickness skin explants kept in organ cultures and stimulates

deposition of new elastic fibers.


P a g e | 33

We further investigated whether SA would penetrate through the skin and induce a

similar elastogenic effect as that observed in monolayer cultures of dermal fibroblasts. We

utilized the organ culture model that is more relevant to the in vivo environment, and the

obtained results would further justify the clinical (topical) use of SA. The obtained results

indicated that a 10 day-long treatment with 200 µM SA induced formation of new elastic fibers

in cultured full thickness skin explants (2 x 2 mm) derived from the skin biopsies of normal

human skin or from dermal scars (Fig. 4). We observed that the applied treatment stimulated

migration of resident stem cells, which are normally located in the basal layer of epidermis, into

the papillary zone of the dermis and a consecutive production of new elastic fibers that could be

detected by the Movat’s pentachrome staining (Fig. 4). It has previously been established that

black elastic fibers detected with this routine histochemical method fully overlap with structures

detected with the anti-elastin antibody.194, 195

The elastogenic effect of SA can be also observed in cultures of fat tissue-derived fibroblasts;

the magnitude of SA-induced elastogenic stimulation exceeds effects observed in parallel

cultures treated with an elastogenic growth factor, IGF-1.

Because the latest reports indicate the usefulness of fat tissue-derived fibroblasts in

regenerative medicine,6 we also investigated whether such cells, obtained in high numbers from

liposuction procedures performed during cosmetic surgery, would also respond to SA in a

similar elastogenic manner as SDFs. Indeed, we have established that treatment with SA also

induced production of new elastic fibers in monolayer cultures of FDFs maintained in the

presence of FBS (Fig. 5). This observation further confirmed the usefulness of FDFs in


P a g e | 34

regenerative medicine. It also indicated that eventual treatment of these cells with SA before

their commitments to the resin scaffolds of injection to badly healing wounds would induce their

full capabilities of ECM production including normal elastic fibers.

This conclusion was further enforced by results of further experiments indicating that the

magnitude of SA-induced upregulation of elastin deposition in both SDFs and FDFs cultures

maintained in the presence of 2% FBS actually exceeded elastogenic effect of a natural

elastogenic factor applied in its optimal concentrations (50 ng/ml IGF-1) (Fig.5). Interestingly,

the results of immunostaining and metabolic labeling of insoluble elastin also indicated that the

addition of 100 µM SA to cultures treated with 50 ng/ml IGF-1 induced a more potent net

elastogenic effect than those observed in parallel cultures maintained in the presence of these

growth factors alone (Fig. 6).

Statistical analysis showed that the level of significance between control and SA

treatment (P < 0.001) is higher than the level between control and a growth factor treatment (P <

0.05 or P < 0.01).

SA induces both elastogenesis and collagenogenesis in cultures of aortic smooth muscle cells

and cardiac fibroblasts, but the addition of spironolactone to SA-treated cultures exclusively

reduces formation of new collagen fibers.

While the regeneration of the connective tissue frameworks requires the well-balanced

production of collagen and elastic fibers, overzealous production of collagen fibers (mostly

stimulated by aldosterone) and insufficient production of elastic fibers, after mechanical or

metabolic arterial injuries and during remodeling of ischemic hearts, contributes to the


P a g e | 35

pathogenesis of cardiovascular diseases.146, 152

Therefore, we also explored whether the

additional treatment of cultured AoSMCs and CFs with an inhibitor of mineralocorticoid

receptors, spironolactone, would shift the ultimate balance of ECM production and increase the

production of elastic fibers by those cells. Indeed, our result indicated that while the addition of

SA upregulated production of both elastic fibers and collagen fibers in cultures of AoSMCs and

CFs, parallel cultures of these cells pretreated with 2 µM spironolactone one hour prior to the

addition of 100 µM SA demonstrated decreased deposition of collagen fibers and a marked

upregulation in elastic fiber deposition, as compared with the control counterparts (Fig 7).

In contrast to 100 µM SA, identical concentrations of sodium ions applied in the forms of

NaCl or ascorbic acid do not upregulate deposition of elastic fibers in cultures of skin-derived

fibroblasts.

In the next series of experiments, we aimed to elucidate mechanisms by which SA

induces elastogenesis. Since treatment with ascorbic acid alone did not stimulate deposition of

elastic fibers (Fig. 1), we rationalized that the elastogenic effect of SA is not likely due to an

action of ascorbate ions. Furthermore, now we demonstrate that the super-low concentration of

sodium ions (100 μM) added to the conditioned media that already contain 148 mM of sodium

did not change the production of elastin by cultured fibroblasts (Fig.8).

Thus, we assumed that the elastogenic effect is induced by the unique chemical

properties exhibited by SA. Results depicted in Figure 8 demonstrate that cultures of SDF did

not increase their deposition of immune-detectable elastic fibers after additions of 100 µM NaCl

or 100 µM AA.


P a g e | 36

SA exercises its elastogenic effect only after being transported into the cell interior.

Since in sodium ascorbate molecules, sodium cations and ascorbate anions coexist in

close proximity, we hypothesized that this salt might be transported into the cell interior via the

sodium-dependent vitamin C transporters (SVCTs). To test this hypothesis, we utilized the

anion transport inhibitor probenecid, which also blocks SVCTs uptaking SA.175

Importantly, we

found that preincubation of cultured SDF with probenecid eliminated the SA-induced

upregulation of net elastogenesis (Fig. 9). This strongly suggests that SA needs to be transported

into the cell interior in order to exercise its elastogenic effect.

Cultures of skin-derived fibroblasts maintained in the presence of 2% FBS revealed two peaks

of transient upregulation in the levels of intracellular tropoelastin protein occurring between

3-6 hours and 18-24 hours after the addition of SA.

The results of our next experiment, in which we monitored the time course production of

tropoelastin in SA-treated cultures, demonstrated that this compound induces two separate peaks

in levels of intracellular tropoelastin protein (detected by Western blots) that occur 3-6 hours

and 18-24 hours from the beginning of the treatment (Fig. 10). We therefore conclude that SA

may exercise its net elastogenic effect via at least two independent mechanisms.

SA-treated fibroblasts demonstrate a heightened level of intracellular tropoelastin even after

their translation machinery has been inhibited with cycloheximide. This elastogenic effect

occurs only after SA is transported into the cell interior.


P a g e | 37

The elastogenic process is not 100% efficient. Thus, even in optimal conditions, a

significant fraction of newly produced tropoelastin (49-50%) protein is subjected to the

proteolytic degradation and not secreted.194, 195

Therefore, we investigated whether treatment

with SA would possibly decrease the process of intracellular degradation of the already

produced tropoelastin. First, we established that fibroblasts treated with SA for 3-6 hours

contain more intact (70 kDa) tropoelastin than those in untreated controls (Fig. 10). Then we

found that cultures of SDFs in which the effective production of new tropoelastin has been

stopped by pre-incubation with the specific inhibitor of translation process, cycloheximide,196

still demonstrate heightened levels of intracellular tropoelastin when treated with SA for 2-6

hours (Fig. 11). This observation indicates that the first peak of tropoelastin level observed in

cells treated with SA for only 2 to 6 hours is not due to any enhancement of protein synthesis,

but rather reflects the better preservation of the already existing tropoelastin protein. This

beneficial effect (a higher level of Western blot-detected tropoelastin) was not visible in parallel

cultures in which intracellular influx of SA has been inhibited by probenecid (Fig. 12).

Cultures of skin-derived fibroblasts maintained in the presence of 2% FBS demonstrate a

transient upregulation in the net level of tropoelastin mRNA occurring 18 hours after SA

addition.

Once we have established that SA induce higher levels of preservation of the already

produced tropoelastin (―fast effect‖ observed in cultured treated for 2-6 hours), we further

investigated the nature of the ―late effect‖ of SA visible as a significant upregulation of

intracellular tropoelastin protein in cultures treated for 24 hours. We found that this ―late effect‖

of SA was preceded by the increase in levels of tropoelastin mRNA transcripts. Results of RT-


P a g e | 38

PCR demonstrated that treatment with SA stimulated a transient increase in levels of

tropoelastin mRNA transcripts 18 hours after the initial SA treatment (Fig. 13).

Moreover, we also detected that cultures of SDFs treated jointly for 18 hours with SA

and optimal concentrations of IGF-1 demonstrate even higher levels of tropoelastin mRNA than

cultures treated with either of these growth factors alone (Fig. 14). This indicates that the

observed additive effect of SA and IGF-1 might either occur on the level of the elastin gene’s

transcription or reflect the increased stability of tropoelastin mRNA (Fig. 14).

The “late effect” of SA leading to upregulation of tropoelastin mRNA levels is executed

through the enhancement of the primary elastogenic signals triggered by IGF-1 receptor.

In addition to the important observation that the treatment with SA leads to upregulation

of tropoelastin mRNA levels in cultures of SDFs, we also confirmed that this effect is not

present in cells that have been pretreated with probenecid; hence, it is also executed by SA that

has been actively transported to the cell interior (Fig. 15).

Since cultures treated with SA demonstrated upregulation in levels of tropoelastin

mRNA, and cultures jointly treated with IGF-1 and SA showed even higher levels of

tropoelastin mRNA than their counterparts treated with IGF-1 alone, we hypothesized that the

observed synergistic effect of SA could be attained through the enhancement of elastogenic

signals triggered by the IGF-1 receptor. To test whether the IGF-1R signaling could be involved

in the observed upregulation in tropoelastin mRNA levels, we cultured dermal fibroblasts in the

presence of the specific inhibitor of the IGF-1R tyrosine kinase, PPP188

, and eventually

established that such functional elimination of IGF-1R signaling abolished the SA-induced


P a g e | 39

upregulation of tropoelastin mRNA in cultures maintained in the presence of 2% FBS, which,

among other growth factors, also contain traces of IGF-1 (Fig. 15).

Jointly, results of the above-mentioned experiments allow for speculation that SA

transported into the cell interior might activate another kinase-involving pathway causing

additional phosphorylation of IGF-IR, and eventual enhancement of the downstream signals

leading to activation of the elastin gene and an increase in tropoelastin mRNA levels.68, 78

Indeed, further results revealed that treatment with SA enhanced levels of phosphorylated IGF-

1R (immunoprecipitated with anti-phospho-tyrosine antibody, Py99), but only in cultures

maintained in the presence of FBS (Fig. 16). Treatment with SA also enhanced levels of IGF-IR

phosphorylation, induced by treatment with IGF-1 in cultures kept in serum-free media (Fig.

16). Significantly, the mentioned effects of SA were eliminated in cultures pretreated and

maintained in the presence of PPP (Fig. 16).

The previous studies from our laboratory demonstrated that the additional

phosphorylation of IGF-1R, which eventually leads to elastogenic signaling, can be induced by

aldosterone, and that this mechanism involves activation of c-Src.68, 78

Therefore, we

investigated whether a similar mechanism could be induced by intracellular SA. Our results

revealed that incubation with a selective inhibitor of Src-family tyrosine kinase, PP2, eliminated

the upregulation of IGF-1R phosphorylation that has been observed in cultures maintained in the

presence of SA and 2% FBS (Fig. 17). These results confirmed that c-Src tyrosine kinase

activity is engaged in the SA-induced pathway leading to super-phosphorylation of IGF-1R that,

in turn, triggers the down-stream elastogenic pathways (Fig. 17).


P a g e | 40

Treatment with SA also stimulates the net deposition of immune-detectable elastic fibers and

enhances levels of insoluble elastin in cultures of skin-derived fibroblasts isolated from

patients with Loeys-Dietz syndrome.

In the next series of experiments, we investigated whether treatment with SA would also

induce better deposition of elastic fibers in cultures of fibroblasts derived from Loeys-Dietz

Syndrome (LDS), a genetic disease caused by a mutation in the TGFβ receptor-1 (LDS1) or

TGFβ receptor-2 (LDS2) and characterized by the impaired deposition of elastic fibers. We

found that cultures of LDS1-derived fibroblasts treated with SA contained more immuno-

detected elastic fibers and deposited more metabolically labeled insoluble elastin than the

respective control cultures that were maintained without SA. It is important to mention that cells

derived from LDS2 were always more responsive to SA treatment. Moreover, we have also

established that addition of SA to cultures maintained in the presence of IGF-1 further

upregulated the net elastogenic effect of these growth factors (Figs. 18 and 19).

Addition of SA to cultures of skin fibroblast isolated from Williams-Beuren Syndrome

patients upregulates the level of tropoelastin mRNA transcripts and the ultimate deposition of

elastic fibers.

WBS is another genetic disease that expresses improper deposition of ECM caused by

the deletion of about 27 genes from the long arm of chromosome 7, including the elastin

gene.197

Haplosufficiency of the elastin gene results in inadequate production of elastic fiber,

which causes cardiovascular diseases.198

Here, we tested whether treatment with SA-increased

tropoelastin mRNA expression from one locus. RT-PCR analysis indicated that cultures of WBS


P a g e | 41

SDFs with SA treatment expressed significantly more tropoelastin mRNA transcripts than their

parallel cultures without SA treatment in the presence or absence of IGF-1 (Fig 20). In addition,

SA induced upregulation of tropoelastin mRNA levels and increased the ultimate deposition of

elastic fibers in cultures of WBS SDFs (Fig. 20). Therefore, SA, which is safe and harmless, can

be a therapeutic agent to induce elastogenesis in WBS patients.


P a g e | 42

Figure 1

Treatment with sodium ascorbate (SA) upregulates the deposition of both collagen and

elastic fibers in monolayer cultures of skin-derived fibroblasts. In contrast, treatment with

ascorbic acid (AA) enhances the deposition of collagen fibers only.

Representative photomicrographs of 6-day-old cultures of skin-derived fibroblasts immuno-

stained with antibodies to tropoelastin or collagen type I. (Original magnification 600x)

Cells were treated with 100 µM AA or 100 µM SA at day 1 and day 4.


P a g e | 43


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Figure 2

Treatment with SA upregulates a net deposition of fibrillin-1, a major component of the

microfibrillar scaffold of elastic fibers.

Representative photomicrographs of 6-day-old cultures of skin-derived fibroblasts immuno-

stained with antibodies to tropoelastin and fibrillin-1. (Original magnification 600x)

Cells were treated with 100 µM SA at day 1 and day 4.


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Figure 3

Treatment with SA, using concentrations between 50-400 µM, stimulates elastic fiber

production in a dose-dependent manner.

(A) Representative photomicrographs of 6-day-old cultures of skin-derived fibroblasts showing

elastic fibers detected with anti-tropoelastin antibody. (Original magnification 600x)

Cultures were treated at day 1 and day 4 with 0 μM to 800 µM SA.

(B) Statistically-evaluated results of a quantitative assay of insoluble elastin in 3-day-old

quadruplicate cultures of skin-derived fibroblasts that were metabolically labeled with [3H]-

Valine.

SDFs were also treated with the indicated concentrations of SA.


P a g e | 46


P a g e | 47

Figure 4

Treatment with SA upregulates a net deposition of elastic fibers in full thickness skin

tissue explants derived from biopsies of normal skin and dermal scars.

Representative photomicrographs depicting black-stained elastic fibers visualized with Movat’s

pentachrome method in 10-day-old cultures of dermal explants derived from normal skin and

dermal scar tissue. (Original magnification 200x)

The explants were maintained for 10 days in the presence or absence of 200 µM SA, which was

added daily.


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Figure 5

Cultures of skin-derived fibroblasts (SDF) and fat-derived fibroblasts (FDF) treated with

100 µM SA produce more immuno-detectable elastic fibers and metabolically labeled

insoluble elastin than their respective counterparts treated with IGF-1.

(A) Representative photomicrographs of 6-day-old cultures of FDFs and SDFs immuno-stained

with an anti-tropoelastin antibody. (Original magnification 600x)

Cells were treated at day 1 and day 4 with 100 µM SA or 50 ng/ml IGF-1.



Valine.

Cells were treated for 72 hours with 100 µM SA or 50 ng/ml IGF-1.


P a g e | 49


P a g e | 50

Figure 6

Cultures of skin-derived fibroblasts jointly treated with SA and IGF-1 produce

significantly more elastic fibers than cultures treated with each factor individually.

(A) Representative photomicrographs of 6-day-old cultures of skin-derived fibroblasts immune-

stained with the antibody to tropoelastin. (Original magnification 600x)

Cultures were treated at day 1 and day 4 with 50 ng/ml IGF-1 in the presence or absence of 100

μM SA.



Valine.

Cultures of SDFs were treated once with 50 ng/ml IGF-1 in the presence or absence of 100 μM

SA.


P a g e | 51


P a g e | 52

Figure 7

Treatment with sodium ascorbate (SA) upregulates deposition of both collagen and elastic

fibers in monolayer cultures of both aortic smooth muscle cells (AoSMCs) and human fetal

cardiac fibroblasts (CFs). However, pretreatment with the mineralocorticoid receptor

inhibitor spironolactone exclusively reduces production of collagen fibers in SA-treated

cultures.

Representative photomicrographs of 6-day-old cultures of (A) AoSMCs and (B) CFs immuno-

stained with antibodies to tropoelastin or collagen type I. (Original magnification 600x)

Cells were pretreated with 2 μM spironolactone 1 hour prior to addition of 100 μM SA at day 1

and day 4.


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P a g e | 54

Figure 8

While treatment with 100 μM SA stimulates deposition of elastic fibers in cultures of skin-

derived fibroblasts, the parallel cultures treated with the same concentrations of sodium

ions engaged in other salt (100 μM NaCl) or free ascorbic acid (AA) did not stimulate

elastogenesis.

Representative photomicrographs of 6-day-old cultures of skin-derived fibroblasts immune-

stained with anti-tropoelastin antibody. (Original magnification 600x)

Cells were treated on day 1 and day 4 with 100 µM SA, 100 µM NaCl or 100 µM AA.


P a g e | 55

Figure 9

Pretreatment with probenecid (an inhibitor of organic anion transporters that also blocks

sodium-dependent vitamin C transporters (SVCTs)) eliminates the elastogenic effect of SA

observed in cultures of skin-derived fibroblasts.

(A) Representative photomicrographs of 6-day-old cultures of skin-derived fibroblasts immune-

stained with anti-tropoelastin antibody. (Original magnification 600x)

Cells were pretreated with 400 μM probenecid 1 hour prior to the addition of 100 μM SA on day

1 and day 4.



Valine.

Cultures were pretreated for 1 hour either with or without probenecid prior to 72-hour

treatments with 100 μM SA.


P a g e | 56


P a g e | 57

Figure 10

The Western blot analysis performed in the time course manner revealed that skin-

derived fibroblast cultures kept in the presence of 2% FBS and 100 µM SA demonstrate

two peaks of transient upregulation in levels of intracellular tropoelastin, occurring 3 to 6

hours and 18 to 24 hours after the addition of SA.

Representative Western blots with anti-tropoelastin antibody and statistically-evaluated results

from three separate experiments, in which quadruplicate cultures of skin-derived fibroblasts

were maintained either in the presence or absence of 100 μM SA for indicated periods of time.


P a g e | 58

Western Blot


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Figure 11

Cultures of SDFs maintained in the presence of a translation inhibitor, cycloheximide, also

demonstrate heightened levels of intracellular tropoelastin when treated with SA for only

2 to 6 hours. This indicates that SA may protect the already synthesized tropoelastin

molecules.


from three separate experiments, in which quadruplicate cultures of skin-derived fibroblasts,

pretreated for 30 minutes with 10 μg/ml cycloheximide, were subsequently maintained with this

translation inhibitor in either the presence or absence of 100 μM SA for the indicated periods of

time.


P a g e | 60

Western Blot


P a g e | 61

Figure 12

Pretreatment with probenecid eliminated the tropoelastin-protecting effect of SA observed

in cultures of skin-derived fibroblasts in which the translation of new proteins has been

inhibited with cycloheximide.


from three separate experiments, in which quadruplicate cultures of skin-derived fibroblasts,

pre-incubated for 30 minutes with or without 400 µM probenecid and/or 10 µg/ml of

cycloheximide, were subsequently treated for 3 hours with or without 100 μM SA with these

inhibitors.


P a g e | 62

Western Blot


P a g e | 63

Figure 13

The time course examination of skin-derived fibroblasts maintained in the presence of 2%

FBS demonstrates a transient upregulation in the net level of tropoelastin mRNA

occurring 18 hours after addition of SA.

Representative one-step RT-PCR demonstrating levels of tropoelastin mRNA transcripts and

statistically-evaluated results from three separate experiments, in which quadruplicate cultures

of skin-derived fibroblasts were maintained in the presence or absence of 100 μM SA for

indicated periods of time.


P a g e | 64

RT-PCR


P a g e | 65

Figure 14

Confluent cultures of skin-derived fibroblasts jointly treated for 18 hours with SA and

IGF-1 demonstrate higher levels of tropoelastin mRNA than cultures treated with these

reagents alone


statistically evaluated results from three separate experiments, in which quadruplicate cultures

of skin-derived fibroblasts were maintained for 18 hours in the presence and absence of 50

ng/ml IGF-1 and 100 μM SA, applied alone or together.


P a g e | 66

RT-PCR


P a g e | 67

Figure 15

One hour-long pretreatment of cultured skin-derived fibroblasts with probenecid or with

an inhibitor of IGF-1R tyrosine kinase, PPP, eliminated the SA-induced upregulation of

tropoelastin mRNA levels observed in 18-hour old cultures.


statistically-evaluated results from three separate experiments, in which quadruplicate cultures

of skin-derived fibroblasts were pretreated with 400 μM probenecid for 1 hour followed by 18-

hour treatment with or without 100 μM SA.


P a g e | 68

RT-PCR


P a g e | 69

Figure 16

Addition of SA stimulates phosphorylation of IGF-1R detected in cultures of skin-derived

fibroblasts maintained in the presence of 2% FBS. The pretreatment with an inhibitor of

IGF-1R kinase, PPP, abolishes the SA-stimulated phosphorylation of IGF-1R.

The effect of SA described above cannot be observed in the parallel cultures maintained in

the serum-free conditions.

SDF cultures were pre-incubated in 2% FBS media with or without 0.5 μM PPP for 30 minutes

prior to the indicated treatments: 100 μM SA, 50 ng/ml IGF-1. Cell lysates were

immunoprecipitated (IP) with anti-PY99 antibodies that recognize phosphorylation on tyrosine,

were electrophoresed, and were probed with the anti-IGF-1R antibodies.

Immuno-Precipitation with Anti-PY99 Antibody

Followed by Western Blots with anti-IGF-1R Antibody


P a g e | 70

Figure 17

Pretreatment of cultured skin-derived fibroblasts with c-Src tyrosine kinase inhibitor, PP2,

eliminates the SA-induced phosphorylation of IGF-1R.

SDF cultures were pre-incubated in 2% FBS media with or without 10 μM PP2 for 30 minutes

prior to treatment with 100 μM SA. Cell lysates were immunoprecipitated (IP) with anti-PY99

antibodies that recognize phosphorylation on tyrosine. The IP products were resolved on SDS-

PAGE and immunoblotted with the anti-IGF-1R antibody.

Immuno-Precipitation with anti-PY99 antibody

Followed by Western Blots with anti-IGF-1R


P a g e | 71

Figure 18

Treatment with SA also stimulates the net deposition of immunodetectable elastic fibers

and enhances levels of insoluble elastin in cultures of skin-derived fibroblasts isolated from

patients with Loeys-Dietz Syndrome-1.

(A) Representative photomicrographs of 6-day-old cultures of SDFs isolated from LDS1

patients immuno-stained with the anti-tropoelastin antibody. (Original magnification 600x)

Cells were treated at day 1 and day 4 with 50 ng/ml IGF-1 in the presence or absence of 100 μM

SA.



Valine.

Cultures of LDS1 skin-derived fibroblasts were treated for 72 hours with or without 50 ng/ml

IGF-1 in the presence or absence of SA.


P a g e | 72


P a g e | 73

Figure 19

Treatment with SA significantly increases deposition of immuno-detectable elastic fibers

and deposition of metabolically labeled insoluble elastin in cultures of skin-derived

fibroblasts isolated from patients with Loeys-Dietz syndrome-2. Addition of SA also

significantly enhances the net elastogenic effects of IGF-1.

(A) Representative photomicrographs depicting deposition of immuno-detectable elastic fibers

in 6-day-old cultures of skin-derived fibroblasts isolated from patient with LDS2. (Original

magnification 600x)

50 ng/ml IGF-1 and 100 μM SA were added twice at day 1 and 4.



Valine.

Cultures of LDS2 skin-derived fibroblasts were treated for 72 hours with 50 ng/ml IGF-1 in the

presence or absence of SA.


P a g e | 74


P a g e | 75

Figure 20

Treatment with SA also significantly upregulates production of immuno-detectable elastic

fibers and level of tropoelastin mRNA detected in cultures of fibroblasts isolated from

patients with Williams-Breuren syndrome maintained in the presence of 2% FBS, and

mildly enhances the elastogenic effects of IGF-1.

(A) Representative photomicrographs of 6-day-old cultures of SDFs isolated from WBS patients

immuno-stained with anti-tropoelastin antibody. (Original magnification 600x)

Cells were treated at day 1 and day 4 with 50 ng/ml IGF-1 in the presence or absence of 100 μM

SA.

(B) Representative one-step RT-PCR demonstrating levels of tropoelastin mRNA transcripts

and statistically-evaluated results from three separate experiments, in which quadruplicate

cultures of WBS skin-derived fibroblasts were maintained for 18 hours in the presence and

absence of 50 ng/ml IGF-1 and 100 μM SA, applied alone or together.


P a g e | 76


P a g e | 77

CONCLUSION

Human skin-derived fibroblasts (SDFs) maintained in monolayer cultures and those

present in the full-thickness explants of human skin produce more elastic fibers when

treated in vitro with sodium ascorbate (SA), applied in concentrations ranging from 50-

200 µM.

The optimal concentration of sodium ascorbate (100 µM) also upregulates the net

deposition of elastic fibers in monolayer cultures of fibroblasts derived from human fat

tissue (FDFs), as well as in cultures of smooth muscle cells isolated from human aorta

(AoSMCs) and human cardiac fibroblasts (CFs).

Treatment with SA upregulates elastogenesis in cultures of SDFs derived from patients

with such genetic diseases as Loyes-Dietz Syndrome and Williams-Beuren Syndrome,

which are characterized by inadequate elastogenesis.

In contrast to 100 µM SA, the treatment with similar concentration of separate ions

making the SA salt, 100 µM of NaCl or 100 µM ascorbic acid does not induce any

upregulation in the net deposition of elastic fibers.

SA exercises its net elastogenic effects only after being actively transported into the cell

interior in two separate mechanisms:

1- The ―fast effect,‖ which reflects the greater stability of intracellular tropoelastin

already synthesized in response to elastogenic growth factors present in the serum.

2- The ―late effect,‖ which reflects the enhancement of the elastin gene expression

induced after the parallel activation of IGF-1R tyrosine kinase by IGF-1 and

activation of c-src tyrosine kinase, occurring in the presence of intracellular SA.


P a g e | 78

DISCUSSION

There is a clear need for more research in order to have a better understanding of the

elastogenic processes, and for the discovery of a pharmacologically accepted elastogenic factor

devoid of major side effects. The presented investigations introduced in this thesis were aimed

at this goal.

This is the first study clearly demonstrating that sodium ascorbate (SA) can induce

formation of new elastic fibers by such cell types as skin-derived fibroblasts (SDFs), fat-derived

fibroblasts (FDFs), human aortic smooth muscle cells (AoSMCs) and human fetal cardiac

fibroblasts (CFs).

After establishing that SA stimulates elastogenesis in cultures of various cell types, we

further investigated mechanisms by which SA induces elastogenesis. First of all, we found that

cells maintained in the serum-free medium did not produce more elastin when treated with 100

µM SA (data not shown). This was in contrast to cells maintained in the presence of 2% FBS;

these cells demonstrate a significant upregulation of elastogenesis when treated with 100 µM

SA. This observation indicated that SA might accelerate elastogenic signals triggered by certain

factors present in the serum and does not act as a primary stimulator of such signals. This

assumption was strongly validated by results of further experiments demonstrating that 100 µM

of SA induced a higher ultimate elastogenic effect than a known stimulator of elastogenesis,

IGF-1, applied in a low physiological concentration of 50 ng/ml; simultaneous treatment with

100 µM of SA and the low concentration of IGF-1 further enhanced the elastogenic effect of

these factors.


P a g e | 79

Previously, our laboratory discovered that raising the sodium ion concentration from the

physiological level (148mM) to 160 mM enhances deposition of elastic fibers. We therefore

initially hypothesized that sodium ions released from the SA would be solely responsible for the

observed elastogenic effect of this ascorbate derivative. This initial hypothesis was quickly

verified and discarded after we learned that 100 μM of NaCl or 100 µM of AA did not induce

elastogenesis. This result prompted speculation that the unique conformation of SA can be

responsible for triggering the ultimate elastogenic effect within relatively narrow window of

concentration (50-200 µM) of this compound.

Most importantly, results of our other set of experiments indicating that the ultimate

elastogenic effect of 100 µM SA cannot be observed in cultures pretreated with probenecid

prompted another possibility: that the µM concentration of sodium would activate the sodium-

dependent vitamin C transporters (SVCTs) to quickly translocate SA to the cell interior. We

may further hypothesize that such a quick transportation of the SA prevents the imminent

oxidization of ascorbate anions to the inactive dehydroascorbate, which can be rapidly

transported into cells and hydrolyzed if not reduced back to ascorbate.199

It has been

documented that the regeneration of elastin in SOD-deficient mice with skin atrophy was

achieved by oral administration of a vitamin C derivative, Ascorbyl 2-phosphate 6-palmitate,

which can efficiently permeate cell membranes followed by its conversion to an ascorbate

anion.164, 180

This supports the hypothesis that only intracellular unoxidized ascorbate anions

induce elastogenesis.

Once we established that only intracellular SA would induce a chain of events eventually

leading to the ultimate increase of the net deposition of elastic fibers, we aimed at a dissection of

the intracellular mechanism(s) that can be positively modulated after intracellular influx of SA.


P a g e | 80

First of all, we found that cultures of dermal fibroblasts cultured in the presence of FBS-treated

with SA showed two peaks in levels of intracellular tropoelastin. The first ―fast‖ effect was

observed in as quickly as 3 hours after addition of SA and the second one was apparent in

cultures 18 to 24 hours old. We then established that the ―fast‖ elevation of intracellular

tropoelastin levels, documented by Western blotting, has not been achieved by any increase in

levels of tropoelastin mRNA, nor inhibited in cells pretreated with the translation inhibitor

cycloheximide. These observations indicate that the intracellular uptake of SA would somehow

protect the already synthesized 72-kDa tropoelastin protein.

However, the particular mechanism of this phenomenon remains speculative at this time.

We may hypothesize that intracellular unoxidized ascorbate ions might bind to tropoelastin

molecules similarly to the previously described effect of tannic acid, thus protecting them from

proteolytic degradation.195

Based on the results of previous studies,200, 201

we may not exclude the plausible

possibility that the intracellular increase of unoxidized ascorbate achieved after SA treatment

would cause reduction in level of free radicals (reactive oxygen species, ROS). This might

prevent the ROS-dependent inactivation of 1-antitrypsin, an extremely potent inhibitor of

elastolytic serine proteinases.

We did not measure whether a short treatment with SA would significantly increase

levels of hydroxylation of tropoelastin-resided proline and lysine residues. Therefore, we cannot

confirm the putative link between the observed increase in levels of intracellular tropoelastin

and the previously suggested phenomenon that the heightened tropoelastin hydroxylation would

enhance the physical resistance of this protein to the enzymatic degradation.202


P a g e | 81

Most importantly, our data showed that the ―late‖ elastogenic effect of SA, observed

only in cells maintained in the presence of FBS, could be linked with a significant increase in

tropoelastin mRNA. This upregulation was eliminated in the presence of the IGF-R tyrosine

kinase inhibitor, PPP, indicating prerequisite involvement of IGF-1R downstream signaling in

the SA-induced elastogenesis. Indeed, we further demonstrated this effect with SA-enhanced

phosphorylation on IGF-1R and that this effect could be inhibited in cells pre-incubated with the

c-Src inhibitor (PP2). Interestingly, this observation correlates well with results of the recent

study from our laboratory, which reported that the mechanism that selectively triggers the IGF-

1-mediated elastogenic signals induced by aldosterone is propagated only after activation of c-

Src. It, in turn, causes additional phosphorylation of the IGF-1R on Tyr-1316, which leads to

subsequent activation of PI3 kinase/Akt and ultimate upregulation of the elastin gene.68, 80, 203

While collagen provides the skin with mechanical strength, elastic fibers are responsible

for elasticity and resiliency in tissues.204

Therefore, a well-balanced production of new collagen

and elastic fibers is required during normal embryonic development and during the regeneration

of post-natal tissues affected by numerous diseases or mechanical damage. However, studies

aimed at wound healing or regeneration of wrinkled and stretch-marked skin have indicated that

the remodeling of the skin’s connective tissue framework is mostly associated with poor

deposition of new elastic fibers.205-207

Among the other factors that can potentially stimulate production of new ECM, ascorbic

acid (AA) has been widely tested and is recommended as an agent to stimulate collagen

deposition in vivo and in vitro.208

At the same time, this collagenogenic agent has been shown to

negatively interfere with the production of elastic fiber.45

In contrast, the results of our

experiments presented in this thesis document for the first time that a low concentration of SA


P a g e | 82

potently upregulates the production of both collagen type 1 and elastic fibers in cultures of all

tested cell types capable of ECM production. We also found that SA penetrates into the full

thickness of skin explants and upregulates the deposition of new elastic fibers. These particular

observations indicated that SA, which can penetrate into the skin, should be further tested as a

topically applied factor for reducing the formation of wrinkles and stretch marks or as a factor

capable of accelerating wound healing.

Fibroblasts isolated from human skin are also often used in manufacturing artificial skin,

which would at least temporarily cover the chemically or thermally damaged skin or replace

skin destroyed by ischemia or infection. While an optimal solution would be the use of

autologous fibroblasts obtained from the same patient, the additional harvesting of large

numbers of dermal fibroblasts derived from normal skin would add to the burden on the patient.

Since liposuction, a relatively new and widely used technique, allows for the safe

harvesting of a larger number of ECM-producing fibroblasts than the traditional punch skin

biopsy, we also tested whether fibroblasts harvested from the subdermal fat tissue would display

elastogenic potential that could be further stimulated by SA. In fact, fat-derived fibroblast (FDF)

transplantation has already been used to regenerate damaged skin, since FDFs were capable of

stimulating a resumption in the production of collagen and fibronectin after being injected into

subdermal tissues.209

The therapeutic effect of locally-injected FDFs has been also reported in

the healing of difficult wounds.210

To date, the elastogenic potential of FDFs has not been evaluated. Therefore, our results

demonstrating that treatment with SA enhances production of elastic fibers in cultures of FDFs

maintained in the presence of FBS constitute a real novelty. The results also encourage future


P a g e | 83

applications of SA for the production of skin replacements based on FDFs supported by

biopolymers, which would be less rigid and more elastic.

Vascular smooth muscle cells (SMCs) are the main types of cells residing in the tunica

media of arteries and veins. They are responsible for producing and organizing components of

elastic fibers and lamellae during development, providing resiliency of the vascular wall. SMCs

are also major players in the pathogenesis of atherosclerosis in which SMCs degrade elastic

fibers and migrate to intima, followed by a pathological thickening and occlusion of vessels.144

Currently, angioplasty followed by anti-proliferative and anti-thrombogenic drug-eluting stent

implantation is widely used to prevent the occlusion of the injured arteries caused by an

overzealous healing process.

Unfortunately, the overall success rate is no greater than 60% because of the overgrowth

of the stent mesh with activated SMCs leading to secondary in-stent stenosis.211

Numerous

papers, including those from our laboratory, documented that heightened SMC migration and

proliferation coincides with low deposition of elastic fibers, and that this inverse relationship

could be revered after the stimulation of new elastogenesis, either in vitro or in intra-arterial

stents.212-214

While pro-elastogenic action of selected factors has been documented, numerous

side effects of those factors (TGFβ-1, IGF-1, aldosterone or dexamethasone) prevent the wide

use of these factors in clinic.66, 67, 69

Previous study has demonstrated that the addition of 200 μM of SA in SMC-seeding on

the hyaluronan-based constructs increased elasticity and reduced stiffness of artificial blood

vessel constructs tested in vitro.165

However, the mechanism driving these physical changes has

not been investigated. Thus, our findings of the elastogenic effect of SA in cultures of AoSMCs

may explain the mechanism responsible for the reported increased elasticity of these constructs


P a g e | 84

maintained in the presence of SA. Beyond the application of SA in the production of more

resilient artificial vessel constructs, our results also encourage future testing on whether local,

oral or systemic application of SA can also be used to enforce the action of stents applied for the

prevention of coronary re-occlusions or for supporting the structure of arterial walls afflicted by

aneurysms.

Cardiac fibroblasts (CFs) are the most abundant cell type of the myocardium responsible

for regulating levels of various ECM proteins.148

However, in injured hearts, these stromal cells

usually respond to numerous hormones and cytokines with the overzealous production of

collagen that leads to the formation of rigid post-infarct scars and myocardial fibrosis.152

It has

recently been shown that the blockade of the mineralocorticoid receptors with eplerenone or

spironolactone leads to the exclusive propagation of an elastogenic effect of aldosterone that

counterbalances the production of collagen fibers after cardiac infarction.68, 78, 155, 156

Formation

of the more resilient ECM containing new elastic fibers resulted in the substantial improvement

of cardiac function and with the significant decrease (30 %) of post-infarct mortality.152-154

Results of recent studies from our laboratory indicated that aldosterone applied in the presence

of mineralocorticoid receptor inhibitors actually exclusively stimulates the production of new

elastin by the heart stromal fibroblasts, in the mechanism that involves cross-activation of c-Src

and consecutive phosphorylation of the IGF-1 receptor, which in turn triggers the downstream

elastogenic pathway.

Any increase in aldosterone levels that is not counter-balanced with MR blockers may

induce severe side effects including cardiac fibrosis. However, results presented in this study

showing that application of SA may also activate c-Src, phosphorylation of IGF-1 receptor and

the downstream elastogenic pathway strongly encourage future studies testing whether such a


P a g e | 85

cheap and well-tolerated compound as SA might also be used in the treatment of post-infarct

patients. We may also hypothesize that using this compound together with anti-fibrotic agents

would particularly improve the production of the balanced ECM and improve the cardiac

function after myocardial infarction.

Our results also encourage another therapeutic application of SA in genetically-

challenged patients afflicted by elastin gene insufficiency (i.e. Williams-Beuren syndrome,

WBS) or Loeys-Dietz syndrome, in which loss/disarray of elastic fibers has been connected to

the mutations of the TGFβ receptors-1/2.138, 215

Since SA induced elastogenesis in cultures of SDFs isolated from children diagnosed

with LDS and WBS, our results also encourage therapeutic applications of SA in children with

these genetic syndromes, characterized by arterial dissections, wrinkled skin and deep dermal

creases at young ages.136, 216

Since gene therapy is not yet clinically available due to safety issues, relatively safe

topical treatments with SA are therapeutic candidates that may result in alleviation of impaired

formation of elastic fibers, leading to reinforcement of arterial walls and enhancement of skin

elasticity in these patients.


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