About Hyaluronan in the Hypertrophic Heart344730/FULLTEXT01.pdf · 2010. 8. 20. · General characteristics 35 Quantitative RT-PCR 35 Quantification of hyaluronan in heart tissue

About Hyaluronan in the Hypertrophic Heart

Studies on coordinated regulation of extracellular matrix signalling

Urban Hellman

Department of Public Health and Clinical Medicine,

Medicine

901 87 Umeå

Umeå 2010

U. Hellman: About Hyaluronan in the Hypertrophic Heart

Responsible publisher under Swedish law – the Dean of the Faculty of Medicine

Copyright© 2010 by Urban Hellman

New series No. 1359

ISBN: 978-91-7459-043-2

ISSN: 0346-6612

Cover by:Urban Hellman

E-version available at http://umu.diva-portal.org/

Printed by: Print och Media

Umeå, Sweden 2010

Till Magdalena, Daniel och Simon

How could you describe this heart in words

without filling a whole book?

Note written by Leonardo da Vinci beside

an anatomical drawing of the heart, c. 1513


4

Table of Contents Abstract 6 List of papers 7 Abbreviations 8 Introduction 10

The heart 10 Cardiac hypertrophy 10

Fetal gene program 12 Intercellular signalling 13 Extracellular matrix 14 Glycosaminoglycans 14 Hyaluronan 15

Structure 15 Hyaluronan synthesis and catabolism 16

Hyaluronan synthesis 16 Hyaluronidases 16 Hyaluronan turnover 17 Regulation of hyaluronan synthesis 17

Hyaluronan function 18 High molecular weight hyaluronan 18 Low molecular weight hyaluronan 18 Hyaluronan oligosaccharides 18 Intracellular hyaluronan 19

Hyaladherins 19 Hyaluronan receptors 19 Hyalectans 20

Hyaluronan in tissue growth 21 Hyaluronan in the cardiovascular system 22

Cardiac development 22 Myocardial infarction 23 Cardiac vessels 23

Aims 24 Presentation of papers 25 Methodology 26

Rat hypertrophy model 26 Cell culture 26 Stimulation of cells 26 RNA preparation 27 DNA preparation 27 Real-time PCR 27 Hyaluronan purification 28

Table of contents

5

Quantitative analysis of hyaluronan 29 Histochemistry staining for hyaluronan and CD44 29 Dynamic Light Scattering size analysis of hyaluronan 29 Microarray gene expression analysis 30 Microarray gene expression data analysis 30 Correlation between hyaluronan concentration and gene transcription 31 Preparation of microvesicles 31 Cross-talk between cardiomyocytes and fibroblasts 32 Hyaluronan stimulation of cardiomyocytes 32 Flow cytometry analysis 33 Electron microscopy 33 Identification of microvesicular contents 33 Microvesicular DNA transfer into target fibroblasts 34 Microvesicular induced effects on target cells 34

Results 35 Paper I and II: 35

General characteristics 35 Quantitative RT-PCR 35 Quantification of hyaluronan in heart tissue 35 Histochemical analysis of hyaluronan and CD44 36 Microarray gene expression 36

Paper III: 37 Quantitative analysis of hyaluronan in cell media 37 Real-time polymerase chain reaction analysis of hyaluronan synthases 37 Dynamic Light Scattering Size analysis of hyaluronan 37 Crosstalk between cardiomyocytes and fibroblasts 38 Hyaluronan stimulation of cardiomyocytes 38

Paper IV: 39 Preparation and characteristics of microvesicles 39 Identification of cardiosomal contents 39 Cardiosomal DNA transfer into target fibroblasts 39 Cardiosome induced effects on fibroblasts 39

Discussion 40 Conclusions 44 Sammanfattning på svenska 46 Acknowledgments 48 References 49


6

Abstract Background. Myocardial hypertrophy is a risk factor for cardiovascular morbidity and

mortality. Independent of underlying disease, the cardiac muscle strives in different ways to

compensate for an increased workload. This remodelling of the heart includes changes in the

extracellular matrix which will affect systolic and diastolic cardiac function. Furthermore, signal

transduction, molecular diffusion and microcirculation will be affected in the hypertrophic

process. One important extracellular component is the glycosaminoglycan hyaluronan. It has

been shown to play a major role in other conditions that feature cellular growth and

proliferation, such as wound healing and malignancies. The aim of this thesis was to investigate

hyaluronan and its role in both an experimental rat model of cardiac hypertrophy as well as in

cultured mouse cardiomyocytes and fibroblasts.

Methods. Cardiac hypertrophy was induced in rats by aortic ligation. Hyaluronan

concentration was measured and expression of genes coding for hyaluronan synthases were

quantified after 1, 6 and 42 days after operation, in cardiac tissue from the left ventricular wall.

Localization of hyaluronan and its receptor CD44 was studied histochemically. Hyaluronan

synthesis was correlated to gene transcription using microarray gene expression analysis.

Cultures of cardiomyocytes and fibroblasts were stimulated with growth factors. Hyaluronan

concentration was measured and expression of genes coding for hyaluronan synthases were

detected. Hyaluronan size was measured and crosstalk between cardiomyocytes and fibroblasts

was investigated.

Results. Increased concentration of hyaluronan in hypertrophied cardiac tissue was

observed together with an up-regulation of two hyaluronan synthase genes. Hyaluronan was

detected in the myocardium and in the adventitia of cardiac arteries whereas CD44 staining was

mainly found in and around the adventitia. Hyaluronan synthesis correlated to the expression of

genes, regulated by transcription factors known to initiate cardiac hypertrophy. Stimulation of

cardiomyocytes by PDGF-BB induced synthesis of hyaluronan. Cardiomyocytes also secreted a

factor into culture media that after transfer to fibroblasts initiated an increased synthesis of

hyaluronan. When stimulated with hyaluronan of different sizes, a change in cardiomyocyte

gene expression was observed. Different growth factors induced production of different sizes of

hyaluronan in fibroblasts. The main synthase detected was hyaluronan synthase-2.

Cardiomyocytes were also shown to secrete microvesicles containing both DNA and RNA.

Isolated microvesicles incubated with fibroblasts were observed by confocal microscopy to be

internalized into fibroblasts. Altered gene expression was observed in microvesicle stimulated

fibroblasts.

Conclusion. This study shows that increased hyaluronan synthesis in cardiac tissue during

hypertrophic development is a part of the extracellular matrix remodelling. Cell cultures

revealed the ability of cardiomyocytes to both synthesize hyaluronan and to convey signals to

fibroblasts, causing them to increase hyaluronan synthesis. Cardiomyocytes are likely to express

receptors for hyaluronan, which mediate intracellular signalling causing the observed altered

gene expression in cardiomyocytes stimulated with hyaluronan. This demonstrates the extensive

involvement of hyaluronan in cardiac hypertrophy.

List of papers

7

List of papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I. Hellman U, Hellström M, Mörner S, Engström-Laurent A, Åberg AM, Oliviero P, Samuel JL, Waldenström A. Parallel up-regulation of FGF-2 and hyaluronan during development of cardiac hypertrophy in rat. Cell Tissue Res. 2008 Apr;332(1):49-56. Epub 2008 Jan 15.

II. Hellman U, Mörner S, Engström-Laurent A, Samuel J-L,

Waldenström A. Temporal regulation of extracellular matrix genes in experimental cardiac hypertrophy. Genomics. 2010 Apr 21. [Epub ahead of print]

III. Hellman U, Malm L, Ma L-P, Larsson G, Mörner S, Fu M, Engström-

Laurent A, Waldenström A. Hyaluronan is both a product and stimulator of cardiomyocytes: A study in cell cultures of cardiomyocytes and fibroblasts. Manuscript: Submitted

IV. Hellman U, Ronquist G, Waldenström A. Cardiomyocyte

microvesicles convey bioinformatic messages to target cells. Manuscript


8

Abbreviations

ABCC5/MRP5 ATP-binding cassette, subfamily C, member 5/

multidrug resistance transporter protein

ABCC7/CFTR ATP-binding cassette, subfamily C, member

5/ cystic fibrosis transmembrane conductance

regulator

ACE angiotensin-converting enzyme

ACTS/Acta1 alpha 1 skeletal muscle actin

α-MHC/Myh6 alpha-myosin heavy chain

AngII angiotensin II

ANP/Nppa atrial natriuretic peptide

AO acridin orange

ATP adenosine triphosphate

BGN biglycan

β-MHC/Myh7 β-myosin heavy chain

BNP/Nppb B-type natriuretic peptide

CCN1/CYR61 cystein rich protein 61

CCN2/CTGF connective tissue growth factor

CRTL1 cartilage link protein

DAPI 4'-6-Diamidino-2-phenylindole

DMEM Dulbecco’s modified Eagle’s medium

DLS Dynamic Light Scattering

ECM extracellular matrix

EGR1 early growth response 1

ET1 endothelin 1

ERBB erythroblastic leukemia viral oncogene

ERK1 extracellular signal-regulated kinase1 (MAPK3)

ERK2 extracellular signal-regulated kinase2 (MAPK1)

FBN1 fibrillin 1

FGF2 fibroblast growth factor 2

FGFR1 FGF receptor 1

FOS FBJ murine osteosarcoma viral oncogene

homolog

GAG glycosaminoglycan

Gapdh D-glyceraldehyde-3-phosphate dehydrogenase

GlcUA D-glucuronic acid

GlcNAc D-N-acetylglucosamine

GPCR G-protein-coupled receptor

GPI glycophosphatidylinositol

HA hyaluronan

Abbreviations

9

HAS hyaluronan synthase

HAPLN HA and proteoglycan link protein

HARE HA receptor for endocytosis

HBW heart-to-body weight

HYAL hyaluronidase

IEG immediate early genes

IL-1B interleukin-1β

JAK janus kinase

JUNB jun B proto-oncogene

LTBP2 latent transforming growth factor binding

protein 2

LYVE-1 lymphatic vein endothelium receptor-1

MAPK mitogen-activated protein kinase

MEF2 myocyte enhancer factor 2

MWCO molecular weight cut off

MYC myelocytomatosis viral oncogene homolog

NFAT nuclear factor of activated T-cells

NFκB nuclear factor κB

PDGF-BB platelet-derived growth factor BB

PI3K phosphatidyl inositol 3-kinase

RAS rat sarcoma viral oncogene

RAS renin angiotensin system

RHAMM receptor for HA-mediated motility

SP1 specificity protein 1

SPAM1 sperm adhesion molecule 1

SRF serum response factor

STAT Signal Transducer and Activator of

Transcription

TGFB transforming growth factor beta

TP53 tumour protein Tp53

UDP uridine-diphosphate


10

Introduction

The heart An adult human inhales 6-8 litres of air per minute. This adds up to more

than 6 billion tonnes of oxygen inhaled by all humans per year, a lot of it

used in the oxidative phosphorylation to form adenosine triphosphate (ATP)

in the mitochondria. The oxygen is distributed through the circulatory

system, to all tissues and cells in the body, by the heart. After the oxygenated

blood from the lungs is collected in the left atrium it passes to the left

ventricle, which pumps it out throughout the body. The de-oxygenated blood

is collected in the right atrium and is via the right ventricle pumped to the

lungs, where carbon dioxide is exchanged for oxygen. This continuous blood

flow must be maintained for many decades, which make a flawless heart

performance vital. To be able to meet changes in workload, the heart is a

dynamic organ that can grow and change in response to altered demands.

Cardiac hypertrophy The word “hypertrophy” is derived from the Greek hyper (above, more

than normal) and trophe (nutrition) and is defined as “the enlargement or

overgrowth of an organ or part due to an increase in size of its constitute

cells”1.

Figure 1. Cardiac response to physiological and pathological stimuli. Principal adaptive

physiological growth with maintained cardiac function and maladaptive pathological growth.

Introduction

11

Increased cardiac workload will lead to an enlargement of the heart in an

attempt to manage the hemodynamic demand. The growth is an adaptive or

maladaptive response to physiological or pathological stimuli (figure 1).

In healthy individuals cardiac growth may occur during chronic exercise,

pregnancy and maturation. This is commonly referred to as physiological

hypertrophy but since this is a normal cardiac growth it has been proposed

to be defined as adaptive (figure 2)2. Growth during exercise and pregnancy

is reversible without any adverse effect on cardiac function.

Figure 2. Proposed criteria for

classifying cardiac

hypertrophy2.

Pathological cardiac hypertrophy is induced by stress signals, e.g. long

standing hypertension, neurohormonal activation, myocardial infarction,

valvular heart disease, hypertrophic and dilated cardiomyopathy. This

conditions may initially be adaptive to normalize wall stress and preserve

contractile performance3. However sustained myocardial hypertrophy

predisposes the individual to heart failure, arrhythmia and sudden death4.

Pathological cardiac hypertrophy has a severe prognosis and is a predictor of

progressive heart disease. It is a common condition which makes it a major

health problem worldwide.

Based on the ratio of left ventricular wall thickness to left ventricular

chamber dimension, hypertrophic growth develops in two ways. Concentric

hypertrophy is caused by chronic pressure overload in which addition of

contractile sarcomeres in parallel results in increased cell and wall thickness

with a reduced left ventricular volume. Eccentric hypertrophy is mainly due

to volume overload in which addition of sarcomeres in series cause cell


12

elongation, dilation and sometimes thinning of the heart wall. In both types

of growth development, the increase in cardiomyocyte size and

cardiomyocyte disarray is accompanied by an increase in the number of

cardiac fibroblasts, causing fibrosis and increased myocardial stiffness5. This

is followed by further hemodynamic overload and hypertrophy resulting in a

detrimental cycle of cardiac enlargement and myocyte loss. This can

contribute to diastolic dysfunction and predisposition to arrhythmias6.

Increase and activation of fibroblasts with subsequent fibrosis have been

associated to increase of circulating hormones such as angiotensin II (Ang

II) and endothelin 1 (ET1) and cytokines/proteins such as transforming

growth factor β TGFB), connective tissue growth factor CCN2/CTGF) and

platelet-derived growth factor (PDGF)7.

Fetal gene program

After birth, when the heart is exposed to an oxygen rich environment,

there is a shift of substrates for energy provision from lactate and glucose

oxidation to fatty acid oxidation8. This shift is accompanied by expression of

“adult” isoforms of proteins. However, the ability to re-activate the fetal gene

program is preserved in the heart9. The heart senses hypertrophy-inducing

stimuli either directly through biomechanical stretch sensitive receptors, like

the integrins, or neurohormonal stimuli, like angiotensin II (AngII),

endothelin-1 and adrenalin. This leads to the expression of immediate early

genes (e.g. Jun, Fos, Myc, Egr1) and genes considered to be markers for the

fetal gene program, Nppa, Nppb, Myh7 and Acta1 (coding for atrial

natriuretic peptide (ANP), B-type natriuretic peptide (BNP), β-myosin heavy

chain (β-MHC) and α1 skeletal muscle actin (ACTS)). Hormones, cytokines,

chemokines and peptide growth factors in the circulation or the extracellular

matrix interacts with G-protein-coupled receptors (GPCRs), tyrosine kinase

receptors, serine/threonine receptors and gp130 linked receptors, thus

activating intracellular signalling pathways. Intracellular transduction

through phosphorylation and dephosphorylation events are mediated by an

array of pathways, e.g. the mitogen-activated protein kinase (MAPK)

pathway, the calcineurin-nuclear factor of activated T-cells (NFAT) circuit,

the phosphatidyl inositol 3-kinase (PI3K)-Akt pathway, the renin

angiotensin system (RAS) and the JAK-STAT pathway (Janus Kinase - Signal

Transducer and Activator of Transcription)10.

A number of transcription factors coordinate cardiac development and

differentiation of myocytes. Many of these, such as myocyte enhancer factor

2 (MEF2), GATA4, NFAT, serum response factor (SRF), Nkx2.5, nuclear

factor κB (NFκB), Hand1/2, EGR1 and CREB are re-activated during cardiac

Introduction

13

hypertrophy by the signal transduction pathways mentioned above. Their

involvement in the fetal gene program is exemplified by GATA4, which is a

transcriptional regulator of ANP, BNP, α-MHC and β-MHC11, 12. EGR1 has

also been shown to be a transcriptional mediator of cardiac hypertrophy13.

Figure 3. General flowchart over signal

transduction in the process of

cardiomyocyte hypertrophy.

Pressure and/or volume overload, will through extracellular factors via

intracellular pathways that affect gene expression and protein translation,

ultimately alter myocyte shape to adopt to the increased burden of the heart

(figure 3).

Intercellular signalling The myocardial cells can receive signals in several different ways. One

way is through direct contact with another cell, where the signal molecule is

bound to the membrane of the signalling cells or with gap-junction

connexons, where the cytoplasms of the two cells are connected through a

continuous aqueous channel.

Cells can also secrete signal molecules. This includes paracrine signalling,

which only affects local cells, and endocrine signalling, where endocrine cells

secrete hormones into the bloodstream, thus transmitting the signal

throughout the body.

Intercellular communication can also be facilitated by exocytosis of

membrane microvesicles called exosomes. They are about 40-100 nm in


14

diameter and are secreted when multivesicular bodies, containing exosomes,

fuse with the cell membrane and release their content into the extracellular

environment. Exosomes can be released by most cell types in vitro and they

have also been found in vivo in several body fluids. Exosomes can contain

proteins, RNA, microRNA and DNA14-16. Both mRNA and microRNA have

been shown to be functional after transfer to target cells and protein content

may be linked to functions associated with the originating cell-type.

Detection of tumor cell-derived exosomes in blood, with tumor cell

biomarkers, suggests a role of exosomes in diagnosis and therapeutic

decisions15.

Extracellular matrix Different tissues differ not only by their cell types. Their extracellular

environment is built up by a complex network of proteins and

polysaccharides constituting the extracellular matrix (ECM). Cells synthesize

and secrete the components locally, where they assemble in a network.

Glycosaminoglycans (GAGs) and fibrous proteins (e.g. collagens,

fibronectin, fibrillin, laminin, elastin) are the most abundant classes of

molecules in the ECM. The diverse forms of tissues are enabled by variations

of the ECM composition. It varies from rock hard to soft and transparent and

the ECM can be the major component, as in cartilage, or a minor component,

as in the brain. The ECM serves as physical structure and a scaffold for cell

adhesion and cell movements. It also transduces signals into cells, thus

regulating cellular functions and can bind soluble growth factors, regulating

their distribution, activation and presentation to cells17.

Glycosaminoglycans GAGs are highly negatively charged, unbranched polysaccharides

consisting of repeating disaccharide units. The members of the GAG family

are defined according to their disaccharide structure and number and

location of sulphate groups; hyaluronan, chondroitin sulphate, dermatan

sulphate, heparin sulphate and keratin sulphate. GAGs form gels at very low

concentrations and are highly osmotic due to their negative charge and

attract large amounts of water. This enables the ECM to withstand

compressive forces. All GAGs except HA are attached to a core protein,

forming proteoglycans. Proteoglycans have an important role in cell-cell

signalling. They can bind growth factors, proteases and protease inhibitors

and thus block the activity of the protein, provide a reservoir of the protein

for delayed release and present the protein to cell surface receptors.

Introduction

15

Hyaluronan In 1934, Karl Meyer and John Palmer isolated from bovine vitreous

humour a previously undescribed polysaccharide18. They named it

hyaluronic acid from hyaloid (vitreous) and uronic acid. However, in 1894,

Carl Thore Mörner isolated a “mucin” from the vitreous humour, presumably

consisting of protein contaminated hyaluronan19. The name hyaluronan

(HA) was introduced in 1986. HA is the only GAG that is unsulphated and

not forming proteoglycans by binding to a core protein. HA is present in all

vertebrates and most tissues differentiate in an ECM where HA is a major

constituent. It can also be found in the pericellular coat called glycocalyx.

Skin tissue harbours about 50% of the total amount of HA in the body and it

is also a major part of, for example, the vitreous humour of the human eye

and synovial joint fluid20.

Structure

HA is an unbranched polysaccharide which assumes a stiffened random

coil in solution. It is a polydisperse population of molecules at varying chain

lengths, occupying a large hydrated volume, up to 1000 times greater than

its own dry volume. HA is soluble up to very high concentrations and shows

no evidence of chain-chain association21. The chemical structure of repeating

D-glucuronic acid and D-N-acetylglucosamine disaccharides was solved in

the 1950s (figure 5)22. The number of repeated disaccharides can reach 105,

representing a molecular mass of ~20,000 kDa23.

Figure 4. Hyaluronan structure. HA is composed of repeating disaccharides of D-glucuronic acid

(GlcUA) and D-N-acetylglucosamine (GlcNAc) linked by a glucuronic β(13) bond. The

disaccharide units are then linearly polymerized by hexosaminidic β(14) linkages.


16

Hyaluronan synthesis and catabolism

Hyaluronan synthesis

Most cell types that have been investigated can synthesize HA. One

exception is erythrocytes and the ability of cardiomyocytes to synthesize HA

has never been investigated.

In contrast to the other GAGs, which are synthesized and covalently

linked to core proteins in the Golgi apparatus and secreted by exocytosis, HA

is synthesized on the cytosolic side of the cell membrane. In mammals there

are three hyaluronan synthases (HASs) with multiple transmembrane

domains, which utilize UDP-glucuronic acid (uridine-diphosphate) and

UDP-N-acetylglucosamine to assemble the HA chain24. The growing chain is

transported through the cell membrane into the extracellular space. It have

been shown that the transporter proteins ATP-binding cassette, subfamily C,

member 5 (ABCC5/MRP5) and member 7 (ABCC7/CFTR) possess the ability

to translocate HA to the ECM through the cell membrane25-27. Atomic force

microscopy observations of HA have revealed its capability to form many

different conformations, e.g. extended chains, relaxed coils, condensed

rods28. HA can also form fibers/cables, networks and stacks through self-

association.

Hyaluronidases

HA is degraded by a group of enzymes called hyaluronidases (HYALs)

through hydrolysis. There are six HYAL-like sequences found in the human

genome. Hyal1, 2 and 3 are clustered on chromosome 3p21.3. Hyal4, HyalP1

and Spam1 (sperm adhesion molecule 1) are clustered on chromosome

7q31.3. HyalP1 is a pseudogene, transcribed but not translated in humans.

SPAM1 codes for PH-20, the enzyme that facilitates penetration of a sperm

through the HA-rich cumulus mass surrounding the ovum, necessary for

fertilization29. In mouse, Hyal1 is often co-transcribed with Hyal3.

Three of the enzymes can be GPI (glycophosphatidylinositol) -linked to

the outer cell membrane, HYAL2, HYAL4 and PH-20, but they also exist in

free processed form. HYAL1, HYAL2 and PH-20 have known enzymatic

activities but no enzymatic activity of HYAL3 and HYAL4 on HA has been

detected, so far. However HYAL-4 have been shown to have hydrolytic

activity on chondroitin sulfate30.

HYAL1 and HYAL2 are the major HYALs in somatic tissue31. In a complex

with the HA receptor CD44 and HYAL2, HA is bound and then cleaved by

HYAL2 to 20kDa fragments, about 50 disaccharide units. The complex is

internalized in caveolin and flotillin rich caveolae, delivered to endosomes

Introduction

17

and then the HA fragments are degraded by HYAL1 in lysosomes to tetra-

and hexasaccharides followed by degradation to monosaccharides by

exoglycosidases (figure 5)31.

Figure 5. A putative metabolic

pathway of HA degradation.

HA is bound to CD44 and

internalized. Hyaluronidase-

2, -1 and exoglycosidases are

subsequently degrading HA to

monosaccharides.

Hyaluronan turnover

It has been estimated that almost a third of all HA in the human body is

degraded in 24 h. The half time of HA in the blood is between two and five

minutes. In the epidermis it is between one and two days and in cartilage

about one to three weeks32, 33. A large proportion of HA is captured by

receptors on reticulo-endothelial cells in lymph nodes and in the liver, which

internalize and degrade HA in lysosomes.

Regulation of hyaluronan synthesis

The HASs differs in enzymatic properties. HAS3 is the most catalytically

active, followed by HAS2, and then HAS 1, the least active 34. It has been

shown that they also differ in the size of HA synthesized. HAS3 synthesized

the smallest, 100-1000 kDa whereas HA synthesized by HAS1 and HAS2

ranged between 200-2000 kDA. It has later been shown that all HASs are

capable of synthesizing high molecular sizes of HA, depending on cell type

and regulatory factors35. The HASs are using a cytosolic pool of substrate and

synthesis may be regulated by availability of the UDP-sugars36, 37. The level of

HA synthesis is also regulated by the expression of the HAS genes and the


18

subsequent translation of HAS proteins. HAS expression is regulated by

several growth factors and cytokines, such as PDGF-BB, TGFB, FGF2 and IL-

1B38-44.

Hyaluronan function

High molecular weight hyaluronan

High molecular size HA, defined as 400-2000 kDa (200-10000

saccharides), is extracellular and space-occupying. It has several regulatory

and structural functions. In the fluid of the joint capsule it is a lubricant and

shock absorber. In the vitreous humour of the eye, it is a space-occupying

material. It also functions in organizing the ECM23, 45, 46.

HA networks show a high resistance towards water flow, thus forming

flow barriers in tissue, even though water can freely diffuse in the network.

Movements of macromolecules are also hindered in a HA network, whereas

low-molecular weight molecules can diffuse more easily. The HA chains are

constantly moving in the solution and thus, the pore sizes in the network

change, allowing molecules to pass with different degrees of retardation

depending on their volume.

High molecular HA has been shown to be anti-inflammatory and

immunosuppressive47, 48. It also suppresses angiogenesis and synthesis of

HA49, 50.

Low molecular weight hyaluronan

Low molecular size HA ranges between 10 kDa and 500 kDa. These

molecules share their biological functions with HA oligosaccharides, such as

induction of NFκB, increase of cyto- and chemokines and increase of nitric

oxide51.

Hyaluronan oligosaccharides

HA sizes up to 10 kDa refers to as oligosaccharides, which have many

novel functions that are not shared by either low or high molecular weight

HA52. When HA oligosaccharides of various sizes bind to cell surface

receptors, such as CD44 and RHAMM (receptor for HA-mediated motility),

they mediate intracellular signal transduction pathways which affect gene

expression and cell function. In several cases they suppress the actions of

high molecular HA, e.g. they stimulate angiogenesis and can induce an

inflammatory response53-55. HA oligosaccharides are also found in most

malignancies, where they facilitate tumour cell motility and invasion56.

Introduction

19

It is unknown if the smaller sizes of HA is only derived from degradation

of high molecular HA or if the HASs can be regulated to synthesize them.

Intracellular hyaluronan

HA seems to be internalized for more reasons than to be degraded. HA

has been detected in various cell types, e.g. smooth muscle cells, endothelial

cells, eptithelial cells and fibroblasts. The variation of morphology and

distribution suggests different purposes for HA inside the cells. It has been

detected both in the cytoplasm and the nucleus. Intracellular HA may be

involved in growth regulation and mitosis57, 58. The increase of both

extracellular and intracellular HA is simultaneous during mitosis and

proliferation. How HA is transported to the cytoplasm and nucleus is

unknown, however the possibility that it is synthesized and deposited

directly into the cytoplasm, instead of being extruded to the outside of the

cell, has not been completely ruled out.

Hyaladherins

HA can bind to different proteins. These proteins with HA binding

domains have been named hyaladherins. Differences in their tissue

expression, cell localisation and regulation explain how HAs simple structure

can display such a wide range of functional activities. Most of the HA

receptors and binding proteins are members of the link module

superfamily59. These HA receptors have one link module and all other link

module superfamily members possess two link modules in tandem.

Hyaluronan receptors

HA-receptor interactions mediate at least three important physiological

processes; signal transduction, HA internalization and pericellular matrix

assembly60, 61. There are four HA cell surface receptors, mediating

intracellular signalling, with the link module motif; lymphatic vein

endothelium receptor-1 (LYVE-1), stabilin-1, stabilin-2 and CD44.

LYVE-1, a homologue to CD44 with unique transmembrane and

cytoplasmic domains, is expressed by lymphatic vein endothelium and

internalizes HA62.

Stabilin-1 has a possible role in angiogenesis63. Stabilin-2, or HARE, (HA

receptor for endocytosis) clears HA from vascular and lymphatic

circulations64.

CD44 is a transmembrane glycoprotein, coded in humans by a gene with

19 exons. Exons 6-14 are alternatively spliced and can generate a multitude

of variant CD44 isoforms65, 66. The most common form, CD44s/CD44H


20

includes none of the variant exons67 but all isoforms contain the HA-binding

link module. CD44 has been shown to be involved in many biological

functions, e.g. retention and endocytosis of HA, angiogenesis, tumour

invasion and metastasis, adhesion and rolling of lymphocytes and also cell

migration68. The main ligand of CD44 is HA, however collagen, fibronectin,

osteopontin and several other molecules can also bind to CD4466.

RHAMM (receptor for HA-mediated motility) is a cytoplasmic protein

with the ability to translocate outside the cell via unknown routes. As an

extracellular HA-binding protein, RHAMM contributes to normal wound

healing69, 70. The HA-CD44-RHAMM complex mediates, through the

intracellular domain of CD44, an increased expression of CD44 and

increased activation of ERK1/2, thus activating migration and invasion

functions71.

Hyalectans

Also parts of the link module superfamily are the hyalectans or lecticans,

a family of large aggregating chondroitin sulphate proteoglycans. They

consist of versican, aggrecan, neurocan and brevican and they all have the

HA-bindning link module motif at the N-terminal of their core protein72, 73.

The middle part of the core protein is a GAG-binding region.

The hyalectan binding to HA is stabilized by a link protein, also

possessing the link module motif, which binds HA and another part binds

the hyalectan. There are four members in the HA and proteoglycan link

protein (HAPLN) family (HAPLN1-4). HAPLN1, or cartilage link protein

(CRTL1) is predominantly expressed in cartilage. HAPLN2 and HAPLN4 are

expressed in the brain and central nervous system and HAPLN3 is widely

expressed74.

Versican is the hyalectan that is the most versatile in structure and tissue

distribution. Versican binds many other molecules than HA and in vitro

studies suggest that versican is involved in cell adhesion, proliferation and

migration, thus playing a role in development and maintenance of the

ECM75, 76.

Aggrecan and HA form huge complexes in the chondrocyte ECM (figure

6), providing cartilage with its load bearing properties. Some of these

aggregates are bound to the cell surface via CD4477.

Neurocan and brevican are expressed in brain and the nervous system

and found in HA complexes in the ECM78.

Introduction

21

Figure 6. Extracellular matrix aggregates of HA and proteoglycans.

Hyaluronan in tissue growth

The ability of HA to co-regulate cell behaviour during embryonic

development, healing processes, inflammation and tumour development

makes HA very important in tissue growth. HA concentration and

organization changes when tissues and organs differentiate. Cells divide and

migrate in an ECM rich in HA79, 80. HA promotes proliferation by providing a

hydrated pericellular zone that enables cell rounding during mitosis.

Inhibition of HA synthesis leads to cell cycle arrest at mitosis before cell

rounding and detachment57.

HA concentration may be increased in malignant tumours compared to

benign and normal tissue. In some tumours HA levels are predictive of

malignancy81. Malignant cells have the ability to survive in anchorage-

independent conditions in which normal cells would undergo apoptosis82.

Cells require cell-survival signals from growth factors and ECM components,

such as fibronectins, laminins and collagens, mediated through integrins.

HA strongly promotes anchorage-independent growth83-86 and malignant

cells resistance to growth arrest and apoptosis is dependent on HA-CD44

interaction87, 88. Through remodelling of the ECM, a growth-adapted cellular

environment can be created that induces cells for survival and proliferation.

Increased HAS expression and subsequent HA synthesis caused increased

metastasis formation or growth of tumours in xenograft models of

fibrosarcoma, prostate, colon and breast cancer83, 89-91. In contrast, reduced

HAS levels suppresses tumour growth84, 92.

Another example of tissue growth where HA is involved is wound

healing93 and there are more similarities between wounds and tumours.

Tumours, in particular carcinomas activate a latent wound healing program


22

but in a prolonged and over expressed manner. Most genes that regulate the

wound healing process are also important regulators of cancer growth94.

In adult wound healing, tissues are restored but with the addition of

fibrosis and scar. In contrast, fetuses heal skin wound without leaving a scar.

The fetal wound matrix contains huge amounts of HA which remains longer

than in adult wounds. There are also more HA receptors on fetal cells than

on adult cells. This might cause the fetal wound matrix to be more

permissive for fibroblast migration and thus accelerate repair and avoid

fibrosis and scar tissue95.

Hyaluronan in the cardiovascular system

Cardiac development

The retinoic acid and the neuregulin/ERBB (erythroblastic leukemia viral

oncogene) signalling are two major pathways involved in formation of the

trabeculated myocardium during cardiac development. The nuclear retinoic

acid receptor has been found to be a transcription factor regulating the Has2

gene96. Mice embryos without Has2 expression (Has2-/-) synthesize no HA

and display severe cardiac and vascular abnormalities, caused by impaired

transformation of the cardiac endothelium to mesenchyme, and die during

embryonic development97. This phenotype is rescued by activating the ERBB

signalling pathway, either through addition of exogeneous HA or active RAS

(rat sarcoma viral oncogene). This shows that for a normal cardiac

development, HA signalling mediated through the ERBB and RAS signalling

pathways is necessary98, 99.

HA signalling also mediates MAP3K1 (MEKK1) phosphorylation by

CD44, which subsequently activates both ERK1/2 (extracellular signal-

regulated kinase1, MAPK3/extracellular signal-regulated kinase2, MAPK1)

and NFκB. This induces differentiation in epicardial cells, which plays an

important role in the formation of coronary vasculature100.

HA demonstrates an active role in regulation of cardiac development and

analysis of HAPLN1, and two versican deficient mouse models demonstrate

similar cardiac malfunctions in the models101. This suggests that a complete

HA-HAPLN1-versican complex is necessary for normal cardiac development.

Endocardial cushion development and remodelling of the atrioventricular

septal complex during cardiogenesis is regulated by CCN1/CYR61 (cystein

rich protein 61) and CCN2/CTGF (connective tissue growth factor) and HA-

induced signalling102.

HYAL2-deficient mice develop right or left atrium dilation and

enlargements of valves. Left ventricular cardiomyocytes displayed significant

Introduction

23

hypertrophy in acutely affected mice. This demonstrates the importance HA

degradation in the heart103.

Neonatal CD44 null mice have shown the necessity of CD44 for

development of right ventricular hypertrophy induced by hypoxia, indicating

a role of CD44 in the hypertrophic process104.

Myocardial infarction

The increased presence of HA after myocardial infarction has been shown

in rats indicating involvement of HA in the healing process105. Injection of a

HA-based hydrogel into the epicardium of the infarcted area in rat hearts,

increased wall thickness, reduced infarcted area, increased vasculature and

improved cardiac function was achieved, compared to controls106.

Cardiac vessels

As mentioned above, short HA fragments have been found to induce

angiogenesis, via induction of endothelial cell proliferation in contrast to

high molecular size HA107. The localisation of HA in the vessels differ.

Arteries and veins differ in localisation of HA in the vessel walls and the

vessels of newborn rats contain more HA compared to adult rats108. In adult

human aorta, HA is found in all three wall layers109.

Aims

24

Aims

The overall aim of this thesis was to investigate if the GAG hyaluronan is

part of the hypertrophic process in the heart.

The aims were:

To investigate the temporal expression of the genes coding for the

HASs, the growth factor FGF2 and their receptors CD44 and

FGFR1, in an experimental rat model of cardiac hypertrophy.

To study the correlation between myocardial HA concentration

and gene expression over time.

To investigate if cardiomyocytes are capable of synthesizing HA.

To study possible cell signalling between fibroblasts and

cardiomyocytes leading to HA synthesis and if so, in which

manner the signal is transduced.

To study if the size of the HA produced can be related to its

biological effects.

To investigate if cardiomyocytes posses intercellular

communication capability via exocytosis of exosomes.

Methodology

25

Presentation of papers

Paper I

In this study the expression of the genes coding for HAS 1, 2, 3, CD44,

fibroblast growth factor 2 (FGF2) and FGF receptor1 (FGFR1) as well as

histological evidence for an increase of HA and CD44 were investigated in an

experimental rat model of cardiac hypertrophy.

The abdominal aorta was banded to induce cardiac hypertrophy and

mRNAs, prepared from heart tissue, were analysed after 1, 6 and 42 days.

Total concentration of myocardial HA was quantified. HA and CD44 were

investigated histochemically.

Paper II

To further investigate the role of hyaluronan and regulation of its

synthesis in the same experimental rat model of cardiac hypertrophy as in

paper I, quantitative measurements of myocardial hyaluronan concentration

was correlated to gene transcription in hypertrophic cardiac tissue. Factor

analysis was used to study this correlation over time.

Paper III

Cardiomyocytes capability to synthesize HA was studied and as well as if

the different HA synthases are activated in the different cell types.

Mouse cardiomyocytes (HL-1) and fibroblasts (NIH 3T3) were cultivated

in absence or presence of the growth factors FGF2, PDGF-BB and TGFB2.

HA concentration was quantified and the size of HA was estimated using

dynamic light scattering. Cardiomyocytes were also stimulated with HA to

detect presence of HA receptors mediating intracellular signalling which

affect gene transcription.

Paper IV

Microvesicles from media of cultured cardiomyocytes derived from adult

mouse heart were isolated by differential centrifugation and preparative

ultracentrifugation, followed by characterization with transmission electron

microscopy and flow cytometry.

Identification of microvesicular mRNA and DNA was performed and

transfer of cardiac microvesicles into target fibroblasts was analyzed with

gene expression analysis and confocal microscopy.


26

Methodology

Rat hypertrophy model

To obtain tissue from hypertrophied hearts, a cardiac hypertrophy rat

model was used. A titanium clip with 0.15 mm inner diameter was put

around the aorta just proximal to the renal arteries. Age-matched control

rats were sham operated and subjected to exactly the same procedure except

for placing the clip around the aorta. The rats were sacrificed at 1, 6 and 42

days postoperatively. To determine if cardiac hypertrophy occurred in the

aortic banded animals the heart-to-body weight (HBW) ratio was calculated.

The hearts were excised after pentobarbital anaesthesia and immediately

washed in NaCl 0.9%, weighed and placed in liquid nitrogen or RNAlater

(Qiagen). (Paper I, II)

Cell culture

HL-1, a cell line derived from adult mouse heart, displaying phenotypic

features typical of adult cardiomyocytes110, was used. Cardiomyocytes plated

in T-75 flasks coated with fibronectin (Sigma)-gelatin (Fisher Scientific) were

maintained in Claycomb Medium (JRH. Biosciences). During culture, the

medium was changed routinely every 24 h.

Fibroblasts, NIH 3T3 cells111 (LGC Standards AB) were cultured and

passaged following the standard procedure in Dulbecco’s modified Eagle’s

medium (DMEM, Fisher Scientific).

HL-1 and NIH3T3 cells were passaged twice per week. All cultures were

kept in an atmosphere of 95% air-5% CO2, 37ºC and at a relative humidity of

approximately 95%. (Paper III, IV)

Stimulation of cells

NIH 3T3 cells alone and HL-1cells alone were plated at a

concentration of 0.3106 cells/mL into 6-well plates. In a monolayer co-

culture of HL-1 with NIH 3T3, 0.24106 HL-1 cells/mL were mixed with

0.06106/mL of NIH 3T3 cells to a 6-well plate. After 72 h, cells were grown

to confluence and all media were replaced with serum-free and antibiotic-

free media for 24 h. In the treatment groups, cells were then stimulated with

FGF2 (5ng/mL, 10ng/mL), PDGF-BB (50ng/mL, 100ng/mL) and TGFB2

(5ng/mL, 10ng/mL) (Biosource, Invitrogen), respectively, six replicates of

each. Plates with no growth factor addition were used as controls. After 24 h,

the media from each growth condition was collected and the cells were then

harvested and placed in RNAlater (Qiagen). (Paper III)

Methodology

27

RNA preparation

For each rat group, total RNA was isolated from heart tissue from 6 aortic

banded rats and 6 sham operated rats, using the RNeasy Fibrous Tissue Kit

(Qiagen). (Paper I and II)

For each cell group, total RNA was isolated from three wells using the

RNeasy Mini Kit (Qiagen). (Paper III)

Total RNA was isolated with RNEASY Mini Kit (Qiagen) from

cardiomyocytes (n=2) and microvesicular pellets (n=2) prepared from 18 mL

Claycomb medium after 48 h incubation with cardiomyocytes. (Paper IV)

The concentration of the RNA was measured in a NanoDrop ND-1000

Spectrophotometer (NanoDrop Technologies Inc.) and the integrity of the

RNA was analyzed in a 2100 Bioanalyser (Agilent Technologies Inc.).

Omniscript RT Kit (Qiagen) was used to synthesize cDNA. (Paper I).

Aliquots of total RNA were converted to biotinylated double-stranded cRNA

according to the specifications of the Illumina Totalprep RNA Amplification

Kit (Ambion). (Paper II-IV)

DNA preparation

DNA was isolated with GenElute Mammalian Genomic DNA Miniprep

Kit (Sigma-Aldrich) from a microvesicular pellet prepared from 18 mL

Claycomb medium after 48 h incubation with cardiomyocytes. To add a poly-

T tail, DNA was incubated with 25 µL 100 mM dGTP (Gibco BRL, Life

Technologies) and terminal deoxynucleotidyl transferase (TdT) (Invitrogen)

for 30 min at 37ºC, according to manufacturer’s protocol. The constructed

cDNA was purified and transcribed to synthesize biotinylated cRNA with

Illumina Totalprep RNA Amplification Kit (Ambion). (Paper IV)

Real-time PCR

Relative quantitation of gene expression changes was performed using an

Applied Biosystems Prism 7900HT Sequence Detection System according to

the manufacturer’s specifications.

The gene-specific minor groove binder probes were FAM labelled and the

GAPDH-specific minor groove binder probe was VIC labelled. All samples

were run in triplicates and amplification were analyzed using Applied

Biosystems Prism Sequence Detection Software version 2.2-2.3 Relative

quantification was calculated according to the comparative CT method

(Applied Biosystems) using a statistical confidence of 99.9%. The amount of

target gene mRNA, normalized to an endogeneous control and relative to a calibrator, is given by 2-ΔΔCT. The gene expression fold change of the aorta


28

banded animals is the average 2-ΔΔCT value relative to the average 2-ΔΔCT value

for the sham operated animals. (Paper I-III)

Rat cDNA-specific TaqMan Gene Expression Assays for Has1, Has2,

Has3, CD44, Fgf2, Fgfr-1, Gata4, alpha-myosin heavy chain (Myh6) and

beta-myosin heavy chain (Myh7) from Applied Biosystems were used in

paper I. The rat D-glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene

was used as an endogeneous control. (Paper I)

Relative quantification of mRNA expression of four genes differentially

expressed was performed to confirm the validity of the microarray

expression data in paper II. Rat cDNA-specific TaqMan Gene Expression

Assays for Cspg2, Itgb1, Ccn2/Ctgf, and Tgfb2 from Applied Biosystems

were used in the study. The rat Gapdh gene was used as an endogeneous

control. (Paper II)

Mouse cDNA-specific TaqMan Gene Expression Assays for Has1, Has2

and Has3 from Applied Biosystems were used in paper III. The mouse

Gapdh gene was used as an endogenous control. (Paper III)

Hyaluronan purification

Heart tissue

The wet weight of the rat heart tissue samples was measured before being

dried in a rotary vacuum pump for 3 hours. Dry weights were measured, and

then the samples were homogenized. The samples were suspended in a

solution of pronase, 5 Units/ml (Pronase from Streptomyces griseus,

BioChemica, Fluka). Thereafter, 0.1 units protease per mg tissue was added

to each tissue sample before they were incubated in a water bath at 55ºC for

16 hours. The temperature was then raised to 100ºC, and samples were

boiled for 10 minutes. 50-100µl phenylmethanesulfonyl fluoride

(BioChemika, Fluka) was added, and samples were centrifuged for 15

minutes at 15,000 G. The supernatant was diluted 20-100 times in PBS.

(Paper I, II)

Cell media

Hyaluronan was purified from cell media of cultured cardiomyocytes and

fibroblasts prior to size analysis. Four mL of cell media were concentrated to

50 µL using 10 kDa cut-off Ultra Cell filter-unit (Millipore) by 15 min

centrifugation at 4,000 x g. A total digestion of proteins, present in the

growth medium, was achieved using pronase (Boehringer Mannheim) at a

concentration of 2 mg/mL in each sample. The protein digestion was allowed

to continue for 72 h at 40°C and subsequently quenched by increasing the

temperature to 100°C for 3 minutes. Digested peptides and amino acids, as

Methodology

29

well as CaCl2, were removed using a 10kDa cut-off Ultra Cell filter-unit by 3

x 15 min centrifugation at 4,000 x g.

Desalted HA samples were loaded on a DEAE-FF anion-exchange column

with a bead volume of 1 mL (GE-Life Science). HA was eluted from the

column with 8 column volumes of 0.4 M NaCl 20 mM TRIS-HCl pH 7.4

The eluted fraction was collected and concentrated to approximately 1.5

mL. The volume of the concentrated samples was further reduced to 30 µL

using a 0.5 mL 10 kDa cut-off Ultra Cell filter-unit. The buffer was changed

by 3 steps of 15 min centrifugation followed by dilution with 0.5 mL of 100

mM NaCl in D2O using the same concentration device. The purity of the

samples was controlled by SDS-page, for detection of residual proteins after

pronase digestion, and by NMR to detect any other impurities, such as lipids

and DNA. (Paper III)

Quantitative analysis of hyaluronan

HA samples purified from rat heart tissue and cell media from cell

cultures were analyzed for HA concentration using an enzyme-linked

binding protein assay (Corgenix). Absorbance was read at 450 nm with

correction at 650 nm on a spectrophotometer (Multiscan Ascent, Thermo

Labsystems). All analyses were performed with SPSS statistical analysis

package (version 13.0, SPSS Inc.). Data were expressed as mean±S.D.

Differences between two groups were compared using Mann-Whitney U test.

Statistical significance was set to P<0.05. (Paper I-III)

Histochemistry staining for hyaluronan and CD44

Hearts from the animals used for gene expression analysis were also used

for histochemical analysis with the addition of 2 hearts in the 6 and 42 days

groups. Approximately 2 mm thick slices were dissected just below the bi-

and tricuspid valves and fixed in glutaraldehyde, dehydrated and embedded

in paraffin112. The HA and CD44 stainings were performed on serial sections.

For the localization of HA, the same hyaluronan binding protein probe as in

the quantification analysis, was used (Corgenix)113. For the localisation of

CD44 a purified mouse anti-rat (Pgp-1, H-CAM) monoclonal antibody

(Pharmingen) was used. All slides were blindly evaluated by two of the

authors. For photo documentation a Canon EOS 10D camera and a 100 mm

macro lens with accessories were used (Canon Inc.). (Paper I)

Dynamic Light Scattering size analysis of hyaluronan

The size of HA synthesized by fibroblasts, cardiomyocytes or a co-culture

of the two cells types was estimated by Dynamic Light Scattering (DLS). The


30

Z-average value, which is the mean hydrodynamic diameter weighted against

the intensity of the DLS signal, was used to estimate the mean hydrodynamic

size of the different HA molecules. Due to the hygroscopic properties of HA,

the hydrodynamic diameter of HA is highly dependent on the buffer and salt

conditions used in the DLS measurements. Therefore, the hydrodynamic

diameters of HA purified from cell media were compared with those of HA

with known molecular weights of 70 kDa, 450 kDa and 2420 kDa (Hyalose,

L.L.C.) recorded at identical conditions.

Each HA sample was intensively shaken at 4oC for 20 minutes, to dissolve

possible entanglements. To sediment remaining entanglements, the samples

were centrifuged for 20 minutes at 14,000 rpm, 50 µL of sample was added

into a disposable low volume cuvette with a 10 mm path length. DLS

measurements were conducted at 20o C using a Nano Zetasizer (Malvern

Instruments) equipped with a HeNe-laser with a wave length of 633 nm.

Before each measurement the temperature was allowed to equilibrate for 10

minutes. The back scattered light was detected at an angle of 173 degrees.

For each cell condition the light scattering was measured for 200 seconds

with 10 replicate measurements. The DLS data was analyzed using the

Dispersion Technology Software v.5.10 (Malvern). Z-average, the mean

intensity weighted diameter, was collected for each HA sample. Mean values

and standard deviations from each measurement were calculated and

compared using GrapPad Prism v.5. Outliers in Z-average were detected and

rejected by calculation of the Dixon’s Q ratio, using P = 0.05114. (Paper III)

Microarray gene expression analysis

Aliquots of total RNA were converted to biotinylated double-stranded

cRNA with the Illumina Totalprep RNA Amplification Kit (Ambion). The

labelled cRNA samples were hybridized to RatRef-12 Expression Beadchip

(paper II), MouseRef-8 v2 (paper III, IV) (Illumina, San Diego, CA, USA),

incubated with streptavidin-Cy3 and scanned on the Illumina Beadstation

GX (Illumina, San Diego, CA, USA). (Paper II-IV)

Microarray gene expression data analysis

To determine differentially expressed genes, microarray data were

analyzed using Illumina Beadstudio software (version 3.2.1). Intensity data

were normalized using Beadstudios cubic spline algorithm. A number of

filtering steps were applied to avoid false positives. Significant differential

expression was calculated using the Beadstudio software by applying

multiple testing corrections using Benjamini and Hochberg False Discovery

Rate (FDR) 115, 116. The gene expression fold change was calculated as the

average signal value of aorta ligated animals/stimulated cells relative to the

Methodology

31

average signal value of the sham operated animals/control cells. A significant

up-regulation was defined as a foldchange ≥1.5 and a significant down-

regulation was defined as foldchange ≤0.67. Statistical significance was set

to P < 0.05. To avoid selecting genes with high foldchange due to low signal

intensity a minimum signal intensity value was utilized. For up-regulated

genes the signal intensity was set at >50 in the ligated aorta

group/stimulated cells, 2.5 times the highest background signal. For down-

regulated genes the signal intensity was set at >50 in the sham group/control

cells. (Paper II-IV)

Correlation between hyaluronan concentration and gene

transcription

Linear correlation between HA concentration in the left ventricle and

expression levels of significantly differentially expressed genes was tested to

identify a set of genes whose expression changes are associated with changes

in HA concentration. Correlation between HA concentration and gene

expression levels were calculated with SPSS (version 16.0, SPSS Inc.). The

Pearson correlation coefficient was used and statistical significance was set

to P<0.05.

Factor analysis was performed to examine the change in correlation over

time for the genes with expression levels found to correlate with HA

concentration. Principal components method was used to analyze correlation

matrix and 2 factors were extracted. Since the differentially expressed genes

in growing hearts are regulated according to a fetal gene program117,

activated by the increased afterload the sham operated animals could not be

included in the correlation analysis. MetaCore™ (GeneGo Inc.) was used to

generate a network between the correlating genes with transcription factors

to elucidate common transcriptional relationships.

To further increase the knowledge of the environment in which HA is

active, genes coding for proteins associated to HA and the ECM were

investigated. Genes coding for proteins such as structural ECM molecules,

cell membrane receptors that bind to ECM structures, molecules that

interact between cell surface and ECM, growth factors that interact with the

ECM and enzymes that regulate the turnover and remodeling of ECM

molecules were filtered from the lists of significantly differentially expressed

genes at the three time points. These genes were also used to generate a

network with transcription factors. (Paper II)

Preparation of microvesicles

Cell media were centrifuged to remove cell debris, 3,000 x g for 20 min at

4ºC, repeated three times, followed by 10,000 x g for 20 min at 4ºC,


32

repeated three times. The acquired supernatants were ultracentrifuged at

130,000 x g (49,000 rpm) for 2 h at 4ºC in an MLS-50 rotor and a Beckman

Optima™ MAX-E Ultracentrifuge (Beckman Coulter) to separate the

microvesicle pellet from the supernatant containing soluble molecules. Pellet

was dissolved in PBS. A similar ultracentrifugation of cell culture medium

alone was carried out to rule out the presence of any microvesicles in the

culture medium.

Cross-talk between cardiomyocytes and fibroblasts

Measurements of HA concentrations in cell media and gene expression

analysis were used to elucidate the existence of signalling crosstalk between

cardiomyocytes and fibroblasts.

Three general ways of mediating signalling crosstalk between cells were

considered. Direct cell to cell contact, extracellular signalling transferred by

microvesicles released into media 14 or extracellular signaling transferred

through exocytosis of soluble molecules into media.

After 24 h incubation, media from cardiomyocytes, fibroblasts and co-

cultured cardiomyocytes and fibroblasts were transferred to both

cardiomyocyte and fibroblast cultures. After additional 24 h incubation,

media were collected and the cells were harvested and placed in RNAlater.

Concentrations of HA in media from cardiomyocytes and fibroblasts were

measured as described above.

To distinguish between extracellular signaling through microvesicles and

exocytosis of soluble molecules, media that induced increased HA synthesis

in cells was centrifuged to separate pellet and media supernatant containing

soluble molecules (described above). Cells were then incubated with pellet

dissolved in new media or with media supernatant. The concentration of HA

in media was measured and compared to controls incubated with fresh

media. Gene expression in cells (n=3, except fibroblasts incubated with

media supernatant, where n=2) was analyzed with Illumina Beadstation

(Illumina).

Hyaluronan stimulation of cardiomyocytes

To investigate the presence of HA interaction with cardiomyocytes,

native-HA, 10 µg/mL, 800-1200 kDa (Select-HA™ 1000) or oligo-HA, 50

µg/mL, 6-mer (HYA-OLIGO6EF-1) (Hyalose, L.L.C.) was added to

cardiomyocytes in Claycomb media, followed by 24 h incubation. The cells

were harvested and placed in RNAlater.

Methodology

33

Differential gene expression, analyzed with Illumina Beadstation, was

used to detect intracellular signaling resulting in changed transcription,

compared to control cells (n=2 in each group). (Paper III)

Flow cytometry analysis

Fluorescence-activated cell sorter (FACS) was used to detect proteins on

microvesicle surfaces. Isolated microvesicles were stained with 250 ng

mouse anti-annexin-II, mouse anti-clathrin heavy chain, mouse anti-

flotillin-1 and mouse anti-caveolin-3 (BD Biosciences) for 20 min, in the

dark on ice. After an additional ultracentrifugation to wash the pellet, it was

resuspended in 100 µL PBS and 1 µL rat anti-mouse IgG PE and incubated

for 20 min, in the dark on ice. The ultracentrifugation was repeated and the

pellet resuspended in PBS. Microvesicles were analyzed on FACSCalibur

(Becton Dickinson). (Paper IV)

Electron microscopy

For electron microscopy, the microvesicles were fixed in a solution

containing 3% glutaraldehyde in 75 mM sodium cacodylate buffer (pH 7.4)

with 4% polyvinylpyrolidone and 2 mM CaCl2, for 6 h. They were

subsequently rinsed in the same buffer for one hour, and then post fixed in

1% osmium tetroxide over night at 4ºC. After another rinse in buffer the

sample was dehydrated in a graded series of acetone and then embedded in

an epoxy resin.

Ultrathin sections (70 nm) were cut, and collected on formvar coated

copper grids and then contrasted with uranyl acetate and lead citrate for

electron microscopy performed with a JEOL 1200-EX (Jeol Ltd.). (Paper IV)

Identification of microvesicular contents

Biotinylated cRNA was converted from microvesicle DNA and RNA

(described above). The labeled cRNA samples were hybridized to MouseRef-

8 Expression Beadchip (Illumina), incubated with streptavidin-Cy3 and

scanned on the Illumina Beadstation GX

Identified mRNAs have to be detected both in cardiomyocytes and

microvesicles to be considered as positively detected. Since cardiomyocytes

reasonably are the source of microvesicle mRNA, they should themselves

contain the same mRNA.

Detected DNA sequences correspond to the 50 nucleotide long probe

sequences on the gene expression chip. It does not necessarily mean that the

microvesicle DNA is derived from that gene. That 50 nucleotide long

sequence could possible also exist in non-coding parts of the genome. (Paper

IV)


34

Microvesicular DNA transfer into target fibroblasts

Microvesicles were stained with 20 µmol/L acridin orange (AO)

(Invitrogen) for 90 min, in the dark at room temperature. The sample was

diluted to 4 mL and ultracentrifuged at 130,000 x g (49,000 rpm) for 2 h at

4ºC. The supernatant was removed to eliminate contamination of

unincorporated AO. The AO-stained microvesicles suspended in 1 mL PBS

were then put in a dialysis bag with a 3,500 MWCO (molecular weight cut

off) dialysis membrane (Spectra/Por) and dialysed against 300 mL PBS for

24 h with one change of dialysis buffer after 5 h. The sample was

ultracentrifuged and the pellet dissolved in DMEM and incubated for 3 h

with fibroblasts, grown for 24 h on a cell culture microscope slide (Falcon).

The slide was subsequently mounted with 4'-6-Diamidino-2-phenylindole

(DAPI) to stain fibroblast nuclei and studied in a Nikon Eclipse E800

confocal microscope. Light microscope was used to add a layer in images to

visualize cell borders. (Paper IV)

Microvesicular induced effects on target cells

Fibroblasts, grown on 6-well plates, were incubated for 48 h Claycomb

medium, previously incubated for 24 h with cardiomyocytes (n=3).

A part of the same Claycomb medium was ultracentrifuged, (described

above) and the supernatant was also incubated for 48 h with fibroblasts

(n=2).

Isolated microvesicles from cardiomyocytes incubated for 48 h in

Claycomb media were dissolved in DMEM and incubated with fibroblasts for

48 h (n=2).

The stimulated fibroblasts were compared to control fibroblasts

incubated in fresh Claycomb medium (n=3) and DMEM (n=2), respectively.

RNA was prepared from fibroblasts, labelled and hybridized to

MouseRef-8 Expression Beadchip (Illumina), as described above.

To determine differentially expressed genes microarray data were

analyzed using gene expression module in Beadstudio software, version

3.3.7. (described above). (Paper IV)

Results

35

Results

Paper I and II:

General characteristics

The heart weight increased in the aorta ligated group compared to the

sham group. At day 1 after operation, the heart weight of the aorta ligated

animals was 15% higher than in the sham operated group. At day 42 the

weight had increased by 43% compared to sham operated animals. The

operation initially caused all rats, in particular the aorta ligated group, to

lose weight which affected the HBW (heart-to-body weight) ratio

calculations. Genes traditionally used as markers for cardiac hypertrophic

growth and the fetal gene program (Nppa, Nppb, Acta1, Myh6 and Myh7)

showed an increased expression as early as 1 day after surgery in the aorta

ligated rat hearts. As expected, expressional changes in early genes was

observed only in the acute phase.

Quantitative RT-PCR

Hyaluronan synthases and CD44.

HAS1 expression had increased six-fold in the aorta banded rats at day 1

and 6 and then dropped to three-fold after 42 days.

HAS2 had a similar expression as HAS1 at day 1 but dropped earlier to

three-fold by day 6 and was expressed at basal level after 42 days.

HAS3 did not show any detectable cardiac expression at all either in aorta

ligated or sham operated rats.

CD44 expression was up-regulated eight-fold at day 1 in the aorta banded

group, its expression progressively decreased, to reach control values at day

42.

Fibroblast growth factor-2 and fibroblast growth factor receptor-1.

The expression of FGF-2 had increased almost five-fold at day 1 and was

further up-regulated at day 6. After 42 days the level decreased but was still

significantly up-regulated two-fold as compared with sham-operated

animals.

The expression of FGFR-1 was transiently up-regulated two-fold only at

day 1.

Quantification of hyaluronan in heart tissue

The average HA concentration in the hearts of the sham operated animals

correlated well to earlier studies105. The average HA concentration in the

hearts at day 1 was 473±262 μg/g dry weight in the aorta ligated animals and


36

the concentration was significantly increased compared to sham operated

animals (P=0.037). The concentrations at day 6 and day 42 were not

significantly increased compared to sham operated animals.

Histochemical analysis of hyaluronan and CD44

HA was detected in the myocardium and in the adventitia of cardiac

arteries in all rats in both aorta ligated and sham operated animals. No

difference in staining intensity was detected between the groups.

With one exception, CD44-staining was detected only in the hearts of the

aorta ligated animals. CD44 staining was mainly found in and around the

adventitia, in some arteries also in the media-intima, and in right ventricular

trabeculi.

Microarray gene expression

The number of genes that were significantly up- or down-regulated on the

chip at day 1, 6 and 42 was 742, 216 and 371 respectively. Out of these genes

there were 106 (14%), 4 (2%) and 31 (8%) that correlated with the

concentration of HA in the left ventricle in the hearts. Of the 106 genes, 81

were identified and have a known function. The MetaCore™ (GeneGo Inc.)

bioinformatics software was used to generate a network between the

correlating genes and transcription factors. This analysis revealed an

enrichment of genes regulated by 6 transcription factors, JUNB, FOS, MYC,

TP53, SP1 and EGR1. Four of these transcription factors, JunB, Fos, Myc and

Egr1 were up-regulated at day 1.

The change in correlation with HA concentration over time with the 106

genes at day 1 and the 31 genes at day 42, were analyzed with factor analysis.

The 106 genes from day 1 formed two tight clusters, representing positive

correlation to HA concentration and negative correlation. The same genes in

animals from day 6 showed clustering but less tight. At day 42 the

correlation was lost. The 31 genes correlating to HA concentration at day 42

showed an inverse development over time. On day 42 a tight correlation was

evident but at day 1 and 6 no cluster was seen. In the sham operated animals

no cluster of correlating genes was observed.

Genes with association to HA specifically and the ECM in general were

filtered from the lists of significantly differentially expressed genes at the

three time points. These 37 genes were also used to generate a network with

transcription factors. Most of the genes were also bioinformatically found to

be transcriptionally regulated by TP53, SP1, AP-1 and MYC. Four of these

genes, CD44, biglycan (Bgn), latent transforming growth factor binding

protein 2 (Ltbp2) and fibrillin 1 (Fbn1), correlated significantly with HA

concentration levels.

Results

37

Paper III:

Quantitative analysis of hyaluronan in cell media

The only growth factor that caused cardiomyocytes to synthesize HA was

PDGF-BB at 100 ng/mL in the media.

Fibroblasts on the other hand synthesize HA with or without any addition

of growth factors. The addition of growth factors showed that FGF2 and

PDGF-BB at higher concentrations (10 and 100 ng/mL) induced a lower

concentration of HA than at lower concentrations (5 and 50 ng/mL). TGFB2

induced the highest concentration of HA but no difference was seen in

relation to its concentration.

Co-culturing cardiomyocytes and fibroblasts resulted in increased HA

concentration compared to cells cultured separately, especially when taking

into account the lower amount of fibroblasts in the co-culture.

In contrast, FGF2 and PDGF-BB stimulated co-cultured cells resulted in

lower HA concentration in the media compared to cells cultured alone.

Media from co-cultured cells stimulated with TGFB2 contained twice as

much HA as in TGFB2 stimulated fibroblasts.

Real-time polymerase chain reaction analysis of hyaluronan

synthases

The endogenous control Gapdh, used in real-time PCR analysis to

normalize for differences in the amount of total RNA added, were in some

cell/growth factor combinations not at a consistent level between samples

and controls. Therefore, the β-actin and 36B4 genes were also tested as

endogenous controls but with the same result. For this reason the real-time

PCR results have only been used as a detection of expression of the different

Has’s, without the possibility to calculate foldchange and significance of

changes in expression.

Dynamic Light Scattering Size analysis of hyaluronan

The size of HA synthesized by fibroblasts, cardiomyocytes or co-culture of

the two cell types was estimated by DLS.

Cardiomyocytes produced HA with a diameter of 135 ±15 nm when

stimulated with a high concentration of PDGF-BB. Under other conditions

cardiomyocytes did not produce any detectable levels of HA.

Non-stimulated fibroblasts produced HA with a hydrodynamic diameter

of 175 ±25 nm.

Upon stimulation by low levels of FGF2, fibroblasts released HA with a

diameter of 140 ±20 nm, whereas fibroblasts cultured with high levels of

FGF2 produced HA with a diameter of 60 ±15 nm.

Fibroblasts stimulated by the low dose of PDGF-BB produced HA with a

diameter of 100 ±15 nm. When the fibroblasts were stimulated by a high


38

dose of PDGF-BB the concentration of HA became too low to ensure reliable

DLS measurements.

The different concentrations of TGFB2 showed little effect on the size of

produced HA, with a hydrodynamic diameter of 110 ±15 nm and 100 ±5 nm

for the low and high concentration of this growth factor, respectively.

When cardiomyocytes and fibroblasts were co-cultured without any

growth factors they secreted HA with a diameter of 160 ±20 nm.

The addition of low levels of FGF2 induced HA of 115 ±45 nm, whereas

the high level of FGF2 yielded no detectable HA.

Low levels of PDGF-BB did not induce a HA concentration high enough

for reliable DLS measurements, whereas a high dose of PDGF-BB yielded HA

with a size of 220 ±80 nm.

When a low dose of TGFB2 was added to co-cultured cells, HA with a size

of 170 ±35 nm was produced, whereas the high concentration of TGFB2

resulted in HA with a diameter of 85 ±15 nm.

Crosstalk between cardiomyocytes and fibroblasts

Synthesis of HA by fibroblasts increased after incubation with Claycomb

media from either co-cultured cells or cardiomyocytes alone, compared to

both fibroblasts incubated in DMEM and fibroblasts incubated in fresh,

unused Claycomb media. A lower HA concentration was obtained after

incubation of fibroblasts with fresh, unused Claycomb media.

Fibroblasts incubated with pellet from ultracentrifugation of Claycomb

media from cardiomyocytes, dissolved in DMEM showed similar HA

concentration to fibroblast controls while fibroblasts incubated in the

supernatant of ultracentrifuged Claycomb media from cardiomyocytes

demonstrated a two-fold increase in HA concentration. Cardiomyocytes

showed no synthesis of HA irrespective of media added.

Gene expression analysis of fibroblasts incubated for 24 h with Claycomb

media transferred from cardiomyocytes, resulted in 333 differentially

expressed genes compared to fibroblasts incubated with unused Claycomb

media. The same analysis of fibroblasts incubated for 24 h with

ultracentrifuged Claycomb media supernatant transferred from

cardiomyocytes, resulted in 96 differentially expressed genes. In both cases

the Tgfb2 gene was up-regulated 3-fold.

Hyaluronan stimulation of cardiomyocytes

The addition of native-HA and oligo-HA to Claycomb media before

incubation for 24h, affected cardiomyocytes gene expression. When native-

HA was added to Claycomb media 35 differentially expressed genes were

detected and 63 genes were detected with oligo-HA added.

Results

39

Paper IV:

Preparation and characteristics of microvesicles

Transmission electron microscopy of microvesicles secreted from

cardiomyocytes revealed small, closed exosome-like vesicles (40-300 nm)

surrounded by a bilayered membrane. Since the microvesicles are

cardiomyocyte-derived they are called cardiosomes to denote their origin.

Some of them had a distinct electron dense appearance while others

displayed an electron lucent interior. Furthermore, approximately 80% of

the cardiosome population was positive for flotillin-1 and 30% for caveolin-3

in the flow cytometry analysis, indicative of a certain degree of heterogeneity.

Identification of cardiosomal contents

The cardiosomes contained 1595 detected mRNAs of which 1520 also

were detected in cardiomyocytes. It was possible to connect 423 of these

genes/proteins in a network by using the MetaCore™ (GeneGo Inc.)

bioinformatics software. Furthermore, mRNA from 35 genes coding for

proteins in the small and large ribosomal subunit and 8 additional genes

were detected and could be connected in a network. Finally 33 genes coding

for proteins in mitochondria could be detected.

Analysis of cardiosomal DNA converted to biotinylated cRNA detected

signals from gene probes corresponding to 343 different chromosomal DNA

sequences.

Cardiosomal DNA transfer into target fibroblasts

Examination by confocal microscopy of fibroblasts incubated with AO-

stained cardiosomes revealed intracellular AO-stained spots, localized in

cytoplasm and inside the nuclear membrane.

Cardiosome induced effects on fibroblasts

Incubation of fibroblasts with cardiosomes resuspended in fresh DMEM

medium induced 161 differently expressed genes compared to incubation

with control medium.


40

Discussion This study has focused on investigating the role of HA in the development

of cardiac hypertrophy. Both an experimental rat model of cardiac

hypertrophy and cultured cardiomyocytes and fibroblasts have been used.

An early and pronounced increase in the expression of Has1 and Has2 in

the hearts of the aorta ligated animals was observed, paralleled by a similar

increase in the expression of CD44 and Fgf2. This was accompanied by an

increase in HA concentration in the rat hearts. The induced expressions of

Has1 and Fgf2 were an early (day 1) and sustained response (day 6 and 42).

This pattern contrasted with that of Has2 and CD44, the expressions of

which were transient, the levels decreasing as soon as after day 6 to reach

control levels by day 42.

This indicates that HA derived from HAS1 might be synthesized for a

different purpose in the late phase of the hypertrophic development of the

heart and therefore regulated by other factors than Has2 is regulated by.

Assuming that genes involved in the same biological chain of events have

similar temporal expression patterns, then HA, synthesised by HAS2, is

linked to the expression of CD44. HA concentration and CD44 expression

levels correlated day 1. The CD44 receptor was mostly localised around the

arteries in the hearts from aorta ligated animals. It is known that CD44 has a

role during angiogenesis53, 118 and the HA synthesised by HAS2 may be

correlated to a vascular response to either hypertension and/or adaptation to

a growing heart.

In contrast, in cultured cardiomyocytes and fibroblasts, Has2, but not

Has1 was detected both in controls and all growth factor treated

combinations. One reason for Has1 not being expressed, as it was in heart

tissue, could be the absence of vascular cells in the cell cultures. This would

mean that HA synthesized by HAS1 is associated with vessels which is

contradictory to the assumption made from the results from cardiac tissue

where Has2 is associated to the vasculature.

Obviously the regulation of the HASs is very complex.

The addition of growth factors in cell media of cultured cardiomyocytes

and fibroblasts consistently affected the HA size, demonstrating that growth

factors not only influence the amount of synthesized HA but the processes

initiated by growth factors also induced a change in HA size. In most cases,

the cells produced HA in smaller size when stimulated with growth factors.

The predominant synthase expressed was Has2, suggesting that HA size was

not controlled by shift in type of HAS expressed.

Discussion

41

Which type of cells that contributes to HA synthesis and expression of

HASs in the cardiac tissue in the aorta ligated rats is unknown. Cell cultures

were used to investigate the capability of cardiomyocytes to synthesize HA.

The addition of PDGF-BB, at the higher concentration (100 ng/ml), to

cultured cardiomyocytes, activated synthesis of HA in the cells. Thus

cardiomyocytes must express HAS and indeed, real-time PCR showed both

Has2 and Has3 transcription.

Adding HA to cardiomyocyte media showed that both oligo-HA and

native HA influenced transcription of cardiomyocyte genes. This suggests

that cardiomyocytes express HA binding receptors on the surface, capable of

triggering gene transcription through intracellular signalling. Hence,

cardiomyocytes can produce, detect and be affected by HA in their

environment.

An interesting finding was the distinctly affected HA concentration levels

found both in untreated and TGFB2 treated co-cultured cells compared to

cardiomyocytes and fibroblasts cultured alone. This was explained by the

observation that cardiomyocytes secrete through exocytosis soluble

molecule(s), a still unknown factor which after transfer to fibroblasts up-

regulated the Tgfb2 gene and increased the synthesis of HA. HA seems to be

requested by the growing and proliferating cardiomyocytes, which is in

concordance with the observations of many other cell types needing

increased HA synthesis for growth79, 80

The lower concentration of PDGF-BB induced no synthesis of HA. This

may indicate a threshold that must be exceeded before synthesis is initiated

in cardiomyocytes. It is therefore tempting to speculate that fibroblasts are

the primary source of HA synthesis in the myocardium and cardiomyocytes

are a secondary producer of HA that start their synthesis when PDGF-BB

rises to a certain level. The range in sizes of HA synthesized in

cardiomyocytes overlapped sizes of HA seen in fibroblasts and co-cultured

cell controls. Thus, cardiomyocytes might produce HA in similar size to

fibroblasts when an acute need for high amounts of HA arises to support the

HA synthesis derived from fibroblasts.

Cardiomyocytes are also capable of intercellular signalling via exosomes,

denoted cardiosomes. The cardiosome population observed was not

homogenous in size, expressed surface proteins or electron density observed

in electron microscopy. This suggests that cardiomyocytes secrete

cardiosomes with different purpose and perhaps intended for different

targets cells.

They contained both mRNA and DNA, which could be transferred to

fibroblasts with subsequent change in gene transcription. This

internalization of cardiosomes, even into the nucleus, was visualized by

confocal microscopy.


42

Cardiosomal mRNA coding for both small and large ribosomal subunits

as well as proteins involved in mitochondrial energy generation were

identified. This implies that cardiosomes are carrying means to support

protein production and energy generation in the targets cells for the

transcription of their delivered genetic material.

To further investigate the changes in HA synthesis in this animal model,

correlation between HA synthesis and gene expression changes was

investigated.

Subsets of the differentially expressed genes that correlated with HA

concentrations at day 1, 6 and 42 were identified. Thus, HA synthesis and

such genes are likely to be governed by a common regulatory pathway.

With factor analysis it was possible to demonstrate that genes, which

expression levels correlated with HA concentration at day 1 in aorta ligated

animals, also correlated in a similar way after 6 days. No correlation was

found between these genes in the sham operated animals at any time point.

One explanation for the temporal differences seen in gene correlation

could be the regulation and expression of different HA synthases.

Also the genes coding for angiotensin-converting enzyme (ACE) and

ACE2 showed distinct temporal patterns of expression with acute up-

regulation of Ace at day 1 and a late response of Ace2 at day 42.

The renin angiotensin system (RAS) is a known regulator of cardiac

growth. Angiotensin II (Ang II) is a prohypertrophic effector peptide, while

angiotensin 1-7 has the opposite effect, activated by angiotensin-converting

enzyme (ACE) and ACE2 respectively119, 120.

Most likely the observed co-regulation of genes with HA concentrations at

day 1 and 6 reflects the acute hypertrophic process while the genes being

active at day 42 shows a shift to a steady state program mirrored by the

expression levels seen in Has1 and Has2 as well as Ace and Ace2.

Differentially expressed genes with expression levels correlating with HA

concentration are likely to be regulated by the same transcription factors.

These genes were analyzed in the bioinformatic software MetaCore™

(GeneGo Inc. USA) and an enrichment of genes regulated by 6 transcription

factors, JUNB, FOS, MYC, TP53, SP1 and EGR1 were identified. Four of

these transcription factors, JunB, Fos, Myc and Egr1 were up-regulated at

day 1, indicating strong association with the transcriptional changes in the

aorta ligated animals.

JunB, Fos Myc and Egr1 are immediate early genes (IEG), activated in

response to stimuli mediated via Ang II and/or mechanical factors121-124.

They are well known as early regulators of cell growth and to precede the

expression of cardiac hypertrophy markers, e. g. ACTA1 and ANP. The

correlation of HA synthesis to the expression of ANP, which is regulated by

Discussion

43

the ACE and IEG’s, opens the possibility that HA also is part of the fetal gene

program activated in cardiac hypertrophy.

When this bioinformatic analysis was repeated on differently expressed

genes associated to HA specifically and to the ECM in general, the same six

transcription factors, JUNB, FOS, MYC, TP53, SP1 and EGR1 were found to

regulate these genes. The physical proximity of these ECM proteins and

receptors to HA and correlation to HA synthesis reveals a collective change

in response to aortic ligation and cardiac hypertrophy.


44

Conclusions

The aim of this thesis was to investigate HA and its role in cardiac

hypertrophy, using both an experimental rat model of cardiac hypertrophy

as well as cultured mouse cardiomyocytes and fibroblasts.

Increased concentration of HA in hypertrophied cardiac tissue was

observed together with an up-regulation of two Has genes. The different

expression patterns of the Has1 and Has2 genes in both the cardiac

hypertrophy rat model and cultured cardiomyocytes and fibroblasts, may

indicate that HA is needed for two different purposes in the hearts exposed

to aortic ligation, one for an acute and transient response and one for a

sustained response. One could be for cardiomyocyte growth, one for vascular

growth.

In cultured fibroblasts it was shown that different growth factors induced

different concentrations and sizes of HA, even though the main synthase

expressed was Has2. FGF2 and PDGF-BB induced HA synthesis in

fibroblasts and co-cultivated cells in a reverse dose dependent response, the

highest dose induced least HA production and smallest size.

This indicates that different growth factors and their concentrations

regulate HA synthesis and size in other ways than just switching between

different HASs.

Future research needs to investigate the localisation of HAS1 and HAS2

in the cardiac tissue from aorta ligated animals, as well as the growth factor

regulation of HA size.

Four transcription factors, Myc, Fos, JunB and Egr1 were up-regulated in

the aorta ligated animals. These immediate early genes are known to be early

regulators of the re-expression of fetal genes (e.g. Acta1 and Nppa) in cardiac

hypertrophy. They are also known to regulate both several of the differently

expressed genes coding for structural and regulatory molecules interacting

within the ECM and several of a subset of differently expressed genes which

expression levels correlated to HA concentration, in cardiac tissue of the

aorta ligated animals.

The coordinated synthesis of HA and expression of genes regulated by

immediate early genes, suggests the involvement of immediate early genes in

the regulation of synthesis of HA and that HA is a part of the fetal gene

program. Functional studies should be performed to elucidate the

involvement of immediate early genes in the regulation of HAS expression

and HA synthesis.

Cell cultures revealed the ability of cardiomyocytes to both synthesize and

detect HA in their environment with subsequent altered gene expression.

Conclusions

45

Cardiomyocytes also secreted a factor into culture media that after transfer

to fibroblasts increased synthesis of HA. This ability of cardiomyocytes to

send out a request to fibroblasts to synthesize HA indicates a need for HA of

the cardiomyocytes. When fibroblasts deliver the request, receptors on the

cardiomyocytes surface can detect the HA signal and after signal

transduction, a changed gene transcription in the cardiomyocytes is

achieved.

This demonstrates the close involvement between HA and

cardiomyocytes. A more detailed study in the changes in gene expression in

cardiomyocytes, when stimulated by HA, could reveal the reason for

demanding the fibroblasts to synthesize HA.

It was also demonstrated that cardiomyocytes possess an intercellular

way of communication via secreted microvesicles. These cardiomyocyte-

derived exosomes, cardiosomes, contained both DNA and mRNA and were

shown to be internalized when transferred to fibroblasts with subsequent

altered gene expression. Through the transfer of the cardiomyocytes own

mRNA to target cells, where they are translated to proteins, the

cardiomyocyte can influence behaviour and function of target cells in their

neighbourhood. The destinations and purposes of secreted cardiosomes need

to be clarified in the future.

This study shows that increased HA synthesis in cardiac tissue during

hypertrophic development is a part of the extracellular matrix remodelling.

Assuming that growing cells need a certain ECM composition for growth,

the observed changes in transcription may in part represent a demand for a

remodelling of the ECM. The new ECM, together with growth factors

transduce intracellular signalling through several receptors simultaneously.

Subsequently when cell receptors respond to the remodelled ECM, the

growth can progress.

It has in many cases been demonstrated that HA enriched extracellular

matrices are necessary for cells to be able to grow and proliferate. The

discovery that cardiomyocytes under certain circumstances can synthesize

HA themselves opens up for interesting speculations. Recently, it has been

shown that the myocardium has the ability to regenerate, although very

slowly125, 126. HA might be an important factor in this process.


46

Sammanfattning på svenska Myokardiell hjärtmuskelhypertrofi är en riskfaktor för kardiovaskulär

morbiditet och mortalitet. Oberoende av underliggande orsak, strävar

hjärtmuskeln på olika sätt att kompensera för en förändring av den

hemodynamiska belastningen. Förändringar i hjärtat inkluderar även

förändringar i extracellulär matrix, vilket stör den systoliska och diastoliska

funktionen. Dessutom kommer signaltransduktion, molekylär diffusion och

mikrocirkulation påverkas. En viktig extracellulär komponent är

glykosaminoglykan hyaluronan. Den har visat sig spela en viktig roll i andra

vävnader som uppvisar cellulär tillväxt och proliferering, såsom sårläkning

och cancer.

Syftet med denna avhandling var att undersöka hyaluronan och dess roll

både i en experimentell hjärthypertrofimodell samt i odlade

muskardiomyocyter och fibroblaster.

Hjärthypertrofi inducerades hos råttor genom ligering av bukaorta.

Hyaluronankoncentration mättes och uttryck av gener som kodar för

hyaluronan syntaser kvantifierades i hjärtvävnad från vänstra kammarens

vägg efter 1, 6 och 42 dagar efter operation. Lokalisering av hyaluronan och

CD44-receptorn studerades histokemiskt. Hyaluronansyntesens korrelation

till transkription av gener studerades med hjälp av microarray

genexpressionsanalys.

Odlingskulturer med kardiomyocyter och fibroblaster stimulerades med

olika tillväxtfaktorer. Hyaluronan-koncentration mättes och uttryck av gener

som kodar för hyaluronansyntaser analyserades. Hyaluronanstorlek mättes

och cell-till-cell-kommunikation mellan kardiomyocyter och fibroblaster

undersöktes med avseende på hyaluronansyntes.

Ökad koncentration av hyaluronan i hypertrofisk hjärtvävnad sågs

tillsammans med uppreglering av två hyaluronansyntasgener. Hyaluronan

upptäcktes i hjärtmuskeln och i hjärtartärernas adventitia medan färgning av

CD44 huvudsakligen fanns i och runt adventitia. Hyaluronansyntes

korrelerade till uttrycket av gener som regleras av transkriptionsfaktorer

som är kända för att inducera hjärthypertrofi.

Stimulering av kardiomyocyter med PDGF-BB inducerade syntes av

hyaluronan. Kardiomyocyter utsöndrade också en faktor i odlingsmediumet

som efter överföring till fibroblaster initierade en ökad syntes av hyaluronan

hos fibroblasterna. När kardiomyocyter stimulerades med hyaluronan i olika

storlekar, observerades en förändring i deras genuttryck. Olika

tillväxtfaktorer inducerade olika storlekar av hyaluronan i fibroblaster. Det

huvudsakliga syntas som detekterades i de odlade cellerna var

hyaluronansyntas-2. Kardiomyocyter påvisades utsöndra mikrovesiklar som

Sammanfattning på svenska

47

innehöll både DNA och RNA. Isolerade mikrovesiklar inkuberades med

fibroblaster varefter en internalisering observerades i fibroblaster med

konfokalmikroskopi. Förändrat genuttryck observerades i fibroblaster efter

stimulering med mikrovesiklar.

Denna studie visar att en ökad hyaluronansyntes i hjärtvävnad under

utveckling av hypertrofi är en del av anpassningen av extracellulär matrix.

Cellkulturer visade att kardiomyocyter kan både syntetisera hyaluronan och

förmedla signaler till fibroblaster, vilket får dem att öka sin

hyaluronansyntes. Kardiomyocyter kan också uttrycka receptorer för

hyaluronan, vilka förmedlar intracellulära signaler som orsakar de

observerade förändringarna i genuttryck hos kardiomyocyter som

stimulerats med hyaluronan. Detta påvisar en omfattande medverkan av

hyaluronan i den process som leder till hjärthypertrofi.


48

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