Fibroblasts and ECM

Question 1

the functions of ECM

Answer 1

• provides structural support and acts as adhesion, chemical and mechanical substrates.
• dynamic system that alters its compositions and organization in response to environmental stimuli.
• functions as a cell signaling system via surface receptors influencing cellular properties.

Question 2

collagen type I

Answer 2

thick perimysial fibres forming a fibrillar network to help withstand shear-stress and co-ordinate contraction.

Question 3

collagen type III

Answer 3

thinner and less rigin fibres conferring elasticity to the myocardium

Question 4

the collagenous make up of the heart, turnover

Answer 4

• Collagen I and III account for 85-90% and it’s not only the QUANTITY but also the QUALITY and RATIO of these two isoforms that influence compliance and diastolic relaxation.
• The specific orientation of the collagen fibers relative to the cardiac myocytes is thought to orient the cells within the myocardium thereby preventing excessive stretch during diastole and contributing to elastic recoil during systole
• Collagen is a stable protein degraded at a rate of 0.6% per day.
• The proteins have a half-life of 80-120 days.

Question 5

Fibronectin
structure
intercations
produced by

Answer 5

• Multi-domain adhesive glycoprotein that can interact and cross-link with multiple proteins within the ECM such as collagen.
• The protein is a dimer composed 2 large subunits joined by disulfide bonds.
• There are several splice variants differentially expressed in development and adulthood. The substrate specificity is determined by the splice variant.
• They contribute to the organizing and stability of the matrix by forming cross-links and in cell adhesion and migration.
• Fibronectin expression can be stimulated by mechanical and growth factors such as TGF-b.

Question 6

Laminin

Answer 6

• Is an adhesive glycoprotein formed by the association of three chains: a, b & g.
• It contains different functional domains that can interact with collagen IV, proteoglycans and transmembrane receptors.
• It is largely produced by cardiac myocytes, vascular smooth muscle cells and endothelial cells.

Laminin is a major component of the basement membrane.

Question 7

Elastin

Answer 7

• They have a flexible conformation due to the numerous proline residues and they do form crosslinks with each other.
• When the fibers stretch these crosslinks keep the fibers connected.
• Elastin is the dominant ECM present in the walls of the blood vessels and the interstitium.

Question 8

Periostin

Answer 8

• Periostin is secreted mostly by fibroblasts or cells that acquire a fibroblast like-phenotype during development or following injury.
• The protein can interact directly with ECM proteins and several integrins.
• Periostin is a TGF-b-3 responsive gene and is thought to mediate the maturation of the atrioventricular valve promoting mesenchymal-fibroblast differentiation.
• Mice deficient in the protein display abnormalities in cell differentiation and valve formation.

Question 9

Osteopontin

Answer 9

• Is a Ca2+ binding protein with an RGD (arginine-glycine-aspartate) binding sequence.
• It is able forms complexes with collagen I, II, III, IV and membrane receptors.
• The protein is synthesised by smooth muscle cells and fibroblasts and is involved in cell adhesion, migration and cellular growth.
• Osteopontin also contributes to vascular remodelling.

Question 10

proteoglycans

Answer 10

Proteoglycans are composed of a core protein and associated with glycosaminoglycans (GAG).
There are 4 classes of GAGs:
1) hyaluronan
2) chondroitin sulfate/ dermatan sulfate
3) heparan sulfate/heparin
4) keratin sulfate

During their synthesis the GAGs are covalently linked to the core proteins and sulfated
before being secreted into the ECM as proteoglycans.

Question 11

what cells produce ECM?

Answer 11

fibroblasts are the mains source but other cells also contribute@
Myocytes, endothelial cells, VSMCs

Question 12

what are fibroblasts?

Answer 12

• Cells of mesenchymal origin and account for the largest cell population in the heart.
• They are able to secrete and breakdown proteins that form the ECM.
• Their regulation of ECM composition is also dependent on chemical (e.g. growth factors, cytokines) and mechanical (e.g. stretch) stimuli.
• Degradation of the ECM is modulated through the expression of matrix metalloproteinases and the natural inhibitors.

Question 13

regulation of ECM turnover PARTICIPANTS

Answer 13

MMP - matrix metalloproteinase
MT-MMP = membrane type-MMP
TIMP - tissue inhibitor matrix metalloproteinase
PAI plasminogen activator inhibitors
uPA urokinase plasminogen activator

MMPs and TIMPs have several isoforms each with different substrate specificity

Question 14

ECM turnover process (regulation)

Answer 14

fibroblasts secrete TIMPs, which inhibit MMP activity
they also secrete PAI-1, which inhibits uPA and prevents uPA from converting plasminogen to active plasmin.
Plasmin activates proMMPs into active MMPs and overall increases ECM degradation and activation of TGF-beta and other growth factors, such as VEGF which stimulates MMP activation as well

MT-MMPs on firoblasts cleave pro MMPs into active MMPs

Question 15

MMPs
what are they
function
structure
regulation

Answer 15

A family of zinc-containing endoproteinases that share structural domains but differ in substrate specificity, cellular sources, and inducibility.
All MMPs share the following functional features:
1. they degrade ECM components;
2. they are secreted in a latent proform and require activation for proteolytic activity;
3. they contain Zn2+at their active site;
4. they need calcium for stability;
5. they function at neutral pH;

they are inhibited by specific tissue inhibitors of metalloproteinases (TIMPs).

Question 16

Integrins

Answer 16

• Heterodimeric cell surface receptors composed of two subunits, a and b.
• They provide the dynamic interaction between the environmental cues (e.g. ECM or mechanical stress) and intracellular events (e.g. cardiac myocytes or fibroblasts).
• each subunit has a large extracellular domain, transmembrane domain and short cytoplasmic tail.
• A single receptor can bind to several ligands and a single ligand can bind to several receptors.
• Integrins have an RGD (arginine-glycine-aspartate) attachment site for ECM proteins.

beta subunits connect to actin filaments via ‘adaptor’ or ‘bridging’ proteins such as actinin, talin, paxillin and vinculin regulating cell shape, orientation and migration.

Question 17

How can integrins act as signal transducers (inside-out)

Answer 17

An agonist binds to non-integrin receptor eg Growth factors such as PDGF that affects proliferation and cell survival binds to a receptor (inside out signalling). Its activation triggers downstream signalling molecules such as tyrosine kinase and focal adhesion kinase/ this leads to cytoskeleton reorganisation (re organisation of talin-paxilin-vinculin-actin-alpha-actinin complex). In turn, this leads to binding of an integrin activation complex to the cytoplasmic domain of the integrin subunits.
In (1), binding of the integrin activation complex causes a conformational change in the integrin subunits leading to increased affinity of matrix binding (laminin [yellow)].
In (2), binding of the integrin activation complex leads to integrin clustering and increased avidity of integrinmatrix binding, perhaps causing a more permanent binding of integrins to matrix (here depicted by laminin).

Question 18

Integrin outside-in signalling

Answer 18

After ligand binding (eg, of laminin, shown in yellow), the integrin conformation is altered and subsequently the heterodimer can participate in events critical for organization of the  cytoskeleton and other intracellular signaling events that might be important for cell survival or initiation/propagation of cardiac myocyte hypertrophic events. 
#Thus, mechanical tension outside the cell could be converted to intracellular biochemical signals through the integrins.

For this process, the cytoplasmic domain of the integrin subunits signals through a host of molecules such as kinase (FAK, Akt, Raf, MEK, ERK [shown in olive]), cytoskeletal organizers (eg, paxillin), small GTPases (eg, Rho, Rac, Ras [shown in blue]), and other molecules.

Question 19

Force transmission between the ECM and cell interior:

Answer 19

ECM binds to integrins through the RGD peptide. Once the interaction is established focal complexes begin to form.
When a mechanically stable contact is established, focal complexes mature into larger focal adhesions. The size of the adhesions is approx proportional to the size of the mechanical signal which in turn affects stiffness and pulling forces.

Question 20

ECM remodelling in the failing heart

Answer 20

• In the healthy heart, maintenance of tissue architecture requires a fine balance between synthesis and degradations.
• Disturbances in ECM turnover in either of these two processes could lead to cardiac dysfunction.
• For example alterations in the extracellular collagen matrix plays a crucial role in left ventricular (LV) remodelling whereby a decrease, disruption or defective composition of the ECM promote LV dilation and rupture.

Question 21

In a sentence, what characteristic describe the LV remodelling after MI?

Answer 21

the LV remodeling process after MI is complex, dynamic, and time dependent, and progresses in parallel with healing over months.

Question 22

LV remodelling after MI nvolves differential changes between the IZ and NIZ with
respect to the following:

Answer 22

(1) LV structure, shape, and topography
(2) cell type, such as myocytes and nonmyocytes;
(3) proteins, cytokines, and growth factors; and
(4) the ECCM

Question 23

What maintains the normal structure of cardiac tissue?

Answer 23

a fine balance, between matrix metalloproteinases (MMPs) that degrade ECCM and endogenous tissue inhibitors
of MMPs (TIMPs) that inhibit MMPs, maintains normal remodeling and function, and an imbalance can result in adverse remodeling.

Question 24

what different changes in collagen levels in cardiac remodelling can result in?

Answer 24

although a 2- to 3-fold increase in myocardial collagen above the normal level results in increased LV stiffness and mild dysfunction, a very small decrease in collagen below normal can lead to drastic consequences including LV dilation and rupture. In reperfused MI, decreased or damaged ECCM in the IZ is associated with cardiac rupture.

Question 25

how many cells in the heart are nonmyocytes? how many of those are fibroblasts?

Answer 25

First, nearly 75% of the cells in the healthy heart are nonmyocytes, which include fibroblasts that account for ~90% of nonmyocyte cell mass

Question 26

briefly outline the process of LV r emodelling after MI

Answer 26

MI results in time-dependent damage to myocytes, nonmyocytes, and the ECCM in the IZ; ventricular dysfunction followed by volume overload and progressive dilation; reactive hypertrophy with interstitial fibrosis and increased collagen in the NIZ; gradual reparative fibrosis in the IZ; and vascular remodeling in the IZ and NIZ.

Question 27

substances stimulating ECCM deposition:

Answer 27

endogenous
The key enzyme, prolyl-4-
hydroxylase (P4H), catalyzes the hydroxylation of proline
on alpha monomers to yield stable protocollagen molecules
that are secreted into the ECCM. P4H requires several
cofactors, including ascorbic acid (vitamin C)
Aldosterone
Fibronectin
Growth hormone
TIMP
TGF-beta
AT1R
Synthetic/drugs
MMP-inihibitors
Adenosine inhibitors
Anti-TNF-alpha

Question 28

substances decresing ECCM deposition:

Answer 28

endogenous
Chymase
MMP
Adenosine 
TNFalpha
Synthetic/drugs:
ACE-I
ARBs
Mineralocorticoid antagonists

Question 29

therapeutic targets to prevent fibrosis:

Answer 29

growth factors
• Modulation of the renin-angiotensin (Ang) system using Ang converting enzyme (ACE) inhibitors or Ang receptor antagonists (e.g. losartan) appear to be effective in reducing cardiac fibrosis in a variety of models in animals and humans.
• TGF-b plays a central role in fibroblast activation inducing ECM deposition by supressing MMP and inducing TIMP expression. Wounds treated with anti-TGF-b showed reduced ECM synthesis and scarring. Inhibitors of TGF-b receptors are also considered as potential anti-fibrotic compounds.
• Endothelin secreted by endothelial cells induces ECM production and fibroblast differentiation. Endothelin receptor antagonism may be considered as an appropriate therapy for fibrosis. Currently endothelin receptor blockers (bosentan) are used for the treatment of pulmonary arterial hypertension.
• Platelet derived growth factor (PDGF) is responsible for inflammatory and smooth muscle cell migration and proliferation to the site of injury. PDGF receptor inhibitor imatnib mesylate delays wound closure together with reduction in myofibroblast number and fibronectin and collagen I.

TNF-a is a pro-inlfammatory cytokine expressed at the site of injury could also be a potential target since its overxpression in animal models develop heart failure.

Question 30

what is the aim of antifibrotic therapy after MI?

Answer 30

The aim of antiremodeling therapy after MI is to prevent, limit, or reverse adverse structural remodeling and thereby interrupt the sequence of LV dilation, LV dysfunction, disability, and death.
An important aspect of this goal is to protect the ECCM during remodeling after MI.

Question 31

Collective
evidence emerging from experimental and clinical studies using antiremodeling strategies after MI suggest that careful

attention should also be given to timing. why?

Answer 31

careful
attention should also be given to timing (Figure 2), especially because antifibrotic agents exert global actions that can affect both the IZ and NIZ . Experimental data on the temporal evolution of healing and ECCM remodeling suggest that these agents could potentially enhance adverse ECCM remodeling in the IZ during the highly vulnerable periods of very early and early stages of healing after MI (Figure 3; Table 3). Pending further safety data.

Question 32

The ability of fibroblasts to also establish functional heterocellular gap junctional coupling with caridomyocytes ?

Answer 32

The ability of fibroblasts to also establish functional heterocellular gap junctional coupling with caridomyocytes

in vivo is still under debate.

Question 33

What is a prerequisite for fibroblasts to accomplish its main task in the heart, that is, the generation and maintenance of a regular 3D network of extracellular structural proteins

Answer 33

The formation of 3D cellular network bby fibroblasts

Question 34

This heart skelton has two main functions:

Answer 34

(1) serves as a scaffold for cardiomyocytes that integrates contractile forces of individual cells as to result in an efficent pump function of the entire organ and
(2) ensures long-term integrity of the orderly 3D cellular architecture of cardiomyocytes, which itself is a prerequisite for the fast and uniform electric activation of the heart

Question 35

Is the heart skeleton static?

Answer 35

This heart skeleton does not represent a static entity but is subject to continuous turnover, which is largely under the control of fibroblasts that synthesise and secrete collagens, matrix metalloproteinasas, and tissue inhibitors of MMPs.

Question 36

There exist at least 4 distinct possibilities of interactions between fibroblasts and cardiomyocytes that may result in arrhythmogenesis:

Answer 36

1) Abnormal increase in connective tissue
2) Paracrine interactions
3) Mechano-eelctrical feedback
4) Direct electrical interactions

There is a possibility of arrhythmogenic cardiomyocyte-fibroblast interactions.

Question 37

How does cardiac fibrosis play a role in the development of electric and mechanical dysfunctions of the heart?

Answer 37

Cardiac fibrosis plays a central roel in the development of electric and mechanical dysfunctions of the heart. The main causes of cardiac fibrosis include mechanical overload of the heart, infarction, genetic predisposition, old age.
The large depositions of extacellular matrix disrupt the orderly arrangement of the cells, affecting both the functional efficacy of the heart as a pump and electrical coupling, predisposing to aberrant and proarrhythmic conduction.

Excess ECM proteins un the fibrotically remodelled myocardium form electrically insulating laminae that force impulse conduction to follow tortuous pathways reculting in slow overall conduction, discontinuous conduciton and blocks.
The severity of these disruptions depends on the amount and spatial distribution of connective tissue, and it is categorised into interstitial, compact, diffuse, and patchy fibrosis.

Question 38

Compact fibrosis

Answer 38

ormed by healed infarct scars is in fact the least arrhythmogenic despite its gross structural appearance, because reentry can only occur around the area of fibrosis and accordingly is only rarely observed in humans.

Question 39

Patchy fibrosis and severe interstitial fibrosis

Answer 39

bundles of cardiomyocytes are separated over extended distances by colalgenous septa, resultin in tortuous activations of the electric substrate. This fibrotic texture is highly vulnerab;e to arrhythmias.

Question 40

diffuse fibrosis

Answer 40

collagenous septa are relatively short and thus have a lesser impact on conduction. In sever fibrosis, however, this texture can induce slow conduction that favours spiral wave formation.

Question 41

Apart from causing abnormal conduction patterns, fibrosis also does what?

Answer 41

ibrosis also eases abnormal impulse generation because the success of a putative ectopic focus to drive the surrounding myocardium is dependent on the degree of electronic coupling of this focus to the surrounding normally polarised myocardium.

Question 42

Myofibroblasts:

what are they

Answer 42

Myofibroblasts (aka activated fibroblasts) contribute substantially to fibrinogenesis. Myofibroblasts are not normally present in the wokring myocardium but typically appear in the wake of insults to the heart such as mechanical overload and infarction. Myofibroblasts are thought to derive from resident fibroblasts undergoing phenotype swithc. Myofibroblasts differ from fibroblasts by the expression of alpha-smooth muscle actin.
Myofibroblasts synthesise collagen types I and II, and also produce cytokines and growth factors.

Question 43

what do myofibroblasts do? their regulation?

Answer 43

Myofibroblasts synthesise collagen types I and II, and also produce cytokines and growth factors.
Among these factors, transforming growth factor-Beta1 (TGF-Beta1) plays a central role by actin as a potent activator of fibroblasts and inducing their transforamtion into myofibroblasts.
Other factors involved in fibroblast activation and collagen overprduction include angiotensin II, endothelin-1, fibroblast growth factor 2, insulin-like growth factor, several interleukins as well as other molecues.

Question 44

fibroblasts paracrine effects might have a role in arrhythmogenesis?

Answer 44

Because of the close proximity of stromal and parenchymal cells, paracrine effects might have a role in arrhythmogenesis. Currently there exists little evidence in favour of such theory. However, studies in vitro suggest that paracrine fibroblast-cardiomyocyte interactions can cause alterations in cardiomyocyte electrophysiology. When cultured in the presence of cardiac fibroblast conditioned medium, it was found that gene expression for the fast sodium inward current channel Nav1.5 and the inward rectifier potassium chanel Kir2.1 was substantially reduced, resulting in slowed conduction. Trypsin treatment abolished this effect, suggesting that it was due to a protein secreted by fibroblasts.

Question 45

therapeutic efforts to address cardiac fibrosis

Answer 45

Therapeutic efforts are geared towards either suppressing fibroblast collagen production or ??? Interfering with collagen degradation. Central to these effors is the RAAS (both local and systemic) as it plays a eky role in excess collage production by fibroblasts. Therapeutic interventions towards RAAS have demonstrated beneficial effects on fibrosis and attenuation of arrhythmogenesis in anaimal models of senscence and in clinical studies, Statins represent a further class of drugs that have been shown to attenuate cardiac fibrosis in animal models of HCM, infarction, HTN. MOA of statins probably includespleiotropic effects ranging form improvement of endothelial dysfunction to anti-inflammatory and antiapoptotic activity as well as a modualtion of TGF-Beta1 levels.

Question 46

who cultured cardiomyocytes with cardiac fibroblasts? what’s the experiemtn

Answer 46

In 1969, Hyde and colleagues published a
meticulous study in which they measured membrane potentials
of cultured cardiac fibroblasts (10 to 20 mV), of
cardiomyocytes (55 to 75 mV), and of cardiomyocytes in
mixed cultures (50 mV).83 From the intermediate membrane
potential in mixed cultures and determinations of
length constants, they concluded that fibroblasts and cardiomyocytes
are functionally coupled and influence each other
by electrotonic interactions. This interpretation was supported
by the observation that fibroblasts connecting spatially disjunct
cardiomyocytes caused synchronization of spontaneous
activity, a phenomenon also reported in the same year by a
Japanese researcher.84 After many years of relative silence,
the topic of electrotonic cross-talk between fibroblasts and
cardiomyocytes has regained substantial attention over the
last decade when it was shown, as summarized below, that
myofibroblasts coupled by gap junctions to cardiomyocytes
have the potential to elicit arrhythmias by inducing slow

conduction and ectopic activity

Question 47

diac fibrotic
remodeling resulting from fibroblast activation not
only compromises mechanical pump function but is a powerful
and well-established arrhythmogenic condition on its
own due to its capacity to disrupt the normally uniform
electric substrate for propagation.
myofibroblasts have the potential to exert direct arrhythmogenic

effects on cardiomyocytes by

Answer 47

1) by causing electric
remodeling-based on paracrine interactions, (2) by affecting
cardiomyocyte electrophysiology through electro-mechanical
feedback, and (3) by acting as a source of injury current for
cardiomyocytes. Whereas there exists circumstantial evidence
that these 3 mechanisms might be operational in intact
hearts, unequivocal proof is still missing on account of the
current dearth of methodologies appropriate for directly

investigating these mechanisms in intact tissue.

Question 48

what are the unanswered Qs re fibroblast arrhythmogenesis

Answer 48

(1) What is the
relative contribution of myofibroblasts versus fibroblasts to
excess ECM deposition in fibrosis? (2) Is there a correlation
between the density/spatial distribution of myofibroblasts and
the frequency of occurrence of cardiac arrhythmias? (3) Do
myofibroblasts also establish functional heterocellular gap
junctions with cardiomyocytes in vivo? What is the conductance
of these junctions? (4) What ion channel repertoire do
myofibroblasts exhibit in vivo? Is it different from fibroblasts,
and is it appropriate to give rise to injury currents of
sufficient magnitude as to successfully induce slow conduction
and ectopic activity?