Cardiovascular Research 72 (2006) 375–383www.elsevier.com/locate/cardiores

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Review

Effects of static and cyclic loading in regulating extracellular matrixsynthesis by cardiovascular cells

Vishal Gupta, K. Jane Grande-Allen ⁎

Department of Bioengineering, MS-142, Rice University, PO Box 1892, Houston, TX 77251-1892, USA

Received 28 October 2005; received in revised form 18 August 2006; accepted 25 August 2006Available online 1 September 2006Time for primary review 17 days

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Abstract

Extracellular matrix (ECM) provides several structural and functional characteristics to tissues including cell support, mechanical integrityand biological signaling. In cardiovascular tissues, cells produce various ECM components such as collagen, elastin, proteoglycans, matrixmetalloproteinases, growth factors and signaling molecules. The cardiovascular cells (cardiac fibroblasts, cardiomyocytes, endothelial cells,and vascular smooth muscle cells) sense the changes in mechanical strains applied to them, through cell-surface receptors such as integrinsand ion channels, and adjust their expression and synthesis of ECM molecules in order to adapt their environment to these changes. ECMchanges due to altered mechanics are evident in numerous pathological situations including hypertension, cardiac hypertrophy, myocardialinfarction, myxomatous heart valve disease, and atherosclerosis. In hypertrophic conditions, for example, increased mechanical loading isinvolved with enhanced collagen synthesis, whereas in myxomatous and atherosclerotic conditions reduced mechanical strains areaccompanied by an accumulation of proteoglycans. Therefore, investigating the effects of various strain patterns on cardiovascular cells canenhance our understanding of ECM regulation and pathologies. This review focuses on the in vitro modulation of the synthesis of variousECM molecules through static or cyclic stretching of cardiovascular cells.© 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.

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Keywords: Connective tissue; Mechanotransduction; Extracellular matrix; Matrix metalloproteinases; Stretch

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1. Introduction

Mechanical stimulation is an important modulator of cellfunction and plays a critical role during tissue developmentand repair. Mechanical stimuli are transmitted to cells via theextracellular matrix (ECM), which provides an adhesivesurface for cells and structural organization to tissue. Cellssensing mechanical strains will then reciprocate by remodel-ing their surrounding ECM. The role of mechanical stimuliwas described first in bone remodeling and is now beingactively investigated for many tissue types. Cells within thecardiovascular tissues have been shown to respond tomechanical stimuli by modulating the synthesis of almostall major components of the ECM, including collagen,elastin, proteoglycans (PGs), glycosaminoglycans (GAGs),

⁎ Corresponding author. Tel.: +1 713 348 3704; fax: +1 713 348 5877.E-mail address: grande@rice.edu (K.J. Grande-Allen).

0008-6363/$ - see front matter © 2006 European Society of Cardiology. Publishedoi:10.1016/j.cardiores.2006.08.017

matrix metalloproteinases (MMPs), glycoproteins, andvarious soluble proteins such as growth factors.

The major cell types found in cardiovascular tissuesinclude cardiac fibroblasts, cardiomyocytes, endothelial cells(ECs), and smooth muscle cells (SMCs); all of these cellsinteract dynamically with the ECM in response to mechanicalstrains during development and disease [1,2]. Fibroblasts arethe major cell type in cardiac muscle, representing two thirdsof cardiac cells in number, and are mainly responsible forcardiac matrix production [3]. The other cell type in thecardiac muscle is cardiomyocytes, which primarily have acontractile role [4]. Blood vessels, in contrast, predominantlycontain SMCs and fibroblasts in concentric layers. All thesecardiovascular tissues are lined with endothelial cells, whichact as a semi-permeable barrier between the tissues and bodyfluids.

These various types of cardiovascular cells experiencecomplex mechanical strains, which are either mainly static or

d by Elsevier B.V. All rights reserved.

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cyclic in nature with myriad amplitudes and frequencies.Because distinctive strain patterns differentially affect ECMsynthesis according to the structural and functional needs ofthe tissues, it is important to understand the role of varioustypes of mechanical stimulation. This review will focus onthe modulation of ECM synthesis by cardiovascular cells inresponse to mechanical stresses and strains, with a particularemphasis on the effects of static vs. cyclic mechanical strains.

2. Extracellular matrix and non-matrix proteinsrelevant to mechanical loading of cardiovascular cells

Each ECM component fulfills a different structural orfunctional need in connective tissues but has also been shownto influence cell growth and migration. Collagen is the mostabundant protein in cardiovascular tissues; it is secreted bycells to provide tensile strength and serve as an organizationalscaffold. The other major fibrillar ECM protein is elastin,which provides elastic recoil and is therefore an essentialcomponent of arteries. PGs consist of one or more GAGchains attached to a core protein; PGs and GAGs servediverse biological functions, including as acting as “spacefillers,” within cardiovascular tissues [5]. The GAG hyalur-onan and the large PG versican, in particular, sequester largevolumes of water and provide resistance to repeatedcompressive loading, while the small leucine rich PGs suchas decorin and biglycan have been shown to contribute tocollagen fibrillogenesis [6,7]. In many pathological condi-tions of the cardiovascular system (i.e., hypertension,atherosclerosis, and myxomatous mitral valve disease) signi-ficantly altered profiles of GAGs and PGs have been found toaccompany alterations in mechanical strains within the tissue,which has spurred investigations into the effects of mecha-nical strains on GAG and PG synthesis [8,9]. PGs alsoinfluence cell proliferation, migration, and phenotype andtheir synthesis is in turn regulated by growth factors andmechanical strains [10]. Tissue homeostasis is maintained bythe synthesis of new matrix by cells and the degradation ofmatrix by MMPs and other proteases. Mechanical stimulinormally vary physiologically, but increased strains resultingfrom stenting, hypertension, or atherosclerosis may lead toenhanced matrix degradation and remodeling such as byMMPs [11]. The gelatinases MMP-2 and MMP-9 are theeasiest to study using zymography and hence are the mostcommonly characterized MMPs in these reports. Other pro-teases such as MMP-1, -8 and -14, which have collagenaseactivity, have also been investigated. In addition tosynthesizing ECM proteins and proteases, cells also secretea variety of signaling and adhesion molecules includinggrowth factors. Of these non-matrix mediators, transforminggrowth factor (TGF-β), which can influence proliferation,differentiation, and many other cell functions, and fibronec-tin, a glycoprotein involved in cell-matrix attachment andthus cell growth and migration, have been the most widelystudied with respect to mechanical loading [12–14]. Themechanical modulation of cardiovascular cells' secretion of

vascular endothelial growth factor (VEGF), platelet derivedgrowth factor (PDGF), fibroblast growth factor-2 (FGF-2),endothelin-1 and angiotensin-II has also been investigatedsince these proteins mediate cell growth, migration andsignaling [15,16]. The ECM itself directs the gene expressionof many transcribed proteins [17] by changing the physicaland chemical environment of the cells and the cytoskeletonassociation with mRNA through transmembrane receptors.

3. In vivo mechanical strains experienced bycardiovascular tissues and relation to ECM remodeling

Cells in the cardiovascular system are exposed to a com-plex variety of shear, tensile, and compressive strains.Vascular endothelial cells constantly experience shear strainsfrom blood flow, whereas pulsatile pressures result in bothtensile and compressive strains on the subendothelial cellswithin vascular and cardiac tissues. The nonhomogeneousand multiaxial strains experienced by the cardiac wall cellsare about 10% in magnitude on average [18,19]. Allcardiovascular cells experience these strains with everyheartbeat, i.e., at pulsatile frequencies close to 1 Hz.

Alterations in these physiological mechanical loads maycause compensatory remodeling of the ECM, a commonprocess in cardiovascular pathologies such as hypertension,cardiac hypertrophy, myocardial infarction, myxomatousheart valve disease, and atherosclerosis [2,20]. In hyperten-sion, the arterial mechanical strains reportedly increase by15% with a corresponding increase in collagen [21]. Cardiachypertrophy is also associated with excess collagen deposi-tion (fibrosis) due to mechanical overload. Atherosclerosis,which develops in part due to reduced and disturbed shearstresses, is characterized by accumulation of collagen, GAGsand cholesterol [8]. Myxomatous mitral heart valves, whichare enlarged and flail and thus are speculated to be underreduced tissue tension, have an overabundance of GAGs [9].To understand these pathologies in more depth, manyinvestigators have studied the effects of mechanical strainson ECM synthesis in vitro to clarify the relationships betweentissue microstructure, cell mechanotransduction, and loadingconditions using either commercial devices such as theFlexcell system [22] or custom built devices to apply mecha-nical strains in two-dimensional (2D) [23–26] or three-dimensional (3D) culture [27–29].

Mechanical strains regulate ECM synthesis through cell-matrix interactions, cytoskeletal rearrangements, and byopening stretch-activated ion channels, thereby activatingmembrane-bound enzymes or releasing growth factors in anautocrine or paracrine manner [13,30]. The transmembranereceptor integrins, which connect the ECM to the cytoskel-eton, have been shown to play key roles in transducingmechanical signals to the cell interior [31,32]. The subject ofcell mechanotransduction pathways has been widelyreviewed [15,16,33–35]. Mechanical stimuli also affectother aspects of cell phenotype such as their orientation,growth and differentiation [33]; the overall effects of

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mechanical strains on these characteristics of vascularsmooth muscle cells (SMCs) and cardiac fibroblasts havebeen reviewed elsewhere [33,36].

4. Effect of various types of mechanical strains on ECMsynthesis by cardiovascular cells

4.1. Cardiac fibroblasts

Cardiac fibroblasts experience high mechanical loads andremodel the ventricular and atrial ECM during development,growth, and pathogenesis. These cells are always under cyclicstrains whose magnitude and frequency vary with heart rateand pressure load. Cardiac chamber walls contain bothcardiomyocytes and fibroblasts with the latter serving asscaffolding primarily through maintaining a network of col-lagen fibers. Correspondingly, cyclic strains applied to cardiacfibroblasts have been found to modulate collagen synthesisand to cause the secretion of various growth factors into theECM.When these cells were subjected to cyclic loading, theircollagen I gene expression increased up to 4-fold compared tono loading conditions [37–40]. This response was similarwhether the cells were grown on untreated elastomericmembranes or membranes coated with collagen, fibronectinor laminin [40]; collagen type III mRNA expression by thesesame cells, however, did not change appreciably. Cyclicstretching has been shown to accelerate the degradation ofprocollagen, but not to the same extent that new procollagen isexpressed, resulting in a net increase [37]. In this same study,the exogenous addition of TGF-β to these stretchingconditions further increased procollagen mRNA expressionby 4.3-fold. In a different study, the cyclic-strain-inducedincrease in collagen synthesis was accompanied by increasedsecretion of TGF-β [38]. TGF-β can enhance collagenexpression in a direct or indirect manner, often via the renin-angiotensin system, which is highly relevant to collagenousscarring in the myocardium. There has been only one directcomparison of the effect of static and cyclic strains on collagensynthesis by cardiac fibroblasts [41]. In this study, 5% cyclicstrain (0.33 Hz) induced a 70% increase in the ratio of collagentype III to type I, whereas 5% static strain increased the ratio byonly 5% compared to nonstretched controls [41].

One additional report investigated the effect of staticstrains alone on ECM synthesis. Cardiac fibroblasts subjectedto uniaxial static strains showed a significant increase incollagen I, collagen III, and fibronectin expression with 10%strain, while 20% strain decreased collagen III andfibronectin expression when compared to nonstretchedcontrols [42]. Similarly, when tensile equibiaxial strainswere applied, cells subjected to 3% strain expressed morecollagen III and fibronectin but at 6% strain the mRNA levelsfor collagen III decreased as compared to nonstretchedcontrols [42]. In contrast, compressive equibiaxial strains ofeither 3% or 6% caused decreased expression of collagen IIIand fibronectin, suggesting that tensile strains might be theprimary signal for stretch-induced matrix synthesis by

cardiac fibroblasts. In the same study, TGF-β activity wasincreased at both 10% or 20% uniaxial strain and 6% biaxialstrain (either tensile or compressive); 3% biaxial strain had noeffect on TGF-β activity.

In general, mechanical strains increase the synthesis ofcollagen and release of growth factors by cardiac fibroblasts,which can cause myocardial hypertrophy [43]. This regula-tion of collagen synthesis is mainly mediated by TGF-β andMAP kinase pathways [38,39]. However, there is a paucity ofinformation regarding cardiac fibroblasts' synthesis of otherECM components and proteases in response to mechanicalstrains.

4.2. Cardiomyocytes

The cardiomyocytes are mainly involved with musclecontraction [4] yet they also produce various ECM moleculesand release growth factors. Physiologically, cardiomyocytesare exposed to cyclic loading but can also experiencehemodynamic (pressure) overload manifested as an increasein baseline static stretch. This hemodynamic overload causescardiac hypertrophy (increase in cell size), which cansignificantly affect cell structure and function [16]. Therefore,the effects of both cyclic and static strain are relevant to thesecells. Interestingly, much less attention has been given tocardiomyocytes than to other cardiovascular cells; only threestudies have investigated the effect of mechanical strains ontheir synthesis of extracellular proteins relevant to ECM andtissue remodeling, likely because chemical (altered bloodchemistry) and electrical (heart contraction) stimulations aremore relevant to cardiac muscle. Regardless, mechanicalstimulation has been shown to be important in hypertrophy. Intwo in vitro models of load-induced hypertrophy, staticuniaxial strains of 20% were applied to cardiomyocytes,resulting in significant increases in the secretion of endothelin-1 and angiotensin-II [44,45], both growth-promoting factorswhose release would activate phosphorylation cascades,initiate cell growth, and subsequently cause hypertrophy[45]. A third study reported that 20% cyclic stretch caused anincrease in MMP-2 and MMP-14 expression via theangiotensin II-JAK-STAT1 pathway; the JAK/STAT pathwayhas been demonstrated to be involved in cell-specific MMPexpression [46]. Although mechanotransduction pathwaysmay initiate signal transduction through many possiblemechanisms, growth factor secretion results in the release ofsecond messengers in stretch-induced cardiac hypertrophy[15,16,47]. At this time, no study has explored howother ECMconstituents such as collagen, elastin, and PGs are synthesizedby cardiomyocytes in mechanical stretch-induced hypertro-phic conditions, presumably because cardiac fibroblasts bearthe majority of this function.

4.3. Endothelial cells

All cardiovascular tissues are lined with a layer of en-dothelial cells, which experience both pulsatile pressures

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(normal tensile stresses) and shear stresses imposed by theflowing blood in the vasculature [48]. Shear stresses aretransmitted to the ECs through the glycocalyx (a thin layer ofglycoproteins and PGs surrounding the plasma membrane),stretch-activated ion channels, or integrin binding amongother possible pathways [49]. Because cyclic strains andshear stresses on ECs regulate vascular architecture, tone,and remodeling, their effects on the synthesis of variousECM molecules have been widely investigated. ECs mainlysynthesize GAGs, PGs, MMPs, fibronectin and a variety ofgrowth factors such as PDGF and FGF. These ECM com-ponents and growth factors are critical to anticoagulation,anti-atherogenicity, and growth of the underlying SMCs.

The range of shear stresses on ECs, which normally varywith the flow rate of blood during diastole and systole, canaffect the synthesis of various proteins. Shear stressesequivalent to laminar flow (1 dyn/cm2) decreased PGsecretion whereas high shear stresses (5 to 40 dyn/cm2)increased GAG and PG synthesis compared to static no-flowconditions. These results were shown both in non-pulsatileconditions for durations of 24 h or less [50–52] and inpulsatile conditions of 72 h duration [53]. These ranges ofhigh shear stresses were similar to those of veins (∼5 dyn/cm2) and arteries (∼23 dyn/cm2) [53], which indicates thathigh shear stresses are necessary for normal synthesis ofGAGs and PGs and to maintain homeostasis within vasculartissue. When pulsatile shear stresses were applied bidirec-tionally, as compared to static or unidirectionally, ECsincreased MMP-9 expression, which has been shown topromote atherosclerosis [54].

Cyclic strains have also been shown to modulate ECMsynthesis by ECs. High strains (4.9–12.5%) increased totalprotein synthesis and decreased fibronectin secretion into themedium [24]; the authors speculated that these high strainspossibly caused cell injury. In other studies, cyclic strains of10–24% decreased collagen and non-collagenous protein,but increased PDGF-B expression [55,56]. The expression ofMMP-2 and membrane type I MMP, partially mediated byp38 and ERK-dependent pathways, was found to increasewith cyclic strains in a magnitude and time-dependentmanner [57,58]. When aortic ECs were co-cultured withSMCs in tubular constructs, the cells decreased collagen andGAG deposition after 15 days under pulsatile shear stressconditions [59]. This study demonstrated the significance ofcell–cell interactions as the presence of ECs caused SMCs toexpress more of a contractile phenotype compared to a syn-thetic phenotype. Similar to growth factors, the cytoskeletalproteins are major intermediates in transmitting the strainsthrough integrins and regulating gene expression; Davies hascomprehensively reviewed hemodynamic mechanotransduc-tion within ECs [48]. Hemodynamics are also important inthe development of pathogeneses such as atheroscleroticlesions. The heparan sulfate GAGs that are synthesized byECs, located in their glycocalyx and underlying basementmembrane, have anticoagulation activity; hence, the ECslining vessels that are subject to high shear stresses are each

surrounded by a thick glycocalyx for improved anti-atherogenicity [60].

There has been only one study to directly compare theeffects of shear and cyclic strains on ECs. This study foundthat shear stress increased PDGF-B and bFGF expression,whereas cyclic strains had no effect [61]. These resultsindicate the importance of fluid shear in regulating ECsecretion of growth factors, which then regulate mitogenicactivity and govern the vascular structural response duringatherosclerosis.

Overall, investigators have shown that low shear stressesand cyclic strains decrease the synthesis of ECM moleculesby ECs. Static strains, which are less relevant to ECs, have notbeen investigated in this context. Cyclic strains on ECsdecrease matrix building and cell-matrix binding proteins(collagen, fibronectin) and increase matrix degrading pro-teins (MMPs), consequently reducing net ECM. In contrast,high shear stresses variably regulate ECM expression andsynthesis and maintain arterial wall tone and architecture.

4.4. Vascular smooth muscle cells

Smooth muscle cells, located in the medial layer of thevasculature, are responsible for vessel contractility and remo-deling during growth and pathogenesis. In vivo, SMCsmainly experience cyclic tensile strains due to pressure forcesof the blood and compression due to thinning of the vesselwall during inflation. Shear stresses experienced by the ECsin the blood vessels can also be transferred to SMCs but themagnitudes of these transferred stresses are low compared tothe tensile stresses [48]. As SMCs are the predominant celltype in blood vessels, there are abundant studies on themechanical regulation of ECM synthesis by SMCs.

Cyclic strains tend to increase collagen and elastin syn-thesis by vascular SMCs but these responses are sensitive tothe strain magnitude, frequency and duration [28,62–64]. In astudy of very high strains (25%) at 0.05 Hz, a significantincrease in total protein and collagen synthesis could be seenonly after 5 days of stretching [65]. The authors attribute thisdelay to the extremely high strain, which might have da-maged the cells; the frequency may have also been too low toprovide proper mechanical stimulus. Another study showedthe dependence of total protein and collagen synthesis onstrain magnitude; synthesis was increased between 5% and10% strain, but then was unchanged between 10% and 20%strain, compared to nonstretched controls [66]. In a differentinvestigation using 4% cyclic strain, collagen I expressionincreased to a maximum at 12 h, then remained constant up to48 h [67]. Although the majority of these reports noted thatstrain increased overall protein synthesis, there is oneexception. Kulik and Alvarado reported a slight decrease inprotein and collagen synthesis when pulmonary arterialSMCs were subjected to cyclic stretch using strains of 10 or20% and frequencies of 0.33–0.5 Hz [26]. Possibleexplanations for these unexpected findings included theloss of cell surface receptors, a lack of cell adhesion

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molecules, or insufficient stretching time (3 or 6 h only). Theculture environment is also highly relevant. For example, theaddition of serum did not cause any increase in total protein orcollagen synthesis by cyclically stretched aortic SMCs, where-as in serum-free medium the total protein and collagen syn-thesis doubled over stationary controls [68]. When seeded in3D tubular collagen scaffolds and cyclically stretched (2.5–10%, 0.5 Hz), SMCs demonstrated no change in collagensynthesis, but elastin synthesis increased enormously [27]. Inanother 3D study, when SMCs were seeded together withECs on a polyglycolic acid (PGA) scaffold, collagen contentincreased under pulsatile conditions (5% radial distension,2.75 Hz) compared to non-pulsatile conditions [69]. Thesevariations in elastin and collagen contents alter tissue materialproperties, emphasizing the critical role of mechanical strain intissue remodeling and tissue-engineering applications.

Cyclic mechanical strains generally increase the GAG andPG synthesis by cardiovascular cells but the increases areoften specific to certain types of GAGs. For example, cyclicstretching of aortic SMCs in 2D culture tripled the synthesisof the GAGs hyaluronan and chondroitin 6-sulfate overstationary cultures, but did not affect the synthesis ofchondroitin 4-sulfate and dermatan sulfate [62]. Similarly,cyclic stretching increased the expression of the PGsversican, biglycan, and perlecan, but decreased the expres-sion of the PG decorin [67]. These two reports are consistentas versican contains abundant chondroitin 6-sulfate andaggregates with hyaluronan, whereas decorin is associatedwith dermatan sulfate. SMC expression of the heparan sulfatePG syndecan-4, a cell-adhesion molecule colocalized withintegrins in focal adhesions, was increased after 1 h of biaxialcyclic stretching (10%, 1 Hz) but then decreased over 24 h[70]. The expression of syndecan-4 did not change whenstrain was lowered from 10% to 3%. Mechanical stretch alsocaused syndecan-4 shedding from the cell surface, whichserved to promote cell motility via decreased focal adhesions.

MMP synthesis by SMCs has also been modulated withthe application of cyclic strains, but different types of MMPsare regulated in distinct ways. One study found a significantdownregulation of MMP-1 (collagenase) with 4% cyclicstretching [71] whereas another study reported no changes inthe expression of MMP-2 or MMP-9 (both gelatinases) [72].Two other studies, however, did find an increase in MMP-2with cyclic strains ranging from 10% to 16% [64,73]. In atubular collagen construct, which provides more of an invivo-like 3D environment, the application of 10% cyclicstretch to the embedded vascular SMCs increased MMP-2levels more than 5-fold compared to nonstretched controls[29].

Cyclic stretching has almost uniformly been found toincrease SMC synthesis of growth factors and signalingmolecules. The expression and secretion of TGF-β, PDGF,and VEGF increase substantially with cyclic stretching[64,71,74–76]. Cyclic strain-induced secretion of TGF-β andFGF-2 into the culture medium was found to be time and straindependent [25,77]. These growth factors regulate SMCgrowth,

differentiation and gene expression in an autocrine or paracrinemanner. For example, secreted TGF-β promoted L-prolinetransport and hence collagen synthesis and cell growth duringarterial remodeling in hypertension [77].

Although the role of static stretching on ECM synthesishas received less attention than cyclic stretching, severalreports have examined its effect on SMCs. Static stretch hasbeen reported to increase SMC synthesis of tropoelastin, theprecursor of elastin [78]. Three studies in particular havedirectly compared the effects of static and cyclic strains[11,79,80]. In the first study, cyclic strain (10%, 0.86 Hz),compared to 10% static strain, caused a 3-fold increase intotal protein and collagen synthesis [79]. Moreover, artifi-cially generating an increase in intracellular cAMP levels (byadding theophylline, inhibitor of cAMP degrading enzyme)inhibited the collagen synthesis in cyclically stretchedcultures only, suggesting that cAMP was involved in theresponse to cyclic stimuli but not static stimuli. cAMPmediates protein synthesis by SMCs; its levels decreaseduring hypertension and are inversely related to increasedcollagen synthesis [81]. In the second study, a 50-foldincrease in MMP-2 mRNAwas found after 24 h of 5% staticstretch but no change was found in the absence of stretch or incyclic stretch (1 Hz) [11]. Additionally, secretion of MMP-2and MMP-9 increased with static stretch, but decreased withcyclic stretch. The stretching protocol of the third studysimulated an arterial balloon injury to explain the tightregulation of syndecan-4 expression and cell migrationduring the injury process [80]; the high (30%) static strainsrapidly increased syndecan-4 expression compared to 10%cyclic strains but subsequent application of 5% cyclic strainsdownregulated the expression. All three of these studiesclearly demonstrated that static strains promote ECMdegradation and adhesion molecules whereas cyclic strainsenhance SMC synthesis of fibrillar ECM proteins.

Alternative mechanical loading conditions such as centri-fugal force and shear stresses have also been used to inves-tigate ECM synthesis by SMCs. Although centrifugal forcesare less physiologically relevant to SMCs than the methodsdiscussed above, they serve as a simple tool to applycircumferential stresses, as found in hypertension. Centrifu-gal forces applied to vascular SMCs increased total GAGsynthesis, predominantly affecting heparan sulfate (found inthe glycocalyx and basement membrane) but minimallyaffecting hyaluronan (which can be present in either thepericellular matrix or in the ECM) [82,83]. These are thesame trends found with the application of static strains, whichshow some mechanical equivalence to centrifugal forces.Shear stresses have also been investigated because they aretransferred from ECs to SMCs in vivo. Shear stresses (10–20 dyn/cm2) applied to SMCs decreased MMP-2 activationand significantly inhibited cell migration; SMC migrationfrom the media is a characteristic finding in intimalhyperplasia [84,85].

Overall, cyclic strains applied to SMCs increase ECMbuilding proteins, degrading proteases, and signaling molecules

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whereas static strains and shear stresses downregulateECM degrading proteases. ECM synthesis also depends onstrain magnitude and durations. The results were also foundto be sensitive to cyclic frequency and the culture condi-tions such as 2D vs. 3D or biaxial vs. uniaxial strain.

5. Summary and future directions

Cardiovascular cells show selective responses to differenttypes of strains (Table 1). In general, the literature reviewedhere reports that strains enhance the synthesis of most ECMcomponents in cell culture. Although cells tend to respondrapidly to strains in a magnitude dependent manner, there areno overwhelming trends regarding time dependence. Cyclicstrains are the obvious choice to apply mechanical stimuli tocardiovascular cells, which experience cyclic stresses underpulsatile blood flow. However, in certain circumstances suchas when failing hearts are supported by continuous-flow leftventricular assist devices (LVADs) [86], the cardiac cells willexperience predominantly static tensile stresses. The growinguse of these continuous-flow LVADs mandates understand-ing the effect of static strains on ECM synthesis. From thelimited investigations regarding static strains published todate, the comparative effects of static vs. cyclic strains appearto vary for different ECM macromolecules. Cyclic strainstend to affect collagen and elastin synthesis more profoundlythan do static strains whereas the reverse is true for MMP-2synthesis. Given that MMP expression increases in heartfailure and that many patients are put on LVAD support [87],the potential for alterations in MMP expression warrantscontinued in vivo and in vitro research to understand theeffect of various loading conditions on ECM synthesis anddegradation. Furthermore, although SMCs and collagen werethe focus of most of these reports, changes in other ECM

Table 1Overview of ECM modulation studies for cardiovascular cell types

Cell type ECM protein changes with strain

Static Cyclic

Cardiacfibroblasts

↑ Collagen [41,42] ↑↑ Collagen [37–41]↑↓ Others [42] ↑↑ Others [38]

Cardiomyocytes ↑↑ Others [44,45] ↑↑ MMPs [46]Endothelial

cells↑ GAGs and PGs [50–52] ↓↓ Collagen [55,59]↑↑ Others [61] ↑↓ GAGs and PGs [53,59]

↑↑ MMPs [54,57,58]↑↓ Others [24,56]

SMC ↓ Collagen [79] ↑ Collagen[62,26–28,64–69,79]

↑↑ Elastin [78] ↑↑ Elastin [27,28,63]↑↑GAGs and PGs [80,82,83] ↑↓ GAGs and PGs

[62,67,70,80]↓ MMPs [11,84,85] ↑ MMPs [29,64,71–73]

↑↑ Others[25,64,71,74–77]

“Others” include total protein, growth factors and signaling molecules.Double arrow = 100%, single arrow = 60–99%, combined arrow = 40–60%,contradictory studies, or different responses to various strain magnitudes.

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components such as GAGs, PGs and MMPs are prominent inmany cardiovascular pathologies, so the effects of strains ontheir synthesis should be given more emphasis in the future.

Many culture conditions have been used to apply mecha-nical strains to cardiovascular cells. 2D cell culture is ap-pealing due to easy handling, maintenance, and manipulationof the mechanical strains. In addition, tissue-engineeringtechniques have recently been used to characterize vascularcell phenotype and modulation of ECM synthesis [88,89].The cell shape and pericellular environment in 3D cultures ismore like those found in native tissues and cells may utilizecell-matrix interactions (i.e., focal adhesions) and mechan-otransduction signaling differently than when they are in 2Dcultures, although admittedly mechanical strains can be morecomplex to apply in 3D [90]. In the future, 3D cultures maybecome more widely used to characterize cellular responsesto mechanical strains.

The effect of cyclic strains on ECM synthesis appears to bespecific to the different cell types in cardiovascular tissues.For example, collagen synthesis increased with strain inSMCs and cardiac fibroblasts, whereas the body of responsesin ECs is less uniform. Another cardiovascular cell type isheart valve interstitial cells, which resemble myofibroblasts[91]. Heart valve cells' responses to mechanical strains haveonly recently been explored in 2D and 3D culture systems[92,93] although no study has focused exclusively on ECMmodulation. Porcine aortic valve interstitial cells producedmore protein and GAGs than porcine aortic SMCs in 3Dculture [93]; clearly, valve cells require further investigationas they are responsible for valvular remodeling and disease.Among all the reports of cardiovascular cells, inconsistenciesregarding the matrix production elicited by mechanicalstimuli may be due to different breeds, species, and ages ofanimals from which the cells were derived. Furthermore,variations in ECMgene expression with stretchmay be due tothe effects of pure tensile or compressive strains imposed bydifferent stretch devices. In addition to the stretch applied tothe cultured cells, their metabolic state, culture conditions,and accumulation of secretory products likely also affect theirsynthesis of matrix components and matrix mediators.

While the role of integrins in mechanotransduction is wellknown [48], other membrane-bound proteins such as theADAMs (a disintegrin and a metalloproteinase domain) alsosupport integrin-mediated cell adhesion because they cancleave ECM proteins by their metalloproteinase domains[94]. Recently, a new mechanism of mechanotransductionwas proposed in which mechanical stimulation encouragesgrowth factor shedding into the ECM [95]. Because ADAMshave the ability to shed many cell-adhesion molecules andcell-surface proteins including cytokines and growth factors,they may be key regulators in mechanotransduction signalingpathways and their synthesis by cardiovascular cells certainlymerits further investigation. The relevant intracellular signal-ing pathways, however, may differ for various types ofstrains. Numerous mechanotransduction pathways (mostinvolving MAP kinase) for specific cell types have been

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proposed [15,16,20,32–35,48,49], but only a few reportshave indicated which pathways are specifically activated bystatic stresses. The application of static strains to cardiacfibroblasts, for example, activated G protein subunits [96]and the ERK2 or JNK1 pathways [97], whereas release ofendothelin-1 from cardiomyocytes under static strains acti-vated MAP kinases and Raf-1 in stretch-induced cardiachypertrophy [45]. It has yet to be determined which particularpathway is required for regulation of the synthesis of each ofthe ECMmolecules under the diversity of mechanical stimuliexperienced in vivo. Overall, determining the precise role ofstatic and cyclic mechanical stimulations on the regulatedsynthesis of various cardiovascular matrix macromoleculesshould remain an intriguing area of research for many years tocome.

Acknowledgement

This work was supported by the Whitaker Foundation.

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