Cardiovascular Research

Cardiovascular Research 1999 42(1):27-44; doi:10.1016/S0008-6363(99)00021-8
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Regulation of cardiovascular collagen synthesis by mechanical load

Jill E. Bishop^* and Gisela Lindahl

Cardiovascular Research Group, Centre for Cardiopulmonary Biochemistry and Respiratory Medicine, University College London Medical School, The Rayne Institute, 5 University Street, London, WC1E 6JJ, UK

Jill.Bishop{at}ucl.ac.uk

^* Corresponding author. Tel.: +44-171-209-6972; fax: +44-171-209-6973

Received 22 October 1998; accepted 10 January 1999

KEYWORDS Collagen; Heart; Artery; Mechanical load

	1 Introduction

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
The cardiovascular system is constantly exposed to mechanical perturbation from shear and tensile stresses. During development cardiovascular cells respond to changes in mechanical load; growing, dividing and laying down extracellular matrix. Changes in the normal levels of these forces then have further profound effects on these cells resulting in abnormal changes in cardiovascular structure and consequently function. These remodelling processes suggest that the mechanical environment is a key modulator of cell function.

The importance of mechanical forces in the regulation of tissue growth, development and disease has been appreciated for many years. Early studies in the 1960–70s demonstrated, for example, the importance of mechanical load in skeletal muscle growth and development. It was determined that even in the presence of adequate nutrition, and with hormonal and neuronal control, skeletal muscle would not grow without mechanical stimulation [1–3]. The reverse is also true – disuse of a skeletal muscle leads to atrophy [4]. Similarly bone growth and remodelling is dependent on continuous stimuli of pressure and tension [5]. Growth of the lung is also partly regulated by mechanical forces [6]. The response of the cardiovascular system to mechanical stimuli is therefore not unique, however the ability of the cardiovascular system to respond to changes in physical forces by changing the physical properties of the cardiovascular tissues in an attempt to normalise these forces (see Fig. 1) – makes this reciprocal interaction between structure and function and the mechanical environment an extremely fascinating area of study.

View larger version (20K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Reciprocal relation between mechanical forces and cardiovascular remodelling The cardiovascular system responds to changes () in haemodynamics by cell hypertrophy, proliferation and extracellular matrix deposition. This tissue remodelling results in changes in the physical properties of the tissues which may be sufficient to counteract the altered haemodynamics. If additional stimuli, such as tissue injury, are present the cellular response may be more severe resulting in an over-production of matrix components. This excessive deposition of matrix may then have a further deleterious effect on the haemodynamics.

 
Research into the role of mechanical force in tissue growth has been advanced considerably over the last 10 years or so with the development of devices designed to subject isolated cells to a variety of mechanical loads including shear stress, mechanical strain, tension and compression. These systems have enabled us to explore the direct effects of defined forces on the function of specific cell types including cells of the cardiovascular system, bone, tendon, lung and skeletal muscle in the absence of humoral, neuronal or nutritional regulation.

The aim of this review is to outline physiological and pathological situations in which mechanical forces have an impact on cardiovascular extracellular matrix composition – the extracellular matrix being important in determining the structure and function of the cardiovascular system. In vitro studies that support the concept that mechanical forces directly influence cell function will be described including the advances being made in the elucidation of the pathways of mechano-signal transduction. Interaction between growth factors, extracellular matrix and mechanical load, in terms of the mechano-transduction process, will be highlighted specifically. Readers interested in the regulation of myocyte growth by mechanical load are directed to other review articles [7,8]. This subject will be discussed peripherally, particularly in relation to signal transduction where studies have been more extensive than in matrix-producing mesenchymal cells.

	2 Stimulation of cardiovascular matrix remodelling in the heart and vasculature

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
Extracellular matrix remodelling occurs in both blood vessels and the heart in response to increases in mechanical forces such as raised blood pressure and flow. Although there is strong evidence for a direct effect of mechanical load on myocyte growth, the regulation of collagen deposition appears more complex. Mechanical load may directly affect mesenchymal cell activity and/or the stimulus may come from the release of profibrotic growth factors produced either in direct response to the mechanical load, in a paracrine or autocrine manner, or as a result of the underlying pathology that produces the increase in blood pressure. A key player here may be angiotensin II which is further discussed below. For the sake of simplicity, reference is made here only to the early compensatory growth which is more likely to involve a direct stimulus by mechanical load. Later pathological consequences, although clearly still influenced by the mechanical environment, are likely to include tissue repair responses to cell injury and necrosis, and may be further complicated by confounding factors such as hypoxia/anoxia and by cardiovascular risk factors and ageing.

2.1 Vascular remodelling
During the development of hypertension there is rapid cellular activation involving changes in cell morphology and phenotype, leading to cell hypertrophy, hyperplasia and the deposition of extracellular matrix proteins, particularly collagen and elastin. These events lead to a thickening of the blood vessel wall. All cells of the vasculature are involved, with increased replication of endothelial cells in the intima, smooth muscle cell hypertrophy, hyperplasia and matrix production in the media, and replication, recruitment and increased matrix production by fibroblasts in the adventitia [9–12]. Increased synthesis of collagen and elastin occur during the development of hypertension in both the pulmonary [13,14] and the systemic circulation [11,15]. Evidence from the application of mechanical load to vessels in organ culture systems indicate that mechanical load may directly stimulate increased metabolism of these extracellular matrix proteins [16,17] and selectively regulate cell proliferation [18].

Blood vessels are exposed to several forms of mechanical force – shear stress, pressure and tensile stress. The latter has several components including a circumferential stress caused by expansion or dilation of the vessel wall and internal stresses generated by the cells themselves in response to the external forces. Endothelial cells, lining blood vessel walls are exposed to all these forces and, as the biological interface between the blood and the underlying vascular tissue, may function to transmit information regarding changes in these forces to the underlying cells. This may be mediated by the production and release of vasoactive substances and growth factors by endothelial cells in response to the changes in shear and tensile stresses. Shear stress, for example, stimulates the production of polypeptide growth factors by endothelial cells including platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) [19] – factors which enhance mesenchymal cell proliferation and matrix production. These factors may therefore stimulate the vascular remodelling process compensating for the increase in tensile wall stress.

Given the wealth of literature on both shear and mechanical load, the remainder of this review will be limited to the known effects of mechanical load only on cardiovascular cell function. Interested readers are directed to the reviews by Resnick and Gimbrone [20], Reinhart [21] and Nerem et al. [22] for further information on the cellular effects of shear stress.

In vitro, increased cyclic strain also effects production of vasoconstrictor agents by endothelial cells –increasing endothelin-1 production, for example [23]. In addition to its vasoconstrictor effect endothelin-1 is also a vascular smooth muscle cell and fibroblast mitogen and chemoattractant [24–26]. Endothelin-1 is increased in the pulmonary arteries during the initial stages of hypoxia-induced hypertension, and, since it stimulates pulmonary artery fibroblast procollagen synthesis, replication and chemotaxis, may have a dual role in the hypertensive remodelling process [26].

Increased transmural pressure also leads to an increase in the movement of blood-borne substances across the vessel wall [27]. Even molecules as large as fibrinogen (the 340 000 protein cleaved to form fibrin during clot formation) pass through the vessel wall to the adventitia. This movement of macromolecules may occur either as a direct effect of the pressure increasing mass flow, or through perturbation of the endothelium leading to increased permeability. Transport across the vessel wall of other blood-borne factors, such as plasma fibronectin, also increases during the development of hypertension [28]. Such blood-borne factors may then directly influence the vascular remodelling process. Thrombin and angiotensin II, for example, have been shown to stimulate procollagen synthesis and mesenchymal cell replication [29–32]. The accumulation of these factors in the vessel wall, may provide additional stimuli for the development of vascular remodelling, including the perivascular fibrosis that occurs during the development of pressure-overload induced cardiac hypertrophy.

There are several pathological situations where inappropriate mechanical load may cause vessel remodelling. The high rate of failure of vein grafts compared to arterial grafts, for example, may be due to the response of venous smooth muscle cells to pulsatile pressures to which they are not normally subjected. Predel et al. [33] demonstrated that subjecting saphenous vein smooth muscle cells to cyclic load caused an increase in thymidine incorporation and cell proliferation, whereas internal mammary artery smooth muscle cells, which are accustomed to such pulsatile pressure, showed no such response, thus demonstrating that perhaps the stenosis that occurs is partly due to placing the vein in a foreign mechanical environment. Remodelling is then an attempt at an adaptive response necessary to redress the balance between load and vessel architecture. Restenosis following balloon catheterization may also be a response to mechanical stimuli, but in this case the response may be driven by mechanical damage in addition to mechanical perturbation. Glagov [34] recently postulated that restenosis may be related, at least partially, to a reactive–adaptive remodelling process in which the mechanical environment plays a key role.

2.2 Cardiac hypertrophy and fibrosis
The myocardium is exposed to cyclical stresses during the cardiac cycle [35]. Changes in blood pressure or volume affect systolic and/or diastolic wall stress which stimulate ventricular growth. Pressure overload has a significant effect on both contractile and extracellular matrix protein synthesis. Total protein and procollagen fractional synthesis rates increase in response to pressure overload in several models [36–39]. The latter is achieved by an increased procollagen gene expression by fibroblasts – the principle collagen producing cell in the heart [36,40], an increase in the efficiency of protein synthesis [41] and an increase in fibroblast proliferation [39,42,43].

The nature of the force may influence the cellular response, and this certainly appears true for the collagen-producing fibroblast. Both volume and pressure overload stimulate increased collagen synthesis, but in the case of volume overload, or early phases of pressure overload this is matched by the myocyte hypertrophy, resulting in an enlarged ventricle with a normal collagen composition. However in chronic or more severe pressure overload collagen deposition exceeds the hypertrophic response leading to excessive collagen deposition and fibrosis (Fig. 2). Although the precise mechanisms involved in the transition from normal expansion of the collagen mass to fibrosis during the development of cardiac hypertrophy are not fully understood, there is growing evidence to suggest a role for the locally produced growth factors such as TGF-β [40,44] (see Table 1). There is considerable data supporting a role for angiotensin II in the development of cardiac fibrosis [45–47]. In addition, insufficient perfusion and oxygen supply in the more severe instances of pressure overload may lead to regions of myocyte necrosis and subsequent collagen deposition during scar formation. A process initiated by a change in the mechanical environment may therefore progress with additional regulation by locally produced factors (which may initially be produced in response to the mechanical load). During compensatory growth this permits parallel increases in myocyte growth and collagen deposition, whereas later excess deposition of collagen may occur as a result of a combined effect of the mechanical stimulus and a change in the growth factor profile with excessive levels of profibrotic agents derived from the blood, inflammatory cells or produced by resident cells.

View larger version (41K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Myocyte hypertrophy, collagen deposition and cardiac fibrosis. A schematic representation of the variation in the nature of collagen deposition that constitutes the development of fibrosis during cardiac hypertrophy. During the development of cardiac hypertrophy or following ischaemic damage collagen deposition may match the myocyte growth or be excessive. The former represents a compensatory hypertrophy, the latter results in fibrosis. Reprinted with permission from Elsevier Science.

 

View this table: [in this window] [in a new window]   Table 1 Growth factors that regulate cardiovascular fibroblast function

 
Following myocardial infarction, in addition to the collagen deposited at the site of tissue damage, as a wound healing response (reparative fibrosis, [48]) there is enhanced collagen deposition distal to the infarct (reactive fibrosis) [49] (see also Fig. 2). Infarction leads to the production of TGF-β [50] and angiotensin II [51,52] in the surrounding tissue which may serve to stimulate both myocyte hypertrophy and matrix deposition. The fibrosis remote from the infarct site may also be the result of a direct stimulation of cell function by growth factors and/or increased mechanical load. The infarct and subsequent scar will contract, partly due to the presence of myofibroblasts [53], pulling on the surrounding tissue. There will also be a need for an increase work-load by the viable muscle.

	3 Regulation of procollagen gene expression

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
The major fibrillar collagens in the heart and vasculature are types I and III. Type I forms thick bundles of fibres whilst type III forms finer, more reticulate fibres. Both types, synthesized by fibroblasts in the myocardium and fibroblasts and smooth muscle cells in the vessel wall, may co-exist within bundles of fibres. These collagens impart stiffness on the ventricle and vasculature, and the concentration and relative proportions of these types may subsequently affect myocardial and vascular compliance. Changes occur in the composition of the collagen network during the development of cardiac hypertrophy and fibrosis [38,54–56]. In animal models, an increase in type III collagen is often seen early on [55], followed by a large and sustained increase in type I [38].This may reflect the extent of tissue damage and wound healing, since early deposition of type III followed by type I is characteristic of the processes observed during tissue repair.

The syntheses of these collagens are regulated by separate genes which in vitro appear differentially regulated in response to mechanical load and growth factors. With respect to the effect of mechanical load, Carver et al. [57] found an increase in only type III with 24 h of cyclical mechanical load in cardiac fibroblasts in vitro, whereas we observed an increase in type I later, after 48 h [58]. The mechanisms involved in the differential regulation of the two collagen types during the development of cardiac hypertrophy and fibrosis are poorly understood, however this may reflect the diverse regulatory signals acting in different cell types in vivo, combined with the considerable sequence differences that exist in the regulatory regions of these co-expressed genes [59,60]. For example, the heterodimeric CCAAT binding factor shown to activate the proximal promoters of the 1(I) and 2(I) genes, does not bind the equivalent segment of the 1(III) procollagen promoter [61].

Despite the fact that these sequences have been known for a long time, and there have been extensive studies over the past 10 years, relatively little is known about the regulation of the procollagen genes. This can perhaps be explained by the great complexity by which these genes seem to be controlled. The transcriptional regulation of the two procollagen type I genes, 1(I) and 2(I), which are the better characterized, is necessarily complex since they are spatially and temporally expressed at widely different levels during, for example, tissue remodelling and wound healing, and in a cell- and tissue-specific fashion during development. In general, the analyses suggest that the regulatory sequences of these two co-ordinately expressed genes are organised somewhat differently. For example, transcription of the murine 1(I) collagen gene in distinct mesenchymal cell lineages appears to be under the control of separate cis-acting elements spread within 3.5 kilo base pairs of sequence 5' (upstream) of the transcription start site [62,63]. However, the equivalent specificity of the mouse 2(I) collagen gene seems to be confined to the 350 base pair proximal promoter [64,65], although enhancer regions have been identified further upstream [66]. In addition to the proximal promoter region and sequences further upstream, the expression of the 1(I) procollagen gene has been shown to be under the influence of sequences in the first intron, however again this is an area of some controversy [67,68]. The intronic regions in the 2(I) gene also appear to be important, since a DNaseI hypersensitive site similar to that seen in the 1(I) collagen gene is indicative of regulatory activity. Through the work by de Crombrugghe and coworkers, who have recently cloned a negative regulator designated BFCOL1 (previously IF1) [69], evidence is accumulating to support the notion that although structurally different and distinctly arranged, some regulatory sequences of the type I collagen genes bind the same trans-acting factors, which could account for their co-ordinate expression.

The stimulation of the two genes in response to the growth factor TGFβ1, appears to be regulated at different sites [70–72], although there have been conflicting reports as to which precise elements and transcription factors are involved [73,74]. To further demonstrate the complexity of these studies, the control of the TGFβ1 response in the 1(I) gene appears to be organised distinctly in different species [75,76]. Taken together, DNA binding sites for the transcription factors AP1, NF-I, Sp1 have all been implicated in the TGFβ1 response. The mechanisms involved in the response to mechanical load and growth factors other than TGFβ1 (e.g. serum and PDGF), on the transcription of the 1(I) and 2(I) genes, are as yet unexplored (further, see Section 6.6). Studies elucidating these would greatly enhance our understanding of the pathways and factors involved in the regulation of expression of procollagens during pressure overload.

	4 Influence of the extracellular matrix on the mechanical loading of cardiovascular cells

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
Cells in the vessel wall and within the myocardium are surrounded by a highly organised network of extracellular matrix proteins which provide the tissue with strength, flexibility and structural definition. The most abundant components of the extracellular matrix are the fibrillar collagens (types I, III and V), the basement membrane collagen type IV, elastin, fibronectin, laminin and proteoglycans. The relative concentration and organization of these components influences the physical characteristics of the cardiovascular tissue. The composition of the matrix also influences how the cells perceive the forces applied to the tissue. This role takes several forms: firstly the three dimensional structure of the matrix and the resulting ‘stiffness’ will influence the extent to which the cells within the matrix may be physically deformed, for example, cells within a highly collagenous vessel may be less susceptible to deformation than those in a more elastic vessel. Secondly, the matrix may influence the alignment of cells within the matrix and thus determine which aspect of the cell is exposed to the force, which will bias the extent and direction of force to which the cells are exposed. Thirdly, all cells, not only myocytes and smooth muscle cells, are able to generate tension, through their cytoskeleton. The ability to contract and the subsequent tension generated depend on the nature of the matrix in which the cells lie and are adhered to. Rigid matrices resist deformation and therefore permit force generation within the cells as they contract, whereas a more malleable matrix deforms as a result of cell contraction and little tension will develop. Forces generated by a cell as it contracts greatly influence cellular activity, and more specifically, alter its response to growth factors [77]. Cell–matrix interactions are central to the mechanisms of load perception and load transmission. The role of integrins in these processes are discussed in some detail below.

What is most interesting, regarding the extracellular matrix is that not only does it influence the mechanical load that the cells perceive, but such load regulates the amount and composition of extracellular matrix proteins that the cells produce. Thus there exists a ‘dynamic reciprocity’ between matrix and load – the term originally coined by Bissell et al. [78] to describe the effect of matrix composition on the cell’s matrix production (see Fig. 1).

	5 Effect of mechanical load on cardiovascular fibroblast function

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
In order to study the direct effect of load on cell function, cell straining devices have been developed to subject isolated cells to known mechanical strain. Detailed lists of types of apparatus developed and the nature of the load applied have been produced elsewhere [79,80]. In 2D-straining devices, cells are grown on an elastic membrane (usually a silicone elastomer) coated with an extracellular matrix protein to aid adherence, and the membrane is stretched or distorted uni- or bi-axially producing either a static strain, or with the aid of a motor or vacuum pump, a cyclical load. There are also a number of 3D systems in which the cells are grown within a matrix, often a collagen gel. The gel may be free-floating, permitting the cells to contract the collagen gel [81]; tethered, enabling the cells to develop a contractile force [82]; or stretched unidirectionally producing an equal and opposite force to that developed by the contracting fibroblasts [83]. Measurements of mechano-signal transduction, protein synthesis and cell proliferation are made seconds, minutes, hours or days after the onset of the load. As will become clear, the nature of the matrix and the load applied may differentially modulate cell phenotype. Except where otherwise stated, results given from in vitro data represent experiments in which cells were grown in monolayers and subjected to a 2D cyclical load.

5.1 Stimulation of cell replication by mechanical load
A common in vivo response to increased mechanical tension by mesenchymal cells of the vasculature and myocardium, is cell proliferation. Mechanical load also stimulates cell proliferation in vitro. The response however appears to be cell-type specific, tissue specific and may depend on the cell’s ability to produce autocrine mitogens in response to load. Cyclic load stimulates endothelial [84] and smooth muscle cell proliferation (see Table 2. This is true for aortic [29], human coronary artery [85], and saphenous vein smooth muscle cells [33]. However, Leung et al. [86] found no effect on thoracic aorta smooth muscle cell replication, although this may be due, in part, to the elastin matrix on which the cells were cultured in the latter experiment [86] – in the other experiments cells were grown on collagen. Wilson et al. [87] have demonstrated that vascular smooth muscle cell replication in response to load is dependent on the nature of the matrix on which the cells are grown, with cells grown on a fibronectin or collagen matrix being more responsive than those grown on elastin or laminin. The extent of the load applied may also influence the response. Banes et al. demonstrated that a strain representing greater than 15% elongation was required to stimulate endothelial cell replication [88]. The fibroblast response appears to be tissue specific since pulmonary artery and cardiac fibroblasts appear unresponsive [personal observation], whereas lung fibroblast proliferation is stimulated [89]. This again may be a function of the type of load and the nature of the underlying matrix. As with the smooth muscle cells, replication is observed when fibroblasts are grown on collagen [89] rather than elastin [58].

View this table: [in this window] [in a new window]   Table 2 Effect of mechanical load on cardiovascular cell functiona

 
A vessel culture system has confirmed some of these findings using hilar pulmonary artery segments [18]. BrdU labelling increased in response to static load but only in medial cells, not the adventitia, despite the fact that under non-loaded conditions the adventitial fibroblast were more proliferative than medial smooth muscle cells. Interestingly endothelial denudation decreased the BrdU uptake in the smooth muscle cells suggesting that the response may be mediated through the production of smooth muscle mitogens by the endothelium [18]. However, de-endothelialization does not affect enhanced collagen or elastin synthesis in response to load [16,90].

5.2 Enhancement of procollagen synthesis by mechanical load
As described earlier, mechanical loading in vivo applied to both the heart and vasculature leads to an increased deposition of extracellular matrix proteins, in particular collagen. This can occur through both an increase in the number of collagen-producing cells (as described above) or an increase in procollagen synthesis per cell. When mechanical load is applied to cells in vitro again the response appears to be cell type and tissue specific (see Table 2). Cyclic mechanical load enhances the effect of serum growth factors on procollagen synthesis in cardiovascular and pulmonary artery fibroblasts after 48 h, whereas in the absence of serum load had no effect [58,91]. However, urethral fibroblast procollagen and fibronectin synthesis is stimulated by static strain in the absence of serum [92] although here the changes in collagen where only small compared to those seen for fibronectin. Mesangial cells, the mesenchymal derived cells in the kidney that synthesize collagen and fibronectin to support the glomerular capillary loop, show an increase in procollagen synthesis after 48 h in 0.4% serum [93]. Carver et al. [57], using neonatal rat cardiac fibroblasts saw no change in type I collagen synthesis after 24 h of cyclical load, whereas type III collagen synthesis increased by 70% with a similar increase in type III procollagen mRNA levels. These experiments were performed on laminin coated plates and in the presence of 5% foetal bovine serum and 10% newborn calf serum. The cyclic regime used produced a larger effect than that of a static load of similar magnitude. So once again cell source and the nature of the underlying matrix may influence the response, as well as the magnitude of the load applied. We have further defined the influence of the underlying extracellular matrix on the procollagen synthesis response to load by growing human cardiac fibroblasts on different matrix-coated flexible bottomed plates [94]. Fibroblasts grown on fibronectin responded more rapidly and under serum-free conditions compared to cells grown on collagen or elastin.

As far as other cell types are concerned, no change in procollagen synthesis was seen in pulmonary artery smooth muscle cells [95] whereas aortic smooth muscle cell procollagen synthesis was increased by load [96]. Procollagen synthesis was decreased by cyclic strain in endothelial cells [97]. Using human skin fibroblasts cultured in 3D collagen gels collagen synthesis was decreased in free retracting lattices and increased in bound lattices (i.e. under tension) compared to rates in monolayer cultures [98,99]. It is difficult to draw definitive conclusions from these studies, regarding the cell specificity of the response, given that such a small number of studies have been performed, and again different methods of loading cells, including the extent and frequency of the load, were employed.

5.3 Mechanical stimulation of autocrine and paracrine growth factors
It is known in vivo that the development of hypertension and cardiac hypertrophy are associated with the expression of polypeptide growth factors such as TGF-β [40], bFGF [100] and PDGF [101] and the local production of angiotensin II [52]– see Table 1. Many responses to mechanical load described above in in vitro studies are associated with autocrine growth factor generation by the loaded cells or interactions with an exogenous source of growth factors. These observations suggests that in vivo growth may be mediated by the combined effect of load and growth factors. In response to static, unidirectional mechanical load in vitro, myocytes selectively release angiotensin II (AngII) [102,103] which appears to be, at least partially, responsible for the stimulation of the subsequent hypertrophy [102–104].

Similarly, endothelin-1 release is increased by endothelial cells in response to load in vitro. This occurs as an initial increase in the release of stored endothelin-1, followed by a more prolonged enhancement of synthesis [23]. Endothelin-1, as well as angiotensin, may therefore be important growth factors in the vascular and cardiac remodelling process in addition to their known vasoconstrictor effects.

The stimulation of growth factor production by shear occurs through a direct effect on gene transcription. A shear stress response element has been identified on the PDGF B chain promoter containing a core sequence GAGACC [19] which has also been found in other gene promoters of proteins up-regulated by shear such as TGF-β, tissue plasminogen activator, intercellular adhesion molecule-1 and monocyte chemotactic protein-1 [19]. This sequence binds the transcription factor heterodimer Nf-B p50–p65 [105]. PDGF production is also enhanced by pulmonary fibroblasts and aortic smooth muscle cells in response to cyclic load [87,89,106]. Wilson et al. [87] demonstrated an increased synthesis and release of PDGF A chain in response to cyclic load by aortic smooth muscle cells, an effect that could be blocked with RGD peptides, suggesting a role for integrins in mechano-signal transduction (see below). Wilson et al. have recently identified the stretch response element in the proximal promoter of the PDGF-A gene, and shown that it involves the transcription factor Egr-1, and possibly Sp1 [107]. Interestingly, the same region had previously been shown to be involved in the shear stress response [108], which suggests identical mechanisms of regulation by these distinct mechanical stimuli. The stimulation of PDGF release is necessary for the effect of load on smooth muscle cell replication, thus PDGF has an autocrine role in these cells.

To date these is no evidence for a role for autocrine growth factors in the load induced stimulation of cardiac or vascular fibroblast procollagen synthesis. On the contrary, we have demonstrated that no autocrine factors are released by loaded vascular fibroblast that stimulate procollagen synthesis [91]. However we were able to show an autocrine mitogenic factor (possibly PDGF) released by loading lung fibroblasts [89,106]. Thus from the limited evidence available thus far we would hypothesize that cardiac and vascular fibroblasts respond either directly to load, or in vivo may respond to paracrine factors released by the loading of other cells. Indeed, there appears to be a synergistic effect of load and growth factors on fibroblast function.

	6 How are mechanical forces perceived by the cells? mechanisms of mechano-signal transduction

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
Having determined the response of mesenchymal cells to mechanical load in terms of gene expression and cell replication, the next goal is to understand the mechanisms by which mechanical load regulates these processes, i.e. how a mechanical stimulus is transduced into a biochemical event. Although most information has been gained from the study of myocytes, limited data from other cell types suggests that there may be cell-specific responses, involving specific signalling pathways for the stimulation of the expression of a particular gene. When looking at the signalling processes it is therefore imperative to keep focused on the final gene product of interest, given that many pathways may be simultaneously activated by mechanical load.

Mechano-transduction has been examined at a number of levels and these are summarized in Table 3 and Fig. 3. These may be classified as the initial site of mechano-signal transduction – the site of mechano-sensing; secondary signalling pathways involving the phosphorylation and activation of enzymes involved in signal transduction; and tertiary responses such as increased synthesis of transcription factors. Activation at each level is likely to be required before the later increases in the synthesis and phosphorylation of contractile proteins, matrix proteins or growth factors, or cell division, are observed.

View this table: [in this window] [in a new window]   Table 3 Early signalling events and the resulting stimulation of gene expression induced by mechanical load

 

View larger version (50K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Intracellular signalling mechanisms that translate extracellular mechanical signals into a biochemical response. Both cytoskeletal and second messenger pathways are shown to demonstrate the diversity of mechanisms that may be activated. The cytoskeletal involvement may take the form of converting a physical ‘pull’ into chemical energy thus fuelling the synthetic machinery. It may also lead to a distortion of the nuclear matrix directly effecting DNA structure and the process of transcription. On the other hand an activation of membrane bound enzymes by the distortion of the membrane may lead to the phosphorylation and activation of many intracellular signalling pathways which consequently regulate the activity and expression of nuclear transcription factors. The data was obtained from a combination of both myocyte studies and those from other cell types and therefore does not indicate a single transduction sequence, nor does it reflect the need for all signalling pathways to be activated before a response is determined. A more comprehensive, though speculative figure can be found in the recent review by Banes et al. [171].

 
6.1 Mechano-sensory mechanisms
There are three key potential mechano-sensing mechanisms, involving the following:

(i) the cytoskeleton (intracellular network of cables of microtubules, actin filaments and intermediate filaments in determining cell shape and tensile forces within cells);

(ii) integrins (transmembrane extracellular matrix receptors linked to intracellular signalling machinery);

(iii) stretch-activated ion channels (SACs, channels in the plasma membrane that open (or close) in response to mechanical stimuli).

6.2 Cytoskeletal deformation
There is considerable evidence to suggest that cell shape is an important determinant of cellular activity. Changes in cell shape occur as a result of changes in cell attachment (distance between attachment sites or number of sites). Such changes are known to influence cell behaviour, such as the likelihood of cells undergoing apoptosis [109]. The cell is a tensile structure [110–112] due to the presence of the actin containing myofilaments and incompressible microtubules which hold the cell in its lowest energy state when in suspension. A cell will adhere to a 2D layer of extracellular matrix via cell surface receptors which ‘pull’ the cell into an elongated and flattened shape, creating tension within the cell. The cytoskeleton therefore ensures a continuity of information from the extracellular environment to the nucleus in the form of structural forces (Fig. 3). Such a model has been described by Ingber and colleagues in which the cell is defined as a tensegrity structure such that changes in the distance between attachment sites is transmitted through deformation of the cytoskeletal elements (closely associated with the attachment sites), which results in changes in the shape of the nuclear cytomatrix thereby directly stimulating gene activity [110,113]. Likewise in 3D, cell shape is determined by the composition, arrangement and tensile state of the extracellular matrix in which the cell are embedded. In a tethered collagen gel, actin stress fibres develop within the cell and the cell becomes elongated along the lines of tension [114]. However, detaching the gel causing the cells to ‘relax’, leads to disruption of the cytoskeletal network and the cells develop a stellate morphology [81]. Cells maintained under mechanical stress in 3D undergo replication and are synthetically active, however if the tension is relieved the cells become non-proliferative, entering G0, and lose their synthetic phenotype. The two conditions also result in cells which respond very differently to growth factors [115].

Mechanical load stimulates actin polymerization and the development of stress fibres [116,117]. mRNA, polysomes and rough endoplasmic reticulum (rER) are all attached to the actin cytomatrix during active translation of protein and it is postulated that such interactions stabilize mRNA and increase the efficiency of protein synthesis [118,119]. This is analogous to the in vivo situation in which pressure overload leads to an increased efficiency of protein synthesis [41]. In addition to its effect on translation, the cytoskeleton may also influence transcription. Changes in the arrangement of the nuclear matrix may enhance interactions between specific genes and transcription factors leading to an unfolding of the DNA and a de-repression of DNA synthesis. DNA regulatory proteins (such as DNA polymerase) appear to be physically associated with the nuclear protein matrix [119].

However, despite the fact that there is cytoskeletal alignment in response to load [120] and given all the circumstantial evidence suggesting a role for the cytoskeleton and cell shape in mechano-signal transduction, direct experimental evidence is less forthcoming. Sadoshima et al. [121] demonstrated little effect of the cytoskeleton as the initial transducer of the mechanical signal in cardiac myocytes. Cytochalasin D which depolymerizes actin microfilaments had no effect on the load induced c-fos expression. The effect on protein synthesis was a little more difficult to define given that cytochalasin D itself, as expected from the evidence described above, decreased normal protein synthesis. Similarly colchicine, which disrupts microtubules had no effect on protein synthesis or c-fos expression in response to load, but again attenuated normal levels of protein synthesis.

6.3 Integrins as mechano-receptors
Cells adhere to the extracellular matrix principally via interactions with integrins. Integrins are heterodimeric transmembrane proteins consisting of one and one β chain. Sixteen and eight β chains have been identified, but the number of heterodimers is restricted by the limited number of combinations of and β chains that are able to dimerize (twenty have been identified so far). Specific matrix components bind specific integrin dimers (reviewed by Hynes [122]), for example, 1β1 binds to collagen and laminin, 5β1 binds to fibronectin, 3β1 binds both fibronectin and collagen. The fibronectin-binding integrins are particularly important in the assembly and development of the heart and vasculature, demonstrated using knockout mutations of fibronectin or the 5b1 and v integrins [123]. 1β1 has been identified on cardiac fibroblasts and is important for angiotensin II-mediated contraction in collagen gels [124]. Other integrin subunits identified on cardiac fibroblasts include 2β1 and 3β1 [125]. In culture, binding to the matrix causes integrin clustering at the site of focal adhesion complexes. Although integrins appear to lack intrinsic enzymatic activity, following cell adhesion there is a stimulation of tyrosine phosphorylation leading to a series of second messenger signals which may ultimately influence cell function.

Integrin-mediated cell adhesion and cell spreading lead to intracellular signalling and the regulation of cell function in a manner not dissimilar to that observed in response to mechanical load, suggesting that there may be a common theme for cellular responses to all types of mechanical deformation. The principle kinase activated upon integrin clustering is focal adhesion kinase (FAK) [126] and we have recently shown that there is increased FAK phosphorylation in human cardiac fibroblasts in response to mechanical load [127] This kinase is associated with the cytoskeleton and becomes localized to the cytoplasmic domain of integrins at the site of focal adhesions. This process triggers a cascade of second messenger events (reviewed by Craig and Johnson [128]) including autophosphorylation of FAK, binding of c-Src and the Grb-2 adaptor protein and activation of ERK through the Grb2–Sos–Ras pathway. Also critical to integrin clustering is the presence of two functionally active GTP-binding proteins, rac and rho [129] which regulate the bundling of actin filaments and the appearance of stress fibres [130]. These events influence ultimately, not only normal cell function, but their response to other external factors such as the presence of growth factors. Integrins are closely associated with growth factors receptors, indeed it has been demonstrated that stimulation of integrins can lead to the autophosphorylation of the PDGF receptor independent of PDGF receptor occupancy [131].

Since it is recognized that the nature of the extracellular matrix can influence cell behaviour, this would suggest that binding by specific integrins may have specific functional consequences. This has now been demonstrated in a number of studies [87,132]. As the site linking the extracellular matrix being deformed by the load and the cellular signalling machinery, the role of integrins as first line mechano-transducers is now receiving considerable attention (see below), not only in a passive capacity, linking the extracellular matrix with the cytoskeleton, but also through an active role in mechanosignal transduction.

Evidence supporting a role for integrins in transmitting the mechanical signal of cellular distortion to the nucleus comes from the elegant studies of Ingber and colleagues who have demonstrated, using ligand-coated magnetic microbeads to distort the cell surface integrins, that there is a rearrangement of the cytoskeletal filaments leading to an increase in cytoskeletal stiffness, and a distortion of the nucleus along the axis of the applied tension field [113,133]. Thus there is a continuum of ‘mechanical’ information from the cell surface through to the nucleus – the integrins providing the link between the extracellular mechanical signal and the intracellular response.

Evidence is also emerging for a role for integrins as cardiac fibroblast mechanosensors and interestingly there appears to be specific integrins involved. We have recently demonstrated that the fibronectin specific integrins 5β1 and vβ3 are essential for the load induced stimulation of procollagen synthesis [94]. MacKenna et al. have demonstrated the importance of integrins in the load mediated activation of the MAPkinases, ERK2 and JNK1 [134]. Fibronectin integrins also appear important in the replicative response of aortic smooth muscle cells to mechanical load [87]. Cells grown on fibronectin respond more dramatically, in terms of replication, than cells grown on collagen, laminin or elastin. Blocking RGD peptides – a principle extracellular matrix binding domain of many integrins, prevents the load-induced response [87]. The limited data available using neonatal myocytes however, suggests that mechanical load is not transduced through the RGD peptide binding integrins in these cells [121].

6.4 Stretch-activated ion channels
One of the first responses to mechanical load (occurring within milliseconds) is the opening of selective ion channels [135]. Specific stretch-activated ion channels (SACs) have been identified on most cells [135]. SACs are distinct from ion channels that respond to voltage changes or ligand–receptor interactions. The energy required to open these channels is provided by membrane strain rather than metabolic stimulation. The channels are then kept open by energy derived from the flux of ions. SACs are mostly cation channels such as K⁺, Na⁺ or Ca²⁺. They are transmembrane proteins, linked to cytoskeletal strands of the spectrin/fodrin family [136]. In addition to responding to external mechanical force, they can also respond to internal mechanical forces (i.e. contraction and osmotic pressure) and thus may regulate cell size by activating feed-back mechanisms to adjust metabolism and cell division.

Although the existence of SACs has been known for some time the physiological role of these channels is not fully understood (for reviews see [137,138]). A SAC in vascular smooth muscle cells has been identified which may be involved in pressure-induced vascular remodelling [139]. Load induced stimulation of aortic elastin synthesis appears to be dependent on stretch-activated L-type calcium channels [16]. Fluxes of monovalent cations Na⁺, K⁺ and H⁺ may also be important in this response. Sadoshima et al. [121] determined that although stretch activated non-selective cation channels were opened in response to load, blocking these channels with gadolinium had no significant effect on the load-induced stimulation of immediate early genes c-fos and c-jun (see below). Furthermore, gadolinium had no effect on the load-induced stimulation of protein synthesis. It was also determined that depleting extracellular calcium stores did not affect the stretch-induction of c-fos expression, although depletion of intracellular stores significantly attenuated the c-fos induction by load [140] suggesting a role for intracellular calcium ions rather than the need for calcium uptake.

6.5 Second messenger pathways
Evidence is now emerging of the possible intracellular signalling pathways triggered by mechanical load. The pathways determined to date are summarized in Fig. 3. Although many pathways may be triggered it is not yet clear which events are required for the stimulation of the expression of specific genes. The development of more specific inhibitors of signalling pathways should increase our understanding of the important steps involved for our genes of interest. There is little information currently available on the mechanosignalling pathways important in cardiac fibroblasts. The data described here is consequently a summary of what is known from experiments on a number of different cell types.

Unidirectional static loading of myocytes stimulates signalling molecules within seconds to minutes of the onset of load. There is an activation of the membrane-associated phospholipases – phospholipase D, C and A₂ leading to an increase in inositol tris–phosphate (IP₃), diacylglycerol (DAG) and arachidonic acid metabolites [140]. Activation of these phospholipases by mechanical load may be caused by a perturbation of the plasma membrane causing a spatial reorganization of these membrane bound enzyme complexes, and an increased accessibility of the enzyme to substrates in the plasma membrane. Increased levels of DAG and IP₃ then stimulate protein kinase C (PKC) activity and intracellular calcium release respectively. Increased levels of IP₃ and DAG have also been demonstrated in response to mechanical load in vitro in endothelial cells [141] and periodontal ligament fibroblasts [142].

Stimulation of PKC by mechanical load is required for both myocyte hypertrophy [143] and smooth muscle cell replication [144]. In the cardiac myocyte, stimulation of PKC by mechanical load leads to the phosphorylation of proteins in the MAP kinase signalling cascade [140,145]. Raf-1, MAPKKK and MAPKK activities are all increased [145]. MAPKK activation is followed by the phosphorylation of both threonine and tyrosine residues on the 42 000 MAP kinase, resulting in its activation [145]. Activation of MAP kinase appears to be via both PKC-dependent and PKC-independent pathways since inhibitors of PKC or PKC depletion removes only 60–70% of the MAP kinase activity stimulated by mechanical load [145]. The stimulation of the MAP kinase pathway may be due in part to AngII secretion by myocytes in response to load since AngII also stimulates this pathway. The AngII type I receptor antagonist, CV-11974, partly inhibits the stretch-induced activation of Raf-1 and the MAP kinases [102]. Receptor tyrosine kinases do not appear to be involved here, since the inhibitors tyrphostin and genistein do not change MAP kinase activity in response to load [145]. MacKenna et al. [134] have demonstrated matrix and integrin specific activation of ERK2 and JNK1 in response to load in cardiac fibroblasts. We have also shown a rapid stimulation of ERK1 and 2 in cardiac fibroblasts [146]. Furthermore we have been able to demonstrate that inhibition of ERK phosphorylation prevents the load induced stimulation of procollagen synthesis [147] thus demonstrating for the first time a direct link between mechanical load, MAPkinase and procollagen synthesis. Mucsi and coworkers showed that blocking the MAPK (ERK) pathway partially blocked the induction of the procollagen ₁(I) gene by TGFβ1, further demonstrating the importance of MAPK in procollagen type I regulation [148]. Ras/Raf and MAPK activity have been mapped to different sites in the promoter region of procollagen ₁(I) [149].

The role of cAMP in mechanosignal transduction in vascular cells is unclear. Wierbitsky et al. [150] demonstrated a decrease in adenylate cyclase activity in response to load in coronary smooth muscle cells, associated with a decrease in the level of the stimulatory G protein subunit (G_s45). Modulation of cAMP production had no effect on the stress-induced increase in elastin metabolism in isolated vessels [16]. Cyclic mechanical strain has been shown to increase cAMP levels in arterial and microvascular endothelial cells [151] but not in endothelial cells derived from umbilical or saphenous veins [151,152]. No change was observed in the cAMP levels in cardiac myocytes in response to load [140]. Thus the effect of mechanical forces on cAMP is variable and cell type specific and may depend on the nature of the load applied. Changes in the levels of cAMP may be observed but the consequence of this is not yet clear.

6.6 Tertiary signals: activation of transcription factors
Activated MAP kinases translocate to the nucleus and phosphorylate nuclear factors such as c-myc and c-jun leading to increased transactivation of gene expression [104,153]. c-Fos and c-jun form a heterodimer that binds to AP-1 sites present on a number of gene promoters. Such consensus sequences are found in the promoters of matrix metalloproteinases and interleukins as well as in the promoter and the first intron of the procollagen type I [ ₁(I)] gene. Activated MAPK also phosphorylates the transacting factor p62^TCF which, together with the serum response factor (SRF) binds at the serum response element, a significant component of many genes including c-fos [154] and atrial naturietic factor [155]. Furthermore pp90rsk which activates SRF is phosphorylated by ERK, leading to enhanced c-fos transcription [140]. MAP kinases may therefore induce transactivation at AP1 sites in two ways – by directly activating c-Jun through phosphorylation and by enhancing c-jun and c-fos expression. The ribosomal S6 kinase that phosphorylates the S6 protein, which plays a role in facilitating translation, is also activated by MAP kinase which may therefore act to aid further translocation of secondary and tertiary messengers to the nucleus [145].

In isolated cardiac myocytes subjected to static load, enhanced expression of c-fos was dependent on the stretch-activation of PKC and production of arachidonic acid metabolites, through PLA₂ and cytochrome P450 monooxygenase activity [140]. Although the links between c-fos expression and increased protein synthesis described thus far are slightly tenuous, there is increasing evidence to suggest that this increase in the expression of the immediate early genes is an important event prior to enhanced protein synthesis. A stretch response element in the c-fos promoter has been mapped to the serum response element and expression is induced after just 1 min of mechanical load [140,156]. c-Jun, c-myc and egr-1 are also transiently up-regulated by mechanical load in myocytes [157]. A role for c-fos and other transcription factors such as c-myc and c-jun in myocyte hypertrophy has also been suggested in vivo. c-Fos and c-jun expression is increased in isolated heart preparations when subjected to physiological levels of systolic pressure [158]. These oncogenes are up-regulated in vivo in response to pressure overload [159,160]. It has been suggested that a decrease in the ability to respond to mechanical loading in the elderly is due in part to a decreased ability to express c-fos in response to such a stimulus [161]. Other oncogenes have also demonstrated increased expression in response to mechanical forces. NFB, AP-1 and CRE binding proteins are all induced in response to cyclic load in endothelial cells [162]. In addition to binding sites for AP1, the human procollagen ₁(I) gene also contains putative binding sites for Egr-1, NF-B and p62^TCF, however their significance in stretch regulation in fibroblasts has to be determined. To this end we have begun to investigate the regulation of gene expression by mechanical load in cardiac fibroblasts and have observed an increase in NF-1 and NFkB like binding activity – two consensus sequences identified in the ₁(I) procollagen gene [163].

	7 Synergistic interactions between growth factors and mechanical load in the stimulation of cardiovascular cell growth

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
The role of growth factors in the response of cardiovascular cells to mechanical load can be divided into two principle mechanisms: firstly, as described above, mechanical load may stimulate the release of growth factors (enhanced synthesis or release of stored growth factor) which then stimulates protein synthesis or cell proliferation; secondly, growth factors (exogenous or endogenous) may act synergistically with load to enhance a growth response. These observations have important implications in terms of the hypertrophic/remodelling response since it suggests that mechanical load may not only stimulate the production of growth factors but may also modulate the effect of growth factors present in hypertrophying tissues.

A synergistic interaction between mechanical load and growth factors has been demonstrated in the stimulation of fibroblast procollagen synthesis. Mechanical load alone has no effect on cardiac or pulmonary artery fibroblast procollagen synthesis in the absence of serum or polypeptide growth factors, but enhances the stimulatory effect of profibrotic mediators including serum, PDGF and TGFβ [58,91]. The mechanisms whereby mechanical load enhances the effect of growth factors are not yet known. These studies have interesting parallels, however, with the observation that during cell adhesion, both the extracellular matrix and activation of intracellular rho/rac GTPases (through addition of growth factors) are required for the assembly of integrin adhesion complexes [129]. The important study by McNamee and coworkers, mentioned earlier, shows that fibroblasts binding to fibronectin directly stimulate the synthesis of PIP₂, the lipid substrate needed for PDGF signalling [164]. Mechanically loading may affect cells in a similar manner to cell adhesion – through integrin clustering, and, as described for adhesion, growth factors may be required to produce maximal stimulation of cell function. Thus mechanical load may enhance the generation of second messengers following ligand binding leading to a possible convergence with growth factor second messenger pathways. Many of the pathways stimulated by mechanical load, described above, are also stimulated during growth factor signalling. In addition, as described above, integrins may also trigger growth factor signalling in the absence of the growth factor ligand [131].

Support for a link between the mechanical environment and growth factor signalling also comes from Lin and Grinnell [165] who demonstrated a decrease in DNA synthesis in response to PDGF in mechanically relaxed collagen matrices. Fibroblasts were cultured in anchored collagen matrices during which time mechanical stresses develop, the matrices were then dislodged allowing the stresses to dissipate. The decrease in response was attributed to a decrease in PDGF receptor autophosphorylation as a result of mechanical relaxation. PDGF is also known to stimulate the expression and rate of synthesis of the 2 integrin subunit [166] which may enhance integrin signalling triggered by mechanical load – further evidence that integrins may be involved in the synergy. Alternatively mechanical load may increase the number of growth factor receptors or the efficiency of growth factor receptor binding.

Another interesting link between growth factors and integrins, which may be particularly relevant to the cardiac fibroblast, was the observation that osteopontin, an acidic phosphoprotein with RGD sequences, is produced by cardiac fibroblasts and mediates angiotensin-induced DNA synthesis and collagen gel contraction [167].

	8 Future directions

Top 1 Introduction 2 Stimulation of cardiovascular... 3 Regulation of procollagen... 4 Influence of the... 5 Effect of mechanical... 6 How are mechanical... 7 Synergistic interactions... 8 Future directions Acknowledgments References

 
An understanding of the mechanisms of mechano-signal transduction will provide a new area of therapeutic targeting to prevent undesired hypertrophy or remodelling. Rather than attempting to block the action of growth factors or hormonal influences, which has consequences for unaffected organs whose normal growth and function depend on such autocrine, paracrine or endocrine agents, one may be able to target the specific tissue under enhanced mechanical load.

Studies using cardiac myocytes may lead to the elucidation of the mechanisms involved in the switch from a compensatory hypertrophy to a decompensating, pathological state. Such an understanding would then permit the development of agents that specifically block this switch, allowing a healthy compensatory growth, which may be beneficial or a necessity, whilst preventing the progression to failure. It is known that following ischaemia, there is a degree of eccentric hypertrophy in the surrounding viable myocytes. It may be possible to utilize mechanical load to induce such a compensatory hypertrophy to minimize the effect of the infarction on the performance of the remaining viable myocardium.

Application of mechanical load may be used in other proactive ways to promote hypertrophy. The development of the procedure of myoplasty, where skeletal muscle (from the anterior latissimus dorsi muscle) is used as a replacement for damaged myocardium, incorporates a mechanical loading of the skeletal muscle to achieve cardiac muscle characteristics. Previously this was achieved by electrical stimulation which lead to the conversion of the skeletal muscle from a slow oxidative muscle to a fast glycolytic muscle which is less susceptible to fatigue. However, this procedure alone causes a degree of atrophy to the muscle thus losing power. To combat this, experimental training procedures are now incorporating a mechanical stretch which not only prevents the atrophy but causes hypertrophy [168]. These training regimes also influence the composition and distribution of collagen in the tissue. Thus a combined training of skeletal stretch and electrical stimulation would improve the condition of the muscle prior to the surgical procedure. A mechanical loading regime is also used in the induction of ventricular hypertrophy prior to the two-stage arterial switch operation for transposition of the great arteries, in this case by banding the pulmonary artery [169]. However, the contractile properties may be impaired in these ventricles and recent experimental evidence suggests that a combined pressure-overload and drug treatment may improve the ‘quality’ of the hypertrophied myocardium in terms of both mechanical properties and collagen composition [170].

In summary, cells respond to mechanical load via interactions with the extracellular matrix, through activation of second messenger pathways and stimulation of gene expression. Enhanced cell growth, replication and matrix production is determined through additive or synergistic effects of the pathways of mechano-signal transduction and growth factor signalling resulting in a remodelling of vascular and cardiac tissue. Thus mechanical forces play key roles as regulators of cardiovascular structure and function.

Time for primary review 20 days.

	Acknowledgments

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We wish to thank The Wellcome Trust and The British Heart Foundation for their financial support.

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