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Non-fibrillar collagens: Key mediators of post-infarction cardiac remodeling?
Journal of Molecular and Cellular Cardiology 48 (2010) 530–537
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Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Review article
Non-fibrillar collagens: Key mediators of post-infarction cardiac remodeling? Patricia E. Shamhart, J. Gary Meszaros ⁎ Department of Integrative Medical Sciences, Northeastern Ohio Universities College of Medicine (NEOUCOM), 4209 State Route 44, Rootstown, OH 44272-0095, USA
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Article history: Received 17 April 2009 Received in revised form 9 June 2009 Accepted 21 June 2009 Available online 30 June 2009 Keywords: Myocardial infarction Wound healing Myofibroblasts Matrix metalloproteinases Serine proteases Integrins
a b s t r a c t Cardiac remodeling is accelerated during pathological conditions and several anabolic and catabolic regulators work in concert to repair the myocardium and maintain its functionality. The fibroblasts play a major role in this process via collagen deposition as well as supplying the degradative matrix metalloproteinases. During the more acute responses to a myocardial infarction (MI) the heart relies on a more aggressive wound healing sequence that includes the myofibroblasts, specialized secretory cells necessary for infarct scar formation and thus, rescue of the myocardium. The activated fibroblasts and myofibroblasts deposit large amounts of fibrillar collagen during the post-MI wound healing phase, type I and III collagen are the most abundant collagens in the heart and they maintain the structural integrity under normal and disease states. While collagen I and III have been the traditional focus of the myocardial matrix, recent studies have suggested that the non-fibrillar collagens (types IV and VI) are also deposited during pathological wound healing and may play key roles in myofibroblast differentiation and organization of the fibrillar collagen network. This review highlights the potential roles of the non-fibrillar collagens and how they work in concert with the fibrillar collagens in mediating myocardial remodeling. Published by Elsevier Ltd.
Contents 1. 2. 3. 4. 5. 6. 7. 8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular and extracellular composition of the myocardium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Type V collagen: Potential regulator of fibril assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The importance of non-fibrillar collagens: The interaction with other ECM components . . . . . . . . . . . . . . . . . . . . . . . . . . Type IV collagen: The ubiquitous non-fibrillar collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Type VI collagen: A more specialized non-fibrillar collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degradation on non-fibrillar collagens: The role of MMPs and serine proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major questions and challenges (see Table 2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Do non-fibrillar collagens play a role in determining the source of the myofibroblasts by promoting differentiation of resident fibroblasts or stem cell homing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. What specific matrix–cell interactions and downstream signaling events mechanistically control differentiation of myofibroblasts? . . 8.3. How much and which types of collagen (fibrillar versus non-fibrillar) are produced by myofibroblasts? . . . . . . . . . . . . . . . 8.4. Does the collagen matrix play a major role in fibroblast migration to sites distal from the infarcted area to induce a global fibrosis? . 8.5. Does the limited breakdown of collagen VI by MMPs yield a more stable collagen that could nucleate organizational sites for collagen I and III fibril assembly? Could non-fibrillar collagens play a role in angiogenesis or VSMC function? . . . . . . . . . . . . . . . . 9. Overall summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The extracellular matrix ECM of the heart is a dynamic environment encompassing both fibrillar and non-fibrillar collagens. Cardiac fibroblasts (CFs) are the major ECM producers in the heart and are ⁎ Corresponding author. Tel.: +1 330 325 6432; fax: +1 330 325 5912. E-mail address: [email protected] (J.G. Meszaros). 0022-2828/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.yjmcc.2009.06.017
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triggered to alter ECM composition during pathophysiological conditions that range from the acute (myocardial infarction, MI) to the chronic (hypertension, heart failure, diabetes). In each of these diseases the CFs adjust their function to maintain architectural integrity within the myocardium in an effort to provide sufficient support for cardiac function. The CFs respond to disease stimuli by adjusting their proliferation, migration, differentiation, collagen deposition and matrix metalloproteinase (MMP) production.
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In the setting of post-MI remodeling, replacement fibrosis and infarct scar formation are necessary events to repair and, depending on the severity of the infarct, potentially rescue the heart from ventricular wall collapse or rupture. The CFs migrate to the ischemic area and border zone and deposit large amounts of ECM to replace the dying myocytes and to “wall off” the injured area via scar formation [1]. CFs are capable of differentiating to the hypersecretory myofibroblast phenotype, which plays an even more critical role in acute post-MI remodeling compared to the more chronic diseases. The myofibroblasts harbor enhanced capabilities to secrete ECM and perform repair functions, and work in concert with the fibroblasts to orchestrate the wound healing process. The major distinguishing feature of the myofibroblast is that they express α-smooth muscle actin, which allows for stress fiber organization and sustained cell contraction to stabilize the wound. The prolonged activity of fibroblasts and myofibroblasts is where matrix deposition can go awry — continued collagen production proceeds from both sources, and of particular concern is the extended life-span of the myofibroblasts. These cells perform the necessary wound healing duties then undergo apoptosis, but if this does not occur then the persistent activity of these cells causes excessive matrix accumulation and detrimental fibrosis. An important question is how long is required for the myofibroblast to complete their beneficial repair duties following the MI? Which types of collagen do they produce? What are the mechanisms that turn CF and myofibroblast activity on and off? Several complex factors work in concert to influence myocardial repair, including humoral factors, mechanical stimuli and matrix–cell interactions. This review will focus on how the matrix changes during remodeling and will ask how the matrix itself is capable of influencing the repair of the myocardium. Historically, fibrillar collagens, mainly types I and III, have been the major focus in the heart, however, significant contributions from nonfibrillar collagens during pathological cardiac remodeling have been reported as well. Although the non-fibrillar collagen types IV and VI are viewed as minor structural components of the myocardium, these collagens anchor and organize the fibrillar collagens and have other roles in controlling CF migration, differentiation and proliferation. Several intriguing findings from in vitro and in vivo studies will be presented to emphasize the interdependence of the collagen components in repair and remodeling of the heart. 2. Cellular and extracellular composition of the myocardium The adult myocardium is made up of a network of fibrillar (I, III, and V) and non-fibrillar (IV and VI) collagens. By far, collagen I is the most abundant ECM protein in the body and is expressed in every major organ and tissue. Mutations in collagen I lead to osteogenesis imperfecta (OI), an autosomal dominant disease that is characterized by weak or brittle bones. OI mouse models have been developed, one of which has been used to study the myocardial matrix composition. The OI murine model has a deficiency in the pro-alpha2(I) chain, and a well-characterized deficit in tendon and bone strength. In the myocardium these animals displayed significant reductions in collagen density and fibril diameter leading to a higher compliance of the LV upon passive inflation [2]. Although it remains to be tested, the reduction in collagen I quality and/or content would likely be detrimental in the case of a severe stresses such as MI or ischemia– reperfusion injury where ventricular rupture could occur. Also of
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interest is whether other collagen types are elevated in the hearts of OI mice in an effort to maintain its biomechanical integrity. Cardiac fibroblasts are the major matrix producing cells in the heart and are the most abundant cell type in terms of overall numbers, whereas the larger myocytes account for the majority of the myocardial mass. The myocytes are arranged in layers woven together by the fibrous ECM which is interspersed with fibroblasts [3]. The fibrillar collagens of the ECM not only provide structure to the heart but also signal to the various cells of the myocardium to alter their activity. We have demonstrated that solubilized collagen types I and III potently stimulate cardiac fibroblast proliferation in vitro [4]. Recently we have established that collagens type I and III also enhance cardiac fibroblast migration relative to type VI (unpublished results). Monomeric type I collagen enhances smooth muscle cell proliferation whereas type I in its native fibrillar form reduces proliferation [5]. Several in vitro effects of the fibrillar and non-fibrillar collagens are displayed in Table 1. The importance of types I and III collagen in cardiac pathologies has been extensively studied, and these collagens increase significantly in response to MI and in several other diseases. It has been debated how the relative proportions or ratios of these collagens change during accelerated remodeling, and it has been suggested that accumulation of collagen I, the more highly organized of the two, is responsible for the decreased compliance and fibrotic stiffening of the myocardium leading to global dysfunction [1]. Following a myocardial infarction, it has been shown that type III collagen is deposited during early remodeling whereas, type I collagen deposition predominates during the intermediate and late remodeling stages [6]. 3. Type V collagen: Potential regulator of fibril assembly Type V collagen is a widely distributed, low abundance fibrillar collagen that has been shown to associate with type I collagen in several diverse tissues. A unique property of type V is the formation of a hybrid fibril with type I collagen in vitro [7]. Evidence from dermal tissue indicates that collagen V is required to initiate collagen fibril nucleation, and further that type V can regulate the size and organization of collagen fibrils, many of which are heterotypic collagen I and V containing structures [8]. Mutations in type V collagen are linked to Ehlers–Danlos syndrome, a connective tissue disorder that features a hyperextensible skin phenotype [9]. Type V knockout mice are embryonic lethal at E10.5, at least in part due to cardiovascular failure. However, the heterozygote (ColVa1+/−) mice are viable and display symptoms similar to Ehlers–Danlos patients [10]. The specific function of type V collagen in the heart is not welldefined, despite its widespread expression and distribution throughout the interstitial matrix [11]. Type V collagen is deposited in the myocardium by cardiac fibroblasts and VSMCs [12,13]. In vitro, type V collagen imparts antiproliferative effects on VSMCs and endothelial cells [14,15]. There are limited studies that describe the effect of type V collagen on VSMC, myocyte, and cardiac fibroblast activation despite reports that describe alterations of type V during several cardiovascular pathophysiological conditions. At 20 and 40 weeks, spontaneously hypertensive rats have significantly elevated levels of type V collagen
Table 1 In vitro effects of fibrillar and non-fibrillar collagen substrates. Collagen type
I (fibrillar)
III (fibrillar)
IV (non-fibrillar)
V (fibrillar)
VI (non-fibrillar)
H9C2 myoblasts Cardiac fibroblasts
↓ Proliferation [34] ↑ Proliferation [4] ↑ Migration Monomeric: ↑ proliferation [5] Fibrillar: ↓ proliferation [5]
? ↑ Proliferation [4] ↑ Migration ?
↑ Proliferation [34] ?
? ?
↑ Differentiation to VSMC [31–33]
↓ Proliferation [14]
? ↑ Myofibroblast differentiation [4] ↓ Migration ?
VSMCs
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compared to control rats [16]. Type V collagen content also increases with atherosclerosis, dilated cardiomyopathy, and the formation of hypertrophic scars [17–19]. The importance and consequence of elevated type V during these pathological conditions have not been established, most likely since the exact function of type V is unknown. Perhaps the most revealing study that links collagen V with cardiac pathology utilized the ColVa1+/− mice. It was determined that cardiac valve development is dependent upon the presence of collagen V, and that type I collagen increases in the heart valves and left ventricles, perhaps as a compensatory mechanism [20]. To date, the role of type V collagen following a myocardial infarction has not been determined. We speculate that collagen V would play a critical role in infarct scar formation as well as replacement fibrosis, due to its apparent importance in type I collagen organization. It would certainly be interesting to utilize the ColVa1+/− mouse in a myocardial infarction study to further define how type V contributes to post-MI remodeling. 4. The importance of non-fibrillar collagens: The interaction with other ECM components The fibrillar collagens are recognized as the structural support for the matrix but the non-fibrillar collagens are key regulators in anchoring and organizing the ECM meshwork. Type IV collagen is the major component of a specialized region of ECM surrounding cells such as the myocyte referred to as the basement membrane (see Fig. 1). In the basement membrane, collagen IV forms a scaffold with laminin, entactin, and perlecan to create a collagen, proteoglycan, and glycoprotein meshwork [21,22]. Type IV interacts predominantly with the α1β1 and α2β2 integrins, with the former having a higher affinity for type IV and the latter for type I collagen. In addition to these integrins, evidence for collagen IV binding to α3β1, α10β1, α11β1, and αvβ3 has also been put forth [23]. The basement membrane is a diverse environment and additional ECM components may be found in the basement membrane in accordance with specific cell types. It has been reported that type VI collagen interacts with type IV collagen in the basement membrane creating an anchoring bridge between the basement membrane and the interstitial matrix [24]. Type VI collagen interacts with multiple interstitial matrix proteins including type I collagen and fibronectin [25]. It is speculated that the α1, α2, α3, and α10 (β1) and the αvβ3 integrins all potentially bind collagen VI in a cell-type specific manner [26,27]. Our laboratory has presented recent evidence that the α3 integrin plays a mechanistic role in the phenotypic transformation of cardiac myofibroblasts [28]. The nonfibrillar collagens are integral components of ECM adhesion throughout the body, however little is known about their role in injury responses, particularly in the myocardium. 5. Type IV collagen: The ubiquitous non-fibrillar collagen Individual cardiac myocytes are surrounded by a specialized basement membrane ECM composed mainly of type IV collagen, glycoproteins, and proteoglycans that is incorporated into the endomysium. Beyond the basement membrane the composition of the matrix changes and type I and III collagen begin to dominate the interstitial matrix. Eghbali et al. revealed that cardiac fibroblasts produce most of the interstitial collagens, types I and III, and basement membrane collagen IV, whereas and that cardiac myocytes are also capable of producing type IV collagen [29]. Blood vessels are tightly surrounded by a basement membrane and an interstitial matrix. Individual matrix components interact with various cells to lend support and structure but they are also capable of stimulating cell differentiation and/or proliferation. Plating adult smooth muscle cells (SMCs) on type IV collagen induces an increase in SMC contractile proteins, α-smooth muscle actin and smooth muscle myosin heavy chain [30]. Stem cells plated on type IV collagen
spontaneously differentiate to smooth muscle cells [31–33]. Furthermore, it was shown that collagen IV induced smooth muscle cell differentiation of Sca-1+ progenitor cells was mediated by α1/β1/αv integrins [32]. In vitro type IV collagen induced proliferation of H9C2 myoblasts, myocyte precursor cells, whereas type I collagen attenuated this response [34]. In culture, type IV collagen induces SMC differentiation and myocyte precursor cell proliferation but few studies have addressed the effect of type IV on cardiac fibroblast activation. Understanding how type IV collagen affects all cardiac cell types is important because it may provide clues as to why type IV collagen composition is altered during several cardiovascular pathophysiological conditions and how these changes affect remodeling and recovery. Following a myocardial infarction, type IV collagen increases expression in the peripheral zone at day 3 post-infarction and reaches its maximal expression in this zone between 7 and 11 days [35]. Interestingly, type IV appearance does not increase in the outer infarct zone until day 4 and in the inner portion of the infarct region at day 10. The accumulation of type IV in the inner portion of the infarct zone is minimal compared to other regions [35]. Type IV collagen was shown to be restricted to the myocyte basement membrane of hypertrophic cardiomyopathy patients as well as being localized in the regions of replacement fibrosis [36]. Streptozotocin-treated diabetic rats likewise exhibit left ventricular type IV collagen accumulation [37]. The consequence of accumulated type IV collagen during these pathophysiological conditions, particularly post-MI, is poorly understood. It is generally thought that collagen IV and the basement membrane are highly adhesive and may serve glue cells and ECM in place, and that they slow the migration of several cell types. The reports that indicate a somewhat delayed appearance of type IV collagen in the middle of an infarct zone may indicate that the initial absence of type IV in the damaged area permits the migration of fibroblast and myofibroblasts to the damaged area, followed next by reparative and replacement fibrosis via fibrillar collagen deposition and the eventual deposition of type IV collagen. In this scenario, the type IV collagen deposition may be a result of bulk collagen deposition rather than contributing to a well-organized basement membrane. These and several other possibilities are still to be to be determined.
6. Type VI collagen: A more specialized non-fibrillar collagen Type VI collagen is expressed in the heart (both embryonic and adult), basement membranes, and in vascular smooth muscle [38–40]. Type VI collagen formation is a unique process that differs from fibrillar collagen assembly. Like all collagen structures, type VI is composed of three α chains in a triple helix, but unique to type VI, the monomer triple helix assembles into a dimer followed by a tetramer both occurring in an anti-parallel, lateral direction [41,42]. The tetramer is secreted from the cell and assembles in an end to end fashion creating the beaded filament appearance of the type VI microfibril [42]. Type VI acts as an adhesive structure in the basement membrane and interacts with other ECM components [43]. Mutations in type VI collagen genes have been linked to two major skeletal muscle defects, Bethlem myopathy and Ullrich congenital muscular dystrophy [44]. Type VI-deficient mice have been generated by the deletion of the α1 chain, preventing formation of the triple helical collagen VI molecule [45]. The type VI knockout mice exhibit a defective muscular phenotype with skeletal muscle necrosis and varying muscle fiber dimensions resembling Bethlem myopathy. Skeletal muscle myofibers from type VI-deficient mice have altered sacroplasmic reticulum and mitochondrial structures causing mitochondrial dysfunction and apoptosis [46]. Mitochondrial function can be restored by placing the isolated myofibers on a type VI collagen matrix [46]. The ECM of the type VI-deficient mice has an altered organization and fibroblasts isolated from these mice have abnormal
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Fig. 1. Non-fibrillar collagens organize the basement membrane. The basement membrane is a complex region composed mainly of non-fibrillar collagens and adhesive glycoproteins and proteoglycans; laminin interacts with type IV collagen, entactin, perlecan, and integrin receptors on the cell membrane. Type IV collagen also interacts with non-fibrillar type VI collagen that spans beyond the basement membrane to interact with the fibrillar collagens.
fibronectin deposition that is very similar to fibroblasts from Bethlem myopathy patients [47]. Our laboratory has recently begun to study how specific collagen substrates influence cardiac fibroblast activity. We have observed that type VI collagen attenuates CF migration whereas types I and III facilitate migration in an in vitro wound healing assay (unpublished results). We have reported that type VI collagen potently induces myofibroblast differentiation in vitro [4]. Since myofibroblasts are crucial mediators of wound healing, we have explored the progressive
increases in myofibroblasts content and type VI collagen deposition following a myocardial infarction. Seven days post-MI, both type VI and myofibroblast content were significantly increased in the infarcted zones of the MI animals [28]. By 14 days the myofibroblast content returned to control levels but type VI collagen was still elevated. During the later stages of remodeling at 16–20 weeks postinfarction, both type VI collagen and myofibroblast content were enhanced in the infarcted areas [4]. It was surprising that the myofibroblast levels were elevated this late in remodeling, leading
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us to speculate that the increase in collagen VI may preserve these cells either by preventing their disappearance via apoptosis or by promoting their in vivo differentiation. Thus, the preservation of the myofibroblast in the infarcted area could significantly contribute to the progression of fibrosis both within the damaged area as well as distal sites. We did see elevations in both collagen VI and myofibroblasts in the non-infarcted samples taken from the posterior wall of the 16 week post-MI samples, indicating that with time the deleterious remodeling will spread beyond the ischemic area. This study [4] was the first to link in vivo evidence for the elevation of both type VI collagen and myofibroblast content, and while it does not clearly demonstrate that collagen VI mechanistically controls the differentiation, this along with our study at earlier time points [28] suggests a strong temporal relationship between the two parameters. In our initial attempts to define the cell–matrix interactions that mediate the differentiation process, we revealed through crosslinking assays that type VI collagen interacts with cardiac fibroblasts by binding the α3 integrin. We then demonstrated that blockade of the α3 integrin with a function blocking antibody attenuated collagen VI induced α-SMA production of fibroblasts in vitro. We therefore concluded that the α3 integrin plays a key role in type VI collageninduced myofibroblast differentiation [28]. We proposed that type VI collagen induces myofibroblast differentiation to mediate the late stage of cardiac remodeling following an infarction (see Fig. 2). Several other cardiac pathologies have been linked to alterations type VI collagen. Polymorphisms and linkage disequilibrium of the collagen type VI gene loci COL6A1 and COL6A2 located on chromosome 21 have been implicated in abnormal development of the cardiac AV valves leading to congenital heart defects in Down's syndrome patients (trisomy 21) [48–50]. Skin fibroblasts isolated from individuals with trisomy 21 had an increased adhesive ability to type VI collagen, an effect that was shown to be mediated by the α3β1 integrin [26]. As discussed above, we determined that type VI collagen-induced myofibroblast differentiation is α3 integrin mediated, therefore it would be interesting to determine whether differentiation is correlated to the increased adhesion of fibroblasts isolated from trisomy 21 individuals plated on type VI collagen. Diabetic patients may develop diabetic cardiomyopathy resulting in an accumulation of the fibrillar collagens and type VI collagen [51]. Interestingly, Shimizu et al. determined the type VI accumulation occurred in the endomysium whereas types I and III increased in the perimysium [51]. Type VI collagen accumulated in the endomysium of patients with atrial fibrillation [52]. An increase in type VI was seen in the hearts of rats with renal clipping-induced hypertension, however no change in the amount of type IV collagen was seen in these animals [53]. In the future it would be interesting to determine whether the
accumulation type VI collagen during the various pathological conditions is correlated or even causative to an increase in the myofibroblast population. 7. Degradation on non-fibrillar collagens: The role of MMPs and serine proteases A key component to cardiac remodeling is the degradation of old matrix that allows for the deposition of new matrix. The myocardial matrix metalloproteinases (MMPs) are the regulators of ECM degradation during remodeling and are classified as collagenases (MMP-1, 8, 13), gelatinases (MMP-2, 9), and stromelysins (MMP-3, 7) [54]. Collagen types I and III are initially degraded by collagenases, which break down the helical structure, and the degradation products are further digested by gelatinases [55]. Interestingly fibrillar type V and non-fibrillar type IV are not targeted by collagenases but rather by gelatinases and stromelysins [54,55]. Type VI collagen does not interact with MMP-9, a gelatinase that degrades most fibrillar collagens [56]. It was demonstrated that type VI collagen is resistant to breakdown by MMPs-1, 2, 3, and 9; instead it is degraded by serine proteases [57]. These enzymes are produced by mast cells and neutrophils during the inflammatory response and are capable of activating TGF β and angiotensin II by cleaving their precursors [58]. We have determined that type VI collagen accumulates in the infarcted region of the heart 7 days post-MI and remains elevated for up to 20 weeks [4,28]. The buildup of collagen VI occurs in spite of serine protease levels being high, since the mast cells are likely producing more of these enzymes during the initial stages of post-MI inflammation and remodeling. The later accumulation of type VI collagen may be explained by the lack of type VI collagen proteases, after the delivery of the proteases decreases in a time-dependent manner. Thus, collagen VI may represent a more stable form of collagen that does not turnover in the same manner as the other myocardial collagens that are subject to breakdown by multiple MMPs. 8. Major questions and challenges (see Table 2) 8.1. Do non-fibrillar collagens play a role in determining the source of the myofibroblasts by promoting differentiation of resident fibroblasts or stem cell homing? Myofibroblasts accumulate in the heart during post-MI remodeling, but the source of the myofibroblasts has yet to be determined. Do changes in the ECM stimulate the resident cardiac fibroblasts to differentiate to the myofibroblast or are stem cells recruited from the
Fig. 2. Temporal progression of non-fibrillar collagen deposition and myofibroblast content during in vivo post-MI remodeling. The time-dependent elevation in type IV collagen begins in the peripheral areas of the heart (distal from the infarct) and takes weeks to become elevated in the infarcted area. Type VI, in contrast, becomes elevated in the infarcted area within days after MI induction, and later accumulates in the peripheral areas of the heart. The myofibroblasts are prevalent in the early phase of remodeling, temporarily disappear, and then reappear in late phase remodeling. We speculate that the early remodeling and myofibroblast appearance in the infarct are largely mediated by the robust inflammatory response; the increase in myofibroblast content in the infarcted and peripheral areas then becomes dependent upon cell–matrix interactions that may involve type VI collagen.
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periphery? Similar questions arise in other organs including the lung and liver. Several candidates are debated as precursors of myofibroblasts in the lung; resident peribronchiolar adventitial fibroblasts have been suggested but others indicate circulating fibrocytes or others have even proposed that endothelial cells transition to a mesenchymal α-SMA expressing cell [59]. In the liver, hepatic stellate cells are the most established myofibroblast precursor but others have suggested portal fibroblasts are the source of myofibroblasts from both bile duct ligation and in chronic viral hepatitis [59]. There are several hormonal and mechanical stimuli that may activate the resident cardiac fibroblasts to differentiate to the myofibroblast phenotype. For example, TGF-β stimulates myofibroblast differentiation in vitro; and following a myocardial infarction the initial inflammatory response provides ample TGF-β to the resident fibroblasts. Interestingly, Kuwahara et al. blocked TGF-β following a myocardial infarction and determined that the loss of functional TGF-β attenuated the appearance of myofibroblasts at day 3 and decreased the activation of the resident fibroblasts during early remodeling [60]. We determined that type VI collagen potently induces myofibroblast differentiation in vitro and that both myofibroblast content and type VI collagen accumulate in the heart post-MI, but we have not determined whether increased type VI is inducing the myofibroblast differentiation during late remodeling [4]. We will utilize the type VI collagen knockout mouse to address this question (Table 2). 8.2. What specific matrix–cell interactions and downstream signaling events mechanistically control differentiation of myofibroblasts? Our laboratory revealed that type VI collagen potently stimulated myofibroblast differentiation, although we still have not elucidated the signaling mechanisms that underlie this process [4]. We have also shown that the α3 integrin mediates the type VI induced differentiation [28]. Two potential pathways are the SMAD 2/3 pathway, induced by TGF-β, and ROCK/LIMK, induced by mechanical stress. Alterations in collagen deposition, integrin expression and downstream signaling should be evaluated to differentiate changes amongst the pathological conditions such as diabetes, hypertension, and hypertrophic cardiomyopathy. 8.3. How much and which types of collagen (fibrillar versus non-fibrillar) are produced by myofibroblasts? Myofibroblasts are crucial mediators of wound healing and are typically referred to as a hypersecretory phenotype. The definition of hypersecretory is quiet vague; how much collagen do the myofibroblasts produce and is there a differential secretion composition compared to cardiac fibroblasts? TGF-β was utilized to induce differentiation, and it was determined that myofibroblasts produce 2-fold more collagen per cell, but the changes in specific collagen isoforms were not evaluated [61]. In another study, TFG-β stimulation significantly increased production of both types I and III [62]. Determining myofibroblast non-fibrillar collagen production would be important to more fully understand the role of myofibroblasts in post-injury remodeling.
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8.4. Does the collagen matrix play a major role in fibroblast migration to sites distal from the infarcted area to induce a global fibrosis? The role of the fibroblasts and myofibroblasts in repairing the infarcted myocardium has been well studied; these cells must migrate to the wound and deposit large amounts of collagen for scar formation and to replace dying myocytes. Many neurohumoral and inflammatory factors come into play during this early, aggressive phase of remodeling. The concern then becomes how is the rest of the healthy tissue either near or distal from the infarct affected by the changes in these circulating factors? The development of a more diffuse, global fibrosis occurs long after the inflammatory response is complete, but the mechanisms that mediate the spreading of the fibrosis throughout the healthy myocardium are not well-defined. The fibroblasts activated during the early phase of remodeling may migrate out of the wound area, or perhaps the neurohumoral factors cause activation of the fibroblasts that are resident in the distal sites. Determining the source, as well as the composition (fibrillar and non-fibrillar) of the matrix as it spreads is an important step to understand how the fibrosis spreads to the healthy tissue. 8.5. Does the limited breakdown of collagen VI by MMPs yield a more stable collagen that could nucleate organizational sites for collagen I and III fibril assembly? Could non-fibrillar collagens play a role in angiogenesis or VSMC function? Collagen VI appears resistant to breakdown by MMPs, and it has been proposed that its degradation depends upon delivery of chymases and serine proteases by inflammatory cells during the early phases of post-injury remodeling. Despite this, the collagen VI accumulates steadily in the days and weeks after MI induction both in infarcted and non-infarcted areas, therefore as the inflammatory response recedes the heart may be left with more stable (type VI) collagen throughout the myocardium. Collagen VI could then form a network capable of binding the fibrillar collagens to promote net deposition and collagen assembly. Thus, co-localization studies of these three collagen types (using in vivo and in vitro models) would provide beneficial information as to the stability of collagen VI and its role in fibrillar matrix assembly and organization. There are several studies to suggest that the type IV collagen network in the myocardium can slow angiogenesis. Matrix degradation must occur for angiogenesis to proceed and depends on the action of the MMPs. In the case of collagen IV, several MMPs are capable of degrading it and clearing the way for new vessels to form. With collagen VI, little is known about its role in angiogenesis. Does it create a pro-angiogenic environment by creating a scaffold on which cells can migrate? Could it function as a barrier against angiogenesis? We have found type VI collagen to inhibit the migration of cardiac fibroblasts in vitro (unpublished results), but little is known about how collagen VI affects VSMC migration and angiogenesis. Performing in vivo studies of vessel formation in the type VI-deficient mouse following MI or ischemia–reperfusion injury will be critical to understanding the role of the matrix environment in controlling angiogenesis. 9. Overall summary
Table 2 Future directions. 1. Do non-fibrillar collagens influence activation (proliferation, differentiation, migration) of the various cardiac cell types and does this activation contribute to cardiac remodeling? 2. What is the role of increased type IV collagen following a myocardial infarction? Why does the increased deposition initially occur in the distal sites? 3. Myofibroblasts are critical mediators of wound healing; how much non-fibrillar collagen do these cells secrete? 4. Are the non-fibrillar collagens, particularly type VI, more resistant to degradation than the fibrillar ones?
The myocardium is a dynamic environment; the ECM is continually regulated by cardiac fibroblasts under normal conditions and heightened during pathological states. The fibrillar collagens create a strong meshwork to support the myocytes and fibroblasts and the non-fibrillar collagens are the glue that holds the matrix together. The individual matrix components are capable of stimulating cell activation. Following a myocardial infarction, deposition both fibrillar types I and III increases to create a fibrotic replacement scar at the site of myocyte apoptosis; overactive fibroblasts deposit excess collagen at
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both the infarct and distal regions. Also following an infarction, type VI deposition increases at both the infarct and distal sites, and myofibroblast content is enhanced in both areas. The distribution of type IV collagen also changes — initially it is found in the noninfarcted area following an MI and slowly increases in the infarcted area. Why these non-fibrillar collagens change in quantity and distribution following an MI is still an open question, however several lines of evidence indicate that they play key roles in cardiac cell activation, infarct scar formation, fibrillar collagen organization, and perhaps several other processes during the acute and prolonged phases of myocardial repair. References [1] Weber KT. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol 1989 Jun;13(7):1637–52. [2] Weis SM, Emery JL, Becker KD, McBride Jr DJ, Omens JH, McCulloch AD. 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Contents lists available at ScienceDirect
Journal of Molecular and Cellular Cardiology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Review article
Non-fibrillar collagens: Key mediators of post-infarction cardiac remodeling? Patricia E. Shamhart, J. Gary Meszaros ⁎ Department of Integrative Medical Sciences, Northeastern Ohio Universities College of Medicine (NEOUCOM), 4209 State Route 44, Rootstown, OH 44272-0095, USA
a r t i c l e
i n f o
Article history: Received 17 April 2009 Received in revised form 9 June 2009 Accepted 21 June 2009 Available online 30 June 2009 Keywords: Myocardial infarction Wound healing Myofibroblasts Matrix metalloproteinases Serine proteases Integrins
a b s t r a c t Cardiac remodeling is accelerated during pathological conditions and several anabolic and catabolic regulators work in concert to repair the myocardium and maintain its functionality. The fibroblasts play a major role in this process via collagen deposition as well as supplying the degradative matrix metalloproteinases. During the more acute responses to a myocardial infarction (MI) the heart relies on a more aggressive wound healing sequence that includes the myofibroblasts, specialized secretory cells necessary for infarct scar formation and thus, rescue of the myocardium. The activated fibroblasts and myofibroblasts deposit large amounts of fibrillar collagen during the post-MI wound healing phase, type I and III collagen are the most abundant collagens in the heart and they maintain the structural integrity under normal and disease states. While collagen I and III have been the traditional focus of the myocardial matrix, recent studies have suggested that the non-fibrillar collagens (types IV and VI) are also deposited during pathological wound healing and may play key roles in myofibroblast differentiation and organization of the fibrillar collagen network. This review highlights the potential roles of the non-fibrillar collagens and how they work in concert with the fibrillar collagens in mediating myocardial remodeling. Published by Elsevier Ltd.
Contents 1. 2. 3. 4. 5. 6. 7. 8.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cellular and extracellular composition of the myocardium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Type V collagen: Potential regulator of fibril assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The importance of non-fibrillar collagens: The interaction with other ECM components . . . . . . . . . . . . . . . . . . . . . . . . . . Type IV collagen: The ubiquitous non-fibrillar collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Type VI collagen: A more specialized non-fibrillar collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Degradation on non-fibrillar collagens: The role of MMPs and serine proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Major questions and challenges (see Table 2). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1. Do non-fibrillar collagens play a role in determining the source of the myofibroblasts by promoting differentiation of resident fibroblasts or stem cell homing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2. What specific matrix–cell interactions and downstream signaling events mechanistically control differentiation of myofibroblasts? . . 8.3. How much and which types of collagen (fibrillar versus non-fibrillar) are produced by myofibroblasts? . . . . . . . . . . . . . . . 8.4. Does the collagen matrix play a major role in fibroblast migration to sites distal from the infarcted area to induce a global fibrosis? . 8.5. Does the limited breakdown of collagen VI by MMPs yield a more stable collagen that could nucleate organizational sites for collagen I and III fibril assembly? Could non-fibrillar collagens play a role in angiogenesis or VSMC function? . . . . . . . . . . . . . . . . 9. Overall summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The extracellular matrix ECM of the heart is a dynamic environment encompassing both fibrillar and non-fibrillar collagens. Cardiac fibroblasts (CFs) are the major ECM producers in the heart and are ⁎ Corresponding author. Tel.: +1 330 325 6432; fax: +1 330 325 5912. E-mail address: [email protected] (J.G. Meszaros). 0022-2828/$ – see front matter. Published by Elsevier Ltd. doi:10.1016/j.yjmcc.2009.06.017
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triggered to alter ECM composition during pathophysiological conditions that range from the acute (myocardial infarction, MI) to the chronic (hypertension, heart failure, diabetes). In each of these diseases the CFs adjust their function to maintain architectural integrity within the myocardium in an effort to provide sufficient support for cardiac function. The CFs respond to disease stimuli by adjusting their proliferation, migration, differentiation, collagen deposition and matrix metalloproteinase (MMP) production.
P.E. Shamhart, J.G. Meszaros / Journal of Molecular and Cellular Cardiology 48 (2010) 530–537
In the setting of post-MI remodeling, replacement fibrosis and infarct scar formation are necessary events to repair and, depending on the severity of the infarct, potentially rescue the heart from ventricular wall collapse or rupture. The CFs migrate to the ischemic area and border zone and deposit large amounts of ECM to replace the dying myocytes and to “wall off” the injured area via scar formation [1]. CFs are capable of differentiating to the hypersecretory myofibroblast phenotype, which plays an even more critical role in acute post-MI remodeling compared to the more chronic diseases. The myofibroblasts harbor enhanced capabilities to secrete ECM and perform repair functions, and work in concert with the fibroblasts to orchestrate the wound healing process. The major distinguishing feature of the myofibroblast is that they express α-smooth muscle actin, which allows for stress fiber organization and sustained cell contraction to stabilize the wound. The prolonged activity of fibroblasts and myofibroblasts is where matrix deposition can go awry — continued collagen production proceeds from both sources, and of particular concern is the extended life-span of the myofibroblasts. These cells perform the necessary wound healing duties then undergo apoptosis, but if this does not occur then the persistent activity of these cells causes excessive matrix accumulation and detrimental fibrosis. An important question is how long is required for the myofibroblast to complete their beneficial repair duties following the MI? Which types of collagen do they produce? What are the mechanisms that turn CF and myofibroblast activity on and off? Several complex factors work in concert to influence myocardial repair, including humoral factors, mechanical stimuli and matrix–cell interactions. This review will focus on how the matrix changes during remodeling and will ask how the matrix itself is capable of influencing the repair of the myocardium. Historically, fibrillar collagens, mainly types I and III, have been the major focus in the heart, however, significant contributions from nonfibrillar collagens during pathological cardiac remodeling have been reported as well. Although the non-fibrillar collagen types IV and VI are viewed as minor structural components of the myocardium, these collagens anchor and organize the fibrillar collagens and have other roles in controlling CF migration, differentiation and proliferation. Several intriguing findings from in vitro and in vivo studies will be presented to emphasize the interdependence of the collagen components in repair and remodeling of the heart. 2. Cellular and extracellular composition of the myocardium The adult myocardium is made up of a network of fibrillar (I, III, and V) and non-fibrillar (IV and VI) collagens. By far, collagen I is the most abundant ECM protein in the body and is expressed in every major organ and tissue. Mutations in collagen I lead to osteogenesis imperfecta (OI), an autosomal dominant disease that is characterized by weak or brittle bones. OI mouse models have been developed, one of which has been used to study the myocardial matrix composition. The OI murine model has a deficiency in the pro-alpha2(I) chain, and a well-characterized deficit in tendon and bone strength. In the myocardium these animals displayed significant reductions in collagen density and fibril diameter leading to a higher compliance of the LV upon passive inflation [2]. Although it remains to be tested, the reduction in collagen I quality and/or content would likely be detrimental in the case of a severe stresses such as MI or ischemia– reperfusion injury where ventricular rupture could occur. Also of
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interest is whether other collagen types are elevated in the hearts of OI mice in an effort to maintain its biomechanical integrity. Cardiac fibroblasts are the major matrix producing cells in the heart and are the most abundant cell type in terms of overall numbers, whereas the larger myocytes account for the majority of the myocardial mass. The myocytes are arranged in layers woven together by the fibrous ECM which is interspersed with fibroblasts [3]. The fibrillar collagens of the ECM not only provide structure to the heart but also signal to the various cells of the myocardium to alter their activity. We have demonstrated that solubilized collagen types I and III potently stimulate cardiac fibroblast proliferation in vitro [4]. Recently we have established that collagens type I and III also enhance cardiac fibroblast migration relative to type VI (unpublished results). Monomeric type I collagen enhances smooth muscle cell proliferation whereas type I in its native fibrillar form reduces proliferation [5]. Several in vitro effects of the fibrillar and non-fibrillar collagens are displayed in Table 1. The importance of types I and III collagen in cardiac pathologies has been extensively studied, and these collagens increase significantly in response to MI and in several other diseases. It has been debated how the relative proportions or ratios of these collagens change during accelerated remodeling, and it has been suggested that accumulation of collagen I, the more highly organized of the two, is responsible for the decreased compliance and fibrotic stiffening of the myocardium leading to global dysfunction [1]. Following a myocardial infarction, it has been shown that type III collagen is deposited during early remodeling whereas, type I collagen deposition predominates during the intermediate and late remodeling stages [6]. 3. Type V collagen: Potential regulator of fibril assembly Type V collagen is a widely distributed, low abundance fibrillar collagen that has been shown to associate with type I collagen in several diverse tissues. A unique property of type V is the formation of a hybrid fibril with type I collagen in vitro [7]. Evidence from dermal tissue indicates that collagen V is required to initiate collagen fibril nucleation, and further that type V can regulate the size and organization of collagen fibrils, many of which are heterotypic collagen I and V containing structures [8]. Mutations in type V collagen are linked to Ehlers–Danlos syndrome, a connective tissue disorder that features a hyperextensible skin phenotype [9]. Type V knockout mice are embryonic lethal at E10.5, at least in part due to cardiovascular failure. However, the heterozygote (ColVa1+/−) mice are viable and display symptoms similar to Ehlers–Danlos patients [10]. The specific function of type V collagen in the heart is not welldefined, despite its widespread expression and distribution throughout the interstitial matrix [11]. Type V collagen is deposited in the myocardium by cardiac fibroblasts and VSMCs [12,13]. In vitro, type V collagen imparts antiproliferative effects on VSMCs and endothelial cells [14,15]. There are limited studies that describe the effect of type V collagen on VSMC, myocyte, and cardiac fibroblast activation despite reports that describe alterations of type V during several cardiovascular pathophysiological conditions. At 20 and 40 weeks, spontaneously hypertensive rats have significantly elevated levels of type V collagen
Table 1 In vitro effects of fibrillar and non-fibrillar collagen substrates. Collagen type
I (fibrillar)
III (fibrillar)
IV (non-fibrillar)
V (fibrillar)
VI (non-fibrillar)
H9C2 myoblasts Cardiac fibroblasts
↓ Proliferation [34] ↑ Proliferation [4] ↑ Migration Monomeric: ↑ proliferation [5] Fibrillar: ↓ proliferation [5]
? ↑ Proliferation [4] ↑ Migration ?
↑ Proliferation [34] ?
? ?
↑ Differentiation to VSMC [31–33]
↓ Proliferation [14]
? ↑ Myofibroblast differentiation [4] ↓ Migration ?
VSMCs
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compared to control rats [16]. Type V collagen content also increases with atherosclerosis, dilated cardiomyopathy, and the formation of hypertrophic scars [17–19]. The importance and consequence of elevated type V during these pathological conditions have not been established, most likely since the exact function of type V is unknown. Perhaps the most revealing study that links collagen V with cardiac pathology utilized the ColVa1+/− mice. It was determined that cardiac valve development is dependent upon the presence of collagen V, and that type I collagen increases in the heart valves and left ventricles, perhaps as a compensatory mechanism [20]. To date, the role of type V collagen following a myocardial infarction has not been determined. We speculate that collagen V would play a critical role in infarct scar formation as well as replacement fibrosis, due to its apparent importance in type I collagen organization. It would certainly be interesting to utilize the ColVa1+/− mouse in a myocardial infarction study to further define how type V contributes to post-MI remodeling. 4. The importance of non-fibrillar collagens: The interaction with other ECM components The fibrillar collagens are recognized as the structural support for the matrix but the non-fibrillar collagens are key regulators in anchoring and organizing the ECM meshwork. Type IV collagen is the major component of a specialized region of ECM surrounding cells such as the myocyte referred to as the basement membrane (see Fig. 1). In the basement membrane, collagen IV forms a scaffold with laminin, entactin, and perlecan to create a collagen, proteoglycan, and glycoprotein meshwork [21,22]. Type IV interacts predominantly with the α1β1 and α2β2 integrins, with the former having a higher affinity for type IV and the latter for type I collagen. In addition to these integrins, evidence for collagen IV binding to α3β1, α10β1, α11β1, and αvβ3 has also been put forth [23]. The basement membrane is a diverse environment and additional ECM components may be found in the basement membrane in accordance with specific cell types. It has been reported that type VI collagen interacts with type IV collagen in the basement membrane creating an anchoring bridge between the basement membrane and the interstitial matrix [24]. Type VI collagen interacts with multiple interstitial matrix proteins including type I collagen and fibronectin [25]. It is speculated that the α1, α2, α3, and α10 (β1) and the αvβ3 integrins all potentially bind collagen VI in a cell-type specific manner [26,27]. Our laboratory has presented recent evidence that the α3 integrin plays a mechanistic role in the phenotypic transformation of cardiac myofibroblasts [28]. The nonfibrillar collagens are integral components of ECM adhesion throughout the body, however little is known about their role in injury responses, particularly in the myocardium. 5. Type IV collagen: The ubiquitous non-fibrillar collagen Individual cardiac myocytes are surrounded by a specialized basement membrane ECM composed mainly of type IV collagen, glycoproteins, and proteoglycans that is incorporated into the endomysium. Beyond the basement membrane the composition of the matrix changes and type I and III collagen begin to dominate the interstitial matrix. Eghbali et al. revealed that cardiac fibroblasts produce most of the interstitial collagens, types I and III, and basement membrane collagen IV, whereas and that cardiac myocytes are also capable of producing type IV collagen [29]. Blood vessels are tightly surrounded by a basement membrane and an interstitial matrix. Individual matrix components interact with various cells to lend support and structure but they are also capable of stimulating cell differentiation and/or proliferation. Plating adult smooth muscle cells (SMCs) on type IV collagen induces an increase in SMC contractile proteins, α-smooth muscle actin and smooth muscle myosin heavy chain [30]. Stem cells plated on type IV collagen
spontaneously differentiate to smooth muscle cells [31–33]. Furthermore, it was shown that collagen IV induced smooth muscle cell differentiation of Sca-1+ progenitor cells was mediated by α1/β1/αv integrins [32]. In vitro type IV collagen induced proliferation of H9C2 myoblasts, myocyte precursor cells, whereas type I collagen attenuated this response [34]. In culture, type IV collagen induces SMC differentiation and myocyte precursor cell proliferation but few studies have addressed the effect of type IV on cardiac fibroblast activation. Understanding how type IV collagen affects all cardiac cell types is important because it may provide clues as to why type IV collagen composition is altered during several cardiovascular pathophysiological conditions and how these changes affect remodeling and recovery. Following a myocardial infarction, type IV collagen increases expression in the peripheral zone at day 3 post-infarction and reaches its maximal expression in this zone between 7 and 11 days [35]. Interestingly, type IV appearance does not increase in the outer infarct zone until day 4 and in the inner portion of the infarct region at day 10. The accumulation of type IV in the inner portion of the infarct zone is minimal compared to other regions [35]. Type IV collagen was shown to be restricted to the myocyte basement membrane of hypertrophic cardiomyopathy patients as well as being localized in the regions of replacement fibrosis [36]. Streptozotocin-treated diabetic rats likewise exhibit left ventricular type IV collagen accumulation [37]. The consequence of accumulated type IV collagen during these pathophysiological conditions, particularly post-MI, is poorly understood. It is generally thought that collagen IV and the basement membrane are highly adhesive and may serve glue cells and ECM in place, and that they slow the migration of several cell types. The reports that indicate a somewhat delayed appearance of type IV collagen in the middle of an infarct zone may indicate that the initial absence of type IV in the damaged area permits the migration of fibroblast and myofibroblasts to the damaged area, followed next by reparative and replacement fibrosis via fibrillar collagen deposition and the eventual deposition of type IV collagen. In this scenario, the type IV collagen deposition may be a result of bulk collagen deposition rather than contributing to a well-organized basement membrane. These and several other possibilities are still to be to be determined.
6. Type VI collagen: A more specialized non-fibrillar collagen Type VI collagen is expressed in the heart (both embryonic and adult), basement membranes, and in vascular smooth muscle [38–40]. Type VI collagen formation is a unique process that differs from fibrillar collagen assembly. Like all collagen structures, type VI is composed of three α chains in a triple helix, but unique to type VI, the monomer triple helix assembles into a dimer followed by a tetramer both occurring in an anti-parallel, lateral direction [41,42]. The tetramer is secreted from the cell and assembles in an end to end fashion creating the beaded filament appearance of the type VI microfibril [42]. Type VI acts as an adhesive structure in the basement membrane and interacts with other ECM components [43]. Mutations in type VI collagen genes have been linked to two major skeletal muscle defects, Bethlem myopathy and Ullrich congenital muscular dystrophy [44]. Type VI-deficient mice have been generated by the deletion of the α1 chain, preventing formation of the triple helical collagen VI molecule [45]. The type VI knockout mice exhibit a defective muscular phenotype with skeletal muscle necrosis and varying muscle fiber dimensions resembling Bethlem myopathy. Skeletal muscle myofibers from type VI-deficient mice have altered sacroplasmic reticulum and mitochondrial structures causing mitochondrial dysfunction and apoptosis [46]. Mitochondrial function can be restored by placing the isolated myofibers on a type VI collagen matrix [46]. The ECM of the type VI-deficient mice has an altered organization and fibroblasts isolated from these mice have abnormal
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Fig. 1. Non-fibrillar collagens organize the basement membrane. The basement membrane is a complex region composed mainly of non-fibrillar collagens and adhesive glycoproteins and proteoglycans; laminin interacts with type IV collagen, entactin, perlecan, and integrin receptors on the cell membrane. Type IV collagen also interacts with non-fibrillar type VI collagen that spans beyond the basement membrane to interact with the fibrillar collagens.
fibronectin deposition that is very similar to fibroblasts from Bethlem myopathy patients [47]. Our laboratory has recently begun to study how specific collagen substrates influence cardiac fibroblast activity. We have observed that type VI collagen attenuates CF migration whereas types I and III facilitate migration in an in vitro wound healing assay (unpublished results). We have reported that type VI collagen potently induces myofibroblast differentiation in vitro [4]. Since myofibroblasts are crucial mediators of wound healing, we have explored the progressive
increases in myofibroblasts content and type VI collagen deposition following a myocardial infarction. Seven days post-MI, both type VI and myofibroblast content were significantly increased in the infarcted zones of the MI animals [28]. By 14 days the myofibroblast content returned to control levels but type VI collagen was still elevated. During the later stages of remodeling at 16–20 weeks postinfarction, both type VI collagen and myofibroblast content were enhanced in the infarcted areas [4]. It was surprising that the myofibroblast levels were elevated this late in remodeling, leading
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us to speculate that the increase in collagen VI may preserve these cells either by preventing their disappearance via apoptosis or by promoting their in vivo differentiation. Thus, the preservation of the myofibroblast in the infarcted area could significantly contribute to the progression of fibrosis both within the damaged area as well as distal sites. We did see elevations in both collagen VI and myofibroblasts in the non-infarcted samples taken from the posterior wall of the 16 week post-MI samples, indicating that with time the deleterious remodeling will spread beyond the ischemic area. This study [4] was the first to link in vivo evidence for the elevation of both type VI collagen and myofibroblast content, and while it does not clearly demonstrate that collagen VI mechanistically controls the differentiation, this along with our study at earlier time points [28] suggests a strong temporal relationship between the two parameters. In our initial attempts to define the cell–matrix interactions that mediate the differentiation process, we revealed through crosslinking assays that type VI collagen interacts with cardiac fibroblasts by binding the α3 integrin. We then demonstrated that blockade of the α3 integrin with a function blocking antibody attenuated collagen VI induced α-SMA production of fibroblasts in vitro. We therefore concluded that the α3 integrin plays a key role in type VI collageninduced myofibroblast differentiation [28]. We proposed that type VI collagen induces myofibroblast differentiation to mediate the late stage of cardiac remodeling following an infarction (see Fig. 2). Several other cardiac pathologies have been linked to alterations type VI collagen. Polymorphisms and linkage disequilibrium of the collagen type VI gene loci COL6A1 and COL6A2 located on chromosome 21 have been implicated in abnormal development of the cardiac AV valves leading to congenital heart defects in Down's syndrome patients (trisomy 21) [48–50]. Skin fibroblasts isolated from individuals with trisomy 21 had an increased adhesive ability to type VI collagen, an effect that was shown to be mediated by the α3β1 integrin [26]. As discussed above, we determined that type VI collagen-induced myofibroblast differentiation is α3 integrin mediated, therefore it would be interesting to determine whether differentiation is correlated to the increased adhesion of fibroblasts isolated from trisomy 21 individuals plated on type VI collagen. Diabetic patients may develop diabetic cardiomyopathy resulting in an accumulation of the fibrillar collagens and type VI collagen [51]. Interestingly, Shimizu et al. determined the type VI accumulation occurred in the endomysium whereas types I and III increased in the perimysium [51]. Type VI collagen accumulated in the endomysium of patients with atrial fibrillation [52]. An increase in type VI was seen in the hearts of rats with renal clipping-induced hypertension, however no change in the amount of type IV collagen was seen in these animals [53]. In the future it would be interesting to determine whether the
accumulation type VI collagen during the various pathological conditions is correlated or even causative to an increase in the myofibroblast population. 7. Degradation on non-fibrillar collagens: The role of MMPs and serine proteases A key component to cardiac remodeling is the degradation of old matrix that allows for the deposition of new matrix. The myocardial matrix metalloproteinases (MMPs) are the regulators of ECM degradation during remodeling and are classified as collagenases (MMP-1, 8, 13), gelatinases (MMP-2, 9), and stromelysins (MMP-3, 7) [54]. Collagen types I and III are initially degraded by collagenases, which break down the helical structure, and the degradation products are further digested by gelatinases [55]. Interestingly fibrillar type V and non-fibrillar type IV are not targeted by collagenases but rather by gelatinases and stromelysins [54,55]. Type VI collagen does not interact with MMP-9, a gelatinase that degrades most fibrillar collagens [56]. It was demonstrated that type VI collagen is resistant to breakdown by MMPs-1, 2, 3, and 9; instead it is degraded by serine proteases [57]. These enzymes are produced by mast cells and neutrophils during the inflammatory response and are capable of activating TGF β and angiotensin II by cleaving their precursors [58]. We have determined that type VI collagen accumulates in the infarcted region of the heart 7 days post-MI and remains elevated for up to 20 weeks [4,28]. The buildup of collagen VI occurs in spite of serine protease levels being high, since the mast cells are likely producing more of these enzymes during the initial stages of post-MI inflammation and remodeling. The later accumulation of type VI collagen may be explained by the lack of type VI collagen proteases, after the delivery of the proteases decreases in a time-dependent manner. Thus, collagen VI may represent a more stable form of collagen that does not turnover in the same manner as the other myocardial collagens that are subject to breakdown by multiple MMPs. 8. Major questions and challenges (see Table 2) 8.1. Do non-fibrillar collagens play a role in determining the source of the myofibroblasts by promoting differentiation of resident fibroblasts or stem cell homing? Myofibroblasts accumulate in the heart during post-MI remodeling, but the source of the myofibroblasts has yet to be determined. Do changes in the ECM stimulate the resident cardiac fibroblasts to differentiate to the myofibroblast or are stem cells recruited from the
Fig. 2. Temporal progression of non-fibrillar collagen deposition and myofibroblast content during in vivo post-MI remodeling. The time-dependent elevation in type IV collagen begins in the peripheral areas of the heart (distal from the infarct) and takes weeks to become elevated in the infarcted area. Type VI, in contrast, becomes elevated in the infarcted area within days after MI induction, and later accumulates in the peripheral areas of the heart. The myofibroblasts are prevalent in the early phase of remodeling, temporarily disappear, and then reappear in late phase remodeling. We speculate that the early remodeling and myofibroblast appearance in the infarct are largely mediated by the robust inflammatory response; the increase in myofibroblast content in the infarcted and peripheral areas then becomes dependent upon cell–matrix interactions that may involve type VI collagen.
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periphery? Similar questions arise in other organs including the lung and liver. Several candidates are debated as precursors of myofibroblasts in the lung; resident peribronchiolar adventitial fibroblasts have been suggested but others indicate circulating fibrocytes or others have even proposed that endothelial cells transition to a mesenchymal α-SMA expressing cell [59]. In the liver, hepatic stellate cells are the most established myofibroblast precursor but others have suggested portal fibroblasts are the source of myofibroblasts from both bile duct ligation and in chronic viral hepatitis [59]. There are several hormonal and mechanical stimuli that may activate the resident cardiac fibroblasts to differentiate to the myofibroblast phenotype. For example, TGF-β stimulates myofibroblast differentiation in vitro; and following a myocardial infarction the initial inflammatory response provides ample TGF-β to the resident fibroblasts. Interestingly, Kuwahara et al. blocked TGF-β following a myocardial infarction and determined that the loss of functional TGF-β attenuated the appearance of myofibroblasts at day 3 and decreased the activation of the resident fibroblasts during early remodeling [60]. We determined that type VI collagen potently induces myofibroblast differentiation in vitro and that both myofibroblast content and type VI collagen accumulate in the heart post-MI, but we have not determined whether increased type VI is inducing the myofibroblast differentiation during late remodeling [4]. We will utilize the type VI collagen knockout mouse to address this question (Table 2). 8.2. What specific matrix–cell interactions and downstream signaling events mechanistically control differentiation of myofibroblasts? Our laboratory revealed that type VI collagen potently stimulated myofibroblast differentiation, although we still have not elucidated the signaling mechanisms that underlie this process [4]. We have also shown that the α3 integrin mediates the type VI induced differentiation [28]. Two potential pathways are the SMAD 2/3 pathway, induced by TGF-β, and ROCK/LIMK, induced by mechanical stress. Alterations in collagen deposition, integrin expression and downstream signaling should be evaluated to differentiate changes amongst the pathological conditions such as diabetes, hypertension, and hypertrophic cardiomyopathy. 8.3. How much and which types of collagen (fibrillar versus non-fibrillar) are produced by myofibroblasts? Myofibroblasts are crucial mediators of wound healing and are typically referred to as a hypersecretory phenotype. The definition of hypersecretory is quiet vague; how much collagen do the myofibroblasts produce and is there a differential secretion composition compared to cardiac fibroblasts? TGF-β was utilized to induce differentiation, and it was determined that myofibroblasts produce 2-fold more collagen per cell, but the changes in specific collagen isoforms were not evaluated [61]. In another study, TFG-β stimulation significantly increased production of both types I and III [62]. Determining myofibroblast non-fibrillar collagen production would be important to more fully understand the role of myofibroblasts in post-injury remodeling.
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8.4. Does the collagen matrix play a major role in fibroblast migration to sites distal from the infarcted area to induce a global fibrosis? The role of the fibroblasts and myofibroblasts in repairing the infarcted myocardium has been well studied; these cells must migrate to the wound and deposit large amounts of collagen for scar formation and to replace dying myocytes. Many neurohumoral and inflammatory factors come into play during this early, aggressive phase of remodeling. The concern then becomes how is the rest of the healthy tissue either near or distal from the infarct affected by the changes in these circulating factors? The development of a more diffuse, global fibrosis occurs long after the inflammatory response is complete, but the mechanisms that mediate the spreading of the fibrosis throughout the healthy myocardium are not well-defined. The fibroblasts activated during the early phase of remodeling may migrate out of the wound area, or perhaps the neurohumoral factors cause activation of the fibroblasts that are resident in the distal sites. Determining the source, as well as the composition (fibrillar and non-fibrillar) of the matrix as it spreads is an important step to understand how the fibrosis spreads to the healthy tissue. 8.5. Does the limited breakdown of collagen VI by MMPs yield a more stable collagen that could nucleate organizational sites for collagen I and III fibril assembly? Could non-fibrillar collagens play a role in angiogenesis or VSMC function? Collagen VI appears resistant to breakdown by MMPs, and it has been proposed that its degradation depends upon delivery of chymases and serine proteases by inflammatory cells during the early phases of post-injury remodeling. Despite this, the collagen VI accumulates steadily in the days and weeks after MI induction both in infarcted and non-infarcted areas, therefore as the inflammatory response recedes the heart may be left with more stable (type VI) collagen throughout the myocardium. Collagen VI could then form a network capable of binding the fibrillar collagens to promote net deposition and collagen assembly. Thus, co-localization studies of these three collagen types (using in vivo and in vitro models) would provide beneficial information as to the stability of collagen VI and its role in fibrillar matrix assembly and organization. There are several studies to suggest that the type IV collagen network in the myocardium can slow angiogenesis. Matrix degradation must occur for angiogenesis to proceed and depends on the action of the MMPs. In the case of collagen IV, several MMPs are capable of degrading it and clearing the way for new vessels to form. With collagen VI, little is known about its role in angiogenesis. Does it create a pro-angiogenic environment by creating a scaffold on which cells can migrate? Could it function as a barrier against angiogenesis? We have found type VI collagen to inhibit the migration of cardiac fibroblasts in vitro (unpublished results), but little is known about how collagen VI affects VSMC migration and angiogenesis. Performing in vivo studies of vessel formation in the type VI-deficient mouse following MI or ischemia–reperfusion injury will be critical to understanding the role of the matrix environment in controlling angiogenesis. 9. Overall summary
Table 2 Future directions. 1. Do non-fibrillar collagens influence activation (proliferation, differentiation, migration) of the various cardiac cell types and does this activation contribute to cardiac remodeling? 2. What is the role of increased type IV collagen following a myocardial infarction? Why does the increased deposition initially occur in the distal sites? 3. Myofibroblasts are critical mediators of wound healing; how much non-fibrillar collagen do these cells secrete? 4. Are the non-fibrillar collagens, particularly type VI, more resistant to degradation than the fibrillar ones?
The myocardium is a dynamic environment; the ECM is continually regulated by cardiac fibroblasts under normal conditions and heightened during pathological states. The fibrillar collagens create a strong meshwork to support the myocytes and fibroblasts and the non-fibrillar collagens are the glue that holds the matrix together. The individual matrix components are capable of stimulating cell activation. Following a myocardial infarction, deposition both fibrillar types I and III increases to create a fibrotic replacement scar at the site of myocyte apoptosis; overactive fibroblasts deposit excess collagen at
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both the infarct and distal regions. Also following an infarction, type VI deposition increases at both the infarct and distal sites, and myofibroblast content is enhanced in both areas. The distribution of type IV collagen also changes — initially it is found in the noninfarcted area following an MI and slowly increases in the infarcted area. Why these non-fibrillar collagens change in quantity and distribution following an MI is still an open question, however several lines of evidence indicate that they play key roles in cardiac cell activation, infarct scar formation, fibrillar collagen organization, and perhaps several other processes during the acute and prolonged phases of myocardial repair. References [1] Weber KT. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol 1989 Jun;13(7):1637–52. [2] Weis SM, Emery JL, Becker KD, McBride Jr DJ, Omens JH, McCulloch AD. 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