Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.     

Matrix metalloproteinases and their function in myocardium

A significant number of myocardial diseases are accompanied by increased synthesis and degradation of the extracellular matrix (ECM) as well as by changed maturation and incorporation of ECM components. Important groups of enzymes responsible for both normal and pathological processes in ECM remodeling are matrix metaloproteinases (MMPs). These enzymes share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The most important function of MMPs is the ability to cleave various ECM components; including such rigid molecules as fibrillar collagen molecules. The amount and activity of MMPs in cardiac tissue are regulated by a range of activating and inhibiting processes. Although MMPs play multifarious roles in many myocardial diseases, here we have focused on their function in ischemic cardiac tissue, dilated cardiomyopathy and hypertrophied cardiac tissue. The inhibition of MMPs by means of synthetic inhibitors seems to be a promising strategy in cardiac disease treatment. Their effects on diseased cardiac tissue have been successfully tested in several experimental studies.     

Dynamic expression profiles of MMPs/TIMPs and collagen deposition in mechanically unloaded rat heart: implications for left ventricular assist device support-induced cardiac alterations

Left ventricular assist devices (LVADs) ameliorate heart failure by reducing preload and afterload. However, extracellular matrix (ECM) deposition after application of LVADs is not clearly defined. The purpose of the present study was to investigate ECM remodeling after mechanical unloading in a rat heart transplant model. Sixty male Lewis rats were subjected to abdominal heterotopic heart transplantation, and the transplanted hearts were pressure- and volume-unloaded. The age- and weight- matched male Lewis rats who had undergone open thoracic surgeries were used as the control. Left ventricle ECM accumulation and the expression/activity of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) were measured on the third, seventh, and fourteenth days after transplantation/sham surgery. Compared with the control group, myocardial ECM deposition significantly increased on the seventh and fourteenth days after heart transplantation (P?<?0.05) and peaked on the 14th day. The gelatinase activity as well as mRNA expression of MMP-2 and MMP-9 significantly increased after transplantation (P?<?0.05). Both mRNA and protein levels of TIMP-1 and TIMP-2 significantly increased compared with those of the control group. Mechanical unloading may lead to adverse remodeling of the ECM of the left ventricle. The underlying mechanism may due to the imbalance of the MMP/TIMP system, especially the remarkable upregulation of TIMPs in the pressure and volume unloaded heart.     

Matrix remodeling during endochondral ossification

Endochondral ossification, the process by which most of the skeleton is formed, is a powerful system for studying various aspects of the biological response to degraded extracellular matrix (ECM). In addition, the dependence of endochondral ossification upon neovascularization and continuous ECM remodeling provides a good model for studying the role of the matrix metalloproteases (MMPs) not only as simple effectors of ECM degradation but also as regulators of active signal-inducers for the initiation of endochondral ossification. The daunting task of elucidating their specific role during endochondral ossification has been facilitated by the development of mice deficient for various members of this family. Here, we discuss the ECM and its remodeling as one level of molecular regulation for the process of endochondral ossification, with special attention to the MMPs.     

Interleukin-1 has opposing effects on connective tissue growth factor and tenascin-C expression in human cardiac fibroblasts

Cardiac fibroblasts (CF) play a central role in the repair and remodeling of the heart following injury and are important regulators of inflammation and extracellular matrix (ECM) turnover. ECM-regulatory matricellular proteins are synthesized by several myocardial cell types including CF. We investigated the effects of pro-inflammatory cytokines on matricellular protein expression in cultured human CF. cDNA array analysis of matricellular proteins revealed that interleukin-1α (IL-1α, 10 ng/ml, 6 h) down-regulated connective tissue growth factor (CTGF/CCN2) mRNA by 80% and up-regulated tenascin-C (TNC) mRNA levels by 10-fold in human CF, without affecting expression of thrombospondins 1–3, osteonectin or osteopontin. Western blotting confirmed these changes at the protein level. In contrast, tumor necrosis factor α (TNFα) did not modulate CCN2 expression and had only a modest stimulatory effect on TNC levels. Signaling pathway inhibitor studies suggested an important role for the p38 MAPK pathway in suppressing CCN2 expression in response to IL-1α. In contrast, multiple signaling pathways (p38, JNK, PI3K/Akt and NFκB) contributed to IL-1α-induced TNC expression. In conclusion, IL-1α reduced CCN2 expression and increased TNC expression in human CF. These observations are of potential value for understanding how inflammation and ECM regulation are linked at the level of the CF.     

Interaction between cell and extracellular matrix in heart disease: multiple roles of tenascin-C in tissue remodeling

The heart remodels myocardial tissue in physiological and pathological response. The cell-extracellular matrix (ECM) interaction provides not only structural and mechanical support but also important biological signaling during tissue remodeling. Among various ECM molecules, tenascin-C (TNC) is well known as a regulator of multiple cellular functions during embryogenesis, wound healing or cancer progression. In the heart, TNC appears in several important steps of embryonic development such as the initial differentiation of cardiomyocytes or coronary vasculo/angiogenesis, but it is not detected in a normal adult myocardium. However, TNC is found to re-express after myocardial injury and may regulate cellular behavior during tissue remodeling by modulating the attachment of cardiomyocytes to connective tissue, by enhancing migration and differentiation of myofibroblasts, and by inducing matrix metallo-proteinases. TNC also interacts with other ECM molecules and may modulate progression of fibrosis. Furthermore, transient and site specific expression of TNC closely associated with myocardial injury and inflammation suggests not only its key roles during tissue remodeling but also that TNC can be a marker for myocardial disease activity.     

Microarray-based identification of aminopeptidase N target genes in keratinocyte conditioned medium-stimulated dermal fibroblasts

Of the many processes that affect the outcome of wound repair, epidermal-dermal interactions are essential to extracellular matrix (ECM) remodeling and in particular, soluble factors released by keratinocytes are known to have a direct impact on the production of ECM by dermal fibroblasts. Aminopeptidase N (APN) has recently been proposed as a cell-surface receptor for stratifin and is responsible for the stratifin-mediated matrix metalloproteinase-1 (MMP-1) upregulation in fibroblasts. The present study examines whether modulation of APN gene expression has any impact on the fibroblast ECM gene expression profile. The result reveals that in the presence of keratinocyte-derived soluble factors, transient knockdown of APN in dermal fibroblasts affects the expression of key ECM components such as fibronectin, tenascin-C, MMP-1, MMP-3, and MMP-12. The regulatory effects of APN on fibronectin and selective MMPs appear to be associated with receptor-mediated signal transduction independently of its peptidase activity. On the contrary, inhibition of the APN enzymatic activity by bestatin significantly reduces the tenascin-C expression and enhances the contraction of fibroblast-populated collagen gel, suggesting an activity-dependent regulation of fibroblast contractility by APN. The overall effects of APN on the expression of fibronectin, tenascin-C, and MMPs in fibroblasts propose an important role for APN in the regulation of keratinocyte-mediated ECM remodeling and fibroblast contractile activity.     

Binding to EGF receptor of a laminin-5 EGF-like fragment liberated during MMP-dependent mammary gland involution

Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (DIII) from the ECM macromolecule laminin-5. Binding of a recombinant DIII fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of DIII liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.     

Multilevel regulation of matrix metalloproteinases in tissue homeostasis indicates their molecular specificity in vivo

The matrix metalloproteinases (MMPs) play a crucial role in irreversible remodeling of the extracellular matrix (ECM) in normal homeostasis and pathological states. Accumulating data from various studies strongly suggest that MMPs are tightly regulated, starting from the level of gene expression all the way to zymogen activation and endogenous inhibition, with each level controlled by multiple factors. Recent in vivo findings indicate that cell–ECM and cell–cell interactions, as well as ECM bio-active products, contribute an additional layer of regulation at all levels, indicating that individual MMP expression and activity in vivo are highly coordinated and tissue specific processes.     

Reversion inducing cysteine rich protein with Kazal motifs and cardiovascular diseases: The RECKlessness of adverse remodeling

The Reversion Inducing Cysteine Rich Protein With Kazal Motifs (RECK) is a glycosylphosphatidylinositol (GPI) anchored membrane-bound regulator of matrix metalloproteinases (MMPs). It is expressed throughout the body and plays a role in extracellular matrix (ECM) homeostasis and inflammation. In initial studies, RECK expression was found to be downregulated in various invasive cancers and associated with poor prognostic outcome. Restoring RECK, however, has been shown to reverse the metastatic phenotype. Downregulation of RECK expression is also reported in non-malignant diseases, such as periodontal disease, renal fibrosis, and myocardial fibrosis. As such, RECK induction has therapeutic potential in several chronic diseases. Mechanistically, RECK negatively regulates various matrixins involved in cell migration, proliferation, and adverse remodeling by targeting the expression and/or activation of multiple MMPs, A Disintegrin And Metalloproteinase Domain-Containing Proteins (ADAMs), and A Disintegrin And Metalloproteinase With Thrombospondin Motifs (ADAMTS). Outside of its role in remodeling, RECK has also been reported to exert anti-inflammatory effects. In cardiac diseases, for example, it has been shown to counteract several downstream effectors of Angiotensin II (Ang-II) that play a role in adverse cardiac and vascular remodeling, such as Interleukin-6 (IL-6)/IL-6 receptor (IL-6R)/glycoprotein 130 (IL-6 signal transducer) signaling and Epidermal Growth Factor Receptor (EGFR) transactivation. This review article focuses on the current understanding of the multifunctional effects of RECK and how its downregulation may contribute to adverse cardiovascular remodeling.     

Dynamic induction of ADAMTS1 gene in the early phase of acute myocardial infarction

Extracellular matrix (ECM)-degrading enzymes such as matrix metalloproteases (MMPs) play an essential role in the repair of infarcted tissue, which affects ventricular remodeling after myocardial infarction. ADAMTS1 (A disintegrin and metalloprotease with thrombospondin motifs), a newly discovered metalloprotease, was originally cloned from a cancer cell line, but little is known about its contribution to disease. To test the hypothesis that ADAMTS1 appears in infarcted myocardial tissue, we examined ADAMTS1 mRNA expression in a rat myocardial infarction model by Northern blotting, real-time RT-PCR and in situ hybridization. Normal endothelium expressed little ADAMTS1 mRNA, while normal myocardium expressed no detectable ADAMTS1 mRNA. Up-regulation of ADAMTS1 was demonstrated by Northern blot analysis and real-time RT-PCR at 3 h after coronary artery ligation. In situ hybridization revealed strong ADAMTS1 mRNA signals in the endothelium and myocardium in the infarcted heart, mainly in the infarct zone, at 3 h after myocardial infarction. The rapid and transient up-regulation of the ADAMTS1 gene in the ischemic heart was distinct from the regulatory patterns of other MMPs. Our study demonstrated that the ADAMTS1 gene is a new early immediate gene expressed in the ischemic endothelium and myocardium.     

Matrix remodeling in experimental and human heart failure: a possible regulatory role for TIMP-3

In the failing heart, an imbalance in matrix metalloproteinases (MMPs) and their biological regulators, the tissue inhibitors of MMPs (TIMPs), may result in cardiac dilatation from matrix degradation. We hypothesized that a reduction of myocardial TIMP-3 is associated with adverse matrix remodeling in both human and experimental heart failure. Cardiomyopathic hamsters at age 15 wk (normal), 25 wk (compensated stage), and 35 wk (overt failure) were compared with age-matched normal controls. MMP activity (gelatinase bioassay) was increased in cardiomyopathic hearts (P = 0.03) and peaked during the transition to overt heart failure. TIMP-3 content (immunoblot) was decreased compared with normal controls (74 +/- 5% at 25 wk, 69 +/- 10% at 35 wk; P = 0.001) and its reduction was associated with increased MMP activity (r = -0.6; P = 0.004). TIMP-1 increased progressively (P = 0.001), whereas TIMP-2, TIMP-4, and MMP protein levels were unchanged. Myocardial collagen (hydroxyproline content) increased with time during the progression to end-stage cardiac failure (P < 0.0001). Collagen synthesis ([(14)C]proline uptake) was elevated in cardiomyopathy at 15 and 25 wk (P < 0.05). The collagen cross-linking ratio (insoluble:soluble collagen) was reduced (P = 0.003) as the left ventricle dilated. By confocal microscopy restricted to viable myocardium, collagen content was reduced (P = 0.04) with fragmentation (P < 0.0001) and thinning (P = 0.003) of perimysial collagen fibers. Similarly, patients with end-stage congestive heart failure (n = 7) compared with nonfailing controls (n = 2) had elevated gelatinase MMP activity (P = 0.02) associated with isolated reductions in TIMP-3 (55 +/- 5% of normal; P = 0.003). Reductions of TIMP-3 parallel adverse matrix remodeling in the cardiomyopathic hamster and the failing human heart. TIMP-3 may contribute to the regulation of myocardial remodeling and its reduction may promote a transition from compensated to end-stage congestive heart failure.     

Heart extracellular matrix gene expression profile in the vitamin D receptor knockout mice

1,25-Dihydroxyvitamin D₃ [1,25D] deficiency and vitamin D receptor [VDR] genotypes are risk factors for several diseases and disorders including heart diseases. Extracellular matrix (ECM) remodeling mediated by matrix metalloproteinases [MMPs] contributes to progressive left ventricular remodeling, dilation, and heart failure. In the present study, we used high-density oligonucleotide microarray to examine gene expression profile in wild type [WT] and vitamin D receptor knockout mice (VDR KO) which was further validated by RT-PCR. Microarray analysis revealed tissue inhibitors of metalloproteinases [TIMP-1 and TIMP-3] were significantly under expressed in VDR KO mice as compared to WT mice which was further validated by RT-PCR. Zymography and RT-PCR showed that MMP-2 and MMP-9 were up regulated in VDR KO mice. In addition, cross-sectional diameter and longitudinal width of the VDR KO heart myofibrils showed highly significant cellular hypertrophy. Trichrome staining showed marked increase in fibrotic lesions in the VDR KO mice. Heart weight to body weight ratio showed 41% increase in VDR KO mice when compared to WT mice. This data supports a role for 1,25D in heart ECM metabolism and suggests that MMPs and TIMPs expression may be modulated by vitamin D.     

Stretch-induced membrane type matrix metalloproteinase and tissue plasminogen activator in cardiac fibroblast cells

In the normal heart, cardiomyocytes are surrounded by extracellular matrix (ECM) and latent matrix metalloproteinases (MMPs), which are produced primarily by cardiac fibroblasts. An activator of latent MMPs might be induced by ischemic conditions or pressure-induced stretching. To test the hypothesis that an activator of latent MMP is induced in the ischemic heart during transformation of a compensatory hypertrophic response to a decompensatory failing response in cardiac fibroblast cells, we stretched the human cardiac fibroblasts at 25 cycles/min in serum-free or 5% serum culture condition. The membrane type (MT)-MMP activity in stretched cells was measured by zymography and immuno-blot analyses using MT-MMP-2 antibody. The MT-MMP activity was further characterized by transverse-urea gradient (TUG)-zymography. The results suggested that stretch induced a membrane MMP in the fibroblasts that was similar to the MT-MMP induced in ischemic heart. Furthermore, we observed that membrane MMP has distinct mobility in TUG-zymography. To localize the MT-MMP and tissue plasminogen activator (tPA) of latent MMPs, the membrane and cytosol were separated by a method employing a detergent and sedimentation. The MT-MMP and tPA activities of cytosol and membrane fractions were measured by gelatin- and plasminogen-zymography, respectively. Differential-display mRNA analysis was performed on control and stretched cells. In situ immuno-labelling was performed to localize the MT-MMP. The results indicate that induction of MT-MMP occurred in the membrane fractions. The secretion of tPA was elevated in the stretched cells. The MT-MMP activity was inhibited by prior incubation with an antibody generated to membrane MMP. The tPA activity was inhibited by using tPA antibody. These results suggest that, under stretched conditions, neutral transmembrane matrix proteinases are induced in the cardiac fibroblasts. This may lead to activation of adverse ECM remodeling, cardiac dilatation, and failure. J. Cell. Physiol. 176:374–382, 1998. © 1998 Wiley-Liss, Inc.     

Localizing matrix metalloproteinase activities in the pericellular environment

Matrix metalloproteinases (MMPs) are a group of structurally related proteolytic enzymes containing a zinc ion in the active site. They are secreted from cells or bound to the plasma membrane and hydrolyze extracellular matrix (ECM) and cell surface-bound molecules. They therefore play key roles in morphogenesis, wound healing, tissue repair and remodeling in diseases such as cancer and arthritis. Although the cell anchored membrane-type MMPs (MT-MMPs) function pericellularly, the secreted MMPs have been considered to act within the ECM, away from the cells from which they are synthesized. However, recent studies have shown that secreted MMPs bind to specific cell surface receptors, membrane-anchored proteins or cell-associated ECM molecules and function pericellularly at focussed locations. This minireview describes examples of cell surface and pericellular partners of MMPs, as well as how they alter enzyme function and cellular behaviour.     

Matrix metalloproteinase stromelysin-3 in development and pathogenesis

The extracellular matrix (ECM) serves as a medium for cell-cell interactions and can directly signal cells through cell surface ECM receptors, such as integrins. In addition, many growth factors and signaling molecules are stored in the ECM. Thus, ECM remodeling and/or degradation plays a critical role in cell fate and behavior during many developmental and pathological processes. ECM remodeling/degradation is, to a large extent, mediated by matrix metalloproteinases (MMPs), a family of extracellular or membrane-bound, Zn2+-dependent proteases that are capable of digesting various proteinaceous components of the ECM. Of particular interest among them is the MMP11 or stromelysin-3, which was first isolated as a breast cancer associated protease. Here, we review some evidence for the involvement of this MMP in development and diseases with a special emphasis on amphibian metamorphosis, a postembryonic, thyroid hormone-dependent process that transforms essentially every organ/tissue of the animal.     

Peroxisome proliferator-activated receptor-γ ligands attenuate brain natriuretic peptide production and affect remodeling in cardiac fibroblasts in reoxygenation after hypoxia

Cardiac fibroblasts (CFs) participate in cardiac remodeling after hypoxic cardiac damage, and remodeling is thought to be mediated by CF synthesis of brain natriuretic peptide (BNP). It is unknown whether the peroxisome proliferator-activated receptors (PPARs), which mediate cellular signaling for growth and migration, affect BNP synthesis and whether PPARs participate in regulation of extracellular matrix protein (ECM) expression for remodeling. We examined the production of BNP in cultured neonatal ventricular CFs and its signaling system on collagen synthesis and on activation of matrix metalloproteinases (MMPs) in reoxygenation after hypoxia. BNP mRNA was detected in CFs, and a specific BNP protein, BNP1-32, was secreted into the media. Abundance of collagen I and III was increased in the media at reoxygenation. mRNA and protein levels for MMP-2 and the tissue inhibitor of metalloproteinase (TIMP)-1 were enhanced in CFs at reoxygenation. These observations also were noted in CFs after incubation with angiotensin II (10 μM) for 24 h. Pretreatment with pioglitaozone (0.1–10 μM) attenuated BNP mRNA and protein abundance of collagen III, MMP-2, and TIMP-1 in CFs at reoxygenation. The secreted BNP was also decreased by pioglitaozone in the media. Furthermore, PPAR activators inhibited reoxygenation-induced activation of nuclear factor (NF)-kB. These results demonstrate that PPAR activators inhibit BNP synthesis in CFs and imply that PPAR activators may regulate ECM remodeling partially through the NF-kB-mediated pathway.     

Matrix metalloproteinases and angiogenesis

Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell-cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand-binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer-associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.     

Extracellular matrix and heart development

The extracellular matrix (ECM) of the developing heart contains numerous molecules that form a dynamic environment that plays an active and crucial role in the regulation of cellular events. ECM molecules found in the heart include hyaluronan, fibronectin, fibrillin, proteoglycans, and collagens. Tight regulation of the spatiotemporal expression, and the proteolytic processing of ECM components by proteases including members of the ADAMTS family, is essential for normal cardiac development. Perturbation of the expression of genes involved in matrix composition and remodeling can interfere with a myriad of events involved in the formation of the four-chambered heart and result in prenatal lethality or cardiac malformations as seen in humans with congenital heart disease. In this review, we summarize what is known about the specific importance of some of the components of the ECM in relation to the cardiovascular development.     

Matrix Metalloproteinase in Mammary Gland Remodeling-Modulation by Glycosaminoglycans

Mammary gland which undergoes proliferation, differentiation and involution in adult life is a useful model system to study the role of extracellular matrix (ECM) in regulating tissue specific functions. The involution that follows weaning results in the suppression of casein gene expression, collapse of alveolar structures and degradation of basement membrane as evidenced by biochemical analysis of matrix components like proteoglycans and collagen. Differential expression of three different MMPs viz. 130 K, 68 K and 60 K with varying specificity to Col IV of basement membrane and Col I of stroma, their selective inhibition by TIMP and proteoglycans and modulation by estrogen highlight the importance of these in the remodeling of the ECM in the mammary gland. The inhibition of these MMPs by glycosaminoglycans, particularly CS and change in the concentration of CS at different stages of mammary gland development suggests the existence of a novel mechanism for the regulation of the activity of MMPs at extracellular sites.