The plasminogen-MMP system is more activated in the scar than in viable myocardium 3 months post-MI in the rat

Left ventricular (LV) remodeling following myocardial infarction (MI) is a complex process involving extracellular matrix degradation and fibrosis. While early remodeling is beneficial, chronic remodeling leads to decompensated heart failure (HF). We assessed the hypothesis that activation of the plasminogen-MMP system is involved in the remodeling of the infarct scar and compared it to the remaining viable myocardium. MI was induced by coronary artery ligature in 42 male Wistar rats. Three months following surgery, animals were divided into compensated (n=26) or decompensated (n=16) groups and compared to sham-operated rats (n=17). Scar and remaining viable LV myocardium (LVM) were separately analyzed for MMP-2, -7, -9, urokinase type and tissue type plasminogen activator (uPA and tPA) mRNA levels by RT-PCR. Their protein or activity levels, plus those of plasminogen/plasmin, tissue inhibitor of metalloproteinase-1, -2, -4 (TIMP-1, -2, -4) and plasminogen activator inhibitor-1 (PAI-1) were analyzed in tissue conditioned media by Western blot, ELISA and/or zymography. MMP and plasmin proteolytic activities were increased in the scar as compared to paired LVM thus indicating that activation of plasminogen and pro-MMPs is a key event in scar tissue remodeling. MMP and plasminogen activators (uPA, tPA) mRNAs were increased accordingly. Furthermore, inhibitors of the proteolytic enzymes, TIMP-1 and PAI-1 were increased in the scars from failing hearts and LVM thus suggesting a dynamic interplay between proteolysis and its inhibitors. This study shows a high degree of activation of the MMP-plasminogen system and the balance with their inhibitors in the infarcted myocardium, and suggests that this activation participates more to the remodeling of the scar tissue than to the remaining myocardium.     

Myocardial extracellular matrix remodeling in transgenic mice overexpressing tumor necrosis factor alpha can be modulated by anti-tumor necrosis factor alpha therapy

Myocardial fibrosis caused by maladaptive extracellular matrix (ECM) remodeling is implicated in the dysfunction of the failing heart. Matrix metalloproteinases (MMPs) regulate ECM remodeling, and are regulated by cytokines. Transgenic mice with cardiac-specific overexpression of tumor necrosis factor alpha (TNF-alpha) (TNF1.6) develop heart failure. We hypothesized that modulation of TNF-alpha and/or MMP activity might alter the myocardial ECM remodeling process and the development of heart failure. To test this hypothesis, we took advantage of the TNF1.6 mice and studied soluble and total collagens and collagen type profiling by using hydroxyproline quantification, Sircol collagen assay, Northern blot analysis, and immunohistochemistry and studied myocardial function by using echocardiography. Progressive ventricular hypertrophy and dilation in the TNF1.6 mice were accompanied by a significant increase in MMP-2 and MMP-9 activity, an increase in collagen synthesis, deposition, and denaturation, and a decrease in undenatured collagens. In young TNF1.6 mice, these changes in the ECM were associated with marked diastolic dysfunction as demonstrated by significantly reduced transmitral Doppler echocardiographic E/A wave ratio. Anti-TNF-alpha treatment with adenoviral vector expressing soluble TNF-alpha receptor type I attenuated both MMP-2 and MMP-9 activity, prevented further collagen synthesis, deposition and denaturation, and preserved myocardial diastolic function in young, but not old, TNF1.6 mice. The results suggest a critical role of TNF-alpha and MMPs in myocardial matrix remodeling and functional regulation and support the hypothesis that both TNF-alpha and MMPs may serve as potential therapeutic targets in the treatment of heart failure.     

Matrix Metalloproteinases: Pathways of Induction by Bioactive Molecules

Regulation of the extracellular matrix (ECM) is an important therapeutic target that can potentially attenuate the adverse ventricular remodeling seen in the progression of heart failure. Matrix metalloproteinases (MMPs) degrade numerous ECM proteins. Importantly, the activation of MMPs and their endogenous inhibitors (TIMPs) are associated with ventricular remodeling. Bioactive-molecules (vasoactive peptides) become activated in proportion to the magnitude of heart failure and have been demonstrated to affect directly collagen degradation as well as collagen synthesis in the myocardium. Pro-fibrotic factors such as norepinephrine, angiotensin II, and endothelin-1 stimulate fibrosis by modulating collagen synthesis and MMP/TIMP activity. Antagonism of these bioactive-molecules has produced improved hemodynamic performance concomitant with modulation of MMP/TIMP activity and in association with reverse remodeling. The natriuretic peptides and nitric oxide, both of which function via the second messenger cGMP, demonstrate anti-fibrotic actions by inhibiting collagen synthesis and by stimulating MMP activity. Furthermore, bioactive-molecules along with certain cytokines are reported to amplify MMP activity, suggesting that different signaling systems work together to modulate ECM turnover. Taken together, the evidence supports an important functional role for bioactive-molecules in the regulation of ECM turnover and suggests that pharmacological intervention at the level of such bioactive molecules may provide potential therapeutic strategies for attenuation of the adverse ventricular remodeling associated with the progression of heart failure.     

Impaired extracellular matrix degradation in aortic vessels of cirrhotic rats

BACKGROUND/AIMS: Thinning of the vascular wall occurs in conductance vessels of cirrhotic rats. Increased nitric oxide synthase (NOS) activity has been involved in the pathogenesis of this phenomenon. Therefore, we assessed the NO-regulated cell signaling pathways participating in vascular remodeling in cirrhosis. METHODS: Aortas were obtained from 15 control and 15 cirrhotic rats. Phosphorylated p38 MAPK and ERK1/2 were used to evaluate the activation of cell MAPK signaling pathways. Extracellular matrix (ECM) turnover was estimated by measuring matrix metalloproteinases (MMPs) activity and protein expression of collagen IV, MMP-2, MMP-9 and tissue inhibitor of MMPs (TIMP)-2. Thereafter, 12 control and 12 cirrhotic rats received Nomega-nitro-L-arginine-methyl-ester or vehicle daily for 11 weeks. RESULTS: Cirrhotic vessels showed a reduction in ERK1/2 phosphorylation, lower MMP activity, decreased MMPs expression and higher collagen IV and TIMP-2 abundance, compared to control rats. Chronic NOS inhibition normalized ERK1/2 phosphorylation and MMPs activity, increased MMPs abundance and decreased TIMP-2 expression in cirrhotic rats. CONCLUSIONS: Vascular remodeling in cirrhotic rats is mediated by down-regulation of cell growth and impaired ERK1/2 activation and subsequent imbalance of ECM turnover. These results further stress the importance of vascular NO overactivity in the reduction of vascular wall thickness in cirrhosis.     

IGF-II/mannose 6-phosphate receptor activation induces metalloproteinase-9 matrix activity and increases plasminogen activator expression in H9c2 cardiomyoblast cells

The IGF-II/mannose 6-phosphate receptor (IGF2R) function in extracellular matrix (ECM) remodeling is known to occur as a result of transforming growth factor-beta (TGF-beta) activation and plasmin in the proteolytic cleavage level caused by the interaction between latent TGF-beta and urokinase plasminogen activator receptor (uPAR) respectively. In one of our previous studies, we found IGF-II and IGF2R dose-dependently correlated with the progression of pathological hypertrophy remodeling following complete abdominal aorta ligation. However, how this IGF2R signaling pathway responds specifically to IGF-II and regulates the myocardial ECM remodeling process is unclear. We found that IGF2R was aberrantly expressed in myocardial infarction scars. The matrix metalloproteinase-9 (MMP-9) zymographic activity was elevated in H9c2 cardiomyoblast cells treated with IGF-II, but not IGF-I. Treatment with Leu27IGF-II, an IGF2R specifically binding IGF-II analog, resulted in significant time-dependent increases in the MMP-9, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA); and a reduction in the tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) protein expression. Furthermore, IGF2R expression inhibition by siRNA blocked the IGF-II-induced MMP-9 activity. We hypothesize that after IGF-II is bound with IGF2R, the resulting signal disrupts the balance in the MMP-9/TIMP-2 expression level and increases plasminogen activator (PAs) expression involved in the development of myocardial remodeling. If so, IGF2R signaling inhibition may have potential use in the development of therapies preventing heart fibrosis progression.     

Changes in matrix metalloproteinase (MMP) and tissue inhibitors of metalloproteinases (TIMP) expression profile in Crohn's disease after immunosuppressive treatment correlate with histological score and calprotectin values

Background  

Matrix metalloproteinases (MMPs) constitute a family of enzymes capable of degrading various extracellular matrices (ECM) and basement membrane components playing a role in ECM turnover. They activate and degrade signaling molecules, such as cytokines and chemokines. MMPs are involved in inflammation and have been implicated in tissue degradation and repair occurring in inflammatory bowel disease. The aim of this study was to investigate the MMP profile of intestinal Crohn's disease (CD) patients before and after immunosuppressive treatment (anti-TNF-α agents or corticosteroids and conventional immunosuppressants azathioprine or methotrexate) to learn more about the therapeutic pathways for immunosuppressive agents.     

Inhibition of matrix metalloproteinases prevents the synthesis of insulin-like growth factor binding protein-1 during decidualization in the baboon

During pregnancy in the primate, uterine stromal fibroblasts are transformed into decidual cells. Decidualization is associated with extensive remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) play a pivotal role in ECM degradation. We hypothesized that MMPs also contribute to regulation of IGF binding protein-1 (IGFBP-1), a biochemical marker of primate decidual cells. We reported that IL-1beta (10 ng/ml) with steroid hormones [36 nm estradiol-17beta, 1 microm medroxyprogesterone acetate (P), and 100 ng/ml relaxin] induces in vitro IGFBP-1 synthesis. This study demonstrates that IL-1beta also induces stromelysin-1 (MMP-3) mRNA and synthesis of the latent form of MMP-3 (pro-MMP-3) protein in baboon stromal fibroblasts. In contrast, hormones (particularly P) negatively regulate MMP-3 because their addition decreases IL-1beta-induced pro-MMP-3 protein. The ERK and p38 MAPK pathways induced by IL-1beta regulate pro-MMP-3 because inhibitors PD98059 (20 microm) and SB203580 (1 microm) prevent its synthesis. The nuclear factor-kappaB inhibitory peptide, SN50 (50 microg/ml), or proteasome inhibitor, MG-132 (1 microm), did not inhibit pro-MMP-3 synthesis but appeared to enhance it. The role of MMPs in IGFBP-1 induction was investigated using a broad-spectrum MMP inhibitor, doxycycline, and specific MMP-3 inhibitor, N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH). Both inhibitors caused the dose-dependent decrease of IGFBP-1. alpha-Smooth muscle actin, which is down-regulated during decidualization, was partially up-regulated by doxycycline or N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid. This suggests that alpha-smooth muscle actin is modulated by changes in ECM caused by the action of MMPs/MMP-3. Disruption of actin filaments enhances IGFBP-1 induction. Thus, our data imply that IL-1beta-induced MMPs and particularly MMP-3 may up-regulate IGFBP-1 by disrupting the actin cytoskeleton as a result of ECM degradation.     

Matrix metalloproteinase inhibition and the prevention of heart failure

Matrix metalloproteinases (MMPs) are members of a large family of enzymes that can degrade extracellular matrix as well as other molecules. MMPs participate in a broad variety of normal and pathologic states, and recent evidence implicates the MMP family as potential mediators of cardiac dilation and progression to heart failure. This evidence is based on several lines of investigation. First, members of the MMP family are overexpressed in the myocardium in both experimental and human myocardial injury, infarction, and dilation. Second, overexpression of at least one MMP (MMP-1) in the hearts of transgenic mice can cause cardiac hypertrophy, dilation, and systolic dysfunction. Third, studies from multiple laboratories with different experimental models indicate that inhibition of MMPs through small molecules or gene transfer of endogenous inhibitors favorably affects cardiac remodeling. Fourth, targeted deletion of MMP genes in mice attenuates cardiac remodeling. These compelling results appear to fulfill Koch's Postulates as they may be applied to a non-infectious mediator of a disease, and thus current evidence supports MMP inhibition as a promising strategy for preventing heart failure. However, the crucial question of whether MMP inhibition benefits long-term left ventricular function and survival should be answered.     

Matrix metalloproteinase inhibition after myocardial infarction: a new approach to prevent heart failure?

Increased activity of matrix metalloproteinases (MMPs) has been implicated in numerous disease processes, including tumor growth and metastasis, arthritis, and periodontal disease. It is now becoming increasingly clear that extracellular matrix degradation by MMPs is also involved in the pathogenesis of cardiovascular disease, including atherosclerosis, restenosis, dilated cardiomyopathy, and myocardial infarction. Administration of synthetic MMP inhibitors in experimental animal models of these cardiovascular diseases significantly inhibits the progression of, respectively, atherosclerotic lesion formation, neointima formation, left ventricular remodeling, pump dysfunction, and infarct healing. This review focuses on the role of MMPs in cardiovascular disease, in particular myocardial infarction and the subsequent progression to heart failure. MMPs, which are present in the myocardium and capable of degrading all the matrix components of the heart, are the driving force behind myocardial matrix remodeling. The recent finding that acute pharmacological inhibition of MMPs or deficiency in MMP-9 attenuates left ventricular dilatation in the infarcted mouse heart led to the proposal that MMP inhibitors could be used as a potential therapy for patients at risk for the development of heart failure after myocardial infarction. Although these promising results encourage the design of clinical trials with MMP inhibitors, there are still several unresolved issues. This review describes the biology of MMPs and discusses new insights into the role of MMPs in several cardiovascular diseases. Attention will be paid to the central role of the plasminogen system as an important activator of MMPs in the remodeling process after myocardial infarction. Finally, we speculate on the use of MMP inhibitors as potential therapy for heart failure.     

MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: differential regulation by inflammatory cytokines

Human mesenchymal stem cells (hMSCs) represent promising tools in various clinical applications, including the regeneration of injured tissues by endogenous or transplanted hMSCs. The molecular mechanisms, however, that control hMSC mobilization and homing which require invasion through extracellular matrix (ECM) barriers are almost unknown. We have analyzed bone marrow-derivedhMSCs and detected strong expression and synthesis of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. The ability of hMSCs to traverse reconstituted human basement membranes was effectively blocked in the presence of synthetic MMP inhibitors. Detailed studies by RNA interference revealed that gene knock-down of MMP-2, MT1-MMP, or TIMP-2 substantially impaired hMSC invasion, whereas silencing of TIMP-1 enhanced cell migration, indicating opposing roles of both TIMPs in this process. Moreover, the inflammatory cytokines TGF-beta1, IL-1beta, and TNF-alpha up-regulated MMP-2, MT1-MMP, and/or MMP-9 production in these cells, resulting in a strong stimulation of chemotactic migration through ECM, whereas the chemokine SDF-1alpha exhibited minor effects on MMP/TIMP expression and cell invasion. Thus, induction of specific MMP activity in hMSCs by inflammatory cytokines promotes directed cell migration across reconstituted basement membranes in vitro providing a potential mechanism in hMSC recruitment and extravasation into injured tissues in vivo.     

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

Among the large matrix metalloproteinases (MMPs) family, MMP-12, also referred to as macrophage elastase, plays a significant role in chronic pulmonary pathologies characterized by an intense tissue remodeling such as asthma and COPD. This review will summarize knowledge about MMP-12 structure, functions and mechanisms of activation and regulation, including potential MMP-12 modulation by microRNA. As MMP-12 is involved in many tissue remodeling diseases, efforts have been made to develop specific synthetic inhibitors. However, at this time, very few chemical inhibitors have proved to be efficient and specific to a particular MMP. The relevance of silencing MMP-12 by RNA interference is highlighted. The specificity of this approach using siRNA or shRNA and the strategies to deliver these molecules in the lung are discussed.     

Topology of protease activities reflects atherothrombotic plaque complexity

The pathological remodeling of the arterial wall in atherosclerosis involves protease activities, which play a major role in complications, through plaque rupture. Here, we investigated the release of active proteases by human carotid plaques in relation to (1) the degree of lesion complexity and (2) their compartmentalization between cap, core and media. Eighty human carotid endarterectomy specimens were dissected into culprit stenosing (CPs) and adjacent non-complicated/non-stenosing plaques (NPs). Thirty-five additional CPs were microdissected into cap, core and media. All specimens were compared to control non-atherosclerotic endarteries for the release of components of the plasminogen/plasmin system and matrix metalloproteinases (MMPs). Results show a greater release of the plasminogen activators (PAs), plasmin and active MMPs by CPs compared to NPs, whereas healthy arteries released even lower levels. Furthermore, we highlight a functional interaction between these proteases in human atherosclerotic tissues and more importantly, we demonstrate that the core constitutes the main source of protease activities within CPs. Together, these results suggest that CPs generate plasmin, mainly in the core, which could in turn participate in MMP activation and the onset of complications.     

Expression of matrix metalloproteinases in cardiac allograft vasculopathy and its attenuation by anti MMP-2 ribozyme gene transfection

OBJECTIVE: Proliferation and migration of vascular smooth muscle cells (SMCs) causes intimal thickening during cardiac allograft vasculopathy (CAV). This process requires the degradation or remodeling of extracellular matrix (ECM) surrounding the cells. Imbalance between degradation and accumulation of ECM also contributes to the development of CAV. In this study, we investigated the contribution of matrix metalloprotenases (MMPs), enzymes regulating ECM turnover, to the development of CAV. METHODS: Donor hearts from male DBA mice were heterotopically transplanted to male B10.D2 recipient mice, and harvested at days 15 and 30 post transplantation. We examined expression MMP-2, -3, -9 and -13 of graft vessels using immunohistochemistry. To clarify the role of MMP-2 in CAV, anti MMP-2 ribozyme was delivered into donor hearts just before transplantation, mediated by a hemagglutinating virus of Japan-liposome complex to specifically suppress MMP-2 activity. RESULTS: All MMPs were immunopositive in SMCs from the slightly thickened neointima at day 15. In the advanced stage of intimal thickening at day 30, in addition to increased number of SMCs, accumulation of collagenous fibers was observed; expression of MMP-3, -9 and -13 was decreased. In contrast, MMP-2 expression remained distinctly positive throughout the progression of the vascular remodeling. After the gene transfer of MMP-2 ribozyme, luminal occlusion was significantly decreased compared to non-treated allografts [25.0+/-6.5 vs. 55.1+/-7.0% (P<0.05)] at day 30 post transplantation. CONCLUSION: MMP-2 is a principle MMP throughout the progression of the vascular remodeling in CAV. Anti MMP-2 therapy could therefore be one of the candidates for a supplemental therapy for CAV.     

MMP Induction and Inhibition in Myocardial Infarction

Short-term survival following a myocardial infarction (MI) has greatly improved, due in part to therapeutic interventions that restore blood flow and limit infarct size. The increased incidence of infarct-stimulated left ventricular (LV) remodeling that advances to congestive heart failure (CHF), however, is a significant long-term complication and a leading cause of mortality. Changes to ECM structure and function are primary components of LV remodeling and are precipitated by the early increase in infarct area collagen levels that replace necrotic myocytes and form a scar. ECM turnover is coordinated through the synthesis and degradation of ECM and non-ECM components, particularly the matrix metalloproteinases (MMP), a family of proteolytic enzymes that cleave ECM. MMPs have multiple roles in remodeling events that lead to LV dilation. The inhibition or targeted deletion of specific MMPs attenuates LV remodeling events post-MI. MMP inhibitors have been used in animal models to delineate LV remodeling mechanisms and to evaluate the pharmacologic potential of targeting the ECM to modify LV remodeling post-MI. This review summarizes the current knowledge and limitations of MMP inhibition in the post-MI myocardium.     

Effects of early and late chronic pressure overload on extracellular matrix remodeling

The left ventricle (LV) remodels with age and in response to pressure overload. While aging and pressure overload are superimposed in the clinical context, the structural and functional consequences of the individual processes are not well-understood. Accordingly, the objective of this study was to compare the effects of both early and late chronic hypertension on extracellular matrix (ECM) remodeling. The following groups of Dahl rats were studied: 1) young salt-resistant (control, n=6); 2) young salt-sensitive (early phase of chronic hypertension, n=6); 3) middle-aged salt-resistant (aging, n=5); and 4) middle-aged salt-sensitive (late phase of chronic hypertension, n=6). We measured LV mass (LVM) and body weight (BW) and immunoblotted a panel of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and ECM proteins. Total collagen increased, several MMPs decreased, and TIMP-1 increased in the early phase of hypertension, consistent with fibrosis. Active MMP-8 decreased from 8,010+/-81 U in young salt-resistant to 5,260+/-313 U in young salt-sensitive (p<0.05) rats. During the late phase, chronic hypertension decreased total collagen levels and increased MMP-8 and MMP-14 (all p<0.05). Based on good-fit modeling analysis, MMP-14 (45 kDa) correlated positively with changes in LVM/BW during the early phase. In conclusion, this is the first study to evaluate MMP levels during both early and late chronic phases of hypertension. Our results highlight that ECM remodeling in response to pressure overload is a dynamic process involving excessive ECM accumulation and degradation.