Matrix metalloproteinase-9 gene deletion facilitates angiogenesis after myocardial infarction

Matrix metalloproteinases (MMPs) are postulated to be necessary for neovascularization during wound healing. MMP-9 deletion alters remodeling postmyocardial infarction (post-MI), but whether and to what degree MMP-9 affects neovascularization post-MI is unknown. Neovascularization was evaluated in wild-type (WT; n = 63) and MMP-9 null (n = 55) mice at 7-days post-MI. Despite similar infarct sizes, MMP-9 deletion improved left ventricular function as evaluated by hemodynamic analysis. Blood vessel quantity and quality were evaluated by three independent studies. First, vessel density was increased in the infarct of MMP-9 null mice compared with WT, as quantified by Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I) immunohistochemistry. Second, preexisting vessels, stained in vivo with FITC-labeled GSL-I pre-MI, were present in the viable but not MI region. Third, a technetium-99m-labeled peptide (NC100692), which selectively binds to activated alpha(v)beta3-integrin in angiogenic vessels, was injected into post-MI mice. Relative NC100692 activity in myocardial segments with diminished perfusion (0-40% nonischemic) was higher in MMP-9 null than in WT mice (383 +/- 162% vs. 250 +/- 118%, respectively; P = 0.002). The unique finding of this study was that MMP-9 deletion stimulated, rather than impaired, neovascularization in remodeling myocardium. Thus targeted strategies to inhibit MMP-9 early post-MI will likely not impair the angiogenic response.     

Selective hydrolysis of triple-helical substrates by matrix metalloproteinase-2 and -9

The role of proteases in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of extracellular matrix (ECM) components. Differentiating between the up-regulation of MMP production and the presence of activated MMPs can be difficult but may well dictate which MMPs are critical to invasion. Because the hydrolysis of collagens is one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, alpha1(V)436-450 THP and alpha1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val bond, analogous to the bond cleaved by MMP-9 in the corresponding native collagen. Kinetic analyses showed k(cat)/K(m) values of 14,002 and 5,449 s(-1)m(-1) for MMP-2 and -9 hydrolysis of alpha1(V)436-447 fTHP, respectively. These values, along with individual k(cat) and K(m) values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14. alpha1(V)436-447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells. Binding of the alpha(2)beta(1) integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.     

The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis

Matrix metalloproteinases (MMPs) are a large group of enzymes responsible for matrix degradation. Among them, the family of gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) is overproduced in the joints of patients with rheumatoid arthritis. Because of their degradative effects on the extracellular matrix, gelatinases have been believed to play an important role in progression and cartilage degradation in this disease, although their precise roles are yet to be defined. To clarify these roles, we investigated the development of Ab-induced arthritis, one of the murine models of rheumatoid arthritis, in MMP-2 or MMP-9 knockout (KO) mice. Surprisingly, the MMP-2 KO mice exhibited severe clinical and histologic arthritis than wild-type mice. The MMP-9 KO mice displayed milder arthritis. Recovery from exacerbated arthritis in the MMP-2 KO mice was possible by injection of wild-type fibroblasts. These results indicated a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease.     

Immunocharacterization of matrix metalloproteinase-2 and matrix metalloproteinase-9 in canine transmissible venereal tumors

Matrix metalloproteases (MMPs) are endogenous proteases that are responsible for degradation of extracellular matrix (ECM) proteins and cell surface antigens. The breakdown of ECM participates in the local invasion and distant metastases of malignant tumors. Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round cell neoplasm of dogs that affects mainly the external genitalia of both sexes. CTVT generally is a locally invasive tumor, but distant metastases also are common in puppies and immunocompromised dogs. We investigated the immune expressions and activities of MMP-2 and MMP-9 in CTVT. The presence of these enzymes in tumor cells and tissue homogenates was demonstrated by immunohistochemistry and western blotting. We used gelatin substrate zymography to evaluate the activities of MMP-2 and MMP-9 enzymes in tumor homogenates. We found that tumor cells expressed both MMP-2 and MMP-9. Electrophoretic bands corresponding to MMP-9 and MMP-2 were identified in immunoblots and clear bands that corresponded to the active forms of MMP-2 and MMP-9 also were detected in gelatin zymograms. Our study is the first detailed documentation of MMPs in CTVT.     

A residue in the S2 subsite controls substrate selectivity of matrix metalloproteinase-2 and matrix metalloproteinase-9

Matrix metalloproteinase (MMP)-2 and MMP-9 are closely related metalloproteinases that are implicated in angiogenesis. The two proteins have a similar domain structure and highly homologous catalytic domains, making them an excellent comparative model for understanding the structural basis of substrate recognition by the MMP family. Although the two MMPs exhibit some overlap in substrate recognition, our recent work showed that MMP-2 can cleave a set of peptide substrates that are only poorly recognized by MMP-9 (Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., and Smith, J. W. (2002) J. Biol. Chem. 277, 4485-4491). Mutations at the P(2) position of these peptide substrates dramatically reduced their selectivity for MMP-2. Inspection of the corresponding S(2) pocket of the substrate-binding cleft of the protease reveals that MMP-9 contains an Asp, whereas MMP-2 contains Glu. Here, we test the hypothesis that this conservative substitution has a role in substrate selectivity. Mutation of Glu(412) in MMP-2 to Asp significantly reduced the hydrolysis of selective substrates, with only a minor effect on hydrolysis of non-selective substrates. The predominant effect of the mutation is at the level of k(cat), or turnover rate, with reductions reaching as high as 37-fold. The residues that occupy this position in other MMPs are highly variable, providing a potential structural basis for substrate recognition across the MMP family.     

Substrate hydrolysis by matrix metalloproteinase-9

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P(3) through P(2'). This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P(3) and P(1'). This study shows that MMP-9 has a unique preference for Arg at both P(2) and P(1), and a preference for Ser/Thr at P(2'). Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.     

Early induction of matrix metalloproteinase-9 transduces signaling in human heart end stage failure

Extracellular matrix (ECM) turnover is regulated by matrix metalloproteinases (MMPs) and plays an important role in cardiac remodeling. Previous studies from our lab demonstrated an increase in gelatinolytic-MMP-2 and -9 activities in endocardial tissue from ischemic cardiomyopathic (ICM) and idiopathic dilated cardiomyopathic (DCM) hearts. The signaling mechanism responsible for the left ventricular (LV) remodeling, however, is unclear. Administration of cardiac specific inhibitor of metalloproteinase (CIMP) prevented the activation of MMP-2 and -9 in ailing to failing myocardium. Activation of MMP-2 and -9 leads to induction of proteinase activated receptor-1 (PAR-1). We hypothesize that the early induction of MMP-9 is a key regulator for modulating intracellular signaling through activation of PAR and various downstream events which are implicated in development of cardiac fibrosis in an extracellular receptor mediated kinase-1 (ERK-1) and focal adhesion kinase (FAK) dependent manner. To test this hypothesis, explanted human heart tissues from ICM and DCM patients were obtained at the time of orthotopic cardiac transplants. Quantitative analysis of MMP-2 and -9 gelatinolytic activities was made by real-time quantitative zymography. Gel phosphorylation staining for PAR-1 showed a significant increase in ICM hearts. Western blot and RT-PCR analysis and in-situ labeling, showed significant increased expression of PAR-1, ERK-1and FAK in ICM and DCM. These observations suggest that the enhanced expression and potentially increased activity of LV myocardial MMP-9 triggers the signal cascade instigating cardiac remodeling. This early mechanism for the initiation of LV remodeling appears to have a role in end-stage human heart failure.     

Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol

Breast cancer patients frequently develop metastases. This process requires the degradation of extracellular matrix proteins which act as a barrier to tumour cell passage. These proteins can be degraded by proteases, mainly the matrix metalloproteinases (MMPs). MMP-2 and -9 which are frequently detected in breast cancer tissues. ProMMPs are released from cancer cells, and their activation is considered to be a crucial step in metastases development. In breast cancer, estrogen metabolism is altered favouring the accumulation of 2- and 4-hydroxyestradiol (2- and 4-OHE(2)). These estradiol metabolites can generate free radicals. Since reactive species are known activators of proMMPs, this study was designed to determine if the free radicals generated by 2- and 4-OHE(2) can activate proMMP-2 and -9. Activation of MMPs by hydroxyestradiol was determined by monitoring the cleavage of a fluorogenic peptide and by zymography analysis. Both estradiol metabolites activated the MMP-2 and -9. 4-OHE(2) was a more potent activator than 2-OHE(2), which reflects its higher capacity to generate free radicals. ProMMPs activation was mainly mediated through O(2)*-, although the free radical HO* also activated the proMMPs but to a lesser extent. ProMMPs activation was not observed with estrogens that cannot generate free radicals, i.e. estradiol, estrone, 2- and 4-methoxyestradiol, and 16alpha-hydroxyestrone. These results demonstrate that 2- and 4-OHE(2) at a concentration as low as 10(-8)M can activate the proMMP-2 and -9 and might play an important role in the invasion of breast cancer cells.     

Genetic variants in matrix metalloproteinase-9 gene modify metalloproteinase-9 levels in black subjects

Altered matrix metalloproteinases (MMPs) levels are involved in cardiovascular diseases and increased MMP-9 levels enhance the cardiovascular risk in apparently healthy subjects. We investigated the effects of MMP-9 gene polymorphisms and haplotypes on the circulating MMP-9 levels in healthy black subjects and the effects of an MMP-2 polymorphism on the plasma MMP-2 concentrations. We studied 190 healthy subjects, nonsmokers, self-reported as blacks (18-63 years). Genotypes for the MMP-2 C(-1306)T polymorphism and the MMP-9 C(-1562)T, 90(CA)(14-24) and Q279R polymorphisms (rs243865, rs3918242, rs2234681, and rs17576, respectively) were determined by TaqMan(?) Allele Discrimination assay and real-time polymerase chain reaction or restriction fragment length polymorphism. Alleles for the 90(CA)(14-24) polymorphism were grouped as low (L) when there were <21 and high (H) when there were ≥21 CA repeats. The plasma levels of MMP-2 and MMP-9 were determined by gelatin zymography. The software PHASE 2.1 was used to estimate the haplotypes frequencies. Although we found no effects of the MMP-9 C(-1562)T or the Q279R polymorphisms on MMP-9 levels, higher MMP-9 levels were associated with the HH genotype for the -90(CA)(14-24) polymorphism compared with the HL or LL genotypes. Lower MMP-9 levels were found in carriers of the CRL haplotype (combining the C, R, and L alleles for the MMP-9 polymorphisms) compared with the CRH haplotype. Consistent with this finding, the CRL haplotype was more commonly found in subjects with low MMP-9 levels. The MMP-2 C(-1306)T polymorphism had no effects on the plasma MMP-2 levels. Our results show that MMP-9 genetic variations modify MMP-9 levels in black subjects and may offer biochemical evidence implicating MMP-9 in the pathogenesis of cardiovascular diseases in blacks.     

Normobaric hyperoxia inhibits NADPH oxidase-mediated matrix metalloproteinase-9 induction in cerebral microvessels in experimental stroke

Matrix metalloproteinase-9 (MMP-9) and NADPH oxidase contribute to blood-brain barrier (BBB) disruption after ischemic stroke. We have previously shown that normobaric hyperoxia (NBO) treatment reduces MMP-9 and oxygen free radical generation in ischemic brain. In this study, we tested the hypothesis that NBO protects the BBB through inhibiting NADPH oxidase-mediated MMP-9 induction in transient focal cerebral ischemia. Male Sprague-Dawley rats (n = 69) were given NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery. Cerebral microvessels were isolated for analyzing MMP-9 and NADPH oxidase. BBB damage was non-invasively quantified with magnetic resonance imaging. In normoxic rats, both NADPH oxidase catalytic subunit gp91(phox) and MMP-9 expression were up-regulated in ischemic hemispheric microvessels after 90-min middle cerebral artery occlusion with 22.5 h reperfusion. Inhibition of NADPH oxidase with apocynin reduced the MMP-9 increase, indicating a causal link between NADPH oxidase-derived superoxide and MMP-9 induction. NBO treatment inhibited gp91(phox) expression, NADPH oxidase activity, and MMP-9 induction, which led to significantly less BBB damage and brain edema in the ischemic brain. These results suggest that gp91(phox) containing NADPH oxidase plays an important role in MMP-9 induction in ischemic BBB microvasculature, and that NBO treatment may attenuate MMP-9 induction and brain edema through inhibiting NADPH oxidase after transient cerebral ischemia.     

Mechanism of inhibition of matrix metalloproteinase-9 induction by NO in vascular smooth muscle cells.

Vascular smooth muscle (VSM) cell migration is a critical step in the development of a neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and extracellular matrix, facilitating VSM cell migration. Recently, we demonstrated that nitric oxide (NO) inhibits interleukin-1 beta (IL-1 beta)-stimulated MMP-9 induction in rat aortic VSM cells. In this study, we examined the hypothesis that NO inhibits MMP-9 induction by attenuating superoxide generation and extracellular signal-regulated kinase (ERK) activation. Stimulation of VSM cells with IL-1 beta significantly (P < 0.05) increased superoxide production, ERK activation, and MMP-9 induction. Pretreatment of VSM cells with the NO donor DETA NONOate significantly (P < 0.05) decreased IL-1 beta-stimulated superoxide generation. In addition, pretreatment of VSM cells with a specific ERK pathway inhibitor, PD-98059, or DETA NONOate inhibited IL-1 beta-stimulated ERK activation and MMP-9 induction. Direct exposure of VSM cells to increased superoxide levels by treatment with xanthine/xanthine oxidase increased ERK activation and MMP-9 induction, whereas pretreatment of cells with PD-98059 significantly (P < 0.05) inhibited xanthine/xanthine oxidase-stimulated ERK activation and MMP-9 induction. We conclude that NO inhibits IL-1 beta-stimulated MMP-9 induction by inhibiting superoxide generation and subsequent ERK activation.     

Constitutively active lysophosphatidic acid receptor-1 enhances the induction of matrix metalloproteinase-2

Lysophosphatidic acid (LPA) is a simple phospholipid which interacts with at least six G protein-coupled transmembrane LPA receptors (LPA(1)-LPA(6)). In rat neuroblastoma B103 cells, we have recently reported that each LPA receptor indicates the different cellular functions, including cell motility, invasion and tumorigenicity. Especially, mutated and constitutively active LPA(1) enhanced these cellular effects in B103 cells. In the present study, to better understand a role of mutated LPA(1) underlying progression of cancer cells, we measured the expression and activity levels of matrix metalloproteinases (MMPs) in constitutively active mutant Lpar1-expressing B103 cells (lpa1Δ-1), compared with each wild-type LPA receptor-expressing cells. LPA receptor-unexpressing cells were also used as control. In quantitative real time RT-PCR analysis, the expressions of Mmp-9 were detected at the same levels in all cells. By contrast, Mmp-2 expressions of lpa1Δ-1 were significantly higher than those of other cells. In gelatin zymography, proMmp-9 was observed at the same levels in all cells. Interestingly, markedly high levels of proMmp-2 and Mmp-2 were detected in lpa1Δ-1 cells, whereas no activation was in other cells. The increased expression and activity of Mmp-2 in lpa1Δ-1 cells were suppressed by the pretreatment with a Gq protein inhibitor. These results suggest that mutated LPA(1) may involve in the enhancement of Mmp-2 expression and activation in rat neuroblastoma cells.     

Role of matrix metalloproteinase-2 and -9 in the development of diabetic retinopathy

Diabetic retinopathy represents the most common causes of vision loss in patients affected by diabetes mellitus. The cause of vision loss in diabetic retinopathy is complex and remains incompletely understood. One of the earliest changes in the development of retinopathy is the accelerated apoptosis of retinal microvascular cells and the formation of acellular capillaries by unknown mechanism. Results of a recent research suggest an important role of matrix metalloproteinases (MMPs) in the development of diabetic retinopathy. MMPs are a large family of proteinases that remodel extracellular matrix components, and under pathological condition, its induction is considered as a negative regulator of cell survival; and in diabetes, latent MMPs are activated in the retina and its capillary cells, and activation of MMP-2 and -9 induces apoptosis of retinal capillary cells. This review will focus on the MMP-2 and MMP-9 in the diabetic retina with special reference to oxidative stress, mitochondria dysfunction, inflammation and angiogenesis, as well as summarizing the current information linking these proteins to pathogenesis of diabetic retinopathy.     

Mutations in isocitrate dehydrogenase 2 accelerate glioma cell migration via matrix metalloproteinase-2 and 9

The gene encoding isocitrate dehydrogenase (IDH) is somatically mutated predominantly in secondary glioblastoma multiforme. Glioma-specific mutations in IDH1 always produced a single amino acid substitution at R132, but mutations in IDH2 were exclusively at R172 which was the analogous site to R132 in IDH1. Mutations of IDH1 and IDH2 led to simultaneous loss and gain of activities in the production of α-ketoglutarate and 2-hydroxyglutarate, respectively. Matrix metalloproteinases (MMPs) are zinc-dependent endoproteinases involved in the degradation of the extracellular matrix. The exact role of IDH2 mutant on MMPs activity and cell migration has not been fully studied. Here, we show that in response to IDH2 mutations, low levels of α-ketoglutarate increased the stabilization of HIF-1α which can contribute to tumor growth. Moreover, mutant IDH2-induced HIF-1α improved the secretion levels of pro-MMP-2 and pro-MMP-9 as well as the conversion from pro-MMP-2 to its active form, giving C6 glioma cells a higher migration potential. The HIF-1α pathway is probably a critical pathway for release of MMPs in the glioma cancer harboring IDH mutant.