Matrix metalloproteinase expression in vein grafts: role of inflammatory mediators and extracellular signal-regulated kinases-1 and -2

Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-alpha (TNF-alpha), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 days) upregulation of vein graft-associated MMP-9, MMP-2, MT1-MMP, thrombin activity, and TNF-alpha levels with concomitant TIMP-2 downregulation. MMP-2 activation preceded MT1-MMP upregulation. PMN infiltration and vein graft-associated MPO activity increased within hours after arterialization, indicating a prompt, local inflammatory response. In cultured smooth muscle cells, both thrombin and TNF-alpha upregulated MT1-MMP expression; however, only thrombin activated MMP-2. Inhibition of ERK-1/2 activation blocked arterialization-induced upregulation of MMP-2, MMP-9, and MT1-MMP. Thus, thrombin, inflammatory mediators, and activation of the ERK-1/2 pathway control MMP and TIMP-2 expression in arterialized vein grafts.     

Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.     

Reduced NO signaling during pregnancy attenuates outward uterine artery remodeling by altering MMP expression and collagen and elastin deposition

Recent findings indicate that endothelial nitric oxide (NO) plays a key role in uterine artery outward circumferential remodeling during pregnancy. Although the underlying mechanisms are not known, they likely involve matrix metalloproteinases (MMPs). The goal of this study was to examine the linkage among NO inhibition, expansive remodeling, and MMP expression within the uterine vascular wall. Adult female rats were treated with N(G)-nitro-L-arginine methyl ester [L-NAME (LPLN)] beginning on day 10 of pregnancy and until death at day 20 and compared with age-matched controls [late pregnant (LP)]. Mean arterial pressure of LPLN rats was significantly higher than controls. LPLN fetal and placental weights were significantly reduced compared with controls. Main uterine arteries (mUA) were collected to determine dimensional properties (lumen area and wall thickness), collagen and elastin content, and levels of endothelial nitric oxide synthase (eNOS) and MMP expression. Circumferential remodeling was attenuated, as evidenced by significantly smaller lumen diameters. eNOS RNA and protein were significantly (>90%) decreased in the LPLN mUA compared with LP. Collagen and elastin contents were significantly increased in LPLN rats by ～10 and 25%, respectively, compared with LP (P < 0.05). Both MMP-2 and tissue inhibitors of metalloproteinase-2 as assessed by immunofluorescence were lower in the endothelium (reduction of 60%) and adventitia (reduction of 50%) of LPLN compared with LP mUA. Membrane bound MMP-1 (MT1-MMP) as assessed by immunoblot was significantly decreased in LPLN. These data suggest a novel contribution of MMPs to gestational uterine vascular remodeling and substantiate the linkage between NO signaling and gestational remodeling of the uterine circulation via altered MMP, TIMP-2, and MT1-MMP expression and activity.     

Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system

Reactive astrocytes occurring in response to neurodegeneration are thought to play an important role in neuronal regeneration by upregulating the expression of extracellular matrix (ECM) components as well as the ECM degrading metalloproteinases (MMPs). We examined the mRNA levels and cellular distribution of membrane type matrix metalloproteinase 1 (MT1-MMP) and tissue inhibitors 1-4 of MMPs (TIMPs) in brain stem and spinal cord of wobbler (WR) mutant mice affected by progressive neurodegeneration and astrogliosis. MT1-MMP, TIMP-1 and TIMP-3 mRNA levels were elevated, whereas TIMP-2 and TIMP-4 expression was not affected. MT1-MMP was expressed in reactive astrocytes of WR. In primary astrocyte cultures, MT1-MMP mRNA was upregulated by exogeneous tumor necrosis factor alpha. Increased plasma membrane and secreted MMP activities were found in primary WR astrocytes.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.     

Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3

During tissue-invasive events, migrating cells penetrate type I collagen-rich interstitial tissues by mobilizing undefined proteolytic enzymes. To screen for members of the matrix metalloproteinase (MMP) family that mediate collagen-invasive activity, an in vitro model system was developed wherein MDCK cells were stably transfected to overexpress each of ten different MMPs that have been linked to matrix remodeling states. MDCK cells were then stimulated with scatter factor/hepatocyte growth factor (SF/HGF) to initiate invasion and tubulogenesis atop either type I collagen or interstitial stroma to determine the ability of MMPs to accelerate, modify, or disrupt morphogenic responses. Neither secreted collagenases (MMP-1 and MMP-13), gelatinases (gelatinase A or B), stromelysins (MMP-3 and MMP-11), or matrilysin (MMP-7) affected SF/HGF-induced responses. By contrast, the membrane-anchored metalloproteinases, membrane-type 1 MMP, membrane-type 2 MMP, and membrane-type 3 MMP (MT1-, MT2-, and MT3-MMP) each modified the morphogenic program. Of the three MT-MMPs tested, only MT1-MMP and MT2-MMP were able to directly confer invasion-incompetent cells with the ability to penetrate type I collagen matrices. MT-MMP-dependent invasion proceeded independently of proMMP-2 activation, but required the enzymes to be membrane-anchored to the cell surface. These findings demonstrate that MT-MMP-expressing cells can penetrate and remodel type I collagen-rich tissues by using membrane-anchored metalloproteinases as pericellular collagenases.     

Evidence for a cooperative role of gelatinase A and membrane type-1 matrix metalloproteinase during Xenopus laevis development

Matrix metalloproteinases (MMPs) are a large family of extracellular or membrane-bound proteases. Their ability to cleave extracellular matrix (ECM) proteins has implicated a role in ECM remodeling to affect cell fate and behavior during development and in pathogenesis. We have shown previously that membrane-type 1 (MT1)-MMP [corrected] is coexpressed temporally and spatially with the MMP gelatinase A (GelA) in all cell types of the intestine and tail where GelA is expressed during Xenopus laevis metamorphosis, suggesting a cooperative role of these MMPs in development. Here, we show that Xenopus GelA and MT1-MMP interact with each other in vivo and that overexpression of MT1-MMP and GelA together in Xenopus embryos leads to the activation of pro-GelA. We further show that both MMPs are expressed during Xenopus embryogenesis, although MT1-MMP gene is expressed earlier than the GelA gene. To investigate whether the embryonic MMPs play a role in development, we have studied whether precocious expression of these MMPs alters development. Our results show that overexpression of both MMPs causes developmental abnormalities and embryonic death by a mechanism that requires the catalytic activity of the MMPs. More importantly, we show that coexpression of wild type MT1-MMP and GelA leads to a cooperative effect on embryonic development and that this cooperative effect is abolished when the catalytic activity of either MMP is eliminated through a point mutation in the catalytic domain. Thus, our studies support a cooperative role of these MMPs in embryonic development, likely through the activation of pro-GelA by MT1-MMP.     

Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells

Cultured hepatic stellate cells (HSCs) are known to change their morphology and function with respect to the production of extracellular matrices (ECMs) and matrix metalloproteinases (MMPs) in response to ECM components. We examined the regulatory role of the native form of type I collagen fibrils in pro-MMP-2 production and activation in cultured HSCs. Gelatin zymography of the conditioned media revealed that pro- and active form of MMP-2 was increased in the HSCs cultured on type I collagen gel but not on type I collagen-coated surface, gelatin-coated surface, type IV collagen-coated surface, or Matrigel, suggesting the importance of the native form of type I collagen fibrils in pro-MMP-2 production and activation. The induction of active MMP-2 by extracellular type I collagen was suppressed by the blocking antibody against integrin beta1 subunits, indicating the involvement of integrin signaling in pro-MMP-2 activation. RT-PCR analysis indicated that MMP-2, membrane type-1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) mRNA levels were elevated in HSCs cultured on type I collagen gel. The increased MT1-MMP proteins were localized on the cell surface of HSCs cultured on type I collagen gel. In contrast to the expression of MMP-2, HSCs showed a great decline in MMP-13 expression in HSCs cultured on type I collagen gel. These results indicate that the native fibrillar (polymerized) but not monomeric form of type I collagen induced pro-MMP-2 production and activation through MT1-MMP and TIMP-2 in cultured HSCs, suggesting an important role of HSCs in ECM remodeling in the hepatic perisinusoidal spaces.     

Hepatocyte growth factor enhances MMP activity in human endothelial cells

Scatter factor (SF) or hepatocyte growth factor (HGF) has been identified as an angiogenic factor. Angiogenesis requires not only tube formation but also invasion of pericytes and extracellular matrix (ECM) remodeling to promote new vessel stabilization. In the current study, the effect of SF/HGF on endothelial cell (EC) production of matrix metalloproteinases (MMPs) was explored. We showed that SF/HGF enhanced MT1-MMP synthesis and induced MMP-2 activation in two human EC lines: dermal microvessel EC and coronary arterial EC. Furthermore, SF/HGF accelerated EC invasion into matrix, an activity that could be inhibited by a MMP inhibitor. We also demonstrated that the MAP kinase cascade is critical in signal transduction pathway from SF/HGF stimulation to MT1-MMP up-regulation. The current study indicates that MMP activation is a novel effect of SF/HGF on ECs.     

The role of the IGF-I receptor in the regulation of matrix metalloproteinases, tumor invasion and metastasis.

The breakdown of the extracellular matrix (ECM) by proteinases is an essential step in the process of cancer invasion and metastasis. Malignant progression is frequently associated with upregulated production and/or activity of one or several ECM degrading proteinases. Prominent among them are the matrix metalloproteinases (MMPs). The MMPs constitute a family of structurally related, zinc-dependent endopeptidases collectively capable of degrading essentially all the components of the extracellular matrix. At present, 23 members of the human MMP gene family are known. The increased expression and/or activity of one or more members of this family have been documented in essentially all human malignancies and some have been implicated in the process of angiogenesis. Prominent among those are MMP-2 and MT1-MMP, two metalloproteinases that form a cell membrane-associated complex leading to MMP-2 activation and ECM proteolysis. Here, we review our data that identified the type 1 insulin-like growth factor receptor (IGF-IR) as a regulator of tumor invasion and the synthesis of MT1-MMP and MMP-2 and report on the signal transduction pathways that mediate this regulation. These findings are discussed in the context of a broader review of the role of the IGF-IR/IGF axis in the regulation of tumor invasion and metastasis.     

The roles of substrate thermal stability and P2 and P1' subsite identity on matrix metalloproteinase triple-helical peptidase activity and collagen specificity

The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.     

MT1-MMP: a tethered collagenase

Gene ablation in mice offers a powerful tool to assay in vivo the role of selected molecules. Numerous new mouse models of matrix metalloproteinases (MMP) deficiency have been developed in the past 5 years and have yielded a new understanding of the role of MMPs while also putting to rest assumptions based on data predating the days of mouse models. The phenotype of the MT1-MMP deficient mouse is one example which illustrates the sometimes rather surprising insights into extracellular matrix remodeling in development and growth that can be gained with mouse genetics. While MT1-MMP appears to play little or no role in embryonic development, loss of this enzyme results in progressive impairment of postnatal growth and development affecting both the skeleton and the soft connective tissues. The underlying pathologic mechanism is loss of an indispensable collagenolytic activity, which remains essentially uncompensated. Our findings demonstrate that growth and maintenance of the skeleton requires coordinated and simultaneous MT1-MMP-dependent remodeling of all soft tissue attachments (ligaments, tendons, joint capsules). We note that the phenotype of the MT1-MMP deficient mouse bears no resemblance to those of mice deficient in MMP-2 and tissue inhibitors of metallo-proteinase (TIMP)-2 all but dispelling the view that activation of MMP-2 by the MT1-MMP/TIMP-2/proMMP-2 axis plays a significant role in growth and development throughout life. It is of interest to note that loss of a single catabolic function such as selective collagen degradation mediated by MT1-MMP gives rise to profound impairment of a number of both anabolic and catabolic functions.     

Characterization of stromelysin 1 (MMP-3), matrilysin (MMP-7), and membrane type 1 matrix metalloproteinase (MT1-MMP) derived fibrin(ogen) fragments D-dimer and D-like monomer: NH2-terminal sequences of late-stage digest fragments.

Matrix metalloproteinases (MMPs) participate in physiological remodeling of the extracellular matrix. Recently we determined that both fibrinogen (Fg) and cross-linked fibrin (XL-Fb) are substrates for selected MMPs. Specifically, XL-Fb clots were solubilized by MMP-3 (stromelysin 1) by cleavage at gamma Gly 404-Ala 405, resulting in a D-like monomer fragment. Similarly, MMP-7 (matrilysin) and MT1-MMP (membrane type 1 matrix metalloproteinase) solubilized XL-Fb clots. However, the molecular mass of fragment D-dimer, obtained after MMP-7 and MT1-MMP degradation of XL-Fb, is similar to that of fragment D-dimer from plasmin degradation ( approximately 186 kDa). In contrast, fragment D-like monomer, from MMP-3 degradation of both fibrinogen (Fg) and XL-Fb, is similar to fragment D from plasmin degradation of Fg ( approximately 94 kDa). Reduced chains from MMP-3, MMP-7, and MT1-MMP digests of Fg and XL-Fb were subjected to direct sequence analyses and D/D-dimer alpha-chain showed cleavage at both alpha Asp 97-Phe 98 and alpha Asn 102-Asn 103. Degradation of the beta-chain resulted in microheterogeneity of cleavage sites at beta Asp 123-Leu 124, beta Asn 137-Val 138, and beta Glu 141-Tyr 142, whereas all three enzymes cleaved the gamma-chain at gamma Thr 83-Leu 84. In both Fg and XL-Fb, several cleavage sites obtained by proteolysis with MMP-3, MMP-7, and MT1-MMP were found to be in very close proximity to those obtained by plasmin on these same substrates. That does not occur with other MMPs such as MMP-1, -2, and -9 and MT2-MMP. The degradation of XL-Fb by MMPs suggests both plasmin-dependent and independent mechanisms of fibrinolysis that might be relevant in inflammation, angiogenesis, arthritis, and atherosclerosis.     

Oxidative stress and the development of diabetic retinopathy: Contributory role of matrix metalloproteinase-2

Matrix metalloproteinases (MMPs) degrade extracellular matrix and regulate many functions including cell signaling. Oxidative stress is implicated in the development of diabetic retinopathy, and MMP-2, the most ubiquitous member of the MMP family, is sensitive to oxidative stress. This study aimed to determine the regulation of MMP-2 by oxidative stress in the development of diabetic retinopathy and the role of MMP-2 in the apoptosis of retinal capillary cells. The effects of mitochondrial superoxide scavenger on glucose-induced alterations in MMP-2, and its proenzyme activator MT1-MMP and physiological inhibitor TIMP-2, were determined in retinal endothelial cells, and the regulation of their glucose-induced accelerated apoptosis by the inhibitors of MMP-2 was accessed. To confirm in vitro results, the effects of antioxidant supplementation on MMP-2, MT1-MMP, and TIMP-2 were investigated in the retina of streptozotocin-induced diabetic rats. Glucose-induced activation of retinal capillary cell MMP-2 and MT1-MMP and decrease in TIMP-2 were inhibited by superoxide scavengers, and their accelerated apoptosis was prevented by the inhibitors of MMP-2. Antioxidant therapies, which have been shown to inhibit oxidative stress, capillary cell apoptosis, and retinopathy in diabetic rats, ameliorated alterations in retinal MMP-2 and its regulators. Thus, MMP-2 has a proapoptotic role in the loss of retinal capillary cells in diabetes, and the activation of MMP-2 is under the control of superoxide. This suggests a possible use of MMP-2-targeted therapy to inhibit the development of diabetic retinopathy.     

Role of cell surface metalloprotease MT1-MMP in epithelial cell migration over laminin-5

Laminin-5 (Ln-5) is an extracellular matrix substrate for cell adhesion and migration, which is found in many epithelial basement membranes. Mechanisms eliciting migration on Ln-5 need to be elucidated because of their relevance to tissue remodeling and cancer metastasis. We showed that exogenous addition of activated matrix metalloprotease (MMP) 2 stimulates migration onto Ln-5 in breast epithelial cells via cleavage of the gamma2 subunit. To investigate the biological scope of this proteolytic mechanism, we tested a panel of cells, including colon and breast carcinomas, hepatomas, and immortalized hepatocytes, selected because they migrated or scattered constitutively in the presence of Ln-5. We found that constitutive migration was inhibited by BB94 or TIMPs, known inhibitors of MMPs. Limited profiling by gelatin zymography and Western blotting indicated that the ability to constitutively migrate on Ln-5 correlated with expression of plasma membrane bound MT1-MMP metalloprotease, rather than secretion of MMP2, since MMP2 was not produced by three cell lines (one breast and two colon carcinomas) that constitutively migrated on Ln-5. Moreover, migration on Ln-5 was reduced by MT1-MMP antisense oligonucleotides both in MMP2+ and MMP2- cell lines. MT1-MMP directly cleaved Ln-5, with a pattern similar to that of MMP2. The hemopexin-like domain of MMP2, which interferes with MMP2 activation, reduced Ln-5 migration in MT1-MMP+, MMP2+ cells, but not in MT1-MMP+, MMP2- cells. These results suggest a model whereby expression of MT1-MMP is the primary trigger for migration over Ln-5, whereas MMP2, which is activated by MT1-MMP, may play an ancillary role, perhaps by amplifying the MT1-MMP effects. Codistribution of MT1-MMP with Ln-5 in colon and breast cancer tissue specimens suggested a role for this mechanism in invasion. Thus, Ln-5 cleavage by MMPs may be a widespread mechanism that triggers migration in cells contacting epithelial basement membranes.     

Constraining specificity in the N-domain of tissue inhibitor of metalloproteinases-1; gelatinase-selective inhibitors

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal pentapeptide of TIMP and the C-D beta-strand connector which occupy the primed and unprimed regions of the active site. The loop between beta-strands A and B forms a secondary interaction site for some MMPs, ranging from multiple contacts in the TIMP-2/membrane type-1 (MT1)-MMP complex to none in the TIMP-1/MMP-1 complex. TIMP-1 and its inhibitory domain, N-TIMP-1, are weak inhibitors of MT1-MMP; inhibition is not improved by grafting the longer AB loop from TIMP-2 into N-TIMP-1, but this change impairs binding to MMP-3 and MMP-7. Mutational studies with N-TIMP-1 suggest that its weak inhibition of MT1-MMP, as compared to other N-TIMPs, arises from multiple (>3) sequence differences in the interaction site. Substitutions for Thr2 of N-TIMP-1 strongly influence MMP selectivity; Arg and Gly, that generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the N-TIMP-1(AB2) mutant, it produces a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and -9, respectively. Interestingly, the Gly mutant has a Ki of 2.1 nM for MMP-9 and >40 muM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily.     

Morphological and biochemical characterization of remodeling in aorta and vena cava of DOCA-salt hypertensive rats

Arterial remodeling occurs in response to mechanical and neurohumoral stimuli. We hypothesized that veins, which are not exposed to higher pressures in hypertension, would demonstrate less active remodeling than arteries. We assessed remodeling with two standard measures of arterial remodeling: vessel morphometry and the expression/function of matrix metalloproteinases (MMPs). Thoracic aorta and vena cava from sham normotensive and DOCA-salt hypertensive rats (110 +/- 4 and 188 +/- 8 mmHg systolic blood pressure, respectively) were used. Wall thickness was increased in DOCA-salt vs. sham aorta (301 +/- 23 vs. 218 +/- 14 mum, P < 0.05), as was medial area, but neither measure was altered in the vena cava. The aorta and vena cava expressed the gelatinases MMP-2, MMP-9, transmembrane proteinase MT1-MMP, and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemically, MMP-2 localized to smooth muscle in the aorta and densely in endothelium/smooth muscle of the vena cava. Western and zymographic analyses verified that MMP-2 was active in all vessels and less active in the vena cava than aorta. In hypertension, MMP-2 expression and activity in the aorta were increased (59.1 +/- 3.7 and 74.5 +/- 6.1 units in sham and DOCA, respectively, P < 0.05); similar elevations were not observed in the vena cava. MMP-9 was weakly expressed in all vessels. MT1-MMP was expressed by the aorta and vena cava and elevated in the vena cava from DOCA-salt rats. TIMP-2 expression was significantly increased in the aorta of DOCA rats compared with sham but was barely detectable in the vena cava of sham or DOCA-salt hypertensive rats. These findings suggest that large veins may not undergo vascular remodeling in DOCA-salt hypertension.     

Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia

This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.