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Acute venous occlusion enhances matrix metalloprotease activity: Implications on endothelial dysfunction.

ABSTRACT: Venous hypertension is associated with microvascular inflammation, restructuring, and apoptosis, but the cellular and molecular mechanisms underlying these events remain uncertain. In the present study, we tested the hypothesis that elevated venous pressure and reduction of shear stress induce elevated enzymatic activity. This activity in turn may affect endothelial surface receptors and promote their dysfunction. Using a rodent model for venous hypertension using acute venular occlusion, microzymographic techniques for enzyme detection, and immunohistochemistry for receptor labeling, we found increased activity of the matrix metalloproteases (MMPs) -1, -8, and -9 and tissue inhibitors of metalloproteases (TIMPs) -1 and -2 in both high- and low-pressure regions. In this short time frame, we also observed that elevated venule pressure led to two different fates for the vascular endothelial growth factor receptor-2 (VEGFR2); in higher-pressure upstream regions, some animals exhibited higher VEGFR2 expression, while others displayed lower levels upstream compared to their downstream counterparts with lower pressure. VEGFR2 expression was, on average, more pronounced upon application of MMP inhibitor, suggesting possible cleavage of the receptor by activated enzymes in this model. We conclude that venous pressure elevation increases enzymatic activity which may contribute to inflammation and endothelial dysfunction associated with this disease by influencing critical surface receptors.

SUBMITTER: Alsaigh T

PROVIDER: S-EPMC3021174 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Acute venous occlusion enhances matrix metalloprotease activity: Implications on endothelial dysfunction.

Alsaigh Tom T Pocock Elizabeth S ES Bergan John J JJ Schmid-Schönbein Geert W GW

Microvascular research 20101016 1

Venous hypertension is associated with microvascular inflammation, restructuring, and apoptosis, but the cellular and molecular mechanisms underlying these events remain uncertain. In the present study, we tested the hypothesis that elevated venous pressure and reduction of shear stress induce elevated enzymatic activity. This activity in turn may affect endothelial surface receptors and promote their dysfunction. Using a rodent model for venous hypertension using acute venular occlusion, microz ...[more]

PMID: 20923679

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Project description:Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of ?2?1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an ?2?1 integrin. Herein, we used ALT-C as a ?2?1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration.ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The ?2?1 integrin binding properties of ALT-C, its dissociation constant (Kd ) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting.Our data demonstrate that ALT-C, after binding to ?2?1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression.These results demonstrate that ?2?1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.

Project description:An understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intradomain dynamics occurring at picosecond to millisecond timescales. However, the correlation between interdomain dynamics and the function of a protein is poorly understood. Here, we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type 1 collagen fibrils correlate with its activity. Using single-molecule Förster resonance energy transfer, we identified functionally relevant open conformations in which the two MMP1 domains are well separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. We fitted the histograms of single-molecule Förster resonance energy transfer values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We used a two-state Poisson process to describe the dynamics and calculate the kinetic rates from the fit parameters. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an interdomain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single-molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single-molecule tracking can overcome for other insoluble substrates. Interdomain communications are likely important for multidomain proteins.

Dataset Information

Acute venous occlusion enhances matrix metalloprotease activity: Implications on endothelial dysfunction.

Publications

Acute venous occlusion enhances matrix metalloprotease activity: Implications on endothelial dysfunction.

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