Noncanonical matrix metalloprotease-1-protease-activated receptor-1 signaling triggers vascular smooth muscle cell dedifferentiation and arterial stenosis

Abstract

Vascular injury that results in proliferation and dedifferentiation of vascular smooth muscle cells (SMCs) is an important contributor to restenosis following percutaneous coronary interventions or plaque rupture. Protease-activated receptor-1 (PAR1) has been shown to play a role in vascular repair processes; however, little is known regarding its function or the relative roles of the upstream proteases thrombin and matrix metalloprotease-1 (MMP-1) in triggering PAR1-mediated arterial restenosis. The goal of this study was to determine whether noncanonical MMP-1 signaling through PAR1 would contribute to aberrant vascular repair processes in models of arterial injury. A mouse carotid arterial wire injury model was used for studies of neointima hyperplasia and arterial stenosis. The mice were treated post-injury for 21 days with a small molecule inhibitor of MMP-1 or a direct thrombin inhibitor and compared with vehicle control. Intimal and medial hyperplasia was significantly inhibited by 2.8-fold after daily treatment with the small molecule MMP-1 inhibitor, an effect that was lost in PAR1-deficient mice. Conversely, chronic inhibition of thrombin showed no benefit in suppressing the development of arterial stenosis. Thrombin-PAR1 signaling resulted in a supercontractile, differentiated phenotype in SMCs. Noncanonical MMP-1-PAR1 signaling resulted in the opposite effect and led to a dedifferentiated phenotype via a different G protein pathway. MMP-1-PAR1 significantly stimulated hyperplasia and migration of SMCs, and resulted in down-regulation of SMC contractile genes. These studies provide a new mechanism for the development of vascular intimal hyperplasia and suggest a novel therapeutic strategy to suppress restenosis by targeting noncanonical MMP-1-PAR1 signaling in vascular SMCs.

Keywords: Cardiovascular Disease; Cell Differentiation; Matrix Metalloproteinase (MMP); PAR1; Smooth Muscle; Thrombin.

FIGURE 1.

MMP-1 inhibition reduces medial and intimal lesion thickness following carotid artery wire-injury in mice. A, representative photomicrographs (16×) of carotid arteries from the right uninjured contralateral artery or the left injured artery from male C57BL6 mice treated with daily subcutaneous injections of vehicle (20% Me₂SO), 5 mg/kg FN-439, or 10 mg/kg bivalirudin (Bival) for 21 days (n = 14). B, medial plus intimal thickness based on a micromillimeter scale bar of injured carotid arteries harvested from mice in A. Each data point represents four averaged (quadrants) measurements from each mouse artery. Horizontal lines indicate mean medial plus intimal thickness in microns. The mean medial thickness (horizontal lines) for each treatment cohort was calculated from cross-sections of the arteries as described under “Experimental Procedures.” *, p < 0.05 by ANOVA. C, proposed mechanism of divergent signaling and outcomes resulting from PAR1 activation by MMP-1 versus thrombin at two different cleavage sites in arterial injury and restenosis. D, merged immunofluorescence of representative sections from wire-injured carotid arteries of vehicle-treated mice from A stained with Abs for smooth muscle actin (SMA-Cy3; monoclonal), mPAR1 (polyclonal), FITC-2°, or Mmp-1a (polyclonal), FITC-2°. The cell nuclei were counterstained (blue) with DAPI. The insets in the lower left corners represent magnified regions prominent co-localization in the neointima. Autofluorescence of the elastic lamina can be seen as distinct green bands.

FIGURE 2.

MMP-1 cleaves and activates PAR1 on human SMCs. A, immunohistochemistry of sections from human atherosclerotic lesions showing co-localization of SMA (1A4) with MMP-1 (SB12e) and PAR1 (SFLLR-Ab). A, adventitia; M, media; L, lumen. B, IHC of MMP-1, PAR1, and SMA depicting co-localization in the media, neointima, and endothelium of a human atherosclerotic plaque. Arrowheads point to areas of localization in the neointima. The triangles show staining in the endothelium. C, PAR1 surface expression (shaded fill) of three SMC lines analyzed by FACS using the SFLLR-Ab. 2° antibody control is shown as a black line with white fill. D, MMP-1 cleavage of PAR1 on CD314 SMCs after 30 min of treatment with 5 nm MMP-1 ± 5 μm FN-439. The Span12 antibody spanning the PAR1 cleavage site was used to recognize full-length receptor. Loss of antibody binding indicates receptor cleavage by MMP-1 (5 nm); 5 μm FN-439 MMP-1 inhibitor. E, calcium flux measurements of SMCs following challenge with MMP-1 or thrombin (5 nm thrombin or MMP-1). In the bottom traces, the cells were pretreated for 3 min with the PAR1 inhibitor 5 μm RWJ-56110 (RWJ) prior to the addition of agonist.

FIGURE 3.

MMP-1 is a potent chemoattractant for PAR1-dependent SMC migration. A, 16 h of migration in Boyden chambers of AO391 and CD314 SMCs toward either 5 nm thrombin (Thr) or 5 nm MMP-1, with and without 5 μm RWJ-56110 (RWJ). B, CD314 cells were grown to confluency in 6-well plates and scratched using a sterile P1000 polypropylene pipette tip. The cells were then treated with either 5 nm thrombin or MMP-1 with or without 5 μm RWJ-56110 and allowed to migrate for 16 h. Four micrographs (4×) at times 0 and 16 h were used to compare treated and untreated (PBS buffer (vehicle)) wells. The mean numbers of cells migrated in (n = 4 fields) are quantified on the right. The data are shown as means ± S.E. *, p < 0.05 (Student's unpaired t test). C, 16 h migration of AO391, CD314, and HCA SMCs toward agonist peptides (Thr: TFLLRN, SFLLRN; MMP-1: PR-SFLLRN; Reversed MMP-1: RP-SFLLRN) at the concentrations indicated (30 and 300 μm). D, PAR1 surface expression on CD314 SMCs over 60 min as observed by FACS using the SFLLR-Ab following treatment with either 5 nm thrombin or 5 nm MMP-1. The experiments were either performed at 37 or 4 °C. *, p < 0.05; **, p < 0.01 by ANOVA.

FIGURE 4.

Thrombin-PAR1 activates SMC contraction. A, immunofluorescence of FITC-phospho-MLC in CD314 cells treated for 15 min with either 5 nm thrombin (Thr) or 5 nm MMP-1, with and without 5 μm RWJ-56110 (RWJ). Quantification of mean green fluorescence intensity of six fields is shown on the right. DAPI was used as a nuclear counterstain. B, immunofluorescence of FITC-phalloidin in CD314 cells treated for 5 min with either 5 nm thrombin or 5 nm MMP-1, with and without 5 μm RWJ-56110. Quantification of mean green fluorescence intensity of six fields is shown on the right. DAPI was used as a nuclear counterstain. C and D, immunofluorescence of phospho-FAK-FITC (Tyr-397) following treatment with 5 nm thrombin or MMP-1 over 60 min (left). Western blot analysis of phospho-FAK (Tyr-397) following stimulation with either 5 nm thrombin or 5 nm MMP-1 at the time points indicated (right). E, Western blot analysis of RhoA bound to GTP 15 min following stimulation with either 5 nm thrombin or 5 nm MMP-1, with and without 5 μm RWJ-56110 pretreatment for 15 min.

FIGURE 5.

MMP-1-PAR1 drives SMC dedifferentiation. Shown are the results of quantitative RT-PCR in 1° HCA cells of myocardin, SM-22, calponin, and fibronectin following 24 h of treatment with either 5 nm thrombin (Thr) or 5 nm MMP-1, with and without 5 μm RWJ-56110 (RWJ). The data are expressed as relative fold changes of triplicate samples normalized to GAPDH. *, p < 0.05; **, p < 0.01 by ANOVA.

FIGURE 6.

Thrombin and MMP-1 differentially signal through PAR1-G_i pathways in SMC proliferation. A, fold change in proliferation (DNA crystal violet staining, A_{595 nm}) after 3 days of treatment with 5 nm thrombin (Thr), 5 nm MMP-1, 300 μm TFLLRN, or 300 μm PR-SFLLRN, with or without 5 μm RWJ-56110 (RWJ) in AO391 cell line. B, fold change in proliferation of HCA cells after 3 days of treatment with 5 nm thrombin or 5 nm MMP-1, with and without 5 μm RWJ-56110. C, proliferative dose-response curves in CD314 cells to thrombin and MMP-1 over the concentrations (nm) indicated. D, representative calcium traces of AO391 cells treated with either 5 nm thrombin or 5 nm MMP-1. Cells were pretreated with 200 ng/ml PTx for 16 h. E, 24 h of proliferation of CD314 cells treated with either 5 nm thrombin or 5 nm MMP-1, with and without 200 ng/ml PTx. All proliferation assays are representative graphs expressing average fold changes from six individual replicates per treatment. F and G, ERK1/2 phosphorylation in AO391 (F) and CD314 (G) cells 15 min after treatment with either 5 nm thrombin or 5 nm MMP-1 in the presence of 5 μm RWJ-56110 or 200 ng/ml PTx. *, p < 0.05; **, p < 0.01 by ANOVA.