Chronic matrix metalloproteinase inhibition retards age-associated arterial proinflammation and increase in blood pressure

Abstract

Age-associated arterial remodeling involves arterial wall collagen deposition and elastin fragmentation, as well as an increase in arterial pressure. This arterial remodeling is linked to proinflammatory signaling, including transforming growth factor-β1, monocyte chemoattractant protein 1, and proendothelin 1, activated by extracellular matrix metalloproteinases (MMPs) and orchestrated, in part, by the transcriptional factor ets-1. We tested the hypothesis that inhibition of MMP activation can decelerate the age-associated arterial proinflammation and its attendant increase in arterial pressure. Indeed, chronic administration of a broad-spectrum MMP inhibitor, PD166739, via a daily gavage, to 16-month-old rats for 8 months markedly blunted the expected age-associated increases in arterial pressure. This was accompanied by the following: (1) inhibition of the age-associated increases in aortic gelatinase and interstitial collagenase activity in situ; (2) preservation of the elastic fiber network integrity; (3) a reduction of collagen deposition; (4) a reduction of monocyte chemoattractant protein 1 and transforming growth factor-β1 activation; (5) a diminution in the activity of the profibrogenic signaling molecule SMAD-2/3 phosphorylation; (6) inhibition of proendothelin 1 activation; and (7) downregulation of expression of ets-1. Acute exposure of cultured vascular smooth muscle cells in vitro to proendothelin 1 increased both the transcription and translation of ets-1, and these effects were markedly reduced by MMP inhibition. Furthermore, infection of vascular smooth muscle cells with an adenovirus harboring a full-length ets-1 cDNA increased activities of both transforming growth factor-β1 and monocyte chemoattractant protein 1. Collectively, our results indicate that MMP inhibition retards age-associated arterial proinflammatory signaling, and this is accompanied by preservation of intact elastin fibers, a reduction in collagen, and blunting of an age-associated increase in blood pressure.

Figure 1. MMP activation in situ.

A) Fluorescence micrographs of in situ gelatin zymograms (green, 400X), and average intensity of the cleaved gelatin signal (lower panel, n=3/goup). B). Fluorescence micrographs of in situ casein zymograms (red, 400X) and average intensity of the cleaved casein signal (lower panel, n=3/group). C). Fluorescence micrographs of in situ collagenase zymograms (green, 200X) and the average intensity of the cleaved casein signal (lower panel, n=3/group). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media.

Figure 2. Aortic extracellular matrix remodeling

A) Photomicrographs of elastic fibers (dark blue) in E.V.G. stained with elastin protein in aortic sections with (left panels, 400X). Average elastin fraction (EF, right panel, n=5/per group) B) Western blots of Fibrillin-1(left panels) and average data (n=3/group, right panel). C) Photomicrographs of Collagen I immunostaining (brown, 200X, left panels) of aortic sections, and average data (right panel, n=4/group). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media.

Figure 3. Proinflammatory signaling molecules

A) Representative Western blots of the MCP-1 dimer (left panel), and average data (n=3/group, right panel). B) Photomicrographs of aortic wall TGF-β1 staining (brown, 400X). C). Western blots of TGF-β1 (left panel), and average data (n=3/group, right panel). D) Photomicrographs of aortic wall p-SMAD2/3 staining (upper panels, brown color, 400X); and Western blots of p-SMAD2/3 (lower left panels), and average data (n=3/group, lower right panel). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media.

Figure 4. Endothelin-1 expression and cleavage, and transcriptional factor, ets-1, expression

A) Immunolabelling of aortic wall ET-1 (brown color, 200X); B) Western blots of arterial wall ET-1 (left panel), and average data (right panel, n=3/group). #p< 0.05, 24M vs. 24Mi. C) Photomicrographs of aortic wall ets-1 staining (brown, 400X). D) Western blots of ets-1 (left panels), and average data (n=3/group). *p<0.05, vs. 16M group; and # p<0.05, vs. 24M group. L=lumen; and M=media.

Figure 5. Proinflammation cascade within VSMC

A) Western blots of ets-1 (left panels), and average data (right panel, n=3 independent experiments). * p<0.05, vs. control. B). RT-PCR analysis (n=4 independent experiments). vs control; #p<0.05, vs. PD166793 treatment. C) Representative Western blots of ets-1. D). Over-expression of ets-1 in VSMC increases activated MCP-1 and TGF-β1 protein.

Figure 6. Age-associated increases in systolic blood pressure (SBP) and diastolic blood pressure (DBP) are reduced by MMP inhibition

The interaction between treatment groups and age on arterial pressure in linear mixed effects models is highly statistically significant for both SBP (*p = 0.0006); and DBP (#p = 0.0082).