Atorvastatin and sildenafil decrease vascular TGF-β levels and MMP-2 activity and ameliorate arterial remodeling in a model of renovascular hypertension

Abstract

Imbalanced matrix metalloproteinase (MMP)-2 activity and transforming growth factor expression (TGF-β) are involved in vascular remodeling of hypertension. Atorvastatin and sildenafil exert antioxidant and pleiotropic effects that may result in cardiovascular protection. We hypothesized that atorvastatin and sildenafil alone or in association exert antiproliferative effects by down-regulating MMP-2 and TGF-β, thus reducing the vascular hypertrophy induced by two kidney, one clip (2K1C) hypertension. Sham and 2K1C rats were treated with oral atorvastatin 50 mg/kg, sildenafil 45 mg/kg, or both, daily for 8 weeks. Blood pressure was monitored weekly. Morphologic changes in the aortas were studied. TGF-β levels were determined by immunofluorescence. MMP-2 activity and expression were determined by in situ zymography, gel zymography, Western blotting, and immunofluorescence. The effects of both drugs on proliferative responses of aortic smooth muscle cells to PDGF and on on MMP-2 activity in vitro were determined. Atorvastatin, sildenafil, or both drugs exerted antiproliferative effects in vitro. All treatments attenuated 2K1C-induced hypertension and prevented the increases in the aortic cross-sectional area and media/lumen ratio in 2K1C rats. Aortas from 2K1C rats showed higher collagen deposition, TGF-β levels and MMP-2 activity and expression when compared with Sham-operated animals. Treatment with atorvastatin and/or sildenafil was associated with attenuation of 2K1C hypertension-induced increases in these pro-fibrotic factors. However, these drugs had no in vitro effects on hr-MMP-2 activity. Atorvastatin and sildenafil was associated with decreased vascular TGF-β levels and MMP-2 activity in renovascular hypertensive rats, thus ameliorating the vascular remodeling. These novel pleiotropic effects of both drugs may translate into protective effects in patients.

Keywords: Atorvastatin; Hypertension; Matrix metalloproteinase; Sildenafil.

Graphical abstract

Fig. 1

Effects of atorvastatin and sildenafil on cell proliferation. Rat aortic smooth muscle cells (RASMCs) were treated with atorvastatin or sildenafil at different concentrations and PDGF-BB (1 ng/mL) was added 1 h later for 24 h. 3H-thymidine incorporation was measured. Control cells are untreated RASMCs without fetal bovine serum in the medium. Data are expressed as mean±S.E.M. (n=5 in the control group and n=6 in the other groups). *P<0.05 versus Control. **P<0.05 versus PDGF-BB alone.

Fig. 2

Systolic blood pressure (mmHg) measured by tail-cuff method (a) and body weight (b) in the eight experimental groups along 10 weeks of study. Data are shown as mean±S.E.M. *P<0.01 versus Sham Vehicle group; ^#P<0.01 versus 2K1C Vehicle group. (2K1C+Vehicle: n=8; 2K1C+ATORVA: n=7; 2K1C+SILD: n=8; 2K1C+ATORVA+SILD: n=9; Sham+Vehicle: n=8; Sham+ATORVA: n=10; Sham+SILD: n=8; Sham+ATORVA+SILD: n=10).

Fig. 3

Panel (a) shows aortic structural alterations induced by 2K1C hypertension and the effects of treatment with atorvastatin (ATORVA), sildenafil (SILD), or both drugs. Panels (b) and (c) show the cross sectional area (CSA) and media to lumen ratio (M/L), respectively, in each study group. Data are expressed as mean±S.E.M. *P<0.05 versus Sham Vehicle group; **P<0.05 versus 2K1C Vehicle group. (Sham+Vehicle: n=9; Sham+ATORVA: n=9; Sham+SILD: n=14; Sham+ATORVA+SILD: n=11; 2K1C+vehicle: n=9; 2K1C+ATORVA: n=10; 2K1C+SILD: n=11; 2K1C+ATORVA+SILD: n=14).

Fig. 4

Collagen surface area in the media layer of aortas from rats and effects of treatments. Panel (a) shows representative photomicrographs of aortic samples stained by Trichrome (Gomori) (400×). Panel (b) shows quantitative evaluation of the collagen surface area stained in blue. Data are expressed as mean±S.E.M. *P<0.05 versus Sham Vehicle group; **P<0.05 versus 2K1C Vehicle group. (Sham+Vehicle: n=7; Sham+ATORVA: n=6; Sham+SILD: n=7; Sham+ATORVA+SILD: n=5; 2K1C+vehicle: n=5; 2K1C+ATORVA: n=7; 2K1C+SILD: n=7; 2K1C+ATORVA+SILD: n=5).

Fig. 5

TGF-β expression in the media layer of aortas from rats. Panel (a) shows representative photomicrographs (400×) of TGF-β expression by immunofluorescence (in red; A=adventicia; M=media; E=endothelium). Panel (b) shows the quantification of red staining of TGF-β. Data are expressed as mean±S.E.M. *P<0.05 versus Sham Vehicle group; **P<0.05 versus 2K1C Vehicle group. (Sham+Vehicle: n=3; Sham+ATORVA: n=5; Sham+SILD: n=5; Sham+ATORVA+SILD: n=6; 2K1C+vehicle: n=4; 2K1C+ATORVA: n=4; 2K1C+SILD: n=3; 2K1C+ATORVA+SILD: n=4).

Fig. 6

Representative sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gelatin zymogram of aortic samples (Panel (a)). Molecular weights of MMP-2 bands (75, 72 and 64 kDa MMP-2) were identified after electrophoresis on 7% SDS–PAGE. STD: internal standard. Panel (b) shows the quantification of each molecular weight form (75, 72 and 64 kDa) of MMP-2 in the aortic extracts. Data are expressed as mean±S.E.M. *P<0.05 versus Sham Vehicle group; **P<0.05 versus 2K1C Vehicle group. (Sham+Vehicle: n=18; Sham+ATORVA: n=9; Sham+SILD: n=9; Sham+ATORVA+SILD: n=9; 2K1C+vehicle: n=18; 2K1C+ATORVA: n=10; 2K1C+SILD: n=9; 2K1C+ATORVA+SILD: n=10).

Fig. 7

Effects of treatments on in situ gelatinolytic activity and MMP-2 levels detected by immunofluorescence in the aortas. Panel (a) shows representative photographs of in situ gelatinolytic activity (400×), MMP-2 detected by immunofluorescence, and their co-localization (merge) in the aortas. Panel (b) shows the quantification of aortic in situ gelatinolytic activity detected as bright green fluorescence. Panel (c) shows the quantification of aortic MMP-2 levels detected by immunofluorescence as bright red fluorescence. Data are expressed as mean±S.E.M. *P<0.05 versus Sham Vehicle group; **P<0.05 versus 2K1C Vehicle group. (Sham+Vehicle: n=6; Sham+ATORVA: n=7; Sham+SILD: n=8; Sham+ATORVA+SILD: n=7; 2K1C+vehicle: n=5; 2K1C+ATORVA: n=7; 2K1C+SILD: n=8; 2K1C+ATORVA+SILD: n=8).

Fig. 8

Expression of MMP-2 in aortas from Sham and 2K1C rats treated with atorvastatin, sildenafil or the combination of both drugs normalized by β-actin expression. Panel (a) shows representative Western blotting results. Panel (b) shows the quantification in the different study groups. Data are expressed as mean±S.E.M. *P<0.05 versus Sham Vehicle group; **P<0.05 versus 2K1C Vehicle group. (Sham+Vehicle: n=5; Sham+ATORVA: n=6; Sham+SILD: n=6; Sham+ATORVA+SILD: n=7; 2K1C+vehicle: n=7; 2K1C+ATORVA: n=5; 2K1C+SILD: n=6; 2K1C+ATORVA+SILD: n=5).

Fig. 9

Effects of atorvastatin, sildenafil, or the combination of both drugs at different concentrations (from 0.1 to 10 μM) on the activity of human recombinant MMP-2. Phenanthroline (Phen) 0.1 mM was used as a positive control for MMP-2 inhibition. Data are shown as mean±S.E.M. (n=5 per group). *P<0.05 versus Vehicle.