Microvessel vascular smooth muscle cells contribute to collagen type I deposition through ERK1/2 MAP kinase, alphavbeta3-integrin, and TGF-beta1 in response to ANG II and high glucose

Abstract

This study determines that vascular smooth muscle cell (VSMC) signaling through extracellular signal-regulated kinase (ERK) 1/2-mitogen-activated protein (MAP) kinase, alphavbeta(3)-integrin, and transforming growth factor (TGF)-beta1 dictates collagen type I network induction in mesenteric resistance arteries (MRA) from type 1 diabetic (streptozotocin) or hypertensive (HT; ANG II) mice. Isolated MRA were subjected to a pressure-passive-diameter relationship. To delineate cell types and mechanisms, cultured VSMC were prepared from MRA and stimulated with ANG II (100 nM) and high glucose (HG, 22 mM). Pressure-passive-diameter relationship reduction was associated with increased collagen type I deposition in MRA from HT and diabetic mice compared with control. Treatment of HT and diabetic mice with neutralizing TGF-beta1 antibody reduced MRA stiffness and collagen type I deposition. Cultured VSMC stimulated with HG or ANG II for 5 min increased ERK1/2-MAP kinase phosphorylation, whereas a 48-h stimulation induced latent TGF-beta1, alphavbeta(3)-integrin, and collagen type 1 release in the conditioned media. TGF-beta1 bioactivity and Smad2 phosphorylation were alphavbeta(3)-integrin-dependent, since beta(3)-integrin antibody and alphavbeta(3)-integrin inhibitor (SB-223245, 10 microM) significantly prevented TGF-beta1 bioactivity and Smad2 phosphorylation. Pretreatment of VSMC with ERK1/2-MAP kinase inhibitor (U-0126, 1 microM) reduced alphavbeta(3)-integrin, TGF-beta1, and collagen type 1 content. Additionally, alphavbeta(3)-integrin antibody, SB-223245, TGF-beta1-small-intefering RNA (siRNA), and Smad2-siRNA (40 nM) prevented collagen type I network formation in response to ANG II and HG. Together, these data provide evidence that resistance artery fibrosis in type 1 diabetes and hypertension is a consequence of abnormal collagen type I release by VSMC and involves ERK1/2, alphavbeta(3)-integrin, and TGF-beta1 signaling. This pathway could be a potential target for overcoming small artery complications in diabetes and hypertension.

Fig. 1.

A: pressure-passive diameter relationship of mesenteric resistance arteries from control (sham), hypertensive (HT), Type 1 diabetic (D1), HT + transforming growth factor (TGF)-β1 antibody (TGF-β1-AB), and D1 + TGF-β1 antibody (TGF-β1-AB) mice. *P < 0.05 statistically significant differences between sham vs. HT; sham vs. D1, HT vs. HT + TGF-β1-AB; and D1 vs. D1 + TGF-β1-AB. B: Western blot analysis and cumulative data showing the increased collagen type 1 of mesenteric resistance arteries from control (sham), hypertensive (HT), Type 1 diabetic (D1), HT + TGF-β1-AB, and D1 + TGF-β1-AB mice. *P < 0.05 statistically significant differences between: sham vs. HT; sham vs. D1; HT vs. HT + TGF-β1-AB; and D1 vs. D1 + TGF-β1-AB; n = 7 mice for each experiments.

Fig. 2.

A: Western blot analysis showing the expression of α-actin in primary cultured vascular smooth muscle cells (VSMC) prepared from mice mesenteric resistance arteries and aorta (top) and Von Willebrand factor (bottom); data are representative of n = 5. B: Western blot analysis and cumulative data of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 mitogen-activated protein (MAP) kinase in response to acute stimulation of VSMC with ANG II (100 nM) or high glucose (HG, 22 mM). *P < 0.05, statistically significant differences between control (CTR) vs. ANG II and CTR vs. HG; n = 6 for each experiment. C: immunoprecipitation/Western blot analysis and cumulative data of collagen type 1 in the conditioned media of cultured VSMC stimulated for 48 h with ANG II or HG. *P < 0.05, CTR vs. ANG II and CTR vs. HG; n = 6. D: immunostaining showing collagen type 1 network formation on VSMC, growth on cover slip, stimulated with ANG II or HG for 48 h. Data are representative of n = 5.

Fig. 3.

A: immunoprecipitation (IP)/Western blot analysis and cumulative data showing αvβ₃-integrin shedding from VSMC surface in response to ANG II or HG for 48 h measured in the conditioned media. *P < 0.05, statistically significant differences between CTR vs. ANG II and CTR vs. HG; n = 6. B: IP/Western blot analysis and cumulative data showing the release of TGF-β1 measured in the conditioned media of VSMC stimulated with ANG II or HG for 48 h. *P < 0.05 statistically significant differences between CTR vs. ANG II and CTR vs. HG; n = 6. C: TGF-β1 bioactivity measured in the conditioned media of VSMC stimulated with ANG II or HG for 48 h. P < 0.05, statistically significant differences between CTR vs. ANG II and CTR vs. HG (*) and between ANG II or HG vs. ANG II + β₃-integrin antibody, ANG II + SB-223245, HG + β₃-integrin antibody, or HG + SB-223245 (#); n = 6 for each experiment.

Fig. 4.

A: Western blot analysis and cumulative data showing phosphorylated and total Smad2 of VSMC stimulated with ANG II or HG for 48 h. *P < 0.05, statistically significant between CTR vs. ANG II and CTR vs. HG; n = 6. B: Western blot analysis and cumulative data showing phosphorylated and total Smad2 of VSMC pretreated with β₃-integrin antibody (1:100 dilution) or SB-223245 (10 μM) and stimulated with ANG II or HG for 48 h. NS, statistically no significance between CTR vs. ANG II or HG ± β₃-integrin antibody or SB-223245; n = 6.

Fig. 5.

Western blot analysis and cumulative data showing the release of collagen type 1 in the conditioned media from VSMC pretreated with mitogen-extracellular signal-regulated kinase (MEK) inhibitor (U-0126, 1 μM) and stimulated with ANG II or HG (A). NS, no statistical significance between CTR + U-0126 vs. ANG II or HG + U-0126; n = 6. B: β₃-integrin specific antibody (1:100 dilution) and stimulated with ANG II or HG. NS, no statistical significance between CTR + β₃-integrin antibody vs. ANG II or HG + β₃-integrin antibody; n = 6. C: SB-223245 (10 μM) and stimulated with ANG II or HG. NS, no statistical significance between CTR + SB-223245 vs. ANG II or HG + SB-223245; n = 6.

Fig. 6.

A: Western blot analysis and cumulative data showing the release of collagen type 1 in the conditioned media from VSMC transfected with small-interfering RNA (siRNA)-TGF-β1 (40 nM) and stimulated with ANG II or HG. NS, no statistical significance between CTR + siRNA-TGF-β1 vs. ANG II or HG + siRNA-TGF-β1; n = 4. B: Western blot analysis showing the expression of TGF-β1 and α-actin in VSMC transfected with siRNA-TGF-β1, n = 4. C: Western blot analysis and cumulative data showing the release of collagen type 1 in the conditioned media from VSMC transfected with siRNA-Smad2 (40 nM) and stimulated with ANG II or HG. NS, no statistical significance between CTR + siRNA-Smad2 vs. ANG II or HG + siRNA-Smad2; n = 4. D: Western blot analysis showing the expression of TGF-β1 and β-actin of VSMC transfected with siRNA-Smad2; n = 4.

Fig. 7.

Model illustrating the mechanism leading to the abnormal collagen type 1 deposition in resistance artery wall from hypertensive and Type 1 diabetic mice.