Rho Kinase Regulates Aortic Vascular Smooth Muscle Cell Stiffness Via Actin/SRF/Myocardin in Hypertension

Figures

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  Fig. 1.. Effects of Y-27632 on the blood pressure and aortic stiffness in SHR and WKY rats. (A-B) Time dependent changes of systolic blood pressure (SBP) (A) and diastolic blood pressure (DBP) (B) measured by tail cuff at conscious status. (C) Representative waveform of aortic blood pressure measured invasively by a Millar catheter at the end of two-week treatment. (D) Systolic Aortic blood pressure (SAP), diastolic blood pressure (DAP) and pulse pressure (PP) under the treatments. (E) Reduction rate of blood pressure versus vehicle control. (F-G) Aortic stiffness in SHR and WKY rats in vivo evaluated by echography before (D0) or at day 7 (D7) and day 14 (D14) after the initiation of the treatments with Y-27632 or vehicle (DMSO), reflected by the change of arterial com-pliance (F) and arterial strain (G). Data are shown as mean ± SEM, *P<0.01 vs. corresponding WKY. #P<0.05, ##P< 0.01 vs corresponding vehicle. n= 5 rats/group.

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  Fig. 2.. Effect of Y-27632 on TA VSMC stiffness. (A) Thickness of medial layer of aortic wall and the ratio of lumen diameter. (B) Collagen deposition in medial layer of aortic wall. Scale bar: 100μm, n = 5 rats/group. (C) Representative force curves during advance from atomic force microscopy (AFM) nanoindentation of vascular smooth muscle cells (VSMCs) from the thoracic aorta (TA). (D) Representative of the reversible changes of elastic modulus with or without the presence of Y-27632 in SHR TA VSMC. (E) Representative dose response of SHR TA VSMC stiffness to Y-27632. (F) Average apparent elastic modulus of TA VSMCs. n= 4 rats/group. Data are shown as mean ± SEM, *P<0.01 vs. corresponding WKY. #P<0.05, ##P< 0.01 vs corresponding vehicle. NS: no significant difference.

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  Fig. 3.. The ROCK activity in TA VSMCs. (A) The Rho kinase (ROCK) activity of VSMCs in thoracic aorta (TA) and renal artery (RA) detected by ROCK activity kit. (B) Western blots and the quantified data of phosphorylation of myosin phosphatase target subunit 1 (MYPT1) (Thr696) in TA and RA. *P<0.01 vs WKY treated with vehicle, #P< 0.01 vs corresponding TA. (C) The ROCK activity of TA VSMCs under the treatment of Y-27632 or vehicle detected by ROCK activity kit. (D) Effect of Y-27632 on phosphorylation of MYPT1 (Thr696) in TA. GAPDH was used as loading control. Data are shown as mean ± SEM, *P<0.05 vs WKY, #P<0.05 vs corresponding vehicle. NS: no significant difference.

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  Fig. 4.. Effect of Y-27632 on actin/SRF/myocardin pathway in TA VSMCs. (A) Effects of Y-27632 on ratio of F/G actin detected by western blotting. (B) Fluoresce staining of F- and G-actin in primary WKY rat thoracic aorta (WTA) and SHR thoracic aorta (STA) VSMCs. Scale bar: 50μm. (C) The mRNA level of myocardin in WTA and STA VSMCs. (D) Representative western blots and the quantified data of myocardin in nuclear fractions and total cell lysates from WTA and STA VSMCs and the ratio of nuclear/total protein. (E) The mRNA level of SRF in WTA and STA VSMCs. (F) Representative Western blots and the quantified data of SRF in nuclear fractions from WTA and STA VSMCs. GAPDH and HDAC1 were used as loading control of total cell lysates and nuclear fractions, respectively. Data are shown as mean ± SEM, **P<0.01 vs WTA, ##P<0.01 vs corresponding vehicle. NS: no significant difference.

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  Fig. 5.. Effects of Y-27632 on the expression of stiff-ness-associated genes in VSMCs. (A) The relative mRNA levels of SRF/myocardin target genes in VSMCs, α-SMA: al pha-smooth muscle ac-tin; MYH11: myosin heavy chain 11; SM22: smooth muscle 22; SMTN: smoothelin. (B) Represen-tative western blots and the quantified data of α-SMA in total cell lysates. GAPDH was used as loading control. (C) Immunostainings of α-SMA in VSMCs. Scale bar: 50μm. Data are shown as mean ± SEM, **P<0.01 vs WTA, ##P<0.01 vs corresponding vehicle. All, n=5 rats/group.

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  Fig. 6.. Effect of the inhibition of SRF/myocardin by CCG-100602. (A) The ROCK activity of TA VSMCs detected by ROCK activity kit. (B) Representative western blots and the quantified data of ROCK substrate, phosphorylation of MYPT1 (Thr696) in TA VSMCs. (C). Representative western blots and the quantified data of myocardin in nuclear fractions and total cell lysates and their ratio. (D) TA VSMC stiffness detected by AFM. (E and F) Aortic stiffness in SHR and WKY rats evaluated by echography before (D0) or at day 7 (D7) and day 14 (D14) after the initiation of the treatments with CCG-100602 or vehicle (DMSO) reflected by the change of arterial compliance (E) and arterial strain (F). Data are shown as mean ± SEM, *P<0.05, **P<0.01 vs corresponding WKY, #P<0.05, ##P<0.01 vs vs corresponding vehicle.

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  Fig. 7.. An illustration of the mechanism. SRF/myocardin pathway acts as an essential regulator of VSMC stiffness and plays a central role in the aortic stiffness associated with hypertension.