The inhibitory effect of BNP on profibrotic gene expression is abolished by the smooth muscle-targeted expression of RhoA^A188. Quantitative RT-PCR analysis of profibrotic gene mRNA expression was performed with the hearts of the indicated mouse groups left untreated or treated with Ang II. Values are means ± SEM for three or four replicates. *, P < 0.05; **, P < 0.01; †, P < 0.01 versus BNP-Tg mice treated with Ang II; §, P < 0.01 versus RhoA^WT/BNP-Tg mice treated with Ang II; NS, not significant.

cGK I hemizygosity recruits ROCK as a major mediator for profibrotic gene expression. (A and B) Quantitative RT-PCR analysis of profibrotic gene mRNA expression in cGK I^+/− and WT mouse aortas (A) (n = 5) and hearts (B) (n = 3 to 7) left untreated or treated with Ang II and Y-27632. (C) Absolute mRNA level reductions by Y-27632 in Ang II-treated cGK I^+/− and WT mouse hearts. (D) Y-27632-inhibitable fractions of incremental gene expression of cGK I^+/− over WT hearts under Ang II treatment, as calculated by the following formula: Y-27632 inhibitability (%) = [(expression in Ang II-treated cGK I^+/− hearts) − (expression in Ang II-plus-Y-27632-treated cGK I^+/− hearts)]/[(expression in Ang II-treated cGK I^+/− hearts) − (expression in Ang II-treated WT hearts)] × 100. *, P < 0.05; **, P < 0.01; #, P < 0.001; NS, not significant.

cGK I hemizygosity facilitates ROCK activation and ROCK-dependent vascular hypertrophy and fibrosis. cGK I^+/− and WT mice were left untreated or treated with Ang II and Y-27632 before analysis. (A) Immunoblot showing levels of P-moesin and moesin in aortas. (B) Bar graphs showing ROCK activity, as determined by the P-moesin/moesin ratio, in the aortas (n = 3). (C) Systolic BPs (sBP) of mice (n = 4 to 10). (D to F) Morphological assessment of coronary arteries (n = 13 to 92). (D) Representative images of Sirius red-stained heart sections. Bar, 50 μm. (E and F) Morphometric analysis of coronary artery MT (E) and PVF (F). (Left) MT and PVF were determined by the following formulas: (i) MT = medial area/luminal area and (ii) PVF = perivascular fibrotic area/(luminal area + medial area + perivascular fibrotic area). (Middle) Absolute values of reduction by Y-27632 in Ang II-treated mice. (Right) Y-27632-inhibitable fractions of Ang II-induced increases of MT and PVF. Values are means ± SEM. *, P < 0.05; **, P < 0.01; #, P < 0.001; NS, not significant.

The inhibitory effect of cGMP on RhoA-mediated protein synthesis and SRE-dependent gene transcription is reversed by the smooth muscle-targeted expression of RhoA^A188. (A and B) MASMCs were derived from NTg, RhoA^WT-Tg, and RhoA^A188-Tg mice. (A) Effects of 8-Br cGMP and Y-27632 on Ang II-induced protein synthesis, as assessed by [³H]leucine uptake (left), and on serum-induced DNA synthesis, as assessed by [³H]thymidine uptake (right). Values are means ± SEM for six replicates. #, P < 0.0001; †, P < 0.05 versus serum-starved NTg cells; §, P < 0.0001 versus Ang II-treated NTg cells; ¶, P < 0.0001 versus Ang II- and Y-27632-treated RhoA^A188-Tg cells; NS, not significant. (B) Effects of 8-Br cGMP and Y-27632 on TGF-β-induced SRE (left)- and AP-1 (right)-dependent transcriptional activity. Values are means ± SEM for triplicates. #, P < 0.0001; †, P < 0.05 versus serum-starved NTg cells; §, P < 0.0001 versus TGF-β-treated NTg cells; ¶, P < 0.0001 versus TGF-β- and Y-27632-treated RhoA^A188-Tg cells. (C) Proposed model showing in vivo pathways whereby pro- and antifibrotic signals converge intracellularly. (D) cGK I is a major determinant of RhoA activity and controls the relative contributions of RhoA to the cellular profibrotic pathways in normal (left) and cGK I-deficient (right) blood vessels.

Smooth muscle-targeted expression of RhoA^A188 augments vascular ROCK activity, hypertrophy, and fibrosis and imparts unresponsiveness to the inhibitory effect of BNP. NTg, RhoA^WT-Tg, RhoA^A188-Tg, BNP-Tg, RhoA^WT/BNP-Tg, and RhoA^A188/BNP-Tg mice were compared for analysis. (A) (Left) Bar graphs showing ROCK activity in the aortas (n = 3). (Right) Fraction of incremental ROCK activity in RhoA^WT-Tg and RhoA^A188-Tg aortas over NTg aortas that is inhibitable by crossbreeding with BNP-Tg mice. The original blot image is found in Fig. S2B in the supplemental material. (B) Systolic BPs (sBP) of mice (n = 5 to 11). (C and D) Morphological assessment of coronary arteries (n = 25 to 53). (C) Representative Sirius red staining of heart sections. Bar, 50 μm. (D) (Left) Morphometric analysis of coronary artery MT (top) and PVF (bottom). The MT and PVF were determined as described in the legend for Fig. 2. (Right) Fraction of incremental MT (top) and PVF (bottom) of RhoA^WT-Tg or RhoA^A188-Tg coronary arteries over NTg coronary arteries that is inhibitable by crossbreeding with BNP-Tg mice. In panels A and D, the BNP inhibitability of the myc-RhoA-induced increase in the indicated parameter was corrected for the baseline effect of BNP by the following formula: BNP inhibitability (%) = [(RhoA^x-Tg value − RhoA^x/BNP-Tg value) − (NTg value − BNP-Tg value)]/(RhoA^x-Tg value − NTg value), where x is WT or A188. Values are means ± SEM. *, P < 0.05; **, P < 0.01; #, P < 0.001; NS, not significant.

Inverse correlation between RhoA serine 188 phosphorylation and ROCK activity in mouse blood vessels. (A) Specificity of P-RhoA antibody (Ser188). Recombinant RhoA protein was phosphorylated or left unphosphorylated by cGK Iα and immunoblotted with P-RhoA antibody. (B) Immunoblot showing concurrent phosphorylation of RhoA Ser188 and VASP Ser237 in VSMCs treated with cGMP analogs. cGK I inh, a membrane-permeating cGK I inhibitory peptide (DT-3). (C to F) Immunoblotting analysis of cGK I^+/− and WT mouse aortas. (C) (Left) cGK I protein level. (Right) Levels of P-RhoA and RhoA in immunoprecipitates with RhoA antibody. (D) Level of P-moesin Thr558, without or with Ang II administration for 2 weeks. (E) ROCK protein level. (F) Level of membrane-associated and cytosolic RhoA separated by cellular fractionation. (G) Spearman's rank correlation test between the P-RhoA/RhoA and P-moesin/moesin ratios in cGK I^+/− and WT mouse aortas. (H and I) Immunoblotting analysis of aortas of BNP-Tg mice and NTg littermates. (H) (Top and middle) Levels of P-RhoA and RhoA in immunoprecipitates with RhoA antibody. IgGL, IgG light chain. (Bottom) cGK I protein level. (I) Level of P-moesin Thr558. Values are means ± SEM for three or four replicates. *, P < 0.05; **, P < 0.01.

Generation of transgenic mice that specifically express either WT or phosphorylation-resistant RhoA in arterial smooth muscle. (A) Transgene constructs for WT RhoA (RhoA^wt) and phosphorylation-resistant RhoA (RhoA^A188). Arrowheads, primer set used for genotyping and RT-PCR analysis in panel C; gray bar, SpeI-SpeI fragment recognized by the XbaI-HindIII probe (open bar) in the Southern blotting analysis in panel B. bGH, bovine growth hormone polyadenylation signal; 4F2, human 4F2 enhancer. (B) Genomic Southern blot analysis was performed with the F1 and F2 offspring of the RhoA^WT (line 39) and RhoA^A188 (line 30) transgenic mice. The transgenic vector (A) was used to quantify the copy number (first and second lanes). (C) Transgene mRNA expression was assessed by semiquantitative RT-PCR analysis of aortas between different founder lines (F2 offspring) of RhoA^WT-Tg and RhoA^A188-Tg mice. (D) Representative immunostaining with anti-myc-tag antibody showing transgene protein expression in NTg, RhoA^WT-Tg (line 39), and RhoA^A188-Tg (line 30) mouse heart and aorta sections (brownish signal). L, lumen. Bars, 50 μm. (E) Immunoblot with anti-RhoA and anti-myc-tag antibodies showing the ∼24-kDa transgene product expression in NTg, RhoA^WT-Tg, and RhoA^A188-Tg mouse aortas. Arrowhead (myc-RhoA), transgene product; arrow, endogenous RhoA. (F) Immunoblot showing the P-moesin level in NTg, RhoA^WT-Tg, and RhoA^A188-Tg mouse aorta lysates. Values are means ± SEM for triplicates. *, P < 0.05.