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International audience; BACKGROUND: We have previously shown that angiotensin II type 2 receptor (AT(2)R) stimulation causes endothelium-dependent vasodilation that does not desensitize after chronic angiotensin II type 1 receptor (AT1R) blockade, suggesting a role for AT2R in antihypertensive treatment. METHODS AND RESULTS: We recorded mean arterial pressure (MAP) and investigated AT2R by Western blot analysis, immunohistochemistry, and function in isolated mesenteric resistance arteries (205 microm in diameter) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) receiving the following for 4 weeks in drinking water: placebo, AT1R blockade (candesartan; 2 mg/kg per day), ACE inhibitor (perindopril; 3 mg/kg per day), nonselective vasodilator (hydralazine; 16 or 24 mg/kg per day), or candesartan plus hydralazine (16 mg/kg per day). In precontracted isolated arteries, AT2R stimulation (angiotensin II in the presence of candesartan) caused vasodilation in WKY rats (MAP=118 mm Hg) and vasoconstriction in SHR (MAP=183 mm Hg). In SHR treated with candesartan (MAP=146 mm Hg) or hydralazine (16 mg/kg per day; MAP=145 mm Hg), AT2R-induced contraction was reduced by 50%. In SHR treated with perindopril (MAP=125 mm Hg), AT2R stimulation induced vasodilation. In SHR treated with hydralazine (24 mg/kg per day; MAP=105 mm Hg) and in SHR treated with hydralazine (16 mg/kg per day) plus candesartan (MAP=102 mm Hg), an AT2R-mediated vasodilation was restored. Immunochemistry and Western blot analysis showed that AT2R expression, lower in SHR than in WKY rats, was restored to normal levels by treatments reducing arterial pressure in SHR. CONCLUSIONS: Our results suggest that in resistance arteries of SHR, (1) AT2R is downregulated by hypertension, and (2) specific and nonspecific antihypertensive treatments restore AT(2)R expression and vasodilator functions.

International audience; BACKGROUND: We have previously shown that angiotensin II type 2 receptor (AT(2)R) stimulation causes endothelium-dependent vasodilation that does not desensitize after chronic angiotensin II type 1 receptor (AT1R) blockade, suggesting a role for AT2R in antihypertensive treatment. METHODS AND RESULTS: We recorded mean arterial pressure (MAP) and investigated AT2R by Western blot analysis, immunohistochemistry, and function in isolated mesenteric resistance arteries (205 microm in diameter) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) receiving the following for 4 weeks in drinking water: placebo, AT1R blockade (candesartan; 2 mg/kg per day), ACE inhibitor (perindopril; 3 mg/kg per day), nonselective vasodilator (hydralazine; 16 or 24 mg/kg per day), or candesartan plus hydralazine (16 mg/kg per day). In precontracted isolated arteries, AT2R stimulation (angiotensin II in the presence of candesartan) caused vasodilation in WKY rats (MAP=118 mm Hg) and vasoconstriction in SHR (MAP=183 mm Hg). In SHR treated with candesartan (MAP=146 mm Hg) or hydralazine (16 mg/kg per day; MAP=145 mm Hg), AT2R-induced contraction was reduced by 50%. In SHR treated with perindopril (MAP=125 mm Hg), AT2R stimulation induced vasodilation. In SHR treated with hydralazine (24 mg/kg per day; MAP=105 mm Hg) and in SHR treated with hydralazine (16 mg/kg per day) plus candesartan (MAP=102 mm Hg), an AT2R-mediated vasodilation was restored. Immunochemistry and Western blot analysis showed that AT2R expression, lower in SHR than in WKY rats, was restored to normal levels by treatments reducing arterial pressure in SHR. CONCLUSIONS: Our results suggest that in resistance arteries of SHR, (1) AT2R is downregulated by hypertension, and (2) specific and nonspecific antihypertensive treatments restore AT(2)R expression and vasodilator functions.

International audience; Myogenic tone (MT), a fundamental stretch-sensitive vasoconstrictor property of resistance arteries and veins, is a key determinant of local blood flow regulation. We evaluated the pathways involved in MT development. The role of the RhoA/Rho kinase, p38 MAP kinase, and HSP27 in MT was investigated in the rabbit facial vein (RFV), previously shown to possess MT at a pressure level equivalent to 20 mm Hg. Venous MT is poorly understood, although venous diseases affect a large proportion of the population. Stretched RFV are characterized by a temperature-sensitive MT, which is normal at 39 degrees C but fails to develop at 33 degrees C. This allows for the discrimination of the pathways involved in MT from the multiple pathways activated by stretch. Isolated RFV segments were mounted in organ baths and stretched. Temperature was then set at 33 degrees C or 39 degrees C. MT was associated to the translocation of RhoA to the plasma membrane and the Rho kinase inhibitor Y27632 decreased stretch-induced MT by 93.1+/-4.9%. MT was also associated to an increase in p38 (131.0+/-12.5% at 39 degrees C versus 100% at 33 degrees C) and HSP27 phosphorylation (196.1+/-13.3% versus 100%), and the p38 MAP kinase inhibitor SB203580 decreased MT by 36.5+/-8.1%. (39 degrees C, compared with RFV stretched at 33 degrees C). Finally, phosphorylation of p38 was blocked by Y27632 and HSP27 phosphorylation was inhibited by SB203580 and Y27632. Thus, MT and the associated p38 and HSP27 phosphorylation seem to depend on RhoA/Rho kinase activation in stretch RFV.

cAMP and cyclic GMP-dependent kinases (PKA and PKG) phosphorylate the small G protein RhoA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RhoA-dependent functions and positively regulates RhoA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase pathway plays an essential role, both in vitro and in vivo, in the regulation of RhoA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RhoA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RhoA-S188E) and active RhoA proteins (Q63L-RhoA-S188E), we show that phosphorylation of Ser188 of RhoA protects RhoA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RhoA to proteasome-mediated degradation is because of its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin, induce nonadditive increases in RhoA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RhoA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RhoA level and RhoA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-ω -nitro-l-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RhoA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.

Background and Purpose— CADASIL is an inherited small-vessel disease responsible for lacunar strokes and cognitive impairment. The disease is caused by highly stereotyped mutations in Notch3 , the expression of which is highly restricted to vascular smooth muscle cells (VSMCs). The underlying vasculopathy is characterized by degeneration of VSMCs and the accumulation of granular osmiophilic material (GOM) and Notch3 protein within the cell surface of these cells. In this study, we assessed early functional changes related to the expression of mutant Notch3 in resistance arteries. Methods— Vasomotor function was examined in vitro in arteries from transgenic mice that express a mutant Notch3 in VSMC. Tail artery segments from transgenic and normal wild-type male mice were mounted on small-vessel arteriographs, and reactivity to mechanical (flow and pressure) forces and pharmacological stimuli were determined. Mice were studied at 10 to 11 months of age when VSMC degeneration, GOM deposits, and Notch3 accumulation were not yet present. Results— Passive arterial diameter, contraction to phenylephrine, and endothelium-dependent relaxation to acetylcholine were unaffected in transgenic mice. By contrast, flow-induced dilation was significantly decreased and pressure-induced myogenic tone significantly increased in arteries from transgenic mice compared with wild-type mice. Conclusions— This is the first study to our knowledge providing evidence that mutant Notch3 impairs selectively the response of resistance arteries to flow and pressure. The data suggest an early role of vascular dysfunction in the pathogenic process of the disease.