Role Of RhoE/Rnd3 In Regulating Vasoconstriction Response Of Mice And Potential Mechanisms

Background:RhoE/Rnd3,a member of the small GTPase protein family,has been demonstrated involved in regulating the pathogenesis of various diseases,including cancer,neurodegenerative diseases,and cardiovascular diseases.The RhoA/ROCK signaling plays a key role in different pathophysiological processes,such as contraction,proliferation migration,and stiffness of vascular smooth muscle cells,which leads to increased peripheral vascular resistance and subsequently an increase in blood pressure.It has been proved that the activation of the RhoA/ROCK signaling pathway is significantly augmented in different kinds of hypertensive animal models and patients with hypertension.As an endogenous RhoA/ROCK inhibitor,RhoE can inhibit the activation of RhoA/ROCK signaling pathway via directly binding to ROCK1,or inhibiting the activation of RhoA.However,the role of RhoE in regulating vasoconstriction response of mice has not been explored so far.Objective:To investigate the role of RhoE on vascular function and arterial blood pressure and its underlying molecular mechanisms.Methods:The expression of RhoE in the thoracic aorta from hypertensive animal models and patients with hypertension was detected by immunohistochemistry.Smooth muscle cell-specific RhoE knockout(RhoE^flox/flox;SMMHC-Cre)mice were generated for the study.The RhoE^flox/flox mice were used as a control.Basal blood pressure was monitored using the radio telemetry method.Cardiac function and pulse wave velocity of the aorta were analyzed by echocardiography.HE staining was performed on thoracic aorta,carotid artery and mesenteric artery in each group.The vascular vasoconstriction of the aortic ring was analyzed by the Myograph experiment.KCl,PE,U46619,and 5-HT were added for evaluating vasoconstriction response,endothelium-dependent relaxation factor（Ach）and endothelium-independent relaxation factor（SNP）were added for evaluating vasodilatation response.Next,to identify whether VSMC-specific knockout RhoE can change acute pressor response,we monitored blood pressure under the stimulation of different dosages of PE（0.1μg/Kg,1μg/Kg,and 10μg/Kg）.The cell contraction analysis kit was used to assess the cell contraction response under the stimulation of PE with or without ROCK inhibitors.Results:1.The expression of RhoE in hypertensive animal models and hypertensive patients is lower than in the control mice of Non-hypertensive patients（p<0.05）.2.Deficiency of RhoE in VSMCs had no significant effect on cardiac function,arterial morphology,or blood pressure.However,aortic PWV of RhoE^flox/flox;SMMHC-Cre mice was significantly higher than that of control mice（224.5±98.6 vs 134.0±54.3 mm/s,p=0.009）.Myograph results showed an augmented vasoconstriction response to PE in RhoE^flox/flox;SMMHC-Cre（p<0.05）.Besides,the transient increase of blood pressure in RhoE^flox/flox;SMMHC-Cre mice induced by 3 different concentrations of PE is much higher than in control mice.The delta mean systolic blood pressure caused by stimulation of 0.1μg/Kg and 1μg/Kg had a significant difference compared with control mice（0.1μg/Kg:12.4±5.2 vs 4.6±2.9 m N,p=0.013;1μg/Kg:16.4±0.5 vs 6.7±2.5 m N,p=0.001;10μg/Kg:29.5±8.2vs 19.5±8.8 m N,p=0.062）.3.After being transfected with si RNA-RhoE,the contractility of human aortic smooth muscle cells was significantly increased,and the activity of ROCK was increased indicated by western blot data.Pre-treated with Y-27632 or Fasudil,the vasoconstriction response under PE stimulation was significantly inhibited（p<0.05）.（3）After 3 days injection of fasudil,the acute pressor response stimulated by PE was also inhibited.The increase of mean arterial pressure of RhoE^flox/flox;SMMHC-Cre mice was reversed.The absolute value of mean arterial pressure increment was no difference compared with the control group.Conclusion:The expression of RhoE in the aorta of hypertensive patients tends to be decreased compared with non-hypertensive patients.The deficiency of RhoE in vascular smooth muscle cells has no effects on basal blood pressure,however,it promotes the occurrence of aortic stiffness.RhoE regulates the contraction of the vascular smooth muscle cells and vascular function mainly by activating the RhoA/ROCK signaling pathway.