Wang Jiawen, Yuan Zhen, Yu Na, Jiao Qian, Zhou Honglei, Liao Wenjie, Shan Jiwei, Ruan Shanshan, Zhao Yi, Mo Ya, Qi Luyao, Li Tiejun, Fu Jianjun, Ke Bowen, Xu Yufang, Qian Xuhong, Zhang Jian, Zhao Zhenjiang, Li Shiliang, Wang Rui, Li Honglin
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science & Technology, Shanghai, China (J.W., Z.Y., N.Y., Q.J., H.Z., W.L., J.S., S.R., Y.M., L.Q., J.F., Y.X., X.Q., J.Z., Z.Z., S.L., R.W., H.L.).
Department of Anesthesiology, Laboratory of Anesthesia and Critical Care Medicine, National-Local Joint Engineering Research Centre of Translational Medicine of Anesthesiology, West China Hospital, Sichuan University, Chengdu, China (Y.Z., B.K.).
Circulation. 2025 Aug 26;152(8):519-536. doi: 10.1161/CIRCULATIONAHA.124.071884. Epub 2025 Jun 16.
The RhoA (Ras homolog family member A) signaling pathway is pivotal in regulating vascular smooth muscle cells (VSMCs) function and blood pressure homeostasis. Current inhibitors of the RhoA signaling pathway are limited in hypertension treatment, suffering from poor efficacy, insufficient specificity, and developmental challenges.
Cryo-electron microscopy (EM), proximity ligation assay (PLA), and site-directed mutagenesis were used to explore the mechanism of RhoA activity regulation. VSMC, hypertensive animal models, and Myh11-CRE (smooth muscle-specific RhoGDI1 knockout) mice were used to investigate the role of the TRPV4 (transient receptor potential cation channel subfamily V member 4)-RhoA-RhoGDI1 (Rho GDP dissociation inhibitor 1) axis in hypertension.
AH001 (()-1-(3-ethylphenyl) ethane-1,2-diol) was identified as a novel inhibitor of the RhoA signaling pathway. It targets the TRPV4-RhoA-RhoGDI1 axis to effectively sequester inactive RhoA-GDP in the plasma membrane and cytoplasm, which is distinct from typical RhoA inhibition modes. The cryo-EM structure of the TRPV4-RhoA complex showed that AH001-bound TRPV4 adopts a closed state with RhoA in an inactive GDP-bound state. Functional studies further revealed that AH001 reduced the pool of active RhoA by enhancing TRPV4-RhoA binding and facilitating RhoGDI1-RhoA interaction in VSMC. This inhibition notably decreased both acute and long-term blood pressure and prevented vascular remodeling in Ang II-induced hypertensive mice and spontaneously hypertensive rats. However, these antihypertensive effects were weakened in and Myh11-CRE mice. Additionally, AH001 effectively inhibited VSMC contraction via the RhoA/ROCK (Rho-associated protein kinase)/MYPT1 (myosin phosphatase target subunit 1)/MLC (myosin light chain 2) signaling pathway and suppressed VSMC phenotype switching to myofibroblasts through the RhoA/ROCK/LIMK1 (LIM domain kinase)/cofilin/MRTF-A (myocardin-related transcription factor A)/SRF (serum response factor) signaling cascade. TRPV4 and RhoGDI1 knockdown attenuated AH001's inhibition of VSMC contraction and phenotypic switching to myofibroblasts.
This study revealed a novel mode of RhoA signaling inhibition targeting the TRPV4-RhoA-RhoGDI1 axis, offering new insights for future antihypertensive drug development and proposing innovative strategies for targeting challenging Rho GTPases.
RhoA（Ras同源家族成员A）信号通路在调节血管平滑肌细胞（VSMC）功能和血压稳态中起关键作用。目前RhoA信号通路抑制剂在高血压治疗中存在局限性，疗效不佳、特异性不足且面临开发挑战。
采用冷冻电子显微镜（EM）、邻近连接分析（PLA）和定点诱变来探索RhoA活性调节机制。利用VSMC、高血压动物模型和Myh11-CRE（平滑肌特异性RhoGDI1基因敲除）小鼠来研究瞬时受体电位阳离子通道亚家族V成员4（TRPV4）-RhoA-RhoGDI1（Rho GDP解离抑制剂1）轴在高血压中的作用。
AH001（()-1-(3-乙基苯基)乙烷-1,2-二醇）被鉴定为一种新型RhoA信号通路抑制剂。它靶向TRPV4-RhoA-RhoGDI1轴，有效地将无活性的RhoA-GDP隔离在质膜和细胞质中，这与典型的RhoA抑制模式不同。TRPV4-RhoA复合物的冷冻电镜结构显示，结合AH001的TRPV4处于关闭状态，RhoA处于无活性的结合GDP状态。功能研究进一步表明AH001通过增强VSMC中TRPV4-RhoA结合并促进RhoGDI1-RhoA相互作用来减少活性RhoA池。这种抑制作用显著降低了急性和长期血压，并预防了血管紧张素II诱导的高血压小鼠和自发性高血压大鼠的血管重塑。然而，在Myh11-CRE小鼠中这些降压作用减弱。此外，AH001通过RhoA/ROCK（Rho相关蛋白激酶）/MYPT1（肌球蛋白磷酸酶靶亚基1）/MLC（肌球蛋白轻链2）信号通路有效抑制VSMC收缩，并通过RhoA/ROCK/LIMK1（LIM结构域激酶）/cofilin/MRTF-A（心肌相关转录因子A）/SRF（血清反应因子）信号级联抑制VSMC向肌成纤维细胞的表型转换。TRPV4和RhoGDI1基因敲低减弱了AH001对VSMC收缩和向肌成纤维细胞表型转换的抑制作用。
本研究揭示了一种靶向TRPV4-RhoA-RhoGDI1轴的新型RhoA信号抑制模式，为未来抗高血压药物开发提供了新见解，并为靶向具有挑战性的Rho GTPases提出了创新策略。
