Breslin Jerome W, Daines Dayle A, Doggett Travis M, Kurtz Kristine H, Souza-Smith Flavia M, Zhang Xun E, Wu Mack H, Yuan Sarah Y
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL
Department of Biological Sciences, Old Dominion University, Norfolk, VA.
J Am Heart Assoc. 2016 Apr 5;5(4):e003336. doi: 10.1161/JAHA.116.003336.
Microvascular leakage of plasma proteins is a hallmark of inflammation that leads to tissue dysfunction. There are no current therapeutic strategies to reduce microvascular permeability. The purpose of this study was to identify the role of Rnd3, an atypical Rho family GTPase, in the control of endothelial barrier integrity. The potential therapeutic benefit of Rnd3 protein delivery to ameliorate microvascular leakage was also investigated.
Using immunofluorescence microscopy, Rnd3 was observed primarily in cytoplasmic areas around the nuclei of human umbilical vein endothelial cells. Permeability to fluorescein isothiocyanate-albumin and transendothelial electrical resistance of human umbilical vein endothelial cell monolayers served as indices of barrier function, and RhoA, Rac1, and Cdc42 activities were determined using G-LISA assays. Overexpression of Rnd3 significantly reduced the magnitude of thrombin-induced barrier dysfunction, and abolished thrombin-induced Rac1 inactivation. Depleting Rnd3 expression with siRNA significantly extended the time course of thrombin-induced barrier dysfunction and Rac1 inactivation. Time-lapse microscopy of human umbilical vein endothelial cells expressing GFP-actin showed that co-expression of mCherry-Rnd3 attenuated thrombin-induced reductions in local lamellipodia that accompany endothelial barrier dysfunction. Lastly, a novel Rnd3 protein delivery method reduced microvascular leakage in a rat model of hemorrhagic shock and resuscitation, assessed by both intravital microscopic observation of extravasation of fluorescein isothiocyanate-albumin from the mesenteric microcirculation, and direct determination of solute permeability in intact isolated venules.
The data suggest that Rnd3 can shift the balance of RhoA and Rac1 signaling in endothelial cells. In addition, our findings suggest the therapeutic, anti-inflammatory potential of delivering Rnd3 to promote endothelial barrier recovery during inflammatory challenge.
血浆蛋白的微血管渗漏是炎症的一个标志，会导致组织功能障碍。目前尚无降低微血管通透性的治疗策略。本研究的目的是确定一种非典型Rho家族GTP酶Rnd3在控制内皮屏障完整性中的作用。还研究了递送Rnd3蛋白改善微血管渗漏的潜在治疗益处。
利用免疫荧光显微镜观察，发现Rnd3主要存在于人类脐静脉内皮细胞核周围的细胞质区域。用异硫氰酸荧光素标记的白蛋白的通透性和人脐静脉内皮细胞单层的跨内皮电阻作为屏障功能的指标，并用G-LISA分析测定RhoA、Rac1和Cdc42的活性。Rnd3的过表达显著降低了凝血酶诱导的屏障功能障碍的程度，并消除了凝血酶诱导的Rac1失活。用小干扰RNA耗尽Rnd3表达显著延长了凝血酶诱导的屏障功能障碍和Rac1失活的时间进程。对表达绿色荧光蛋白-肌动蛋白的人脐静脉内皮细胞进行延时显微镜观察显示，共表达mCherry-Rnd3可减弱凝血酶诱导的伴随内皮屏障功能障碍的局部片状伪足减少。最后，一种新的Rnd3蛋白递送方法减少了失血性休克和复苏大鼠模型中的微血管渗漏，这是通过对肠系膜微循环中异硫氰酸荧光素标记的白蛋白外渗进行活体显微镜观察以及直接测定完整分离小静脉中的溶质通透性来评估的。
数据表明Rnd3可以改变内皮细胞中RhoA和Rac1信号的平衡。此外，我们的研究结果表明，在炎症刺激期间递送Rnd3以促进内皮屏障恢复具有治疗、抗炎潜力。
