Rnd3 as a Novel Target to Ameliorate Microvascular Leakage

Abstract

BackgroundMicrovascular 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.Methods and ResultsUsing 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.ConclusionsThe 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.

Sphingosine‐1‐phosphate protects against brain microvascular endothelial junctional protein disorganization and barrier dysfunction caused by alcohol

“…We assessed endothelial barrier function using two distinct methods. The first was to determine TER of confluent HBMEC monolayers using the ECIS system (Applied Biophysics, Troy, NY, USA) as previously described . The advantages of this method are its sensitivity to detect small changes in barrier function due to nanometer‐scale changes in the junctions between cells, and the ability to make moment‐to‐moment measurements, typically every 1‐10 seconds apart .…”

“…The second method we used to evaluate barrier function was to determine the apparent solute permeability ( P solute ) of HBMEC monolayers to molecules of different sizes, as previously described . Briefly, HBMEC were seeded on the upper chamber of gelatin‐coated transwell membranes (0.4 μm; Corning, Corning, MA), and allowed to grow until 3 days post‐confluence so that they formed mature junctions.…”

“…The first was to determine TER of confluent HBMEC monolayers using the ECIS system (Applied Biophysics, Troy, NY, USA) as previously described. 24,25 The advantages of this method are its sensitivity to detect small changes in barrier function due to nanometerscale changes in the junctions between cells, and the ability to make moment-to-moment measurements, typically every 1-10 seconds apart. 26 Cells were subcultured onto gelatin-coated 8W1E ECIS electrode arrays and grown until 3 days post-confluence, allowing for junctional maturation.…”

Endothelial Protrusions in Junctional Integrity and Barrier Function

“…These include myosin light chain (MLC) kinase (MLCK) and RhoA/ROCK signaling, which control myosin activity (Moy et al , 1993; Sheldon et al , 1993; Garcia et al , 1995; Verin et al , 1995; Yuan et al , 1997; Garcia et al , 1999; Tinsley et al , 2000; Yuan et al , 2002; Huang et al , 2003; Birukova et al , 2004; Breslin & Yuan, 2004; Tinsley et al , 2004b; Breslin et al , 2006; Reynoso et al , 2007; Kumar et al , 2009; Dudek et al , 2010; Beard et al , 2014). The balance of activities among members of the Rho family of small GTPases such as RhoA, Rac1, and Cdc42 have also been shown to be of importance for both disruption and stabilization of the endothelial barrier (Waschke et al , 2004a; Waschke et al , 2004b; Waschke et al , 2006; Baumer et al , 2008a; Schlegel et al , 2009; Schlegel & Waschke, 2009; Spindler et al , 2010; Breslin et al , 2015; Breslin et al , 2016; Zhang et al , 2016). In addition, there is increasing evidence that in addition to phosphorylation, other posttranslational modifications such as S-nitrosation of junctional proteins (Marin et al , 2012; Sánchez et al , 2013; Guequén et al , 2016), or specific palmitoylation of PKCβ by palmitoyl acyltransferase DHHC21 (Beard et al , 2016) can lead to elevated microvascular leakage.…”

“…S1P, which enhances endothelial barrier function, elicited an increase in lamellipodia protrusion frequency (Breslin et al , 2015). Overexpression of the atypical Rho GTPase Rnd3 decreased local lamellipodia protrusion frequency in association with its ability to attenuate increases in endothelial permeability (Breslin et al , 2016). In addition to this correlative data, the impact of (−)blebbistatin on endothelial barrier function was tested because it specifically inhibits local lamellipodia formation without affecting actin stress fibers.…”