ELUCIDATING THE ROLE OF R-RAS IN REGULATING ENDOTHELIAL BARRIER FUNCTION DURING SEPSIS PROGRESSION

Abstract

Increased vascular permeability is a driving factor in the progression of sepsis to its more severe form, septic shock. Sepsis-induced vascular dysfunction and hyperpermeability lead to edema, organ failure and often death, as sepsis is the leading cause of death in ICUs globally. Currently, there are no sepsis-specific therapies and medical treatment remains supportive in the form of antibiotics, vasopressors, and fluid resuscitation. We have previously shown that the small GTPase R-Ras, in its active form, is a key mediator of endothelial barrier function and that pharmacological inhibition or siRNA knockdown of R-Ras greatly increases permeability through an increase in Src kinase and vascular endothelial (VE)-cadherin tyrosine phosphorylation. Here, we investigated whether R-Ras signaling was affected by tumor necrosis factor alpha (TNF-alpha), the main permeability-inducing cytokine in sepsis, to drive vessel leakiness. Using a microfluidic 2D endothelial barrier model that incorporates the hemodynamic force of fluid shear stress (FSS), we found that TNF-alpha inactivates R-Ras to drive vessel permeability through an increase in Src, focal adhesion kinase (FAK), and VE-cadherin signaling. Under FSS, expression of active R-Ras mutants blocked endothelial permeability induced by serum from sepsis patients or purified TNF-alpha, and re-expression of R-Ras in knockdown cells restored barrier function. Using molecular biology and immunofluorescence techniques such as GST pulldowns, protein phosphorylation analysis and proximity ligation assays, we found that R-Ras rapidly formed a complex with Src and FAK under FSS to modulate endothelial barrier function. Treatment with TNF-alpha increased the interaction between Src and FAK, and FAK and VE-cadherin to induce endothelial permeability through the phosphorylation and subsequent destabilization of VE-cadherin. Re-expression of active R-Ras blocked the ability of TNF-alpha to induce Src/FAK/VE-cadherin signaling. Intriguingly, we found that FSS significantly activated R-Ras, while TNF-alpha treatment blocked this activation. We next investigated which of the following components of flow onset was responsible for the activation of R-Ras: the mechanical force of fluid shear stress, a ligand-activated signaling pathway, or a combination of both. We found that sphingosine 1-phosphate (S1P), a potent endothelial barrier enhancer detected in abundance in the bloodstream, directly activated R-Ras to maintain barrier function and knockdown of R-Ras blocked the ability of S1P to enhance barrier function under FSS. Moreover, we found that this effect was mediated through S1P receptor 1 (S1PR1) as treatment with an S1PR1-specific antagonist abrogated the barrier enhancing effects of S1P under FSS. Finally, we investigated the effects of R-Ras in regulating sepsis-mediated permeability in more physiologically relevant models such as sepsis mouse models and 3D blood vessel organoids. Using a fecal-induced peritonitis (FIP) murine model, we found that R-Ras activity in the lung vasculature was significantly diminished in septic mice with increased TNF-alpha levels and vessel permeability. This finding indicates a direct role for R-Ras in sepsis progression. Moreover, we developed physiologically relevant blood vessel organoids with inducible expression of active and dominant negative forms of R-Ras to investigate the effects of R-Ras in regulating endothelial permeability in human-derived 3D cultures. We found that expression of active R-Ras blocked TNF-alpha-mediated endothelial junction permeability, while the expression of dominant negative R-Ras induced junctional permeability even in the absence of TNF-alpha. Taken together, this study demonstrates the importance of R-Ras regulation of vessel permeability in sepsis and may provide an opportunity for developing targeted therapies for endothelial barrier dysfunction.