Harnessing homeostatically active RhoC at cell junctions preserves human endothelial barrier function during inflammation

Abstract

The GTPase RhoA plays a critical role in generating actomyosin-mediated contractile forces that cause endothelial hyperpermeability during systemic inflammatory diseases. RhoA is almost identical to RhoB and RhoC, all of which are molecular targets of specific bacterial toxins. Searching for new treatments to modulate Rho-dependent endothelial integrity, we demonstrate that the selective and simultaneous activation of these three Rho GTPases with a chimeric recombinant toxin does not induce cell contraction but enhances microvascular endothelial barrier function in response to pathological inflammatory challenges in vitro and in vivo. This pro-barrier effect is specifically mediated by RhoC, whose activity is increased by cell confluence. The uniqueness of RhoC relies on an arginine residue (R188) within its hypervariable region that determines its junctional localization, high homeostatic activity, and barrier-protective function, in contrast to the permeability-inducing, low homeostatic activity of RhoA and RhoB. Quantitative proteomics and high-resolution confocal microscopy revealed that RhoC regulates perijunctional actomyosin and the expression of myosin light chain proteins. Thus, harnessing the activity of RhoC represents a potential therapy for strengthening endothelial barriers during pathological inflammation.