Epoxycyclopentenone-Containing Oxidized Phospholipids Restore Endothelial Barrier Function via Cdc42 and Rac

Abstract

After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.

Prostaglandins PGE2 and PGI2 promote endothelial barrier enhancement via PKA- and Epac1/Rap1-dependent Rac activation

“…To deplete endogenous GEF-H1, Tiam1, Vav2, or Rac HPAEC were treated with gene-specific siRNA duplexes, as described elsewhere [13,[40][41][42]. To deplete endogenous Epac1 and Rap1, Stealth™ Select 3 RNAi (Homo sapiens) sets were used.…”

“…To deplete endogenous Epac1 and Rap1, Stealth™ Select 3 RNAi (Homo sapiens) sets were used. Pre-designed Stealth siRNAs of standard purity were ordered from Invitrogen (Carlsbad, CA), and transfection of EC with siRNA was performed as previously described [13,40]. Nonspecific, non-targeting siRNA was used as a control treatment.…”

“…Rac and Rap activation assays were performed using commercially available assay kits purchased from Upstate Biotechnology (Lake Placid, NY), as previously described [12,13]. In brief, cells were treated with vehicle or stimulated with PGE 2 or beraprost for various periods of time.…”

“…In addition, both barrier-protective and barrier-disruptive processes in EC are differentially regulated by small GTPases Rac and Rho, which induce distinct patterns of cytoskeletal and cell contact remodeling leading to EC barrier protection or compromise [9][10][11][12][13].…”

“…Rap1 activation induces localization of Rac-specific GEFs Tiam1 and Vav2 to the membrane areas at the cell periphery [35,37], which causes local activation of small GTPase Rac. In turn, activated Rac stimulates effector signaling kinase PAK1 as well as cytoskeletal and cell contact-associated downstream effectors and induces rearrangements of actin cytoskeleton, focal adhesions and adherens junctions, which determine increased EC monolayer barrier properties [7,10,13,38,39].…”