Wnt5A/Ryk signaling critically affects barrier function in human vascular endothelial cells

Abstract

Satisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.

WNT Signaling in Cardiac and Vascular Disease

“…The observed enhanced endothelial permeability induced by WNT5A could translate into an early inflammatory stage of the endothelium. Moreover, recent studies focused on endothelial barrier dysfunction are pointing toward an important role of WNT5A in the regulation of endothelial permeability ( Skaria et al, 2016 , 2017 ).…”

The Protective Effect of Total Flavones from Rhododendron simsii Planch. on Myocardial Ischemia/Reperfusion Injury and Its Underlying Mechanism

“…ROCK is one of the best characterized effectors of small GTPase RhoA, which belongs to the AGC (protein kinases A, G, and C) family of serine/threonine protein kinases. As a major downstream effector of RhoA, ROCK promotes actin-myosin-mediated contractile force generation by phosphorylating a variety of downstream target proteins and thus plays an important role in many cardiovascular pathogeneses, such as arterial hypertension, atherosclerosis, heart attack, vascular remodeling, and myocardial hypertrophy [ 32 , 33 ]. ROCK has two isoforms, ROCK1 and ROCK2, which account for 65% overall identity and 92% identity in the kinase domain, respectively.…”

Discovery of a ROCK inhibitor, FPND, which prevents cerebral hemorrhage through maintaining vascular integrity by interference with VE-cadherin

“… 5 Activation of ROCK induces actin cytoskeletal rearrangement and disrupts VEC-mediated intercellular adhesion. 32 Our group has initiated a research program to identify new drug candidates targeting ROCK for the prevention of hemorrhagic stroke, which combined docking-based virtual screening with a zebrafish model. 15 , 16 We demonstrated that ROCK1 inhibitors effectively inhibited the phosphorylation of downstream targets in the ROCK signaling pathway in vitro , and protected against statin-induced cerebral hemorrhage in vivo .…”