WIF1 prevents Wnt5A mediated LIMK/CFL phosphorylation and adherens junction disruption in human vascular endothelial cells

Phosphorylation of LIMK/CFL were significantly increased in Wnt5A-treated HCAEC compared with non-treated cells (Fig. 1), thereby confirming previous findings [8]. Here, we further tested the antagonizing effects of WIF1 on Wnt5A-induced phosphorylation of LIMK/CFL. As shown herein by western blot analysis, combining Wnt5A with WIF1 significantly suppressed Wnt5A-triggered phosphorylation of both LIMK and CFL while combination with sFRP was not effective (Fig. 1). When activated by phosphorylation, LIMK phosphorylates the actin depolymerisation factor CFL1. Phosphorylation inactivates CFL1 and prevents its ability to depolymerise actin filaments leading to the formation of actin stress fibers. Increased stress fiber formation disrupts the AJ assembly at inter-endothelial junctions [12]. Here, we further investigated the antagonizing effects of WIF1 on Wnt5A-induced disruption of AJ. In non-treated cells, β-catenin and VE-cadherin were aligned at the cellular periphery forming intercellular boarders. Consistent with our previous finding [8], treatment with Wnt5A notably disrupted both β-catenin and VE-cadherin at intercellular boarders, forming large inter-endothelial gaps (Fig. 2, white arrow heads). Additionally, this study has shown that combining Wnt5A with WIF1 notably prevented Wnt5A-induced disassembly of both β-catenin and VE-cadherin while sFRP failed to exert any antagonistic effects (Fig. 2, Additional file 2: Figure S2A, B). This finding is supported by functional measurements of HCAEC monolayer permeability. Treatment with Wnt5A significantly decreased the resistance of HCAEC monolayers to alternating current, thereby indicating enhanced permeability of Wnt5A-treated HCAEC monolayers (Additional file 3: Figure S3), confirming previous findings [8]. Combining Wnt5A with WIF1 significantly prevented Wnt5A-induced endothelial hyperpermeability while no significant antagonistic effect was observed with sFRP1 (Additional file 3: Figure S3).

Ryk was previously identified as the Wnt5A receptor on HCAEC [8]. The current study demonstrates the potency of WIF1 to prevent Wnt5A-induced cytoskeleton remodelling and monolayer barrier dysfunction in human vascular endothelial cells. WIF1 is a 371 amino acid protein composed of a distinct WIF domain homologous to the Wnt binding WIF domain of the Ryk receptor, five EGF-like domains, and a hydrophilic tail [13]. Secreted Wnt antagonists including WIF1 and sFRP sequester Wnt proteins away from the receptors, thereby inhibiting signalling pathways [10, 13] (see Scheme, Fig. 3). It has been shown that the antagonistic effect of WIF1 is mainly mediated by its WIF domain. In an attempt to map the Wnt binding sites in WIF domain, previous studies revealed the presence of an alkyl-binding site that is capable of interacting with essential lipid groups of Wnts [14‐16]. Although WIF1 is expressed in different tissues, higher levels are reported in cartilage, lung, retina and brain [13, 17‐20]. Several studies link elevated expression level of Wnt5A to inflammatory response in sepsis and atherosclerosis [4‐6, 21]. Inflammatory activated leucocytes are the major source of Wnt5A [4, 7] and in this context, the observation that elevated Wnt5A levels correlate positively with the leucocyte count in sepsis is of particular interest [5]. The presence of lower levels of sFRP observed during worsening of disease in sepsis patients [5] is of further interest as various sFRP are shown to attenuate Fzd receptor-mediated inflammatory Wnt5A signalling in leucocytes [4, 7]. A previous study showed the upregulation of WIF1 mRNA in pro-inflammatory activated human monocytes [4]. Presently, it is not clear if monocytes release WIF1 to counter regulate the adverse effects of Wnt5A on VEC. In light of recent in vitro findings supporting an inhibitory effect of WIF1 on Wnt5A signalling in human VEC, the altered expression and antagonistic effects of WIF1 in diseases showing dysregulated Wnt5A signalling is worthy of future investigations.

In conclusion, this is the first report identifying antagonistic effects of WIF1 on Wnt5A mediated actin cytoskeleton signalling pathway in primary human vascular endothelial cells. Our data suggest that the Wnt5A pathway leading to barrier dysfunction of vascular endothelial cells is a target for the natural Wnt5A antagonist WIF1. This finding could offer novel therapeutic options for diseases associated with severe vascular leakage such as sepsis and septic shock.