Frizzled-4 regulates β-catenin in endothelial cells exposed to disturbed flow via an atypical Wnt pathway leading to proinflammatory activation and increased permeability

Abstract

ObjectiveEndothelial cells are regulated by hemodynamic wall shear stress and multidirectional shear stress is known to promote endothelial dysfunction, although the molecular mechanisms are poorly defined. Wnt pathways play an important role in non-vascular mechanoresponsive cells. Here we investigated their role in endothelial mechanosignalling and endothelial dysfunction.Approach & ResultsHuman aortic endothelial cells were exposed to shear stress using an orbital shaker. The expression of Frizzled-4 receptors was significantly increased in endothelial cells exposed to low magnitude multidirectional flow (LMMF) relative to high magnitude uniaxial flow (HMUF). Increased expression was also detected in regions of the murine aortic arch exposed to LMMF. The increased Frizzled-4 expression in cultured cells was abrogated following knockdown of R-spondin-3 (RSPO-3) using RNA interference. LMMF also increased the stabilisation and nuclear localisation of β-catenin, an effect that was dependent on Frizzled-4 and RSPO-3. Inhibition of β-catenin using a small molecule inhibitor (iCRT5), or knockdown of Frizzled-4 or R-spondin-3 resulted in a significant reduction of pro-inflammatory gene expression in endothelial cells exposed to LMMF. Stabilisation of the β-catenin destruction complex using IWR-1 under LMMF also reduced pro-inflammatory gene expression, as did inhibition of Wnt5a signalling. Interestingly, inhibition of the canonical Wnt pathway had no effect. Inhibition of β-catenin signalling also reduced endothelial permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling.ConclusionsThese data suggest the presence of an atypical Wnt-β-catenin pathway in endothelial cells that promotes inflammatory activation and barrier disruption in response to LMMF.