IL-33 via PKC mu/PRKD1 Mediated alpha-Catenin Phosphorylation Regulates Endothelial Cell-Barrier Integrity and Ischemia-Induced Vascular Leakage

Angiogenesis, neovascularization, and vascular remodeling are highly dynamic processes, where endothelial cell-cell adhesion within the vessel wall controls a range of physiological processes, such as growth, integrity, and barrier function. The cadherin-catenin adhesion complex is a key contributor to inner blood-retinal barrier (iBRB) integrity and dynamic cell movements. However, the pre-eminent role of cadherins and their associated catenins in iBRB structure and function is not fully understood. Using a murine model of oxygen-induced retinopathy (OIR) and human retinal microvascular endothelial cells (HRMVECs), we try to understand the significance of IL-33 on retinal endothelial barrier disruption, leading to abnormal angiogenesis and enhanced vascular permeability. Using electric cell-substrate impedance sensing (ECIS) analysis and FITC-dextran permeability assay, we observed that IL-33 at a 20 ng/mL concentration induced endothelial-barrier disruption in HRMVECs. The adherens junction (AJs) proteins play a prominent role in the selective diffusion of molecules from the blood to the retina and in maintaining retinal homeostasis. Therefore, we looked for the involvement of adherens junction proteins in IL-33-mediated endothelial dysfunction. We observed that IL-33 induces alpha-catenin phosphorylation at serine/threonine (Ser/Thr) residues in HRMVECs. Furthermore, mass-spectroscopy (MS) analysis revealed that IL-33 induces the phosphorylation of alpha-catenin at Thr(654) residue in HRMVECs. We also observed that PKC mu/PRKD1-p38 MAPK signaling regulates IL-33-induced alpha-catenin phosphorylation and retinal endothelial cell-barrier integrity. Our OIR studies revealed that genetic deletion of IL-33 resulted in reduced vascular leakage in the hypoxic retina. We also observed that the genetic deletion of IL-33 reduced OIR-induced PKC mu/PRKD1-p38 MAPK-alpha-catenin signaling in the hypoxic retina. Therefore, we conclude that IL-33-induced PKC mu/PRKD1-p38 MAPK-alpha-catenin signaling plays a significant role in endothelial permeability and iBRB integrity.