Downregulation of mitofusin2 disrupts vascular endothelial barrier function by modulating connexin43 redistribution

Abstract Vascular leakage is a hallmark pathological feature of acute lung injury and sepsis. Elucidating the underlying mechanisms governing vascular leakage and identifying novel therapeutic targets are of critical importance. To investigate the role of mitofusin2(mfn2) in sepsis-associated vascular leakage, a cecal ligation and puncture -induced septic rat model, and vascular endothelial-specific mfn2 knockdown or overexpression mouse models was used. Microcirculation microscopy was used to assess vascular leakage. Confocal microscopy and coimmunoprecipitation were used to analyze protein‒protein interactions. Our findings revealed that mfn2 modulated vascular leakage through a nonmitochondrial pathway. Sepsis-induced downregulation of mfn2 led to the aberrant aggregation of connexin43 at the plasma membrane, which enhanced the interaction of Cx43 with Zonula Occludens-1 (ZO-1). This interaction disrupted the continuous distribution of ZO-1 and impaired the integration of claudin-5 and VE-cadherin into endothelial junctions, thereby compromising barrier integrity.α-Carboxyl terminus 1(α-CT1) blocked the Cx43-ZO-1 interaction, restoring ZO-1 continuity and the proper localization of claudin-5 and VE-cadherin. Concomitantly, α-CT1 enhanced the barrier function of human microvascular endothelial cell monolayers. Mechanistically, mfn2 downregulation increased Cx43 phosphorylation at Ser368 via the protein kinase C pathway and inhibited Rab5/Rab7-mediated endosomal activity; these two synergistic effects promoted Cx43 accumulation at the plasma membrane. Collectively, our results demonstrate that mfn2 regulates vascular leakage by modulating Cx43 redistribution. Plasma membrane aggregation of Cx43 increases its interaction with ZO-1, leading to dysfunction of tight junctions and adherens junctions, and ultimately exacerbating vascular leakage in sepsis.