TMEM16B mediates angiogenesis through TRPV4/VEGFA pathway in rat cerebral ischemia-reperfusion injury

Ischemic stroke remains a leading cause of mortality and long-term disability worldwide. Angiogenesis serves as a core mechanism underlying neurovascular protection following cerebral ischemia, highlighting the urgent need to identify key regulators of post-ischemic angiogenesis. TMEM16B, a calcium-activated chloride channel abundantly expressed in vascular endothelial cells, is functionally implicated in angiogenic regulation. This study aimed to investigate the role of TMEM16B in angiogenesis after cerebral ischemia-reperfusion injury and further elucidate its functional linkage with the TRPV4/VEGFA signaling pathway. A rat model of middle cerebral artery occlusion followed by reperfusion (MCAO/R) was established. Two independent sets of in vivo experiments were performed. First, adeno-associated virus (AAV) transduction was conducted 14 days prior to MCAO induction to achieve TMEM16B overexpression or knockdown. Second, the selective TRPV4 inhibitor HC067047 was administered 2 h before model induction, with or without concurrent TMEM16B overexpression. Neurological function was assessed using a battery of behavioral tests at multiple postoperative time points. The expression profiles of TMEM16B, TRPV4, VEGFA, and CD34 were determined via immunohistochemistry, western blot, and quantitative real-time PCR (qPCR). Additionally, transmission electron microscopy (TEM) was used to examine the ultrastructural morphology and injury of vascular endothelial cells within ischemic brain tissue. TMEM16B overexpression significantly improved neurological function, upregulated TRPV4, VEGFA, and CD34 expression, increased microvessel density, and attenuated vascular endothelial damage in MCAO/R rats. In contrast, TMEM16B knockdown aggravated neurological deficits and suppressed endogenous angiogenic responses. Pharmacological inhibition of TRPV4 exacerbated cerebral ischemic injury and partially abrogated the pro-angiogenic and neuroprotective effects conferred by TMEM16B overexpression. TMEM16B upregulation promotes angiogenesis and ameliorates neurological deficits following cerebral ischemia-reperfusion injury in rats by activating the TRPV4/VEGFA signaling pathway. These findings identify TMEM16B as a promising therapeutic target for ischemic stroke.