TMEM215 Prevents Endothelial Cell Apoptosis in Vessel Regression by Blunting BIK-Regulated ER-to-Mitochondrial Ca Influx.

Abstract

In developmental and pathological tissues, nascent vessel networks generated by angiogenesis require further pruning/regression to delete nonfunctional endothelial cells (ECs) by apoptosis and migration. Mechanisms underlying EC apoptosis during vessel pruning remain elusive. TMEM215 (transmembrane protein 215) is an endoplasmic reticulum-located, 2-pass transmembrane protein. We have previously demonstrated that TMEM215 knockdown in ECs leads to cell death, but its physiological function and mechanism are unclear. We characterized the role and mechanism of TMEM215 in EC apoptosis using human umbilical vein endothelial cells by identifying its interacting proteins with immunoprecipitation-mass spectrometry. The physiological function of TMEM215 in ECs was assessed by establishing a conditional knockout mouse strain. The role of TMEM215 in pathological angiogenesis was evaluated by tumor and choroidal neovascularization models. We also tried to evaluate its translational value by delivering a <i>Tmem215</i> small interfering RNA (siRNA) using nanoparticles in vivo. <i>TMEM215</i> knockdown in ECs induced apoptotic cell death. We identified the chaperone BiP as a binding partner of TMEM215, and TMEM215 forms a complex with and facilitates the interaction of BiP (binding immunoglobin protein) with the BH (BCL-2 [B-cell lymphoma 2] homology) 3-only proapoptotic protein BIK (BCL-2 interacting killer). <i>TMEM215</i> knockdown triggered apoptosis in a BIK-dependent way and was abrogated by BCL-2. Notably, <i>TMEM215</i> knockdown increased the number and diminished the distance of mitochondria-associated endoplasmic reticulum membranes and increased mitochondrial calcium influx. Inhibiting mitochondrial calcium influx by blocking the IP₃R (inositol 1,4,5-trisphosphate receptor) or MCU (mitochondrial calcium uniporter) abrogated <i>TMEM215</i> knockdown-induced apoptosis. <i>TMEM215</i> expression in ECs was induced by physiological laminar shear stress via <i>EZH2</i> downregulation. In EC-specific <i>Tmem215</i> knockout mice, induced <i>Tmem215</i> depletion impaired the regression of retinal vasculature characterized by reduced vessel density, increased empty basement membrane sleeves, and increased EC apoptosis. Moreover, EC-specific <i>Tmem215</i> ablation inhibited tumor growth with disrupted vasculature. However, <i>Tmem215</i> ablation in adult mice attenuated lung metastasis, consistent with reduced <i>Vcam1</i> expression. Administration of nanoparticles carrying <i>Tmem215</i> siRNA also inhibited tumor growth and choroidal neovascularization injury. <i>TMEM215</i>, which is induced by blood flow-derived shear stress via downregulating <i>EZH2</i>, protects ECs from BIK-triggered mitochondrial apoptosis mediated by calcium influx through mitochondria-associated ER membranes during vessel pruning, thus providing a novel target for antiangiogenic therapy.