Ca2+-activated Cl- channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells

Abstract

Introduction: Ca²⁺-activated Cl^- channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells.

Objective: This study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis.

Methods: Whole-cell patch clamp techniques were used to record Ca²⁺-activated Cl^- currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively.

Results: TMEM16A mediates the Ca²⁺-activated Cl^- channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC₅₀ of 0.1209 μM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased in vitro tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect.

Conclusion: This study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.

Keywords: 5-aza, 5-Aza-2′-deoxycytidine; ANOVA, analysis of variance; Angiogenesis; CCK-8, Cell Counting Kit-8; CaCCs, Ca2+-activated Cl− currents; Cholesterol; DMEM, Dulbecco’s Modified Eagle Medium; DNMT1, DNA methyltransferase 1; EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid; Endothelial cells; FBS, fetal bovine serum; HAECs, human aortic endothelial cells; HEPES, N-2-hydroxyethil-piperazine-N'-2-ethanesulfonic acid; MβCD, methyl-β cyclodextrin; NMDG, N-methyl-D-glucamine; PVDF, polyvinylidene fluoride; RIPA, radio immunoprecipitation assay; ROS, reactive oxygen species; SE, standard error; TMEM16A; shRNAs, short hairpin RNAs.