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.

中文翻译：

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Ca2+激活的Cl-通道TMEM16A被胆固醇抑制促进内皮细胞血管生成

介绍

Ca ²⁺激活的Cl ^-通道TMEM16A在内皮细胞中表达，并导致许多疾病，如高血压、血脑屏障功能障碍和肺动脉高压。目前尚不清楚 TMEM16A 是否调节内皮血管生成，内皮血管生成参与许多生理和病理过程。胆固醇调节包括 TMEM16A 在内的许多离子通道，而高胆固醇水平会导致内皮功能障碍。胆固醇是否调节内皮细胞中 TMEM16A 的表达和功能仍有待确定。

客观的

本研究旨在研究胆固醇是否调节内皮血管生成中的 TMEM16A 表达和功能。

方法

全细胞膜片钳技术用于记录用 TMEM16A 过表达质粒转染的人主动脉内皮细胞 (HAEC) 和 HEK293 细胞中Ca ²⁺激活的 Cl ^-电流。蛋白质印迹用于检查 TMEM16A 和 DNA 甲基转移酶 1 (DNMT1) 在 HAEC 中的表达。CCK-8试验、愈合试验和管形成试验分别用于测试内皮细胞增殖、迁移和血管生成。

结果

TMEM16A 介导 HAEC 中 Ca ²⁺激活的 Cl ^-通道。胆固醇处理通过上调 HAEC 中 DNMT1 来抑制 TMEM16A 表达，并且胆固醇对 TMEM16A 表达的抑制作用被 DNMT1 抑制剂 5-aza 阻断。此外，胆固醇的直接应用抑制了异源 HEK293 细胞中的 TMEM16A 电流，IC ₅₀为 0.1209 μM。同样，胆固醇直接抑制 HAEC 中的 TMEM16A 电流。此外，TMEM16A 敲低增加了体外管形成、细胞迁移和 HAEC 增殖，而 TMEM16A 过表达产生了相反的效果。

结论

这项研究揭示了胆固醇介导的 TMEM16A 抑制的新机制，胆固醇通过 DNMT1 介导的甲基化降低 TMEM16A 表达并直接抑制通道活性。TMEM16A 通道抑制促进内皮细胞血管生成。