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Ca2+-activated Cl− channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells
- Source :
- Journal of Advanced Research, Journal of Advanced Research, Vol 29, Iss, Pp 23-32 (2021)
- Publication Year :
- 2020
- Publisher :
- Elsevier, 2020.
-
Abstract
- Graphical abstract Cholesterol reduces TMEM16A expression via DNMT1-mediated promoter methylation and directly inhibits channel activities. TMEM16A inhibition promotes endothelial angiogenesis.<br />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.
- Subjects :
- 0301 basic medicine
Patch-Clamp Techniques
Angiogenesis
Endothelial cells
CCK-8, Cell Counting Kit-8
0302 clinical medicine
Cell Movement
5-aza, 5-Aza-2′-deoxycytidine
Endothelial dysfunction
Aorta
ANOVA, analysis of variance
Tube formation
lcsh:R5-920
Multidisciplinary
Neovascularization, Pathologic
Chemistry
Cell migration
Transfection
Cell biology
Endothelial stem cell
Cholesterol
Blood-Brain Barrier
030220 oncology & carcinogenesis
Hypertension
Medicine
lcsh:Medicine (General)
HEPES, N-2-hydroxyethil-piperazine-N'-2-ethanesulfonic acid
DMEM, Dulbecco’s Modified Eagle Medium
DNA (Cytosine-5-)-Methyltransferase 1
HAECs, human aortic endothelial cells
PVDF, polyvinylidene fluoride
RIPA, radio immunoprecipitation assay
03 medical and health sciences
EGTA, ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid
ROS, reactive oxygen species
Downregulation and upregulation
FBS, fetal bovine serum
Chloride Channels
medicine
DNMT1, DNA methyltransferase 1
NMDG, N-methyl-D-glucamine
Humans
lcsh:Science (General)
shRNAs, short hairpin RNAs
Anoctamin-1
Cell Proliferation
MβCD, methyl-β cyclodextrin
TMEM16A
SE, standard error
HEK 293 cells
medicine.disease
HEK293 Cells
030104 developmental biology
Calcium
CaCCs, Ca2+-activated Cl− currents
lcsh:Q1-390
Subjects
Details
- Language :
- English
- ISSN :
- 20901224 and 20901232
- Volume :
- 29
- Database :
- OpenAIRE
- Journal :
- Journal of Advanced Research
- Accession number :
- edsair.doi.dedup.....4c199e8fb29bfa734f960b1e2a8383f5