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. 2023 Dec:6:100055.
doi: 10.1016/j.jmccpl.2023.100055. Epub 2023 Nov 19.

Angiotensin II induces endothelial dysfunction and vascular remodeling by downregulating TRPV4 channels

Narendra Babu Kondapalli  1 , Venkatesh Katari  1 , Kesha Dalal  1 , Sailaja Paruchuri  1 , Charles K Thodeti  1
Affiliations

Angiotensin II induces endothelial dysfunction and vascular remodeling by downregulating TRPV4 channels

Narendra Babu Kondapalli et al. J Mol Cell Cardiol Plus. 2023 Dec.

Abstract

Angiotensin II (Ang II) is a potent vasoconstrictor of vascular smooth muscle cells (VSMC) and is implicated in hypertension, but it's role in the regulation of endothelial function is not well known. We and others have previously shown that mechanically activated ion channel, Transient Receptor Potential Vanilloid 4 (TRPV4) mediates flow- and/or receptor-dependent vasodilation via nitric oxide (NO) production in endothelial cells. Ang II was demonstrated to crosstalk with TRPV4 via angiotensin 1 receptor (AT1R) and β-arrestin signaling in epithelial and immortalized cells, however, the role of this crosstalk in endothelial cell function is not fully explored. Ang II treatment significantly downregulated TRPV4 protein expression and TRPV4-mediated Ca2+ influx in human EC without altering TRPV4 mRNA levels. Further, TRPV4-induced eNOS phosphorylation and NO production were significantly reduced in Ang II-treated human EC. Importantly, Ang II infusion in mice revealed that, TRPV4/p-eNOS expression and colocalization was reduced in endothelium in vivo. Finally, Ang II infusion induced vascular remodeling as evidenced by decreased lumen to wall ratio in resistant mesenteric arteries. These findings suggest that Ang II induces endothelial dysfunction and vascular remodeling via downregulation of TRPV4/eNOS pathway and may contribute to hypertension, independent of or in addition to its effect on vascular smooth muscle contraction.

Keywords: Angiotensin II; Ca2+; Endothelial cells; TRPV4; Vascular remodeling; eNOS.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Angiotensin II treatment downregulates TRPV4 protein expression in HMECs. Cells were treated with Ang II (200 nM for 48 h) and the expression of TRPV4 was assessed using western blot and quantitative PCR (qPCR). A) Western blots showing TRPV4 protein expression. B) Relative TRPV4 expression levels in control and Ang II treated HMEC. GAPDH was used as loading control and for normalization (n = 4 biological replicates/group). C) qPCR analysis of TRPV4 mRNA levels in control and Ang II treated HMEC. (n = 5 biological replicates/group). All data points represented are the mean ± SEM, Statistical significance (unpaired t-test; nonparametric, Mann-Whitney test) is indicated by * p ≤ 0.05.
Fig. 2
Fig. 2
Angiotensin II treatment attenuates TRPV4 mediated calcium influx in HMECs. A) Representative fluorescence images of cytosolic Ca2+ influx in response to TRPV4 agonist GSK1016790A (GSK1) (n = 3 biological replicates/group, representative images are from ∼5 fields/group/experiment). (Magnification: 40×; Scale bar: 75 μm). B) Representative traces showing relative changes in cytosolic Ca2+ in response to GSK1 in the fluo-4/AM-loaded HMECs treated with (n = 3 biological replicates/group, data points represent an average of 128 cells/group) and without Ang II (200 nM/48 h) (n = 3 biological replicates/group, data points represent an average of 143 cells/group). The arrow represents the time when the cells were stimulated with GSK1. C) Quantitative analysis of cytosolic Ca2+ influx induced by GSK1 in HMEC (n = 3 biological replicates/group). All data points represented are the mean ± SEM. Statistical significance (unpaired t-test, nonparametric, Mann-Whitney test) is indicated by * p ≤ 0.01.
Fig. 3
Fig. 3
TRPV4 mediated eNOS phosphorylation is inhibited by Angiotensin II. HMEC were treated with Ang II and/or GSK1 and the expression of phospho-eNOS (S1177) was determined by immunofluorescence and western blot. A) Representative confocal fluorescence images of human EC stained with phospho-eNOS (Ser1177) (n = 3 biological replicates/group, representative images are from 5 fields/group/experiment). (Magnification: 40×, scale bar: 75 μm). B) Quantification of mean fluorescence intensity (MFI) of phospho-eNOS in HMEC (n = 3 biological replicates/group, data points represent average of 5 fields/group/experiment). C) Western blots showing phospho-eNOS expression (n = 3 biological replicates/group). D) Relative expression of phospho-eNOS. Total eNOS was used as a loading control and for normalization (n = 3 biological replicates/group). All data points represented are the mean ± SEM. ANOVA with Tukey's multiple comparisons test was performed. Statistical significance is indicated by * p ≤ 0.05, ** p ≤ 0.01.
Fig. 4
Fig. 4
TRPV4 mediated NO production is inhibited by Angiotensin II. HMEC were treated with Ang II and/or GSK1 and NO levels were determined by loading with DAF-FM. A) Representative images of HMEC showing DAF fluorescence indicative of NO levels. (n = 3 biological replicates/group, representative images are from 5 fields/group/experiment). (Magnification: 40×, scale bar: 50 μm). B) Quantification of DAF fluorescence in HMEC (n = 3 biological replicates/group, data points represent an average of 5 fields/group/experiment). All data points represented are the mean ± SEM from at least three independent experiments. ANOVA with Tukey's multiple comparisons test was performed. Statistical significance is indicated by * p ≤ 0.05.
Fig. 5
Fig. 5
Angiotensin II infusion reduces TRPV4/p-eNOS expression in aortic endothelium and vascular remodeling in mesenteric vessels in vivo. A) Representative confocal immunofluorescence images of aorta (cross-section) depicting the p-eNOS/TRPV4 staining in endotheium with and without Ang II infusion (Red: TRPV4; Green: p-eNOS) (n = 5 (control) and n = 3 (Ang II) biological replicates/group). (Magnification: 40×, scale bar: 75 μm). B) Representative images of mesenteric (cross-section) arteries showing the vessel diameter. C) Quantification of mean lumen/wall ratio in mesenteric vessels (n = 5 (control) and n = 6 (Ang II) biological replicates/ group). (Magnification: 20×; Scale bar:100um). All data points represented are the mean ± SEM. Statistical significance (unpaired t-test; nonparametric, Mann-Whitney test) is indicated by ** p ≤ 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Angiotensin II induces endothelial dysfunction via downregulation of TRPV4/eNOS/NO signaling axis. In normal endothelium, TRPV4 activation leads to calcium influx in EC which in turn results in the phosphorylation of eNOS (endothelial nitric oxide synthase) at S1177and NO (nitric oxide) production leading to vasodilation. However, infusion of Ang II in mice may cause internalization and downregulation of TRPV4 (via β-arrestin) and mediated calcium influx, eNOS phosphorylation and NO production resulting in decreased vasodilation (Created with Biorendor.com).
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