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

2.1. Cell Culture

Human microvascular endothelial cells (HMEC-1) were purchased from ATCC (Manassas, VA, United States) and were cultured as previously described (Guarino et al., 2019). Briefly, HMEC-1 were cultured in MCDB-131 media (Corning, United States), supplemented with 10 % FBS, 1 % penicillin-streptomycin, 1 % l-glutamine, 1 % hydrocortisone, and 10 ng/mL human EGF.

2.2. Western blot analysis

Cells were serum starved and treated with and without Ang II (200 nM) for 48 h. After, cells were lysed in RIPA buffer containing 1× protease and phosphatase inhibitor cocktails (Millipore Sigma and Roche, Basel, Switzerland). Lysates were loaded into precast polyacrylamide gels (Bio-Rad) for electrophoresis. Gels were transferred onto a PVDF membrane and blocked in 5 % milk powder in tris-buffered saline (TBS) with 0.1 % Tween-20. Membranes were incubated overnight at 4 °C with primary antibodies: TRPV4 (1:300; Alomone Labs, Jerusalem, Israel); eNOS-phospho Ser1177 (1:1000; BD Bioscience, New Jersey); eNOS (1:1000; Cell Signaling Technology, USA) and GAPDH (1:5000; Cell Signaling Technology, USA). After incubation, membranes were washed 3 times with 1× TBST for 5 min each, followed by 1 h incubation at room temperature in appropriate secondary antibody, anti-rabbit or anti-mouse (1:5000; Cell Signaling Technology, USA) conjugated with horseradish peroxidase (Cell Signaling Technology, USA). Signals were detected with western blot imager (Azure 500 Fluorescent Western Blot Imager, Dublin, United States), and developed with an immobilon ECL ultra-western HRP substrate (Darmstadt, Germany) or Azure biosystem chemiluminescent substrates (Dublin, CA, United States). Quantification was performed using Image J software (NIH).

2.3. Quantitative PCR (qPCR)

RNA was extracted from HMEC-1 treated with and without Ang II (200 nM/48 h) by using the RNeasy mini kit (Qiagen, Hilden, Germany) and quantified with a BioTek micro plate reader. Revert aid first strand cDNA synthesis kit (Thermo Fisher Scientific, USA) was used to synthesize cDNA, and fast SYBR green master mix (Thermo Fisher Scientific, USA) was used for qPCR on Real-Time PCR Systems (Applied Biosystems, StepOnePlus). The following real-time primers were obtained from Integrated DNA Technologies (Coralville, IA, United States): hGAPDH (forward-5′-TGC ACC ACC AAC TGC TTA GC-3′, reverse: 5′-GGC ATG GAC TGT GGT CAT GAG −3′) and hTRPV4 (forward-5′-TCA CTC TCA CCG CCT ACT ACC A-3′, reverse: 5′-CCC AGT GAA GAG CGT AAT GAC C-3′). Gene expression was normalized to GAPDH and ΔΔCt values were expressed as a fold change.

2.4. Calcium imaging

HMEC-1 were cultured as described previously, on MatTek glass bottom dishes (MatTek, Ashland, MA, United States). After treatment with Ang II as described above, cells were loaded with Fluo-4/AM (4 μM) for 25 min and were washed in previously described Ca²⁺ media [8,14]. Ca²⁺ imaging was performed on an Olympus FluoView 300 microscope (Olympus, Shinjuku, Tokyo, Japan)/Leica SP5 confocal microscope after stimulation with the TRPV4 agonist, GSK1016790A (100 nM).

2.5. Immunofluorescence of phospho-endothelial nitric oxide synthase

HMEC-1 were cultured on glass cover slips in six-well plates until they were ∼ 70–80 % confluent. After this, the complete media was replaced with serum free media, left for overnight, followed by treatment with and without Ang II (200 nM for 48 h). Cells were then washed with PBS (3 times) and fixed for 20 min at room temperature in PBS containing 4 % paraformaldehyde. Cells were then rinsed and permeabilized with 0.25 % Triton X-100 in PBS and blocked with serum-containing media for 20 min. Afterwards, cells were incubated with the primary phospho-endothelial nitric oxide synthase (p-eNOS) antibody (1:100; BD Bioscience, New Jersey) at room temperature for 1 h. The cells were then washed with PBS and incubated with Alexa Fluor-conjugated secondary antibodies (1:500). Cells were mounted on glass slides using Fluoromount-G™ mounting medium containing DAPI (Invitrogen, USA). Images were obtained using multiphoton microscope (Leica Microsystems) with a 40× objective and processed using Image J software (NIH).

2.6. NO measurement

HMEC-1 were seeded on glass cover slips in six-well plates at the appropriate densities. At ∼70–80 % confluency, complete media was replaced with serum free media overnight, and cells were treated with and without Ang II (200 nM/48 h), followed by stimulation with known TRPV4 activator GSK-1016790A(100 nM/30 min) and incubation with 10 μM diaminofluorescein-FM diacetate (DAF-FM DA; NO-sensitive fluorescent dye) without phenol red at 37 °C in 5 % CO₂ for 30 min. Cells were then washed with PBS (3 times) and fixed for 20 min at room temperature in PBS containing 4 % paraformaldehyde. Cells were then washed with PBS (3 times) and mounted on glass slides using Fluoromount-G™ mounting medium containing DAPI (Invitrogen, USA). Images were obtained using multiphoton microscope (Leica Microsystems) with a 40× objective and processed using Image J software (NIH).

2.7. Animals and angiotensin II infusion

3–5 months old male mice (C57BL/6 J) were used in this study. All experiments were performed according to the guidelines and approval of the Institutional Animal Care and Use Committee of The University of Toledo. Mice were housed in a temperature-controlled room with a 12:12 h light-dark cycle and maintained with access to food and water ad libitum. Mice were divided into two groups, control (n = 8) and experimental (Ang II) (n = 8) group. Control mice received saline, while the experimental group received 0.2 μg/kg of Ang II daily by the intraperitoneal (i.p.) route for a period of 28 days. On day 29, mice were euthanized, aorta, and mesenteric arteries were collected.

2.8. Immunofluorescence

Aorta sections (5 μm) were incubated with the following primary antibodies: mouse anti-eNOS-phospho Ser1177 (1:200; BD Bioscience, New Jersey), mouse anti-CD31 (1:50; Invitrogen, Carlsbad, CA, USA), and anti-TRPV4 (1:50; Alomone Labs, Jerusalem, Israel). After the samples were washed with PBS, the tissue sections were incubated with appropriate secondary antibodies coupled to Alexa Fluor-488 or Alexa Fluor-594 (Invitrogen, USA) and mounted using Fluoromount-G™ mounting medium containing DAPI. Slides were imaged with 40× objective using Leica SP5 multiphoton microscope and processed using NIH ImageJ software. Paraffin sections of mesenteric arteries were exposed to antigen retrieval, stained with TRPV4 and CD31 antibodies and imaged as described above. For TRPV4 internalization, cells were transfected with TRPV4-EGFP [11], serum starved and stimulated with Ang II (200 nM/2 h), fixed and imaged with 63× objective using Leica SP5 multiphoton microscope and processed using NIH ImageJ software.

2.9. Mesenteric isolation and histological analysis

A second or third branch of the mesenteric arterial bed of 130 to 280 μm in lumen diameter and about 3 mm in length was carefully dissected out and was used for histological examination. Mesenteric artery morphometric analysis was performed with ImageJ software.

2.10. Statistical analysis

Statistical analyses were carried out using GraphPad Prism 8 statistical software. Analysis was performed using one-way ANOVA with Tukey's multiple comparisons test and student's t-test (unpaired, nonparametric) with Mann-Whitney test was used. All values reported are the Mean ± SEM. A value of *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, was considered significant.

3.1. Angiotensin II treatment downregulates TRPV4 protein expression and TRPV4- mediated calcium influx without altering TRPV4 mRNA levels in HMECs

To determine if Ang II cross-talks with TRPV4 in human EC, we first measured the TRPV4 protein expression after Ang II treatment (200 nM for 48 h). Western blot analysis revealed that Ang II treatment significantly (p ≤ 0.05) downregulated TRPV4 protein expression in human EC (Fig. 1A and B). Next, we measured the TRPV4 mRNA levels. qPCR analysis showed that there was no significant difference in TRPV4 mRNA in control and Ang II treated human EC (Fig. 1C). Further, we found that Ang II treatment induced internalization of TRPV4 as evidenced by disappearance of TRPV4-EGFP from the plasma membrane, with distinct puncta like appearance in the cell indicating targeting to intracellular vesicles (Supplemental Fig. 1).

Next, to determine the functional consequence of TRPV4 downregulation, we measured TRPV4-mediated Ca²⁺ influx in control and Ang II treated human EC. Stimulation with TRPV4 specific agonist, GSK-1016790A (GSK1; 100 nM) induced robust Ca²⁺ influx that was significantly (p ≤ 0.01) abolished when human EC were pretreated with Ang II (200 nM/48 h) (Fig. 2A-C), suggesting that Ang II treatment results in functional downregulation of TRPV4 in human EC.

3.2. TRPV4 mediated eNOS phosphorylation and NO production are inhibited by Angiotensin II

We next investigated if Ang II mediated TRPV4 downregulation modulates endothelial function. To achieve this, we measured eNOS phosphorylation as well as NO production in the human EC using phospho-specific antibodies against eNOS Ser1177 and DAF-FM DA, respectively. Immunofluorescence analysis revealed that cells stimulated with GSK1 showed robust staining of phospho-eNOS, which was inhibited by pretreatment of the cells with Ang II (Fig. 3A). Quantification of the fluorescence intensity revealed significant reduction in eNOS phosphorylation (Fig. 3B). Western blot analysis also showed increased phosphorylation of eNOS in response to GSK1, which was inhibited by Ang II treatment (Fig. 3C and D). We then measured NO generation, where cells stimulated with GSK1 showed significant NO production, which was inhibited by pretreatment of the cells with Ang II (Fig. 4A and B).

3.3. Angiotensin II infusion downregulates TRPV4/p-eNOS expression in endothelium in vivo and induces vascular remodeling

To investigate if Ang II induces endothelial dysfunction in vivo via downregulation of TRPV4/eNOS pathway, we infused Ang II for 28 days. Immunohistochemical analysis of aortic cross sections from vehicle-treated mice showed robust TRPV4 staining in the endothelium (red; Fig. 5A) which is co-localized (yellow) with p-eNOS (green Fig. 5A). As a control, we found that TRPV4 was co-localized with endothelial marker CD31 (Supplemental Fig. 2A). In contrast, TRPV4 and p-eNOS expression and co-localization was significantly attenuated in the aorta of Ang II-infused mice (green; Fig. 5A). Similarly, we found co-localization of TRPV4 with CD31 in the endothelium of control mesenteric arteries which was significantly reduced in Ang II-treated arteries (Supplemental Fig. 2B).

Finally, to determine the pathophysiological significance of TRPV4/eNOS/NO downregulation by Ang II, we measured lumen to wall ratio in histological sections of mesenteric arteries. We found that lumen to wall area ratio was significantly reduced in mesenteric arteries of Ang II-infused mice compared to vehicle controls (Fig. 5B and C).

4.1. Limitations

The major limitation of this study is that blood pressure was not measured before or after Ang II infusion in mice. However, we do not expect any change in the blood pressure as TRPV4 appears to have contrasting roles in endothelium and VSMC [23]. We used only male mice for the remodeling studies which needs to be investigated in female mice to confirm if there is sex/gender difference in Ang II induced endothelial dysfunction and vascular remodeling.

4.2. Perspectives

Ang II has been implicated as a designated vasoconstrictor majorly through its action on VSMC via Gq/Rho/Rho kinase pathway. Although Ang II was demonstrated to inhibit TRPV4-dependent vasodilation in cerebral parenchymal arteries and mesenteric arteries, the molecular mechanisms are not known. Our findings that Ang II induce endothelial dysfunction and vascular remodeling via downregulation of TRPV4 and dependent eNOS/NO pathway uncovered hitherto VSMC independent role for Ang II in the regulation of vascular tone. This, coupled with the recent findings that TRPV4 activation inhibits Ang II signaling [7], indicates that TRPV4 acts as an endogenous regulator of Ang II and identifies Ang II-TRPV4 crosstalk as a novel target for the treatment of hypertension.