TRPC5 is essential in endothelium-dependent contraction of aorta from diet-induced obese mice

The role of the Ca2+-permeable ion channel TRPC5 in regulating vasocontraction in obesity is poorly understood. Here, we investigated whether TRPC5 contributes to vascular dysfunction in obesity by promoting endothelium-dependent contraction via activation of cytosolic phospholipase A2 (cPLA2) in the aortic endothelial cells of obese mice. Acetylcholine-induced endothelium-dependent relaxation and contraction in the aorta were measured using wire myography. PLA2 activity was measured by the fluorogenic PLA2 substrate Bis-BODIPY™ FL C11-PC. The intracellular Ca2+ level in response to acetylcholine was measured by Fluo-4 fluorescence. Endothelium-derived contracting factors were assessed by enzyme immunoassay. Diet-induced obesity (DIO) attenuated endothelium-dependent vasodilation, enhanced endothelium-dependent contraction (EDC), and increased the expression of TRPC5 in the mouse aorta. Activation of TRPC5 promoted EDC in the wild-type mouse aorta, whereas pharmacological inhibition and genetic knockout of TRPC5 decreased EDC in the DIO mouse aorta. Moreover, cPLA2 phosphorylation and activity were higher in aortic endothelial cells from DIO mice, and this was attenuated by inhibition and knockout of TRPC5. Cyclooxygenase 2 (COX-2) expression was increased in DIO mouse endothelium and was decreased by a TRPC5 inhibitor and knockout of TRPC5. Release of prostaglandins F2α (PGF2α) and E2 (PGE2) was involved in TRPC5-regulated EDC in DIO mice. This study demonstrated that TRPC5 contributes to endothelial and vascular dysfunction and is involved in EDC through activation of cPLA2 and enhanced COX-2-PGF2α/PGE2 levels in DIO mice.


Introduction
The mechanisms by which high-fat diet-induced obesity (DIO) increases the risk of cardiovascular disease are not completely understood.However, it is known that DIO is related to endothelial dysfunction and impaired endothelium-dependent vascular tone [1] which lead to the development of atherosclerosis [2] .Identifying the molecular mechanisms of obesity-induced endothelial dysfunction will help to establish new strategies for therapy.
Endothelium regulates vascular tone by keeping the balance between beneficial endothelium-derived relaxing factors (EDRFs) and harmful endothelium-derived contracting factors (EDCFs) in response to substances in the blood and the shear stress of blood flow [3] .Endotheliumdependent contraction (EDC) induced by EDCFs is usually augmented under disease conditions such as hypertension and hyperlipidemia [4] .EDC can be induced by acetylcholine (ACh) in arteries pre-treated with a nitric oxide synthase inhibitor due to extracellular Ca 2 + influx via nonselective cation channels [5] .The underlying mechanisms of intracellular Ca 2 + ([Ca 2 + ] i ) signal regulation associated with ACh need further study.EDCFs such as vasoconstrictor prostanoids cause EDC associated with an elevated endothelial [Ca 2 + ] i level [6] .The [Ca 2 + ] i rise triggers cytosolic phospholipase A2 (cPLA 2 ), which turns arachidonic acid into prostanoids via cyclooxygenase [6] .A recent study has shown the involvement of the transient receptor potential vanilloid 4 channel in EDCFs release in the hypertensive mouse aorta [3] .
Transient receptor potential channel canonical family member 5 (TRPC5) is a Ca 2 + -permeable channel expressed in the cardiovascular system that plays a key role in pressure sensing by aortic baroreceptors [7] .A previous study has shown that TRPC5 is able to regulate angiogenesis under hypoxia-ischemia [8] .Recent studies have indicated its involvement in endothelial cell senescence and carotid artery contraction under physiological conditions [ 9 , 10 ].However, it is not clear whether TRPC5 is involved in regulating EDC in the aorta of obese mice, and the underlying mechanisms remain elusive.
In the present study, we hypothesized that TRPC5 contributes to vascular dysfunction in obesity by promoting EDC.We demonstrated that TRPC5 enhances EDC in the DIO mouse aorta through the activation of cytosolic cPLA 2 in endothelial cells.

Animals
TRPC5-knockout (TRPC5 − / − ) and wild-type (WT) mice on the C57BL/6J background were a kind gift from Professor David E. Clapham (Harvard Medical School, USA) and maintained in Jiangnan University.All mice were kept at 22, 23 °C with a 12 h light/dark cycle.At the age of 6-8 weeks, TRPC5 − / − and WT mice were fed standard chow (normal-fat diet, NFD) or a high-fat diet (HFD, 45% kCal from fat; TP23200, Trophic Animal Feed High-tech Co., Ltd., Nantong, China) for > 3 months to develop DIO.Metabolic parameters, including body weight, fat pad mass, and blood glucose, as well as the lipid profile, were confirmed in HFD and NFD mice (Table S1).Age-and sex-matched TRPC5 − / − and WT littermates were selected by an established polymerase chain reaction genotyping method (Fig. S1) for this study.CO 2 inhalation was used for euthanasia.All animal experiments were performed in accordance with the guidelines issued by the National Institutes of Health, USA, and approved by the Animal Experiments Ethics Committee of Jiangnan University (approval number: JN. 20201115t0121231[298]).

Primary cells
Primary mouse aortic endothelial cells (MAoECs) from NFD and HFD mice were cultured in EC medium (ScienCell, Carlsbad, CA, USA).Confluent cells after < 3 passages were used.To obtain the cells, mice were euthanized with CO 2 , and the aorta was acutely dissected, placed in sterile PBS, and digested in 0.02% collagenase type 1A for 25 min at 37 °C.The digested tissue was centrifuged at 1200 g for 5 min and the pelleted cells were re-suspended and cultured at 37 °C with 5% CO 2 .Non-adherent cells were removed 2 h later.

Wire myography
The aorta was removed to cold Krebs solution, divided into 2-mm segments, and mounted in a myograph (model 610M, Danish Myo Tech-nology, Aarhus, Denmark).The chamber contained 95% O 2 and 5% CO 2 and was maintained at 37 °C.After equilibration at 0 mN for 30 min, a ring was stretched to an optimal baseline force of 3 mN for 30 min.KCl (60 mmol/L) was applied to confirm the contractility of the artery.For EDR measurement, aortic rings were pre-contracted with 1 mol/L phenylephrine (phe) for ∼5 min before exposure to progressively increasing concentrations of ACh.In EDC experiments, the segments were pre-incubated with L-NAME (100 mol/L) for 30 min before application of ACh.Aortas were pre-treated with AM237, clemizole (Aladdin), methylarachidonyl fluorophosphonate (MAFP, Cayman), NS-398 (Sigma), or VAS-2870 (Sigma) as necessary.AM237 efficiency was confirmed by patch clamp (Fig. S2) as previously described [11] .In some experiments, the endothelium was mechanically denuded by rubbing the lumen gently with a steel wire.Successful removal was confirmed by the lack of a relaxant response to bradykinin (100 nmol/L).

PLA 2 activity
A fluorogenic PLA 2 substrate (Bis-BODIPY TM FL C 11 -PC) (Thermo Fisher Scientific) was used for PLA 2 activity measurement in the en-face aorta.Aortas were incubated with Bis-BODIPY TM FL C 11 -PC in normal physiological saline containing (in mmol/L): 140 NaCl, 5 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, 5 HEPES, and 2% BSA (pH 7.4) for 20 min at 37 °C in the dark.An Andor confocal microscope (Dragonfly 200) was used to capture images with 480 nm excitation and 520 nm emission.The fluorescence intensity was analyzed by ImageJ.Aortas were pretreated with activator or inhibitor for 30 min if necessary, and then stimulated with ACh for 10 min.

Enzyme immunoassay (EIA)
Aortic rings were isolated from NFD and HFD mice and incubated with AM237, clemizole, MAFP, NS-398, or VAS-2870 as necessary.ACh (10 mol/L for 5-10 min) was used to stimulate the production of contraction factors.Then, the tissue was removed and the solution was centrifuged at 5000 rpm for 5 min at 4 °C.The solutions were frozen and stored at -80 °C until EIA assay.PGF 2  , 8-isoprostanes, PGE 2 , PGI 2 , and PGD 2 were measured using EIA kits (Meimian, China) according to the manufacturer's instructions.

Statistics
Data are represented as the mean ± standard error of the mean (SEM).Statistics were analyzed using GraphPad Prism 6.0 software.Concentration-response curves were constructed by two-way analysis of variance with repeated measures followed by the Bonferroni test.Comparisons between two groups were analyzed by Student's unpaired two-tailed t -test or the Mann-Wallis test.Differences among three or more groups were measured by one-way analysis of variance followed by Dunnett's or Tukey's multiple comparison test or Kruskal-Wallis and Dunn's post hoc non-parametric test.P -values < 0.05 were considered to be significantly different.

Role of diet-induced obesity in ACh-induced relaxation and contraction of mouse aorta
A wire myograph was used to measure vascular tone in the mouse aorta.In the phe-precontracted aorta, ACh induced a concentrationdependent relaxation in the range of 0.001-0.3mol/L in both NFD control and DIO mice in the absence of L-NAME.Further increases of ACh concentration from 1 to 100 mol/L led to contraction in the control and DIO mouse aorta ( Fig. 1 a).The magnitude of ACh-induced relaxation was significantly lower and ACh-induced contraction was stronger in the DIO mouse aorta than in the NFD control ( Fig. 1 a-c).These results suggested that DIO is associated with vascular dysfunction.

Activation of TRPC5 enhances endothelium-dependent vasoconstriction in mouse aorta
Western blot analysis and immunostaining were used to investigate the expression of TRPC5 in endothelial cells from the NFD control and DIO mouse aorta.As expected, TRPC5 was expressed in these cells.Moreover, the expression level of TRPC5 was much higher in DIO endothelial cells ( Fig. 2 a and b).To investigate whether TRPC5 is involved in the decreased vasodilation and enhanced vasocontraction, we assessed the response of vascular tone to AM237, a potent and selective TRPC5 activator [11] , by measuring ACh-induced concentrationdependent vasodilation and contraction in the NFD control mouse aorta preconstricted with phe.The results showed that ACh-induced relaxation from 0.001 to 0.3 mol/L did not change in aortas treated with 100 nmol/L AM237.However, further increases in ACh concentration induced contraction that was significantly enhanced in AM237treated NFD control aortas ( Fig. 2 c-e).We then investigated the effect of AM237 on ACh-induced vasoconstriction in the presence of L-NAME (100 mol/L) without phe pretreatment.In this case, the magnitude of ACh-induced contraction was significantly higher in the AM237treated intact aorta, while removal of the endothelium inhibited the ACh-induced contraction with or without AM237 ( Fig. 2 f-h).These results showed that TRPC5 activation increases ACh-induced EDC in the mouse aorta.

TRPC5 regulates endothelium-dependent contraction in aorta from obese mice
We used TRPC5 − / − mice and clemizole, a TRPC5 inhibitor, to study the role of TRPC5 in DIO mouse EDC.Wire myography was used to measure vascular tone.We found that ACh-induced contraction in the presence of L-NAME was attenuated by the inhibitor and by knockout of TRPC5 ( Fig. 3 a and b).As expected, removal of endothelium abolished the ACh-elicited contraction.These results demonstrated a vital role of TRPC5 in EDC of the obese mouse aorta.

Role of cPLA 2 activation in TRPC5-regulated contraction of DIO mouse aorta
To investigate the mechanism underlying TRPC5-regulated vasocontraction, we studied cytosolic phospholipase A2 (cPLA 2 ), a Ca 2 +sensitive vasocontractile agonist [3] .Western blot analysis and enzyme immunoassays showed that, compared to the NFD control, cPLA 2 phosphorylation and PLA 2 activity was increased in DIO MAoECs while the cPLA 2 level did not change ( Fig. 4 a and d).Importantly, AM237 treatment significantly increased cPLA 2 phosphorylation in a dose-dependent manner and enhanced PLA 2 activity but did not alter the expression of cPLA 2 in NFD control MAoECs ( Fig. 4 a, b  and d).As expected, both pharmacological inhibition of TRPC5 by clemizole and knockout of TRPC5 significantly attenuated the cPLA 2 phosphorylation and PLA 2 activity in DIO MAoECs ( Fig. 4 a and d).We also found that clemizole decreased cPLA 2 phosphorylation in a concentration-dependent manner ( Fig. 4 c).MAFP (10 mol/L), an inhibitor of cPLA 2 , significantly attenuated the ACh-induced vasocontraction in both the intact DIO and AM237-treated NFD control mouse aorta ( Fig. 4 e and f).
Notably, the Ca 2 + influx induced by 10 mol/L ACh was much higher in DIO MAoECs than in the NFD control ( Fig. 5 a and b).Moreover, AM237 treatment enhanced the ACh-induced Ca 2 + entry in NFD control MAoECs ( Fig. 5 a and b).As expected, the [Ca 2 + ] i was markedly reduced in clemizole-treated DIO and TRPC5 − / − DIO MAoECs ( Fig. 5 a  and b).
Together, these results demonstrated that TRPC5-regulated vascular contraction is associated with an intracellular Ca 2 + rise and cPLA 2 activation.

Role of COX-2 in TRPC5-regulated vasoconstriction in the DIO mouse
We then investigated the involvement of COX in the TRPC5-cPLA 2regulated vasocontraction in obesity ( Fig. 6 ).Immunoblot data showed that COX-2 expression was much higher in DIO MAoECs than in the NFD control while there was no difference of COX-1 expression between the two groups ( Fig. 6 a).Moreover, activation of TRPC5 by AM237 (100 nmol/L) markedly increased the COX-2 level in NFD control MAoECs ( Fig. 6 a).As expected, the TRPC5 inhibitor clemizole (20 mol/L) and knockout of TRPC5 attenuated COX-2 expression in DIO MAoECs ( Fig. 6 a).In contrast, the COX-1 level was similar in these groups ( Fig. 6 a).To identify the specific COX isoform involved Mean ± SEM; a, * P < 0.05 vs NFD, # P < 0.05 vs HFD, NS, no significant difference, Kruskal-Wallis and Dunn's post hoc non-parametric test (p-cPLA 2 ) and one-way ANOVA followed by Turkey's multiple comparisons test (cPLA 2 ); b, * P < 0.05, NS, no significant difference vs no AM237, one-way ANOVA followed by Dunnett's multiple comparisons test; c, * P < 0.05, NS, no significant difference vs no clemizole, one-way ANOVA followed by Dunnett's multiple comparisons test; d, * P < 0.05 vs NFD, # P < 0.05 vs HFD, one-way ANOVA followed by Turkey's multiple comparisons test; e, * P < 0.05 vs CTL, two-way ANOVA followed by Bonferroni test (left) and Student's unpaired two-tailed t test (right); f, * P < 0.05 vs CTL, # P < 0.05 vs AM237, two-way ANOVA followed by Bonferroni test (left) and one-way ANOVA followed by Turkey's multiple comparisons test (right).in TRPC5-regulated vascular contraction in obesity, we exposed aortas to the nonselective COX inhibitor indomethacin (1 mol/L), the COX-1 selective inhibitor VAS-2870 (30 mol/L), and the COX-2 selective inhibitor NS-398 (3 mol/L), before ACh stimulation.We found significant blockade of ACh-induced contraction in the indomethacin-and NS-398 treated DIO mouse aorta, while the COX-1 inhibitor VAS-2870 did not affect ACh-induced contraction ( Fig. 6 b).Similarly, indomethacin and NS-398 also inhibited the AM237 (100 nmol/L)-elevated contractile response to ACh (3-100 mol/L) in the NFD control mouse aorta ( Fig. 6 c).

PGF 2 𝛼 and PGE 2 as endothelium-derived contracting factors in DIO mouse
As we had demonstrated the involvement of COX-2 in the TRPC5regulated contractile response in DIO mice, we then investigated the involvement of EDCF candidates, the COX-2-derived prostanoid products.Five possible EDCF candidates, PGF 2  , PGE 2 , PGD 2 , PGI 2 , and 8isoprostanes were assayed using EIA kits.The ACh (10 mol/L)-induced release of PGF 2  and PGE 2 , but not PGD 2 , PGI 2 , and 8-isoprostanes was higher in the DIO mouse aorta than in the NFD control ( Fig. 7 a).Importantly, the enhanced production of PGF 2  and PGE 2 were inhibited by treatment with clemizole (20 mol/L), knockout of TRPC5, treatment with MAFP, NS-398, and the removal of endothelium in the DIO mouse aorta ( Fig. 7 b).Beside, the AM237-elevated ACh-induced PGF 2  and PGE 2 production was also markedly attenuated by treatment with MAFP, NS-398, and removal of endothelium in the NFD control mouse  (d) Schematic of the mechanism of TRPC5 regulation of endothelium-dependent contraction in the DIO mouse aorta.Mean ± SEM; a, * P < 0.05, NS, no significant difference vs NFD, Student's unpaired two-tailed t test; b, * P < 0.05 vs CTL, one-way ANOVA followed by Dunnett's multiple comparisons test; c, * P < 0.05 vs CTL, # P < 0.05 vs AM237 group, one-way ANOVA followed by Turkey's multiple comparisons test.aorta ( Fig. 7 c).In sum, these data suggested that PGF 2  and PGE 2 are involved in vascular contraction regulated by TRPC5-cPLA 2 in DIO mice.

Discussion
The main findings of the present study are as follows: (1) TRPC5 plays a critical role in endothelium-dependent vascular contraction in obese mice.(2) Activated cPLA 2 is involved in TRPC5-mediated contraction in the obese mouse aorta.(3) COX-2 contributes to the regulation of vascular contraction by TRPC5-cPLA 2 under obese conditions.(4) PGF 2  and PGE 2 are EDCFs released during vascular contraction regulated by TRPC5 under obesity.In sum, this study demonstrated a key role of the TRPC5-cPLA 2 -COX2-PGF 2  /PGE 2 pathway in vascular contraction under obese conditions ( Fig. 7 d).
Arterial endothelial cells are both the source and target of factors associated with cardiovascular disease, including obesity; they show an imbalance between beneficial EDRFs and harmful EDCFs [ 2 , 12 , 13 ].Although endothelial TRPC5 has been shown to contribute to contraction in mouse carotid artery [9] , its role in vascular tone control under pathophysiological conditions such as obesity remained elusive.Furthermore, the molecular mechanism of its contribution to obesity had not been determined.EDC plays a fundamental role in the maintenance and regulation of vascular tone and blood pressure [14] .Augmented EDC contributes to endothelial dysfunction and aggravates the progression of vascular diseases, including obesity [15] .An early signal in the EDC response is a cytosolic Ca 2 + rise in endothelial cells, and this stimulates the production of contractile prostanoids, leading to vascular contraction [16] .A recent study showed that direct activation of the Ca 2 + -permeable ion channel TRPV4 induces endothelial-dependent contraction in the hypertensive mouse aorta [3] .However, very little was known about the molecular identity of endothelial Ca 2 + entry channels that contribute to EDC in obesity.
The present study was designed to answer the following questions: (1) whether TRPC5 plays an essential role in the harmful effects of ED-CFs to override its beneficial effects in obesity; and (2) whether the inhibition or knockout of TRPC5 ameliorates EDC in obesity.Our data revealed that TRPC5 expression is increased in DIO MAoECs.Activation of TRPC5 by the potent and selective pharmacological agonist AM237 markedly enhanced ACh-induced EDC in the NFD control mouse aorta, while pharmacological inhibition and genetic knockout of TRPC5 sig-nificantly ameliorated EDC in the DIO mouse aorta.We demonstrated that increased TRPC5 expression in obesity augmented the EDC response, which further promoted endothelial dysfunction.TRPC5 knockout and inhibition alleviated the endothelial dysfunction induced by obesity.
It has been reported that the phosphorylation of Ca 2 + -dependent cPLA 2 is required for arachidonic acid release [ 17 , 18 ].Arachidonic acid is metabolized by COX-1 and -2 to prostaglandins that are vasoconstrictor metabolites in endothelial and vascular dysfunction [16] .Our study suggested that the balance between vasodilator and vasoconstrictor metabolites of arachidonic acid is disturbed in obesity and is regulated by increased TRPC5 and activated cPLA 2 .

Conclusion
In sum, this study demonstrated that TRPC5 plays a role in vascular dysfunction in obesity by enhancing EDC in the DIO mouse aorta via activation of cytosolic cPLA 2 in endothelial cells.We believe that this study establishes a generalized scheme for the role of different TRP channels in mediating EDC in different arteries in cardiovascular disease.Our study fills an important gap in the knowledge about EDC under disease conditions, and lays out a blueprint for the future development of TRPC5-based therapeutic options against obesity.

Declaration of Competing Interest
The authors declare that they have no conflicts of interest in this work.