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NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels

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Abstract

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

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References

  1. Adapala RK, Kanugula AK, Paruchuri S, Chilian WM, Thodeti CK (2020) TRPV4 deletion protects heart from myocardial infarction-induced adverse remodeling via modulation of cardiac fibroblast differentiation. Basic Res Cardiol 115:14. https://doi.org/10.1007/s00395-020-0775-5

    Article  CAS  Google Scholar 

  2. Adapala RK, Talasila PK, Bratz IN, Zhang DX, Suzuki M, Meszaros JG, Thodeti CK (2011) PKCalpha mediates acetylcholine-induced activation of TRPV4-dependent calcium influx in endothelial cells. Am J Physiol-Heart Circul Physiol 301:H757-765. https://doi.org/10.1152/ajpheart.00142.2011

    Article  CAS  Google Scholar 

  3. Allaqaband H, Gutterman DD, Kadlec AO (2018) Physiological consequences of coronary arteriolar dysfunction and its influence on cardiovascular disease. Physiology 33:338–347. https://doi.org/10.1152/physiol.00019.2018

    Article  CAS  Google Scholar 

  4. Aoyama T, Paik YH, Watanabe S, Laleu B, Gaggini F, Fioraso-Cartier L, Molango S, Heitz F, Merlot C, Szyndralewiez C, Page P, Brenner DA (2012) Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent. Hepatology 56:2316–2327. https://doi.org/10.1002/hep.25938

    Article  CAS  Google Scholar 

  5. Bubolz AH, Mendoza SA, Zheng X, Zinkevich NS, Li R, Gutterman DD, Zhang DX (2012) Activation of endothelial TRPV4 channels mediates flow-induced dilation in human coronary arterioles: role of Ca2+ entry and mitochondrial ROS signaling. Am J Physiol-Heart Circul Physiol 302:H634-642. https://doi.org/10.1152/ajpheart.00717.2011

    Article  CAS  Google Scholar 

  6. Buus NH, Simonsen U, Pilegaard HK, Mulvany MJ (2000) Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in acetylcholine relaxation of isolated human small arteries. Br J Pharmacol 129:184–192. https://doi.org/10.1038/sj.bjp.0703041

    Article  CAS  Google Scholar 

  7. Cai H, Griendling KK, Harrison DG (2003) The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 24:471–478. https://doi.org/10.1016/S0165-6147(03)00233-5

    Article  CAS  Google Scholar 

  8. Camici PG, Crea F (2007) Coronary microvascular dysfunction. N Engl J Med 356:830–840. https://doi.org/10.1056/NEJMra061889

    Article  CAS  Google Scholar 

  9. Cao S, Anishkin A, Zinkevich NS, Nishijima Y, Korishettar A, Wang Z, Fang J, Wilcox DA, Zhang DX (2018) Transient receptor potential vanilloid 4 (TRPV4) activation by arachidonic acid requires protein kinase A-mediated phosphorylation. J Biol Chem 293:5307–5322. https://doi.org/10.1074/jbc.M117.811075

    Article  CAS  Google Scholar 

  10. Cenac N, Altier C, Motta JP, d’Aldebert E, Galeano S, Zamponi GW, Vergnolle N (2010) Potentiation of TRPV4 signalling by histamine and serotonin: an important mechanism for visceral hypersensitivity. Gut 59:481–488. https://doi.org/10.1136/gut.2009.192567

    Article  CAS  Google Scholar 

  11. Chidgey J, Fraser PA, Aaronson PI (2016) Reactive oxygen species facilitate the EDH response in arterioles by potentiating intracellular endothelial Ca(2+) release. Free Radic Biol Med 97:274–284. https://doi.org/10.1016/j.freeradbiomed.2016.06.010

    Article  CAS  Google Scholar 

  12. Craige SM, Chen K, Pei Y, Li C, Huang X, Chen C, Shibata R, Sato K, Walsh K, Keaney JF Jr (2011) NADPH oxidase 4 promotes endothelial angiogenesis through endothelial nitric oxide synthase activation. Circulation 124:731–740. https://doi.org/10.1161/CIRCULATIONAHA.111.030775

    Article  CAS  Google Scholar 

  13. Djordjevic T, Pogrebniak A, BelAiba RS, Bonello S, Wotzlaw C, Acker H, Hess J, Gorlach A (2005) The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells. Free Radic Biol Med 38:616–630. https://doi.org/10.1016/j.freeradbiomed.2004.09.036

    Article  CAS  Google Scholar 

  14. Edwards DH, Li Y, Griffith TM (2008) Hydrogen peroxide potentiates the EDHF phenomenon by promoting endothelial Ca2+ mobilization. Arterioscler Thromb Vasc Biol 28:1774–1781. https://doi.org/10.1161/ATVBAHA.108.172692

    Article  CAS  Google Scholar 

  15. Edwards G, Feletou M, Weston AH (2010) Endothelium-derived hyperpolarising factors and associated pathways: a synopsis. Pflugers Arch 459:863–879. https://doi.org/10.1007/s00424-010-0817-1

    Article  CAS  Google Scholar 

  16. Everaerts W, Zhen X, Ghosh D, Vriens J, Gevaert T, Gilbert JP, Hayward NJ, McNamara CR, Xue F, Moran MM, Strassmaier T, Uykal E, Owsianik G, Vennekens R, De Ridder D, Nilius B, Fanger CM, Voets T (2010) Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis. Proc Natl Acad Sci U S A 107:19084–19089. https://doi.org/10.1073/pnas.1005333107

    Article  Google Scholar 

  17. Fan HC, Zhang X, McNaughton PA (2009) Activation of the TRPV4 ion channel is enhanced by phosphorylation. J Biol Chem 284:27884–27891. https://doi.org/10.1074/jbc.M109.028803

    Article  CAS  Google Scholar 

  18. Filosa JA, Yao X, Rath G (2013) TRPV4 and the regulation of vascular tone. J Cardiovasc Pharmacol 61:113–119. https://doi.org/10.1097/FJC.0b013e318279ba42

    Article  CAS  Google Scholar 

  19. Ford TJ, Rocchiccioli P, Good R, McEntegart M, Eteiba H, Watkins S, Shaukat A, Lindsay M, Robertson K, Hood S, Yii E, Sidik N, Harvey A, Montezano AC, Beattie E, Haddow L, Oldroyd KG, Touyz RM, Berry C (2018) Systemic microvascular dysfunction in microvascular and vasospastic angina. Eur Heart J 39:4086–4097. https://doi.org/10.1093/eurheartj/ehy529

    Article  CAS  Google Scholar 

  20. Gao X, Wu L, O’Neil RG (2003) Temperature-modulated diversity of TRPV4 channel gating: activation by physical stresses and phorbol ester derivatives through protein kinase C-dependent and -independent pathways. J Biol Chem 278:27129–27137. https://doi.org/10.1074/jbc.M302517200

    Article  CAS  Google Scholar 

  21. Groschner K, Graier WF, Kukovetz WR (1994) Histamine induces K+, Ca2+, and Cl- currents in human vascular endothelial cells. Role of ionic currents in stimulation of nitric oxide biosynthesis. Circ Res 75:304–314. https://doi.org/10.1161/01.res.75.2.304

    Article  CAS  Google Scholar 

  22. Guler AD, Lee H, Iida T, Shimizu I, Tominaga M, Caterina M (2002) Heat-evoked activation of the ion channel, TRPV4. J Neurosci 22:6408–6414. https://doi.org/10.1523/JNEUROSCI.22-15-06408.2002

    Article  CAS  Google Scholar 

  23. Heathcote HR, Lee MD, Zhang X, Saunter CD, Wilson C, McCarron JG (2019) Endothelial TRPV4 channels modulate vascular tone by Ca(2+) -induced Ca(2+) release at inositol 1,4,5-trisphosphate receptors. Br J Pharmacol 176:3297–3317. https://doi.org/10.1111/bph.14762

    Article  CAS  Google Scholar 

  24. Heusch G (2019) Coronary microvascular obstruction: the new frontier in cardioprotection. Basic Res Cardiol 114:45. https://doi.org/10.1007/s00395-019-0756-8

    Article  CAS  Google Scholar 

  25. Heusch G (2022) Coronary blood flow in heart failure: cause, consequence and bystander. Basic Res Cardiol 117:1. https://doi.org/10.1007/s00395-022-00909-8

    Article  Google Scholar 

  26. Hirano K, Chen WS, Chueng AL, Dunne AA, Seredenina T, Filippova A, Ramachandran S, Bridges A, Chaudry L, Pettman G, Allan C, Duncan S, Lee KC, Lim J, Ma MT, Ong AB, Ye NY, Nasir S, Mulyanidewi S, Aw CC, Oon PP, Liao S, Li D, Johns DG, Miller ND, Davies CH, Browne ER, Matsuoka Y, Chen DW, Jaquet V, Rutter AR (2015) Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor. Antioxid Redox Signal 23:358–374. https://doi.org/10.1089/ars.2014.6202

    Article  CAS  Google Scholar 

  27. Ho WS, Zheng X, Zhang DX (2015) Role of endothelial TRPV4 channels in vascular actions of the endocannabinoid, 2-arachidonoylglycerol. Br J Pharmacol 172:5251–5264. https://doi.org/10.1111/bph.13312

    Article  CAS  Google Scholar 

  28. Jow F, Numann R (2000) Histamine increases [Ca(2+)](in) and activates Ca-K and nonselective cation currents in cultured human capillary endothelial cells. J Membr Biol 173:107–116. https://doi.org/10.1007/s002320001012

    Article  CAS  Google Scholar 

  29. Kalari KR, Nair AA, Bhavsar JD, O’Brien DR, Davila JI, Bockol MA, Nie J, Tang X, Baheti S, Doughty JB, Middha S, Sicotte H, Thompson AE, Asmann YW, Kocher JP (2014) MAP-RSeq: mayo analysis pipeline for RNA sequencing. BMC Bioinform 15:224. https://doi.org/10.1186/1471-2105-15-224

    Article  CAS  Google Scholar 

  30. Khuddus MA, Pepine CJ, Handberg EM, Bairey Merz CN, Sopko G, Bavry AA, Denardo SJ, McGorray SP, Smith KM, Sharaf BL, Nicholls SJ, Nissen SE, Anderson RD (2010) An intravascular ultrasound analysis in women experiencing chest pain in the absence of obstructive coronary artery disease: a substudy from the National Heart, Lung and Blood Institute-Sponsored Women’s Ischemia Syndrome Evaluation (WISE). J Interv Cardiol 23:511–519. https://doi.org/10.1111/j.1540-8183.2010.00598.x

    Article  Google Scholar 

  31. Kohler R, Heyken WT, Heinau P, Schubert R, Si H, Kacik M, Busch C, Grgic I, Maier T, Hoyer J (2006) Evidence for a functional role of endothelial transient receptor potential V4 in shear stress-induced vasodilatation. Arterioscler Thromb Vasc Biol 26:1495–1502. https://doi.org/10.1161/01.ATV.0000225698.36212.6a

    Article  CAS  Google Scholar 

  32. Korishettar AM, Nishijima Y, Wang Z, Xie Y, Fang J, Wilcox DA, Zhang DX (2021) Endothelin-1 potentiates TRPV1-mediated vasoconstriction of human adipose arterioles in a protein kinase C-dependent manner. Br J Pharmacol 178:709–725. https://doi.org/10.1111/bph.15324

    Article  CAS  Google Scholar 

  33. Larsen BT, Bubolz AH, Mendoza SA, Pritchard KA Jr, Gutterman DD (2009) Bradykinin-induced dilation of human coronary arterioles requires NADPH oxidase-derived reactive oxygen species. Arterioscler Thromb Vasc Biol 29:739–745. https://doi.org/10.1161/ATVBAHA.108.169367

    Article  CAS  Google Scholar 

  34. Lassegue B, Clempus RE (2003) Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol 285:R277-297. https://doi.org/10.1152/ajpregu.00758.2002

    Article  CAS  Google Scholar 

  35. Liedtke W, Choe Y, Marti-Renom MA, Bell AM, Denis CS, Sali A, Hudspeth AJ, Friedman JM, Heller S (2000) Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 103:525–535. https://doi.org/10.1016/s0092-8674(00)00143-4

    Article  CAS  Google Scholar 

  36. Martin E, Dahan D, Cardouat G, Gillibert-Duplantier J, Marthan R, Savineau JP, Ducret T (2012) Involvement of TRPV1 and TRPV4 channels in migration of rat pulmonary arterial smooth muscle cells. Pflugers Arch 464:261–272. https://doi.org/10.1007/s00424-012-1136-5

    Article  CAS  Google Scholar 

  37. Meitzler JL, Makhlouf HR, Antony S, Wu Y, Butcher D, Jiang G, Juhasz A, Lu J, Dahan I, Jansen-Durr P, Pircher H, Shah AM, Roy K, Doroshow JH (2017) Decoding NADPH oxidase 4 expression in human tumors. Redox Biol 13:182–195. https://doi.org/10.1016/j.redox.2017.05.016

    Article  CAS  Google Scholar 

  38. Mendoza SA, Fang J, Gutterman DD, Wilcox DA, Bubolz AH, Li R, Suzuki M, Zhang DX (2010) TRPV4-mediated endothelial Ca2+ influx and vasodilation in response to shear stress. Am J Physiol-Heart Circul Physiol 298:H466-476. https://doi.org/10.1152/ajpheart.00854.2009

    Article  CAS  Google Scholar 

  39. Mohapatra DP, Nau C (2005) Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase. J Biol Chem 280:13424–13432. https://doi.org/10.1074/jbc.M410917200

    Article  CAS  Google Scholar 

  40. Munoz M, Lopez-Oliva ME, Rodriguez C, Martinez MP, Saenz-Medina J, Sanchez A, Climent B, Benedito S, Garcia-Sacristan A, Rivera L, Hernandez M, Prieto D (2020) Differential contribution of Nox1, Nox2 and Nox4 to kidney vascular oxidative stress and endothelial dysfunction in obesity. Redox Biol 28:101330. https://doi.org/10.1016/j.redox.2019.101330

    Article  CAS  Google Scholar 

  41. Munoz M, Martinez MP, Lopez-Oliva ME, Rodriguez C, Corbacho C, Carballido J, Garcia-Sacristan A, Hernandez M, Rivera L, Saenz-Medina J, Prieto D (2018) Hydrogen peroxide derived from NADPH oxidase 4- and 2 contributes to the endothelium-dependent vasodilatation of intrarenal arteries. Redox Biol 19:92–104. https://doi.org/10.1016/j.redox.2018.08.004

    Article  CAS  Google Scholar 

  42. Murthy VL, Naya M, Foster CR, Hainer J, Gaber M, Di Carli G, Blankstein R, Dorbala S, Sitek A, Pencina MJ, Di Carli MF (2011) Improved cardiac risk assessment with noninvasive measures of coronary flow reserve. Circulation 124:2215–2224. https://doi.org/10.1161/CIRCULATIONAHA.111.050427

    Article  Google Scholar 

  43. Nilius B, Schwartz G, Oike M, Droogmans G (1993) Histamine-activated, non-selective cation currents and Ca2+ transients in endothelial cells from human umbilical vein. Pflugers Arch 424:285–293. https://doi.org/10.1007/BF00384354

    Article  CAS  Google Scholar 

  44. Nilius B, Viana F, Droogmans G (1997) Ion channels in vascular endothelium. Annu Rev Physiol 59:145–170. https://doi.org/10.1146/annurev.physiol.59.1.145

    Article  CAS  Google Scholar 

  45. Nishijima Y, Cao S, Chabowski DS, Korishettar A, Ge A, Zheng X, Sparapani R, Gutterman DD, Zhang DX (2017) Contribution of KV1.5 channel to hydrogen peroxide-induced human arteriolar dilation and its modulation by coronary artery disease. Circ Res 120:658–669. https://doi.org/10.1161/CIRCRESAHA.116.309491

    Article  CAS  Google Scholar 

  46. Nishijima Y, Zheng X, Lund H, Suzuki M, Mattson DL, Zhang DX (2014) Characterization of blood pressure and endothelial function in TRPV4-deficient mice with l-NAME- and angiotensin II-induced hypertension. Physiol Rep 2:e00199. https://doi.org/10.1002/phy2.199

    Article  CAS  Google Scholar 

  47. Nisimoto Y, Diebold BA, Cosentino-Gomes D, Lambeth JD (2014) Nox4: a hydrogen peroxide-generating oxygen sensor. Biochemistry 53:5111–5120. https://doi.org/10.1021/bi500331y

    Article  CAS  Google Scholar 

  48. Ottolini M, Hong K, Cope EL, Daneva Z, DeLalio LJ, Sokolowski JD, Marziano C, Nguyen NY, Altschmied J, Haendeler J, Johnstone SR, Kalani MY, Park MS, Patel RP, Liedtke W, Isakson BE, Sonkusare SK (2020) Local peroxynitrite impairs endothelial transient receptor potential vanilloid 4 channels and elevates blood pressure in obesity. Circulation 141:1318–1333. https://doi.org/10.1161/CIRCULATIONAHA.119.043385

    Article  CAS  Google Scholar 

  49. Parrinello R, Sestito A, Di Franco A, Russo G, Villano A, Figliozzi S, Nerla R, Tarzia P, Stazi A, Lanza GA, Crea F (2014) Peripheral arterial function and coronary microvascular function in patients with variant angina. Cardiology 129:20–24. https://doi.org/10.1159/000362380

    Article  Google Scholar 

  50. Pendyala S, Gorshkova IA, Usatyuk PV, He D, Pennathur A, Lambeth JD, Thannickal VJ, Natarajan V (2009) Role of Nox4 and Nox2 in hyperoxia-induced reactive oxygen species generation and migration of human lung endothelial cells. Antioxid Redox Signal 11:747–764. https://doi.org/10.1089/ARS.2008.2203

    Article  CAS  Google Scholar 

  51. Pepine CJ, Anderson RD, Sharaf BL, Reis SE, Smith KM, Handberg EM, Johnson BD, Sopko G, Bairey Merz CN (2010) Coronary microvascular reactivity to adenosine predicts adverse outcome in women evaluated for suspected ischemia results from the National Heart, Lung and Blood Institute WISE (Women’s Ischemia Syndrome Evaluation) study. J Am Coll Cardiol 55:2825–2832. https://doi.org/10.1016/j.jacc.2010.01.054

    Article  CAS  Google Scholar 

  52. Rath G, Saliez J, Behets G, Romero-Perez M, Leon-Gomez E, Bouzin C, Vriens J, Nilius B, Feron O, Dessy C (2012) Vascular hypoxic preconditioning relies on TRPV4-dependent calcium influx and proper intercellular gap junctions communication. Arterioscler Thromb Vasc Biol 32:2241–2249. https://doi.org/10.1161/ATVBAHA.112.252783

    Article  CAS  Google Scholar 

  53. Ray R, Murdoch CE, Wang M, Santos CX, Zhang M, Alom-Ruiz S, Anilkumar N, Ouattara A, Cave AC, Walker SJ, Grieve DJ, Charles RL, Eaton P, Brewer AC, Shah AM (2011) Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo. Arterioscler Thromb Vasc Biol 31:1368–1376. https://doi.org/10.1161/ATVBAHA.110.219238

    Article  CAS  Google Scholar 

  54. Reutens AT, Jandeleit-Dahm K, Thomas M, Salim A, De Livera AM, Bach LA, Colman PG, Davis TME, Ekinci EI, Fulcher G, Hamblin PS, Kotowicz MA, MacIsaac RJ, Morbey C, Simmons D, Soldatos G, Wittert G, Wu T, Cooper ME, Shaw JE (2020) A physician-initiated double-blind, randomised, placebo-controlled, phase 2 study evaluating the efficacy and safety of inhibition of NADPH oxidase with the first-in-class Nox-1/4 inhibitor, GKT137831, in adults with type 1 diabetes and persistently elevated urinary albumin excretion: Protocol and statistical considerations. Contemp Clin Trials 90:105892. https://doi.org/10.1016/j.cct.2019.105892

    Article  Google Scholar 

  55. Rigo F, Pratali L, Palinkas A, Picano E, Cutaia V, Venneri L, Raviele A (2002) Coronary flow reserve and brachial artery reactivity in patients with chest pain and “false positive” exercise-induced ST-segment depression. Am J Cardiol 89:1141–1144. https://doi.org/10.1016/s0002-9149(02)02292-0

    Article  Google Scholar 

  56. Sorop O, van de Wouw J, Chandler S, Ohanyan V, Tune JD, Chilian WM, Merkus D, Bender SB, Duncker DJ (2020) Experimental animal models of coronary microvascular dysfunction. Cardiovasc Res 116:756–770. https://doi.org/10.1093/cvr/cvaa002

    Article  CAS  Google Scholar 

  57. Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD (2000) OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2:695–702. https://doi.org/10.1038/35036318

    Article  CAS  Google Scholar 

  58. Takac I, Schroder K, Zhang L, Lardy B, Anilkumar N, Lambeth JD, Shah AM, Morel F, Brandes RP (2011) The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4. J Biol Chem 286:13304–13313. https://doi.org/10.1074/jbc.M110.192138

    Article  CAS  Google Scholar 

  59. Taqueti VR, Hachamovitch R, Murthy VL, Naya M, Foster CR, Hainer J, Dorbala S, Blankstein R, Di Carli MF (2015) Global coronary flow reserve is associated with adverse cardiovascular events independently of luminal angiographic severity and modifies the effect of early revascularization. Circulation 131:19–27. https://doi.org/10.1161/CIRCULATIONAHA.114.011939

    Article  Google Scholar 

  60. Thompson JA, Larion S, Mintz JD, Belin de Chantemele EJ, Fulton DJ, Stepp DW (2017) Genetic deletion of NADPH Oxidase 1 rescues microvascular function in mice with metabolic disease. Circ Res 121:502–511. https://doi.org/10.1161/CIRCRESAHA.116.309965

    Article  CAS  Google Scholar 

  61. Thorneloe KS, Sulpizio AC, Lin Z, Figueroa DJ, Clouse AK, McCafferty GP, Chendrimada TP, Lashinger ES, Gordon E, Evans L, Misajet BA, Demarini DJ, Nation JH, Casillas LN, Marquis RW, Votta BJ, Sheardown SA, Xu X, Brooks DP, Laping NJ, Westfall TD (2008) N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I. J Pharmacol Exp Ther 326:432–442. https://doi.org/10.1124/jpet.108.139295

    Article  CAS  Google Scholar 

  62. Vriens J, Owsianik G, Janssens A, Voets T, Nilius B (2007) Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4. J Biol Chem 282:12796–12803. https://doi.org/10.1074/jbc.M610485200

    Article  CAS  Google Scholar 

  63. Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, Vriens J, Cairns W, Wissenbach U, Prenen J, Flockerzi V, Droogmans G, Benham CD, Nilius B (2002) Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives. J Biol Chem 277:13569–13577. https://doi.org/10.1074/jbc.M200062200

    Article  CAS  Google Scholar 

  64. Weiss WA, Taylor SS, Shokat KM (2007) Recognizing and exploiting differences between RNAi and small-molecule inhibitors. Nat Chem Biol 3:739–744. https://doi.org/10.1038/nchembio1207-739

    Article  CAS  Google Scholar 

  65. Wissenbach U, Bodding M, Freichel M, Flockerzi V (2000) Trp12, a novel Trp related protein from kidney. FEBS Lett 485:127–134. https://doi.org/10.1016/s0014-5793(00)02212-2

    Article  CAS  Google Scholar 

  66. Xie J, Hong E, Ding B, Jiang W, Zheng S, Xie Z, Tian D, Chen Y (2020) Inhibition of NOX4/ROS suppresses neuronal and blood-brain barrier injury by attenuating oxidative stress after intracerebral hemorrhage. Front Cell Neurosci 14:578060. https://doi.org/10.3389/fncel.2020.578060

    Article  CAS  Google Scholar 

  67. Xue K, Wang Y, Wang Y, Fang H (2021) Advanced oxidation protein product promotes oxidative accentuation in renal epithelial cells via the soluble (Pro)renin receptor-mediated intrarenal renin-angiotensin system and Nox4-H2O2 signaling. Oxid Med Cell Longev. https://doi.org/10.1155/2021/5710440

    Article  Google Scholar 

  68. Yang XR, Lin AH, Hughes JM, Flavahan NA, Cao YN, Liedtke W, Sham JS (2012) Upregulation of osmo-mechanosensitive TRPV4 channel facilitates chronic hypoxia-induced myogenic tone and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 302:L555-568. https://doi.org/10.1152/ajplung.00005.2011

    Article  CAS  Google Scholar 

  69. Zhang DX, Borbouse L, Gebremedhin D, Mendoza SA, Zinkevich NS, Li R, Gutterman DD (2012) H2O2-induced dilation in human coronary arterioles: role of protein kinase G dimerization and large-conductance Ca2+-activated K+ channel activation. Circ Res 110:471–480. https://doi.org/10.1161/CIRCRESAHA.111.258871

    Article  CAS  Google Scholar 

  70. Zhang DX, Gutterman DD (2011) Transient receptor potential channel activation and endothelium-dependent dilation in the systemic circulation. J Cardiovasc Pharmacol 57:133–139. https://doi.org/10.1097/FJC.0b013e3181fd35d1

    Article  CAS  Google Scholar 

  71. Zhang DX, Mendoza SA, Bubolz AH, Mizuno A, Ge ZD, Li R, Warltier DC, Suzuki M, Gutterman DD (2009) Transient receptor potential vanilloid type 4-deficient mice exhibit impaired endothelium-dependent relaxation induced by acetylcholine in vitro and in vivo. Hypertension 53:532–538. https://doi.org/10.1161/hypertensionaha.108.127100

    Article  CAS  Google Scholar 

  72. Zhang Y, Wernly B, Cao X, Mustafa SJ, Tang Y, Zhou Z (2021) Adenosine and adenosine receptor-mediated action in coronary microcirculation. Basic Res Cardiol 116:22. https://doi.org/10.1007/s00395-021-00859-7

    Article  CAS  Google Scholar 

  73. Zheng X, Zinkevich NS, Gebremedhin D, Gauthier KM, Nishijima Y, Fang J, Wilcox DA, Campbell WB, Gutterman DD, Zhang DX (2013) Arachidonic acid-induced dilation in human coronary arterioles: convergence of signaling mechanisms on endothelial TRPV4-mediated Ca2+ entry. J Am Heart Assoc 2:e000080. https://doi.org/10.1161/JAHA.113.000080

    Article  CAS  Google Scholar 

  74. Zinkevich NS, Fancher IS, Gutterman DD, Phillips SA (2017) Roles of NADPH oxidase and mitochondria in flow-induced vasodilation of human adipose arterioles: ROS-induced ROS release in coronary artery disease. Microcirculation. https://doi.org/10.1111/micc.12380

    Article  Google Scholar 

  75. Zinkevich NS, Gutterman DD (2011) ROS-induced ROS release in vascular biology: redox-redox signaling. Am J Physiol Heart Circ Physiol 301:H647-653. https://doi.org/10.1152/ajpheart.01271.2010

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Elmbrook Memorial Hospital and Froedtert Hospital for providing human tissues for the study. We also thank Dr. James H. Doroshow (NCI, NIH)) for kindly providing NOX4 antibody and NOX4 overexpressed HEK293 cell lysate and Dr. Charles K. Thodeti (NEOMED, OH) for providing PCR primer sequence information of NOX isoforms. This work was supported by the National Heart, Lung and Blood Institute Grant RO1-HL 096647 (to D.X.Z.), a generous gift from John B. and Judith A. Gardetto to the Children’s Research Foundation (to D.A.W.), and the funding for a joint Ph.D. program from the China Scholarship Council (to Y.X, contract 201708340067)

Funding

National Heart, Lung and Blood Institute Grant RO1-HL 096647 (to D.X.Z.), a generous gift from John B. and Judith A. Gardetto to the Children’s Research Foundation (to D.A.W.), and the funding for a joint Ph.D. program from the China Scholarship Council (to Y.X, contract 201708340067).

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Authors and Affiliations

  1. School of Basic Medical Sciences and Biopharmaceutical Institute, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China

    Yangjing Xie & Yuxian Shen

  2. Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, China

    Yangjing Xie

  3. Department of Medicine, Cardiovascular Center, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI, 53226, USA

    Yangjing Xie, Yoshinori Nishijima, Natalya S. Zinkevich, Ankush Korishettar, David D. Gutterman & David X. Zhang

  4. Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI, USA

    Ankush Korishettar, David D. Gutterman & David X. Zhang

  5. Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

    Juan Fang & David A. Wilcox

  6. Children’s Research Institute, Children’s Wisconsin, Milwaukee, WI, USA

    Juan Fang & David A. Wilcox

  7. Bioinformatics Research and Development Laboratory, Department of Surgery, Genomic Sciences and Precision Medicine Center (GSPMC), Medical College of Wisconsin, Milwaukee, WI, USA

    Angela J. Mathison & Michael T. Zimmermann

  8. Biopharmaceutical Institute, Anhui Medical University, 81 Meishan Road, Hefei, 230032, China

    Yangjing Xie & Yuxian Shen

  9. Department of Biology, College of Liberal Arts and Sciences, University of Illinois at Springfield, Springfield, IL, USA

    Natalya S. Zinkevich

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  1. Yangjing Xie
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  2. Yoshinori Nishijima
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  4. Ankush Korishettar
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  5. Juan Fang
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  9. David D. Gutterman
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Contributions

YX, YN, NZ, AK, AM, MZ and JF planned and performed the experiments. DWA provided the intellectual and technical advice. YX, YN, and DXZ designed the study and analyzed the data. YX wrote the manuscript. YX, YN, AK, DDG, YS, and DXZ reviewed and revised the manuscript and provided critical input throughout the study.

Corresponding authors

Correspondence to Yuxian Shen or David X. Zhang.

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Conflicts of interest/Competing interests

The authors declare that they have no competing interests.

Ethical approval

All protocols were approved by the Institutional Review Board of the Medical College of Wisconsin and Froedtert Hospital on the use of human subjects in research.

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Supplementary Information

Below is the link to the electronic supplementary material.

395_2022_932_MOESM1_ESM.tif

Supplemental Fig S1 Role of H2O2, NO, PGI2, and NOX4 in acetylcholine (ACh)-induced vasodilation in human adipose arterioles (HAA). a) In HAA, cell-permeable H2O2 scavenger peg-catalase (peg-CAT) (500 U/ml) reduced ACh induced vasodilation at log -7 M (n=4 vessels in each group). b) A combination of peg-CAT and TRPV4 inhibitor HC067047 (1 μM) induced a similar inhibition of ACh-induced vasodilation at log -7 - -5 M (n=4 vessels in each group). c) A combination of peg-CAT and NOX1/4- inhibitor GKT137831 (1 μM) also induced a similar inhibition of ACh-induced vasodilation at log -7 M (n=4 vessels in each group). d) In HAA, preincubation with eNOS inhibitor L-NAME (100 μM) and cyclooxygenase inhibitor indomethacin (Indo, 10 μM) significantly reduced the ACh induced vasodilation (n=6 vessels in each group). e) In the presence of L-NAME and indomethacin, NOX1/4- inhibitor GKT137831 (GKT137, 1 μM) further reduced the ACh-induced vasodilation (n=6 vessels in each group). *P < 0.05 vs control or L-NAME +Indo

395_2022_932_MOESM2_ESM.tif

Supplemental Fig S2 Effects of NO and PGI2 in GSK1016790A-induced vasodilation in human adipose arterioles (HAA). a) In HAA, preincubation with eNOS inhibitor L-NAME (100 μmol/L) and cyclooxygenase inhibitor indomethacin (Indo, 10 μmol/L), significantly reduced GSK1016790A-induced vasodilation (n=5 vessels in each group). b) The TRPV4-selective blocker HC067047 (2 μM) significantly reduced GSK1016790A-induced vasodilation (n=5 vessels in each group). *P < 0.05 vs control

395_2022_932_MOESM3_ESM.tif

Supplemental Fig S3 Both wild-type (WT) and mutant TRPV4 proteins are expressed on the cell surface of HEK293 cells. a) Cell surface proteins were captured by a cell surface biotinylation kit, and total protein lysates were used as control. Western blotting with anti-GFP antibody shows that TRPV4-GFP WT, S824A, S823A/S824A (S823A/4A) are expressed on the cell surface and in total lysates. β-actin as a cytosolic protein was detected only in total lysates. b) No significant difference in cell surface/total lysate ratio between WT, S824A, and S823A/S824A (S823A/4A) mutants (n=3 independent experiments)

395_2022_932_MOESM4_ESM.tif

Supplemental Fig S4 Detection of Ser824 phosphorylation in TRPV4- wild type (WT) and mutants expressed in HEK293 cells. HEK293 cells were transfected with TRPV4-GFP wildtype (WT), S823A, or S823A/S824A (S823A/4A) mutants. TRPV4 Ser824 phosphorylation was analyzed by Western blotting with a phosphoserine motif antibody against the motif RXRXXS*/T* (pS824 antibody). After stimulation with PKC activator PMA (1 μM) for 30min, p-S824 were detected in WT and S823A groups, but not in S823A/4A group, confirming the specificity of the pS824 antibody. Total GFP was used as an internal reference (n=3 independent experiments)

395_2022_932_MOESM5_ESM.tif

Supplemental Fig S5 NOX family gene expression in human arteries. a) GTEx V7 RNA sequencing of NOX family genes in human coronary arteries shows that NOX2 and NOX4 are the most abundant isoforms, while NOX1 expression level is low. GTEx RNA sequencing data (n=173) were downloaded from NCBI dbGaP repository, which can also be viewed from the GTEx Portal (gtexportal.org). b) A similar expression profile of NOX family genes was found in human adipose arterioles freshly isolated from patients without coronary artery disease or ≤2 risk factors (n=3). Illumina mRNA sequencing libraries were prepared and sequenced using NovaSeq 6000 by the on-campus core laboratory

395_2022_932_MOESM6_ESM.tif

Supplemental Fig S6 Effect of PKA inhibitor PKI on GSK1016790A-induced Ca2+ influx in TRPV4 wild-type (WT)-overexpressing human coronary artery endothelial cells (HCAEC). a) In HCAEC overexpressing TRPV4-WT (control), GSK1016790A induced Ca2+ influx in a concentration-dependent manner, the arrows indicate the administration of GSK1016790A (GSK). The intracellular Ca2+ concentrations were monitored by the ratiometric calcium indicator fura-2 and the results are presented as F340/F380 ratios. b) In the same WT TRPV4-overexpressing HCAEC, GSK1016790A-induced Ca2+ response at 3 nM was inhibited by the preincubation with PKI (1 μM). c) Summary data of concentration-dependent Ca2+ responses induced by GSK1016790A in the absence and presence of PKI. * P < 0.05 vs control (n=6 independent experiments in each group)

395_2022_932_MOESM7_ESM.tif

Supplemental Fig S7 Effect of NOX2 and NOX4 siRNA on the phosphorylation of Ser824 in TRPV4 overexpressed HCAEC. TRPV4-AN-HIS-overexpressed HCAEC were treated with siControl (siCtrl), NOX2 siRNA (siNOX2), and NOX4 siRNA (siNOX4), and immunoprecipitated with anti-HIS (serum, Sigma H1029). a) TRPV4 Ser824 phosphorylation was analyzed with a phosphoserine motif antibody against the motif RXRXXS*/T* (p-S824 antibodies, Cell Signaling #10001), and the same blot was re-probed with TRPV4 antibody (Cell Signaling #65893) to detect total cellular TRPV4 proteins. b) Immunoblot of NOX4 protein and β-actin. c) Quantitative analysis of pS824 in siRNA treated HCAEC. * P < 0.05 vs control (n=3 independent experiments in each group)

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Xie, Y., Nishijima, Y., Zinkevich, N.S. et al. NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels. Basic Res Cardiol 117, 24 (2022). https://doi.org/10.1007/s00395-022-00932-9

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  • DOI: https://doi.org/10.1007/s00395-022-00932-9

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