In the vasculature, physiological levels of nitric oxide (NO) protect against various stressors, including mechanical stretch. While endothelial NO production in response to various stimuli has been studied extensively, the precise mechanism underlying stretch-induced NO production in venous endothelial cells remains incompletely understood. Using a model of continuous cellular stretch, we found that stretch promoted phosphorylation of endothelial NO synthase (eNOS) at Ser1177, Ser633 and Ser615 and NO production in human umbilical vein endothelial cells. Although stretch activated the kinases AMPKα, PKA, Akt, and ERK1/2, stretch-induced eNOS activation was only inhibited by kinase-specific inhibitors of PKA and PI3K/Akt, but not of AMPKα and Erk1/2. Similar results were obtained with knockdown by shRNAs targeting the PKA and Akt genes. Furthermore, inhibition of PKA preferentially attenuated eNOS activation in the early phase, while inhibition of the PI3K/Akt pathway reduced eNOS activation in the late phase, suggesting that the PKA and PI3K/Akt pathways play distinct roles in a time-dependent manner. Finally, we investigated the role of these pathways in stretch-induced endothelial exocytosis and leukocyte adhesion. Interestingly, we found that inhibition of the PI3K/Akt pathway increased stretch-induced Weibel-Palade body exocytosis and leukocyte adhesion, while inhibition of the PKA pathway had the opposite effects, suggesting that the exocytosis-promoting effect of PKA overwhelms the inhibitory effect of PKA-mediated NO production. Taken together, the results suggest that PKA and Akt are important regulators of eNOS activation in venous endothelial cells under mechanical stretch, while playing different roles in the regulation of stretch-induced endothelial exocytosis and leukocyte adhesion.
References
[1]
Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, et al. (1995) Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation 91: 1314–1319.
[2]
Murohara T, Asahara T, Silver M, Bauters C, Masuda H, et al. (1998) Nitric oxide synthase modulates angiogenesisin response to tissue ischemia. J Clin Invest 101: 2567–2578.
[3]
Wang GR, Zhu Y, Halushka PV, Halushka PV, Lincoln TM, et al. (1998) Mechanism of platelet inhibition by nitric oxide: in vivo phosphorylation of thromboxane receptor by cyclic GMP-dependent protein kinase. Proc Natl Acad Sci U S A 95: 4888–4893.
[4]
Kubes P, Suzuki M, Granger DN (1991) Nitric oxide: An endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A 88, 4651–4655.
[5]
Feliers D, Chen X, Akis N, Choudhury GG, Madaio M, et al. (2005) VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells. Kidney Int 68: 1648–59.
[6]
Motley ED, Eguchi K, Patterson MM, Palmer PD, Suzuki H, et al. (2005) Mechanism of endothelial nitric oxide synthase phosphorylation and activation by thrombin. Hypertension 49: 577–83.
[7]
Sartoretto JL, Kalwa H, Shiroto T, Sartoretto SM, Pluth MD, et al.. (2012) Role of Ca2+ in the control of H2O2-modulated phosphorylation pathways leading to eNOS activation in cardiac myocytes. PLoS One 7; e44627.
[8]
Lauer T, Kleinbongard P, Preik M, Rauch BH, Deussen A, et al. (2003) Direct biochemical evidence for eNOS stimulation by bradykinin in the human forearm vasculature. Basic Res Cardiol. 98: 84–89.
[9]
Jin ZG, Wong C, Wu J, Berk BC (2005) Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells. J Biol Chem 280, 12305–12309.
[10]
Kuebler WM, Uhlig U, Goldmann T, Schael G, Kerem A, et al.. (2003) Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ. Am J Respir Crit Care Med 168, 1391–1398.
[11]
Kawai M, Naruse K, Komatsu S, Kobayashi S, Nagino M, et al.. (2002) Mechanical stress-dependent secretion of interleukin 6 by endothelial cells after portal vein embolization: clinical and experimental studies. Journal of Hepatology 37, 240–246.
[12]
Meng X, Mavromatis K, Galis ZS (1999) Mechanical stretching of human saphenous vein grafts induces expression and activation of matrix-degrading enzymes associated with vascular tissue injury and repair. Exp Mol Pathol 66: 227–237.
[13]
Cheng J, Du J (2007) Mechanical stretch simulates proliferation of venous smooth muscle cells through activation of the insulin-like growth factor-1 receptor. Arterioscler Thromb Vasc Biol 27: 1744–1751.
[14]
Colombo PC, Rastogi S, Onat D, Zacà V, Gupta RC, et al. (2009) Activation of endothelial cells in conduit veins of dogs with heart failure and veins of normal dogs after vascular stretch by acute volume loading. J Card Fail 15: 457–463.
[15]
Eriksson EE, Karlof E, Lundmark K, Rotzius P, Hedin U, et al. (2005) Powerful inflammatory properties of large vein endothelium in vivo. Arterioscler Thromb Vasc Biol 25: 723–728.
[16]
Kobayashi S, Nagino M, Komatsu S, Naruse K, Nimura Y, et al. (2003) Stretch-induced IL-6 secretion from endothelial cells requires NF-kappaB activation. Biochem Biophys Res Commun 308: 306–312.
[17]
Ali MH, Pearlstein DP, Mathieu CE, Schumacker PT (2004) Mitochondrial requirement for endothelial responses to cyclic strain: implications for mechanotransduction. Am J Physiol Lung Cell Mol Physiol 287: 486–496.
[18]
Lowenstein CJ, Morrell CN, Yamakuchi M (2005) Regulation of Weibel-Palade body exocytosis. Trends Cardiovasc Med 15: 302–308.
[19]
Valentijn KM, van Driel LF, Mourik MJ, Hendriks GJ, Arends TJ, et al. (2010) Multigranular exocytosis of Weibel-Palade bodies in vascular endothelial cells. Blood 116: 1807–1816.
[20]
Xiong Y, Hu Z, Han X, Jiang B, Zhang R, et al.. (2013) Hypertensive stretch regulates endothelial exocytosis of Weibel-Palade bodies through VEGF receptor 2 signaling pathways. Cell Res doi: 10.1038/cr.2013.56. [Epub ahead of print].
[21]
Pawlak R CE, Golatowski J, Azzadin A, Buczko W (1998) Nitric oxide and prostacyclin are involved in antithrombotic action of captopril in venous thrombosis in rats. Thromb Haemost 79: 1208–1212.
[22]
Wu J, Wadsworth RM, Kennedy S (2011) Inhibition of inducible nitric oxide synthase promotes vein graft neoadventitial inflammation and remodelling. J Vasc Res 48: 141–149.
[23]
Matsushita K, Yamakuchi M, Morrell CN, Ozaki M, O’Rourke B, et al. (2005) Vascular endothelial growth factor regulation of Weibel-Palade-body exocytosis. Blood 105: 207–214.
[24]
Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, et al. (1999) Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601–605.
[25]
Dixit M, Loot AE, Mohamed A, Fisslthaler B, Boulanger CM, et al. (2005) Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress. Circ Res 97: 1236–1244.
[26]
Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, et al. (2002) Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem. 277: 3388–96.
[27]
Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R (2001) Phosphorylation of Thr (495) regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88: 68–75.
[28]
Butt E, Bernhardt M, Smolenski A, Kotsonis P, Fr？hlich LG, et al. (2002) Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 275: 5179–87.
[29]
Bernier SG, Haldar S, Michel T (2000) Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase. J Biol Chem 275: 30707–15.
[30]
Juliano L, Sartorettoa HK, Michael D, Lippard SJ, Michel T (2011) Hydrogen peroxide differentially modulates cardiac myocyte nitric oxide synthesis. Proc Natl Acad Sci U S A 108: 15792–15797.
[31]
Boo YC, Hwang J, Sykes M, Michell BJ, Kemp BE, et al. (2002) Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism. Am J Physiol Heart Circ Physiol 283: 1819–1828.
[32]
Michell BJ, Harris MB, Chen ZP, Ju H, Venema VJ, et al. (2002) Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. J Biol Chem 277: 42344–42351.
[33]
Zhang Y, Lee TS, Kolb EM, Sun K, Lu X, et al. (2006) AMP-activated protein kinase is involved in endothelial NO synthase activation in response to shear stress. Arterioscler Thromb Vasc Biol 26: 1281–1287.
[34]
Rondaij MG, Bierings R, van Agtmaal EL, Gijzen KA, Sellink E, et al. (2008) Guanine exchange factor RalGDS mediates exocytosis of Weibel-Palade bodies from endothelial cells. Blood 112: 56–63.
[35]
Chachisvilis M, Zhang YL, Frangos JA (2006) G protein-coupled receptors sense fluid shear stress in endothelial cells. Proc Natl Acad Sci U S A 103: 15463–15468.
[36]
Jin ZG, Ueba H, Tanimoto T, Lungu AO, Frame MD, et al. (2003) Ligand-independent activation of vascular endothelial growth factor receptor 2 by fluid shear stress regulates activation of endothelial nitric oxide synthase. Circ Res 93: 354–363.
[37]
Liu J, Agarwal S (2010) Mechanical signals activate vascular endothelial growth factor receptor-2 to upregulate endothelial cell proliferation during inflammation. J Immunol 185: 1215–1221.
[38]
Gélinas DS, Bernatchez PN, Rollin S, Bazan NG, Sirois MG (2002) Immediate and delayed VEGF-mediated NO synthesis in endothelial cells: role of PI3K, PKC and PLC pathways. Br J Pharmacol137: 1021–1030.
[39]
Gudi S, Clark CB, Frangos JA (1996) Fluid flow rapidly activates G-proteins in human endothelial cells: involvement of G-proteins in mechanochemical signal transduction. Circ Res 79: 834–839.
[40]
Reich KM, Gay CV, Frangos JA (1990) Fluid shear stress as a mediator of osteoblast cyclic adenosine monophosphate production. J Cell Physiol 143: 100–4.
[41]
Brouet A, Sonveaux P, Dessy C, Balligand JL, Feron O (2001) Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric oxide synthase in vascular endothelial growth factor-exposed endothelialcells. J Biol Chem 276: 32663–32669.
[42]
Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, et al. (1999) AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285–289.
[43]
Dimmeler S, Hermann C, Galle J, Zeiher AM (1999) Upregulation of superoxide dismutase and nitric oxide synthase mediates the apoptosis-suppressive effects of shear stress on endothelial cells. Arterioscler Thromb Vasc Biol 19: 656–664.
[44]
Thors B, Halldorsson H, Jonsdottir G, Thorgeirsson G (2008) Mechanism of thrombin mediated eNOS phosphorylation in endothelial cells is dependent on ATP levels after stimulation. Biochim Biophys Acta 1783: 1893–1902.
[45]
Xiong Y, Huo Y, Chen C, Zeng H, Lu X, et al. (2009) Vascular endothelial growth factor (VEGF) receptor-2 tyrosine 1175 signaling controls VEGF-induced von Willebrand factor release from endothelial cells via phospholipase C-gamma 1- and protein kinase A-dependent pathways. J Biol Chem 284: 23217–23224.
[46]
Lu Y, Xiong Y, Huo Y, Han J, Yang X, et al. (2011) Grb-2-associated binder 1 (Gab1) regulates postnatal ischemic and VEGF-induced angiogenesis through the protein kinase A-endothelial NOS pathway. Proc Natl Acad Sci U S A 108: 2957–2962.
[47]
Liao XD, Wang XH, Jin HJ, Chen LY, Chen Q (2004) Mechanical stretch induces mitochondria -dependent apoptosis in neonatal rat cardiomyocytes and G2/M accumulation in cardiac fibroblasts. Cell Res 14: 16–26.
