Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1

Andreas Petry; Rachida S. BelAiba; Michael Weitnauer; Agnes Görlach

doi:10.1160/TH12-04-0277

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2012; 108(05): 849-862
DOI: 10.1160/TH12-04-0277

Theme Issue Article

Schattauer GmbH

Inhibition of endothelial nitric oxyde synthase increases capillary formation via Rac1-dependent induction of hypoxia-inducible factor-1α and plasminogen activator inhibitor-1

Andreas Petry^*

¹Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany

,

Rachida S. BelAiba^*

¹Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany

,

Michael Weitnauer

¹Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany

,

Agnes Görlach

¹Experimental and Molecular Pediatric Cardiology, German Heart Center Munich at the Technical University Munich, Munich, Germany

²Munich Heart Alliance, Munich, Germany

› Author Affiliations

Abstract

Summary

Disruption of endothelial homeostasis results in endothelial dysfunction, characterised by a dysbalance between nitric oxide (NO) and reactive oxygen species (ROS) levels often accompanied by a prothrombotic and proproliferative state. The serine protease thrombin not only is instrumental in formation of the fibrin clot, but also exerts direct effects on the vessel wall by activating proliferative and angiogenic responses. In endothelial cells, thrombin can induce NO as well as ROS levels. However, the relative contribution of these reactive species to the angiogenic response towards thrombin is not completely clear. Since plasminogen activator inhibitor-1 (PAI-1), a direct target of the proangiogenic transcription factors hypoxia-inducible factors (HIFs), exerts prothrombotic and proangiogenic activities we investigated the role of ROS and NO in the regulation of HIF-1α, PAI-1 and capillary formation in response to thrombin. Thrombin enhanced the formation of NO as well as ROS generation involving the GTPase Rac1 in endothelial cells. Rac1-dependent ROS formation promoted induction of HIF-1α, PAI-1 and capillary formation by thrombin, while NO reduced ROS bioavailability and subsequently limited induction of HIF-1α, PAI-1 and the angiogenic response. Importantly, thrombin activation of Rac1 was diminished by NO, but enhanced by ROS. Thus, our findings show that capillary formation induced by thrombin via Rac1-dependent activation of HIF-1 and PAI-1 is limited by the concomitant release of NO which reduced ROS bioavailability. Rac1 activity is sensitive to ROS and NO, thereby playing an essential role in fine tuning the endothelial response to thrombin.

Keywords

Thrombin - angiogenesis - nitric oxide - reactive oxygen species - PAI-1

Full Text

References

References
1 Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 2000; 87: 840-844.
2 Raij L. Nitric oxide in the pathogenesis of cardiac disease. J Clin Hypertens (Greenwich) 2006; 8: 30-39.
3 Munzel T, Sinning C, Post F. et al. Pathophysiology, diagnosis and prognostic implications of endothelial dysfunction. Ann Med 2008; 40: 180-196.
4 Hirano K. The roles of proteinase-activated receptors in the vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 2007; 27: 27-36.
5 Wilkins MR. Pulmonary hypertension: the science behind the disease spectrum. Eur Respir Rev 2012; 21: 19-26.
6 Borissoff JI, Spronk HM, Heeneman S. et al. Is thrombin a key player in the 'coagulation-atherogenesis' maze?. Cardiovasc Res 2009; 82: 392-403.
7 Mann KG. Thrombin formation. Chest 2003; 124: 4S-10S.
8 Herkert O, Djordjevic T, BelAiba RS. et al. Insights into the redox control of blood coagulation: role of vascular NADPH oxidase-derived reactive oxygen species in the thrombogenic cycle. Antioxid Redox Signal 2004; 6: 765-776.
9 Michel T, Vanhoutte PM. Cellular signaling and NO production. Pflugers Arch 2010; 459: 807-816.
10 Gorlach A, Brandes RP, Nguyen K. et al. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res 2000; 87: 26-32.
11 Drummond GR, Selemidis S, Griendling KK. et al. Combating oxidative stress in vascular disease: NADPH oxidases as therapeutic targets. Nat Rev Drug Discov 2011; 10: 453-471.
12 Petry A, Djordjevic T, Weitnauer M. et al. NOX2 and NOX4 mediate proliferative response in endothelial cells. Antioxid Redox Signal 2006; 8: 1473-1484.
13 Forstermann U. Nitric oxide and oxidative stress in vascular disease. Pflugers Arch 2010; 459: 923-939.
14 Ushio-Fukai M, Nakamura Y. Reactive oxygen species and angiogenesis: NADPH oxidase as target for cancer therapy. Cancer Lett 2008; 266: 37-52.
15 Ziche M, Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw 2009; 20: 164-170.
16 Semenza GL. Involvement of hypoxia-inducible factor 1 in pulmonary pathophysiology. Chest 2005; 128: 592S-594S.
17 Stefansson S, McMahon GA, Petitclerc E. et al. Plasminogen activator inhibitor-1 in tumor growth, angiogenesis and vascular remodeling. Curr Pharm Des 2003; 9: 1545-1564.
18 Heaton JH, Dame MK, Gelehrter TD. Thrombin induction of plasminogen activator inhibitor mRNA in human umbilical vein endothelial cells in culture. J Lab Clin Med 1992; 120: 222-228.
19 Herkert O, Diebold I, Brandes RP. et al. NADPH oxidase mediates tissue factor-dependent surface procoagulant activity by thrombin in human vascular smooth muscle cells. Circulation 2002; 105: 2030-2036.
20 Kietzmann T, Roth U, Jungermann K. Induction of the plasminogen activator inhibitor-1 gene expression by mild hypoxia via a hypoxia response element binding the hypoxia-inducible factor-1 in rat hepatocytes. Blood 1999; 94: 4177-4185.
21 Kietzmann T, Cornesse Y, Brechtel K. et al. Perivenous expression of the mRNA of the three hypoxia-inducible factor alpha-subunits, HIF1alpha, HIF2alpha and HIF3alpha, in rat liver. Biochem J 2001; 354: 531-537.
22 Djordjevic T, Pogrebniak A, Belaiba RS. et al. The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells. Free Radic Biol Med 2005; 38: 616-630.
23 Belaiba RS, Djordjevic T, Petry A. et al. NOX5 variants are functionally active in endothelial cells. Free Radic Biol Med 2007; 42: 446-459.
24 Gorlach A, Berchner-Pfannschmidt U, Wotzlaw C. et al. Reactive oxygen species modulate HIF-1 mediated PAI-1 expression: involvement of the GTPase Rac1. Thromb Haemost 2003; 89: 926-935.
25 Diebold I, Petry A, Djordjevic T. et al. Reciprocal regulation of Rac1 and PAK-1 by HIF-1alpha: a positive-feedback loop promoting pulmonary vascular remodeling. Antioxid Redox Signal 2010; 13: 399-412.
26 Finkenzeller G, Torio-Padron N, Momeni A. et al. In vitro angiogenesis properties of endothelial progenitor cells: a promising tool for vascularization of ex vivo engineered tissues. Tissue Eng 2007; 13: 1413-1420.
27 Diebold I, Djordjevic T, Petry A. et al. Phosphodiesterase 2 mediates redox-sensitive endothelial cell proliferation and angiogenesis by thrombin via Rac1 and NADPH oxidase 2. Circ Res 2009; 104: 1169-1177.
28 Kojima H, Nakatsubo N, Kikuchi K. et al. Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem 1998; 70: 2446-2453.
29 Kerwin JF. Advances in NOS Inhibitors and NO-Based Therapeutics. Curr Pharm Des 1995; 1: 507-532.
30 Tojo T, Ushio-Fukai M, Yamaoka-Tojo M. et al. Role of gp91phox (Nox2)-containing NAD(P)H oxidase in angiogenesis in response to hindlimb ischemia. Circulation 2005; 111: 2347-2355.
31 Wojciak-Stothard B, Tsang LY, Haworth SG. Rac and Rho play opposing roles in the regulation of hypoxia/reoxygenation-induced permeability changes in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 2005; 288: L749-L760.
32 Bonello S, Zahringer C, BelAiba RS. et al. Reactive oxygen species activate the HIF-1alpha promoter via a functional NFkappaB site. Arterioscler Thromb Vasc Biol 2007; 27: 755-761.
33 Diebold I, Flugel D, Becht S. et al. The hypoxia-inducible factor-2alpha is stabilized by oxidative stress involving NOX4. Antioxid Redox Signal 2010; 13: 425-436.
34 Swiatkowska M, Szemraj J, Al-Nedawi KN. et al. Reactive oxygen species upregulate expression of PAI-1 in endothelial cells. Cell Mol Biol Lett 2002; 7: 1065-1071.
35 Yoshimoto T, Gochou N, Fukai N. et al. Adrenomedullin inhibits angiotensin II-induced oxidative stress and gene expression in rat endothelial cells. Hypertens Res 2005; 28: 165-172.
36 Diebold I, Kraicun D, Bonello S. et al. The 'PAI-1 paradox' in vascular remodeling. Thromb Haemost 2008; 100: 984-991.
37 Tsopanoglou NE, Maragoudakis ME. Role of thrombin in angiogenesis and tumor progression. Semin Thromb Hemost 2004; 30: 63-69.
38 Devy L, Blacher S, Grignet-Debrus C. et al. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. Faseb J 2002; 16: 147-154.
39 Soga N, Connolly JO, Chellaiah M. et al. Rac regulates vascular endothelial growth factor stimulated motility. Cell Commun Adhes 2001; 8: 1-13.
40 Sawada N, Salomone S, Kim HH. et al. Regulation of endothelial nitric oxide synthase and postnatal angiogenesis by Rac1. Circ Res 2008; 103: 360-368.
41 D'Amico G, Robinson SD, Germain M. et al. Endothelial-Rac1 is not required for tumor angiogenesis unless alphavbeta3-integrin is absent. PLoS One 2010; 5: e9766.
42 Selvakumar B, Hess DT, Goldschmidt-Clermont PJ. et al. Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac. FEBS Lett 2008; 582: 2195-2202.
43 Cooke JP, Losordo DW. Nitric oxide and angiogenesis. Circulation 2002; 105: 2133-2135.
44 Cattaneo MG, Cappellini E, Benfante R. et al. Chronic deficiency of nitric oxide affects hypoxia inducible factor-1alpha (HIF-1alpha) stability and migration in human endothelial cells. PLoS One 2011; 6: e29680.
45 Ziche M, Morbidelli L. Nitric oxide and angiogenesis. J Neurooncol 2000; 50: 139-148.
46 Isenberg JS. Nitric oxide modulation of early angiogenesis. Microsurgery 2004; 24: 385-391.
47 Hood JD, Meininger CJ, Ziche M. et al. VEGF upregulates ecNOS message, protein, and NO production in human endothelial cells. Am J Physiol 1998; 274: H1054-H1058.
48 Dulak J, Jozkowicz A, Dembinska-Kiec A. et al. Nitric oxide induces the synthesis of vascular endothelial growth factor by rat vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2000; 20: 659-666.
49 Jozkowicz A, Cooke JP, Guevara I. et al. Genetic augmentation of nitric oxide synthase increases the vascular generation of VEGF. Cardiovasc Res 2001; 51: 773-783.
50 Kimura H, Weisz A, Kurashima Y. et al. Hypoxia response element of the human vascular endothelial growth factor gene mediates transcriptional regulation by nitric oxide: control of hypoxia-inducible factor-1 activity by nitric oxide. Blood 2000; 95: 189-197.
51 Lee PC, Salyapongse AN, Bragdon GA. et al. Impaired wound healing and angiogenesis in eNOS-deficient mice. Am J Physiol 1999; 277: H1600-H1608.
52 Tsurumi Y, Murohara T, Krasinski K. et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity. Nat Med 1997; 3: 879-886.
53 Pipili-Synetos E, Sakkoula E, Haralabopoulos G. et al. Evidence that nitric oxide is an endogenous antiangiogenic mediator. Br J Pharmacol 1994; 111: 894-902.
54 Silvestre JS, Tamarat R, Ebrahimian TG. et al. Vascular endothelial growth factor-B promotes in vivo angiogenesis. Circ Res 2003; 93: 114-123.
55 Ziche M, Morbidelli L, Choudhuri R. et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J Clin Invest 1997; 99: 2625-2634.
56 Sakkoula E, Pipili-Synetos E, Maragoudakis ME. Involvement of nitric oxide in the inhibition of angiogenesis by interleukin-2. Br J Pharmacol 1997; 122: 793-795.
57 Shimizu S, Kageyama M, Yasuda M. et al. Stimulation of in vitro angiogenesis by nitric oxide through the induction of transcription factor ETS-1. Int J Biochem Cell Biol 2004; 36: 114-122.
58 Kook H, Ahn KY, Lee SE. et al. Nitric oxide-dependent cytoskeletal changes and inhibition of endothelial cell migration contribute to the suppression of angiogenesis by RAD50 gene transfer. FEBS Lett 2003; 553: 56-62.
59 Chinje EC, Stratford IJ. Role of nitric oxide in growth of solid tumours: a balancing act. Essays Biochem 1997; 32: 61-72.
60 Murohara T, Asahara T. Nitric oxide and angiogenesis in cardiovascular disease. Antioxid Redox Signal 2002; 4: 825-831.
61 Kvietys PR, Granger DN. Role of reactive oxygen and nitrogen species in the vascular responses to inflammation. Free Radic Biol Med 2012; 52: 556-592.
62 Chigurupati S, Mughal MR, Chan SL. et al. A synthetic uric acid analog accelerates cutaneous wound healing in mice. PLoS One 2010; 5: e10044.
63 Prakash R, Somanath PR, El-Remessy AB. et al. Enhanced cerebral but not peripheral angiogenesis in the goto-kakizaki model of type 2 diabetes involves VEGF and peroxynitrite signaling. Diabetes 2012; 61: 1533-1542.
64 Rabbani ZN, Spasojevic I, Zhang X. et al. Antiangiogenic action of redox-modulating Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+), via suppression of oxidative stress in a mouse model of breast tumor. Free Radic Biol Med 2009; 47: 992-1004.
65 Berchner-Pfannschmidt U, Tug S, Kirsch M. et al. Oxygen-sensing under the influence of nitric oxide. Cell Signal 2010; 22: 349-356.
66 Kozhukhar AV, Yasinska IM, Sumbayev VV. Nitric oxide inhibits HIF-1alpha protein accumulation under hypoxic conditions: implication of 2-oxoglutarate and iron. Biochimie 2006; 88: 411-418.
67 Yasuda H, Nakayama K, Watanabe M. et al. Nitroglycerin treatment may enhance chemosensitivity to docetaxel and carboplatin in patients with lung adenocarcinoma. Clin Cancer Res 2006; 12: 6748-6757.
68 Wellman TL, Jenkins J, Penar PL. et al. Nitric oxide and reactive oxygen species exert opposing effects on the stability of hypoxia-inducible factor-1alpha (HIF-1alpha) in explants of human pial arteries. Faseb J 2004; 18: 379-381.
69 Li F, Sonveaux P, Rabbani ZN. et al. Regulation of HIF-1alpha stability through S-nitrosylation. Mol Cell 2007; 26: 63-74.
70 Kaya A, Boztosun A, Seckin H. et al. The evaluation of hypoxia-inducible factor 1 in N-nitro-L-arginine methyl ester preeclampsia model of pregnant rats. J Investig Med 2011; 59: 1268-1272.
71 Bonello S, Zaerhinger C, Bel Aiba RS. et al. Redox sensitive activation of nuclear factor-kappa B is requiered for hypoxia factor-1alpha transcription in pulmonary artery smooth muscle cells. Circulation 2005; 112: 1246.
72 Katoh M, Egashira K, Mitsui T. et al. Angiotensin-converting enzyme inhibitor prevents plasminogen activator inhibitor-1 expression in a rat model with cardiovascular remodeling induced by chronic inhibition of nitric oxide synthesis. J Mol Cell Cardiol 2000; 32: 73-83.
73 Bouchie JL, Hansen H, Feener EP. Natriuretic factors and nitric oxide suppress plasminogen activator inhibitor-1 expression in vascular smooth muscle cells. Role of cGMP in the regulation of the plasminogen system. Arterioscler Thromb Vasc Biol 1998; 18: 1771-1779.

Supplementary Material

Online Supplementary Material (PDF)