Endothelial NO Synthase Is Increased in Regenerating Endothelium After Denuding Injury of the Rat Aorta

From the Department of Medicine, Division of Cardiology, University of Washington, Seattle.

Correspondence to Bradford C. Berk, MD, PhD, University of Washington, Division of Cardiology, Box 357710, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu

Abstract—Endothelial nitric oxide synthase (eNOS) has been shown to be regulated both transcriptionally and posttranslationally in cultured endothelial cells, but eNOS regulatory mechanisms in vivo have not been elucidated. Because one of the strongest stimuli for eNOS expression in tissue culture is cell proliferation and because increased NO production would be beneficial in the setting of arterial injury, we hypothesized that eNOS expression should be increased in regenerating endothelium after a denuding injury. Rat aortas underwent partial endothelial denudation by passage of a deflated balloon catheter, and eNOS expression was studied 48 hours after injury. Immunohistochemistry with eNOS monoclonal antibody, NADPH diaphorase activity assay under conditions specific for eNOS, and mRNA hybridization were performed in situ on perfusion-fixed rat aortic segments. The vessels were studied en face to enhance visualization compared with cross sections. eNOS protein and mRNA expression were significantly increased in regenerating and migrating endothelial cells at the wound edge, with translocation of eNOS to the plasma membrane at the leading edge. Similar results were obtained when endothelial cells were studied in a tissue culture wound model. An important role for transforming growth factor (TGF)-ß₁ in regulating eNOS expression was suggested by the ability of a TGF-ß₁–neutralizing antibody to limit induction of eNOS at the wound edge. Increased eNOS expression after wounding appears to be related to signal events associated with cell migration as well as proliferation, because eNOS expression in vivo increased in nonproliferating cells and TGF-ß₁–neutralizing antibody inhibited eNOS expression but stimulated proliferation. The current study is the first to suggest an important role in vivo for increased eNOS, and perhaps NO production, in the process of endothelial regeneration and wound repair.

Key Words: endothelial nitric oxide synthase • endothelial regeneration • balloon injury • transforming growth factor-ß₁

This article has been cited by other articles:

				S. Pan, C. J. World, C. J. Kovacs, and B. C. Berk Glucose 6-Phosphate Dehydrogenase Is Regulated Through c-Src-Mediated Tyrosine Phosphorylation in Endothelial Cells Arterioscler Thromb Vasc Biol, June 1, 2009; 29(6): 895 - 901. [Abstract] [Full Text] [PDF]

				J. M. van Golde, M. S. Ruiter, N. C. Schaper, S. Voo, J. Waltenberger, W. H. Backes, M. J. Post, and M. S. Huijberts Impaired Collateral Recruitment and Outward Remodeling in Experimental Diabetes Diabetes, October 1, 2008; 57(10): 2818 - 2823. [Abstract] [Full Text] [PDF]

				C. Filipe, L. Lam Shang Leen, L. Brouchet, A. Billon, V. Benouaich, V. Fontaine, P. Gourdy, F. Lenfant, J.-F. Arnal, A.-P. Gadeau, et al. Estradiol accelerates endothelial healing through the retrograde commitment of uninjured endothelium Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2822 - H2830. [Abstract] [Full Text] [PDF]

				A. Billon, S. Lehoux, L. Lam Shang Leen, H. Laurell, C. Filipe, V. Benouaich, L. Brouchet, C. Dessy, P. Gourdy, A.-P. Gadeau, et al. The Estrogen Effects on Endothelial Repair and Mitogen-Activated Protein Kinase Activation Are Abolished in Endothelial Nitric-Oxide (NO) Synthase Knockout Mice, but Not by NO Synthase Inhibition by N-Nitro-L-arginine Methyl Ester Am. J. Pathol., March 1, 2008; 172(3): 830 - 838. [Abstract] [Full Text] [PDF]

				E. Grad, M. Golomb, I. Mor-Yosef, N. Koroukhov, C. Lotan, E. R. Edelman, and H. D. Danenberg Transgenic expression of human C-reactive protein suppresses endothelial nitric oxide synthase expression and bioactivity after vascular injury Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H489 - H495. [Abstract] [Full Text] [PDF]

				C. D. Searles Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression Am J Physiol Cell Physiol, November 1, 2006; 291(5): C803 - C816. [Abstract] [Full Text] [PDF]

				L. Moldovan, K. Mythreye, P. J. Goldschmidt-Clermont, and L. L. Satterwhite Reactive oxygen species in vascular endothelial cell motility. Roles of NAD(P)H oxidase and Rac1 Cardiovasc Res, July 15, 2006; 71(2): 236 - 246. [Abstract] [Full Text] [PDF]

				X. Pi, G. Garin, L. Xie, Q. Zheng, H. Wei, J.-i. Abe, C. Yan, and B. C. Berk BMK1/ERK5 Is a Novel Regulator of Angiogenesis by Destabilizing Hypoxia Inducible Factor 1{alpha} Circ. Res., June 10, 2005; 96(11): 1145 - 1151. [Abstract] [Full Text] [PDF]

				E. Lopez-Rivera, T. R. Lizarbe, M. Martinez-Moreno, J. M. Lopez-Novoa, A. Rodriguez-Barbero, J. Rodrigo, A. P. Fernandez, A. Alvarez-Barrientos, S. Lamas, and C. Zaragoza Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration PNAS, March 8, 2005; 102(10): 3685 - 3690. [Abstract] [Full Text] [PDF]

				S. C. Tai, G. B. Robb, and P. A. Marsden Endothelial Nitric Oxide Synthase: A New Paradigm for Gene Regulation in the Injured Blood Vessel Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 405 - 412. [Abstract] [Full Text]

				R. Komers and S. Anderson Paradoxes of nitric oxide in the diabetic kidney Am J Physiol Renal Physiol, June 1, 2003; 284(6): F1121 - F1137. [Abstract] [Full Text] [PDF]

				J. Ibrahim, J. K. Miyashiro, and B. C. Berk Shear Stress Is Differentially Regulated Among Inbred Rat Strains Circ. Res., May 16, 2003; 92(9): 1001 - 1009. [Abstract] [Full Text] [PDF]

				N. Peunova, V. Scheinker, H. Cline, and G. Enikolopov Nitric Oxide Is an Essential Negative Regulator of Cell Proliferation in Xenopus Brain J. Neurosci., November 15, 2001; 21(22): 8809 - 8818. [Abstract] [Full Text] [PDF]

				J. M. Edelberg, P. D. Christie, and R. D. Rosenberg Regulation of Vascular Bed-Specific Prothrombotic Potential Circ. Res., July 20, 2001; 89(2): 117 - 124. [Abstract] [Full Text] [PDF]

				L. C.P. Azevedo, M. d. A. Pedro, L. C. Souza, H. P. de Souza, M. Janiszewski, P. L. da Luz, and F. R.M. Laurindo Oxidative stress as a signaling mechanism of the vascular response to injury: The redox hypothesis of restenosis Cardiovasc Res, August 18, 2000; 47(3): 436 - 445. [Abstract] [Full Text] [PDF]

				C. Tsuji, S. Shioya, Y. Hirota, N. Fukuyama, D. Kurita, T. Tanigaki, Y. Ohta, and H. Nakazawa Increased production of nitrotyrosine in lung tissue of rats with radiation-induced acute lung injury Am J Physiol Lung Cell Mol Physiol, April 1, 2000; 278(4): L719 - L725. [Abstract] [Full Text] [PDF]

				C. D. Searles, Y. Miwa, D. G. Harrison, and S. Ramasamy Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase During Cell Growth Circ. Res., October 1, 1999; 85(7): 588 - 595. [Abstract] [Full Text] [PDF]

				C. A. E. MASON, P. CHANG, C. FALLERY, and M. RABINOVITCH Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation FASEB J, August 1, 1999; 13(11): 1423 - 1434. [Abstract] [Full Text]

				R. D. Rosenberg and W. C. Aird Vascular-Bed-Specific Hemostasis and Hypercoagulable States N. Engl. J. Med., May 20, 1999; 340(20): 1555 - 1564. [Full Text] [PDF]