Abstract
Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO2•-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO•-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.
Similar content being viewed by others
References
Lee, P., Wang, C.C. & Adamis, A.P. Ocular neovascularization: an epidemiologic review. Surv. Ophthalmol. 43, 245–269 (1998).
Hardy, P. et al. Oxidants, nitric oxide and prostanoids in the developing ocular vasculature: a basis for ischemic retinopathy. Cardiovasc. Res. 47, 489–509 (2000).
Campochiaro, P.A. Retinal and choroidal neovascularization. J. Cell. Physiol. 184, 301–310 (2000).
Alon, T. et al. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nat. Med. 1, 1024–1028 (1995).
Pierce, E.A., Foley, E.D. & Smith, L.E. Regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity. Arch. Ophthalmol. 114, 1219–1228 (1996).
Smith, L.E. et al. Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor. Nat. Med. 5, 1390–1395 (1999).
Shih, S.C., Ju, M., Liu, N. & Smith, L.E. Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity. J. Clin. Invest. 112, 50–57 (2003).
Wang, S., Wu, Z., Sorenson, C.M., Lawler, J. & Sheibani, N. Thrombospondin-1-deficient mice exhibit increased vascular density during retinal vascular development and are less sensitive to hyperoxia-mediated vessel obliteration. Dev. Dyn. 228, 630–642 (2003).
Spierer, A., Rabinowitz, R., Pri-Chen, S. & Rosner, M. An increase in superoxide dismutase ameliorates oxygen-induced retinopathy in transgenic mice. Eye 19, 86–91 (2005).
Kowluru, R.A., Tang, J. & Kern, T.S. Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes 50, 1938–1942 (2001).
Penn, J.S., Tolman, B.L. & Bullard, L.E. Effect of a water-soluble vitamin E analog, trolox C, on retinal vascular development in an animal model of retinopathy of prematurity. Free Radic. Biol. Med. 22, 977–984 (1997).
Raju, T.N., Langenberg, P., Bhutani, V. & Quinn, G.E. Vitamin E prophylaxis to reduce retinopathy of prematurity: a reappraisal of published trials. J. Pediatr. 131, 844–850 (1997).
Squadrito, G.L. & Pryor, W.A. Oxidative chemistry of nitric oxide: the roles of superoxide, peroxynitrite, and carbon dioxide. Free Radic. Biol. Med. 25, 392–403 (1998).
Kroncke, K.D. Mechanisms and biological consequences of nitrosative stress. Biol. Chem. 384, 1341 (2003).
Gu, X. et al. Hyperoxia induces retinal vascular endothelial cell apoptosis through formation of peroxynitrite. Am. J. Physiol. Cell Physiol. 285, C546–C554 (2003).
Beauchamp, M.H. et al. Redox-dependent effects of nitric oxide on microvascular integrity in oxygen-induced retinopathy. Free Radic. Biol. Med. 37, 1885–1894 (2004).
El-Remessy, A.B., Abou-Mohamed, G., Caldwell, R.W. & Caldwell, R.B. High glucose-induced tyrosine nitration in endothelial cells: role of eNOS uncoupling and aldose reductase activation. Invest. Ophthalmol. Vis. Sci. 44, 3135–3143 (2003).
Brooks, S.E. et al. Reduced severity of oxygen-induced retinopathy in eNOS-deficient mice. Invest. Ophthalmol. Vis. Sci. 42, 222–228 (2001).
El-Remessy, A.B. et al. Experimental diabetes causes breakdown of the blood-retina barrier by a mechanism involving tyrosine nitration and increases in expression of vascular endothelial growth factor and urokinase plasminogen activator receptor. Am. J. Pathol. 162, 1995–2004 (2003).
Jiang, H. et al. Nitrogen dioxide induces cis-trans-isomerization of arachidonic acid within cellular phospholipids. Detection of trans-arachidonic acids in vivo. J. Biol. Chem. 274, 16235–16241 (1999).
Balazy, M. & Poff, C.D. Biological nitration of arachidonic acid. Curr. Vasc. Pharmacol. 2, 81–93 (2004).
Balazy, M. Trans-arachidonic acids: new mediators of inflammation. J. Physiol. Pharmacol. 51, 597–607 (2000).
Kirsch, M., Korth, H.G., Sustmann, R. & de Groot, H. The pathobiochemistry of nitrogen dioxide. Biol. Chem. 383, 389–399 (2002).
Prutz, W.A., Monig, H., Butler, J. & Land, E.J. Reactions of nitrogen dioxide in aqueous model systems: oxidation of tyrosine units in peptides and proteins. Arch. Biochem. Biophys. 243, 125–134 (1985).
Chan-Ling, T., Gock, B. & Stone, J. The effect of oxygen on vasoformative cell division. Evidence that 'physiological hypoxia' is the stimulus for normal retinal vasculogenesis. Invest. Ophthalmol. Vis. Sci. 36, 1201–1214 (1995).
Smith, L.E. Pathogenesis of retinopathy of prematurity. Growth Horm. IGF Res. 14 Suppl A, 140–4 (2004).
Caffe, A.R. et al. Mouse retina explants after long-term culture in serum free medium. J. Chem. Neuroanat. 22, 263–273 (2001).
Beauchamp, M.H. et al. Role of thromboxane in retinal microvascular degeneration in oxygen-induced retinopathy. J. Appl. Physiol. 90, 2279–2288 (2001).
Sennlaub, F. et al. Cyclooxygenase-2 in human and experimental ischemic proliferative retinopathy. Circulation 108, 198–204 (2003).
Roy, U., Loreau, O. & Balazy, M. Cytochrome P450/NADPH-dependent formation of trans epoxides from trans-arachidonic acids. Bioorg. Med. Chem. Lett. 14, 1019–1022 (2004).
Nor, J.E. et al. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J. Vasc. Res. 37, 209–218 (2000).
Armstrong, L.C. & Bornstein, P. Thrombospondins 1 and 2 function as inhibitors of angiogenesis. Matrix Biol. 22, 63–71 (2003).
Guo, N., Krutzsch, H.C., Inman, J.K. & Roberts, D.D. Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res. 57, 1735–1742 (1997).
Dawson, D.W. et al. CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J. Cell Biol. 138, 707–717 (1997).
Jimenez, B. et al. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat. Med. 6, 41–48 (2000).
Swerlick, R.A., Lee, K.H., Wick, T.M. & Lawley, T.J. Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. J. Immunol. 148, 78–83 (1992).
Wada, T. & Penninger, J.M. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 23, 2838–2849 (2004).
Ishikawa, Y. & Kitamura, M. Dual potential of extracellular signal-regulated kinase for the control of cell survival. Biochem. Biophys. Res. Commun. 264, 696–701 (1999).
Gauld, S.B., Blair, D., Moss, C.A., Reid, S.D. & Harnett, M.M. Differential roles for extracellularly regulated kinase-mitogen-activated protein kinase in B cell antigen receptor-induced apoptosis and CD40-mediated rescue of WEHI-231 immature B cells. J. Immunol. 168, 3855–3864 (2002).
Zghibeh, C.M., Raj Gopal, V., Poff, C.D., Falck, J.R. & Balazy, M. Determination of trans-arachidonic acid isomers in human blood plasma. Anal. Biochem. 332, 137–144 (2004).
Llorens, S. & Nava, E. Cardiovascular diseases and the nitric oxide pathway. Curr. Vasc. Pharmacol. 1, 335–346 (2003).
Liu, L. et al. Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock. Cell 116, 617–628 (2004).
Radi, R., Rodriguez, M., Castro, L. & Telleri, R. Inhibition of mitochondrial electron transport by peroxynitrite. Arch. Biochem. Biophys. 308, 89–95 (1994).
Tabuchi, A, Oh, E, Taoka, A, Sakurai, H, Tsuchiya, T & Tsuda . Rapid attenuation of AP-1 transcriptional factors associated with nitric oxide (NO)-mediated neuronal cell death. J Biol. Chem. 271, 31061–7 (1996).
Dameron, K.M., Volpert, O.V., Tainsky, M.A. & Bouck, N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265, 1582–1584 (1994).
Okuno, M., Arimoto, E., Nishizuka, M., Nishihara, T. & Imagawa, M. Isolation of up- or down-regulated genes in PPARgamma-expressing NIH-3T3 cells during differentiation into adipocytes. FEBS Lett. 519, 108–112 (2002).
Nielsen, J.C., Naash, M.I. & Anderson, R.E. The regional distribution of vitamins E and C in mature and premature human retinas. Invest. Ophthalmol. Vis. Sci. 29, 22–26 (1988).
Flynn, J.T. et al. A cohort study of transcutaneous oxygen tension and the incidence and severity of retinopathy of prematurity. N. Engl. J. Med. 326, 1050–1054 (1992).
Mann, R.M., Riva, C.E., Stone, R.A., Barnes, G.E. & Cranstoun, S.D. Nitric oxide and choroidal blood flow regulation. Invest. Ophthalmol. Vis. Sci. 36, 925–930 (1995).
Lahaie, I. et al. A novel mechanism for vasoconstrictor action of 8-isoprostaglandin F2 alpha on retinal vessels. Am. J. Physiol. 274, R1406–R1416 (1998).
Acknowledgements
The authors wish to thank H. Fernandez for her technical skills and help. This work was supported by grants from the Canadian Institutes of Health Research, the March of Dimes Birth Defects Foundation, the Heart and Stroke Foundation of Québec, the Fonds de la Recherche en Santé du Québec, Le Réseau de Recherche en Santé de la Vision and La Fondation du NO. E.K.-D. is recipient of a fellowship award from the 'Association des Juniors en Pédiatrie/Gallia' (France). F.S. and S.C. are recipients of fellowship and scientist awards, respectively, from the Canadian Institutes of Health Research. S.B. and M.S. are recipients of studentships from the Canadian Institutes of Health Research and Heart and Stroke Foundation of Canada, respectively. P.H. is supported by grants from the Hospital For Sick Children Foundation and Fonds de la Recherche en Santé du Québec. M.B. is supported by grants from the US National Institutes of Health (R01 GM62453) and Philip Morris USA, Inc. S.C. also holds a Canada Research Chair (perinatology). The authors thank M. Febbraio and J. Lawler, who provided the CD36 and TSP-1 knockout animals, respectively.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Fig. 1
TAA-induced endothelial cell death is independent of classic arachidonic acid pathways. (PDF 53 kb)
Rights and permissions
About this article
Cite this article
Kermorvant-Duchemin, E., Sennlaub, F., Sirinyan, M. et al. Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1–dependent microvascular degeneration. Nat Med 11, 1339–1345 (2005). https://doi.org/10.1038/nm1336
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nm1336
- Springer Nature America, Inc.
This article is cited by
-
Early prediction of severe retinopathy of prematurity requiring laser treatment using physiological data
Pediatric Research (2023)
-
Tetrahydrobiopterin (BH4) deficiency is associated with augmented inflammation and microvascular degeneration in the retina
Journal of Neuroinflammation (2017)
-
Neonatal systemic inflammation in rats alters retinal vessel development and simulates pathologic features of retinopathy of prematurity
Journal of Neuroinflammation (2014)
-
Risk Factors for Retinopathy of Prematurity: Beyond Age, Birth Weight, and Oxygen
Current Ophthalmology Reports (2013)
-
Endothelial nitric oxide synthase, vascular integrity and human exceptional longevity
Immunity & Ageing (2012)