Pulsatile flow increases the expression of eNOS, ET-1, and prostacyclin in a novel in vitro coculture model of the retinal vasculature.

PURPOSE By the development of a novel retinal microvascular endothelial and pericyte cell coculture system, this study determined the effects of pulsatile flow on the activation of the endothelial cell markers nitric oxide (NO), prostacyclin (PGI2), and endothelin (ET)-1. METHODS Monocultured bovine retinal endothelial cells (BRECs) and cocultured BRECs with bovine retinal pericytes (BRPs) were exposed to low flow (flow rate, 0.3 mL/min; pulse pressure, 6 mmHg; shear stress, 0.5 dyne/cm2) or high flow (flow rate, 25 mL/min; pulse pressure, 56 mmHg; shear stress, 23 dynes/cm2) for 24 hours, by using a novel perfused transcapillary culture system. The cells were characterized by immunohistochemistry and electron and confocal microscopy. Endothelial nitric oxide synthase (eNOS) and phosphorylated-eNOSSer1179 (pp-eNOS) were determined by Western blot analysis. Nitrate, PGI2, and ET-1 levels were quantified in the medium perfusate by using fluorometric and enzyme-linked immunosorbent assays, respectively. Activation of cyclooxygenase (COX)-2 in BRECs was determined by measuring COX-2 promoter activity with a luciferase reporter assay. RESULTS The presence of BRPs and BRECs was confirmed by Western blot, immunocytochemistry, and scanning electron microscopy. Phosphorylated eNOS (pp-eNOS) protein levels in BRECs were significantly increased from low to high flow in both mono- and cocultures, concomitant with a significant increase in nitrate levels in the conditioned medium after exposure to pulsatile flow. In parallel cultures, PGI2 levels were also significantly enhanced concomitant with an increase in the transactivation of a COX-2 promoter BREC after exposure to pulsatile flow. ET-1 levels were also increased in both mono- and cocultured cells. conclusions. In this study a novel, functioning, in vitro model of retinal microvascular endothelial and pericyte cells that respond to changes in pulsatile flow was established.

[1]  M. Cellini,et al.  Color Doppler imaging and plasma levels of endothelin-1 in low-tension glaucoma. , 2009, Acta ophthalmologica Scandinavica. Supplement.

[2]  R. Fernandes,et al.  High Glucose Down-regulates Intercellular Communication in Retinal Endothelial Cells by Enhancing Degradation of Connexin 43 by a Proteasome-dependent Mechanism* , 2004, Journal of Biological Chemistry.

[3]  L. Schmetterer,et al.  Reduced response of retinal vessel diameters to flicker stimulation in patients with diabetes , 2004, British Journal of Ophthalmology.

[4]  J. Flammer,et al.  Increased Endothelin-1 Plasma Level in Young Adults with Retinal Vascular Occlusive Diseases , 2004, Klinische Monatsblatter fur Augenheilkunde.

[5]  José Pedro De La Cruz,et al.  Pharmacological approach to diabetic retinopathy. , 2004, Diabetes/metabolism research and reviews.

[6]  An-Fei Li,et al.  High glucose alters connexin 43 expression and gap junction intercellular communication activity in retinal pericytes. , 2003, Investigative ophthalmology & visual science.

[7]  T. Yorio,et al.  Eyeing endothelins: A cellular perspective , 2003, Molecular and Cellular Biochemistry.

[8]  R. Klein,et al.  Retinal vessel diameters and their associations with age and blood pressure. , 2003, Investigative ophthalmology & visual science.

[9]  M. Bartoli,et al.  Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives , 2003, Diabetes/metabolism research and reviews.

[10]  Jing Zheng,et al.  Effects of Pulsatile Shear Stress on Nitric Oxide Production and Endothelial Cell Nitric Oxide Synthase Expression by Ovine Fetoplacental Artery Endothelial Cells1 , 2003, Biology of reproduction.

[11]  L. Schmetterer,et al.  Nitric oxide regulates retinal vascular tone in humans. , 2003, American journal of physiology. Heart and circulatory physiology.

[12]  Y. Courtois,et al.  Cyclooxygenase-2 in Human and Experimental Ischemic Proliferative Retinopathy , 2003, Circulation.

[13]  L. Schmetterer,et al.  Regulation of human retinal blood flow by endothelin-1. , 2003, Experimental eye research.

[14]  M. Arrebola,et al.  Effects of the selective inhibition of platelet thromboxane synthesis on the platelet‐subendothelium interaction , 2002, British journal of pharmacology.

[15]  Richard B. Rosen,et al.  Retinal Blood Flow in the Normal Human Eye Using the Canon Laser Blood Flowmeter , 2002, Ophthalmic Research.

[16]  A. Yoshida,et al.  The effect of nitric oxide on retinal blood flow during hypoxia in cats. , 2002, Investigative ophthalmology & visual science.

[17]  YojiTaba,et al.  Transcriptional and Posttranscriptional Regulation of Cyclooxygenase-2 Expression by Fluid Shear Stress in Vascular Endothelial Cells , 2002 .

[18]  E. Stefánsson,et al.  The impact of ocular blood flow in glaucoma , 2002, Progress in Retinal and Eye Research.

[19]  T. Yorio,et al.  Endothelin: is it a contributor to glaucoma pathophysiology? , 2002, Journal of glaucoma.

[20]  M. Wolzt,et al.  Ocular hyperperfusion following onset of intensified insulin therapy is inversely correlated with plasma endothelin-1 in Type I diabetes , 2002, Diabetologia.

[21]  B. Helmke,et al.  The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis. , 2002, Biorheology.

[22]  S. Cai,et al.  Shear-induced changes in endothelin-1 secretion of microvascular endothelial cells. , 2002, Microvascular research.

[23]  T. Sugiyama,et al.  POSSIBLE INVOLVEMENT OF ENDOTHELIN–1 AND NITRIC OXIDE IN THE PATHOGENESIS OF PROLIFERATIVE DIABETIC RETINOPATHY , 2001, Retina.

[24]  L. Schmetterer,et al.  Role of Nitric Oxide in the Control of Ocular Blood Flow , 2001, Progress in Retinal and Eye Research.

[25]  M. Murakami,et al.  Coupling between Cyclooxygenase, Terminal Prostanoid Synthase, and Phospholipase A2 * , 2001, The Journal of Biological Chemistry.

[26]  L. Kagemann,et al.  Simultaneous management of blood flow and IOP in glaucoma. , 2001, Acta ophthalmologica Scandinavica.

[27]  R. Langenbach,et al.  Why there are two cyclooxygenase isozymes. , 2001, The Journal of clinical investigation.

[28]  G. Chiou.  Review: effects of nitric oxide on eye diseases and their treatment. , 2001, Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics.

[29]  T. Lüscher,et al.  Endothelium-dependent Vasoactive Modulation in the Ophthalmic Circulation , 2001, Progress in Retinal and Eye Research.

[30]  L. Aiello,et al.  Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor. , 2000, The Journal of clinical investigation.

[31]  R. Talanow,et al.  Regulation of the endothelin system by shear stress in human endothelial cells , 2000, The Journal of physiology.

[32]  R. Aebersold,et al.  Identification of Flow-dependent Endothelial Nitric-oxide Synthase Phosphorylation Sites by Mass Spectrometry and Regulation of Phosphorylation and Nitric Oxide Production by the Phosphatidylinositol 3-Kinase Inhibitor LY294002* , 1999, The Journal of Biological Chemistry.

[33]  D. Webb,et al.  Peripheral endothelial dysfunction in normal pressure glaucoma. , 1999, Investigative ophthalmology & visual science.

[34]  B. Sumpio,et al.  Phosphatidylinositol-3 kinase dependent MAP kinase activation via p21ras in endothelial cells exposed to cyclic strain. , 1999, Biochemical and biophysical research communications.

[35]  P. Cahill,et al.  Sustained pulsatile flow regulates endothelial nitric oxide synthase and cyclooxygenase expression in co-cultured vascular endothelial and smooth muscle cells. , 1999, Journal of molecular and cellular cardiology.

[36]  S. Alper,et al.  Endothelin-1 gene suppression by shear stress: pharmacological evaluation of the role of tyrosine kinase, intracellular calcium, cytoskeleton, and mechanosensitive channels. , 1999, Journal of molecular and cellular cardiology.

[37]  T. Gardiner,et al.  Endothelium-derived agents in Pericyte function/dysfunction , 1999, Progress in Retinal and Eye Research.

[38]  M. Murakami,et al.  Functional Coupling Between Various Phospholipase A2s and Cyclooxygenases in Immediate and Delayed Prostanoid Biosynthetic Pathways* , 1999, The Journal of Biological Chemistry.

[39]  K. Umesono,et al.  Glucocorticoid-mediated suppression of the promoter activity of the cyclooxygenase-2 gene is modulated by expression of its receptor in vascular endothelial cells. , 1999, Biochemical and biophysical research communications.

[40]  W. Sessa,et al.  Induction of nitric oxide synthase mRNA by shear stress requires intracellular calcium and G-protein signals and is modulated by PI 3 kinase. , 1999, Biochemical and biophysical research communications.

[41]  A. Tedgui,et al.  Signal transduction of mechanical stresses in the vascular wall. , 1998, Hypertension.

[42]  B. Berk,et al.  Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[43]  S. Orgül,et al.  Endothelium-derived vasoactive substances relevant to normal-tension glaucoma , 1998, Current opinion in ophthalmology.

[44]  P. Cahill,et al.  Enhanced cyclooxygenase‐1 expression within the superior mesenteric artery of portal hypertensive rats: Role in the hyperdynamic circulation , 1998, Hepatology.

[45]  M. Gimbrone,et al.  Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[46]  T. Sugiyama,et al.  Association of endothelin-1 with normal tension glaucoma: clinical and fundamental studies. , 1995, Survey of ophthalmology.

[47]  T. Lüscher,et al.  The vascular endothelium as a regulator of the ocular circulation: a new concept in ophthalmology? , 1994, Survey of ophthalmology.

[48]  M. Currie,et al.  A fluorometric assay for the measurement of nitrite in biological samples. , 1993, Analytical biochemistry.

[49]  Kohner Em.  The retinal blood flow in diabetes. , 1993 .

[50]  D. Harrison,et al.  Hypercholesterolemia increases endothelial superoxide anion production. , 1993, The Journal of clinical investigation.

[51]  R M Nerem,et al.  Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. , 1992, The Journal of clinical investigation.

[52]  B. Haendler,et al.  Molecular Cloning of Human Endothelin (ET) Receptors ETA and ETB , 1992, Journal of cardiovascular pharmacology.

[53]  R. Cohen,et al.  Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C. , 1991, The Journal of clinical investigation.

[54]  M. Yanagisawa,et al.  The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[55]  Sadao Kimura,et al.  A novel potent vasoconstrictor peptide produced by vascular endothelial cells , 1988, Nature.

[56]  Christopher J. Bulpitt,et al.  The retinal blood flow in diabetes , 2004, Diabetologia.

[57]  D. Harrison,et al.  Downloaded from http://circres.ahajournals.org / by guest on February 23, 2013Shear Stress Regulates Endothelial Nitric Oxide Synthase Expression Through c-Src by Divergent Signaling Pathways , 2001 .

[58]  T. Sano,et al.  [Diabetic retinopathy]. , 2001, Nihon rinsho. Japanese journal of clinical medicine.

[59]  H. Herschman,et al.  Function and regulation of prostaglandin synthase 2. , 1999, Advances in experimental medicine and biology.

[60]  P. Cahill,et al.  Flow-mediated regulation of G-protein expression in cocultured vascular smooth muscle and endothelial cells. , 1998, Arteriosclerosis, thrombosis, and vascular biology.

[61]  L V McIntire,et al.  Shear stress and cyclic strain modulation of gene expression in vascular endothelial cells. , 1995, Blood purification.