title: CYP1B1 and endothelial nitric oxide synthase combine to sustain proangiogenic functions of endothelial cells under hyperoxic stress.
abstract: We have recently shown that deletion of constitutively expressed CYP1B1 is associated with attenuation of retinal endothelial cell (EC) capillary morphogenesis (CM) in vitro and angiogenesis in vivo. This was largely caused by increased intracellular oxidative stress and increased production of thrombospondin-2, an endogenous inhibitor of angiogenesis. Here, we demonstrate that endothelium nitric oxide synthase (eNOS) expression is dramatically decreased in the ECs prepared from retina, lung, heart, and aorta of CYP1B1-deficient (CYP1B1(-/-)) mice compared with wild-type (CYP1B1(+/+)) mice. The eNOS expression was also decreased in retinal vasculature of CYP1B1(-/-) mice. Inhibition of eNOS activity in cultured CYP1B1(+/+) retinal ECs blocked CM and was concomitant with increased oxidative stress, like in CYP1B1(-/-) retinal ECs. In addition, expression of eNOS in CYP1B1(-/-) retinal ECs or their incubation with a nitric oxide (NO) donor enhanced NO levels, lowered oxidative stress, and improved cell migration and CM. Inhibition of CYP1B1 activity in the CYP1B1(+/+) retinal ECs resulted in reduced NO levels and attenuation of CM. In contrast, expression of CYP1B1 increased NO levels and enhanced CM of CYP1B1(-/-) retinal ECs. Furthermore, attenuation of CYP1B1 expression with small interfering RNA proportionally lowered eNOS expression and NO levels in wild-type cells. Together, our results link CYP1B1 metabolism in retinal ECs with sustained eNOS activity and NO synthesis and/or bioavailability and low oxidative stress and thrombospondin-2 expression. Thus CYP1B1 and eNOS cooperate in different ways to lower oxidative stress and thereby to promote CM in vitro and angiogenesis in vivo.
authors.affiliation: Univ. of Wisconsin, Dept. of Ophthalmology and Visual Sciences, 600 Highland Ave., K6458 CSC, Madison, WI 53792-4673, USA.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Tang
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Scheef
authors.forename: Zafer
authors.initials: Z
authors.lastname: Gurel
authors.forename: Christine M
authors.initials: CM
authors.lastname: Sorenson
authors.forename: Colin R
authors.initials: CR
authors.lastname: Jefcoate
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D001011:Aorta / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D003331:Coronary Vessels / Q000378:metabolism; D065633:Cytochrome P-450 CYP1B1; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D018496:Hyperoxia / Q000201:enzymology*; D008168:Lung / Q000098:blood supply; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010641:Phenotype; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D012171:Retinal Vessels / Q000378:metabolism; D019699:Thrombospondins / Q000378:metabolism; D000099262:Thrombospondin 2
chemical_list: D000975:Antioxidants; D001189:Aryl Hydrocarbon Hydroxylases; D065633:Cytochrome P-450 CYP1B1; D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D020030:Nitric Oxide Donors; D052250:Nitric Oxide Synthase Type III; D017382:Reactive Oxygen Species; D019699:Thrombospondins; D000099262:Thrombospondin 2; C496315:Nos3 protein, mouse; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse