title: Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells.
abstract: The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1-/-) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1-/- ChEC migration and capillary morphogenesis. TSP1-/- ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1-/- ChEC produced similar levels of VEGF, although TSP1-/- ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Ping
authors.initials: P
authors.lastname: Fei
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Ismail
authors.initials: I
authors.lastname: Zaitoun
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Mitra
authors.initials: M
authors.lastname: Farnoodian
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Debra L
authors.initials: DL
authors.lastname: Fisk
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Shoujian
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America; McPherson Eye Research Institute, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Christine M
authors.initials: CM
authors.lastname: Sorenson
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America; McPherson Eye Research Institute, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America; Department of Biomedical Engineering, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin, United States of America.
authors.forename: Nader
authors.initials: N
authors.lastname: Sheibani
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D049109:Cell Proliferation; D002478:Cells, Cultured; D002829:Choroid / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D018384:Oxidative Stress; D015398:Signal Transduction; D019700:Thrombospondin 1 / Q000235:genetics* / Q000378:metabolism*; D019699:Thrombospondins / Q000378:metabolism; D000099262:Thrombospondin 2
chemical_list: D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D019700:Thrombospondin 1; D019699:Thrombospondins; D000099262:Thrombospondin 2; C585483:Thbs1 protein, mouse