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.

Thrombospondin-2 Expression During Retinal Vascular Development and Neovascularization

“…We have shown that reduced levels of TSP1 expression in EC in culture are generally associated with increased level of TSP2. 22,23 It is not known whether lack of TSP2 results increased level of TSP1. Here, we observed decreased levels of TSP1 expression in TSP2 null retina at various postnatal days when compared with TSP2 + / + mouse retina.…”

“…22,23 However, whether lack of TSP2 results in increased expression of TSP1 remains unknown. Here, we examined the expression of TSP1 in retinas prepared from TSP2…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…This result was subsequently confirmed in choroidal endothelial cells (53). Choroidal capillary endothelial cells from the eyes of Thbs1 -/-mice had elevated phosphorylation of eNOS relative to WT cells, and intracellular NO in the null cells assessed using 4-amino-5-methylamino-2,7-difluorofluorescein (DAF) was sixfold higher than in WT cells.…”

“…However, a recent study reported elevated TSP2 levels produced by Thbs1 -/-choroidal endothelial cells despite having elevated eNOS phosphorylation and NO production as assessed by 4-amino-5-methylamino-2¢,7¢-difluorofluorescein diacetate fluorescence (53).…”

“…Notably, Fei et al also reported that inducible nitric oxide synthase (iNOS) expression was elevated in Thbs1 -/-choroidal endothelial cells relative to WT cells, suggesting that negative regulation of NO synthesis by TSP1 is not limited to eNOS (53), although it was not determined whether these findings in null cells were reversed by exogenous TSP1.…”