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N. Sheibani, E. A. Scheef, C. M. Sorenson; Attenuation of Proliferation and Migration of Retinal Pericytes Cultured From Thrombospondin-1-Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2931.
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Establish primary cultures of retinal pericytes (PC) from wild type (TSP1+/+) and thrombospondin-1-deficient (TSP1-/-) mice and determine the impact lack of TSP1 has on the properties of retinal PC.
Using TSP1+/+ and TSP1-/- immortomice we isolated retinal PC by culturing dissociated retinal cells on un-coated tissue culture plates. The expression of various perivascular and mesenchymal stem cell markers were determined by FACScan analysis. Cell proliferation was determined by counting the number of cells for two weeks and EdU- labeling. The rates of apoptosis were determined by TUNEL labeling.
We show, for the first time, the successful isolation and culture of primary retinal PC from TSP1+/+ and TSP1-/- mice. We show these cells express early and mature markers of PC, including NG2, PDGF-receptor β (PDGF-Rβ), and smooth muscle actin, as well as desmin, calbindin, and mesenchymal stem cell markers such as Sca1, CD11b, CD45, and CD90.1. TSP1+/+ PC proliferated at a faster rate compared to TSP1-/- PC. In addition, TSP1+/+ PC responded to PDGF-BB with enhanced migration and proliferation. In contrast, TSP1-/- PC failed to respond to the promigratory and proliferative activity of PDGF-BB. We observed no significant differences in the rates of apoptosis in these cells. TSP1-/- PC were also less adherent, expressed increased levels of TSP2 and fibronectin, and had decreased amounts of N-cadherin and vβ3 integrin on their surface.
The expression of TSP1 is essential for PC’s proliferative and migratory response to PDGF. Thus, TSP1 may play a significant role in retinal vascular development and homeostasis.
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