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JM Doherty, B Turpie, S Masli, JW Streilein; A Potential Role For Thombospondin In Corneal Endothelial Cell Injury . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2265.
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Purpose: Thrombospondin (TSP), a matrix glycoprotein is recognized for its potent anti-angiogenic properties. For example, TSP while present in normal aqueous humor (AqH) is decreased in AqH obtained from diseased diabetic eyes suggesting a role for TSP in ocular damage. The goal of the present study is to examine the role of TSP in the regulation of corneal endothelial cell responses to inflammatory cell injuries/assaults. Methods: Immortalized BALB/c corneal endothelial cells (CE) were grown to confluence in RPMI medium + 10% FBS. Cells were incubated for 24 hrs in serum-free, phenol-free RPMI or in RPMI containing the following inflammatory agents: LPS (100 ng/ml); IFN-γ (500 U/ml); LPS/IFN-γ in combination; or TNF-α (50 ng/ml). TSP levels in CE were measured by RT-PCR and indirect immunocytochemistry. In separate experiments, exogenous TSP (1.0 µg and 0.1 µg/ml) was added to parallel cultures and CE status determined by 3H-Thymidine (3H) uptake and positive staining for Ki67, a marker of cell proliferation. Results: Cultured CE expressed TSP constitutively. Neither LPS nor IFN-γ added singularly affected TSP levels in CE. The addition of TNF-α to CE resulted in their decreased expression of TSP. In contrast, the addition of LPS/IFN-γ in combination elicited an increase in CE TSP expression. Both the addition of LPS/IFN-γ and TNF-α to CE resulted in a 41 and 29.2 % inhibition of CE proliferation, respectively when assessed by 3H-Thymidine uptake. The addition of exogenous TSP had no effect on 3H uptake either in control or LPS/IFN-γ treated CE. However, TSP restored proliferation of TNF-α treated CE to a level 1.75 fold greater than that observed with control CE. Conclusion: Corneal endothelial cells constitutively express TSP. Expression of TSP is altered upon exposure of CE to inflammatory agents such as LPS/IFN-γ and TNF-α. We suggest that CE may use thrombospondin to maintain cellular homeostasis.
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