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Y. Tan, C. A. Medina-Mendez, R. E. Martinez, E. J. Ewald, G. Puig, P. P. Truong, V. L. Perez; Subconjuctival Administration of Endostatin Improves Corneal Allograft Survival. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1966.
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Inflammation and neovascularization predispose to corneal allograft rejection. Endostatin, an endogenous inhibitor of angiogenesis, is a 20 kDa physiologic cleavage product of collagen XVIII . We propose the hypothesis that Endostatin plays an important role in the inhibition of corneal neovascularizaion and promotes increased corneal allograft survival.
MHC mismatched corneal allotransplants (C57BL/6 →Balb/c) as well as MHC matched syngenic transplants (Balb/c →Balb/c) were performed. Endostatin or PBS were injected subconjuctivally every 3 day from day 0 after transplantation. Corneal grafts were evaluated twice a week and a clinical score for opacification was given using slit lamp biomicroscopy. By day 40 mice were sacrificed and DiI blood perfusion was performed to stain the vessels. Real Time PCR and immunostaining were conducted to determine the VEGFR2, CD3, CD4, and CD8 of the corneas. To test the role of endostatin in T cell activation, in vitro CD4+ T cells were stimulated with CD3/CD28 antibodies and co-cultured with Endostatin. CFSE proliferation assay, ELISA and Flow Cytometry were conducted to determine proliferation, cytokine production and activation of CD4+ T cells.
By day 27 after transplantation PBS treated allografts started to show evidence of rejection in contrast to Endostatin treated grafts that survived up to 40 days during treatment (P=0.0077, 10 mice per group). Corneal graft vascularization measured by confocal microscopy of DiI stained blood vessesl confirmed that the absence of neovascularization in accepted Endostatin treated corneal allografts, while the PBS treated group was rejected and vascularized. Real Time PCR of corneal RNA extracts showed that VEGFR2, CD3, CD4 and CD8 was inhibited in Endostatin treated allografts. Immunohistochemical analysis confirmed the lack of recruitment of T cells in surviving grafts. Furthermore, in vitro analysis of T cell activation showed that Endostatin didn’t alter the proliferation, cytokine production and activation of CD4+ T CD28/CD3 stimulation.
Local administration of Endostatin can prolong corneal allograft survival. This effect appears to be secondary to inhibition of corneal graft neovascularization and not T cell activation, resulting in a significant decrease recruitment of effector T cells into the site of transplantation.
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