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R. Arita, Y. Hata, T. Kita, S. Kawahara, S. Nakao, T. Ishibashi; Critical Role of TNF in Diabetic Retinal Microvascular Damage Mediated Through Rock Pathway. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4977.
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© ARVO (1962-2015); The Authors (2016-present)
Inflammation is thought to play an important role in the pathogeneses of diabetic retinopathy. Particularly, the involvement of a pro-inflammatory cytokine tumor neorosis facter (TNF)-a has been indicated. However, its precise mechanisms are not fully understood. We previously revealed the importance of Rho/Rho-kinase (ROCK) pathway in adherent leukocytes-induced diabetic microvascular damage. We herein investigated the involvement of TNF- in diabetic retinal microvascular damage mediated through Rho/ROCK pathway.
In cultured human microvascular endothelial cells and neutrophils isolated from human peripheral blood, we investigated phosphorylation of MYPT-1, a downstream mediator of ROCK, and the surface expression of CD11b/CD18 on neutrophils in the presence of recombinant TNF- or serum from diabetic retinopathy (DR) patients with or without fasudil (ROCK inhibitor), SB203580 (p38MAPK inhibitor) or PD98059 (MEK1 inhibitor) by western blotting and flow cytometry. Moreover, in co-culture system of these cells, we investigated the effects of TNF- neutralizing antibody by measuring the number of adherent neutrophils and TUNEL-positive endothelial cells. In vivo, we investigated the effect of soluble TNF- receptor (sTNFR: 500 µg/kg subcutaneously 3 times a week for a month) on MYPT-1 phosphorylation, CD11b/18 expression on neutrophils, leukocyte adhesion and endothelial damage in diabetic rats’ retinal tissue.
In vitro, TNF- and serum from DR patients promoted MYPT-1 phosphorylation in endothelial cells and neutrophils. TNF- also increased the expression of CD11b/18 on neutrophils. In addition, fasudil completely and PD98059 partially suppressed TNF-a induced MYPT-1 phosphorylation and CD11b/18 expression on neutrophils. TNF- neutralizing antibody significantly suppressed serum induced MYPT-1 phosphorylation and decreased the number of adherent neutrophils and TUNEL-positive endothelial cells in co-culture system.In vivo, sTNFR similarly suppressed diabetes-induced MYPT-1 phosphorylation in neutrophils and retinal tissue, CD11b/18 expression on neutrophils, leukocyte adhesion and endothelial damage.
These data suggest that TNF- has a potential to activate ROCK pathway in diabetic retinal microvascular system through p44/42MAPK at least in part, and cause diabetic microvascular damage. TNF- blockade could be a novel target for the prevention of diabetic retinopathy in its early phase.
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