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Albert A. Mondragon, Kalpana Parvathaneni, Mary Navarro, Chi F. Lee, Hong S. Kim, Richard G. LeBaron, Reto Asmis, Jeff G. Grigsby, Andrew T. Tsin; BIGH3 Induced Apoptosis of Human Retinal Pericytes. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2411.
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Diabetic retinopathy (DR) is the leading cause of blindness in America. One of the earliest hallmarks of DR is the loss of retinal pericytes (RPC), which help to maintain the integrity of the blood vessels. The cause is unknown, but RPC dropout can lead to leakage, hypoxia, and angiogenesis seen in proliferative DR. We propose a novel pathway where macrophage-derived TGFβ induces expression of the pro-apoptotic protein TGFβ-Induced Gene Human Clone 3 (BIGH3), thereby inducing RPC apoptosis.
Immunohistochemistry (IHC) was conducted using BIGH3 antibody and fluorescence microscopy. RPCs were treated for 24 hours in conditioned media from healthy macrophages (MCM) and macrophages cultured under diabetes-like conditions (25 mM glucose and 100 ug/ml LDL) (dMCM). The conditioned media of RPCs was probed for BIGH3 by Western blot analysis. RPCs were treated with 5 ng/mL TGFβ, and q-PCR and Western blot analysis were performed to analyze BIGH3 expression in the RPCs and protein secretion into the growth medium. A comparative apoptosis assay using BIGH3 was performed on Rhesus retinal endothelial cells (RhREC) and RPCs.
IHC showed BIGH3 localized in the extracellular spaces in the RPC monolayer. Western blot analysis showed increased BIGH3 secretion by RPCs treated with TGFβ and dMCM as compared to control cells. BIGH3 expression was elevated 3.5-fold after TGFβ stimulation. 60% of the RPCs underwent apoptosis induced by 5 µg/mL recombinant BIGH3, 24% by dMCM and 18% by exogenous TGFβ. Importantly, BIGH3 antibody blocked TGFβ and dMCM induced apoptosis. Additionally RPCs are more sensitive to BIGH3 induced apoptosis in comparison to RhREC.
We found that TGFβ secreted by macrophages up-regulated BIGH3 expression and promoted BIGH3-dependent apoptosis in RPCs. This mechanism may contribute to pericyte loss and microvascular damage in the initial stages of diabetic retinopathy and may lead to better understanding of the disease mechanism.
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