BIGH3: A New Protein in Diabetic Retinopathy Complications

Abstract

Purpose: : Diabetic retinopathy, initiated by retinal pericyte drop-out, is accompanied by increased infiltration of activated macrophages and their secretion of TGFβ. The extracellular matrix molecule TGFβ-Induced Gene Human Clone 3 (BIGH3) is a proapoptotic protein that is upregulated by TGFβ. We investigated BIGH3 mRNA and protein upregulation by cultured monkey retinal endothelial cells (MREC) in response to macrophage-derived TGFβ and exogenous TGFβ1 and 2, apoptosis of cultured human retinal pericytes (HRP) in response to recombinant BIGH3, and used IHC to compare levels of BIGH3 in the eyes of diabetic and normal mice.

Methods: : Cultured MREC were treated for 24 hours in conditioned media from macrophages cultured in media containing high glucose and high LDL (macrophage-conditioned media - MCM), The conditioned media and cell lysate were analyzed by q-PCR and Western blot for increased BIGH3 expression and protein. MREC were also treated in MEM with 5 ng/ml TGFβ1 and 2 and Western Blot was performed for BIGH3. Immunohistochemistry was performed on slices from the eyes of normal and diabetic mice. Cultured HRP were treated for 24 hours with 10 µg/ml recombinant BIGH3 protein and assayed for apoptosis using the ssDNA method. MCM was analyzed by ELISA for the presence of TGFβ1 and 2.

Results: : Elevated levels of active TGFβ1 and 2 were found in the MCM from macrophages activated by high glucose and high LDL as compared with non-activated MCM. BIGH3 expression was increased 3-fold in cultured monkey retinal endothelial cells in response to 24 hour treatment with MCM, and BIGH3 protein was increased 3.5-fold by 24 hour treatment with 5 ng/ml TGFβ1 or 2. Recombinant BIGH3 induced increased apoptosis in retinal pericytes (RP). Elevated levels of secreted BIGH3 were detected by IHC in eyes from diabetic mice as compared with the eyes from normal mice.

Conclusions: : We found that macrophage-secreted TGFβ1 and 2 upregulated BIGH3 message and protein by MREC, and that BIGH3 treatment of human retinal pericytes led to increased apoptosis. This may contribute to pericyte loss associated with diabetic retinopathy.

Keywords: diabetic retinopathy