Macrophage-Derived TGFβ Induces BIGH3 Secretion and Promotes Apoptosis of Human Retinal Endothelial Cells

Abstract

Purpose: : Diabetic retinopathy (DR) is accompanied by infiltration of activated macrophages and their secretion of TGFβ. TGFβ upregulates the expression of TGFβ - Induced Gene Human Clone 3 (BIGH3). BIGH3 is an extracellular matrix molecule that causes apoptosis of retinal microvascular cells. Apoptosis of these cells leads to leakage and vessel destruction leading to hypoxia which triggers angiogenesis an important pathological event in DR.

Methods: : Immunohistochemistry (IHC) was performed to compare levels of BIGH3 in the eyes of healthy control and diabetic mice. We studied BIGH3 expression by qPCR and protein upregulation by Western blot using Rhesus retinal endothelial cells (RhREC). This was studied in response to exogenous TGFβ and conditioned media from macrophages cultured in either regular culture media (MCM) or culture media containing high glucose and high LDL (dMCM). Apoptosis of RhREC by recombinant BIGH3, exogenous TGFβ or dMCM were measured using TUNEL assay.

Results: : IHC showed increased macrophage infiltration and BIGH3 localization in eyes from diabetic mice as compared with the eyes from normal mice. BIGH3 expression was increased in RhREC in response to dMCM and BIGH3 protein was increased with TGFβ. Recombinant BIGH3 induced apoptosis in RhREC after 24 hrs. BIGH3 antibody was able to negate this effect. TGFβ and dMCM after 48 hrs caused apoptosis.

Conclusions: : We found that TGFβ upregulates BIGH3 expression and secretion by RhREC, and that BIGH3 leads to increased apoptosis in an auto- and paracrine manner. This mechanism may contribute to the initial microvascular damage that occurs during the onset of DR.

Keywords: diabetic retinopathy • immunohistochemistry • apoptosis/cell death