Modulation of retinal capillary endothelial cells by Müller glial cell-derived factors

Abstract

Purpose: The inner blood-retinal barrier (BRB) is a gliovascular unit in which macroglial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. The purpose of the present study was to identify genes of retinal capillary endothelial cells whose expression is modulated by Müller glial cell-derived factors.

Methods: Conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) and Müller (TR-MUL5) cell lines were chosen as an in vitro model. TR-iBRB2 cells were incubated with conditioned medium of TR-MUL5 (MUL-CM) for 24 h and subjected to microarray and quantitative real-time PCR analysis.

Results: TR-MUL5 cell-derived factors increased alkaline phosphatase activity in TR-iBRB2 cells, indicating that paracrine interactions occurred between TR-iBRB2 and TR-MUL5 cells. Microarray analysis demonstrated that MUL-CM treatment leads to a modulation of several genes including an induction of plasminogen activator inhibitor 1 (PAI-1) and a suppression of an inhibitor of DNA binding 2 (Id2) in TR-iBRB2 cells. Treatment with TGF-beta1, which is incorporated in MUL-CM, also resulted in an induction of PAI-1 and a suppression of Id2 in TR-iBRB2 cells.

Conclusions: In vitro inner BRB model study revealed that Müller glial cell-derived factors modulate endothelial cell functions including the induction of anti-angiogenic PAI-1 and the suppression of pro-angiogenic Id2. Therefore, Müller cells appear to be one of the modulators of retinal angiogenesis.

Figure 1

In vitro cell culture models. These models were used for analyzing paracrine interactions between retinal capillary endothelial and Müller cells. A: In the transfilter co-culture, TR-MUL5 cells were situated on the basolateral aspect of TR-iBRB2 cells monolayer as might occur in vivo. B: The fivefold concentrated conditioned medium of TR-MUL5 cells (MUL-CM) was applied to TR-iBRB2 cells.

Figure 2

Alkaline phosphatase activity in TR-iBRB2 cells. Effects of transfilter co-culture with TR-MUL5 cells (A) and soluble factors secreted from TR-MUL5 cells (B) on the activity of alkaline phosphatase in TR-iBRB2 cells. A: TR-MUL5 cells were situated on the basolateral aspect of TR-iBRB2 cell monolayers as might occur in vivo and cells were co-cultured for six days. B: TR-iBRB2 cells were cultured in the conditioned medium of TR-MUL5 cells (MUL-CM) for 24 h. Each column represents the mean±SEM (n=3). Asterisk represents p<0.01, significantly different from the control.

Figure 3

TGF-β, PAI-1, and Id2 expressions. Expression of TGF-β1 in the conditioned medium of TR-MUL5 cells (MUL-CM) (A) and modulation of PAI-1 (B) and Id2 (C) mRNA expressions by recombinant human TGF-β1 (rhTGF-β1) and MUL-CM in TR-iBRB2 cells. A: The expression of TGF-β1 was determined by immunoblot analysis. B, C: The expression levels of PAI-1 and Id2 mRNA were determined by quantitative real-time PCR analysis and normalized to β-actin mRNA expression. Each column represents the mean±SEM (n=4–12). Asterisk represents p<0.01, significantly different from the control.