Recombinant VEGF₁₆₅b inhibits TNF-α-induced ICAM-1 expression and monocyte adhesion in primary human retinal pigment epithelial cells

Abstract

 

Purpose

 

Local inflammation at the retinal pigment epithelial (RPE) cell layer is associated with inflammatory cell migration and secretion of pro-inflammatory cytokines such as Tumor necrosis factor (TNF)-α. TNF-α up-regulates Intercellular Cell Adhesion Molecule (ICAM)-1 expression on RPE that allows binding of lymphocyte function-associated antigen-1 (LFA-1) on leukocytes contributing to leukocyte adhesion at sites of inflammation. VEGF₁₆₅b is generated by alternative splicing of VEGF-A in the terminal exon 8. VEGF₁₆₅b is cytoprotective and anti-angiogenic but its effects on inflammation have not been elucidated. Therefore, our aim was to investigate the effects of VEGF₁₆₅b on TNF-α-induced ICAM-1 expression and monocyte adhesion in RPE cells.

 

Methods

 

Primary RPE cells were pretreated with 20 ng/ml TNF-α alone, 100 ng/ml VEGF₁₆₅b alone, 100 ng/ml VEGF₁₆₅b with 100 ng/ml anti-VEGF₁₆₅b, 100 ng/ml VEGF₁₆₅b or 10 nM ZM323881 (VEGFR2 inhibitor) prior to exposure to 20 ng/ml TNF-α for 24 h. After the experiment, cells were lysed for Western blotting and monocyte adhesion assays were performed.

 

Results

 

VEGF₁₆₅b and ZM323881 inhibited TNF-α-induced upregulation of ICAM-1 in RPE cells. VEGF₁₆₅b was neutralized by antibody to VEGF₁₆₅b (Figure 1). VEGF₁₆₅b ameliorated the TNF-α-induced monocyte-RPE adhesion (Figure 2).

 

Conclusions

 

These findings indicate that VEGF₁₆₅b inhibits the TNF-α-mediated upregulation of ICAM-1 expression and increases monocyte-RPE cell adhesion, suggesting an anti-inflammatory property of VEGF₁₆₅b in the eye.

 

 

Figure 1. Western blotting showing effects of VEGF₁₆₅b and ZM323881 on TNF-α-induced upregulation of ICAM-1 in RPE cells.

 

 

Figure 2. Decreased adhesion of monocytes to RPE cells after inhibition of ICAM-1 expression by VEGF₁₆₅b.

 

Keywords: 557 inflammation • 701 retinal pigment epithelium • 748 vascular endothelial growth factor