Vascular endothelial growth factor expression and secretion by retinal pigment epithelial cells in high glucose and hypoxia is protein kinase C-dependent

Abstract

Retinal pigment epithelial (RPE) cells express vascular endothelial growth factor (VEGF) in response to high glucose or hypoxia. We hypothesised that VEGF expression and secretion by RPE cells in high glucose and hypoxia are regulated by protein kinase C (PKC). Primary cultured RPE cells from Sprague–Dawley rats were growth-arrested for 48 hr in 0.5% FBS in 5.6 or 30 mm d-glucose. Cells were exposed to hypoxic conditions (<1% O₂, 5% CO₂) for the last 15–18 hr of growth-arrest. PKC -α, -β₁, -δ, -ε, and -ζ were expressed by RPE cells and exposure to high glucose for 48 hr had no effect on expression as demonstrated by Western immunoblotting. High glucose, hypoxia or VEGF stimulated translocation of a number of the PKC isozymes to the membrane or particulate fractions implying activation. In response to high glucose or acute phorbol myristate acetate (PMA) stimulation, VEGF mRNA analysed by RT-PCR was increased. Intracellular VEGF protein identified by immunoblotting and confocal immunofluorescence imaging was significantly increased by high glucose, hypoxia or acute PMA stimulation. Calphostin C or a specific inhibitor of PKC-ζ prevented high glucose-stimulated VEGF expression in high glucose. VEGF secretion, as measured by ELISA in the culture medium, was enhanced in hypoxia but not in high glucose. Following exposure of RPE cells to PMA for 24 hr, PKC-δ was significantly down regulated, whereas PKC-α, -β, -ε and -ζ remained unchanged. Secretion of VEGF in normal or high glucose, or hypoxia was significantly reduced following treatment with PMA for 24 hr but not with the PKC-ζ inhibitor. We conclude that in high glucose and hypoxia PKC isozymes are activated and are necessary for VEGF expression. Secretion of VEGF is enhanced in hypoxia and appears to be regulated by PKC-δ. RPE cells may contribute to the pathogenesis of retinopathy caused by high glucose and hypoxia through the expression and secretion of VEGF that are regulated by PKC isozymes.

Introduction

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the choroid, form the outer blood retinal barrier and are essential for the nutritional and metabolic support of the photoreceptors. Among the growth factors produced and secreted by RPE cells, vascular endothelial growth factor (VEGF) is expressed in response to mechanical stretch (Seko et al., 1999), hypoxia (Mousa et al., 1999) and high glucose (Sone et al., 1996). VEGF receptors are expressed on the RPE cell surface likely indicating important homeostatic and autocoid regulation (Kociok et al., 1998). VEGF is a potent endothelial cell mitogen and permeability factor contributing to angiogenesis (Aiello et al., 1997, Aiello et al., 1994). RPE cells may contribute to the pathogenesis of diabetic retinopathy through their production of VEGF.

Hyperglycemia initiates a cascade of target cell signaling responses and consequent cellular dsyfunction that result in ocular, renal, cardiovascular and other end-organ damage associated with diabetes mellitus (Mooradian and Thurman, 1999). Retinal capillary damage from high glucose-induced pericyte loss (Kern and Engerman, 1996, Agardh et al., 1997) and alterations in retinal hemodynamics (Bursell et al., 1997, Grunwald et al., 1995, Kunisaki et al., 1998) cause a hypoxic state in both the early and late stages of diabetic retinopathy. In retinal endothelial cells and pericytes, high glucose causes activation of protein kinase C (PKC) and consequent expression of VEGF (Enaida et al., 1999, Hata et al., 1999). Exposure to hypoxia and advanced-glycation end products causes additive VEGF expression by RPE cells (Lu et al., 1998). The PKC-dependency of VEGF expression has been shown in several cell types, including mesangial (Cha et al., 2000), vascular smooth muscle (Williams et al., 1997), vascular endothelial cells (Xia et al., 1996) and retinal endothelial cells (Ishii et al., 1998). Hypoxia mediates VEGF expression by increased binding of the active hypoxia inducible factor (HIF)-1α to the hypoxic response element of the VEGF promoter and by increasing the stability of the VEGF mRNA transcript through mitogen-activated protein kinase and Akt pathways, respectively (Suzuma et al., 2000). Hypoxia is the strongest stimulus of VEGF expression and it is likely that the signaling effects of hyperglycemia and hypoxia contribute to the progression of diabetic retinopathy. VEGF activates several PKC isozymes through stimulation of its tyrosine kinase receptor and the phospholipase Cγ pathway in aortic endothelial cells (Xia et al., 1996). The mechanisms by which these effects may regulate VEGF expression and secretion in the RPE cell type remain incompletely understood.

In this study, we hypothesised that high glucose, hypoxia and VEGF would activate specific PKC isozymes in primary cultured RPE cells. Furthermore, we postulated that high glucose and hypoxia-induced production and secretion of VEGF in RPE cells may be PKC dependent. PKC isozyme translocation/activation patterns in response to VEGF and hypoxia, with or without high glucose, were analysed by Western immunoblotting of subcellular fractions and confocal immunofluorescence imaging. Diacylglycerol (DAG)-sensitive PKC isozymes were acutely stimulated by phorbol myristate acetate (PMA) and all PKC isozymes were inhibited with Calphostin C. PKC-δ was selectively down regulated following 24 hr exposure to PMA compared to the other DAG-sensitive PKC isozymes that required 48 hr for down regulation, and PKC-ζ function was blocked using a specific cell-permeable peptide inhibitor. VEGF transcription, translation and secretion into the culture medium were measured. We found that high glucose, hypoxia and VEGF stimulated specific DAG-sensitive PKC isozymes. VEGF expression appears to be dependent on PKC isozymes including PKC-ζ and secretion appears to require PKC-δ.