Protein Kinase CK2 and Retinal Angiogenesis

Abstract

Abstract: : Purpose: To characterize signaling intermediates involved in angiogenic responses of retinal endothelial cells to the extracellular matrix and growth factors using specific inhibitors. Methods: Bovine and human cultured retinal endothelial cells (REC) were used. Assays included tube-like structure formation and development of secondary sprouts on basement membrane (BM) matrix, cell proliferation, and cell migration. Retinas were dissected from postmortem eyes obtained from both diabetic and nondiabetic donors and processed for CK2 immunohistochemistry. Specific inhibitors were tested for inhibition of retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Results: In initial studies, broad-spectrum protein kinase inhibitors, H-7 and H-89, stabilized REC tubes on BM matrix, inhibited secondary sprouting, cell migration and proliferation. More specific inhibitors were then used to known kinases inhibited by H-7 and H-89 in order to identify a kinase target of H-7 and H-89 that played a role in these events. Only inhibitors of protein kinase CK2 (formerly, casein kinase II), emodin and DRB (5,6-dichloro-1-ß-o-ribofuranosyl benzimidazole), were able to efficiently block basal and growth factor-stimulated REC secondary sprouting, proliferation, cell migration, and to stabilize tubes. Actinomycin D caused only minor changes in angiogenic assays, suggesting that CK2 effects on REC did not involve its known impact on transcription. Weak nuclear staining for CK2 α subunit was seen in REC from normal and diabetic donors. In REC from donors with diabetic retinopathy (DR), nuclear staining was stronger than in normal cells and distinct cytoplasmic staining was also visible. In isolated retinas CK2 antibodies mostly stained Muller cells, often around blood vessels. In DR retinas the staining was stronger. The extent of retinal neovascularization in mouse OIR model was reduced over 70% (vs. untreated or vehicle-treated groups) after emodin treatment (6 days at 30 mg/kg body weight/day), and by about 60% after DRB treatment at the same dose. In the treated retinas, the main vascular tree had little changes but the neovascular tufts were greatly reduced in number or absent. Conclusions: This is the first demonstration of the involvement of a ubiquitous protein kinase CK2 in angiogenesis. Naturally derived CK2 inhibitors may prove useful for treatment of proliferative retinopathies.

Keywords: diabetic retinopathy • retinal neovascularization • signal transduction