RESUMO
Diabetic retinopathy is the primary cause of vision loss in working-age adults. Previous work in the eye has shown that surgical sympathectomy in the retina leads to increased capillary numbers. The goal of this study was to investigate the cellular signaling involved in beta-adrenergic receptor regulation of apoptosis in serum-starved human microvascular retinal endothelial cells (HMREC) cultured in high and low glucose conditions. Cells cultured in high (25 mM) and low glucose (5 mM) conditions were serum starved for 18-24 h, followed by treatment with a beta-1-adrenergic receptor agonist, xamoterol (10 microM), for 15, 30, and 45 min. Non-treated controls were also collected. In addition, high glucose or low glucose serum-starved cells were treated with xamoterol and a Fas inhibitor. Immunoblotting showed a decrease in pro-apoptotic proteins upon xamoterol treatment of cells cultured in high glucose. Caspase-3 levels showed a significant decrease in cells treated with Fas inhibitor alone with further reduction when xamoterol was added, indicating that the apoptosis occurs through Fas signaling. TUNEL labeling confirmed the cleaved caspase-3 data indicating increased apoptosis in cells cultured alone in high glucose compared to a decrease in fluorescence in xamoterol-treated cells. These results indicate that beta-adrenergic receptors regulate protein levels of the Fas signaling cascade in HMREC. This study suggests that maintaining beta-adrenergic receptor signaling in diabetic-like conditions may be protective for the retina.
Assuntos
Endotélio Vascular/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais/fisiologia , Receptor fas/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Apoptose/fisiologia , Western Blotting , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Proteína Ligante Fas/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Glucose/farmacologia , Humanos , Hiperglicemia/metabolismo , Marcação In Situ das Extremidades Cortadas , Fosforilação , Vasos Retinianos/citologia , Xamoterol/farmacologiaRESUMO
Diabetic retinopathy is the leading cause of blindness in working-age adults. Recently, data has suggested that some of the pathological changes that occur in the diabetic retina may be due to increased expression of inflammatory markers. We have previously reported that human retinal endothelial cells cultured in high glucose show increased protein levels of iNOS, which were reduced when cells were pre-treated with a beta-1-adrenergic receptor agonist, xamoterol. The cellular signaling involved in this response is not known. The hypothesis of this study was that modulation of PKA activity through beta-adrenergic receptor stimulation can alter members of the mitogen-activated protein kinase (MAP kinase) family to regulate iNOS protein levels. To address this hypothesis, human retinal endothelial cells were grown in high glucose medium, treated with xamoterol, and immunoblotting and ELISA analyses were done to evaluate cellular signaling of PKA, p38 MAP kinase, and p42/p44 MAP kinase. The results indicate that treatment with xamoterol significantly reduced PKA levels in the retinal endothelial cells cultured in high glucose. This reduction in PKA was followed by decreased ratios of phosphorylated p42/p44 and p38 MAP kinases. Blockade of p42/p44 or p38 signaling could restore iNOS protein levels to those prior to xamoterol stimulation, suggesting that beta-adrenergic receptor regulates iNOS protein levels through regulation of PKA and MAP kinase signaling. These results offer new avenues for therapeutic intervention for retinal diseases that involve altered inflammatory marker expression.