Beta-adrenergic receptor stimulation modulates iNOS protein levels through p38 and ERK1/2 signaling in human retinal endothelial cells.

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.

Normal aging involves altered expression of growth factors in the rat choroid.

Normal aging of the choroid results in morphological and physiological changes. The growth factors that mediate these changes are unclear. Vascular endothelial growth factor (VEGF) binds its receptor 2, VEGFR2, to mediate vascular remodeling. Pigment epithelium-derived factor (PEDF) is an inhibitor of angiogenesis produced by retinal pigmented epithelial (RPE) cells. Angiopoietin1 (Ang-1) binds its receptor Tie-2 to recruit mural cells to stabilize vessels. To investigate age-related changes in growth factor activities in aged choroidal vasculature, real-time polymerase chain reaction and protein analysis of VEGF, VEGFR2, PEDF, Ang-1, and Tie-2 were completed on rat choroid/RPE complexes at 8, 22, and 32 months. VEGF messenger RNA (mRNA) peaked at 22 months, whereas protein levels were significantly decreased by 32 months. Both mRNA and protein levels of PEDF were significantly decreased with age. Ang-1 protein levels were not altered, whereas Tie-2 had increased protein levels with age. These results indicate that normal aging involves temporal changes in many of the growth factors common in age-related disease.