Proliferation Of Choroidal Endothelial Cells Research Articles

Advanced glycation end products induce choroidal endothelial cell proliferation, matrix metalloproteinase-2 and VEGF upregulation in vitro.

Advanced glycation end products (AGEs) are considered to be important modulators of angiogenesis and accumulate in choroidal neovascularization (CNV). Their effects regarding cells involved in proliferation of CNV [retinal pigment epithelial (RPE) cells, Müller cells and choroidal endothelial cells (CECs)] were investigated. Furthermore, the effects of AGEs on expression of matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) by CECs were explored. RPE cells, CECs and Müller cells were exposed to AGEs (10 microg/ml, 50 microg/ml and 100 microg/ml) for a time course of three days in their desired medium and proliferation was estimated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. MMP-2 expression of AGE-stimulated CECs was determined by zymography and reverse-transcription polymerase chain reaction (RT-PCR) after 36 h of exposure. Furthermore, VEGF expression of AGE-stimulated CECs (50 microg/ml and 100 microg/ml) was determined by RT-PCR after an exposure time of 36 h. AGEs in a concentration of 50 microg/ml and 100 microg/ml increased the proliferation of CECs (41% vs 46.1%; P<0.005). No AGE effect on RPE cell and Müller cell proliferation was seen. AGEs in all concentrations used upregulated the VEGF mRNA expression of CECs. Zymography and RT-PCR demonstrated the upregulation of MMP-2 by CECs after AGE exposure. AGEs stimulate CEC proliferation, MMP-2 secretion and VEGF upregulation and may be important promoters of CNV formation in exudative AMD in vivo.

Role of PLCγ and Ca2+ in VEGF- and FGF-induced choroidal endothelial cell proliferation

Although both vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors have been shown to be important in the regulation of vascular endothelial cell growth, the roles of phospholipase C (PLC)gamma and Ca(2+) in their downstream signaling cascades are still not clear. We have examined the effects of VEGF and FGF on PLCgamma phosphorylation and on changes in intracellular Ca(2+) levels in primary endothelial cells. VEGF stimulation leads to PLCgamma activation and increases in intracellular Ca(2+), which are correlated with mitogen-activated protein (MAP) kinase (MAPK) activation and cell growth. Inhibition of Ca(2+) increases by the Ca(2+) chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM resulted in marked inhibition of MAPK activation, which was shown to be linked to regulation of cell growth in these cells. In contrast, FGF stimulation did not lead to PLCgamma activation or to changes in intracellular Ca(2+) levels, although MAPK phosphorylation and stimulation of cell proliferation were observed. Neither BAPTA-AM nor the PLC inhibitor U-73122 had an effect on these FGF-stimulated responses. These data demonstrate a direct role for PLCgamma and Ca(2+) in VEGF-regulated endothelial cell growth, whereas this signaling pathway is not linked to FGF-mediated effects in primary endothelial cells. Thus endothelial cell-specific factors regulate the ability of VEGF receptors and FGF receptors to couple to this signaling pathway.

Direct Measurement of VEGF-Induced Nitric Oxide Production by Choroidal Endothelial Cells

Vascular endothelial growth factor (VEGF) and nitric oxide (NO) seem to be involved in the process of angiogenesis, but their interactions are not clearly understood. The aim of this study was to investigate the influence of VEGF on NO production of choroidal endothelial cells (CEC) and its importance in angiogenesis. Experiments were performed using cultured bovine CEC. Basal NO release of unstimulated CEC was measured and compared to NO release of VEGF-stimulated CEC (1, 10, and 100 ng/ml). Further, cells were pretreated with Nω-nitro-l-arginine methyl ester (l-NAME, 1 and 2 mM) and incubated with and without VEGF (10 ng/ml) to investigate the effect of blocking NO synthase. NO release into the medium was assessed by an amperometric NO sensor. To show the importance of NO in angiogenesis, proliferation and migration of CEC were measured after VEGF stimulation and in the presence or absence of l-NAME (1 and 2 mM). Unstimulated CEC continuously produced low levels of NO. Stimulation of the cells with VEGF resulted in a dose-dependent increase in NO release. The time course after stimulation with 10 ng/ml VEGF was characterized by a prompt initial rise up to 140% of unstimulated levels and a subsequent sustained increase over 120 min. Pretreatment with l-NAME attenuated the VEGF-induced response. l-NAME incubation alone led to a reduction in basal NO release. l-NAME also significantly diminished the VEGF-enhanced CEC proliferation and migration.The results demonstrate that VEGF enhances the formation of NO in cultured CEC. The blockade of NO production reduces CEC proliferation and migration, an effect which may be important for controlling angiogenesis, especially in reducing neovascularization in age-related macular degeneration in the eye.

Hypericin inhibits choroidal endothelial cell proliferation and cord formation in vitro

PURPOSE. To evaluate the effect of hypericin on bovine choroidal endothelial cell proliferation and cord formation and on protein kinase C activity. METHODS. The effect of hypericin (0.1–5 µM) on bovine choroidal endothelial cell proliferation was determined by cell number counting and a 3 H-thymidine uptake assay in media containing 1, 5 or 10% serum. For the cord formation assay, bovine choroidal endothelial cells were seeded on basement membrane matrix, and the lengths of the capillary-like structures (cords) formed were quantified by image analysis. The effect of hypericin on cord formation was evaluated in the presence of serum or vascular endothelial growth factor. The effect of hypericin on protein kinase C activity was also measured in the presence or absence of light. RESEULTS. Hypericin inhibited bovine choroidal endothelial cell proliferation in a dose-dependent manner in the presence of light but not in the dark. Serum dose-dependently masked the inhibition of DNA synthesis by hypericin. Cord formation by bovine choroidal endothelial cells was stimulated by serum or vascular endothelial growth factor and inhibited by hypericin in the presence of light. Protein kinase C activity was completely inhibited by hypericin in the presence of light but only mildly inhibited in the absence of light. CONCLUSIONS. Hypericin inhibits bovine choroidal endothelial cell proliferation and cord formation and choroidal endothelial cell protein kinase C activity. These results suggest that hypericin should be further investigated in animal models for its potential to inhibit subretinal neovascularization.