Proliferation Of Choroidal Endothelial Cells Research Articles

IL1R2 promotes retinal angiogenesis to participate in retinopathy of prematurity by activating the HIF1α/PFKFB3 pathway

Retinopathy of prematurity (ROP) is the leading cause of blindness in children, but there is no safe and effective treatment available. Interleukin-1 receptor type 2 (IL1R2) acts as a decoy receptor for IL-1 may affect ROP progression. This study aimed to investigate the role of IL1R2 in ROP. A microglial cell model was established under hypoxia conditions and co-cultured with choroidal endothelial cells, while an oxygen-induced retinopathy (OIR) model was also established. Microglial activation and IL1R2 levels in retinal tissues were analyzed using immunofluorescence assay. Endothelial cell migration was evaluated by Transwell assay and scratch test, angiogenesis was assessed using ELISA and tube formation assay, and proliferation was evaluated by EdU assay. The HIF1α/PFKFB3 pathway was analyzed by western blot. We observed that IL1R2 expression was predicted to be upregulated in ROP and was increased in hypoxia-treated BV2 cells. Additionally, IL1R2 levels were upregulated in the retinal tissues of OIR mice and correlated with microglial activation. In vitro experiments, we found that hypoxia promoted endothelial cell migration, angiogenesis, proliferation, and activated the HIF1α/PFKFB3 pathway, which were rescued by IL1R2 knockdown. Moreover, NHWD-870 (a HIF1α/PFKFB3 pathway inhibitor) suppressed endothelial cell migration, angiogenesis, and proliferation induced by IL1R2 overexpression. In conclusion, IL1R2 facilitates the migration, angiogenesis, and proliferation of choroidal endothelial cells by activating the HIF1α/PFKFB3 pathway to regulate ROP progression.

Inhibition of AIF-1 alleviates laser-induced macular neovascularization by inhibiting endothelial cell proliferation via restrained p44/42 MAPK signaling pathway

Age-related macular degeneration (AMD) is a leading blinding disease worldwide, and macular neovascularization (MNV) is a common complication encountered in the advanced stages of AMD. While the underlying causes of MNV remain elusive, aberrant multiplication of choroidal endothelial cells (CECs) and increased vascular endothelial growth factor (VEGF) are thought to play significant roles in the occurrence and development of MNV. Allograft inflammatory factor-1(AIF-1) is a crucial regulatory factor of vascular tubular structure formation and growth, involving the proliferation and migration of vascular endothelial cells and various tumor cells. This study aimed to understand how AIF-1 effects the proliferation of CECs and the subsequent progression of MNV. To study this, a mouse MNV model was established through laser injury, and the AIF-1 expression levels were then measured using western blot and immunohistochemistry. AIF-1 siRNA was intravitreally injected to silence AIF-1 gene expression. Western blot and choroidal flat mount were performed to measure the progression of MNV and proliferation of the CECs. These results showed that the protein expression of AIF-1 was significantly elevated in the laser-induced mouse MNV model, and the expression trend was consistent with VEGF. The protein level of AIF-1 was significantly decreased after the intravitreal injection of AIF-1 siRNA, the damage range of laser lesions was significantly reduced, and the proliferation of endothelial cells was inhibited. Knockdown of the AIF-1 gene significantly inhibited the expression of mitogen-activated protein kinase p44/42 in MNV lesions. In summary, this research demonstrates that AIF-1 promoted MNV progression by promoting the proliferation of CECs and that silencing AIF-1 significantly ameliorates MNV progression in mouse models, which may act through the p44/42 MAPK signaling pathway. AIF-1 could be a new potential molecular target for MNV.

HGF and VEGF-A and Their Receptors Show Expression and Angiogenic Effects on Human Choroidal Endothelial Cells: Implications for Treatments of Neovascular Age-Related Macular Degeneration

Intraocular neovascularisation is associated with common blinding conditions including neovascular age-related macular degeneration (nAMD). Vascular endothelial growth factor (VEGF) is central in driving choroidal neovascularisation in this disease. Many clinical therapies target VEGF-A with intravitreal anti-VEGF drugs, which, however, have limited efficacy and require repeated, prolonged treatment. Other cytokines are known to be involved, including hepatocyte growth factor (HGF), which is shown to have a role in the early stages of nAMD. We investigated the effect of HGF and its co-operation with VEGF-A on human choroidal endothelial cells (CEC). The expression of HGF and related molecules in CEC was investigated using immunofluorescence, Western blotting and flow cytometry. In vitro assays for proliferation, tubule formation and migration were used to assess the potential role of HGF in neovascularisation. Primary human CEC expressed HGF, VEGF-A and their receptors MET and VEGF receptor 2 (VEGFR2). HGF increased CEC proliferation, tubule formation and migration; the increased proliferation and migration appeared to be additive with that achieved with VEGF-A. This study provides insight into growth factor co-operation in CEC signalling and indicates that simultaneous blockage of multiple growth factors or common downstream signalling pathways may provide a more sustained treatment response, enhancing treatments in nAMD.

Glycine-Conjugated Bile Acids Protect RPE Tight Junctions against Oxidative Stress and Inhibit Choroidal Endothelial Cell Angiogenesis In Vitro

We previously demonstrated that the bile acid taurocholic acid (TCA) inhibits features of age-related macular degeneration (AMD) in vitro. The purpose of this study was to determine if the glycine-conjugated bile acids glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and glycoursodeoxycholic acid (GUDCA) can protect retinal pigment epithelial (RPE) cells against oxidative damage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in choroidal endothelial cells (CECs). Paraquat was used to induce oxidative stress and disrupt tight junctions in HRPEpiC primary human RPE cells. Tight junctions were assessed via transepithelial electrical resistance and ZO-1 immunofluorescence. GCA and GUDCA protected RPE tight junctions against oxidative damage at concentrations of 100–500 µM, and GDCA protected tight junctions at 10–500 µM. Angiogenesis was induced with VEGF in RF/6A macaque CECs and evaluated with cell proliferation, cell migration, and tube formation assays. GCA inhibited VEGF-induced CEC migration at 50–500 µM and tube formation at 10–500 µM. GUDCA inhibited VEGF-induced CEC migration at 100–500 µM and tube formation at 50–500 µM. GDCA had no effect on VEGF-induced angiogenesis. None of the three bile acids significantly inhibited VEGF-induced CEC proliferation. These results suggest glycine-conjugated bile acids may be protective against both atrophic and neovascular AMD.

Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration.

PurposeThis study addresses the hypothesis that age-related stresses upregulate Thy-1 in choroidal endothelial cells (CECs) and contribute to CEC activation and migration, processes important in choroidal neovascularization (CNV).MethodsMeasurements were made of Thy-1 protein (Western blot) in CECs and Thy-1 mRNA (real time quantitative PCR) in CECs treated with VEGF, CCL11, or PBS or in RPE/choroids from young or old donors or lasered or nonlasered mice. Immunolabeled Thy-1 in ocular sections was compared from young versus old human donor eyes or those with or without neovascular AMD or from lasered versus nonlasered mice. Choroidal endothelial cells transfected with Thy-1 or control siRNA or pretreated with Thy-1 blocking peptide or control were stimulated with VEGF or 7-ketocholesterol (7-KC). Choroidal endothelial cell migration, proliferation, cytoskeletal stress fibers, Rac1 activation, and phosphorylated VEGF receptor 2 (VEGFR2), integrin β3, and Src were measured. Statistics were performed using ANOVA.ResultsThy-1 was expressed in retinal ganglion cells and in vascular endothelial-cadherin–labeled choroid and localized to human or mouse laser-induced CNV lesions. Thy-1 protein and mRNA were significantly increased in CECs treated with VEGF or CCL11 and in RPE/choroids from aged versus young donor eyes or from lasered mice versus nonlasered controls. Knockdown or inhibition of Thy-1 in CECs significantly reduced VEGF-induced CEC migration and proliferation, stress fiber formation and VEGFR2, Src, integrin β3 and Rac1 activation, and 7-KC–induced Rac1 and Src activation.ConclusionsThy-1 in CECs regulates VEGF-induced CEC activation and migration and links extracellular 7-KC to intracellular signaling. Future studies elucidating Thy-1 mechanisms in neovascular AMD are warranted.

MicroRNA-18a Decreases Choroidal Endothelial Cell Proliferation and Migration by Inhibiting HIF1A Expression.

BackgroundThe aim of this study was to investigate the effect of hypoxia on the level of microRNA-18a (miR-18a) and hypoxia-inducible factor 1A (HIF1A) expressed by choroidal endothelial cells through cytological analysis.Material/MethodsAfter culturing choroidal endothelial cells (CECs) under normoxia or hypoxia, microRNAs, expressed in different ways, were screened by using GeneChip microRNA array, and the results were confirmed by real-time PCR. The bioinformatics approach was used to screen target genes of target miRNA. In addition, CECs were transfected with target miRNA mimic or inhibitor and then expression levels of targeted miRNA and genes were observed.ResultsThe GeneChip microRNA Array detected 14 miRNAs that were expressed differently. Among these miRNAs, 12 miRNAs were identified as being upregulated and 2 miRNAs as being down-regulated. MiR-18a was most significantly down-regulated. Bioinformatics analysis showed that HIF1A was the target gene of miR-18a. In CECs transfected with miR-18a mimic, there was a remarkable decrease in gene expression level and protein level of HIF1A, and thereby, significant reduction in proliferation and migration of CECs. In CECs transfected with miR-18a inhibitor, there was a significant increase in gene and protein expression levels of HIF1A, and enhanced proliferation and migration of CECs.ConclusionsWe conclude that miR-18a affects the function of CECs by inhibiting the expression of HIF1A.

Interleukin-17 induces angiogenesis in human choroidal endothelial cells in vitro.

The proinflammatory cytokine interleukin-17 (IL-17) has recently been shown to promote angiogenesis. In addition, a receptor for IL-17, IL-17 receptor C (IL-17RC), is enriched in patients with wet, age-related macular degeneration (AMD), a disease characterized by the formation of choroidal neovascularization. However, the role of IL-17 in choroidal endothelial cells (CECs) angiogenesis has not been defined. This study was conducted to determine the effect of IL-17 on proliferation, migration, and tube formation of human CECs. Expression patterns of IL-17 receptor A (IL-17RA) and IL-17RC on isolated human CECs were analyzed by flow cytometry and immunofluorescent staining. Proangiogenic effects of IL-17 on CECs was determined by proliferation assays with a water-soluble tetrazolium cell proliferation reagent kit, wound healing migration assays, and tube formation assays using basement membrane matrix. Cytoskeletal changes were observed by F-actin immunofluorescent staining. Activated Rac1 and RhoA levels were analyzed by pull-down assays. Interleukin-17RA and IL-17RC were present on human CECs. Interleukin-17 enhanced migration and tube formation but did not affect proliferation. Moreover, IL-17 induced rearrangement of the actin cytoskeleton and upregulated activated Rac1 and RhoA in CECs. The PI3K inhibitor wortmannin suppressed CEC migration, cytoskeleton rearrangement, and upregulation of activated Rac1 and RhoA induced by IL-17. Interleukin-17 elicits a proangiogenesis effect on human CECs in vitro by promoting migration and tube formation. The promoted migration effect was dependent on PI3K-Rac1 and RhoA-mediated actin cytoskeleton remodeling.

Hepatocyte growth factor in neovascular age related macular degeneration: In vitro study of its effects on choroidal endothelial cells

AbstractPurpose Intraocular angiogenesis is associated with a number of common blinding conditions including neovascular age‐related macular degeneration (nAMD). The growth factor vascular endothelial growth factor (VEGF) is reported as being central in driving choroidal neovascularisation in nAMD. Many clinical nAMD therapies target VEGF with anti‐VEGF drugs such as ranibizumab, bevacizumab and Aflibercept. These however have limited efficacy and require repeated, prolonged treatment. Other cytokines are known to be involved in nAMD including hepatocyte growth factor (HGF) which has been shown to have a role in the early stages of nAMD. Our aim was to elucidate the effect of HGF and its cooperation with VEGF on human choroidal endothelial cells (CEC).Methods The expression of HGF and related molecules in CEC was investigated using western blotting and flow cytometry. In vitro assays for proliferation, tubule formation and transendothelial permeability were used to assess the role of HGF in nAMD.Results Expression of HGF, VEGF and their receptors c‐met and VEGFR2 and co‐receptor CD44v6 was found in primary human CEC. Stimulation with HGF increased CEC proliferation and tube formation but did not increase permeability, the increased proliferation was additive with VEGF.Conclusion This study provides insight into the interactions or cross‐talk in CEC signalling and indicates that a more efficient approach to regulate intraocular angiogenesis would aim to simultaneously block the actions of multiple growth factors, or the common downstream site in the angiogenesis pathway and this may provide a more sustained treatment response in order to enhance treatment protocols in nAMD. Commercial interest

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle

BackgroundThe purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established.MethodsMonkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1–2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy.ResultsBevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent.ConclusionBevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Interleukin-1β Inhibition Prevents Choroidal Neovascularization and Does Not Exacerbate Photoreceptor Degeneration

The pro-inflammatory cytokine IL-1β has been shown to promote angiogenesis. It can have a neurotoxic or neuroprotective effect. Here, we have studied the expression of IL-1β in vivo and the effect of the IL-1 receptor antagonist on choroidal neovascularization (CNV) and retinal degeneration (RD). IL-1β expression significantly increased after laser injury (real time PCR) in C57BL/6 mice, in the C57BL/6 Cx3cr1(-/-) model of age-related macular degeneration (enzyme-linked immunoabsorbent assay), and in albino Wistar rats and albino BALB Cx3cr1(+/+) and Cx3cr1(-/-) mice (enzyme-linked immunoabsorbent assay) after light injury. IL-1β was localized to Ly6G-positive, Iba1-negative infiltrating neutrophils in laser-induced CNV as determined by IHC. IL-1 receptor antagonist treatment significantly inhibited CNV but did not affect Iba1-positive macrophage recruitment to the injury site. IL-1β significantly increased endothelial cell outgrowth in aortic ring assay independently of vascular endothelial growth factor, suggesting a direct effect of IL-1β on choroidal endothelial cell proliferation. Inhibition of IL-1β in light- and laser-induced RD models did not alter photoreceptor degeneration in Wistar rats, C57BL/6 mice, or RD-prone Cx3cr1(-/-) mice. Our results suggest that IL-1β inhibition might represent a valuable and safe alternative to inhibition of vascular endothelial growth factor in the control of CNV in the context of concomitant photoreceptor degeneration as observed in age-related macular degeneration.

Evaluation of Differential Toxicity of Varying Doses of Bevacizumab on Retinal Ganglion Cells, Retinal Pigment Epithelial Cells, and Vascular Endothelial Growth Factor–Enriched Choroidal Endothelial Cells

To evaluate in vitro the effects of bevacizumab, an anti-vascular endothelial growth factor (VEGF) antibody, on retinal pigment epithelial cells (RPE) and retinal ganglion cells (RGC), at doses that were inhibitory to VEGF-enriched choroidal endothelial cells (CEC). Monkey CEC (RF6A), human RPE cells (ARPE-19), and rat RGC (RGC-5) were exposed for 24 h to increasing doses of bevacizumab. Cell numbers were quantified with WST-1 assay. Cell death was assessed using propidium iodide (PI) staining via flow cytometry and fluorescent microscopy. Bevacizumab was inhibitory to RF6A at 2.0 mg/mL (P < 0.005). No effect on cell viability was noted on ARPE-19 and RGC-5 cell lines at this particular dose of bevacizumab. These results were supported by fluorescent microscopy of PI-stained cells. VEGF-stimulated proliferation of CEC was inhibited by bevacizumab. Bevacizumab was not cytotoxic to human RPE and rat RGC in vitro at a dose that is inhibitory to monkey CEC.

In Skeletal Muscle Advanced Glycation End Products (AGEs) Inhibit Insulin Action and Induce the Formation of Multimolecular Complexes Including the Receptor for AGEs

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

Growth-related effects of oxidant-induced stress on cultured RPE and choroidal endothelial cells

Mounting evidence suggests that oxidative stress caused by reactive oxygen intermediates is a significant mechanism in the pathogenesis of age-related macular degeneration (AMD). Although vascular endothelial growth factor (VEGF) and other cytokines are involved in choroidal neovascularization (CNV) it is largely unknown whether oxidative stress may predispose the eye to increased levels of proangiogenic factors. In an in vitro study we have determined viability and proliferation of both human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) and assessed the release of basic fibroblast growth factor (bFGF) and VEGF from RPE cells after exposing them to oxidative stress. Permanent presence of tert-butyl-hydroperoxide ( tBH), a pro-oxidative stressor, in the cell cultures resulted in decreasing viability and proliferation of RPE cells and CECs. Loss of RPE cell viability was associated with activation of apoptosis by tBH in a dose-dependent manner. The antioxidant, N-acetyl- l-cysteine (NAC), and secreted soluble mediators of RPE cells were appropriate to attenuate the effects of tBH-mediated oxidative stress. RPE cells exposed to tBH were found to release increasing amounts of bFGF but not VEGF after 24 h of culture, thereby supporting proliferation of CECs. These findings suggest that oxidative stress compromises the viability of RPE cells and CECs. However, increased bFGF levels concomitantly released from RPE cells may attenuate the CEC-directed effect, protect CECs from oxidative insults, and are likely to promote CNV.

Comparative antiproliferative and cytotoxic profile of bevacizumab (Avastin), pegaptanib (Macugen) and ranibizumab (Lucentis) on different ocular cells

To compare the antiproliferative and cytotoxic properties of bevacizumab (Avastin), pegaptanib (Macugen) and ranibizumab (Lucentis) on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5) and pig choroidal endothelial cells (CEC). Monolayer cultures of ARPE19, RGC5 and CEC were used. Bevacizumab (0.1-0.3 mg/ml), pegaptanib (0.025-0.08 mg/ml) or ranibizumab (0.04-0.125 mg/ml) diluted in culture medium were added to the cells. Expression of VEGF-receptors (VEGFR1 and VEGFR2) and von Willebrand factor (a marker for endothelial cells) were analysed by immunohistochemistry. CEC cells were stimulated with VEGF. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays cells were grown to confluence and then cultured in a serum-depleted medium to ensure a static milieu. MTT-test was performed after one day. CEC and ARPE19 cells stained positively for VEGFR1 and VEGFR2. More than 95% of the CEC cells were positive for von Willebrand factor. Ranibizumab reduced CEC cell proliferation by 44.1%, bevacizumab by 38.2% and pegaptanib by 35.1% when the drugs were used at their established clinical doses. The differences, however, between the three drugs in respect to cell growth inhibition were not statistically significant. Only a mild antiproliferative effect of bevacizumab or pegaptanib on ARPE19 cells could be observed. Ranibizumab did not alter ARPE19 cell proliferation. No cytotoxicity on RGC5, CEC and ARPE19 cells could be seen. Bevacizumab, pegaptanib and ranibizumab significantly suppress choroidal endothelial cell proliferation. However, when used at the currently established doses none of the drugs was superior over the others in respect to endothelial cell growth inhibition. The biocompatibility of all three drugs--including the off-label bevacizumab--seems to be excellent when used at the currently recommended intravitreal dose.

Choroidal endothelial cells transmigrate across the retinal pigment epithelium but do not proliferate in response to soluble vascular endothelial growth factor

The purpose of this study was to investigate the effects of soluble VEGF on human choroidal endothelial cell (CEC) transmigration across an RPE monolayer as it relates to choroidal neovascularization in AMD. In coculture assays, ARPE-19 (ARPE) was plated on the undersides of Transwell inserts having 0.4 μm pores. Primary human CECs were then plated into the insert. CECs in the Transwell inserts were counted after 72 hr of growth. CEC proliferation was also measured after culturing CECs in ARPE–CEC coculture-conditioned media or in media with exogenous VEGF 121 and/or VEGF 165 added. Transmigration assays were performed on Transwells with 8.0 μm pores: green-labelled CECs were plated in Transwell inserts with or without red-labelled ARPE plated on the undersides of the insert. In some transmigration assays, ARPE was plated into the wells to provide a chemotactic gradient for CEC transmigration. After 72 hr CECs were plated, green cells were counted either within the well media as CECs that transmigrated the epithelial monolayer, or on the underside of the insert as CECs that transmigrated the Transwell insert to but not beyond the ARPE monolayer. A neutralizing antibody to VEGF was added to the wells of Transwells at the time the CECs were plated in the insert and transmigrated CECs were counted. VEGF protein was measured in the conditioned media of ARPE and CEC coculture and in transmigration assays. Compared to control, CEC proliferation significantly increased when CECs were cultured in coculture conditioned media ( p=0.001) or in coculture assays ( p<0.001). However, there was no effect on CEC proliferation when VEGF 121, VEGF 165, or both were added to solo CECs. Antibody to VEGF did not reduce the proliferative effects of coculture conditioned media on CEC. ARPE plated in the well significantly increased CEC transmigration ( p<0.001) compared to transmigration assays without ARPE in the well. VEGF protein measured in the well media of transmigration assays having ARPE within the well was significantly greater than in the assays without ARPE within the well ( p<0.004). Exogenous neutralizing antibody to VEGF significantly reduced transmigration, and this effect was dose-dependent. VEGF provides a chemotactic gradient for human CECs to transmigrate across a monolayer of ARPE. Neutralization of VEGF in the media partially reduces transmigration. Whereas soluble VEGF does not increase proliferation of solo CECs, coculture conditioned media enhances proliferation, suggesting that growth factors other than VEGF cause CEC proliferation. These findings may have relevance to the transformation of occult CNV into CNV within the neurosensory retina in AMD.

Inhibitory effects of verapamil isomers on the proliferation of choroidal endothelial cells

To evaluate the effects of verapamil isomers on in vitro proliferation of bovine choroidal endothelial cells (CECs). CECs were isolated from bovine eyes and cultured in endothelial growth medium (EGM). For the proliferation assays, CECs were exposed to verapamil isomers (0.1-100 microM) in EGM with 2% fetal bovine serum or basic fibroblast growth factor (bFGF) (10 ng/ml). After 72 h of incubation with the desired drug, the cellular proliferation was determined by an MTT assay and a BrdU assay. In addition, the drug toxicity on CECs stimulated with EGM was evaluated by cell counting with trypan blue. All verapamil isomers inhibited the bFGF- or medium-stimulated growth significantly in a concentration range of 10-40 microM without toxicity. No significant differences were seen between the inhibitory effects of the various isomers. Cell toxicity was detected at a concentration of 100 microM verapamil isomers on EGM-stimulated CECs. The results demonstrate the efficacy of all verapamil isomers in inhibiting CEC proliferation involved in the process of choroidal neovascularization. D: -(+)-Verapamil may be recommended for further in vivo evaluation in an animal model of exudative AMD; it has fewer systemic and local side effects because calcium channels are not blocked.

Impact of Endostatin on bFGF-Induced Proliferation, Migration, and Matrix Metalloproteinase-2 Expression/Secretion of Bovine Choroidal Endothelial Cells

Purpose: To investigate the potential role of endostatin, an endogenous angiogenesis inhibitor, in the prevention of choroidal angiogenesis-related disorders. Methods: Bovine choroidal endothelial cells (CEC) were cultured and treated with basic fibroblast growth factor (bFGF) alone or combined with endostatin at concentrations ranging from 0.1 to 10 μg/ml. The proliferation and migration of CECs were evaluated by using 3, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and modified Boyden chamber assay, respectively. For evaluating expression and secretion of matrix metalloproteinase-2 (MMP-2), CEC-conditioned media were subjected to zymography and/or Western blot analysis, and the cells were used for semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Endostatin did not inhibit bFGF-induced or nonstimulated CEC proliferation (p > 0.05). The bFGF-induced migration was significantly inhibited by endostatin at concentrations of 1 and 10 μg/ml (p < 0.05). The bFGF-upregulated expression of mRNA in CECs and the secretion of MMP-2 protein of CECs were both suppressed by endostatin. Conclusions: Inhibitory effect of endostatin on expression and secretion of MMP-2 and cell migration, but not on proliferation of CECs, could respond to its therapeutic action for choroidal neovascularization-dependent disorders.

A role for connective tissue growth factor in the pathogenesis of choroidal neovascularization.

To evaluate the expression of connective tissue growth factor (CTGF) in choroidal neovascular membranes from patients with age-related macular degeneration and the effect of CTGF on choroidal endothelial cell (CEC) function. Using immunohistochemical methods, we analyzed CTGF expression in 13 surgically excised choroidal neovascular membranes related to age-related macular degeneration. The expression of CTGF in retinal pigment epithelial and CEC cultures was determined by means of reverse transcriptase polymerase chain reaction and Western blot, and its regulation by vascular endothelial growth factor and transforming growth factor beta was determined. The effects of CTGF on bovine CEC proliferation, attachment, migration, and tube formation were measured. Vascularized human choroidal neovascular membranes showed strong CTGF immunoreactivity. Double staining disclosed colocalization of CTGF with retinal pigment epithelial cells and CECs. The CTGF induced a significant increase in attachment and migration of CECs; however, it did not stimulate CEC proliferation. The CTGF protein was up-regulated in retinal pigment epithelial cells and CECs by stimulation with transforming growth factor beta and vascular endothelial growth factor, respectively. The expression of CTGF in choroidal neovascular membranes, its regulation by angiogenic growth factors, and its proangiogenic effects on CEC function suggest that CTGF may play a role in the pathogenesis of choroidal neovascularization. Clinical Relevance Multiple growth factors are involved in the pathogenesis of choroidal neovascularization in age-related macular degeneration.

Nerve growth factor regulates human choroidal, but not retinal, endothelial cell migration and proliferation

We have previously shown that sympathetic denervation results in significant blood vessel growth of the choroid and retina. The mechanism of this growth remains unclear. Since sympathetic denervation can result in increased nerve growth factor (NGF) levels, it was the goal of this study to determine if choroidal and retinal endothelial cells in culture would respond to nerve growth factor and if nerve growth factor promote endothelial cell migration and proliferation, two components of angiogenesis. Western blotting with phospho-specific antibodies, cell migration, and cell proliferation assays were employed to determine NGF effects on both choroidal and retinal cell growth. NGF treatment produced phosphorylation of TrkA in choroidal and retinal endothelial cells. NGF stimulation resulted in activation of ERK1/2, Akt, and Src in choroidal endothelial cells, while little phosphorylation was noted following NGF treatment in retinal endothelial cells. NGF increased choroidal endothelial cell migration by 50% over control and this was inhibited by pretreatment with LY294002 (PI3K inhibitor), Akt inhibitor, and MMP2/9 inhibitor. KT5823, PD98059, and PP2 did not affect choroidal cell migration. NGF also produced a 47% increase in choroidal endothelial cell proliferation, which was blocked by PP2, LY294002, Akt inhibitor, KT5823, and PD98059. NGF stimulation did not alter retinal endothelial cell migration or proliferation. Thus, it appears that increased NGF levels that may be noted after sympathectomy are capable of producing some aspects of vascular remodeling via different signaling cascades in choroidal endothelial cells in culture.

Carboxyamido-triazole inhibits substeps of choroidal neovascularization on retinal pigment epithelial cells and choroidal endothelial cells in vitro

Retinal pigment epithelial cells (RPE cells) and choroidal endothelial cells (CECs) are important cell types in the process of choroidal neovascularization in exudative age-related macular degeneration (AMD). In this study, we evaluated the antiproliferative and antimigratory abilities of carboxyamido-triazole (CAI), a drug modulating calcium-dependent signal transduction on RPE cells and CECs. Human fetal RPE cells and bovine CECs were exposed to CAI in a concentration range of 0.1 to 10 microM. Cell proliferation was stimulated with 10% serum or 10 ng/ml bFGF. The effect of CAIs on cell proliferation was estimated. Furthermore, we evaluated CAI's effects on CEC and RPE cell migration induced by fibronectin. CAI had a stronger inhibitory effect on serum-induced CEC proliferation than on RPE cell proliferation. A much stronger effect was seen on the proliferation of bFGF-stimulated RPE cells and CECs. Furthermore, the fibronectin-stimulated migration of RPE cells and CECs was inhibited by CAI. In this assay, a stronger inhibitory effect was seen on RPE cells than on CECs. CONCLUSION; CAI inhibits important substeps of choroidal neovascularization on RPR cells and CECs. Therefore, CAI may be of value for the treatment of choroidal neovascularization in exudative AMD.