Purpose:Emergency keratoplasties for inflamed eyes are considered to have a worse prognosis because of immunologic graft rejection. Amniotic membranes have antiinflammatory and antiangiogenic abilities. Therefore, amniotic membrane transplantation (AMT) was performed to stabilize the situation of eyes with severe infectious keratitis before elective penetrating keratoplasty (PK).Methods:Retrospective, nonrandomized observational case series. Seven to 41 days (median, 20 days) after the onset of intensive antiinfectious medication, an AMT (6 multigrafts and 6 sandwich) was performed in 12 patients [8 men and 4 women; age 46-80 years (median, 66 years)] with herpetic (n = 5), bacterial keratitis (n = 3), or combinations (n = 4). Three to 12 months (median, 5 months) after cessation of the inflammatory status of the eye, a central elective PK (diameter, 7-8 mm) became feasible in 10 eyes. Follow-up ranged from 4 to 38 months (median, 20 months) after PK.Results:The primary success rate of AMT was 11/12 (92%). Five recurrences (41%) were treated successfully 4 times by repeat AMT (sandwich) and 1 time by emergency PK. In 2 of the 12 eyes, an irreversible endothelial immunologic graft reaction appeared 18 and 21 months after PK. One eye suffered from reversible recurrence of herpetic keratitis on the corneal graft. At the end of the follow-up, 10 of 12 grafts (83%) were clear.Conclusions:A rapid decrease in the inflammatory reaction and a fast reepithelialization because of AMT after intensive antiinfectious medication in case of severe ulcerative keratitis may help to avoid an emergency keratoplasty and improves the prognosis of the elective keratoplasty.

BACKGROUND:We investigated concentrations of total homocysteine (tHcy) in elderly people without and those with age-related macular degeneration (AMD). In addition, we tested the association between plasma tHcy and one glycation marker in aqueous humor.METHODS:People with cataract only (n=48), patients with dry AMD (n=38) and those with wet AMD (n=31) were studied. Blood concentrations of tHcy, and methylation and vitamin markers were measured in 116 blood samples. The concentrations of the extracellular soluble receptor for advanced glycated end products (esRAGE) were measured in 77 aqueous humor samples.RESULTS:Mean aqueous humor concentration of esRAGE and that of plasma tHcy did not differ significantly between the groups. Arterial hypertension but not eye disease explained the tHcy elevation in plasma in this study. In the cataract group, a significant negative correlation was found between plasma tHcy and that of esRAGE in aqueous humor (r=-0.483, p=0.006). In patients with dry AMD, the concentration of esRAGE in aqueous humor correlated negatively to tHcy and positively to serum folate.CONCLUSIONS:Plasma tHcy levels were positively associated with hypertension, but not with AMD in this study. Higher esRAGE in aqueous humor was related to higher folate and lower tHcy in blood. Following studies may assess whether B-vitamins can protect against age-related ocular diseases by reducing glycation.

AIM: To investigate the role of advanced glycation end-products (AGES) in the pathogenesis of age-related macular degeneration (AMD).METHODS: Bovine choroidal endothelial cells (CEC) were isolated by the modified protocol using lycopersicon esculentum agglutinin coated Dynabeads, and identified by immunocytochemical staining with anti-Factor VIII antibody and uptaking of dil-acetylated low-density lipoprotein (dil-ac-LDL). AGEs were prepared by incubating 50g/L bovine serum albumin and 150g/L glucose at 37 degrees C for 6 weeks, which were characterized by dot blot assay with anti-AGEs antibody. CEC proliferation was evaluated by 3, (4,5-dimethylthiazol-2-y1)-2,5- diphenyl tetrazolium bromide (MTT) assay, and tube formation in CEC was determined by a Vitrogen system.RESULTS: More than 90% of the cultured cells were positive to Factor VIII immunostaining and had the ability to uptake dil-ac-LDL, which were the features of endothelial cells. 219AGEs we prepared were affinitive to anti-AGEs antibody. After treatment with AGEs for a time course of 3 days, CEC proliferation was significantly increased in a dose-dependent manner by AGEs at concentrations between 62.5mg/L and 500mg/L. The cytokine, basic fibroblast growth factor (bFGF), enhanced strongly tube-like structure formation in CEC to 124%(P<0.05) above that of untreated controls. In this condition, AGEs at the concentrations of 500mg/L and 50mg/L showed no effect on CEC tube formation (P>0.05).CONCLUSION: The present study demonstrated that CEC proliferation was increased by AGEs. However, there was no statistical effect on CEC tube formation. These findings confirm and extend that AGEs could be a potential initiator in the pathogenesis of choroidal neovascularization in exudative AMD, at least in part, through enhancement of CEC proliferation.

To evaluate the effects of verapamil isomers on in vitro proliferation of bovine choroidal endothelial cells (CECs).

PURPOSE:Matrix metalloproteinases (MMP)-2 and -9 play an important role in the pathogenesis of choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) are an important source of MMPs in the outer retinal environment, however little is known about the local factors that modulate MMP secretion in these cells. The purpose of this study was to determine the effects of CNV involved growth factors and the extracellular matrix molecule fibronectin on MMP-2 and -9 secretion by cultured human RPE. METHODS:MMP-2 and -9 secretion was studied using gelatin zymography, Western blot, and ELISA assay of RPE culture supernatants. The effects of stimulating the cells for 36 hours with vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bGFG), tumor necrosis factor-alpha (TNF-alpha), or fibronectin (FN), all angiogenic factors found in CNV membranes, was determined. RESULTS:Resting RPE cells secreted MMP-2 but not MMP-9. Stimulation with TNF-alpha induced secretion of MMP-9 and increased the secretion of MMP-2. MMP-2 secretion was also increased by stimulation with FN and VEGF, but not bFGF. CONCLUSION:The results indicated that the angiogenic molecules VEGF, FN, and TNF-alpha stimulate MMP-2 and -9 secretion from RPE and thus further promote CNV.

Background Proliferative vitreoretinopathy (PVR) is a leading cause of blindness after failed retinal reattachment surgery. PVR is characterized by the proliferation, migration and contraction of retinal pigmented epithelial cells (RPE), and these cellular responses are influenced by the expression and function of integrin receptors. The effect of a cyclic integrin antagonist containing the amino acid sequence Arg-Gly-Asp-D-Phe-Val (RGDfV), specific for the integrin receptors α v β 3 and α v β 5 , was investigated on basic fibroblast growth factor (bFGF), platelet derived growth factor-BB (PDGF-BB), and serum induced human RPE proliferation, migration, invasion and attachment to the extracellular matrix. Furthermore, the effects of bFGF and PDGF-BB regulated expression of integrins α v β 3 and α v β 5 on RPE cells was examined. Methods The effect of a cyclic integrin antagonist and a control peptide (0.01 μg/ml to 300 μg/ml) was investigated on serum or cytokine (bFGF or PDGF-BB pretreatment) induced human fetal RPE cell proliferation by H 3 -thymidine uptake. The effect of the cyclic integrin antagonist on RPE cell attachment onto different extracellular matrices (laminin, collagen IV, fibronectin), RPE cell invasion stimulated by PDGF-BB or serum, and migration stimulated by PDGF-BB, vascular endothelial growth factor (VEGF) or serum was explored. PDGF-BB and bFGF modulation of the integrin receptors α v β 3 and α v β 5 was evaluated by flow cytometry. Results The integrin antagonist did not inhibit DNA synthesis stimulated by serum, bFGF, or PDGF-BB treatment. RPE attachment onto fibronectin was inhibited in a concentration range of 1–10 μg/ml (p < 0.05). Attachment of the RPE cells onto collagen IV and laminin was inhibited in a range of 3–10 μg/ml (p < 0.05). Serum and PDGF-BB stimulated migration was inhibited by the cyclic integrin antagonist in a concentration range of 1–10 μg/ml (p < 0.05). Furthermore, the cyclic integrin antagonist inhibited PDGF-BB stimulated RPE cell invasion through fibronectin (3μg/ml: 66% inhibition, p < 0.001). In each of these experiments, the control peptides had no significant effects. PDGF-BB and bFGF pretreatment of RPE cells increased the expression of integrin receptors α v β 3 (bFGF: 1.9 fold, PDGF-BB: 2.3 fold) and α v β 5 (bFGF: 2.9 fold, PDGF-BB: 1.5 fold). Conclusion A selective inhibition of the integrin receptors α v β 3 and α v β 5 through a cyclic integrin antagonist is able to inhibit RPE cell attachment, migration and invasion. Since these steps are of importance for the progression of PVR, a cyclic integrin antagonist should be further evaluated for the treatment of this disease.

PURPOSE:To determine the effect of the calcium signaling modulating drug carboxyamido-triazole (CAI) on substeps of exudative age-related macular degeneration (AMD) in vitro. MATERIALS AND METHODS:Zymography and ELISA determined the effect of CAI on MMP-2 production of choroidal endothelial cells (CECs) stimulated by bFGF and VEGF. The effects of CAI on attachment of retinal pigment endothelial (RPE) cells/CECs onto fibronectin, laminin, collagen IV, and migration toward fibronectin were investigated. Proliferation induced by serum and bFGF (10 microg/ml) with and without CAI (0.1-10 microM) was measured by cell counting and 3H-uptake. Viability and apoptosis of the exposed cells was assessed by an MTT and an apoptosis assay. RESULTS:CAI inhibited serum- and bFGF-induced proliferation, cell attachment onto fibronectin and collagen IV, but only CEC attachment onto laminin. Inhibition of MMP-2 production was observed (10 microM CAI). CAI reduced the cellular viability by apoptosis induction. CONCLUSIONS:CAI inhibits substeps of exudative macular degeneration and may be of value for the treatment of the disease.

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.

Retinale Pigmentepithelzellen (RPEs) und choroidale Endothelzellen (CECs) spielen eine wichtige Rolle bei der Ausbildung einer choroidalen Neovaskularisation im Rahmen der altersbedingten exsudativen Makuladegeneration (AMD). In dieser Studie untersuchen wir den Einfluss der die Kalziumsignaltransduktion modulierenden Substanz Carboxyamido-triazole (CAI) auf die Proliferation und Migration dieser Zellarten.

Purpose. Integrin a v ß 3 is predominantly expressed on endothelial cells in choroidal neovascularization (CNV). We evaluated the efficacy of cyclic RGD (Arg-Gly-Asp) peptide, an a v -integrin antagonist, in a rat model of laser-induced CNV. Methods. We evaluated the effect of cyclic RGD on the adhesion and cell viability of bovine choroidal endothelial cells (BCECs) by cell counting and the trypan blue dye exclusion test. CNV was induced by laser photocoagulation in female Long Evans rats (day 0), followed by intravitreal administration of one dose of cyclic RGD of 200 (n = 9), 100 (n = 10), 50 (n = 4), or 0µg (n = 9) on days 9 and 11. We assessed the area of CNV by fluorescein angiography and the thickness microscopically on histologic sections. Neovascular vessels were detected by an antibody against factor VIII. Results. Cyclic RGD (0.02 to 200µg/ml) inhibited adhesion of BCECs in a dose-dependent manner without changing the cell viability (p < 0.01). In eyes treated with two injections of 200 or 100µg of cyclic RGD peptide, the development of CNV was significantly (p < 0.01) inhibited in the area of leakage on fluorescein angiography. Histologically, the CNV membrane was observed beneath the retina and the factor VIII-positive cells and red blood cells were involved. The thickness of the lesions was significantly (p < 0.01) reduced in eyes that received 200 or 100µg of RGD. Conclusions. Cyclic RGD effectively inhibited CNV progression in a rat model of laser-induced CNV, suggesting that this a v -integrin antagonist may be beneficial in the treatment of CNV.

Background. 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. Methods. RPE cells, CECs and Müller cells were exposed to AGEs (10 µg/ml, 50 µg/ml and 100 µg/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 µg/ml and 100 µg/ml) was determined by RT-PCR after an exposure time of 36 h. Results. AGEs in a concentration of 50 µg/ml and 100 µg/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. Conclusion. AGEs stimulate CEC proliferation, MMP-2 secretion and VEGF upregulation and may be important promoters of CNV formation in exudative AMD in vivo.

Background. Nitric oxide (NO) is known as an important mediator of endothelial function. The aim of this investigation was to evaluate the influence of mediators of retinal pathology – vascular endothelial growth factor (VEGF) and advanced glycation end products (AGEs) – on NO release from choroidal endothelial cells (CEC) and retinal pigment epithelial (RPE) cells to elucidate the complex role of NO. Methods. Bovine CEC were stimulated using VEGF (1, 10, and 100 ng/ml), and RPE cells were exposed to interferon-γ (IFN-γ 100 U/ml) and lipopolysaccharide (LPS 1 µg/ml). NO release into the media was assessed by an amperometric NO sensor. The influence of AGEs (10 and 100 µg/ml) on NO release from CEC and RPE cells was investigated. The competitive NO synthase inhibitor L ω -nitro-L-arginine methyl ester (L-NAME 2 nmol) was used to pretreat the cells 2 h before NO measurement. Results. Unstimulated CEC produced low basal levels of NO in vitro (39.1±13.9 nmol), and unstimulated RPE cells produced minimal basal levels of NO (15.7±7.0 nmol). Exposure of CEC to VEGF for 30 min resulted in a dose-dependent rise of NO in the medium, which was significantly inhibited by L-NAME. Stimulation of RPE cells with IFN-γ and LPS resulted in a rise of NO in the bath to 125.9±18.5% of basal values. Basal NO release from CEC, and stimulated NO release from RPE cells, was significantly reduced by AGE treatment and L-NAME. Conclusions. These data demonstrate that AGEs formed from the nonenzymatic glycation of proteins with reducing sugars quench NO activities in vitro. The results implicate AGEs as important modulators of NO activity and may be relevant to the impairment of endothelial functions observed in diabetes and aging.

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 N(omega)-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.

A devastating complication of ischemic retinopathies is retinal neovascularization. We studied the impact on retinal endothelial cell proliferation of soluble factors released from cultured retinal glial (Müller) cells and from retinal explant cultures. Hypoxia strongly stimulated VEGF release by all types of cultures but endothelial cell growth was not further increased by the corresponding conditioned media if compared to supernatants obtained under normoxia. When the final concentration of the hypoxia-conditioned media was adjusted to the VEGF level of normoxia-conditioned media, they even inhibited endothelial cell proliferation. Inhibition may be exerted by TGF-β2 but TGF-β2 mRNA and protein expression in Müller cells were found to be down-regulated under hypoxia. We conclude that retinal endothelial cell proliferation is controlled by the balance of the amount and/or efficacy of several stimulatory and inhibitory factors.

BACKGROUND:Angiostatic drugs might provide desirable modulation of choroidal angiogenesis-related diseases, including histoplasmosis and the exudative form of age-related macular degeneration. However, the precise effects of this class of compounds in the choroidal neovascularization are still unclear. In the present study, we investigated the effects of triamcinolone acetonide (TA), an angiostatic steroid, on choroidal angiogenesis in vitro.METHODS:Bovine choroidal endothelial cells (CEC), which are the critical cellular component of choroidal angiogenesis in vivo, were isolated with Lycopersicon esculentum agglutinin-coated Dynabeads and cultured in EGM medium. CEC were treated with basic fibroblast growth factor (bFGF) and TA at various concentrations ranging from 50 to 300 mg/l. The capacities for CEC migration and tube formation were evaluated with the modified Boyden chamber and the Vitrogen collagen assay, respectively. The activities of matrix metalloproteinases (MMP)-2 and -9 were examined using gelatin zymography.RESULTS:The stimulation of CEC with 50 ng/ml bFGF resulted in an increase of about 100% in migration activity (P<0.01). Preincubation of CEC with TA at the indicated concentrations for 20 min inhibited the bFGF-stimulated migration in a dose-dependent manner (P<0.01). After 5 days, the bFGF-stimulated tube formation in CEC was inhibited by TA at the concentrations 100, 150 and 300 mg/l (P<0.01). Gelatin zymography of the culture media of CEC showed that the bFGF-induced activation of MMP-2 was attenuated by 300 mg/l TA (P<0.05).CONCLUSION:Downregulation of the activation of MMPs in CEC could be one of the mechanisms by which angiostatic steroids inhibit choroidal angiogenesis.