Oral administration of lumiracoxib reduces choroidal neovascular membrane development in the rat laser-trauma model.

PURPOSE: To determine whether lumiracoxib, a highly selective cyclooxygenase-2 (COX-2) inhibitor that exhibits anti-inflammatory and antiangiogenic properties, can inhibit experimental choroidal neovascular membrane (CNVM) development induced by focal laser trauma in a well-characterized Brown Norway rat CNVM model. METHODS: Over a 35-day period, 24 rats received daily oral gavage dosages of 20 mg/kg lumiracoxib in a 0.5% (w/v) suspension of sodium carboxymethylcellulose (CMC), while a control group received the 0.5% CMC suspension only. After 7 days, eight laser photocoagulation sites were placed concentrically around the optic disk to induce CNVMs. Thirty-five days later, fundus photography and fluorescein angiography (FA) were performed and eyes were processed for histopathologic analysis. RESULTS: Masked FA grading of lesion sites revealed a small, but statistically significant difference (P<0.0001) in late stage staining intensity and leakage between the mean group scores of treated (1.4) and control (1.7) eyes. Histopathologic analysis demonstrated that the mean CNVM thickness +/- SD of 38 +/-19 microm (n=24 eyes, 175 photocoagulation sites) in the lumiracoxib-treated animals was reduced by 30% (P<0.001) compared to the CNVM mean thickness+/- SD of 54+/- 20 microm (n=24 eyes, 171 photocoagulation sites) in the control animals. CONCLUSION: Systemic administration of the selective COX-2 inhibitor lumiracoxib results in a partial but significant reduction in CNVM development in the rat laser-trauma model and thus may be clinically beneficial as a potential inhibitor of CNVM formation in exudative age-related macular degeneration.

Effects of unoprostone and endothelin 1 on L-type channel currents in human trabecular meshwork cells.

BACKGROUND: The trabecular meshwork (TM) is a smooth muscle-like tissue with contractile properties and by this mechanisms involved in the regulation of aqueous humor outflow. Isopropyl unoprostone (Rescula, Novartis Ophthalmics), a synthetic docosanoid, reduces intraocular pressure in glaucoma patients and normal subjects. In isolated TM strips, unoprostone reduces TM contractility in the presence of endothelin 1 (ET-1). However, the signal transduction pathway of unoprostone still remains unclear. Since L-type channel currents are known to influence the contractility of TM, we examined the effects of unoprostone and ET-1 on L-type channel currents of TM cells. METHODS: The effects of unoprostone, ET-1 and the tyrosine kinase inhibitor herbimycin A on L-type channel currents of cultured human TM cells were investigated using the perforated patch configuration of the patch-clamp technique. RESULTS: Application of ET-1 had no effect on L-type channel currents. Unoprostone led to a dose-dependent reduction of control currents. The effect of unoprostone is independent of ET-1. After preincubation of cells with herbimycin A, unoprostone had no effect on the L-type channel current amplitude. Human TM cells preincubated with herbimycin A showed a reduced current density compared with control cells. Both substances, unoprostone and herbimycin A, increased the inactivation time constant of L-type channel currents. CONCLUSION: We conclude that unoprostone reduces the activity of L-type Ca2+ channels. This effect seems to be independent of ET-1. The signal transduction pathway seems to be mediated by tyrosine kinases.

Genetic difference in susceptibility to the blood-retina barrier breakdown in diabetes and oxygen-induced retinopathy.

The breakdown of the blood-retina barrier (BRB) is a common feature of diabetic retinopathy. The purpose of the present study is to determine whether there are genetic differences in susceptibility to the breakdown of the BRB in diabetic retinopathy using two rat models. In streptozotocin (STZ)-induced diabetes, Brown Norway (BN) rats developed sustained vascular hyperpermeability in the retina during the entire experimental period (16 weeks of diabetes), while diabetic Sprague Dawley (SD) rats only showed retinal hyperpermeability from 3 to 10 days after the onset of diabetes. The strain difference in permeability was not correlated with the blood glucose levels in these two strains. In oxygen-induced retinopathy (OIR), BN rats developed retinal vascular hyperpermeability from postnatal day 12 (P12) to P22 with a peak at P16, which was 8.7-fold higher than that in the age-matched normal controls. In OIR-SD rats, however, hyperpermeability was observed from P14 to P18, with a peak only 2.2-fold higher than that in the controls. The strain difference in vascular hyperpermeability was correlated with the different overexpression of vascular endothelial growth factor (VEGF) in the retina of these two models. This finding suggests that genetic backgrounds contribute to the susceptibility to diabetic retinopathy.

Hypoxia activates matrix metalloproteinase expression and the VEGF system in monkey choroid-retinal endothelial cells: Involvement of cytosolic phospholipase A2 activity.

PURPOSE: To determine whether the gene expression of matrix metalloproteinases (MMPs) as well as that of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF) and its receptors change in response to hypoxic exposure in a primate choroid-retinal endothelial cell line, and furthermore, whether cytosolic phospholipase A2 (cPLA2) plays a role in this process. METHODS: Rhesus macaque choroid-retinal endothelial (RF/6A) cells were incubated under hypoxic conditions for 1, 2, 4, or 8 h prior to RNA extraction. In some experiments cells were pretreated with the cPLA2 inhibitor AACOCF3 (10 microM) for 30 min prior to hypoxia. Changes in gene expression were determined by RT-PCR and quantified by real-time PCR for urokinase plasminogen activator (uPA), collagenase-1 (MMP-1), membrane type-1 metalloproteinase (MT1-MMP), gelatinases A and B (MMP-2, MMP-9), tissue inhibitor-2 (TIMP-2), VEGF and its receptors, Flt-1 (VEGFR-1), KDR (VEGFR-2), and neuropilin-1 (NP-1). MMP-2 secreted by the cells was evaluated by zymography. VEGF release was measured by ELISA. In tube-formation studies, endothelial cells (EC) were seeded into collagen gel, exposed to hypoxia for 4 h, then incubated under normoxic conditions for 72 h. RESULTS: Hypoxia triggered a three fold increase in the gene expression of MT1-MMP, MMP-2, and TIMP-2, and a ten fold increase in MMP-2 levels. Moreover it also induced tube formation in EC. Expression of uPA, MMP-1, and MMP-9 mRNA was not detected. Pretreatment with AACOCF3 abolished hypoxia-induced tube formation and MT1-MMP, MMP-2, and TIMP-2 transcription. Furthermore, hypoxia produced a significant, sustained increase in the gene expression and release of VEGF-165, the only VEGF-A isoform detected in these cells. AACOCF3 reduced the hypoxia-induced VEGF release at 8 h of hypoxia. VEGF receptors KDR and NP-1 were constitutively expressed in EC and up-regulated under hypoxic conditions. CONCLUSIONS: In monkey choroid-retinal EC, hypoxia selectively induces MMP-2 activity. This induction is preceded by MT1-MMP, MMP-2, and TIMP-2 mRNA expression, as well as that of the VEGF-165 isoform and its receptors KDR and NP1. These increases possibly result from hypoxia-induced activation of cPLA2 and subsequent release of arachidonic acid and its conversion to prostaglandins. These molecular changes in EC could, in part, contribute to the angiogenic response that occurs in the development of ischemic retinopathies and choroidal neovascularization.

Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia.

PURPOSE: Proinflammatory signaling mechanisms are implicated in the induction of retinal neovascularization (NV) during ischemic retinopathies. This study examined transcription factor (TF) AP-1, HIF-1, and NF-kappaB DNA-binding in relation to cyclooxygenase (COX)-2 and VEGF RNA and protein levels in hypoxia-triggered monkey choroidal retinal (RF/6A) endothelial cells. Effects of the carboxamide CGP43182 were tested on COX-2 and VEGF activation and prostaglandin (PG)E(2) release. METHODS: RF/6A cells were subjected to hypoxia for 1 and 3 hours, at which times RNA and proteins were isolated. Potential AP-1, hypoxia-inducible factor (HIF)-1 and NF-kappaB DNA-binding sites were identified using DNA sequence search algorithms and were analyzed using gel-shift assay. COX-2 and VEGF RNA, protein, and PGE(2) levels were quantified by RT-PCR, Western analysis, and enzyme immunoassay, respectively. Tubular morphogenesis was analyzed with phase-contrast imaging microscopy. RESULTS: Nuclear AP-1, HIF-1 and NF-kappaB promoter DNA binding increased 1.5-, 4-, and 3-fold, respectively, after 1 hour of hypoxia. COX-2 RNA was elevated five- and fourfold after 1 and 3 hours of hypoxia, respectively. VEGF RNA and protein abundance lagged behind COX-2 induction but were each increased two- to threefold 3 hours after hypoxia. CGP43182 was found to inhibit NF-kappaB DNA binding, COX-2 and VEGF gene expression, PGE(2) release, and hypoxia-induced tubular morphogenesis. CONCLUSIONS: Maximum HIF-1 and NF-kappaB DNA binding immediately before COX-2 expression suggests that these TFs are important regulators of COX-2 induction in hypoxic RF/6A cells. IL-1beta emulated AP-1, HIF-1, and NF-kappaB DNA binding during hypoxia and may be a novel cytokine trigger for NV. CGP43182 appears to be an effective inhibitor of NV. VEGF expression appears to be regulated through dual interdependent mechanisms involving HIF-1 directly and indirectly through NF-kappaB-mediated COX-2 expression and PGE(2) production.