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Pterygia are histologically composed of proliferating fibrovascular tissue. This study compared expression levels of an angiogenic inhibitor, pigment epithelium-derived factor (PEDF), in pterygia with those in normal corneal and conjunctival tissues.The normal human conjunctival and corneal tissues were obtained from surgery or from donor eyes without ocular diseases. Pterygia were excised by therapeutic surgery under a microscope. Pigment epithelium-derived factor and vascular endothelial growth factor (VEGF) were measured by Western blot analysis. Their cellular localizations were determined by immunohistochemistry.Intensive PEDF immunostaining was detected in all the normal corneal and conjunctival samples analyzed, predominantly in the epithelium and endothelium of the cornea and in the epithelium of the limbus and conjunctiva. Under the same immunostaining conditions, pterygial samples showed negative or faint PEDF staining. In contrast, the same pterygial samples all showed intensive VEGF staining, predominantly in the epithelium and in blood vessels. Western blot analysis confirmed that the average PEDF level in pterygia was drastically lower than those in normal corneal and conjunctival tissues, respectively. In contrast, the VEGF level in pterygia was significantly higher than in the normal tissues.Pterygia exhibit significantly lower PEDF but higher VEGF levels than those in normal corneas and conjunctivae. The decreased PEDF level in pterygia may play a role in the formation and progression of pterygia.

We have previously shown that intravitreal injection of plasminogen kringle 5 (K5), a potent angiogenic inhibitor, inhibits ischemia-induced retinal neovascularization in a rat model. Here we report that K5 down-regulates an endogenous angiogenic stimulator, vascular endothelial growth factor (VEGF) and up-regulates an angiogenic inhibitor, pigment epithelium-derived factor (PEDF) in a dose-dependent manner in vascular cells and in the retina. The regulation of VEGF and PEDF by K5 in the retina correlates with its anti-angiogenic effect in a rat model of ischemia-induced retinopathy. Retinal RNA levels of VEGF and PEDF are also changed by K5. K5 inhibits the p42/p44 MAP kinase activation and nuclear translocation of hypoxia-inducible factor-1alpha, which may be responsible for the down-regulation of VEGF. Down-regulation of endogenous angiogenic stimulators and up-regulation of endogenous angiogenic inhibitors, thus leading toward restoration of the balance in angiogenic control, may represent a mechanism for the anti-angiogenic activity of K5.

The purpose of this study was to investigate the potential role of mitogen-activated protein (MAP) kinase in contraction by monitoring MAP kinase phosphorylation (activation) and contraction during agonist stimulation of cat iris sphincter smooth muscle. Changes in tension in response to prostaglandin F(2alpha), latanoprost, a prostaglandin F(2alpha) analog used as an anti-glaucoma drug, and carbachol were recorded isometrically, and MAP kinase activation was monitored by Western blot using a phosphospecific p42/p44 MAP kinase antibody. We found that treatment of the muscle with 2'-Amino-3'-methoxyflavone (PD98059) (10 microM), a specific inhibitor of MAP kinase kinase (MEK), inhibited significantly prostaglandin F(2alpha)- and latanoprost-induced phosphorylation and contraction, but had little effect on those evoked by carbachol. Prostaglandin F(2alpha) increased MAP kinase phosphorylation in a concentration-dependent manner with EC(50) value of 1.1 x 10(-8) M and increased contraction with EC(50) of 0.92 x 10(-9) M. The MAP kinase inhibitors PD98059, Apigenin and 1,4-Diamino-2,3-dicyano-1, 4bis(2-aminophenylthio)butadiene (UO126) inhibited prostaglandin F(2alpha)-induced contraction in a concentration-dependent manner with IC(50) values of 2.4, 3.0 and 4.8 microM, respectively. PD98059 had no effect on prostaglandin F(2alpha)- or on carbachol-stimulated inositol-1,4,5-trisphosphate (IP(3)) production. In contrast, the MAP kinase inhibitor inhibited prostaglandin F(2alpha)-induced myosin-light chain (MLC) phosphorylation, but had no effect on that of carbachol. N-[2-(N-(4-Chloro-cinnamyl)-N-methylaminomethyl)phenyl]-N-[2- hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93) (10 microM), a Ca(2+)-calmodulin-dependent protein kinase inhibitor, and Wortmannin (10 microM), an MLC kinase inhibitor, inhibited significantly (by 80%) prostaglandin F(2alpha)- and carbachol-induced contraction. It can be concluded that in this smooth muscle p42/p44 MAP kinases are involved in the mechanism of prostaglandin F(2alpha)-, but not in that of carbachol, induced contraction. In addition, these data clearly indicate that the stimulation of the iris sphincter with prostaglandin F(2alpha) and carbachol activate two distinct pathways, the MAP kinase pathway and the Ca(2+) mobilization pathway.

Purpose Without therapeutic intervention, corneal neovascularization rapidly compromises visual acuity, and is a leading cause of blindness. Artesunate was reported to inhibit angiogenesis in tumors, although, the effects of artesunate on nontumor angiogenesis have not been investigated. This study was designed to investigate the effect of artesunate on corneal neovascularization and delineate its underlying mechanism of action. Methods Rats with alkali-burned corneas were treated with artesunate for 11 days. Corneal neovascularization was evaluated by measuring the length and area of corneal vasculature in the rats. Apoptotic cells were stained with AnnexinV and propidine iodide (PI), and measured with flow cytometry analysis. Apoptosis-related and p38 mitogen-activated protein kinases (p38MAPK) signaling were evaluated by Western blot analysis. Results Artesunate significantly inhibited corneal neovascularization and inflammation via specifically inducing apoptosis of vascular endothelial cells. In vascular endothelial cells, artesunate increased the Bax/Bcl-2 ratio, reduced mitochondrial membrane potential, stimulated release of cytochrome C, and cleavage of caspase 9 and 3, suggesting that the mitochondrial apoptotic pathway was involved. Artesunate activated p38MAPK, and specific p38MAPK inhibitors suppressed artesunate-induced apoptosis in endothelial cells. Reactive oxygen species (ROS) levels were increased by artesunate. N-acetyl-L-cysteine blocked p38MAPK activation and protected endothelial cells from artesunate-induced apoptosis. Ferrous salt increased ROS levels and elevated the cytotoxic effect of artesunate on endothelial cells, while the iron chelating agent deferoxamine decreased ROS levels and artesunate-induced apoptosis. Artesunate had no effect on expression of Fas, Fas Ligand, or caspase 8 cleavage. Conclusions These results suggest that artesunate induces apoptosis of endothelial cells via an iron/ROS-dependent p38MAPK-mitochondrial pathway.

PURPOSE. Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. The purpose of the present study was to evaluate the therapeutic effect of K5 on alkali-burn–induced corneal neovascularization (NV) and to investigate its mechanism of action. METHODS. Corneal NV was induced in rabbits by NaOH. The rabbits received eye drops containing K5 or vehicle alone, four times per day. Corneal NV and inflammation were monitored every other day with a slit lamp microscope, and the length of the vessels in the cornea and the area of NV were measured. Vascular endothelial growth factor (VEGF) was determined by immunohistochemical and Western blot analyses. The TUNEL assay was used to assess the apoptosis of endothelial cells. The effects of K5 on primary bovine aortic endothelial cells (BAECs) were determined by MTT assay, flow cytometry, transmission electron microscopy, and DNA fragmentation assay. RESULTS. Alkali-burn–induced progressive corneal NV and inflammation in the cornea. K5 delayed the onset of corneal NV (P 0.05) and decreased NV areas (P 0.05) in a dosedependent manner. K5 treatment, after the formation of corneal NV, induced regression of newly formatted vessels in the cornea. K5 decreased the inflammatory index in the corneas at different time points after the alkali burn. Corneal VEGF levels were reduced by K5 treatment. K5 inhibits proliferation and induces apoptosis in BAECs. CONCLUSIONS. Topical application of K5 may have therapeutic potential for the chemical burn-induced corneal NV and inflammation. The inhibitory effect of K5 on corneal NV may be by downregulation of VEGF expression. (Invest Ophthalmol Vis Sci. 2005;46:4062– 4071) DOI:10.1167/iovs.04-1330

Aims/hypothesis Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes. Methods Cultured retinal pigment epithelial cells and retinal capillary endothelial cells were treated with a lipid peroxidation product, 4-hydroxynonenal (HNE), and an antioxidant, N-acetyl-cysteine (NAC). In vivo, rats with streptozotocin-induced diabetes were treated by NAC for 8 weeks. Activation of the canonical WNT pathway was measured by TOPFLASH assay and by western blot analysis of WNT pathway components and a WNT target gene, Ctgf. Oxidative stress in the retina was evaluated by immunostaining of HNE and 3-nitrotyrosine. Results Levels of phosphorylated and total LDL receptor-related protein (LRP)6, and cytosolic β-catenin, as well as transcriptional activity of T cell factor (TCF)/β-catenin were significantly increased by HNE. The production of connective tissue growth factor (CTGF) was also upregulated by HNE. NAC blocked the WNT pathway activation induced by HNE. Furthermore, LRP6 stability was increased by HNE and decreased by NAC. Retinal levels of HNE and 3-nitrotyrosine were significantly increased in diabetic rats, compared with those in non-diabetic rats. In the same diabetic rat retinas, levels of LRP6, cytosolic β-catenin and CTGF were significantly increased. NAC treatment reduced HNE and 3-nitrotyrosine levels and attenuated the upregulation of LRP6, β-catenin and CTGF in diabetic rat retina. Conclusions/interpretation Lipid peroxidation products activate the canonical WNT pathway through oxidative stress, which plays an important role in the development of retinal diseases.