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To date, diseases affecting vascular structures in the posterior eye are mostly treated by laser photocoagulation and multiple intraocular injections, procedures that destroy healthy tissue and can cause vision-threatening complications. To overcome these drawbacks, we investigate the feasibility of receptor-mediated nanoparticle targeting to capillary endothelial cells in the retina after i.v. application. Cell-binding studies using microvascular endothelial cells showed receptor-specific binding and cellular uptake of cyclo(RGDfC)-modified quantum dots via the αvβ3 integrin receptor. Conversely, Mueller cells and astrocytes, representing off-target cells located in the retina, revealed only negligible interaction with nanoparticles. In vivo experiments, using nude mice as the model organism, demonstrated a strong binding of the ligand-modified quantum dots in the choriocapillaris and intraretinal capillaries upon i.v. injection and 1-h circulation time. Nontargeted nanoparticles, in contrast, did not accumulate to a significant amount in the target tissue. The presented strategy of targeting integrin receptors in the retina could be of utmost value for future intervention in pathologies of the posterior eye, which are to date only accessible with difficulty.

Purpose: Galectin-1 and -3 are β-galactoside binding lectins with varying effects on angiogenesis and apoptosis. Since in retinal pigment epithelial cells high amounts of human recombinant galectin (hr-GAL)1 and 3 inhibit cell adhesion, migration and proliferation, we investigated if hr-GAL1 and 3 have homologous effects on human retinal microvascular endothelial cells (HRMEC) in vitro. Methods: To investigate the effect of galectin-1 and -3 on HRMEC, proliferation, apoptosis and viability were analyzed after incubation with 30, 60 and 120 μg/ml hr-GAL1 or 3 by BrdU-ELISA, histone-DNA complex ELISA, live/dead staining and the WST-1 assay, respectively. Further on, a cell adhesion as well as tube formation assay were performed on galectin-treated HRMEC. Migration was investigated by the scratch migration assay and time-lapse microscopy. In addition, immunohistochemical staining on HRMEC for β-catenin, galectin-1 and -3 were performed and β-catenin expression was investigated by western blot analysis. Results: Incubation with hr-GAL1 or 3 lead to a decrease in proliferation, migration, adhesion and tube formation of HRMEC compared to the untreated controls. No toxic effects of hr-GAL1 and 3 on HRMEC were detected. Intriguingly, after treatment of HRMEC with hr-GAL1 or 3, an activation of the proangiogenic Wnt/β-catenin signaling pathway was observed. However, incubation of HRMEC with hr-GAL1 or 3 drew intracellular galectin-1 and -3 out of the cells, respectively. Conclusion: Exogenously added hr-GAL1 or 3 inhibit angiogenic properties of HRMEC in vitro, an effect that might be mediated via a loss of intracellular endogenous galectins. [ABSTRACT FROM AUTHOR]

Purpose: Choroidal neovascularizations (CNV) are partially stabilized through a coverage of pericytes leading to a partial anti-VEGF resistence. Drugs licensed for neovascular AMD (nAMD) do not take this mechanical and growth factor-driven CNV stability into account. The purpose of this work was to see if inhibiting the mammalian target of rapamycin (mTOR) may successfully block angiogenic cellular pathways in primary human retinal pericytes in an in vitro model of nAMD.Methods: The mTOR inhibitor rapamycin was used to treat human retinal pericytes (HRP) at doses ranging from 0.005 to 15 g/ml. A modified metabolism-based XTT-Assay was used to assess toxicity and anti-proliferative effects. A scratch wound experiment showed the effects on migration. On Cultrex basement membrane gels, the influence of rapamycin on the development of endothelial cell capillary-like structures by human umbilical vein vascular endothelial cells (HUVEC) in the absence and presence of pericytes was investigated.Results: Rapamycin showed no signs of toxicity within its range of solubility. The drug showed dose dependent anti-proliferative activity and inhibited migration into the scratch wound. Endothelial cell tube formation in a HUVEC monoculture was effectively inhibited at 45%. A co-culture of HUVEC with pericytes on Cultrex induced endothelial tube stabilization but was disrupted by the addition of rapamycin leading to degradation of 94% of the tubes.Conclusions: Rapamycin allows for an efficient modulation of aspects of angiogenesis in pericytes via mTOR-modulation in vitro. Further studies are needed to elucidate whether rapamycin may have an impact on CNV in nAMD in vivo. [ABSTRACT FROM AUTHOR]

Low energy stereotactic radiotherapy has been proposed for the treatment of neovascular age related macular degeneration. We investigated the in vitro effect of the radiotherapy on pericytes, retinal pigment epithelium and endothelial cells. Primary human retinal pigment epithelium cells, human umbilical vein endothelial cells and human pericytes from Placenta were cultivated. In a pairwise protocol, one plate was irradiated at a dose of 16 Gy, while the second plate served as a non-irradiated control. Thereafter, cells were cultivated either in serum-free (non-permissive) or serum-stimulated (permissive) conditions. A life/dead assay, an XTT and a BrdU assay were performed up to 7 days after irradiation. No cell death occurred at any timepoint in any cell line after treatment nor in the control. Compared to the unirradiated controls, cell viability and metabolic activity were significantly reduced in irradiated cells in the XTT assay, except for non-permissive RPE cells. In the BrdU assay, proliferation was inhibited. While no cell death was detected in vitro, viability and proliferative capacity of all cell lines were significantly reduced. Therefore, it seems that low energy stereotactic radiotherapy inhibits angiogenesis without a direct induction of apoptosis but influencing microvascular function and stability. [ABSTRACT FROM AUTHOR]

Purpose: To determine whether excimer laser ablation of guttae is a viable strategy for removal of diseased tissue in Fuchs' endothelial corneal dystrophy (FECD) on excised human Descemet membranes and whether an excimer laser‐created wound on healthy human corneas ex vivo is recolonized with corneal endothelial cells. Methods: Descemet membranes of FECD patients and corneal endothelium of normal human corneas were ablated ex vivo using an excimer laser licensed for glaucoma surgery. Specimens were kept in cell culture medium supplemented with 10 μm of rho‐kinase inhibitor ripasudil. Corneal endothelial cell regeneration was observed using light and electron scanning microscopy. Furthermore, the whole corneal samples were evaluated by haematoxylin/eosin staining and immunohistochemical analysis using antibodies against Na+/K+‐ATPase. Results: Guttae and corneal endothelium could be ablated with an excimer laser without total ultrastructural damage to the Descemet membrane or stroma. Nearly complete endothelial wound closure was accomplished after 26–38 days in treated corneas. Light and electron scanning microscopy suggested the establishment of a layer of flat endothelial cells. Additionally, Na+/K+‐ATPase expression could only be observed on the inner side of the Descemet membrane. Conclusion: Our proof of concept study demonstrated that excimer lasers can be used to ablate diseased tissue from excised FECD Descemet membranes ex vivo. Additionally, corneal endothelial cells recolonize a previously ablated endothelial area in healthy human corneas ex vivo under treatment with ripasudil. Thus, our results are the first experimental basis to further investigate the feasibility of an excimer laser ablation as a graftless FECD treatment option. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin signaling induced by the Norrin/Frizzled-4 pathway has been shown to improve capillary repair following oxygen induced retinopathy (OIR) in the mouse, a model for retinopathy of prematurity. Here we investigated if treatment with the monovalent cation lithium that has been shown to augment Wnt/β-catenin signaling in vitro and in vivo has similar effects. In cultured human microvascular endothelial cells, LiCl as well as SB 216763, another small molecule that activates Wnt/β-catenin signaling, induced proliferation, survival and migration, which are all common parameters for angiogenic properties in vitro. Moreover, treatment with both agents caused an increase in the levels of β-catenin and their translocation to nuclei while quercetin, an inhibitor of Wnt/β-catenin signaling, completely blocked the effects of LiCl on proliferation. In mice with OIR, intraperitonal or intravitreal treatment with LiCl markedly increased the retinal levels of β-catenin, but did not improve capillary repair. In contrast, repair was significantly improved following intravitreal treatment with Norrin. The effects of LiCl on HDMEC in vitro have minor relevance for OIR in vivo, and the influence of the Norrin/Frizzled-4 pathway on capillary repair in OIR is not reproducible upon enhancing Wnt/β-catenin signaling by LiCl treatment strongly indicating the presence of additional and essential mechanisms. [ABSTRACT FROM AUTHOR]

Background: Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis. Methodology/Principal Findings: By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas. Conclusions/Significance: Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer. [ABSTRACT FROM AUTHOR]

Wnt/β-catenin signaling induced by the Norrin/Frizzled-4 pathway has been shown to improve capillary repair following oxygen induced retinopathy (OIR) in the mouse, a model for retinopathy of prematurity. Here we investigated if treatment with the monovalent cation lithium that has been shown to augment Wnt/β-catenin signaling in vitro and in vivo has similar effects. In cultured human microvascular endothelial cells, LiCl as well as SB 216763, another small molecule that activates Wnt/β-catenin signaling, induced proliferation, survival and migration, which are all common parameters for angiogenic properties in vitro. Moreover, treatment with both agents caused an increase in the levels of β-catenin and their translocation to nuclei while quercetin, an inhibitor of Wnt/β-catenin signaling, completely blocked the effects of LiCl on proliferation. In mice with OIR, intraperitonal or intravitreal treatment with LiCl markedly increased the retinal levels of β-catenin, but did not improve capillary repair. In contrast, repair was significantly improved following intravitreal treatment with Norrin. The effects of LiCl on HDMEC in vitro have minor relevance for OIR in vivo, and the influence of the Norrin/Frizzled-4 pathway on capillary repair in OIR is not reproducible upon enhancing Wnt/β-catenin signaling by LiCl treatment strongly indicating the presence of additional and essential mechanisms. [ABSTRACT FROM AUTHOR]