The Experts below are selected from a list of 129 Experts worldwide ranked by ideXlab platform

M. Elizabeth Hartnett - One of the best experts on this subject based on the ideXlab platform.

  • 7-ketocholesterol induces Endothelial-mesenchymal transition and promotes fibrosis: implications in neovascular age-related macular degeneration and treatment
    Angiogenesis, 2021
    Co-Authors: Haibo Wang, Eric Kunz, Aniket Ramshekar, M. Elizabeth Hartnett
    Abstract:

    Oxidized cholesterols and lipids accumulate in Bruch’s membrane in age-related macular degeneration (AMD). It remains unknown what causal relationship exists between these substances and AMD pathophysiology. We addressed the hypothesis that a prevalent form, 7-ketocholesterol (7KC), promotes Choroidal Endothelial Cell (CEC) migration and macular neovascularization in AMD. Compared to control, 7KC injection caused 40% larger lectin-stained lesions, but 70% larger lesions measured by optical coherence tomography one week after laser-injury. At two weeks, 7KC-injected eyes had 86% larger alpha smooth muscle actin (αSMA)-labeled lesions and more collagen-labeling than control. There was no difference in Cell death. 7KC-treated RPE/choroids had increased αSMA but decreased VE-cadherin. Compared to control-treated CECs, 7KC unexpectedly reduced Endothelial VE-cadherin, CD31 and VEGFR2 and increased αSMA, fibroblast activation protein (FAP) and transforming growth factor beta (TGFβ). Inhibition of TGFβ receptor-mediated signaling by SB431542 abrogated 7KC-induced loss of Endothelial and increase in mesenchymal proteins in association with decreased transcription factor, SMAD3. Knockdown of SMAD3 partially inhibited 7KC-mediated loss of Endothelial proteins and increase in αSMA and FAP. Compared to control, 7KC-treatment of CECs increased Rac1GTP and migration, and both were inhibited by the Rac1 inhibitor; however, CECs treated with 7KC had reduced tube formation. These findings suggest that 7KC, which increases in AMD and with age, induces mesenchymal transition in CECs making them invasive and migratory, and causing fibrosis in macular neovascularization. Further studies to interfere with this process may reduce fibrosis and improve responsiveness to anti-VEGF treatment in non-responsive macular neovascularization in AMD.

  • Optimal Inhibition of Choroidal Neovascularization by scAAV2 with VMD2 Promoter-driven Active Rap1a in the RPE.
    Scientific reports, 2019
    Co-Authors: Haibo Wang, Eric Kunz, Gregory J. Stoddard, William W. Hauswirth, M. Elizabeth Hartnett
    Abstract:

    Age-related macular degeneration (AMD) is a multifactorial chronic disease that requires long term treatment. Gene therapy is being considered as a promising tool to treat AMD. We found that increased activation of Rap1a in the retinal pigment epithelium (RPE) reduces oxidative signaling to maintain barrier integrity of the RPE and resist neural sensory retinal angiogenesis from Choroidal Endothelial Cell invasion. To optimally deliver constitutively active Rap1a (CARap1a) into the RPE of wild type mice, self-complementary AAV2 (scAAV2) vectors driven by two different promoters, RPE65 or VMD2, were generated and tested for optimal active Rap1a expression and inhibition of Choroidal neovascularization (CNV) induced by laser injury. scAAV2-VMD2, but not scAAV2-RPE65, specifically and efficiently transduced the RPE to increase active Rap1a protein in the RPE. Mice with increased Rap1a from the scAAV2-VMD2-CARap1a had a significant reduction in CNV compared to controls. Increased active Rap1a in the RPE in vivo or in vitro inhibited inflammatory and angiogenic signaling determined by decreased activation of NF-κB and expression of VEGF without causing increased Cell death or autophagy measured by increased LCA3/B. Our study provides a potential future strategy to deliver active Rap1a to the RPE in order to protect against both atrophic and neovascular AMD.

  • Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration.
    Investigative ophthalmology & visual science, 2016
    Co-Authors: Haibo Wang, Xiaokun Han, Eric Kunz, M. Elizabeth Hartnett
    Abstract:

    Purpose This 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). Methods Measurements 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. Results Thy-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. Conclusions Thy-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.

  • Upregulation of CCR3 by age-related stresses promotes Choroidal Endothelial Cell migration via VEGF-dependent and -independent signaling.
    Investigative ophthalmology & visual science, 2011
    Co-Authors: Haibo Wang, Erika S. Wittchen, Yanchao Jiang, Balamurali K. Ambati, Hans E. Grossniklaus, M. Elizabeth Hartnett
    Abstract:

    Neovascular age-related macular degeneration (AMD) accounts for 80% of legal blindness in a rapidly growing percentage of the U.S. population, that is, those older than 60 years of age.1 Outcomes have improved with anti–vascular Endothelial growth factor (VEGF) treatment,2 but visual acuity still improves in only 40% of cases, and repeated intraocular injections pose serious ocular risks, including endophthalmitis and retinal detachment.3 Also, treatment benefit is lost after repeated intravitreal injections of VEGF antibody,4 raising a concern of potential retinal toxicity or recurrent Choroidal neovascularization (CNV) caused by compensatory mechanisms through altered regulation of angiogenic and inhibitory growth factors or other signaling.5–8 Thus, considering pathways besides the VEGF pathway is important in the development of therapies for neovascular AMD. The G-protein–coupled receptor (GPCR), C–C chemokine receptor 3 (CCR3), was reported to be important in neovascular AMD.9 Additional evidence supports its role in CNV.10 However, a recent study reported that CCR3 was not important in CNV development when using a CNV gelatinous protein mixture (Matrigel; BD Biosciences, Franklin Lakes, NJ) model.11 Therefore, further study regarding the potential role of CCR3 in AMD is needed. AMD is a complex disease, influenced by environmental and genetic factors. In this study, we focused on the effects of environmental stresses associated with AMD on CCR3 expression in Choroidal Endothelial Cells (CECs). We studied the mechanisms whereby CCR3 signaling activates CECs to migrate, a key event before the development of vision-threatening sensory retinal CNV. We obtained data from human donor eyes and used cultured human CECs and retinal pigmented epithelium (RPE). We also explored the hypothesis that CCR3 and VEGF work synergistically in triggering downstream signaling pathways. We report that the CCR3 ligand, chemokine (C–C motif) ligand 11 (CCL11), activates VEGF receptor 2 to trigger activation of the small GTPase Rac1, which we previously reported is necessary for CEC transmigration of the RPE.12–14

  • The Role of RPE Cell-Associated VEGF189 in Choroidal Endothelial Cell Transmigration across the RPE
    Investigative ophthalmology & visual science, 2011
    Co-Authors: Haibo Wang, P. Geisen, Erika S. Wittchen, Bradley King, Keith Burridge, Patricia A. D'amore, M. Elizabeth Hartnett
    Abstract:

    Purpose. To determine the role of vascular Endothelial growth factor 189 (VEGF189) in Choroidal Endothelial Cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved.

David R. Hinton - One of the best experts on this subject based on the ideXlab platform.

  • Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro.
    Investigative ophthalmology & visual science, 2009
    Co-Authors: S. Ram Kumar, Stephen J. Ryan, Valery Krasnoperov, Parkash S. Gill, Peng Zhou, David R. Hinton
    Abstract:

    The retinal pigment epithelium plays an important role in maintaining photoreceptor Cell survival and function. Normal retinal pigment epithelial (RPE) Cells are quiescent without migration or proliferation.1–3 RPE Cells in the stationary monolayer start to migrate in response to pathologic changes in the microenvironment associated with conditions such as proliferative vitreoretinopathy (PVR),4 age-related macular degeneration,5 and diabetic retinopathy.6 RPE Cell migration is a complex biological process involving changes in Cell attachment, spreading, and cytoskeletal reorganization and is regulated by Cell matrix, matrix-dependent enzymes, cytokines, and growth factors.7–9 RPE Cell migration is also mediated by Cell membrane-associated signaling, especially receptor tyrosine kinases.10,11 Recent reports show that EphB4 and its ligand EphrinB2 mediate Cell migration in a variety of Cells, such as neurons, retinal vascular Endothelial Cells, and Choroidal Endothelial Cells.12,13 The Ephs and Ephrins constitute the largest of the receptor tyrosine kinase families, with 14 receptors and eight ligands.14–16 This family is subdivided into EphA and EphB groups and regulates a diverse array of the Cellular functions (migration, repulsion, adhesion, and vessel maturation).14–17 The interaction between the Eph receptor and the Ephrin ligand activates forward and reverse signaling through interactions with cytoplasmic signaling proteins. The nature of the downstream signaling pathways and the consequent regulation of Cell migration had been demonstrated in various Cell culture systems.18–22 Kertesz23 has highlighted the role of EphB4 in angiogenesis through its effects on Endothelial Cell migration.23 Steinle12 reported that the agonistic EphB4/Fc chimera can stimulate retinal Endothelial Cell migration by activation of PI3K, Src, and other signaling pathways. In a previous study, we found that sEphB4 inhibits vascular Endothelial growth factor (VEGF)-induced Choroidal Endothelial Cell migration.13 We have also demonstrated that platelet-derived growth factor-BB (PDGF) increases RPE Cell migration through the activation of p42/44 mitogen-activated protein kinase (MAPK).24,25 Furthermore, PDGF has been found to be expressed in fibrotic PVR membranes and Choroidal neovascular membranes, which are associated with RPE migration.26,27 Although previous studies have focused on the roles of EphB4 and EphrinB2 in Endothelial Cell function and cancer Cells, little is known about their role in migration of ocular Cells such as RPE Cells. Therefore, the aim of the present study was to determine the expression of EphB4 and EphrinB2 in retinal pigment epithelium, to evaluate the effect of sEphB4 on RPE Cell migration induced by PDGF, and to investigate the signaling pathways involved.

  • Hypericin inhibits Choroidal Endothelial Cell proliferation and cord formation in vitro
    Current Eye Research, 2009
    Co-Authors: Hideya Kimura, Michael S. Harris, Rayudu Gopalakrishna, Usha Gundimeda, Christine Spee, David R. Hinton, Taiji Sakamoto, Stephen J. Ryan
    Abstract:

    PURPOSE. To evaluate the effect of hypericin on bovine Choroidal Endothelial Cell proliferation and cord formation and on protein kinase C activity. METHODS. The effect of hypericin (0.1–5 µM) on bovine Choroidal Endothelial Cell proliferation was determined by Cell number counting and a 3 H-thymidine uptake assay in media containing 1, 5 or 10% serum. For the cord formation assay, bovine Choroidal Endothelial Cells were seeded on basement membrane matrix, and the lengths of the capillary-like structures (cords) formed were quantified by image analysis. The effect of hypericin on cord formation was evaluated in the presence of serum or vascular Endothelial growth factor. The effect of hypericin on protein kinase C activity was also measured in the presence or absence of light. RESEULTS. Hypericin inhibited bovine Choroidal Endothelial Cell proliferation in a dose-dependent manner in the presence of light but not in the dark. Serum dose-dependently masked the inhibition of DNA synthesis by hypericin. Cord ...

  • Soluble EphB4 regulates Choroidal Endothelial Cell function and inhibits laser-induced Choroidal neovascularization.
    Investigative ophthalmology & visual science, 2005
    Co-Authors: Y. Ding, Stephen J. Ryan, Jiehao Zhou, Valery Krasnoperov, Sergey Zozulya, S. Ram Kumar, Parkash S. Gill, David R. Hinton
    Abstract:

    PURPOSE The purpose of this study was to evaluate the effect of a soluble monomeric form of the EphB4 extraCellular domain (sEphB4) on Choroidal Endothelial Cell (CEC) migration and tube formation and on experimental laser-induced Choroidal neovascularization (CNV). METHODS EphrinB2 and EphB4 expression in CECs was investigated by Western blot analysis and immunohistochemistry. Effects of sEphB4 (0.5-3 microg/mL) on CEC migration were evaluated with a modified Boyden chamber assay. Tube formation was assayed in CEC cultures in collagen gel. CNV was induced in rats by laser photocoagulation. The effects of intravitreal injection of sEphB4 on CNV development were evaluated at day 14 by fluorescein angiography (FA), confocal volumetric analysis of isolectin-B4 labeled flatmounts, and histologic examination of CNV membranes. RESULTS CEC Cells express both EphB4 and EphrinB2, according to Western blot analysis. Immunohistochemical sections of rat eye showed immunoreactivity for both EphB4 and EphrinB2 in the Choroidal endothelium. sEphB4 reduced CEC migration in response to vascular Endothelial growth factor (P < 0.01). Similarly, sEphB4 inhibited CEC tube formation in a dose-dependent manner. EphB4, and to a lesser extent EphrinB2, were detected on vascular channels within laser-induced CNV membranes. Intravitreal injection of sEphB4 inhibited laser-induced CNV formation. CNV membranes showed a reduction in leakage score (P < 0.05), and membrane volumes were reduced in size (P < 0.05). Histologic analysis revealed that vascularity was reduced in sEphB4-treated membranes. CONCLUSIONS Recombinant soluble monomeric EphB4 exerts an inhibitory effect on Choroidal angiogenesis in vitro and in vivo. It should be further evaluated for its potential as a novel therapy for CNV.

  • Neuropilin-1 Expression by Endothelial Cells and Retinal Pigment Epithelial Cells in Choroidal Neovascular Membranes
    American journal of ophthalmology, 2005
    Co-Authors: Jennifer I. Lim, Christine Spee, Stephen J. Ryan, Masanori Hangai, Jorge Rocha, Howard S. Ying, David R. Hinton
    Abstract:

    Purpose To determine if vascular Endothelial growth factor (VEGF) coreceptor neuropilin-1 (NP-1) is expressed in Choroidal neovascularization (CNV) and to localize the expression. Design Laboratory investigation. Methods Six CNV membranes (CNVMs) obtained from patients with subfoveal CNV attributable to age-related macular degeneration (AMD) underwent immunohistochemistry for VEGF receptor-2 (VEGFR-2) and NP-1. The positive Cell types were identified by double staining with anticytokeratin, anti-CD31, and antismooth muscle actin (SMA). Results Immunohistochemical staining revealed positivity for VEGFR-2 and NP-1 in all six CNVMs. Both receptors were strongly expressed by new Choroidal Endothelial Cell forming vessels and CD-31-positive Cells in the nonvascular area. They were also expressed in the pigmented retinal pigment epithelial layer and by cytokeratin-positive, nonpigmented retinal pigment epithelium Cells in the nonvascular area. The nonpigmented retinal pigment epithelium Cells positive for VEGFR-2 and NP-1 were highly colocalized with SMA. Conclusions The presence of both VEGF and NP-1 suggest that NP-1 may play a role in the evolution of CNV in AMD.

Kakarla V. Chalam - One of the best experts on this subject based on the ideXlab platform.

  • Differential Sensitivity of Choroidal Endothelial, Retinal Ganglion, and Retinal Pigment Epithelial Cells In Vitro to Proton Radiation
    American journal of ophthalmology, 2013
    Co-Authors: Sankarathi Balaiya, Ravi K Murthy, Robert S. Malyapa, Sandeep Grover, Kakarla V. Chalam
    Abstract:

    Purpose To evaluate the differential sensitivity of Choroidal Endothelial, retinal pigment epithelial, and retinal ganglion Cells to escalating doses of proton beam radiation and to establish a safe dose range for the management of Choroidal neovascularization associated with age-related macular degeneration (AMD). Design Laboratory investigation. Methods Proliferating simian Choroidal Endothelial Cells (RF/6A), differentiated rat retinal ganglion Cells (RGC-5), and serum-starved human retinal pigment epithelial Cells (ARPE-19) were exposed to 2, 4, 8, and 12 cobalt gray equivalent of proton beam radiation and Cell viability was quantified on day 9. Reactive oxygen species levels were analyzed. Results Significant decline of Choroidal Endothelial Cell viability was noted as dose escalated from 4 to 8 cobalt gray equivalent with maximum effect observed at 12 cobalt gray equivalent. RGC-5 and ARPE-19 Cell count decreased to 95% and 62.7% at 8 cobalt gray equivalent, respectively. Sub-analysis between 4 and 8 cobalt gray equivalent radiation revealed significant decrease in Choroidal Endothelial Cell viability (43.1% at 7 cobalt gray equivalent and 32.3% at 8 cobalt gray equivalent of radiation). Correspondingly, RGC-5 and ARPE-19 Cells did not show decrease in Cell count or viability. Reactive oxygen species levels significantly increased in radiation-treated Choroidal Endothelial Cells (8.3%-11.9%). Conclusions At 6-8 cobalt gray equivalent proton beam radiation, retinal ganglion and retinal pigment epithelial Cells are preserved while Choroidal Endothelial Cells are completely inhibited. This dosage offers optimum therapeutic safety window for treatment using proton beam radiation for exudative AMD.

  • Bevacizumab inhibits proliferation of Choroidal Endothelial Cells by regulation of the Cell cycle
    Clinical Ophthalmology, 2013
    Co-Authors: Raluca Rusovici, Chirag J. Patel, Kakarla V. Chalam
    Abstract:

    Background The 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.

  • Evaluation of Choroidal Endothelial Cell proliferation after exposure to varying doses of proton beam radiation.
    Retina (Philadelphia Pa.), 2011
    Co-Authors: Kakarla V. Chalam, Sankarathi Balaiya, Robert S Malyappa, Wen Hsi, Vikram S Brar, Ravi K Murthy
    Abstract:

    PURPOSE Focal epiretinal radiation has emerged as a promising tool in the management of Choroidal neovascularization associated with age-related macular degeneration. However, the dosages tested are not backed by Cell culture studies used in the clinical setting empirically. METHODS Choroidal Endothelial Cells (RF6A) were maintained in a log scale and exposed to a single fraction of 2, 4, 8, and 12 cobalt gray-equivalent of proton radiation with an internal control. Cell viability was quantified using Vi-Cell XR and neutral red assay at days 5, 9, and 12 after radiation. Mitochondrial viability using WST-1 and reactive oxygen species levels using dihydrorhodamine 123 were measured at similar intervals. RESULTS By using neutral red assay, on day 12, the percentages of viable Cells compared with control were 100.1 ± 5.7%, 96.7 ± 23.3%, 27.6 ± 6.6%, and 19.5 ± 3% at radiation doses of 2, 4, 8, and 12 cobalt gray-equivalent, respectively (P < 0.001). Increase in reactive oxygen species levels correlated with the number of dead Cells implicating reactive oxygen species as an intermediary molecule (r = 0.85-0.96). CONCLUSION Our study shows sensitivity of cultured Choroidal Endothelial Cells to proton beam radiation at doses of 8 and 12 cobalt gray-equivalent in an in vitro model.

Haibo Wang - One of the best experts on this subject based on the ideXlab platform.

  • 7-ketocholesterol induces Endothelial-mesenchymal transition and promotes fibrosis: implications in neovascular age-related macular degeneration and treatment
    Angiogenesis, 2021
    Co-Authors: Haibo Wang, Eric Kunz, Aniket Ramshekar, M. Elizabeth Hartnett
    Abstract:

    Oxidized cholesterols and lipids accumulate in Bruch’s membrane in age-related macular degeneration (AMD). It remains unknown what causal relationship exists between these substances and AMD pathophysiology. We addressed the hypothesis that a prevalent form, 7-ketocholesterol (7KC), promotes Choroidal Endothelial Cell (CEC) migration and macular neovascularization in AMD. Compared to control, 7KC injection caused 40% larger lectin-stained lesions, but 70% larger lesions measured by optical coherence tomography one week after laser-injury. At two weeks, 7KC-injected eyes had 86% larger alpha smooth muscle actin (αSMA)-labeled lesions and more collagen-labeling than control. There was no difference in Cell death. 7KC-treated RPE/choroids had increased αSMA but decreased VE-cadherin. Compared to control-treated CECs, 7KC unexpectedly reduced Endothelial VE-cadherin, CD31 and VEGFR2 and increased αSMA, fibroblast activation protein (FAP) and transforming growth factor beta (TGFβ). Inhibition of TGFβ receptor-mediated signaling by SB431542 abrogated 7KC-induced loss of Endothelial and increase in mesenchymal proteins in association with decreased transcription factor, SMAD3. Knockdown of SMAD3 partially inhibited 7KC-mediated loss of Endothelial proteins and increase in αSMA and FAP. Compared to control, 7KC-treatment of CECs increased Rac1GTP and migration, and both were inhibited by the Rac1 inhibitor; however, CECs treated with 7KC had reduced tube formation. These findings suggest that 7KC, which increases in AMD and with age, induces mesenchymal transition in CECs making them invasive and migratory, and causing fibrosis in macular neovascularization. Further studies to interfere with this process may reduce fibrosis and improve responsiveness to anti-VEGF treatment in non-responsive macular neovascularization in AMD.

  • Optimal Inhibition of Choroidal Neovascularization by scAAV2 with VMD2 Promoter-driven Active Rap1a in the RPE.
    Scientific reports, 2019
    Co-Authors: Haibo Wang, Eric Kunz, Gregory J. Stoddard, William W. Hauswirth, M. Elizabeth Hartnett
    Abstract:

    Age-related macular degeneration (AMD) is a multifactorial chronic disease that requires long term treatment. Gene therapy is being considered as a promising tool to treat AMD. We found that increased activation of Rap1a in the retinal pigment epithelium (RPE) reduces oxidative signaling to maintain barrier integrity of the RPE and resist neural sensory retinal angiogenesis from Choroidal Endothelial Cell invasion. To optimally deliver constitutively active Rap1a (CARap1a) into the RPE of wild type mice, self-complementary AAV2 (scAAV2) vectors driven by two different promoters, RPE65 or VMD2, were generated and tested for optimal active Rap1a expression and inhibition of Choroidal neovascularization (CNV) induced by laser injury. scAAV2-VMD2, but not scAAV2-RPE65, specifically and efficiently transduced the RPE to increase active Rap1a protein in the RPE. Mice with increased Rap1a from the scAAV2-VMD2-CARap1a had a significant reduction in CNV compared to controls. Increased active Rap1a in the RPE in vivo or in vitro inhibited inflammatory and angiogenic signaling determined by decreased activation of NF-κB and expression of VEGF without causing increased Cell death or autophagy measured by increased LCA3/B. Our study provides a potential future strategy to deliver active Rap1a to the RPE in order to protect against both atrophic and neovascular AMD.

  • Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration.
    Investigative ophthalmology & visual science, 2016
    Co-Authors: Haibo Wang, Xiaokun Han, Eric Kunz, M. Elizabeth Hartnett
    Abstract:

    Purpose This 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). Methods Measurements 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. Results Thy-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. Conclusions Thy-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.

  • Upregulation of CCR3 by age-related stresses promotes Choroidal Endothelial Cell migration via VEGF-dependent and -independent signaling.
    Investigative ophthalmology & visual science, 2011
    Co-Authors: Haibo Wang, Erika S. Wittchen, Yanchao Jiang, Balamurali K. Ambati, Hans E. Grossniklaus, M. Elizabeth Hartnett
    Abstract:

    Neovascular age-related macular degeneration (AMD) accounts for 80% of legal blindness in a rapidly growing percentage of the U.S. population, that is, those older than 60 years of age.1 Outcomes have improved with anti–vascular Endothelial growth factor (VEGF) treatment,2 but visual acuity still improves in only 40% of cases, and repeated intraocular injections pose serious ocular risks, including endophthalmitis and retinal detachment.3 Also, treatment benefit is lost after repeated intravitreal injections of VEGF antibody,4 raising a concern of potential retinal toxicity or recurrent Choroidal neovascularization (CNV) caused by compensatory mechanisms through altered regulation of angiogenic and inhibitory growth factors or other signaling.5–8 Thus, considering pathways besides the VEGF pathway is important in the development of therapies for neovascular AMD. The G-protein–coupled receptor (GPCR), C–C chemokine receptor 3 (CCR3), was reported to be important in neovascular AMD.9 Additional evidence supports its role in CNV.10 However, a recent study reported that CCR3 was not important in CNV development when using a CNV gelatinous protein mixture (Matrigel; BD Biosciences, Franklin Lakes, NJ) model.11 Therefore, further study regarding the potential role of CCR3 in AMD is needed. AMD is a complex disease, influenced by environmental and genetic factors. In this study, we focused on the effects of environmental stresses associated with AMD on CCR3 expression in Choroidal Endothelial Cells (CECs). We studied the mechanisms whereby CCR3 signaling activates CECs to migrate, a key event before the development of vision-threatening sensory retinal CNV. We obtained data from human donor eyes and used cultured human CECs and retinal pigmented epithelium (RPE). We also explored the hypothesis that CCR3 and VEGF work synergistically in triggering downstream signaling pathways. We report that the CCR3 ligand, chemokine (C–C motif) ligand 11 (CCL11), activates VEGF receptor 2 to trigger activation of the small GTPase Rac1, which we previously reported is necessary for CEC transmigration of the RPE.12–14

  • The Role of RPE Cell-Associated VEGF189 in Choroidal Endothelial Cell Transmigration across the RPE
    Investigative ophthalmology & visual science, 2011
    Co-Authors: Haibo Wang, P. Geisen, Erika S. Wittchen, Bradley King, Keith Burridge, Patricia A. D'amore, M. Elizabeth Hartnett
    Abstract:

    Purpose. To determine the role of vascular Endothelial growth factor 189 (VEGF189) in Choroidal Endothelial Cell (CEC) migration across the retinal pigment epithelium (RPE) and to explore the molecular mechanisms involved.

Stephen J. Ryan - One of the best experts on this subject based on the ideXlab platform.

  • Soluble EphB4 inhibition of PDGF-induced RPE migration in vitro.
    Investigative ophthalmology & visual science, 2009
    Co-Authors: S. Ram Kumar, Stephen J. Ryan, Valery Krasnoperov, Parkash S. Gill, Peng Zhou, David R. Hinton
    Abstract:

    The retinal pigment epithelium plays an important role in maintaining photoreceptor Cell survival and function. Normal retinal pigment epithelial (RPE) Cells are quiescent without migration or proliferation.1–3 RPE Cells in the stationary monolayer start to migrate in response to pathologic changes in the microenvironment associated with conditions such as proliferative vitreoretinopathy (PVR),4 age-related macular degeneration,5 and diabetic retinopathy.6 RPE Cell migration is a complex biological process involving changes in Cell attachment, spreading, and cytoskeletal reorganization and is regulated by Cell matrix, matrix-dependent enzymes, cytokines, and growth factors.7–9 RPE Cell migration is also mediated by Cell membrane-associated signaling, especially receptor tyrosine kinases.10,11 Recent reports show that EphB4 and its ligand EphrinB2 mediate Cell migration in a variety of Cells, such as neurons, retinal vascular Endothelial Cells, and Choroidal Endothelial Cells.12,13 The Ephs and Ephrins constitute the largest of the receptor tyrosine kinase families, with 14 receptors and eight ligands.14–16 This family is subdivided into EphA and EphB groups and regulates a diverse array of the Cellular functions (migration, repulsion, adhesion, and vessel maturation).14–17 The interaction between the Eph receptor and the Ephrin ligand activates forward and reverse signaling through interactions with cytoplasmic signaling proteins. The nature of the downstream signaling pathways and the consequent regulation of Cell migration had been demonstrated in various Cell culture systems.18–22 Kertesz23 has highlighted the role of EphB4 in angiogenesis through its effects on Endothelial Cell migration.23 Steinle12 reported that the agonistic EphB4/Fc chimera can stimulate retinal Endothelial Cell migration by activation of PI3K, Src, and other signaling pathways. In a previous study, we found that sEphB4 inhibits vascular Endothelial growth factor (VEGF)-induced Choroidal Endothelial Cell migration.13 We have also demonstrated that platelet-derived growth factor-BB (PDGF) increases RPE Cell migration through the activation of p42/44 mitogen-activated protein kinase (MAPK).24,25 Furthermore, PDGF has been found to be expressed in fibrotic PVR membranes and Choroidal neovascular membranes, which are associated with RPE migration.26,27 Although previous studies have focused on the roles of EphB4 and EphrinB2 in Endothelial Cell function and cancer Cells, little is known about their role in migration of ocular Cells such as RPE Cells. Therefore, the aim of the present study was to determine the expression of EphB4 and EphrinB2 in retinal pigment epithelium, to evaluate the effect of sEphB4 on RPE Cell migration induced by PDGF, and to investigate the signaling pathways involved.

  • Hypericin inhibits Choroidal Endothelial Cell proliferation and cord formation in vitro
    Current Eye Research, 2009
    Co-Authors: Hideya Kimura, Michael S. Harris, Rayudu Gopalakrishna, Usha Gundimeda, Christine Spee, David R. Hinton, Taiji Sakamoto, Stephen J. Ryan
    Abstract:

    PURPOSE. To evaluate the effect of hypericin on bovine Choroidal Endothelial Cell proliferation and cord formation and on protein kinase C activity. METHODS. The effect of hypericin (0.1–5 µM) on bovine Choroidal Endothelial Cell proliferation was determined by Cell number counting and a 3 H-thymidine uptake assay in media containing 1, 5 or 10% serum. For the cord formation assay, bovine Choroidal Endothelial Cells were seeded on basement membrane matrix, and the lengths of the capillary-like structures (cords) formed were quantified by image analysis. The effect of hypericin on cord formation was evaluated in the presence of serum or vascular Endothelial growth factor. The effect of hypericin on protein kinase C activity was also measured in the presence or absence of light. RESEULTS. Hypericin inhibited bovine Choroidal Endothelial Cell proliferation in a dose-dependent manner in the presence of light but not in the dark. Serum dose-dependently masked the inhibition of DNA synthesis by hypericin. Cord ...

  • Soluble EphB4 regulates Choroidal Endothelial Cell function and inhibits laser-induced Choroidal neovascularization.
    Investigative ophthalmology & visual science, 2005
    Co-Authors: Y. Ding, Stephen J. Ryan, Jiehao Zhou, Valery Krasnoperov, Sergey Zozulya, S. Ram Kumar, Parkash S. Gill, David R. Hinton
    Abstract:

    PURPOSE The purpose of this study was to evaluate the effect of a soluble monomeric form of the EphB4 extraCellular domain (sEphB4) on Choroidal Endothelial Cell (CEC) migration and tube formation and on experimental laser-induced Choroidal neovascularization (CNV). METHODS EphrinB2 and EphB4 expression in CECs was investigated by Western blot analysis and immunohistochemistry. Effects of sEphB4 (0.5-3 microg/mL) on CEC migration were evaluated with a modified Boyden chamber assay. Tube formation was assayed in CEC cultures in collagen gel. CNV was induced in rats by laser photocoagulation. The effects of intravitreal injection of sEphB4 on CNV development were evaluated at day 14 by fluorescein angiography (FA), confocal volumetric analysis of isolectin-B4 labeled flatmounts, and histologic examination of CNV membranes. RESULTS CEC Cells express both EphB4 and EphrinB2, according to Western blot analysis. Immunohistochemical sections of rat eye showed immunoreactivity for both EphB4 and EphrinB2 in the Choroidal endothelium. sEphB4 reduced CEC migration in response to vascular Endothelial growth factor (P < 0.01). Similarly, sEphB4 inhibited CEC tube formation in a dose-dependent manner. EphB4, and to a lesser extent EphrinB2, were detected on vascular channels within laser-induced CNV membranes. Intravitreal injection of sEphB4 inhibited laser-induced CNV formation. CNV membranes showed a reduction in leakage score (P < 0.05), and membrane volumes were reduced in size (P < 0.05). Histologic analysis revealed that vascularity was reduced in sEphB4-treated membranes. CONCLUSIONS Recombinant soluble monomeric EphB4 exerts an inhibitory effect on Choroidal angiogenesis in vitro and in vivo. It should be further evaluated for its potential as a novel therapy for CNV.

  • Neuropilin-1 Expression by Endothelial Cells and Retinal Pigment Epithelial Cells in Choroidal Neovascular Membranes
    American journal of ophthalmology, 2005
    Co-Authors: Jennifer I. Lim, Christine Spee, Stephen J. Ryan, Masanori Hangai, Jorge Rocha, Howard S. Ying, David R. Hinton
    Abstract:

    Purpose To determine if vascular Endothelial growth factor (VEGF) coreceptor neuropilin-1 (NP-1) is expressed in Choroidal neovascularization (CNV) and to localize the expression. Design Laboratory investigation. Methods Six CNV membranes (CNVMs) obtained from patients with subfoveal CNV attributable to age-related macular degeneration (AMD) underwent immunohistochemistry for VEGF receptor-2 (VEGFR-2) and NP-1. The positive Cell types were identified by double staining with anticytokeratin, anti-CD31, and antismooth muscle actin (SMA). Results Immunohistochemical staining revealed positivity for VEGFR-2 and NP-1 in all six CNVMs. Both receptors were strongly expressed by new Choroidal Endothelial Cell forming vessels and CD-31-positive Cells in the nonvascular area. They were also expressed in the pigmented retinal pigment epithelial layer and by cytokeratin-positive, nonpigmented retinal pigment epithelium Cells in the nonvascular area. The nonpigmented retinal pigment epithelium Cells positive for VEGFR-2 and NP-1 were highly colocalized with SMA. Conclusions The presence of both VEGF and NP-1 suggest that NP-1 may play a role in the evolution of CNV in AMD.

  • Carboxyamido-triazole modulates retinal pigment epithelial and Choroidal Endothelial Cell attachment, migration, proliferation, and MMP-2 secretion of Choroidal Endothelial Cells.
    Current eye research, 2005
    Co-Authors: Stephan Hoffmann, Stephen J. Ryan, Peter Wiedemann, Man Lin Jin, Laura Masiero, Elise C. Kohn
    Abstract:

    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 μ g/ml) with and without CAI (0.1–10 μ M) was measured by Cell counting and H-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 μ M CAI). CAI reduced the Cellular viability by apoptosis induction. Conclusions: CAI inhib...