Choroidal Endothelial Cell Migration Research Articles

A mast cell stabilizer attenuates pathological angiogenesis in a model of choroidal neovascularization

Age‐related macular degeneration (AMD) is one of the leading causes of irreversible blindness in the elderly worldwide. Wet AMD is characterized by pathologic choroidal neovascularization that invades the RPE and compromises retinal function. Although anti‐vascular endothelial growth factor (anti‐VEGF) therapy has been shown to improve symptoms of wet AMD in a large number of people, long‐term treatment is associated with the development of geographic atrophy. The development of new therapeutic alternatives to prevent the progression of exudative AMD is still necessary. Inflammation and deterioration of the RPE are considered to be the main sources of the pathogenesis of AMD. Recent studies have reported accumulation and degranulation of mast cells in the compromised choroid of patients with AMD. When activated, mast cells can secrete a variety of biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. However, the precise role of mast cells in the pathogenesis of AMD remains largely unknown. We are interested in how mast cells participate in the initiation of inflammation in the choroid which results in pathological choroidal neovascularization. We investigated the effect of a mast cell stabilizer on angiogenic response in the choroid. The angiogenic effect of a mast cell stabilizer, ketotifen fumarate, was evaluated in laser‐induced CNV model. Mice received intraperitoneal injections of ketotifen fumarate (5 and 25 mg/kg/d) or vehicle. CNV area and mononuclear phagocytes were determined respectively by using lectin staining and an anti–IBA‐1 antibody on RPE/choroid flat mounts. CNV was attenuated by ketotifen fumarate with no effect on inflammatory cell recruitment. Conditioned media from activated peritoneal mast cells exerted proangiogenic effects on vascular sprouting. Tryptase increased choroidal endothelial cell migration and choroidal vascular sprouting. The study supports possible modulation of mast cells as a therapeutic modality for CNV.

Regulation of Rac1 Activation in Choroidal Endothelial Cells: Insights into Mechanisms in Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is one of the leading causes of blindness worldwide. Vision loss from the neovascular form is associated with the invasion of choroidal endothelial cells into the neural retina to form vision-threatening macular neovascularization (MNV). Anti-angiogenic agents are the current standard of care but are effective in only ~50% of AMD cases. The molecular mechanisms involved in invasive MNV point to the importance of regulating signaling pathways that lead to pathologic biologic outcomes. In studies testing the effects of AMD-related stresses, activation of the Rho GTPase, Rac1, was found to be important for the choroidal endothelial cell invasion into the neural retina. However, current approaches to prevent Rac1 activation are inefficient and less effective. We summarize active Rac1-mediated mechanisms that regulate choroidal endothelial cell migration. Specifically, we discuss our work regarding the role of a multidomain protein, IQ motif containing GTPase activating protein 1 (IQGAP1), in sustaining pathologic Rac1 activation and a mechanism by which active Rap1, a Ras-like GTPase, may prevent active Rac1-mediated choroidal endothelial cell migration.

HGF and VEGF-A and Their Receptors Show Expression and Angiogenic Effects on Human Choroidal Endothelial Cells: Implications for Treatments of Neovascular Age-Related Macular Degeneration

Intraocular neovascularisation is associated with common blinding conditions including neovascular age-related macular degeneration (nAMD). Vascular endothelial growth factor (VEGF) is central in driving choroidal neovascularisation in this disease. Many clinical therapies target VEGF-A with intravitreal anti-VEGF drugs, which, however, have limited efficacy and require repeated, prolonged treatment. Other cytokines are known to be involved, including hepatocyte growth factor (HGF), which is shown to have a role in the early stages of nAMD. We investigated the effect of HGF and its co-operation with VEGF-A on human choroidal endothelial cells (CEC). The expression of HGF and related molecules in CEC was investigated using immunofluorescence, Western blotting and flow cytometry. In vitro assays for proliferation, tubule formation and migration were used to assess the potential role of HGF in neovascularisation. Primary human CEC expressed HGF, VEGF-A and their receptors MET and VEGF receptor 2 (VEGFR2). HGF increased CEC proliferation, tubule formation and migration; the increased proliferation and migration appeared to be additive with that achieved with VEGF-A. This study provides insight into growth factor co-operation in CEC signalling and indicates that simultaneous blockage of multiple growth factors or common downstream signalling pathways may provide a more sustained treatment response, enhancing treatments in nAMD.

Glycine-Conjugated Bile Acids Protect RPE Tight Junctions against Oxidative Stress and Inhibit Choroidal Endothelial Cell Angiogenesis In Vitro

We previously demonstrated that the bile acid taurocholic acid (TCA) inhibits features of age-related macular degeneration (AMD) in vitro. The purpose of this study was to determine if the glycine-conjugated bile acids glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and glycoursodeoxycholic acid (GUDCA) can protect retinal pigment epithelial (RPE) cells against oxidative damage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in choroidal endothelial cells (CECs). Paraquat was used to induce oxidative stress and disrupt tight junctions in HRPEpiC primary human RPE cells. Tight junctions were assessed via transepithelial electrical resistance and ZO-1 immunofluorescence. GCA and GUDCA protected RPE tight junctions against oxidative damage at concentrations of 100–500 µM, and GDCA protected tight junctions at 10–500 µM. Angiogenesis was induced with VEGF in RF/6A macaque CECs and evaluated with cell proliferation, cell migration, and tube formation assays. GCA inhibited VEGF-induced CEC migration at 50–500 µM and tube formation at 10–500 µM. GUDCA inhibited VEGF-induced CEC migration at 100–500 µM and tube formation at 50–500 µM. GDCA had no effect on VEGF-induced angiogenesis. None of the three bile acids significantly inhibited VEGF-induced CEC proliferation. These results suggest glycine-conjugated bile acids may be protective against both atrophic and neovascular AMD.

IQGAP1 causes choroidal neovascularization by sustaining VEGFR2-mediated Rac1 activation.

Loss of visual acuity in neovascular age-related macular degeneration (nAMD) occurs when factors activate choroidal endothelial cells (CECs) to transmigrate the retinal pigment epithelium into the sensory retina and develop into choroidal neovascularization (CNV). Active Rac1 (Rac1GTP) is required for CEC migration and is induced by different AMD-related stresses, including vascular endothelial growth factor (VEGF). Besides its role in pathologic events, Rac1 also plays a role in physiologic functions. Therefore, we were interested in a method to inhibit pathologic activation of Rac1. We addressed the hypothesis that IQGAP1, a scaffold protein with a Rac1 binding domain, regulates pathologic Rac1GTP in CEC migration and CNV. Compared to littermate Iqgap1+/+, Iqgap1-/- mice had reduced volumes of laser-induced CNV and decreased Rac1GTP and phosphorylated VEGFR2 (p-VEGFR2) within lectin-stained CNV. Knockdown of IQGAP1 in CECs significantly reduced VEGF-induced Rac1GTP, mediated through p-VEGFR2, which was necessary for CEC migration. Moreover, sustained activation of Rac1GTP induced by VEGF was eliminated when CECs were transfected with an IQGAP1 construct that is unable to bind Rac1. IQGAP1-mediated Src activation was involved in initiating Rac1 activation, CEC migration, and tube formation. Our findings indicate that CEC IQGAP1 interacts with VEGFR2 to mediate Src activation and subsequent Rac1 activation and CEC migration. In addition, IQGAP1 binding to Rac1GTP results in sustained activation of Rac1, leading to CEC migration toward VEGF. Our study supports a role of IQGAP1 and the interaction between IQGAP1 and Rac1GTP to restore CECs quiescence and, therefore, prevent vision-threatening CNV in nAMD.

Intravitreal injection of resveratrol inhibits laser-induced murine choroidal neovascularization.

To determine the effects of intravitreal resveratrol (RSV) on murine laser-induced choroidal neovascularization (CNV). The toxicity of RSV to choroidal endothelial cell (CEC) was measured using thiazolyl blue tetrazolium bromide (MTT) assay. Effects of RSV on choroidal endothelial cell (CEC) migration were evaluated with a modified Boyden chamber assay, while tube formation was evaluated in a 2-D gel assay. CNV was induced by laser photocoagulation in mice. The effects of intravitreal injection of RSV on CNV development were evaluated by fluorescein angiography (FA), confocal analysis of isolectin B4 labeled choroidal flat mounts, and histologic examination of CNV membranes. Immunostaining was used to analyze the expression and phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2). No significant cell toxicity was observed in CEC if the concentration of RSV was less than 200 µmol/L (P>0.05). RSV inhibited vascular endothelial growth factor (VEGF)-induced CEC migration (P<0.05) and tube formation (P<0.05) in vitro. Furthermore, intravitreal injection of RSV significantly inhibited laser induced CNV formation in mice. The FA leakage, CNV volume and CNV area analysis revealed that there were 41%, 45%, and 58% reduction in RSV-treated eyes (1.691±0.1032, 178 163±78 623 µm3 and 6508±619.0 µm2, respectively) compared with those in control (2.724±0.08447, 379 676±98 382 µm3 and 16 576±2646 µm2, respectively; P<0.05). Phospho-VEGFR2 expression was much weaker in the sections of CNV lesions in RSV injected mice compared with that in control (P<0.05). Intravitreal injection of RSV exerts an inhibitory effect on CNV, which may through suppressing endothelial cell migration, tube formation and VEGFR2 phosphorylation.

Taurocholic acid inhibits features of age-related macular degeneration in vitro

Previous metabolomics studies from our lab found altered plasma levels of bile acids in patients with age-related macular degeneration (AMD) compared to controls. In this study, we investigated the ability of the bile acid taurocholic acid (TCA) to inhibit features of AMD modeled in vitro. Paraquat was used to induce oxidative stress in HRPEpiC primary retinal pigment epithelial (RPE) cells. Cells were treated with 300 μM paraquat alone or with TCA (10, 50, 100, 200, or 500 μM). RPE tight junction integrity was assessed via ZO-1 immunofluorescence and transepithelial electrical resistance (TEER) measurements. RF/6A macaque choroidal endothelial cells were treated with 100 ng/mL vascular endothelial growth factor (VEGF) to induce angiogenesis. The effect of TCA on VEGF-induced angiogenesis was evaluated with cell proliferation, cell migration, and tube formation assays. Addition of TCA at 100 (P = 8.6 × 10−4), 200 (P = 0.0035), and 500 (P = 2.1 × 10−4) μM resulted in significant preservation of TEER in paraquat treated cells. In RF/6A cells, TCA did not significantly affect VEGF-induced cell proliferation. VEGF-induced migration of RF/6A cells was significantly inhibited at TCA concentrations of 100 (P = 0.010), 200 (P = 0.023) and 500 (P = 0.0049) μM. VEGF-induced tube formation was significantly inhibited when treated with 200 (P = 0.014) and 500 (P = 7.1 × 10−4) μM TCA. In vitro, TCA promoted RPE cell integrity and diminished VEGF-induced choroidal endothelial cell migration and tube formation. This suggests that TCA may have protective effects against both degenerative and neovascular AMD.

Antagonistic effects of exogenous Slit2 on VEGF-induced choroidal endothelial cell migration and tube formation.

Vascular endothelial growth factor (VEGF) is involved in the pathogenesis of choroidal neovascularization. The aim of the present study was to assess the effects of exogenous slit guidance ligand 2 (Slit2) on VEGF-induced choroidal endothelial cell (CEC) migration and tube formation. The protein and mRNA expression levels of Slit2, roundabout guidance receptor (Robo) 1 and Robo4 in CECs were evaluated by immunocytochemistry and reverse transcription-polymerase chain reaction analyses, respectively. Western blot analysis was used to assess Robo4 protein levels in CECs exposed to increasing concentrations (0, 50, 75, 100, 125 and 150 ng/ml) of exogenous Slit2. The effects of exogenous Slit2 (125 ng/ml) on VEGF-induced CEC migration and tube formation were also examined. CECs expressed Slit2 and Robo4, but lacked Robo1 expression, at the mRNA and protein levels. Robo4 protein expression increased significantly following treatment with 50–150 ng/ml exogenous Slit2. No significant difference in Robo4 protein expression was observed in CECs treated with 125 and 150 ng/ml Slit2. VEGF-induced CEC migration and tube formation were significantly reduced following treatment with 125 ng/ml exogenous Slit2. In conclusion, these results indicate that Robo4 is expressed in CECs. In addition, exogenous Slit2 may regulate Robo4 expression and partially inhibit VEGF-induced CEC migration and tube formation.

Thy-1 Regulates VEGF-Mediated Choroidal Endothelial Cell Activation and Migration: Implications in Neovascular Age-Related Macular Degeneration.

PurposeThis 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).MethodsMeasurements 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.ResultsThy-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.ConclusionsThy-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.

Rap1 GTPase Inhibits Tumor Necrosis Factor-α–Induced Choroidal Endothelial Migration via NADPH Oxidase– and NF-κB–Dependent Activation of Rac1

Macrophage-derived tumor necrosis factor (TNF)-α has been found in choroidal neovascularization (CNV) surgically removed from patients with age-related macular degeneration. However, the role of TNF-α in CNV development remains unclear. In a murine laser-induced CNV model, compared with un-lasered controls, TNF-α mRNA was increased in retinal pigment epithelial and choroidal tissue, and TNF-α colocalized with lectin-stained migrating choroidal endothelial cells (CECs). Inhibition of TNF-α with a neutralizing antibody reduced CNV volume and reactive oxygen species (ROS) level around CNV. In CECs, pretreatment with the antioxidant apocynin or knockdown of p22phox, a subunit of NADPH oxidase, inhibited TNF-α-induced ROS generation. Apocynin reduced TNF-α-induced NF-κB and Rac1 activation, and inhibited TNF-α-induced CEC migration. TNF-α-induced Rac1 activation and CEC migration were inhibited by NF-κB inhibitor Bay11-7082. Overexpression of Rap1a prevented TNF-α-induced ROS generation and reduced NF-κB and Rac1 activation. Activation of Rap1 by 8-(4-chlorophenylthio)adenosine-2'-O-Me-cAMP prevented TNF-α-induced CEC migration and reduced laser-induced CNV volume, ROS generation, and activation of NF-κB and Rac1. These findings provide evidence that active Rap1a inhibits TNF-α-induced CEC migration by inhibiting NADPH oxidase-dependent NF-κB and Rac1 activation and suggests that Rap1a de-escalates CNV development by interfering with ROS-dependent signaling in several steps of the pathogenic process.

Expression of Thrombospondin‐1 Modulates the Angioinflammatory Phenotype of Choroidal Endothelial Cells

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age‐related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin‐1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation has a significant impact on phenotype of ChEC. ChEC from TSP1‐deficient (TSP1‐/‐) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re‐expression of TSP1 was sufficient to restore TSP1‐/‐ ChEC migration and capillary morphogenesis. TSP1‐/‐ ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS a marker of inflammation, which was associated with significantly higher level of NO in these cells. Wild type and TSP1‐/‐ ChEC produced similar levels of VEGF, although TSP1‐/‐ ChEC exhibited increased levels of VEGF‐R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.

Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells.

The choroidal circulation plays a central role in maintaining the health of outer retina and photoreceptor function. Alterations in this circulation contribute to pathogenesis of many eye diseases including exudative age-related macular degeneration. Unfortunately, very little is known about the choroidal circulation and its molecular and cellular regulation. This has been further hampered by the lack of methods for routine culturing of choroidal endothelial cells (ChEC), especially from wild type and transgenic mice. Here we describe a method for isolation and culturing of mouse ChEC. We show that expression of thrombospondin-1 (TSP1), an endogenous inhibitor of angiogenesis and inflammation, has a significant impact on phenotype of ChEC. ChEC from TSP1-deficient (TSP1−/−) mice were less proliferative and more apoptotic, less migratory and less adherent, and failed to undergo capillary morphogenesis in Matrigel. However, re-expression of TSP1 was sufficient to restore TSP1−/− ChEC migration and capillary morphogenesis. TSP1−/− ChEC expressed increased levels of TSP2, phosphorylated endothelial nitric oxide synthase (NOS) and inducible NOS (iNOS), a marker of inflammation, which was associated with significantly higher level of NO and oxidative stress in these cells. Wild type and TSP1−/− ChEC produced similar levels of VEGF, although TSP1−/− ChEC exhibited increased levels of VEGF-R1 and pSTAT3. Other signaling pathways including Src, Akt, and MAPKs were not dramatically affected by the lack of TSP1. Together our results demonstrate an important autocrine role for TSP1 in regulation of ChEC phenotype.

Aryl hydrocarbon receptor knock-out exacerbates choroidal neovascularization via multiple pathogenic pathways.

The aryl hydrocarbon receptor (AhR) is a heterodimeric transcriptional regulator with pleiotropic functions in xenobiotic metabolism and detoxification, vascular development and cancer. Herein, we report a previously undescribed role for the AhR signalling pathway in the pathogenesis of the wet, neovascular subtype of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly in the Western world. Comparative analysis of gene expression profiles of aged AhR−/− and wild-type (wt) mice, using high-throughput RNA sequencing, revealed differential modulation of genes belonging to several AMD-related pathogenic pathways, including inflammation, angiogenesis and extracellular matrix regulation. To investigate AhR regulation of these pathways in wet AMD, we experimentally induced choroidal neovascular lesions in AhR−/− mice and found that they measured significantly larger in area and volume compared to age-matched wt mice. Furthermore, these lesions displayed a higher number of ionized calcium-binding adaptor molecule 1-positive (Iba1+) microglial cells and a greater amount of collagen type IV deposition, events also seen in human wet AMD pathology specimens. Consistent with our in vivo observations, AhR knock-down was sufficient to increase choroidal endothelial cell migration and tube formation in vitro. Moreover, AhR knock-down caused an increase in collagen type IV production and secretion in both retinal pigment epithelial (RPE) and choroidal endothelial cell cultures, increased expression of angiogenic and inflammatory molecules, including vascular endothelial growth factor A (VEGFA) and chemokine (C–C motif) ligand 2 (CCL2) in RPE cells, and increased expression of secreted phosphoprotein 1 (SPP1) and transforming growth factor-β1 (TGFβ1) in choroidal endothelial cells. Collectively, our findings identify AhR as a regulator of multiple pathogenic pathways in experimentally induced choroidal neovascularization, findings that are consistent with a possible role of AhR in wet AMD. The data discussed in this paper have been deposited in NCBI's Gene Expression Omnibus; GEO Submission No. GSE56983, NCBI Tracking System No. 17021116.

Interleukin-17 induces angiogenesis in human choroidal endothelial cells in vitro.

The proinflammatory cytokine interleukin-17 (IL-17) has recently been shown to promote angiogenesis. In addition, a receptor for IL-17, IL-17 receptor C (IL-17RC), is enriched in patients with wet, age-related macular degeneration (AMD), a disease characterized by the formation of choroidal neovascularization. However, the role of IL-17 in choroidal endothelial cells (CECs) angiogenesis has not been defined. This study was conducted to determine the effect of IL-17 on proliferation, migration, and tube formation of human CECs. Expression patterns of IL-17 receptor A (IL-17RA) and IL-17RC on isolated human CECs were analyzed by flow cytometry and immunofluorescent staining. Proangiogenic effects of IL-17 on CECs was determined by proliferation assays with a water-soluble tetrazolium cell proliferation reagent kit, wound healing migration assays, and tube formation assays using basement membrane matrix. Cytoskeletal changes were observed by F-actin immunofluorescent staining. Activated Rac1 and RhoA levels were analyzed by pull-down assays. Interleukin-17RA and IL-17RC were present on human CECs. Interleukin-17 enhanced migration and tube formation but did not affect proliferation. Moreover, IL-17 induced rearrangement of the actin cytoskeleton and upregulated activated Rac1 and RhoA in CECs. The PI3K inhibitor wortmannin suppressed CEC migration, cytoskeleton rearrangement, and upregulation of activated Rac1 and RhoA induced by IL-17. Interleukin-17 elicits a proangiogenesis effect on human CECs in vitro by promoting migration and tube formation. The promoted migration effect was dependent on PI3K-Rac1 and RhoA-mediated actin cytoskeleton remodeling.

Inhibition of Elastin Peptide-Mediated Angiogenic Signaling Mechanism(s) in Choroidal Endothelial Cells by the α6(IV)NC1 Collagen Fragment

The inhibitory effects and mechanism(s) of type IV collagen α-6 chain-derived noncollagenous domain (α6[IV]NC1 or hexastatin) on elastin-derived peptide (EDP)-activated choroidal endothelial cell migration, kinase signaling, and membrane type 1 metalloproteinase (MT1-MMP) activation are explored. Mouse choroidal endothelial cells (MCECs) were incubated in media with soluble EDPs (kappa elastin, mouse elastin, and Val-Gly-Val-Ala-Pro-Gly [VGVAPG] hexapeptide) for different time intervals with or without α6(IV)NC1. The MCECs proliferation, migration, tube formation, MT1-MMP expression, and angiogenic signaling were analyzed in cells subjected to EDP and α6(IV)NC1 treatments. The MCECs also were subjected to EDPs, and specific inhibitors for evaluation of focal adhesion kinase (FAK) and protein kinase B (Akt) phosphorylation. Kappa elastin, mouse elastin, and VGVAPG enhanced the migration, without affecting the proliferation of MCECs. The α6(IV)NC1 inhibited survival and EDP-activated migration of MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by α6(IV)NC1. Further, EDP-activated MT1-MMP expression and FAK/phosphoinositide-3-kinase (PI-3K)/mammalian target of rapamycin (mToR)/Akt phosphorylation in MCECs, were reduced by α6(IV)NC1. The EDP-induced FAK and Akt phosphorylation was blocked by FAK- and Akt-specific inhibitors. The EDPs and α6(IV)NC1 are identified to exhibit opposing effects on MCEC angiogenic behavior and signaling. The α6(IV)NC1 inhibited cell survival, EDP-mediated migration, MT1-MMP expression and, FAK/PI-3K/mToR/Akt phosphorylation in MCECs. This work demonstrates α6(IV)NC1 as a prospective endogenous molecule for the treatment of diseases involving choroidal neovascularization in the eye.

Non-Collagenous Domain of Type IV Collagen Regulates Elastin and VGVAPG Activated Choroidal Endothelial Cell Migration and Tube Formation

Non-Collagenous Domain of Type IV Collagen Regulates Elastin and VGVAPG Activated Choroidal Endothelial Cell Migration and Tube Formation

Molecular responses of choroidal endothelial cells to elastin derived peptides through the elastin-binding protein (GLB1)

PurposeNeovascular AMD involves the activation of choroidal endothelial cells to increase their inflammatory and angiogenic behaviors. Elastin derived peptides (EDPs) can elicit some of these phenotypic changes in endothelial cells. This investigation was performed to follow up on those findings by determining a receptor for these peptides in the human eye as well as evaluating the effects of elevated EDPs on choroidal cells in vitro and in vivo. MethodsThe expression of elastin receptor genes including GLB1 was analyzed using reverse transcription PCR. Migration of choroidal endothelial cells was quantified in the presence of inhibitors to different EDP binding proteins. C57BL6 mice were injected with EDPs and studied by electroretinography, transmission electron microscopy, and microarray analysis. ResultsAn alternatively spliced form of beta-galactosidase (GLB1) is present on human choroidal endothelial cells and acts as a receptor for EDPs. Elevated levels of circulating EDPs do not affect retinal function in the mouse, but do increase the expression and deposition of collagen IV in the RPE/choroid complex. ConclusionsEDPs may play a role in neovascular AMD by binding to and inducing neovascular phenotypes in choroidal endothelial cells through their receptor, GLB1. These peptides also cause an increased mRNA expression and deposition of collagen IV in the RPE/choroid, which may alter diffusion properties between the retina and choriocapillaris.

Upregulation of CCR3 by Age-Related Stresses Promotes Choroidal Endothelial Cell Migration via VEGF-Dependent and -Independent Signaling

To explore the molecular mechanisms by which the C-C chemokine receptor type 3 (CCR3) and chemokine (C-C motif) ligand 11 (CCL11) regulate choroidal endothelial cell (CEC) migration and the interactions with the vascular endothelial growth factor (VEGF) signaling pathway. Human retinal sections from young and aged donor normal eyes were immunolabeled. By real-time PCR, CCR3 mRNA was measured in retinal pigmented epithelium (RPE)/choroids obtained from young and aged human donor eyes and in cultured CECs exposed to hydrogen peroxide. CCR3 ligand and CCL11- or VEGF-stimulated CEC migration was also measured in the presence of the CCR3 inhibitor or control using fluorescence microscopy. Activation of Rac1, phosphorylated Akt as a readout for phosphoinositol 3-kinase signaling, and VEGFR2 activation were measured in CECs incubated with CCL11, VEGF, or combined CCL11/VEGF. CCR3 was expressed to a greater level in older compared with that in younger human retinas or RPE/choroids. Ligand-activated CCR3 increased CEC migration, which was inhibited by the CCR3 inhibitor. Rac1 activity, p-Akt, and p-VEGFR2 were significantly increased in CECs incubated with CCL11. The CCR3 inhibitor prevented VEGF-induced CEC migration and Rac1 activation in CECs. Rac1 activity was additively increased in CECs treated with CCL11 and VEGF compared with that in cells with CCL11 or VEGF treatment alone. Ligand-activated CCR3 caused VEGFR2 phosphorylation and coimmunoprecipitation of VEGFR2 and CCR3. Activated CCR3 promotes CEC migration and Rac1 activation and causes an association with and activation of VEGFR2. Cross-talk between CCR3 and VEGF signaling exists and may be important in choroidal neovascularization in human age-related macular degeneration.

The Role of RPE Cell-Associated VEGF189in Choroidal Endothelial Cell Transmigration across the RPE

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. Using real-time PCR, the expression of VEGF splice variants VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉ was determined in human RPE from donor eyes, cultured human RPE in contact with CECs exposed to hydrogen peroxide (H₂O₂) or hypoxia, and RPE/choroid specimens from mice treated with laser to induce choroidal neovascularization (CNV). Activation of VEGF receptors (VEGFRs), phosphoinositol 3-kinase (PI-3K) or Rac1 was measured in CECs cocultured in contact with RPE exposed to peroxide or silenced for VEGF₁₈₉ expression. Migration of CECs across the RPE was determined using fluorescence microscopy. VEGF₁₈₉ expression was increased in human RPE from aged compared with young donor eyes and from mouse RPE/choroids after laser to induce CNV. VEGF₁₈₉ was also upregulated in human RPE challenged with peroxide, hypoxia, or cultured in contact with CECs. CEC migration across RPE was greater after RPE exposure to peroxide to induce VEGF₁₈₉; VEGFR2 and Rac1 activities were also increased in these CECs. When CECs were cocultured with RPE silenced for VEGF₁₈₉, VEGFR2 and Rac1 activities in CECs were significantly reduced, as was CEC migration across the RPE. Inhibition of Rac1 activity significantly inhibited CEC transmigration without affecting PI-3K activity. RPE-derived cell-associated VEGF₁₈₉ facilitates CEC transmigration by Rac1 activation independently of PI-3K signaling and may have importance in the development of neovascular AMD.

The Role of Vascular Endothelial Growth Factor-Induced Activation of NADPH Oxidase in Choroidal Endothelial Cells and Choroidal Neovascularization

Rac1, a subunit of NADPH oxidase, plays an important role in directed endothelial cell motility. We reported previously that Rac1 activation was necessary for choroidal endothelial cell migration across the retinal pigment epithelium, a critical step in the development of vision-threatening neovascular age-related macular degeneration. Here we explored the roles of Rac1 and NADPH oxidase activation in response to vascular endothelial growth factor treatment in vitro and in a model of laser-induced choroidal neovascularization. We found that vascular endothelial growth factor induced the activation of Rac1 and of NADPH oxidase in cultured human choroidal endothelial cells. Further, vascular endothelial growth factor led to heightened generation of reactive oxygen species from cultured human choroidal endothelial cells, which was prevented by the NADPH oxidase inhibitors, apocynin and diphenyleneiodonium, or the antioxidant, N-acetyl-L-cysteine. In a model of laser-induced injury, inhibition of NADPH oxidase with apocynin significantly reduced reactive oxygen species levels as measured by dihydroethidium fluorescence and the volume of laser-induced choroidal neovascularization. Mice lacking functional p47phox, a subunit of NADPH oxidase, had reduced dihydroethidium fluorescence and choroidal neovascularization compared with wild-type controls. Taken together, these results indicate that vascular endothelial growth factor activates Rac1 upstream from NADPH oxidase in human choroidal endothelial cells and increases generation of reactive oxygen species, contributing to choroidal neovascularization. These steps may contributed to the pathology of neovascular age-related macular degeneration.