Murine Choroidal Neovascularization Model Research Articles

Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model.

Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD.

Role of IL-4 in bone marrow driven dysregulated angiogenesis and age-related macular degeneration.

Age-associated sterile inflammation can cause dysregulated choroidal neovascularization (CNV) as age-related macular degeneration (AMD). Intraocular fluid screening of 234 AMD patients identified high levels of IL-4. The purpose of this study was to determine the functional role of IL-4 in CNV formation using murine CNV model. Our results indicate that the IL-4/IL-4 receptors (IL4Rs) controlled tube formation and global proangiogenic responses of bone marrow cells. CCR2+ bone marrow cells were recruited to form very early CNV lesions. IL-4 rapidly induces CCL2, which enhances recruitment of CCR2+ bone marrow cells. This in vivo communication, like quorum-sensing, was followed by the induction of IL-4 by the bone marrow cells during the formation of mature CNVs. For CNV development, IL-4 in bone marrow cells are critically required, and IL-4 directly promotes CNV formation mainly by IL-4R. The IL-4/IL-4Rα axis contributes to pathological angiogenesis through communications with bone marrow cells leading to retinal degeneration.

Mechanisms of FH Protection Against Neovascular AMD.

A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.

MTORC1 signaling pathway regulates macrophages in choroidal neovascularization

Macrophages are involved in choroidal neovascularization (CNV). The mechanistic target of rapamycin complex 1 (mTORC1) is a central cell regulator, but mTORC1 function in macrophages in CNV is not fully understood. We explored the effect of mTORC1 pathway regulation on macrophages in CNV. A laser-induced murine CNV model was performed. Expression of phospho-S6 and F4/80 in CNV lesions was analyzed by immunofluorescence. Macrophages in CNV lesions were found at 1 day after laser treatment, reached a peak at 5 days, and decreased at 7 and 14 days. mTORC1 activity of cells in CNV lesions was increased from 3 to 7 days, and deceased at 14 days. Most infiltrating macrophages in CNV lesions had strong mTORC1 activity at 3 and 5 days that subsequently decreased. In vitro, THP-1 macrophages were polarized to M1 or M2 with rapamycin or siRNA treatment. The human retinal pigment epithelium (RPE) cell line ARPE-19 was co-cultured with macrophages. Cytokine expression of macrophages and ARPE-19 cells was detected by quantitative PCR. Inhibiting mTORC1 activity of macrophages reduced M1 and strengthened M2, which was reversed by mTORC1 hyperactivation. Both M1 and M2 macrophages induced RPE cells to express less PEDF and more MMP9, IL-1β and MCP-1. Inhibiting or enhancing mTORC1 activity of macrophages changed cytokine expression of RPE cells. Together, we demonstrated that macrophage functions in CNV were regulated partly by the mTORC1 pathway, and mTORC1 activity of macrophages influenced the expression of cytokines that are associated with CNV development in RPE cells. This study provides more understanding about the regulatory mechanism of macrophages in CNV.

Lactoferrin Has a Therapeutic Effect via HIF Inhibition in a Murine Model of Choroidal Neovascularization.

BackgroundLactoferrin, a type of glycoprotein, is contained in exocrine fluids such as tears, breast milk, sweat, and saliva, and is known to have anti-microbial, antioxidant, and anti-cancer effects. In the ophthalmological field, topical administration of lactoferrin has been reported to have a therapeutic effect in a murine dry eye model. Hypoxia-inducible factor (HIF) regulates various gene expressions under hypoxia, including vascular endothelial growth factor (VEGF), and is considered as an alternative target for neovascular ocular diseases such as age-related macular degeneration (AMD). We previously screened natural products and identified lactoferrin as a novel HIF inhibitor. In this study, we confirmed that lactoferrin has an HIF inhibitory effect and a therapeutic effect in a murine model of neovascular AMD.MethodsHIF inhibitory effects of lactoferrin were evaluated using a luciferase assay and western blotting in vitro. The quantified volume of choroidal neovascularization (CNV) induced by laser irradiation was compared with oral lactoferrin administration or conditional tissue specific Hif1a knockout mice.ResultsLactoferrin administration showed a significant HIF inhibitory effect in the retinal neuronal cells. Oral administration of lactoferrin or conditional Hif1a gene deletion significantly reduced CNV volume compared to controls.ConclusionsLactoferrin has a therapeutic effect in a laser CNV model by suppressing the retinal HIF activity.

Microarray expression profile and functional analysis of circular RNAs in choroidal neovascularization.

Choroidal neovascularization (CNV) is a leading cause of visual loss in age-related macular degeneration (AMD). However, the molecular mechanism for CNV progression is still unclear. This study aimed to identify CNV-related circular RNAs (circRNAs), a novel class of non-coding RNAs with diverse functions. A total of 117 circRNAs were differentially expressed in the murine CNV model by microarrays. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to identify the functions of selected circRNAs. The host genes of these circRNAs were predicted to be targeted to neurogenesis (ontology: biological process), proteinaceous extracellular matrix (ECM) (ontology: cellular component), and binding (ontology: molecular function). Differentially expressed circRNAs-mediated regulatory networks were enriched in ECM receptor interaction. Most of the dysregulated circRNAs could potentially bind to five different miRNAs by TargetScan and miRanda. Specifically, circ_15752 was identified in this circRNAs pool which may facilitate vascular endothelial cell proliferation, migration, and tube formation, suggesting a critical role in endothelial angiogenesis. Our work suggests that dysregulated circRNAs may be involved in CNV pathogenesis and serve as potential biomarkers for CNV.

HTRA1 synergizes with oxidized phospholipids in promoting inflammation and macrophage infiltration essential for ocular VEGF expression.

Understanding oxidative stress and HTRA1 locus in abnormal angiogenesis resulting in wet AMD pathology is an important step in developing a novel therapeutic approach. Using subretinal injection of oxLDL into C57BL/6 mice, we observed a lesion resembling the features of choroidal neovascularization (CNV), including macrophage infiltration, increased VEGF expression, and neovascularization. However, incubating ARPE-19 cells with oxLDL–a carrier of oxidized phospholipids–resulted in increased expression of inflammatory cytokines and chemoattractant proteins that recruited monocytes, but no substantial increase in expression of VEGF. Furthermore, incubation of ARPE-19 with oxLDL induced higher expression of HTRA1, which we showed to synergize with oxLDL in elevating the expression of inflammatory cytokines and chemoattractant factors. To investigate the role of macrophage infiltration on these expression changes, we treated cultured J774 macrophages with oxLDL and applied the conditioned medium onto ARPE-19 cells. This treatment was found to greatly enhance the expression of VEGF in ARPE-19, indicating the necessity of macrophage secretory products to induce increased expression of VEGF in retinal pigment epithelium. Gene expression analysis revealed that oxLDL induced the expression of Wnt3A in macrophages, a key activator of canonical Wnt signaling pathways. In addition, western blot analysis showed that the macrophage conditioned media further enhanced the reduction of phosphorylated β-catenin induced by oxLDL. Lastly, we investigated HTRA1 as a potential target for AMD therapeutics. We demonstrated the ability of anti-HTRA1 antibody in vitro to neutralize the protease activity of HTRA1 and reduce the inflammatory and angiogenic response to oxidative stress. Finally, we validated the neutralizing effect of anti-HTRA1 antibody in vivo by evaluating lesion size and protein expression in a laser-photocoagulation murine model of CNV. We found that the combination of oxLDL and HTRA1 enhanced CNV size, which was reversed by the addition of anti-HTRA1 antibody. This study not only provides preliminary evidence that HTRA1 may be a viable target for AMD therapeutics but also elucidates the biochemical mechanisms by which this therapeutic effect may be mediated.

Inhibition of the alternative complement pathway accelerates repair processes in the murine model of choroidal neovascularization

Age-related macular degeneration (AMD) is the leading cause of blindness in the US. Polymorphisms in complement components are associated with increased AMD risk, and it has been hypothesized that an overactive complement system is partially responsible for AMD pathology. Choroidal neovascularization (CNV) has two phases, injury/angiogenesis and repair/fibrosis. Complement activation has been shown to be involved in the angiogenesis phase of murine CNV, but has not been investigated during repair. Anaphylatoxin (C3a and C5a) signaling in particular has been shown to be involved in both tissue injury and repair in other models. CNV was triggered by laser-induced photocoagulation in C57BL/6 J mice, and lesion sizes measured by optical coherence tomography. Alternative pathway (AP) activation or C3a-receptor (C3aR) and C5a-receptor (C5aR) engagement was inhibited during the repair phase only of CNV with the AP-inhibitor CR2-fH, a C3aR antagonist (N2-[(2,2-diphenylethoxy)acetyl]-l-arginine, TFA), or a C5a blocking antibody (CLS026), respectively. Repair after CNV was also investigated in C3aR/C5aR double knockout mice. CR2-fH treatment normalized anaphylatoxin levels in the eye and accelerated regression of CNV lesions. In contrast, blockade of anaphylatoxin-receptor signaling pharmacologically or genetically did not significantly alter the course of lesion repair. These results suggest that continued complement activation prevents fibrotic scar resolution, and emphasizes the importance of reducing anaphylatoxins to homeostatic levels. This duality of complement, playing a role in injury and repair, will need to be considered when selecting a complement inhibitory strategy for AMD.

Retinal Inhibition of CCR3 Induces Retinal Cell Death in a Murine Model of Choroidal Neovascularization.

Inhibition of chemokine C-C motif receptor 3 (CCR3) signaling has been considered as treatment for neovascular age-related macular degeneration (AMD). However, CCR3 is expressed in neural retina from aged human donor eyes. Therefore, broad CCR3 inhibition may be harmful to the retina. We assessed the effects of CCR3 inhibition on retina and choroidal endothelial cells (CECs) that develop into choroidal neovascularization (CNV). In adult murine eyes, CCR3 colocalized with glutamine-synthetase labeled Műller cells. In a murine laser-induced CNV model, CCR3 immunolocalized not only to lectin-stained cells in CNV lesions but also to the retina. Compared to non-lasered controls, CCR3 mRNA was significantly increased in laser-treated retina. An intravitreal injection of a CCR3 inhibitor (CCR3i) significantly reduced CNV compared to DMSO or PBS controls. Both CCR3i and a neutralizing antibody to CCR3 increased TUNEL+ retinal cells overlying CNV, compared to controls. There was no difference in cleaved caspase-3 in laser-induced CNV lesions or in overlying retina between CCR3i- or control-treated eyes. Following CCR3i, apoptotic inducible factor (AIF) was significantly increased and anti-apoptotic factor BCL2 decreased in the retina; there were no differences in retinal vascular endothelial growth factor (VEGF). In cultured human Műller cells exposed to eotaxin (CCL11) and VEGF, CCR3i significantly increased TUNEL+ cells and AIF but decreased BCL2 and brain derived neurotrophic factor, without affecting caspase-3 activity or VEGF. CCR3i significantly decreased AIF in RPE/choroids and immunostaining of phosphorylated VEGF receptor 2 (p-VEGFR2) in CNV with a trend toward reduced VEGF. In cultured CECs treated with CCL11 and/or VEGF, CCR3i decreased p-VEGFR2 and increased BCL2 without increasing TUNEL+ cells and AIF. These findings suggest that inhibition of retinal CCR3 causes retinal cell death and that targeted inhibition of CCR3 in CECs may be a safer if CCR3 inhibition is considered as a therapy for neovascular AMD.

The Role of LOX and LOXL2 in the Pathogenesis of an Experimental Model of Choroidal Neovascularization.

We investigated whether lysyl oxidase (LOX) and lysyl oxidase-like 2 (LOXL2) play a role in an experimental model of choroidal neovascularization (CNV). The therapeutic potential of antibodies against LOX (M64) and LOXL2 (AB0023) was evaluated in a murine laser-induced CNV model. Expression of LOX and LOXL2 in the posterior eye cups (including retina, retinal pigment epithelium, choroid, and sclera) was studied by qRT-PCR and immunohistochemistry. In the murine model of CNV, both antibodies were administered intraperitoneally every other day until the day killed. On different time points after laser, treatment outcome was studied by immunohistochemical analysis of inflammation, angiogenesis and fibrosis, and by transcript analysis of different cytokines. Levels of LOX and LOXL2 in the posterior eye cups were increased after CNV-induction at different time points after laser. At day 35, their protein expression patterns appeared to correlate with retinal glial cells and endothelial cells, respectively. Both antibodies significantly inhibited fibrosis, whereas AB0023 also significantly reduced angiogenesis and inflammation. Transcript levels of α-1 type I collagen (COL1A1) in the posterior eye cups were significantly decreased in lasered mice treated with either M64 or AB0023. Vascular endothelial growth factor expression was also reduced only after AB0023 treatment, whereas activated fibroblast marker α-smooth muscle actin (αSMA) levels were not significantly changed. This study suggests that LOX and LOXL2 may play an important role in the pathogenesis of AMD. Targeting LOXL2 could have a broader efficacy than targeting LOX, by reducing angiogenesis and inflammation, as well as fibrosis.

The Role of Macrophage Class A Scavenger Receptors in a Laser-Induced Murine Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a widely used model to mimic many features of CNV resulting from wet AMD. Macrophages have been implicated in the pathogenesis of AMD. Class A scavenger receptors, scavenger receptor-A (SR-A) and macrophage receptor with collagenous domain (MARCO), are expressed on macrophages and are associated with macrophage function. The goal of this study is to examine the role of macrophage scavenger receptors in immune cell recruitment and the formation of CNV. Laser photocoagulation was performed in wild-type and knockout mice with deletion of SR-A (SR-A(-/-)), MARCO (MARCO(-/-)), or both SR-A and MARCO double knockout (DKO). Immune cell recruitment at different time points and CNV lesions at 14 days after laser treatment were evaluated through immunostaining and confocal microscopy. Microarray analysis was performed in eyes 1 day after laser injury. Wild-type eyes showed higher chemokine/receptor expression compared with knockout eyes after laser injury. Scavenger receptor deficiency markedly impaired the recruitment of neutrophils and macrophages to CNV lesions at 1- and 3-days post laser injury, respectively. Significantly reduced CNV volumes were found in the eyes from scavenger receptor knockout mice compared with wild-type mice. The deficiency of scavenger receptors impairs the formation of CNV and immune cell recruitment. Our findings suggest a potential role for scavenger receptors in contributing to CNV formation and inflammation in AMD.

228. Adeno-Associated Vector (Type 8)-Mediated Expression of siRNA Targeting Vascular Endothelial Growth Factor Efficiently Inhibits Neovascularization in a Murine Choroidal Neovascularization Model

228. Adeno-Associated Vector (Type 8)-Mediated Expression of siRNA Targeting Vascular Endothelial Growth Factor Efficiently Inhibits Neovascularization in a Murine Choroidal Neovascularization Model

Reduction of choroidal neovascularization in mice by adeno‐associated virus‐delivered anti‐vascular endothelial growth factor short hairpin RNA

Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy.

A Lipid Nanoparticle System Improves siRNA Efficacy in RPE Cells and a Laser-Induced Murine CNV Model

To explore the possibility of the PEGylated liposome-protamine-hyaluronic acid nanoparticles (PEG-LPH-NP) loaded with siRNA (PEG-LPH-NP-S) in ARPE19 cells and a laser-induced rat model for the treatment of choroidal neovascularization (CNV). PEG-LPH-NP-S was characterized by dynamic light scattering and transmission electron microscopy (TEM). The encapsulation efficiency of siRNA in PEG-LPH-NP was analyzed by ultracentrifugation, whereas the protection of siRNA by PEG-LPH-NP was evaluated by electrophoresis. Human RPE cells (ARPE19) were used as the cell model for the studies of cellular uptake and the inhibition of VEGFR1 expression, visualized by a laser scanning confocal microscope. The area of CNV in the laser-induced rat model after intravitreous injection was measured. The distribution of the lipid nanoparticles in the retina after intravitreous administration was investigated by fluorescence microscopy. Finally, the TUNEL test and morphologic observation of the retina were conducted. It was indicated that PEG-LPH-NP-S was approximately 132 nm in particle size with a positive charge of approximately 20 mV, whereas the encapsulation efficiency of siRNA in PEG-LPH-NP was >95%. PEG-LPH-NP could protect the siRNA load and could facilitate the intracellular delivery of fluorescein-labeled siRNA to ARPE19 cells. VEGFR1 expression in ARPE19 cells could be inhibited, and the CNV area in the murine model could be reduced more effectively by PEG-LPH-NP-S compared with naked siRNA and by PEG-LPH-NP with negative siRNA. It seems that the toxicity of PEG-LPH-NP-S on the rat retina is low, based on the results of TUNEL testing and morphologic observation. PEG-LPH-NP may be a promising lipid nanoparticle system for the siRNA treatment of CNV.

162a Update on Kinoids as an Active Immunotherapy to Combat Pathogenic Cytokines in Viral, Cancer and Autoimmune Diseases: Safety, Immunogenicty

Kinoids are a derivative of biologically non toxic but immunogenic cytokines prepared either by chemical inactivation of the cytokine (1rst generation) or by coupling the cytokine to a carrier protein such as KLH (2nd generation). Given that abnormal overproduction of cytokines are involved in the pathogenesis of severe chronic diseases, including AIDS (IFN α and IL 10), autoimmune diseases (TNF α and IFN α) and cancer (VEGF and TNF α), Kinoids have been experimentally assayed and are currently under clinical trials. In AIDS, inactivated IFN α has been efficiently used in a phase 2 EURIS trial (1). In Rheumatoid Arthritis TNF α-Kinoid immunotherapy proved to be safe and effective in transgenic hTNF α mice developing arthritis (2) and is currently under phase 1-2 trial in Crohn's disease and Rheumatoid Arthritis (NCT00808262). VEGF-Kinoid immunotherapy proved to be experimentally safe and effective in preventing the tumor development in a model of SCID or nude mice (3) and the neovessels sprouting in a murine model of choroidal neovascularization as it occurs in Age-related Macular Degeneration (AMD) and in choroidal melanoma.

Role of Ocular Complement Factor H in a Murine Model of Choroidal Neovascularization

The objective of this study was to explore the relationship between local (ie, ocular) complement factor H (CFH) and choroidal neovascularization (CNV) associated with wet age-related macular degeneration (AMD), a leading cause of irreversible blindness, in laser-treated C57BL/6 mice. Immunohistochemical and RT-PCR analysis of retinal pigmented epithelium (RPE)-choroid sclera revealed that the expression of CFH was down-regulated on day 1 with a dramatic increase on days 5 and 7 postlaser injury. Flat mount and Western blot analysis further revealed that membrane attack complex (MAC) expression was up-regulated on days 1 and 3 postlaser injury; however, MAC was down-regulated on days 5 and 7 postinjury but was still higher than in non-injured mice. Similar patterns for CFH and MAC were observed for RPE cells when serial paraffin sections of the laser spots were analyzed. Subretinal injection of siRNA directed against CFH resulted in a threefold suppression of CFH in the RPE and choroid without affecting either CFH levels in the liver or the functional activity of the alternative pathway in the peripheral blood. Ocular knock-down of CFH resulted in increased MAC deposition, which leads to the early onset as well as exacerbation of laser-induced CNV. In conclusion, our findings provide evidence that CFH present on RPE and choroid regulates local MAC formation that is critical for the development of laser-induced CNV.

Adeno-Associated Vector (Type 8)-Mediated Expression of Soluble Flt-1 Efficiently Inhibits Neovascularization in a Murine Choroidal Neovascularization Model

To assess the feasibility of a gene therapeutic approach to treating choroidal neovascularization (CNV), we generated a recombinant adeno-associated viral (AAV) vector (type 8) encoding soluble Flt-1 (AAV-sflt-1), and determined its ability to inhibit angiogenesis. When we treated human umbilical vein endothelial cells (HUVECs) with the supernatant of cells transduced with AAV-sflt-1 or AAV-EGFP (control), we found that tube formation was significantly inhibited by the former but not the latter (area: 25,121 +/- 557 vs. 68,628 +/- 1357 pixels [p < 0.01]; length: 4811 +/- 246 vs. 10,894 +/- 297 pixels [p < 0.01]). CNV was induced in C57BL/6 mice by making four separate choroidal burns around the optic nerve in each eye, using a diode laser. Thereafter, 2 microl (5 x 10(11) vector genomes/ml) of AAV-sflt-1 (n = 11) or control AAV-LacZ (n = 12) was injected into the subretinal space, and 2 weeks later the eyes were removed for flatmount analysis of CNV surface area. Notably, subretinal delivery of AAV-sflt-1 significantly diminished CNV at the laser lesions, as compared with AAV-LacZ (555 +/- 304 vs. 1470 +/- 1000 microm(2); p = 0.007). These results suggest that there was diffusion of the secreted sFlt-1 across the retina and that long-term suppression of CNV is possible through the use of stable rAAV-mediated sflt-1 expression. In vivo gene therapy thus appears to be a feasible approach to the clinical management of CNV in conditions such as age-related macular degeneration.

Production and characterization of monoclonal anti-sphingosine-1-phosphate antibodies

Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid involved in multiple physiological processes. Importantly, dysregulated S1P levels are associated with several pathologies, including cardiovascular and inflammatory diseases and cancer. This report describes the successful production and characterization of a murine monoclonal antibody, LT1002, directed against S1P, using novel immunization and screening methods applied to bioactive lipids. We also report the successful generation of LT1009, the humanized variant of LT1002, for potential clinical use. Both LT1002 and LT1009 have high affinity and specificity for S1P and do not cross-react with structurally related lipids. Using an in vitro bioassay, LT1002 and LT1009 were effective in blocking S1P-mediated release of the pro-angiogenic and prometastatic cytokine, interleukin-8, from human ovarian carcinoma cells, showing that both antibodies can out-compete S1P receptors in binding to S1P. In vivo anti-angiogenic activity of all antibody variants was demonstrated using the murine choroidal neovascularization model. Importantly, intravenous administration of the antibodies showed a marked effect on lymphocyte trafficking. The resulting lead candidate, LT1009, has been formulated for Phase 1 clinical trials in cancer and age-related macular degeneration. The anti-S1P antibody shows promise as a novel, first-in-class therapeutic acting as a "molecular sponge" to selectively deplete S1P from blood and other compartments where pathological S1P levels have been implicated in disease progression or in disorders where immune modulation may be beneficial.

751. Adeno-Associated Vector (Type 8) Mediated Expression of Flt-1 Efficiently Inhibits Neovascularization in a Murine Choroidal Neovascularization Model

751. Adeno-Associated Vector (Type 8) Mediated Expression of Flt-1 Efficiently Inhibits Neovascularization in a Murine Choroidal Neovascularization Model

Inhibition of VEGF secretion and experimental choroidal neovascularization by picropodophyllin (PPP), an inhibitor of the insulin‐like growth factor‐1 receptor

Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration (AMD) and a leading cause of vision loss. Along with other angiogenic factors like vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and its receptor, IGF-1R, have been implicated in CNV. We have previously shown that the cyclolignan picropodophyllin (PPP) efficiently blocks the insulin-like growth factor-1 receptor (IGF-1R) activity and causes cell death in uveal melanoma cell lines and in an in-vivo model. In this study we investigated the effect of PPP on VEGF expression both in vitro and in vivo and whether this effect has anti-angiogenic consequences in a murine CNV model. C57BL/6J mice with laser-induced CNVs were treated with PPP. Effects on CNV area were assayed by image analysis. VEGF levels in choroids and retinal pigment epithelial cells (APRE-19) were measured by Western blot or ELISA. Transcriptional activation of the VEGF promoter was determined by luciferase reporter gene assay. Mice treated with PPP, administered intraperitoneally or orally, showed 22-32% (p = 0.002) decrease in CNV area. Furthermore, VEGF levels in the choroids were significantly reduced. In cultured APRE-19 cells, IGF-1 was shown to increase VEGF secretion. This increase was completely blocked by PPP. We could confirm that PPP reduced the level of transcriptional activity of VEGF promoter. PPP reduces IGF-1 dependent VEGF expression and CNV in vivo. Accordingly, IGF-1R inhibitors may be useful tools in the therapy of conditions associated with CNV including neovascular AMD.