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Inhibition of Choroidal Neovascularization in a Nonhuman Primate Model by Intravitreal Administration of an AAV2 Vector Expressing a Novel Anti-VEGF Molecule

Authors:
Michael Lukason at Sanofi
Michael Lukason
Elizabeth Barry at Sanofi
Elizabeth Barry
Hillard Rubin
Hillard Rubin
Hillard Rubin
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Peter Pechan at Biogen
Peter Pechan
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Inhibition of vascular endothelial growth factor (VEGF) for the management of the pathological ocular neovascularization associated with diseases such as neovascular age-related macular degeneration is a proven paradigm; however, monthly intravitreal injections are required for optimal treatment. We have previously shown that a novel, secreted anti-VEGF molecule sFLT01 delivered by intravitreal injection of an AAV2 vector (AAV2-sFLT01) gives persistent expression and is efficacious in a murine model of retinal neovascularization. In the present study, we investigate transduction and efficacy of an intravitreally administered AAV2-sFLT01 in a nonhuman primate (NHP) model of choroidal neovascularization (CNV). A dose-dependent and persistent expression of sFLT01 was observed by collecting samples of aqueous humor at different time points over 5 months. The location of transduction as elucidated by in situ hybridization was in the transitional epithelial cells of the pars plana and in retinal ganglion cells. AAV2-sFLT01 was able to effectively inhibit laser-induced CNV in a dose-dependent manner as determined by comparing the number of leaking CNV lesions in the treated versus control eyes using fluorescein angiography. Our data suggest that intravitreal delivery of AAV2-sFLT01 may be an effective long-term treatment for diseases caused by ocular neovascularization.
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Inhibition of Choroidal Neovascularization in a Nonhuman Primate Model by Intravitreal Administration of an AAV2 Vector Expressing a Novel Anti-VEGF Molecu
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... capsid, we delivered a cytomegalovirus (CMV) promoter-driven EGFP expression cassette (CMV-EGFP) via systemic, IVT, and SR routes to the murine retina. We compared the tropism and efficacy of AAV-PHP.eB to IVT and SR deliveries of AAV2/2 36À38 and AAV2/8 10,39 capsids, respectively, which are highly efficient at transducing the retina via these routes. ...
... AAV2/2 was selected as a control serotype as it is the serotype most extensively used for IVT delivery to RGCs in rodents, NHPs, and humans. 18,[37][38][39][40][41] As AAV2/2 does not cross the blood-brain barrier, it was not administered via TV. 42 The effective dose of AAV-PHP.eB-CMV-EGFP was based on previous studies, 23,35,43,44 and as it reflected dosage to the whole body, it was significantly higher than the dose range used for direct IVT delivery to the eye. ...
... Overall, the cell-specific tropisms of TV and IVT administration of AAV-PHP.eB-CMV-EGFP were similar (Figures 1 and 2) to each other and to that of IVT AAV2/ 2-CMV-EGFP, a serotype commonly used for IVT delivery to RGCs. 18,[37][38][39]58,59 In contrast to the broad transduction of RGCs via both IVT and TV delivery in our study (Figures 1, 2, and 3), minimal transduction of RGCs was found in the Simpson et al. study. 35 Indeed, Simpson and colleagues 35 found limited transduction of retinal cells, other than horizontal cells (which were transduced efficiently), when AAV-PHP.eB was delivered systemically and minimal transduction of any cells when delivered via IVT. ...
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Article
Full-text available
  • Mar 2022
Recombinant adeno-associated virus (AAV) vectors are one of the main gene delivery vehicles used in retinal gene therapy approaches, however, there is a need to further improve efficacy, tropism and safety of these vectors. In this study, utilising a CMV-EGFP expression cassette, we characterise the retinal utility of AAV-PHP.eB, a serotype recently developed by in-vivo directed evolution, which can cross the blood-brain barrier and target neurons with high efficacy in mice. Systemic and intravitreal delivery of AAV-PHP.eB resulted in high transduction efficacy of retinal ganglion and horizontal cells, with systemic delivery providing pan-retinal coverage of the mouse retina. Subretinal delivery transduced photoreceptors and retinal pigment epithelium cells robustly. EGFP expression (number of transduced cells and mRNA levels) were similar when the retinas were transduced systemically or intravitreally with AAV-PHP.eB or intravitreally with AAV2/2. Notably, in photoreceptors, EGFP fluorescence intensities and mRNA levels were 50-70 times higher, when subretinal injections with AAV-PHP.eB were compared to AAV2/8. Our results demonstrate pan-retinal transduction of ganglion cells and extremely efficient transduction of photoreceptor and retinal pigment epithelium cells as the most valuable features of AAV-PHP.eB in the mouse retina.
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... One challenge is that IVTs in mice are notoriously difficult due to anatomical challenges. To translate our encouraging findings on Jagged1 targeting into clinical practice, the concept must be tested in established larger-animal models for laser-induced CNV and IVTs, preferably in non-human primates, which are extensively used for preclinical evaluation 15,[84][85][86][87] . ...
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Article
Full-text available
  • May 2023
Antibody-based blocking of vascular endothelial growth factor (VEGF) reduces choroidal neovascularization (CNV) and retinal edema, rescuing vision in patients with neovascular age-related macular degeneration (nAMD). However, poor response and resistance to anti-VEGF treatment occurs. We report that targeting the Notch ligand Jagged1 by a monoclonal antibody reduces neovascular lesion size, number of activated phagocytes and inflammatory markers and vascular leakage in an experimental CNV mouse model. Additionally, we demonstrate that Jagged1 is expressed in mouse and human eyes, and that Jagged1 expression is independent of VEGF signaling in human endothelial cells. When anti-Jagged1 was combined with anti-VEGF in mice, the decrease in lesion size exceeded that of either antibody alone. The therapeutic effect was solely dependent on blocking, as engineering antibodies to abolish effector functions did not impair the therapeutic effect. Targeting of Jagged1 alone or in combination with anti-VEGF may thus be an attractive strategy to attenuate CNV-bearing diseases.
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... Neutralizing PlGF in addition to VEGF may improve the antiangiogenic property of sFLT01 and may overcome refractory nAMD [37]. Several preclinical and clinical studies, including subcutaneous (SC) xenograft mouse tumor models, cynomolgus monkey CNV models, and a phase Ι study of AAV2-sFLT01 intravitreal injections administered in multiple dosage ranges to advanced AMD patients, have shown the efficacy and antiangiogenic potency of this recombinant protein [36,38,39]. ...
Design, construction and in vivo functional assessment of a hinge truncated sFLT01
Article
Full-text available
  • Sep 2022
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... Laserle indüklenen KNV oluşturulmuş maymunlarda sFLT0 içeren adeno-ilişkili vektör kullanılmış ve etkinliği bu çalışmada gösterilmiştir. [16] PEDF (AdPEDF.11) adenoviral vektör kullanılarak uygulanan 28 yaş tip YBMD'li olguda sonuçlar yüz güldürücü görünmektedir. ...
Yaş Tip (Neovasküler) Yaşa Bağlı Maküla Dejenerasyonunda Yeni Geliştirilmekte Olan Tedaviler
Article
Full-text available
  • Jun 2017
ABSTRACT Age-related macular degeneration (AMD) is the most common cause of permanent visual loss in persons over 65 years of age in the developed countries. Currently, intravitreal vascular endothelial growth factor (VEGF) inhibitors are the mainstay of the treatment for patients with wet AMD. Despite significant improvements in the visual acuity since beginning of these therapies, challenges in the treatment of wet AMD are still present. Therefore, there are ongoing researches such as sustained release anti-VEGF therapy, novel generation anti-VEGF agents, viral vectors to modify genetic transcription, and combination therapies. In this review, it is aimed to discuss these emerging therapies. Key Words: Age-related macular degeneration, wet AMD, vascular endothelial growth factor, pharmacological agents, novel drugs, radiotherapy, combination therapies.
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... 4 There also is an increasing body of literature indicating that VEGF plays an important neuroprotective role in the retina. [5][6][7][8][9][10][11][12][13][14][15][16] Consequently, alternative avenues are being pursued to treat neovascular pathologies without affecting VEGF function. 16 In a companion article to this editorial, we show that inhibition of VEGF by rAAV2.7m8-mediated ...
Low-Dose Recombinant Adeno-Associated Virus-Mediated Inhibition of Vascular Endothelial Growth Factor Can Treat Neovascular Pathologies Without Inducing Retinal Vasculitis
Article
Full-text available
  • Jul 2021
The wet form of age-related macular degeneration is characterized by neovascular pathologies that, if untreated, can result in edemas followed by rapid vision loss. Inhibition of vascular endothelial growth factor (VEGF) has been used to successfully treat neovascular pathologies of the eye. Nonetheless, some patients require frequent intravitreal injections of anti-VEGF drugs, increasing the burden and risk of complications from the procedure to affected individuals. Recombinant adeno-associated virus (rAAV)-mediated expression of anti-VEGF proteins is an attractive alternative to reduce risk and burden to patients. However, controversy remains as to the safety of prolonged VEGF inhibition in the eye. Here, we show that two out of four rAAV serotypes tested by intravitreal delivery to express the anti-VEGF drug conbercept lead to a dose-dependent vascular sheathing pathology that is characterized by immune cell infiltrates, reminiscent of vasculitis in humans. We show that this pathology is accompanied by increased expression in vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1), both of which promote extravasation of immune cells from the vasculature. While formation of the vascular sheathing pathology is prevented in immunodeficient Rag-1 mice that lack B and T cells, increased expression of VACM1 and ICAM1 still occurs, indicating that inhibition of VEGF function leads to expression changes in cell adhesion molecules that promote extravasation of immune cells. Importantly, a 10-fold lower dose of one of the vectors that cause a vascular sheathing pathology is still able to reduce edemas resulting from choroidal neovascularization without causing any vascular sheathing pathology and only a minimal increase in VCAM1 expression. The data suggest that treatments of neovascular eye pathologies with rAAV-mediated expression of anti VEGF drugs can be developed safely. However, viral load needs to be adjusted to the tropisms of the serotype and the expression pattern of the promoter.
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Adeno-associated virus as a delivery vector for gene therapy of human diseases
Article
Full-text available
  • Apr 2024
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
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AAV-Based Strategies for Treatment of Retinal and Choroidal Vascular Diseases: Advances in Age-Related Macular Degeneration and Diabetic Retinopathy Therapies
Article
Full-text available
  • Oct 2023
  • BIODRUGS
Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are vascular diseases with high prevalence, ranking among the leading causes of blindness and vision loss worldwide. Despite being effective, current treatments for AMD and DR are burdensome for patients and clinicians, resulting in suboptimal compliance and real risk of vision loss. Thus, there is an unmet need for long-lasting alternatives with improved safety and efficacy. Adeno-associated virus (AAV) is the leading vector for ocular gene delivery, given its ability to enable long-term expression while eliciting relatively mild immune responses. Progress has been made in AAV-based gene therapies for not only inherited retinal diseases but also acquired conditions with preclinical and clinical studies of AMD and DR showing promising results. These studies have explored several pathways involved in the disease pathogenesis, as well as different strategies to optimise gene delivery. These include engineered capsids with enhanced tropism to particular cell types, and expression cassettes incorporating elements for a targeted and controlled expression. Multiple-acting constructs have also been investigated, in addition to gene silencing and editing. Here, we provide an overview of strategies employing AAV-mediated gene delivery to treat AMD and DR. We discuss preclinical efficacy studies and present the latest data from clinical trials for both diseases.
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Retinal and Choroidal Neovascularization Antivascular Endothelial Growth Factor Treatments: The Role of Gene Therapy
Article
  • Sep 2022
  • J OCUL PHARMACOL TH
Neovascularization in ocular vessels causes a major disease burden. The most common causes of choroidal neovascularization (CNV) are age-related macular degeneration and diabetic retinopathy, which are the leading causes of irreversible vision loss in the adult population. Vascular endothelial growth factor (VEGF) is critical for the formation of new vessels and is the main regulator in ocular angiogenesis and vascular permeability through its receptors. Laser therapy and antiangiogenic factors have been used for CNV treatment. Bevacizumab, ranibizumab, and aflibercept are commonly used anti-VEGF agents; however, high costs and the need for frequent intraocular injections are major drawbacks of anti-VEGF drugs. Gene therapy, given the potency of one-time treatment and no need for frequent injections offers the real possibility of such a lasting treatment, with fewer adverse effects and higher patient quality of life. Herein, we reviewed the role of gene therapy in the CNV treatment. In addition, we discuss the advantages and challenges of current treatments compared with gene therapy.
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Localized Structural and Functional Deficits in a Nonhuman Primate Model of Outer Retinal Atrophy
Article
Full-text available
  • Oct 2021
Purpose: Cell-based therapy development for geographic atrophy (GA) in age-related macular degeneration (AMD) is hampered by the paucity of models of localized photoreceptor and retinal pigment epithelium (RPE) degeneration. We aimed to characterize the structural and functional deficits in a laser-induced nonhuman primate model, including an analysis of the choroid. Methods: Macular laser photocoagulation was applied in four macaques. Fundus photography, optical coherence tomography (OCT), dye angiography, and OCT-angiography were conducted over 4.5 months, with histological correlation. Longitudinal changes in spatially resolved macular dysfunction were measured using multifocal electroretinography (MFERG). Results: Lesion features, depending on laser settings, included photoreceptor layer degeneration, inner retinal sparing, skip lesions, RPE elevation, and neovascularization. The intralesional choroid was degenerated. The normalized mean MFERG amplitude within lesions was consistently lower than control regions (0.94 ± 0.35 vs. 1.10 ± 0.27, P = 0.032 at month 1, 0.67 ± 0.22 vs. 0.83 ± 0.15, P = 0.0002 at month 2, and 0.97 ± 0.31 vs. 1.20 ± 0.21, P < 0.0001 at month 3.5). The intertest variation of mean MFERG amplitudes in rings 1 to 5 ranged from 13.0% to 26.0% in normal eyes. Conclusions: Laser application in this model caused localized outer retinal, RPE, and choriocapillaris loss. Localized dysfunction was apparent by MFERG in the first month after lesion induction. Correlative structure-function testing may be useful for research on the functional effects of stem cell-based therapy for GA. MFERG amplitude data should be interpreted in the context of relatively high intertest variability of the rings that correspond to the central macula. Sustained choroidal insufficiency may limit long-term subretinal graft viability in this model.
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Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector
Article
Full-text available
  • Mar 2000
Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (F.IX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype1, 2. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (21011 vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of F.IX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.
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Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization
Article
Full-text available
  • Jan 2009
Vascular endothelial growth factor (VEGF) is important in pathological neovascularization, which is a key component of diseases such as the wet form of age-related macular degeneration, proliferative diabetic retinopathy and cancer. One of the most potent naturally occurring VEGF binders is VEGF receptor Flt-1. We have generated two novel chimeric VEGF-binding molecules, sFLT01 and sFLT02, which consist of the second immunoglobulin (IgG)-like domain of Flt-1 fused either to a human IgG1 Fc or solely to the CH3 domain of IgG1 Fc through a polyglycine linker 9Gly. In vitro analysis showed that these novel molecules are high-affinity VEGF binders. We have demonstrated that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in the mouse oxygen-induced retinopathy model. There were no histological observations of toxicity upon persistent ocular expression of sFLT01 for up to 12 months following intravitreal AAV2-based delivery in the rodent eye. Our data suggest that AAV2-mediated intravitreal gene delivery of our novel molecules may be a safe and effective treatment for retinal neovascularization.
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Inner Limiting Membrane Barriers to AAV-mediated Retinal Transduction From the Vitreous
Article
Full-text available
  • Sep 2009
  • MOL THER
Adeno-associated viral gene therapy has shown great promise in treating retinal disorders, with three promising clinical trials in progress. Numerous adeno-associated virus (AAV) serotypes can infect various cells of the retina when administered subretinally, but the retinal detachment accompanying this injection induces changes that negatively impact the microenvironment and survival of retinal neurons. Intravitreal administration could circumvent this problem, but only AAV2 can infect retinal cells from the vitreous, and transduction is limited to the inner retina. We therefore sought to investigate and reduce barriers to transduction from the vitreous. We fluorescently labeled several AAV serotype capsids and followed their retinal distribution after intravitreal injection. AAV2, 8, and 9 accumulate at the vitreoretinal junction. AAV1 and 5 show no accumulation, indicating a lack of appropriate receptors at the inner limiting membrane (ILM). Importantly, mild digestion of the ILM with a nonspecific protease enabled substantially enhanced transduction of multiple retinal cell types from the vitreous, with AAV5 mediating particularly remarkable expression in all retinal layers. This protease treatment has no effect on retinal function as shown by electroretinogram (ERG) and visual cortex cell population responses. These findings may help avoid limitations, risks, and damage associated with subretinal injections currently necessary for clinical gene therapy.
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Human RPE65 Gene Therapy for Leber Congenital Amaurosis: Persistence of Early Visual Improvements and Safety at 1 Year
Article
Full-text available
  • Aug 2009
  • HUM GENE THER
Human gene therapy with rAAV2-vector was performed for the RPE65 form of childhood blindness called Leber congenital amaurosis. In three contemporaneous studies by independent groups, the procedure was deemed safe and there was evidence of visual gain in the short term. At 12 months after treatment, our young adult subjects remained healthy and without vector-related serious adverse events. Results of immunological assays to identify reaction to AAV serotype 2 capsid were unchanged from baseline measurements. Results of clinical eye examinations of study and control eyes, including visual acuities and central retinal structure by in vivo microscopy, were not different from those at the 3-month time point. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months. The retinal extent and magnitude of rod and cone components of the visual sensitivity between 3 and 12 months were also the same. The safety and efficacy of human retinal gene transfer with rAAV2-RPE65 vector extends to at least 1 year posttreatment.
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Treatment of Leber Congenital Amaurosis Due to RPE65 Mutations by Ocular Subretinal Injection of Adeno-Associated Virus Gene Vector: Short-Term Results of a Phase I Trial
Article
Full-text available
  • Sep 2008
  • HUM GENE THER
Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB-hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21-24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 x 10(10) vector genomes in 150 microl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30-90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p < 0.001); whereas, for control eyes, sensitivity changes were not significant (p = 0.99). Comparisons are drawn between the present work and two other studies of ocular gene therapy for RPE65-LCA that were carried out contemporaneously and reported.
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Aiello LP, Pierre EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King GL, and Smith LEH. Suppression of retinal neovascularization in vivo by inhibition of the vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 92:10457-10461
Article
Full-text available
  • Nov 1995
The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.
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Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor (VEGF)
Article
  • Oct 1996
  • VISION RES
Purpose: Vascular endothelial growth factor (VEGF) is a potent and specific angiogenic growth factor, in vitro and in vivo, that may be associated with the development of intraocular neovascularization. In the current study, the authors analyze the expression of VEGF in subfoveal fibrovascular membranes from patients with age-related macular degeneration. Methods: Surgically removed subfoveal fibrovascular membranes from 18 eyes were analyzed for the expression of VEGF mRNA and protein using in situ hybridization and immunohistochemistry, respectively. Results: Most specimens expressed both VEGF mRNA and protein. The VEGF mRNA expression was particularly high in areas with a marked inflammatory response, in which the expression was concentrated to cells resembling fibroblasts and to surrounding inflammatory cells. VEGF protein expression was seen in fibrovascular parts of the membranes and was predominantly localized to the cytoplasm of fibroblastlike cells. In some of these membranes, strong VEGF protein immunoreactivity also was concentrated to extracellular matrix foci within the fibrovascular stroma. Conclusions: Results indicate that VEGF may be of pathogenetic importance for the development of the choroidal neovascularization (age-related macular degeneration) and also may implicate a role of fibroblasts of presumable choroidal origin in this process.
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Increased Vascular Endothelial Growth Factor Levels in the Vitreous of Eyes With Proliferative Diabetic Retinopathy
Article
  • Nov 1994
  • AM J OPHTHALMOL
The vitreous levels of the angiogenic polypeptide vascular endothelial growth factor (also known as vascular permeability factor) were measured and compared in eyes with and without proliferative diabetic retinopathy. Undiluted vitreous samples from 20 eyes were collected at the time of vitrectomy, and vascular endothelial growth factor levels were determined by using a time-resolved immunofluorometric assay. Vitreous vascular endothelial growth factor levels were significantly higher in eyes with proliferative diabetic retinopathy than in eyes without proliferative diabetic retinopathy (P = .006; Wilcoxon Rank Sum Test). The median vitreous concentration in the eyes with proliferative diabetic retinopathy was 29.1 pM and exceeded the known concentration required for the maximal proliferation of vascular endothelial cells in vitro. These data are consistent with vascular endothelial growth factor serving as a physiologically relevant angiogenic factor in proliferative diabetic retinopathy.
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