Sequence- and target-independent angiogenesis suppression by siRNA via TLR3.

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-alpha/beta activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-gamma and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity-the absence of blood vessels in the cornea-is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice.

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.

Loss of SPARC-mediated VEGFR-1 suppression after injury reveals a novel antiangiogenic activity of VEGF-A.

VEGF-A promotes angiogenesis in many tissues. Here we report that choroidal neovascularization (CNV) incited by injury was increased by excess VEGF-A before injury but was suppressed by VEGF-A after injury. This unorthodox antiangiogenic effect was mediated via VEGFR-1 activation and VEGFR-2 deactivation, the latter via Src homology domain 2-containing (SH2-containing) tyrosine phosphatase-1 (SHP-1). The VEGFR-1-specific ligand placental growth factor-1 (PlGF-1), but not VEGF-E, which selectively binds VEGFR-2, mimicked these responses. Excess VEGF-A increased CNV before injury because VEGFR-1 activation was silenced by secreted protein, acidic and rich in cysteine (SPARC). The transient decline of SPARC after injury revealed a temporal window in which VEGF-A signaling was routed principally through VEGFR-1. These observations indicate that therapeutic design of VEGF-A inhibition should include consideration of the level and activity of SPARC.

Inhibition of laser-induced choroidal neovascularization by atorvastatin by downregulation of monocyte chemotactic protein-1 synthesis in mice.

PURPOSE: To determine the effect of atorvastatin, an HMG CoA reductase inhibitor, on experimental choroidal neovascularization (CNV) induced by laser photocoagulation in mice. METHODS: CNV was induced by laser photocoagulation in normal wild-type mice. The mice received either oral atorvastatin 10 (AS10 group) or 20 (AS20 group) mg/kg per day 3 days before and after laser application; 1 (AS1) and 2 (AS2) mg/kg per day were included in the measurement of the parameters of CNV volume and the expression of chemoattractant CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) and intracellular adhesion molecule (ICAM)-1. CNV responses were compared based on volume measurements 2 weeks after laser photocoagulation. Expression of vascular endothelial growth factor (VEGF), CCL2/MCP-1, and ICAM-1 in the RPE and choroid was quantified by ELISA 2 or 3 days after photocoagulation. Macrophage infiltration of the choroid was determined by flow cytometry. RESULTS: The mean CNV volume was significantly smaller in the AS1 (44.16 +/- 4.67 x 10(4) microm(3)), AS2 (36.49 +/- 4.64 x 10(4) microm(3)), AS10 (25.75 +/- 2.41 x 10(4) microm(3)), and AS20 (33.24 +/- 8.42 x 10(4) microm(3)) groups compared with control mice (64.21 +/- 2.27 x 10(4) microm(3); P = 0.0004, P < 0.0001, P < 0.0001, P < 0.0001, respectively). The mean VEGF and CCL2/MCP-1 protein levels decreased significantly (P = 0.001, P = 0.02, respectively) in the treated group compared with the control group. ICAM-1 expression did not differ significantly between the treated and control groups. The number of choroid-infiltrating macrophages decreased markedly in the treated group. CONCLUSIONS: Atorvastatin effectively inhibited laser-induced CNV in mice and was associated with downregulation of CCL2/MCP-1 and VEGF and reduced macrophage infiltration into the RPE/choroid.

Upregulation of VEGF in murine retina via monocyte recruitment after retinal scatter laser photocoagulation.

PURPOSE: This study was conducted to determine changes in the expression of vascular endothelial growth factor (VEGF) in murine retina after retinal scatter laser photocoagulation. METHODS: Photocoagulation (PHC) was performed on wild-type C57BL/6J mice using a diode laser, and the eyes were enucleated 1, 2, 3, 4, 7, and 14 days after laser treatment. VEGF and monocyte chemoattractant protein (MCP)-1 levels in the sensory retina and retinal pigmented epithelial (RPE) cells in both tissues were measured by ELISA. The VEGF mRNA was measured by real-time RT-PCR. Leukocyte infiltration into the RPE-choroid was determined by flow cytometry. VEGF comparisons between mice subjected to PHC and those treated with monocyte recruitment inhibitor (anti-MCP-1) were performed and statistically analyzed. The expression of VEGF and MCP-1 in the retina was determined by immunohistochemistry. RESULTS: VEGF protein levels significantly increased 1 day after PHC in both the RPE-choroid and the sensory retina. VEGF concentrations were maximum at day 3 after photocoagulation and stayed elevated until day 7. The number of choroid-infiltrating macrophages was markedly increased in mice with laser treatment compared with those without laser treatment. VEGF expression decreased after treatment with neutralized antibody to monocyte recruitment. We demonstrate that MCP-1 expression in the retina increased markedly after scatter laser photocoagulation by immunohistochemistry and ELISA. CONCLUSIONS: Retinal scatter laser photocoagulation induced upregulation of VEGF in the sensory retina and RPE-choroid at an early period. The authors speculate that the major source of VEGF in the retina after retinal scatter laser photocoagulation is the recruited monocytes.

Drug delivery from ocular implants.

Developing an intraocular drug delivery system (DDS) is urgently needed because most vitreoretinal diseases are refractory to conventional pharmacological approaches; eye drops and systemically administered drugs cannot deliver therapeutic drug concentrations into vitreoretinal tissue. Intraocular DDSs address this problem. Intraocular sustained-drug release via implantable devices or injectable microparticles has been investigated to treat vitreoretinal diseases. A nonbiodegradable implant was first used in 1996 for cytomegalovirus retinitis secondary to the acquired immunodeficiency syndrome. Biodegradable implants, composed of hydrophilic or hydrophobic polymers, in the shape of rods, plugs, discs or sheets have been investigated. An injectable rod is presently being assessed in a Phase III trial to treat macular oedema secondary to diabetic retinopathy or branch-retinal vein occlusion. Intraocular DDSs using a biodegradable implant may soon be successfully used to treat serious intraocular disorders.

Long-term retention of dye after indocyanine green-assisted internal limiting membrane peeling.

PURPOSE: To evaluate dye retention in the fundus after indocyanine green (ICG)-assisted internal limiting membrane peeling. METHODS: Ten eyes with stage 3 or 4 nondiabetic idiopathic macular hole (MH group) and six eyes with diffuse diabetic macular edema (DM group) were studied. The fundus was examined with 780-nm infrared illumination by a scanning laser ophthalmoscope (SLO) after ICG-assisted internal limiting membrane peeling. The postoperative follow-up period ranged from 6 to 12 months (mean+/-SD, 3.7+/-2.6 months). RESULTS: Fluorescence from ICG was detected in all studied eyes in both groups up to 6 months after surgery. At 9 months after surgery, ICG fluorescence was visible in all eyes of the DM group, but in only one-third of eyes of the MH group. No fluorescence was detected in fellow eyes that had not been operated on. CONCLUSION: The present study using SLO revealed that ICG remains in the fundus for over 6 months after surgery. The results also suggested that a longer time might be required for dye clearance from the diabetic retina than from the nondiabetic retina.

Intraocular sustained drug delivery using implantable polymeric devices.

Vitreoretinal diseases involving age-related macular degeneration (AMD) are refractory to most topical or systemic drugs. The retina and the vitreous cavity have a unique position regarding pharmacokinetics in that the inner and outer blood retinal barriers separate the retina and vitreous from the systemic circulation. Eye drops achieve minimal therapeutic concentrations in the vitreoretinal tissue. Drug delivery systems are a strategy to address this. Intraocular sustained drug release using implantable devices has been investigated to treat vitreoretinal diseases. Possible targeted diseases include those in which repeated intraocular injections are effective (cytomegalovirus retinitis, uveitis), diseases requiring surgery (proliferative vitreoretinopathy), and chronic diseases (AMD, macular edema, retinitis pigmentosa). Hydrophobic or hydrophilic polymers shaped into a sheet, disc, rod, plug, or a larger device can be implanted into the subretinal space, intrascleral space, vitreous space, peribulbar space, or at the pars plana. Many researchers suggest the feasibility of these implants to treat AMD.

Scleral plug of biodegradable polymers containing tacrolimus (FK506) for experimental uveitis.

PURPOSE: To evaluate the efficacy of a biodegradable polymeric scleral plug containing the immunosuppressive agent, FK506, in a rabbit model for experimental uveitis. METHODS: The scleral plugs were prepared by dissolving poly(DL-lactide-co-glycolide; PLGA) and FK506 (weight, 8.5 mg; length, 5 mm; 1% FK506). The release of FK506 was evaluated in vitro by spectrophotometry on days 1, 3, 7, 14, 21, and 35. In vivo, FK506 concentrations of the vitreous were measured by high performance liquid chromatography 2 and 4 weeks after intravitreous plug implantation in pigmented rabbits. Sixteen pigmented rabbits were immunized twice subcutaneously with 10 mg of Mycobacterium tuberculosis H37Ra antigen. Twelve days later, the right eyes of all rabbits were challenged with an intravitreal injection of 50 micro g of antigen. After the first challenge, the 16 eyes of 16 pigmented rabbits were divided into two groups. Scleral plugs were implanted into the vitreous of the right eye of eight rabbits. Eight control rabbits received a sham device. The aqueous protein concentrations and cell counts were determined on postchallenge days 7, 14, and 28. To simulated chronic inflammation, the eyes were rechallenged with intravitreal antigen on day 14 and were observed for 1 month. Inflammation of the anterior chamber and the vitreous were graded clinically by two masked observers. Retinal function was evaluated by electroretinography (ERG) and histologic examination. RESULTS: Clinical scores (anterior chamber cells, flare, and vitreous opacity) showed that treated eyes had significantly less inflammation than untreated eyes (P<0.001). Quantitative analysis of inflammatory cells (P<0.001) and protein concentrations (P<0.0001) in the anterior chamber showed significant decreases in treated eyes. Histopathologic examination showed marked inflammation and tissue disorganization in the untreated eyes. No retinal toxicity was detected, histopathologically and electroretinographically. After antigen rechallenge, inflammation in experimental eyes was still less than in control eyes. CONCLUSIONS: Intravitreal sustained-release of FK506 from a biodegradable polymeric scleral plug was highly effective in suppressing the inflammation of experimental uveitis in a rabbit model for at least 6 weeks. This device may be useful in the management of patients with severe chronic uveitis.

Macrophage depletion inhibits experimental choroidal neovascularization.

OBJECTIVE: To investigate the role of macrophages in the development of laser-induced choroidal neovascularization (CNV) by selective depletion with liposomal clodronate (Cl(2)MDP-LIP). METHODS: Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice. Animals were treated with intravenous (IV) and/or subconjunctival (SC) Cl(2)MDP-LIP or PBS-LIP at the following time points: 2 days before, immediately after, 2 days before and immediately after, or 2 days after laser injury. CNV responses were compared on the basis of en masse volumetric measurements and fluorescein angiography after laser photocoagulation. Macrophages were identified by immunostaining for F4/80, and vascular endothelial growth factor (VEGF) expression was quantified by ELISA. RESULTS: Macrophages invaded the site of laser injury within 1 day of photocoagulation and peaked at 3 days. IV Cl(2)MDP-LIP significantly decreased the volume of CNV and angiographic leakage when administered 2 days before and/or immediately after laser injury, but not when administered 2 days after injury. SC Cl(2)MDP-LIP significantly decreased lesion volume when coadministered with IV PBS-LIP but not IV Cl(2)MDP-LIP. IV Cl(2)MDP-LIP was significantly more beneficial when administered 2 days before laser injury than immediately after, but combining SC Cl(2)MDP-LIP with IV treatment eliminated this difference. Reduction in CNV volume correlated with VEGF protein levels and number of infiltrating macrophages. CONCLUSIONS: Generalized macrophage depletion reduced the size and leakage of laser-induced CNV and was associated with decreased macrophage infiltration and VEGF protein. These findings define the role of the macrophage as a critical component in initiating the laser-induced CNV response.

Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization.

PURPOSE: To investigate the role of the leukocyte adhesion molecules CD18 and intercellular adhesion molecule (ICAM)-1 in the development of choroidal neovascularization (CNV). METHODS: Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice and species-specific counterparts with targeted homozygous disruption of the CD18 or ICAM-1 gene. Expression of CD18 and ICAM-1 after laser injury was assessed by immunostaining. CNV responses were compared on the basis of en masse volumetric measurements obtained by confocal microscopy 2 weeks after laser injury and by determination of fluorescein angiographic leakage at 1, 2, and 4 weeks after laser injury. RESULTS: The site of laser injury showed upregulation of ICAM-1 and invasion by CD18-positive leukocytes within 1 day of laser injury. Significantly fewer lesions exhibited fluorescein leakage defined to be pathologically significant in CD18-deficient mice at weeks 1, 2, and 4 weeks and in ICAM-1-deficient mice at 1 and 4 weeks, compared with the control. There were a significantly greater number of lesions without fluorescein leakage in CD18-deficient mice than in the other two groups at all time points. The volume of CNV in CD18- and ICAM-1-deficient mice was significantly less than in wild type. CONCLUSIONS: These data suggest a nonredundant role for leukocyte adhesion to vascular endothelium in the development of laser-induced choroidal neovascularization.

Late-onset laser in situ keratomileusis (LASIK) flap dehiscence during retinal detachment surgery.

PURPOSE: To report a case of laser in situ keratomileusis (LASIK) flap dehiscence during retinal detachment surgery 7 months after uneventful refractive surgery. DESIGN: Interventional case report. METHODS: A 47-year-old man noticed a defect of the upper visual field in his right eye 7 months after a LASIK procedure. The fundus showed rhegmatogenous retinal detachment, and a scleral buckling procedure was performed. During the buckling procedure, the corneal flap became detached. RESULTS: At completion of the buckling procedure, the detached corneal flap was carefully raised and the exposed corneal stroma was cleansed of any residual epithelial cells or red blood cells with irrigation using balanced salt solution. One day after the operation, the LASIK flap was repositioned, the cornea had cleared, and the retina was reattached. CONCLUSIONS: As LASIK increases in popularity, the complication we have reported may become more common. We suggest that a retinal detachment surgery should be performed with careful avoidance of corneal trauma even if a long time has passed since the LASIK procedure.

Retention of dye after indocyanine Green-assisted internal limiting membrane peeling.

PURPOSE: To describe the long-term retention of indocyanine green (ICG) in the fundus after ICG-assisted internal limiting membrane peeling. DESIGN: Case report. Two patients underwent vitrectomy including ICG-assisted internal limiting membrane peeling. The fundus was examined with a 780-nm infrared illumination of a scanning laser ophthalmoscope after surgery. RESULTS: No ICG staining of the fundus was visible ophthalmoscopically. Examination with a scanning laser ophthalmoscope, however, detected fluorescence from residual ICG until 6 months after surgery in case 1 and 9 months in case 2. No complication related to the residual ICG was observed. CONCLUSIONS: The results suggested that ICG remains in the fundus for a long period after surgery. Clearance of the dye from the diabetic retina may be prolonged.

Suppression of ICAM-1 in retinal and choroidal endothelial cells by plasmid small-interfering RNAs in vivo.

PURPOSE: Leukocytes play a critical role in ocular diseases such as uveitis, diabetic retinopathy, and choroidal neovascularization. Intercellular adhesion molecule (ICAM)-1 is essential for the migration of leukocytes. Control of ICAM-1 expression may lead to therapies for these diseases. Small-interfering ribonucleic acids (siRNAs) are efficient specific modulators of endogenous gene expression. The authors describe the application of siRNA to suppress ICAM-1 expression on the murine neurosensory retina or retinal pigment epithelial (RPE) cells using a hydrodynamics-based transfection technique (HT) and intravitreal injection (IV) in vivo. METHODS: ICAM-1-specific plasmid siRNAs designed from the murine gene sequence were transfected into the retina using HT and IV in vivo. Green fluorescent protein (GFP) expression plasmid vector is used as a transfection marker in the retinal cells. ICAM-1 expression was analyzed by enzyme-linked immunosorbent assay and flow cytometry. ICAM-1 upregulation was induced by retinal laser photocoagulation and streptozotocin (STZ). RESULTS: After the administration of GFP expression plasmid with HT and IV, histologic analysis showed GFP fluorescence in every layer of the murine retina. After photocoagulation, ICAM-1 expression in the neurosensory retina or RPE cells transferred with plasmid ICAM-1 siRNA was significantly decreased compared with cells that were not transfected or cells transferred with scrambled control siRNA. Plasmid siRNAs silenced ICAM-1 expression after STZ administration compared with control or naked siRNA injection. CONCLUSIONS: SiRNA expression mediated by this plasmid causes efficient and specific downregulation of ICAM-1 expression, suggesting that it can be silenced by plasmid siRNA in murine retina in vivo. This technology may lead to novel concepts to reduce retinal neovascular disease by inhibiting leukocyte infiltration.

Intraocular pressure elevation following triamcinolone acetonide administration as related to administration routes.

PURPOSE: To evaluate the incidence and risk factors of intraocular pressure (IOP) elevation following triamcinolone acetonide (TA) administration. METHODS: In this retrospective observational case series, patients (224 eyes of 202 patients) with diffuse diabetic macular edema (66 eyes), branch retinal vein occlusion (39 eyes), central retinal vein occlusion (25 eyes), exudative age-related macular degeneration (49 eyes), myopic choroidal neovascularization (10 eyes), uveitis (30 eyes), or other conditions (5 eyes) were administered an intravitreal or posterior sub-Tenon capsule injection, or both, of TA. Sub-Tenon capsule injection was performed on 106 eyes (STTA group). Intravitreal injection was performed on 118 eyes (IVTA group), of which 85 eyes underwent simultaneous intravitreal and sub-Tenon capsule injections. Mean follow-up after TA administration was 15.9 +/- 10.4 (range, 3-39) months. The sub-Tenon capsule injection and intravitreal injection of TA were compared with respect to the frequency of IOP elevation and the time between TA administration and the initial IOP elevation, and the possible risk factors responsible for IOP elevation were identified. RESULTS: There was no significant difference in frequency of IOP > 21 mmHg between the STTA group and the IVTA group (P = 0.0588). There was, however, a significant difference in the frequency of IOP > 30 mmHg between the two groups (P = 0.0004). In the IVTA group, more patients needed antiglaucoma medication than in the STTA group (P = 0.0052). The incidence rate of IOP elevation within 1 week after TA administration in the IVTA group was significantly higher than in the STTA group (P = 0.0154). Risk factors for IOP elevation included higher baseline IOP (P < 0.0001), younger patients (P = 0.0095), and simultaneous administration of sub-Tenon capsule and intravitreal injections (P = 0.0228). CONCLUSIONS: Careful follow-up of IOP is required after TA injections.

Comparative study on efficacy of a combination therapy of triamcinolone acetonide administration with and without vitrectomy for macular edema associated with branch retinal vein occlusion.

AIMS: To compare the efficacy ofa combination therapy of triamcinolone acetonide (TA) administration with and without vitrectomy in eyes with macular edema associated with branch retinal vein occlusion over a 1-year period. METHODS: A retrospective, case-control study was conducted in 15 eyes of 15 patients with macular edema associated with branch retinal vein occlusion. Eight eyes underwent simultaneous intravitreal and posterior sub-Tenon capsule injections of TA (TA-injected group). Seven eyes underwent vitrectomy with intravitreal or simultaneous posterior sub-Tenon capsule injection of TA (vitrectomy with TA group). Macular thickness and visual acuity were measured before and at 1, 3, 6 and 12 months after the therapy. RESULTS: Twelve months after the therapy, mean visual acuity improved significantly from baseline in both the TA-injected (p = 0.0069) and vitrectomy with TA groups (p = 0.0145). Macular thickness also improved significantly in both the TA-injected (p = 0.0065) and vitrectomy with TA groups (p = 0.0058). At 12 months after the therapy, there was no significant difference in visual acuity and macular thickness between the two groups (p = 0.3308 and 0.3711, respectively). At the early postoperative stage (1 and 3 months after the therapy), the central macular thickness in the TA-injected group was significantly less than that in the vitrectomy with TA group (p = 0.0140 and 0.0275, respectively); there was no significant difference in visual acuity between the two groups (p = 0.0796 and 0.3753, respectively). CONCLUSION: TA injection without vitrectomy was as effective as a combination therapy of TA injection with vitrectomy.

Drusen complement components C3a and C5a promote choroidal neovascularization.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrialized nations, affecting 30-50 million people worldwide. The earliest clinical hallmark of AMD is the presence of drusen, extracellular deposits that accumulate beneath the retinal pigmented epithelium. Although drusen nearly always precede and increase the risk of choroidal neovascularization (CNV), the late vision-threatening stage of AMD, it is unknown whether drusen contribute to the development of CNV. Both in patients with AMD and in a recently described mouse model of AMD, early subretinal pigmented epithelium deposition of complement components C3 and C5 occurs, suggesting a contributing role for these inflammatory proteins in the development of AMD. Here we provide evidence that bioactive fragments of these complement components (C3a and C5a) are present in drusen of patients with AMD, and that C3a and C5a induce VEGF expression in vitro and in vivo. Further, we demonstrate that C3a and C5a are generated early in the course of laser-induced CNV, an accelerated model of neovascular AMD driven by VEGF and recruitment of leukocytes into the choroid. We also show that genetic ablation of receptors for C3a or C5a reduces VEGF expression, leukocyte recruitment, and CNV formation after laser injury, and that antibody-mediated neutralization of C3a or C5a or pharmacological blockade of their receptors also reduces CNV. Collectively, these findings establish a mechanistic basis for the clinical observation that drusen predispose to CNV, revealing a role for immunological phenomena in angiogenesis and providing therapeutic targets for AMD.