Development Of Laser-induced Choroidal Neovascularization Research Articles

MTORC1 and mTORC2 are differentially engaged in the development of laser-induced CNV

BackgroundThe mechanistic target of rapamycin (mTOR) pathway is a potential target to inhibit pathologic processes in choroidal neovascularization. However, the exact role of mTOR signaling in the development of CNV remains obscure. In this study, we assessed the role of mTORC1 and mTORC2 as well as the effect of rapamycin (sirolimus) on choroidal neovascularization (CNV) in a laser-induced mouse model.MethodsIn experiment A, we observed the natural course of CNV development and the dynamics of mTOR-related proteins during the 12 days after the laser injury. The expression of mTOR-related proteins was evaluated using Western blot (WB). Cryosections of CNV-induced mice were immunostained for the visualization of the vascular and extravascular components of the CNV. Experiment B was performed to confirm the critical period of mTOR signaling in the development of laser-induced CNV, we administered rapamycin before and/or during the active period of mTOR complexes. WB and immunofluorescence staining was performed to evaluate the mode of action and the effect of mTOR inhibition on CNV development.ResultsIn experiment A, we detected high levels of p-mTOR S2448 and p-mTOR S2481 from the 5th to 12th day of laser injury. Immunofluorescence imaging of cryosections of mice sacrificed on day 7 revealed greater co-immunoreactivity of p-mTOR S2448 positive cells with CD11b and F4/80, while p-mTOR S2481 positive cells showed colocalization with CD31, α-SMA, and cytokeratin. In experiment B, rapamycin injection during the active period of mTOR signaling demonstrated near-complete inhibition of CNV lesion as well as significant induction of autophagy.ConclusionOur study suggests the mTOR as a critical player during CNV development in laser-induced mouse model through differentially acting with the mTORC1 and mTORC2. mTORC1 activity was high predominantly in inflammatory cells in CNV lesion, while mTORC2 activity was higher in vascular components and the RPE.

Inhibition of development of laser-induced choroidal neovascularization with suppression of infiltration of macrophages in Smad3-null mice

We evaluated the effects of the loss of Smad3 on the development of experimental argon laser-induced choroidal neovascularization (CNV) in mice. An in vitro angiogenesis model was also used to examine the role of transforming growth factor-β1 (TGFβ1)/Smad3 signaling in vessel-like tube formation by human umbilical vein endothelial cells (HUVECs). CNV was induced in eyes of 8–12-week-old B6.129-background Smad3-deficient (KO) mice (n=47) and wild-type (WT) mice (n=47) by argon laser irradiation. Results showed that the size of the CNV induced was significantly smaller in KO mice as compared with WT mice at day 14 as revealed by high-resolution angiography with fluorescein isothiocyanate-dextran. Immunohistochemistry and real-time reverse transcription-polymerase chain reaction of RNA extracted from laser-irradiated choroidal tissues were conducted on specimens at specific timepoints. Invasion of macrophages (F4/80+), but not neutrophils (myeloperoxidase+), and appearance of myofibroblasts (α-smooth muscle actin+) were suppressed in laser-irradiated KO tissues. mRNA expression of inflammation-related factors, that is, vascular endothelial growth factor (VEGF), macrophage-chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and TGFβ1 in choroidal tissues was suppressed by the loss of Smad3. We then examined the effects of adding a Smad3 inhibitor, SIS3, or an ALK5 inhibitor, SB431542, on tube formation promoted by TGFβ1 or VEGF in HUVECs cocultured with fibroblast feeder. Further addition of SIS3 or SB431542 augmented vessel-like tube formation by HUVECs in the presence of TGFβ1 or VEGF. In conclusion, lack of Smad3 attenuated the growth of laser-induced CNV with suppression of inflammation by macrophages in mice. Because blocking TGFβ1/Smad3 signal stimulated the activity of angiogenesis of HUVECs in vitro, the reduction of CNV in vivo in KO mice is attributed to a decrease in growth factor levels in the tissue by the loss of Smad3.

Intravitreal injection of 99Tc-MDP inhibits the development of laser-induced choroidal neovascularization in rhesus monkeys

The aim of this study was to investigate the safety and efficacy of intravitreal injection of (99)Tc-MDP, a decay product of (99m)Tc-MDP, on the development of laser-induced choroidal neovascularization (CNV) in rhesus monkeys. Experimental CNV was induced by argon laser with a small high-energy laser spot. Monkeys were given 50 μL of (99)Tc-MDP at a concentration of 0.005 μg/mL (n = 6) or 0.01 μg/mL (n = 6) by intravitreal injection once a week immediately after laser injury for a period of 56 days. Control animals were treated with the same volume of PBS (n = 6) in the same way. Eyes were monitored by ophthalmic examination, color fundus photography, fluorescence fundus angiography (FFA), optical coherence tomography (OCT) and histology. Incidences of grade 4 CNV lesions as well as the leakage areas of grade 4 CNVs on the late-phase of fluorescein angiograms were measured in a standardized, randomized and masked fashion fortnightly. The maximum widths and heights of grade 4 CNVs were also calculated by histology at the end of the experiment. Toxicity of (99)Tc-MDP on the retina was evaluated by electroretinogram (ERG) and histologic analysis. (99)Tc-MDP reduced the incidences of grade 4 CNVs by 33.33 % and 39.40 % in the 0.005 μg/mL and 0.01 μg/mL groups, respectively, compared with the PBS group on day 28 (P < 0.05; n = 6). The leakage areas of grade 4 CNVs were smaller in the 0.005 μg/mL (0.7136 ± 0.0283 mm(2), p <0.01; n = 6) and 0.01 μg/mL (0.4351 ± 0.0349 mm(2), p < 0.01; n = 6) groups than those in the PBS control group (0.9373 ± 0.0455 mm(2); n = 6) in a dose-dependent manner on day 28. OCT and histology also showed that the sizes of CNVs were smaller in the (99)Tc-MDP treated groups than those in the PBS group. Although intravitreal injection of (99)Tc-MDP led to mild inflammatory reaction in the anterior chamber, histology and ERG findings demonstrated that (99)Tc-MDP did not cause any change in histological structure or function of the retina (p>0.05). Intravitreal injection of (99)Tc-MDP can inhibit the development of laser-induced CNV without toxic effect on retina, suggesting that (99)Tc-MDP has therapeutic potential for CNV related diseases.

Lentivirus Vector-Mediated Knockdown of Erythropoietin-Producing Hepatocellular Carcinoma Receptors B4 Inhibits Laser-Induced Choroidal Neovascularization

To evaluate the efficacy of erythropoietin-producing hepatocellular carcinoma receptors B4 (EphB4) knockdown on the development of laser-induced choroidal neovascularization (CNV) in vivo. We constructed recombinant lentiviral vectors (Lv) Lv-shRNA-EphB4 to specifically knock down the expression of EphB4. The mRNA and protein expression of EphB4 was investigated by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. CNV was induced by laser photocoagulation in C57BL/6 mice. The mice were then randomly assigned to be intravitreally injected with phosphate-buffered saline (PBS), Lv-shRNA-EphB4 recombinant lentivirus, or an unrelated shRNA recombinant lentivirus (pFU LV-shRNA-NC). An uninjected group was used as the control. Fundus fluorescein angiography (FFA), histologic analysis, and choroidal flat mounts analysis were applied to evaluate the inhibition of CNV after an intravitreal injection. Transfection of Lv-shRNA-EphB4 led to the knockdown of EphB4, and EphB4 mRNA was down-regulated by about 80%. FFA and histologic analysis revealed that the leakage areas and the mean thickness of CNV were much smaller in the Lv-shRNA-EphB4 group than in the PBS-treated, pFU Lv-shRNA-NC group and the non-injection group. Choroidal flat mounts showed significantly less leakage and smaller leakage areas in the Lv-shRNA-EphB4 group than those in other groups. Knocking down the expression of EphB4 exerts an inhibitory effect on CNV in vivo. It may provide a potential strategy for the treatment of CNV.

Low-Dose Lipopolysaccharide Pretreatment Suppresses Choroidal Neovascularization via IL-10 Induction

Recent studies have suggested that some kinds of microbial infection may have a crucial role in the development of many diseases such as autoimmune diseases and certain types of cancer. It has been reported that some chronic infections, such as Chlamydia pneumoniae, and immunological dysfunctions are associated with age-related macular degeneration (AMD), a leading cause of blindness. To evaluate the association between systemic low-level inflammation induced by infection and AMD pathogenesis, we investigated whether intraperitoneal injection of lipopolysaccharide (LPS) can modulate the development of laser-induced choroidal neovascularization (CNV), a key feature of AMD. Contrary to our expectations, the sizes of CNV in mice with LPS pretreatment were approximately 65% smaller than those of the control mice. After LPS pretreatment, serum IL-10 concentration and IL-10 gene expression in peritoneal macrophages and in the posterior part of the eye increased. Peritoneal injection of anti-IL10 antibody reduced CNV suppression by LPS pretreatment. Moreover, adoptive transfer of the resident peritoneal macrophages from LPS-treated mice into control littermates resulted in an approximately 26% reduction in the size of CNV compared with PBS-treated mice. We concluded that CNV formation was suppressed by low-dose LPS pretreatment via IL-10 production by macrophages.

Transforming Growth Factor-β Inhibition Decreases Diode Laser–Induced Choroidal Neovascularization Development in Rats: P17 and P144 Peptides

To assess the effect of transforming growth factor (TGF)-β inhibitor peptides (P17 and P144) on the development of laser-induced choroidal neovascularization (LI-CNV) in a rat model. Sixty-one Long-Evans rats underwent diode LI-CNV model. Forty-eight hours later, treatment was administered. The intravenous control group (IV-control) and intravenous P17 group (IV-17) received five doses (0.2 mg every 72 hours) of vehicle and P17, respectively. Four groups received intravitreal injections of P17 low-dose (LD-17; 1 mg/mL) and high-dose (HD-17; 20 mg/mL) and P144 low-dose (LD-144; 1 mg/mL) and high-dose (HD-144; 3 mg/mL), and fellow eyes received vehicle. CNV evolution was assessed weekly by fluorescein angiography (FA). After death, VEGF, TGF-β and PDGF protein levels were measured by ELISA in RPE and retina homogenates. Data were analyzed with commercially available statistical analysis software. The mean CNV area, measured in pixels, was significantly lower at the second and fourth weeks in IV-17 (P < 0.05) and from the second week in HD-17 (P < 0.05), whereas LD-144 and HD-144 showed significant differences at every time point (P < 0.05). LD-17 showed significantly lower protein levels of TGF-β in retina and PDGF in RPE (P < 0.05), whereas HD-17 showed lower levels of VEGF (RPE and retina; P < 0.05), TGF-β (RPE and retina; P < 0.05), and PDGF (RPE; P < 0.05). HD-144 showed lower VEGF levels in the retina (P < 0.05). TGF-β inhibition with these peptides represents a promising new therapeutic line for CNV targeting a different pathway than current therapies. More studies are needed to assess this effect on early CNV, alone or in combination with anti-VEGF.

Relationship between Complement Membrane Attack Complex, Chemokine (C-C Motif) Ligand 2 (CCL2) and Vascular Endothelial Growth Factor in Mouse Model of Laser-induced Choroidal Neovascularization

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

Suppression of Choroidal Neovascularization and Quantitative and Qualitative Inhibition of VEGF and CCL2 by Heparin

To study the effect of heparin on the development of laser-induced choroidal neovascularization (CNV) and to assess the underlying molecular mechanisms. Bone marrow transplantation (BMT) was conducted by intravenous injection of green fluorescence protein (GFP)-labeled bone marrow cells (1 × 10(7) cells) into irradiated (9 Gy) C57BL/6J mice. Laser photocoagulation was applied to induce CNV; subsequently, unfractionated heparin or phosphate-buffered saline was injected into mice that did or did not undergo BMT. The area of CNV, distribution of injected heparin, and quantities of infiltrating cells positive for Griffonia simplicifolia (GS) and GFP inside and outside the CNV were evaluated. Effects of heparin on the secretion of VEGF, CCL2, and TNF-α by ARPE19 cells and on the binding of VEGF, CCL2, TNF-α, and their receptors were analyzed in vitro. Intravitreal injection of heparin at higher doses reduced the size of the CNV. Heparin localized at the vascular structures and photoreceptor layers adjacent to the laser scar. Only GS-positive cells infiltrating outside the CNV were reduced significantly, but not those inside the CNV or those expressing GFP. Relative decreases in VEGF and CCL2 levels were observed in media of ARPE19 cells at higher heparin concentrations. In vitro binding assays revealed that heparin and porcine ocular fluid, respectively, suppressed the binding of VEGF to VEGFR2 and CCL2 to CCR2. Intravitreal heparin injection inhibited CNV development. Reduced VEGF and CCL2 secretion by RPE cells and suppression of VEGF-VEGFR2 and CCL2-CCR2 interactions at the laser site mediated by heparin may contribute to the pharmacologic effect.

Relative Contribution of VEGF and TNF-α in the Cynomolgus Laser-Induced CNV Model: Comparing the Efficacy of Bevacizumab, Adalimumab, and ESBA105

To compare the relative contribution of VEGF and TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in monkeys and to exploit the feasibility of topical use of suitable antibody fragments for the prevention of experimental CNV. To induce experimental CNV, small high-energy laser spots were used to treat several areas of the macula in the retinas of cynomolgus monkeys according to previously published protocols. To prevent abnormalities, bevacizumab (a potent VEGF inhibitor) and adalimumab or ESBA105 (potent TNF-alpha inhibitors) were given by intravitreal injection 1 week before and 1 week and 3 weeks after laser treatment. ESBA105 was also applied topically in a separate group. Control animals were treated with either intravitreal or topical saline. Eyes were monitored by ophthalmic examination, color photography, and fluorescein angiography. Inhibition of VEGF by bevacizumab completely blocked the formation of CNV. Both TNF-alpha inhibitors also significantly reduced laser-induced CNV abnormalities after intravitreal administration. Most important, topical use of the anti-TNF-alpha single-chain antibody fragment ESBA105 also reduced the formation of CNV. TNF-alpha contributes to laser-induced CNV formation, and its inhibition can be a new therapeutic target for CNV. This study suggests TNF-alpha as another therapeutic target for the prevention and treatment of CNV and adds to the emerging clinical data suggesting the therapeutic value of TNF-alpha inhibitors in age-related macular degeneration (AMD). Further, this study shows that topical therapy with suitable antibody fragments has the potential of being introduced to retinal disease treatment regimens.

Dual Role of Intravitreous Infliximab in Experimental Choroidal Neovascularization: Effect on the Expression of Sulfated Glycosaminoglycans

To determine effects of intravitreous anti-TNF-alpha (infliximab) in a laser-induced choroidal neovascularization (CNV) model by fluorescein angiogram (FA), immunofluorescence, ELISA, and glycosaminoglycan analyses. CNV induction was performed using argon laser. Rats were divided into eight groups (no-laser no-infliximab; laser; laser with 10, 20, 40, 80, or 320 microg infliximab; and isotype-matched IgG). After 3 weeks, CNV area was measured by FA and von Willebrand factor (vWF) immunofluorescence. VEGF, TGF-beta, and syndecan-4 were evaluated by ELISA and immunofluorescence. Glycosaminoglycan expression was determined in retina and choroid of animals metabolically labeled with [(35)S]-sulfate. Cytotoxicity was investigated using ARPE-19 and endothelial cells. FA showed significant reduction in the low-dose infliximab groups (10-40 microg), confirmed by vWF immunofluorescence that showed 49% decrease in the CNV. VEGF and TGF-beta decreased expression detected by ELISA and immunofluorescence paralleled these results. Similar data were observed for syndecan-4. The expression of these molecules in the neovascularization area using 320 microg was similar to the no-infliximab laser group or laser with isotype-matched IgG. Heparan sulfate expression in retina and choroid paralleled the observed effects on angiogenesis. Increased expression of chondroitin sulfate in retina and dermatan sulfate in choroid reflects the effects of injury and fibrosis using high doses of anti-TNF-alpha. Infliximab showed no cytotoxic effect in ARPE-19 cells, whereas high doses led to 20% decrease in endothelial cell viability. Intravitreal infliximab shows dual effect on the development of laser-induced CNV. It reduces angiogenesis and glycosaminoglycan expression at low doses, whereas opposite effects are observed at high doses.

Antiangiogenic immunotherapy targeting Flk-1, DNA vaccine and adoptive T cell transfer, inhibits ocular neovascularization

Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases. The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and so there is no satisfactory therapy for ocular NV. Here, we describe a strategy targeting Flk-1, a self-antigen overexpressed on proliferating endothelial cells in ocular NV, by antiangiogenic immunotherapy—DNA vaccine and adoptive T cell therapy. An oral DNA vaccine encoding Flk-1 carried by attenuated Salmonella typhimurium markedly suppressed development of laser-induced choroidal NV. We further demonstrated that adoptive transfer of vaccine-induced CD8+ T cells reduced pathological preretinal NV, with a concomitant facilitation of physiological revascularization after oxygen-induced retinal vessel obliteration. However, physiological retinal vascular development was unaffected in neonatal mice transferred with vaccine-induced CD8+ T cells. These findings suggested that antiangiogenic immunotherapy targeting Flk-1 such as vaccination and adoptive immunotherapy may contribute to future therapies for ocular NV.

Development of Laser-Induced Choroidal Neovascularization in Rats after Retinal Damage by Sodium Iodate Injection

Purpose: To determine the role of the retinal pigment epithelium (RPE) in the development of laser-induced choroidal neovascularization (CNV) by selective damage with sodium iodate (SI). Methods: In group 1, diode laser photocoagulation was applied to the right eye of Long-Evans rats (n = 11). In group 2 (n = 11), SI was administered intravenously at a dose of 40 mg/kg of body weight 2 days after photocoagulation. In group 3 (n = 10), SI was injected 2 days before laser was applied. In group 4 (n = 10), SI was injected immediately after photocoagulation. Results: CNV ingrowth developed in 42 of 93 burns (45%) of the laser group and in 46 of 75 burns (61%) when SI was administered 2 days after laser (p = 0.036). No CNV developed in the other groups. Conclusions: The angiogenic effect of the RPE is essential during the first hours after the onset of the laser-induced CNV process. When the RPE was damaged before or at the time of photocoagulation, CNV was not induced. These results suggest that RPE integrity is a decisive element in CNV formation.

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Choroidal neovascularization (CNV) occurs in a variety of chorioretinal diseases including age-related macular degeneration (AMD), and is the major cause of severe visual loss in patients with AMD. Oxidative stress has been thought to play an important role in the development of CNV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. In this study, we examined the expression of p22phox, an integral subunit in the NADPH oxidase complex, in the mouse eye. We determined that p22phox is expressed in the retinal pigment epithelial (RPE) cells and inner retinal neurons. A small-interfering RNA (siRNA) designed against p22phox efficiently reduced the expression of the protein in the eye when delivered by means of recombinant adeno-associated virus (AAV) vector. Vector treatment inhibited CNV in the mouse when delivered into the subretinal space where RPE cells were transduced. These results suggest that NADPH oxidase-mediated ROS production in RPE cells may play an important role in the pathogenesis of neovascular AMD, and that this pathway may represent a new target for therapeutic intervention in AMD.

Effect of anti-TNF-alpha on laser-induced choroidal neovascularization

To investigate the role of TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in the mouse. Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice by making four separate choroidal bums in each eye. Animals were treated 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept, 5 microg/h, group 1, n = 12), chimeric monoclonal antibody (infliximab, 5 microg/h, group 2, n = 12) for 7 days by intraperitoneally implanted osmotic pumps. PBS was used as control (group 3, n = 12). The left eyes were removed for histopathologic examination and the right eyes were removed for flatmounts immunohistochemistry immediately after fluorescein angiography. In mice treated with medications 3 days before laser injury, left eyes were collected at 1 or 2 weeks after laser injury. In mice treated with medications 3 days after laser injury, left eyes were collected at 10 days after laser injury. CNV responses were compared by flatmount analysis of CNV-related fluorescence area and by determination of fluorescein angiographic leakage. The level of protein expression of TNF-alpha was semiquantitatively evaluated by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. Western blotting demonstrated that TNF-alpha was highly expressed in choroidal and RPE cells of wild type mice 1 week after laser treatment as compared to the control mice without laser treatment. Etanercept and infliximab administrated 3 days before laser-damage significantly reduced CNV size and pathological fluorescein leakage in comparison to the control group one and two weeks after laser injury. Only etanercept administered 3 days after laser injury still significantly reduced the development of CNV lesions. Histopathological examination confirmed that CNV lesions in treated mice had smaller diameter and thinner center as compared to the control animals. Anti-TNF-alpha treatment reduces the size and leakage of laser-induced CNV. These results suggest the involvement of TNF-alpha in the development of laser-induced CNV and its potential use as a therapeutic agent in the age related macular degeneration.

Inhibition of TNF-α reduces laser-induced choroidal neovascularization

To investigate the role of the TNF-α in the development of laser-induced choroidal neovascularization (CNV) in a mouse model. Four separate laser burns were applied to induce ruptures of Bruch's membrane and subsequent choroidal neovascularization in C57BL/6J mice. TNF-α protein expression was semiquantitatively assessed by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. To investigate the effect of TNF-α inhibition on CNV formation, animals were treated for 7 days via intraperitonealy implanted osmotic pumps either 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept), a chimeric monoclonal antibody (infliximab), or a purified rat anti-mouse/rat TNF monoclonal antibody (TNF-mAb), respectively. Fluorescein angiography, flat-mount preparations, and histopathology were performed at day 7, 10, or 14 after laser treatment. Western blotting demonstrated that TNF-α expression was 4.57-fold higher in the choroid and RPE one week after laser injury compared to control mice without laser. When evaluated one and two weeks after laser injury, etanercept and infliximab given from the 3rd day before laser-damage significantly reduced CNV size and pathological fluorescein leakage compared to the control group after laser treatment only. The inhibitory effect of the monoclonal TNF-α antibody on CNV formation was evident two weeks after photocoagulation but not after one week. Only etanercept administered 3 days after laser injury still reduced significantly the development of CNV lesions. Histopathology confirmed that CNV lesions in treated mice were smaller in size compared to the control animals without TNF inhibitor treatment. In conclusion, anti-TNF-α treatment with different inhibitors reduces both the size and the leakage of laser-induced CNV. These results suggest the involvement of TNF-α in the development of laser-induced CNV and its potential use as a therapeutic agent in the age-related macular degeneration.

Complement Activation via Alternative Pathway Is Critical in the Development of Laser-Induced Choroidal Neovascularization: Role of Factor B and Factor H

The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4(-/-) mice developed CNV similar to their wild-type controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5(-/-) mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of membrane attack complex and angiogenic factors-vascular endothelial growth factor and TGF-beta2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.

Macrophage depletion inhibits experimental choroidal neovascularization.

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). 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. 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. 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.

To investigate the role of the leukocyte adhesion molecules CD18 and intercellular adhesion molecule (ICAM)-1 in the development of choroidal neovascularization (CNV). 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. 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. These data suggest a nonredundant role for leukocyte adhesion to vascular endothelium in the development of laser-induced choroidal neovascularization.

Inhibitory effect of bucillamine on laser-induced choroidal neovascularization in rats

Purpose. We investigated the inhibitory effects of bucillamine on formation of laser-induced choroidal neovascularization (CNV) in a rat model. Methods. Bucillamine administration (approximately 150?mg/kg/day) was started 1 week before photocoagulation and continued to the end of the study. Control groups received drinking water. Two weeks after photocoagulation, choroidal neovascularization development was evaluated using simultaneous fluorescein and indocyanine green angiography, and the maximal thickness of the lesions was measured histologically. Results. The incidence of CNV formation was 99.5 ± 0.2% [mean ± standard deviation (SD)] in control rats and 64.3 ± 15.1% with bucillamine (P < 0.01). Histological study showed that the thickness of the CNV lesions was 23.4 ± 6.5 µm (mean ± SD) in the bucillamine-treated rats, which was significantly decreased compared to that in controls (60.8 ± 9.2 µm) (P < 0.01). Conclusions. Our results suggest that bucillamine may inhibit the development of laser-induced CNV in rats.

Inhibitory effect of losartan on laser-induced choroidal neovascularization in rats

PURPOSE: To investigate the inhibitory effects of losartan, an angiotensin receptor antagonist, on angiogenesis in a rat model of laser-induced choroidal neovascularization. METHODS: Experimental study. Fifteen Brown–Norway male rats received losartan (approximately 5 mg/kg/d) in drinking water, and 15 Brown–Norway male rats received unsupplemented drinking water 1 week before photocoagulation, and it was continued to the end of the study. Two weeks after intense laser photocoagulation, choroidal neovascularization was evaluated by fluorescein angiography and histopathologic evaluation. RESULTS: The incidence of choroidal neovascularization formation was 99.5 ± .2% (mean ± standard deviation) in controls and 72.5 ± 8.8% in losartan-treated rats ( P < .01). Quantitative morphometric assessment revealed mean choroidal neovascularization lesion thickness of 54 and 44.8 μm, respectively, in controls and losartan-treated rats ( P < .01). CONCLUSION: Losartan seems to inhibit development of laser-induced choroidal neovascularization. Angiotensin receptor antagonists may be useful as prophylaxis against choroidal neovascularization associated with age-related macular degeneration.