August 2003
Volume 44, Issue 8
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Retina  |   August 2003
Macrophage Depletion Inhibits Experimental Choroidal Neovascularization
Author Affiliations
  • Eiji Sakurai
    From the Department of Ophthalmology, University of Kentucky, Lexington, Kentucky; the
  • Akshay Anand
    From the Department of Ophthalmology, University of Kentucky, Lexington, Kentucky; the
  • Balamurali K. Ambati
    Department of Ophthalmology, Medical College of Georgia, Augusta, Georgia; and the
  • Nico van Rooijen
    Department of Cell Biology and Immunology, Vrije Universiteit, Amsterdam, The Netherlands.
  • Jayakrishna Ambati
    From the Department of Ophthalmology, University of Kentucky, Lexington, Kentucky; the
Investigative Ophthalmology & Visual Science August 2003, Vol.44, 3578-3585. doi:10.1167/iovs.03-0097
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      Eiji Sakurai, Akshay Anand, Balamurali K. Ambati, Nico van Rooijen, Jayakrishna Ambati; Macrophage Depletion Inhibits Experimental Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2003;44(8):3578-3585. doi: 10.1167/iovs.03-0097.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

objective. To investigate the role of macrophages in the development of laser-induced choroidal neovascularization (CNV) by selective depletion with liposomal clodronate (Cl2MDP-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) Cl2MDP-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 Cl2MDP-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 Cl2MDP-LIP significantly decreased lesion volume when coadministered with IV PBS-LIP but not IV Cl2MDP-LIP. IV Cl2MDP-LIP was significantly more beneficial when administered 2 days before laser injury than immediately after, but combining SC Cl2MDP-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.

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among the elderly in most industrialized nations, 1 yet little is known about the molecular mechanisms of choroidal neovascularization (CNV), the angiogenic process responsible for most severe visual loss in patients with AMD. 2  
The presence of macrophages in histologic studies of CNV has elicited interest in their role in the development of neovascular AMD. The spatiotemporal distribution of macrophages correlates with arborizing CNV in humans 3 and in animal models. 4 In patients with AMD, macrophages are in proximity to thinned and perforated areas of Bruch’s membrane 5 6 and participate in digesting the outer collagenous zone of Bruch’s membrane, 6 both of which facilitate the subretinal entry of CNV. 
To test directly the hypothesis that macrophages play a causal rather than coincidental role in the development of CNV, we used the technique of pharmacological macrophage depletion with liposomal clodronate (Cl2MDP-LIP) in the laser-induced model of CNV, which captures many salient pathologic and molecular features of neovascular AMD. Although free Cl2MDP does not penetrate cell membranes and has a short circulating half-life, Cl2MDP-LIP is phagocytosed by macrophages and rapidly induces apoptosis 7 8 without secretion of proinflammatory cytokines by the dying macrophages. 9 Moreover, Cl2MDP-LIP appears to have a very selective effect on macrophages and phagocytic dendritic cells. Neutrophils and lymphocytes are not directly affected by this drug. 10 11 12  
We sought to assess the differential contribution of macrophages in the local (submandibular) lymph nodes versus that of the circulating, splenic, and hepatic macrophages to the development of CNV in this model. We administered Cl2MDP-LIP by intravenous (IV) injection, which leads to near complete depletion of splenic and hepatic macrophages and marginal zone dendritic cells within 24 hours, persisting for 1 to 2 weeks in mice, 10 and/or subconjunctival (SC) injection, which leads to the depletion of macrophages from the draining submandibular lymph nodes. 13  
We attempted to differentiate the contribution of circulating versus resident retinal macrophages to CNV. Cl2MDP-LIP does not cross the blood–brain barrier (and presumably the blood–retinal barrier) until it is damaged by an inflammatory response. 14 Therefore, we administered IV Cl2MDP-LIP before laser injury and/or after laser injury, because the latter but not the former would permit the drug access to resident macrophages in the retina. We also correlated the macrophage response to laser injury with the level of vascular endothelial growth factor (VEGF), which is operative in the development of CNV 15 16 17 18 19 20 21 to deduce a mechanism for the effect of macrophage depletion in this process. 
Methods
Animals
All animal experiments were in accordance with the guidelines of the University of Kentucky IACUC and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Male wild-type C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) between 6 and 8 weeks of age were used to minimize variability, because age 22 and sex (Tanemura M, et al. IOVS 2001;42:ARVO Abstract 530) can influence susceptibility to CNV. For all procedures, anesthesia was achieved by intramuscular injection of 50 mg/kg ketamine HCl (Fort Dodge Animal Health, Fort Dodge, IA) and 10 mg/kg xylazine (Phoenix Scientific, St. Joseph, MO), and pupils were dilated with topical 1% tropicamide (Alcon, Fort Worth, TX). 
Induction of CNV
Laser photocoagulation (532 nm, 200 mW, 100 ms, 75 μm; OcuLight GL, Iridex, Mountain View, CA) was performed on both eyes of each animal on day 0 by a single individual masked to drug group assignment. Laser spots were applied in a standardized fashion around the optic nerve, using a slit lamp delivery system and a coverslip as a contact lens. The morphologic end point of the laser injury was the appearance of a cavitation bubble, a sign thought to correlate with the disruption of Bruch’s membrane. 
Liposomes
Clodronate (dichloromethylene diphosphonate; Cl2MDP) was a gift of Roche Diagnostics GmbH, Mannheim, Germany. Clodronate-liposomes (Cl2MDP-LIP) were prepared 11 as follows. In short, 86 mg phosphatidylcholine (Lipoid EPC; Lipoid, Ludwigshafen, Germany) and 8 mg cholesterol (Sigma-Aldrich, St. Louis, MO) were combined with 10 mL of a clodronate (0.7 M) solution and sonicated gently. The resultant liposomes were then washed to eliminate free drug. Empty liposomes were prepared under the same conditions with phosphate-buffered saline (PBS; Invitrogen/Gibco, Grand Island, NY) instead of the clodronate solution. Animals received 200 μL Cl2MDP-LIP or PBS-LIP through the tail vein with a 30-gauge needle on day −2 (group 1), day 0 (immediately after laser injury; group 2), day –2 and day 0 (group 3), or day +2 (group 4). At these same time points, animals received 10 μL Cl2MDP-LIP in one eye and 10 μL PBS-LIP in the other injected into the subconjunctival space with a syringe (Hamilton, Reno, NV). Injections were performed in a masked fashion. 
Fluorescein Angiography
Fluorescein angiography was performed with a camera and imaging system (TRC 50 IA camera; ImageNet 2.01 system; Topcon, Paramus, NJ) at 1 week after laser photocoagulation. The photographs were captured with a 20-D lens in contact with the fundus camera lens after intraperitoneal injection of 0.1 mL of 2.5% fluorescein sodium (Akorn, Decatur, IL). A retina specialist not involved in the laser photocoagulation or angiography evaluated the fluorescein angiograms at a single sitting in masked fashion. 
Volume of CNV
One week after laser injury, eyes were enucleated and fixed with 4% paraformaldehyde for 30 minutes at 4°C. Eye cups obtained by removing anterior segments were washed three times in PBS, followed by dehydration and rehydration through a methanol series. After blocking twice with buffer (PBS containing 1% bovine serum albumin (BSA; Sigma-Aldrich) and 0.5% Triton X-100 (Sigma-Aldrich) for 30 minutes at room temperature, eye cups were incubated overnight at 4°C with 0.5% FITC-isolectin B4 (Vector Laboratories, Burlingame, CA), which binds to terminal β-d-galactose residues on the surface of endothelial cells and selectively labels the murine vasculature, diluted with PBS containing 0.2% BSA and 0.1% Triton X-100. After two washings with PBS containing 0.1% Triton X-100, the neurosensory retina was gently detached and severed from the optic nerve. Four relaxing radial incisions were made, and the remaining RPE-choroid-sclera complex was flatmounted in antifade medium (Immu-Mount Vectashield Mounting Medium; Vector Laboratories) and coverslipped. 
Flatmounts were examined with a scanning laser confocal microscope (TCS SP; Leica, Heidelberg, Germany). Vessels were visualized by exciting with blue argon laser wavelength (488 nm) and capturing emission between 515 to 545 nm. A 40× oil-immersion objective was used for all imaging studies. Horizontal optical sections (1 μm step) were obtained from the surface of the RPE-choroid-sclera complex. The deepest focal plane in which the surrounding choroidal vascular network connecting to the lesion could be identified was judged to be the floor of the lesion. Any vessel in the laser treated area and superficial to this reference plane was judged as CNV. Images of each section were digitally stored. The area of CNV-related fluorescence was measured by computerized image analysis with the microscope software (TCS SP; Leica). The summation of whole fluorescent area in each horizontal section was used as an index for the volume of CNV. Imaging was performed by an operator masked to treatment group assignment. 
Immunostaining
At various times during the first week after laser injury, animals were injected with 1 mL FITC-isolectin B4 through the tail vein, and retinal-RPE-choroid-scleral or RPE-choroid-scleral flatmounts were prepared 30 minutes later. These were stained with antibodies against F4/80 (5 μg/mL; Serotec, Oxford, UK), expressed by retinal microglia and all mouse macrophages save those in lymphoid organs 23 or leukocyte common antigen CD45.2 (5 μg/mL; eBioscience, San Diego, CA), which also identifies retinal microglia. 24 Flatmounts were examined by scanning laser confocal microscopy. An optical density plot of the selected area was generated by a histogram graphing tool in the image-analysis software (Photoshop, ver. 6.0; Adobe Systems, Mountain View, CA) to obtain a quantitative index of macrophage numbers, as described previously. 25 26 Image analysis was performed by an operator masked to treatment group assignment. 
VEGF ELISA
At 3 days after injury by 12 laser spots, the RPE-choroid complex was sonicated in lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCl2, 10 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na molybdate, and 1 mM EDTA with protease inhibitor; Sigma-Aldrich) on ice for 15 minutes. VEGF protein levels in the supernatant were determined by an ELISA kit (threshold of detection 3 pg/mL; R&D Systems, Minneapolis, MN) that recognizes all splice variants, at 450 to 570 nm (Emax; Molecular Devices, Sunnyvale, CA), and normalized to total protein (Bio-Rad, Hercules, CA). Duplicate measurements were performed in a masked fashion by an operator not involved in photocoagulation, imaging, or fluorescein angiography. 
Statistics
Volume of CNV.
The dependent variable in the analysis was the average lesion volume per eye per mouse. A linear mixed model for a split plot design was constructed with two whole plot (between mice) factors. These are treatments (i.e., timing of the treatments relative to the laser injury) and intravenous (IV) administration (Cl2MDP-LIP or PBS-LIP). The split plot (within mice) factor was subcutaneous (SC) administration (Cl2MDP-LIP or PBS-LIP). Post hoc comparison of means consisted of either a pair-wise comparison of means or a contrast among the means constructed with error terms, depending on whether the contrast was between or within mice, respectively. Because the variability among mice treated with IV Cl2MDP-LIP differed substantially from the variability among mice treated with IV PBS-LIP, the linear mixed model contained different variance components for these groups. Statistical significance was determined at the 0.05 level. 
F4/80 and VEGF.
Quantitative immunostaining and VEGF protein data were analyzed by ANOVA with the Dunnett multiple comparison test. Results were considered significant at P < 0.05. 
Results
Macrophages invaded the site of laser injury within 1 day, with a peak response at 3 days, followed by rapid disappearance by 5 to 7 days (Fig. 1A) . IV Cl2MDP-LIP administration nearly abolished macrophage recruitment, whereas SC Cl2MDP-LIP blunted the macrophage response when administered with IV PBS-LIP, but did not confer added benefit to IV Cl2MDP-LIP (Fig. 1B) . The peak macrophage number paralleled the maximal amount of VEGF protein detected in the laser lesion at 3 days (r 2 = 0.988; Fig. 1C ) and the volume of CNV at 7 days (r 2 = 0.986). 
Confocal planar analysis revealed marked spatial colocalization of macrophages and endothelial cells at the site of laser injury (Fig. 2) . In addition, IV Cl2MDP-LIP, and SC Cl2MDP-LIP to a lesser extent, decreased both the peak number of macrophages and endothelial cell coverage in parallel. We also observed that, whereas macrophages were found in areas without endothelial cells, the converse was rarely the case, supporting the notion that temporally macrophages precede and promote endothelial cell proliferation. During the first 3 days after laser injury, macrophages were concentrated in the choroidal base and central substance of the CNV lesion, but were sparse near its retinal apex (Fig. 3) . Also, there was no difference in the density of macrophages and retinal microglia in the retina adjacent to the laser scar compared with the remainder of the retina, using either F4/80 (Fig. 3C) or CD45.2 (similar data not shown). 
The stereotypical CNV response to laser injury was markedly inhibited by IV Cl2MDP-LIP and to a lesser extent by SC Cl2MDP-LIP (Fig. 4) . Combined IV and SC Cl2MDP-LIP treatment 2 days before laser injury decreased CNV volume by 90.8% ± 3.1% (P < 0.0001), immediately after laser injury by 79.2% ± 11.1% (P < 0.0001), and before and immediately after laser injury by 91.7% ± 4.7% (P < 0.0001), compared with combined IV and SC PBS-LIP treatment (Fig. 5) . However, when IV and SC Cl2MDP-LIP were administered 2 days after laser injury CNV volume was not significantly reduced (P = 0.11). IV Cl2MDP-LIP treatment reduced CNV volumes in eyes treated with SC Cl2MDP-LIP or SC PBS-LIP. When coadministered with IV PBS-LIP, SC Cl2MDP-LIP treatment 2 days before laser injury decreased CNV volumes by 24.5% ± 7.2% (P = 0.05), immediately after laser injury by 27.8% ± 8.2% (P = 0.05), and before and immediately after laser injury by 35.5% ± 7.9% (P = 0.007), compared with SC PBS-LIP; however, SC Cl2MDP-LIP did not augment the antiangiogenic effect of IV Cl2MDP-LIP. 
Pair-wise comparison of the different groups (timing of administration) revealed that IV Cl2MDP-LIP was more effective when administered 2 days before laser injury than immediately after (P = 0.05) or 2 days after (P < 0.0001; Fig. 5E ). Administering IV Cl2MDP-LIP immediately after laser injury did not provide added benefit when it was administered 2 days before as well (P = 0.83). When SC Cl2MDP-LIP was combined with IV Cl2MDP-LIP, drug treatment 2 days before was no more effective than immediately after (P = 0.20), but both were more effective than 2 days after (P < 0.0001). 
At 1 week after laser photocoagulation, fewer lesions in Cl2MDP-treated animals exhibited fluorescein leakage (Fig. 6) . Greater suppression of angiographic leakage was found when Cl2MDP-LIP was administered both before and immediately after laser injury. 
Discussion
To our knowledge, this study is the first to demonstrate that macrophage depletion by Cl2MDP-LIP inhibits the development of laser-induced CNV, validating our hypothesis that macrophages play a pivotal role in this process. Cl2MDP-LIP decreased the peak macrophage response in parallel with VEGF protein levels and total CNV volume. Cl2MDP-LIP administered before and/or immediately after laser injury inhibited CNV, whereas it did not exhibit any effect when administered 2 days after laser injury. This is presumably because macrophage depletion occurs roughly 24 hours after Cl2MDP-LIP exposure, by which time the peak macrophage response at the site of laser injury has occurred. These data show that macrophages, which previous histopathological studies of experimental and clinical CNV have shown to be closely associated with new vessels, play a causal not a coincidental role in the development of laser-induced CNV. 
IV Cl2MDP-LIP–induced inhibition of CNV was not augmented by SC Cl2MDP-LIP, whereas the latter was observed to inhibit CNV partially when coadministered with IV PBS-LIP. The total volume of SC Cl2MDP-LIP administered to any single animal did not exceed 20 μL, which is insufficient to deplete splenic or hepatic macrophages. 13 In a model of experimental autoimmune pigment-epithelial uveitis (EAPU), IV Cl2MDP-LIP, but not SC Cl2MDP-LIP, inhibited EAPU, 27 suggesting that SC delivery of liposomes does not exert a systemic effect. Therefore, the observed beneficial effect of SC Cl2MDP-LIP on CNV may be attributed to depletion of regional lymph node macrophages. In aggregate, these observations suggest that the predominant pool of macrophages that infiltrate areas of laser-induced CNV is derived from the systemic circulation, although submandibular nodes make a minor contribution. 
The origin of macrophages observed after laser injury has been the subject of much inquiry. 4 28 29 30 We found that administering IV Cl2MDP-LIP immediately after laser injury, which provides access to resident macrophages, did not augment the inhibition of CNV induced by IV Cl2MDP-LIP 2 days before injury. We also found no infiltration of macrophages and microglia in the retina adjacent to the laser scar. Our data provide direct anatomic and functional evidence that circulating rather than resident macrophages are the primary culprit in laser-induced CNV. This is consistent with the in vitro finding of polarized secretion of macrophage chemoattractant protein (MCP)-1 from the RPE into the choroid rather than the retina 31 and the in vivo finding of MCP-1 in RPE and choroid, but not in the retina, of eyes with AMD. 32  
A consensus has yet to emerge in quantifying experimental CNV: Both anatomic and functional metrics have been used. The former include measuring thickness and area on serial sections or volumes by confocal microscopy on RPE-choroidal flatmounts, aided by an endothelial cell marker. En masse volumetric measurements are less susceptible to nonorthogonality and loss or poor quality of sections than serial sectioning. Fluorescein angiography, which correlates with visual acuity in patients with AMD 33 34 35 and also permits longitudinal evaluation of the evolution of the laser lesions unlike histopathological examination, reflects on the leakage of these lesions, which presumably correlates with their activity. We used both anatomic and functional metrics of measuring CNV to corroborate our findings: Cl2MDP-LIP inhibited both the anatomic volume and the angiographic leakage of laser-induced CNV. 
We have shown that the leukocyte adhesion molecules CD18 and intercellular adhesion molecule (ICAM)-1 play a key role in laser-induced CNV. 36 Because liposomes do not interfere with leukocyte adhesion 37 or rolling 38 and PBS-LIP did not inhibit CNV, the antiangiogenic effects of Cl2MDP-LIP can be attributed to depletion of macrophages alone. We infer therefore that the paracrine signals produced by macrophages promote the development of CNV. A likely signaling candidate is VEGF, as its levels were suppressed by Cl2MDP-LIP in tandem with the number of macrophages, particularly because VEGF has been shown to be operative in CNV. 15 16 17 18 19 20 21  
We postulate that Cl2MDP-LIP aborted the early-phase response to laser injury, mediated by macrophage migration, perhaps in response to overexpression of MCP-1, a stereotyped wounding response 39 that also occurs in RPE cells of AMD eyes (Spandau U, et al. IOVS 2000;41:ARVO Abstract 4440). 32 In support of this hypothesis, we have demonstrated that genetic ablation of MCP-1 or its cognate receptor CCR2 markedly inhibits laser-induced CNV (data not shown). However, the inhibition of CNV volume in MCP-1– or CCR2-deficient mice (∼75%) did not match the near abolition induced by maximal Cl2MDP-LIP treatment, probably due to depletion by Cl2MDP-LIP of macrophages responsive not only to MCP-1 but to other chemokines, such as macrophage inflammatory proteins-1α and -β, that may play minor roles in recruiting macrophages. 
Administering Cl2MDP-LIP before or immediately after injury sharply reduced the number of macrophages in the site of laser injury, preventing the paracrine effects of these cells on endothelial cell migration and proliferation. VEGF, a major product of activated macrophages was reduced in parallel with the decrease in the number of macrophages. Laser photocoagulation leads to VEGF production by RPE cells, 40 predominantly on the choroidal side, 41 which itself can act as a macrophage chemoattractant. 42 43 However, because VEGF was reduced by Cl2MDP-LIP, it seems that macrophages contribute perhaps more toward upregulation of VEGF than RPE or that RPE secretion of VEGF may be induced, in part, by macrophage–RPE interaction. Through their own VEGF release 44 macrophages can amplify the local VEGF response. Also macrophage-derived cytokines can stimulate VEGF production in RPE cells 45 and choroidal fibroblasts. 46 Macrophages can perpetuate their ingress by stimulating RPE cells to secrete MCP-1 into the choroid in a polarized gradient. 31 In addition to VEGF, macrophages also may produce matrix metalloproteinases (MMPs) directly 47 or through VEGF, which induces MMP expression in endothelial cells, 48 These MMPs, which have been found in CNV in AMD, 49 can facilitate endothelial cell migration during angiogenesis. 
These findings may have some relevance to CNV in AMD, for although the laser injury model may involve processes not relevant to AMD, it captures many of the important features of the human condition. Laser photocoagulation that disrupts Bruch’s membrane can induce CNV in humans. 50 Both in experimental models and in AMD, newly formed vessels that are functionally incompetent 51 52 project into the subretinal space through defects in Bruch’s membrane. Aggregation of leukocytes near arborizing neovascular tufts 3 4 45 is another shared feature of experimental and clinical CNV. Immunostaining has demonstrated the presence of VEGF and its receptors, 40 53 basic fibroblast growth factor, 54 55 transforming growth factor-β, 54 56 tumor necrosis factor-α, 45 Fas, and Fas-ligand 57 58 in cells of the CNV membranes in both conditions. 
Because angiogenesis is a complex process with multiple redundant and intertwined cascades, it is remarkable that macrophage depletion alone nearly abolished CNV. This suggests, at least in this model of CNV, that macrophages and cytokines derived from them are requisite in this process and buttresses the growing body of evidence implicating leukocytes in the initiation of angiogenesis. Although macrophage inactivation could lead to immunosuppression, no overt infection was observed in our study involving transient macrophage depletion or by other investigators. 9 11 Although the clinical implications of transient, partial depletion of macrophages with Cl2MDP-LIP will be apparent only in human trials, MCP-1 or CCR2 may be attractive molecular targets, particularly with local drug delivery. 59  
 
Figure 1.
 
Cl2MDP-LIP inhibited macrophage recruitment and VEGF protein expression after laser injury. (A) F4/80-positive macrophages were detected in the RPE-choroid of laser lesions within 1 day after laser injury and peaked in number at day 3. The index was normalized to peak response. *P < 0.01 and †P < 0.001 compared with day 0. (B) IV Cl2MDP-LIP nearly abolished the peak macrophage response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited macrophage recruitment when combined with IV PBS-LIP treatment but conferred no benefit when added to IV Cl2MDP-LIP. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. (C) VEGF protein expression peaked at day 3 (data not shown). IV Cl2MDP-LIP inhibited the VEGF protein expression response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited VEGF levels when combined with IV PBS treatment but confered no benefit when added to IV Cl2MDP-LIP. Data are expressed as the mean ± SEM. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. n = 5 per group.
Figure 1.
 
Cl2MDP-LIP inhibited macrophage recruitment and VEGF protein expression after laser injury. (A) F4/80-positive macrophages were detected in the RPE-choroid of laser lesions within 1 day after laser injury and peaked in number at day 3. The index was normalized to peak response. *P < 0.01 and †P < 0.001 compared with day 0. (B) IV Cl2MDP-LIP nearly abolished the peak macrophage response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited macrophage recruitment when combined with IV PBS-LIP treatment but conferred no benefit when added to IV Cl2MDP-LIP. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. (C) VEGF protein expression peaked at day 3 (data not shown). IV Cl2MDP-LIP inhibited the VEGF protein expression response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited VEGF levels when combined with IV PBS treatment but confered no benefit when added to IV Cl2MDP-LIP. Data are expressed as the mean ± SEM. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. n = 5 per group.
Figure 2.
 
Macrophages recruited after laser injury colocalized with endothelial cells, and both responses were inhibited by Cl2MDP-LIP. (A) Three days after laser injury in an animal treated with IV PBS-LIP, macrophages (arrows) stained by Cy5-F4/80 (blue) colocalized (arrowheads) with endothelial cells stained by FITC-Isolectin B4 (green). Colocalization by merging yielded a cyan color. (B) SC Cl2MDP-LIP partially inhibited the number of macrophages (arrows) and CNV volume. (C) IV Cl2MDP-LIP nearly abolished macrophage (arrows) and CNV response. The 1-μm sections with the greatest density of F4/80+ staining within laser scars are shown. Scale bar, 50 μm.
Figure 2.
 
Macrophages recruited after laser injury colocalized with endothelial cells, and both responses were inhibited by Cl2MDP-LIP. (A) Three days after laser injury in an animal treated with IV PBS-LIP, macrophages (arrows) stained by Cy5-F4/80 (blue) colocalized (arrowheads) with endothelial cells stained by FITC-Isolectin B4 (green). Colocalization by merging yielded a cyan color. (B) SC Cl2MDP-LIP partially inhibited the number of macrophages (arrows) and CNV volume. (C) IV Cl2MDP-LIP nearly abolished macrophage (arrows) and CNV response. The 1-μm sections with the greatest density of F4/80+ staining within laser scars are shown. Scale bar, 50 μm.
Figure 3.
 
Macrophages in CNV were not recruited from the resident retinal population. (A) Three days after laser injury, numerous macrophages (arrow) stained by Cy5-F4/80 (blue) were present near the choroidal base of the CNV lesion (endothelial cells stained by FITC-Isolectin B4 appear green ⋆). (B) The highest density of macrophages (arrow), many of which colocalized with endothelial cells (⋆) (merge yields cyan color; arrowheads) was present in the middle of the CNV lesion. (C) A paucity of macrophages were found at the retinal surface of the CNV lesion (perimeter outlined in white) and in the adjacent retina (R). One-micrometer-thick sections are shown. Scale bar, 50 μm.
Figure 3.
 
Macrophages in CNV were not recruited from the resident retinal population. (A) Three days after laser injury, numerous macrophages (arrow) stained by Cy5-F4/80 (blue) were present near the choroidal base of the CNV lesion (endothelial cells stained by FITC-Isolectin B4 appear green ⋆). (B) The highest density of macrophages (arrow), many of which colocalized with endothelial cells (⋆) (merge yields cyan color; arrowheads) was present in the middle of the CNV lesion. (C) A paucity of macrophages were found at the retinal surface of the CNV lesion (perimeter outlined in white) and in the adjacent retina (R). One-micrometer-thick sections are shown. Scale bar, 50 μm.
Figure 4.
 
Cl2MDP-LIP inhibited CNV 1 week after laser injury. IV Cl2MDP-LIP administered 2 days before and immediately after laser injury suppressed CNV volume (A) to a greater degree than when administered 2 days after laser injury (B). SC Cl2MDP-LIP administered 2 days before and immediately after laser injury in the presence of IV PBS-LIP partially inhibited CNV volume (C) compared with IV and SC PBS-LIP treatments at the same times (D). Stacked confocal images (1 μm sections) of FITC-isolectin B4 labeled tissue within laser scars are shown. Scale bar, 100 μm.
Figure 4.
 
Cl2MDP-LIP inhibited CNV 1 week after laser injury. IV Cl2MDP-LIP administered 2 days before and immediately after laser injury suppressed CNV volume (A) to a greater degree than when administered 2 days after laser injury (B). SC Cl2MDP-LIP administered 2 days before and immediately after laser injury in the presence of IV PBS-LIP partially inhibited CNV volume (C) compared with IV and SC PBS-LIP treatments at the same times (D). Stacked confocal images (1 μm sections) of FITC-isolectin B4 labeled tissue within laser scars are shown. Scale bar, 100 μm.
Figure 5.
 
CNV volume was markedly diminished in Cl2MDP-LIP–treated mice 1 week after laser injury. Cl2MDP-LIP administered 2 days before laser injury (A), 2 days before and immediately after laser injury (B), and immediately after laser injury (C) demonstrated potent inhibition of CNV volume when Cl2MDP-LIP was administered IV and mild inhibition when it was administered SC. SC Cl2MDP-LIP did not provide added inhibition when administered with IV Cl2MDP-LIP, but provided moderate inhibition when coadministered with IV PBS-LIP. Neither route of administration provided significant inhibition when administered 2 days after laser injury (D). (E) Pair-wise comparison of CNV volumes between group 1 (treatment 2 days before), group 2 (2 days before and immediately after), group 3 (immediately after), and group 4 (2 days after) are presented. IV Cl2MDP-LIP was more effective when administered 2 days before laser injury (group 1 or 2) than immediately after (group 3) or 2 days after (group 4). When SC Cl2MDP-LIP was combined with IV Cl2MDP-LIP, drug treatments 2 days before (group 1 or 2) or immediately after (group 3) were more effective than at 2 days after (group 4). Administering IV Cl2MDP-LIP immediately after laser injury did not provide added benefit when it was administered 2 days before, as well (group 1 versus group 2). *P < 0.05 versus group 3, #P < 0.0001 versus group 4. All other differences NS. n = 5 for all groups.
Figure 5.
 
CNV volume was markedly diminished in Cl2MDP-LIP–treated mice 1 week after laser injury. Cl2MDP-LIP administered 2 days before laser injury (A), 2 days before and immediately after laser injury (B), and immediately after laser injury (C) demonstrated potent inhibition of CNV volume when Cl2MDP-LIP was administered IV and mild inhibition when it was administered SC. SC Cl2MDP-LIP did not provide added inhibition when administered with IV Cl2MDP-LIP, but provided moderate inhibition when coadministered with IV PBS-LIP. Neither route of administration provided significant inhibition when administered 2 days after laser injury (D). (E) Pair-wise comparison of CNV volumes between group 1 (treatment 2 days before), group 2 (2 days before and immediately after), group 3 (immediately after), and group 4 (2 days after) are presented. IV Cl2MDP-LIP was more effective when administered 2 days before laser injury (group 1 or 2) than immediately after (group 3) or 2 days after (group 4). When SC Cl2MDP-LIP was combined with IV Cl2MDP-LIP, drug treatments 2 days before (group 1 or 2) or immediately after (group 3) were more effective than at 2 days after (group 4). Administering IV Cl2MDP-LIP immediately after laser injury did not provide added benefit when it was administered 2 days before, as well (group 1 versus group 2). *P < 0.05 versus group 3, #P < 0.0001 versus group 4. All other differences NS. n = 5 for all groups.
Figure 6.
 
Cl2MDP-LIP decreased angiographic leakage of laser-induced CNV. Representative late phase (6–8 minutes) fluorescein angiograms 1 week after laser injury of mice treated with IV PBS-LIP before and immediately after (A), with IV Cl2MDP-LIP 2 days before (B), and with IV Cl2MDP-LIP 2 days before and immediately after (C) laser injury.
Figure 6.
 
Cl2MDP-LIP decreased angiographic leakage of laser-induced CNV. Representative late phase (6–8 minutes) fluorescein angiograms 1 week after laser injury of mice treated with IV PBS-LIP before and immediately after (A), with IV Cl2MDP-LIP 2 days before (B), and with IV Cl2MDP-LIP 2 days before and immediately after (C) laser injury.
The authors thank Robinette King and Guojin Chen for technical assistance; Richard J. Kryscio, University of Kentucky Biostatistics Consulting Unit, for statistical analyses; and Ambati M. Rao, Delwood C. Collins, and P. Andrew Pearson for ongoing support. 
Smith, W, Assink, J, Klein, R, et al (2001) Risk factors for age-related macular degeneration: pooled findings from three continents Ophthalmology 108,697-704 [CrossRef] [PubMed]
. Macular Photocoagulation Study Group (1991) Argon laser photocoagulation for neovascular maculopathy: five-year results from randomized clinical trials Arch Ophthalmol 109,1109-1114 [CrossRef] [PubMed]
Grossniklaus, HE, Cingle, KA, Yoon, YD, Ketkar, N, L’Hernault, N, Brown, S. (2000) Correlation of histologic 2-dimensional reconstruction and confocal scanning laser microscopic imaging of choroidal neovascularization in eyes with age-related maculopathy Arch Ophthalmol 118,625-629 [CrossRef] [PubMed]
Nishimura, T, Goodnight, R, Prendergast, RA, Ryan, SJ. (1990) Activated macrophages in experimental subretinal neovascularization Ophthalmologica 200,39-44 [CrossRef] [PubMed]
Killingsworth, MC, Sarks, JP, Sarks, SH. (1990) Macrophages related to Bruch’s membrane in age-related macular degeneration Eye 4,613-621 [CrossRef] [PubMed]
van der Schaft, TL, Mooy, CM, de Bruijn, WC, de Jong, PT. (1993) Early stages of age-related macular degeneration: an immunofluorescence and electron microscopy study Br J Ophthalmol 77,657-661 [CrossRef] [PubMed]
Naito, M, Nagai, H, Kawano, S, et al (1996) Liposome-encapsulated dichloromethylene diphosphonate induces macrophage apoptosis in vivo and in vitro J Leukoc Biol 60,337-344 [PubMed]
van Rooijen, N, Sanders, A, van den Berg, TK. (1996) Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine J Immunol Methods 193,93-99 [CrossRef] [PubMed]
van Rooijen, N, Sanders, A. (1997) Elimination, blocking, and activation of macrophages: three of a kind? J Leukoc Biol 62,702-709 [PubMed]
van Rooijen, N, Kors, N, Kraal, G. (1989) Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination J Leukoc Biol 45,97-104 [PubMed]
Van Rooijen, N, Sanders, A. (1994) Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications J Immunol Methods 174,83-93 [CrossRef] [PubMed]
Alves-Rosa, F, Stanganelli, C, Cabrera, J, van Rooijen, N, Palermo, MS, Isturiz, MA. (2000) Treatment with liposome-encapsulated clodronate as a new strategic approach in the management of immune thrombocytopenic purpura in a mouse model Blood 96,2834-2840 [PubMed]
Van der Veen, G, Broersma, L, Van Rooijen, N, Van Rij, G, Van der Gaag, R. (1998) Cytotoxic T lymphocytes and antibodies after orthotropic penetrating keratoplasty in rats treated with dichloromethylene diphosphonate encapsulated liposomes Curr Eye Res 17,1018-1026 [CrossRef] [PubMed]
Huitinga, I, Damoiseaux, JG, van Rooijen, N, Dopp, EA, Dijkstra, CD. (1992) Liposome mediated affection of monocytes Immunobiology 185,11-19 [CrossRef] [PubMed]
Cui, JZ, Kimura, H, Spee, C, Thumann, G, Hinton, DR, Ryan, SJ. (2000) Natural history of choroidal neovascularization induced by vascular endothelial growth factor in the primate Graefes Arch Clin Exp Ophthalmol 238,326-333 [CrossRef] [PubMed]
Spilsbury, K, Garrett, KL, Shen, WY, Constable, IJ, Rakoczy, PE. (2000) Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization Am J Pathol 157,135-144 [CrossRef] [PubMed]
Schwesinger, C, Yee, C, Rohan, RM, et al (2001) Intrachoroidal neovascularization in transgenic mice overexpressing vascular endothelial growth factor in the retinal pigment epithelium Am J Pathol 158,1161-1172 [CrossRef] [PubMed]
Baffi, J, Byrnes, G, Chan, CC, Csaky, KG. (2000) Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor Invest Ophthalmol Vis Sci 41,3582-3589 [PubMed]
Honda, M, Sakamoto, T, Ishibashi, T, Inomata, H, Ueno, H. (2000) Experimental subretinal neovascularization is inhibited by adenovirus-mediated soluble VEGF/flt-1 receptor gene transfection: a role of VEGF and possible treatment for SRN in age-related macular degeneration Gene Ther 7,978-985 [CrossRef] [PubMed]
Kwak, N, Okamoto, N, Wood, JM, Campochiaro, PA. (2000) VEGF is major stimulator in model of choroidal neovascularization Invest Ophthalmol Vis Sci 41,3158-3164 [PubMed]
Krzystolik, MG, Afshari, MA, Adamis, AP, et al (2002) Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment Arch Ophthalmol 120,338-346 [CrossRef] [PubMed]
Espinosa-Heidmann, DG, Suner, I, Hernandez, EP, Frazier, WD, Csaky, KG, Cousins, SW. (2002) Age as an independent risk factor for severity of experimental choroidal neovascularization Invest Ophthalmol Vis Sci 43,1567-1573 [PubMed]
Austyn, JM, Gordon, S. (1981) F4/80, a monoclonal antibody directed specifically against the mouse macrophage Eur J Immunol 11,805-815 [CrossRef] [PubMed]
Chen, L, Yang, P, Kijlstra, A. (2002) Distribution, markers, and functions of retinal microglia Ocul Immunol Inflamm 10,27-39 [CrossRef] [PubMed]
Lehr, HA, van der Loos, CM, Teeling, P, Gown, AM. (1999) Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis J Histochem Cytochem 47,119-126 [CrossRef] [PubMed]
Lehr, HA, Mankoff, DA, Corwin, D, Santeusanio, G, Gown, AM. (1997) Application of Photoshop-based image analysis to quantification of hormone receptor expression in breast cancer J Histochem Cytochem 45,1559-1565 [CrossRef] [PubMed]
Broekhuyse, RM, Huitinga, I, Kuhlmann, ED, Rooijen, NV, Winkens, HJ. (1997) Differential effect of macrophage depletion on two forms of experimental uveitis evoked by pigment epithelial membrane protein (EAPU), and by melanin-protein (EMIU) Exp Eye Res 65,841-848 [CrossRef] [PubMed]
Gloor, BP. (1974) On the question of the origin of macrophages in the retina and the vitreous following photocoagulation (autoradiographic investigations by means of 3H-thymidine) Albrecht Von Graefes Arch Klin Exp Ophthalmol 190,183-194 [CrossRef] [PubMed]
Ishikawa, Y, Momoeda, S, Yoshitomi, F. (1983) Origin of macrophage in photocoagulated rabbit retina Jpn J Ophthalmol 27,138-148 [PubMed]
Tso, MO. (1973) Photic maculopathy in rhesus monkey: a light and electron microscopic study Invest Ophthalmol 12,17-34 [PubMed]
Holtkamp, GM, De Vos, AF, Peek, R, Kijlsta, A. (1999) Analysis of the secretion pattern of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta 2 (TGF-beta2) by human retinal pigment epithelial cells Clin Exp Immunol 118,35-40 [CrossRef] [PubMed]
Grossniklaus, HE, Ling, JX, Wallace, TM, et al (2002) Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization Mol Vis 8,119-126 [PubMed]
Miller, JW, Schmidt-Erfurth, U, Sickenberg, M, et al (1999) Photodynamic therapy with verteporfin for choroidal neovascularization caused by age-related macular degeneration: results of a single treatment in a phase 1 and 2 study Arch Ophthalmol 117,1161-1173 [CrossRef] [PubMed]
. Treatment of Age Related Macular Degeneration with Photodynamic Therapy (TAP) Study Group (1999) Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with verteporfin: one-year results of 2 randomized clinical trials: TAP report Arch Ophthalmol 117,1329-1345 [CrossRef] [PubMed]
. Treatment of Age Related Macular Degeneration with Photodynamic Therapy (TAP) Study Group (2001) Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with verteporfin: two-year results of 2 randomized clinical trials: TAP Report 2 Arch Ophthalmol 119,198-207 [PubMed]
Sakurai, E, Taguchi, H, Anand, A, et al (2003) Targeted disruption of the CD18 or ICAM-1 gene inhibits choroidal neovascularization Invest Ophthalmol Vis Sci 44,2743-2749 [CrossRef] [PubMed]
Baatz, H, Puchta, J, Reszka, R, Pleyer, U. (2001) Macrophage depletion prevents leukocyte adhesion and disease induction in experimental melanin-protein induced uveitis Exp Eye Res 73,101-109 [CrossRef] [PubMed]
Malhotra, R, Taylor, NR, Bird, MI. (1996) Anionic phospholipids bind to L-selectin (but not E-selectin) at a site distinct from the carbohydrate-binding site Biochem J 314,297-303 [PubMed]
DiPietro, LA, Polverini, PJ, Rahbe, SM, Kovacs, EJ. (1995) Modulation of JE/MCP-1 expression in dermal wound repair Am J Pathol 146,868-875 [PubMed]
Wada, M, Ogata, N, Otsuji, T, Uyama, M. (1999) Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization Curr Eye Res 18,203-213 [CrossRef] [PubMed]
Blaauwgeers, HG, Holtkamp, GM, Rutten, H, et al (1999) Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris: evidence for a trophic paracrine relation Am J Pathol 155,421-428 [CrossRef] [PubMed]
Clauss, M, Weich, H, Breier, G, et al (1996) The vascular endothelial growth factor receptor Flt-1 mediates biological activities: implications for a functional role of placenta growth factor in monocyte activation and chemotaxis J Biol Chem 271,17629-17634 [CrossRef] [PubMed]
Barleon, B, Sozzani, S, Zhou, D, Weich, HA, Mantovani, A, Marme, D. (1996) Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1 Blood 87,3336-3343 [PubMed]
Harmey, JH, Dimitriadis, E, Kay, E, Redmond, HP, Bouchier-Hayes, D. (1998) Regulation of macrophage production of vascular endothelial growth factor (VEGF) by hypoxia and transforming growth factor beta-1 Ann Surg Oncol 5,271-278 [CrossRef] [PubMed]
Oh, H, Takagi, H, Takagi, C, et al (1999) The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes Invest Ophthalmol Vis Sci 40,1891-1898 [PubMed]
Kvanta, A. (1995) Expression and regulation of vascular endothelial growth factor in choroidal fibroblasts Curr Eye Res 14,1015-1020 [CrossRef] [PubMed]
Banda, MJ, Werb, Z. (1981) Mouse macrophage elastase: purification and characterization as a metalloproteinase Biochem J 193,589-605 [PubMed]
Lamoreaux, WJ, Fitzgerald, ME, Reiner, A, Hasty, KA, Charles, ST. (1998) Vascular endothelial growth factor increases release of gelatinase A and decreases release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro Microvasc Res 55,29-42 [CrossRef] [PubMed]
Steen, B, Sejersen, S, Berglin, L, Seregard, S, Kvanta, A. (1998) Matrix metalloproteinases and metalloproteinase inhibitors in choroidal neovascular membranes Invest Ophthalmol Vis Sci 39,2194-2200 [PubMed]
Francois, J, De Laey, JJ, Cambie, E, Hanssens, M, Victoria-Troncoso, V. (1975) Neovascularization after argon laser photocoagulation of macular lesions Am J Ophthalmol 79,206-210 [CrossRef] [PubMed]
Tobe, T, Ortega, S, Luna, JD, et al (1998) Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model Am J Pathol 153,1641-1646 [CrossRef] [PubMed]
Sarks, SH, Van Driel, D, Maxwell, L, Killingsworth, M. (1980) Softening of drusen and subretinal neovascularization Trans Ophthalmol Soc UK 100,414-422 [PubMed]
Otani, A, Takagi, H, Oh, H, et al (2002) Vascular endothelial growth factor family and receptor expression in human choroidal neovascular membranes Microvasc Res 64,162-169 [CrossRef] [PubMed]
Amin, R, Puklin, JE, Frank, RN. (1994) Growth factor localization in choroidal neovascular membranes of age-related macular degeneration Invest Ophthalmol Vis Sci 35,3178-3188 [PubMed]
Ogata, N, Matsushima, M, Takada, Y, et al (1996) Expression of basic fibroblast growth factor mRNA in developing choroidal neovascularization Curr Eye Res 15,1008-1018 [CrossRef] [PubMed]
Ogata, N, Yamamoto, C, Miyashiro, M, Yamada, H, Matsushima, M, Uyama, M. (1997) Expression of transforming growth factor-β mRNA in experimental choroidal neovascularization Curr Eye Res 16,9-18 [CrossRef] [PubMed]
Hinton, DR, He, S, Lopez, PF. (1998) Apoptosis in surgically excised choroidal neovascular membranes in age-related macular degeneration Arch Ophthalmol 116,203-209 [PubMed]
Kaplan, HJ, Leibole, MA, Tezel, T, Ferguson, TA. (1999) Fas ligand (CD95 ligand) controls angiogenesis beneath the retina Nat Med 5,292-297 [CrossRef] [PubMed]
Ambati, J, Gragoudas, ES, Miller, JW, et al (2000) Transscleral delivery of bioactive protein to the choroid and retina Invest Ophthalmol Vis Sci 41,1186-1191 [PubMed]
Figure 1.
 
Cl2MDP-LIP inhibited macrophage recruitment and VEGF protein expression after laser injury. (A) F4/80-positive macrophages were detected in the RPE-choroid of laser lesions within 1 day after laser injury and peaked in number at day 3. The index was normalized to peak response. *P < 0.01 and †P < 0.001 compared with day 0. (B) IV Cl2MDP-LIP nearly abolished the peak macrophage response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited macrophage recruitment when combined with IV PBS-LIP treatment but conferred no benefit when added to IV Cl2MDP-LIP. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. (C) VEGF protein expression peaked at day 3 (data not shown). IV Cl2MDP-LIP inhibited the VEGF protein expression response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited VEGF levels when combined with IV PBS treatment but confered no benefit when added to IV Cl2MDP-LIP. Data are expressed as the mean ± SEM. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. n = 5 per group.
Figure 1.
 
Cl2MDP-LIP inhibited macrophage recruitment and VEGF protein expression after laser injury. (A) F4/80-positive macrophages were detected in the RPE-choroid of laser lesions within 1 day after laser injury and peaked in number at day 3. The index was normalized to peak response. *P < 0.01 and †P < 0.001 compared with day 0. (B) IV Cl2MDP-LIP nearly abolished the peak macrophage response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited macrophage recruitment when combined with IV PBS-LIP treatment but conferred no benefit when added to IV Cl2MDP-LIP. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. (C) VEGF protein expression peaked at day 3 (data not shown). IV Cl2MDP-LIP inhibited the VEGF protein expression response at day 3 in the RPE-choroid of laser lesions. SC Cl2MDP-LIP partially inhibited VEGF levels when combined with IV PBS treatment but confered no benefit when added to IV Cl2MDP-LIP. Data are expressed as the mean ± SEM. *P < 0.01 and †P < 0.001 compared with no Cl2MDP-LIP (PBS-LIP) treatment. n = 5 per group.
Figure 2.
 
Macrophages recruited after laser injury colocalized with endothelial cells, and both responses were inhibited by Cl2MDP-LIP. (A) Three days after laser injury in an animal treated with IV PBS-LIP, macrophages (arrows) stained by Cy5-F4/80 (blue) colocalized (arrowheads) with endothelial cells stained by FITC-Isolectin B4 (green). Colocalization by merging yielded a cyan color. (B) SC Cl2MDP-LIP partially inhibited the number of macrophages (arrows) and CNV volume. (C) IV Cl2MDP-LIP nearly abolished macrophage (arrows) and CNV response. The 1-μm sections with the greatest density of F4/80+ staining within laser scars are shown. Scale bar, 50 μm.
Figure 2.
 
Macrophages recruited after laser injury colocalized with endothelial cells, and both responses were inhibited by Cl2MDP-LIP. (A) Three days after laser injury in an animal treated with IV PBS-LIP, macrophages (arrows) stained by Cy5-F4/80 (blue) colocalized (arrowheads) with endothelial cells stained by FITC-Isolectin B4 (green). Colocalization by merging yielded a cyan color. (B) SC Cl2MDP-LIP partially inhibited the number of macrophages (arrows) and CNV volume. (C) IV Cl2MDP-LIP nearly abolished macrophage (arrows) and CNV response. The 1-μm sections with the greatest density of F4/80+ staining within laser scars are shown. Scale bar, 50 μm.
Figure 3.
 
Macrophages in CNV were not recruited from the resident retinal population. (A) Three days after laser injury, numerous macrophages (arrow) stained by Cy5-F4/80 (blue) were present near the choroidal base of the CNV lesion (endothelial cells stained by FITC-Isolectin B4 appear green ⋆). (B) The highest density of macrophages (arrow), many of which colocalized with endothelial cells (⋆) (merge yields cyan color; arrowheads) was present in the middle of the CNV lesion. (C) A paucity of macrophages were found at the retinal surface of the CNV lesion (perimeter outlined in white) and in the adjacent retina (R). One-micrometer-thick sections are shown. Scale bar, 50 μm.
Figure 3.
 
Macrophages in CNV were not recruited from the resident retinal population. (A) Three days after laser injury, numerous macrophages (arrow) stained by Cy5-F4/80 (blue) were present near the choroidal base of the CNV lesion (endothelial cells stained by FITC-Isolectin B4 appear green ⋆). (B) The highest density of macrophages (arrow), many of which colocalized with endothelial cells (⋆) (merge yields cyan color; arrowheads) was present in the middle of the CNV lesion. (C) A paucity of macrophages were found at the retinal surface of the CNV lesion (perimeter outlined in white) and in the adjacent retina (R). One-micrometer-thick sections are shown. Scale bar, 50 μm.
Figure 4.
 
Cl2MDP-LIP inhibited CNV 1 week after laser injury. IV Cl2MDP-LIP administered 2 days before and immediately after laser injury suppressed CNV volume (A) to a greater degree than when administered 2 days after laser injury (B). SC Cl2MDP-LIP administered 2 days before and immediately after laser injury in the presence of IV PBS-LIP partially inhibited CNV volume (C) compared with IV and SC PBS-LIP treatments at the same times (D). Stacked confocal images (1 μm sections) of FITC-isolectin B4 labeled tissue within laser scars are shown. Scale bar, 100 μm.
Figure 4.
 
Cl2MDP-LIP inhibited CNV 1 week after laser injury. IV Cl2MDP-LIP administered 2 days before and immediately after laser injury suppressed CNV volume (A) to a greater degree than when administered 2 days after laser injury (B). SC Cl2MDP-LIP administered 2 days before and immediately after laser injury in the presence of IV PBS-LIP partially inhibited CNV volume (C) compared with IV and SC PBS-LIP treatments at the same times (D). Stacked confocal images (1 μm sections) of FITC-isolectin B4 labeled tissue within laser scars are shown. Scale bar, 100 μm.
Figure 5.
 
CNV volume was markedly diminished in Cl2MDP-LIP–treated mice 1 week after laser injury. Cl2MDP-LIP administered 2 days before laser injury (A), 2 days before and immediately after laser injury (B), and immediately after laser injury (C) demonstrated potent inhibition of CNV volume when Cl2MDP-LIP was administered IV and mild inhibition when it was administered SC. SC Cl2MDP-LIP did not provide added inhibition when administered with IV Cl2MDP-LIP, but provided moderate inhibition when coadministered with IV PBS-LIP. Neither route of administration provided significant inhibition when administered 2 days after laser injury (D). (E) Pair-wise comparison of CNV volumes between group 1 (treatment 2 days before), group 2 (2 days before and immediately after), group 3 (immediately after), and group 4 (2 days after) are presented. IV Cl2MDP-LIP was more effective when administered 2 days before laser injury (group 1 or 2) than immediately after (group 3) or 2 days after (group 4). When SC Cl2MDP-LIP was combined with IV Cl2MDP-LIP, drug treatments 2 days before (group 1 or 2) or immediately after (group 3) were more effective than at 2 days after (group 4). Administering IV Cl2MDP-LIP immediately after laser injury did not provide added benefit when it was administered 2 days before, as well (group 1 versus group 2). *P < 0.05 versus group 3, #P < 0.0001 versus group 4. All other differences NS. n = 5 for all groups.
Figure 5.
 
CNV volume was markedly diminished in Cl2MDP-LIP–treated mice 1 week after laser injury. Cl2MDP-LIP administered 2 days before laser injury (A), 2 days before and immediately after laser injury (B), and immediately after laser injury (C) demonstrated potent inhibition of CNV volume when Cl2MDP-LIP was administered IV and mild inhibition when it was administered SC. SC Cl2MDP-LIP did not provide added inhibition when administered with IV Cl2MDP-LIP, but provided moderate inhibition when coadministered with IV PBS-LIP. Neither route of administration provided significant inhibition when administered 2 days after laser injury (D). (E) Pair-wise comparison of CNV volumes between group 1 (treatment 2 days before), group 2 (2 days before and immediately after), group 3 (immediately after), and group 4 (2 days after) are presented. IV Cl2MDP-LIP was more effective when administered 2 days before laser injury (group 1 or 2) than immediately after (group 3) or 2 days after (group 4). When SC Cl2MDP-LIP was combined with IV Cl2MDP-LIP, drug treatments 2 days before (group 1 or 2) or immediately after (group 3) were more effective than at 2 days after (group 4). Administering IV Cl2MDP-LIP immediately after laser injury did not provide added benefit when it was administered 2 days before, as well (group 1 versus group 2). *P < 0.05 versus group 3, #P < 0.0001 versus group 4. All other differences NS. n = 5 for all groups.
Figure 6.
 
Cl2MDP-LIP decreased angiographic leakage of laser-induced CNV. Representative late phase (6–8 minutes) fluorescein angiograms 1 week after laser injury of mice treated with IV PBS-LIP before and immediately after (A), with IV Cl2MDP-LIP 2 days before (B), and with IV Cl2MDP-LIP 2 days before and immediately after (C) laser injury.
Figure 6.
 
Cl2MDP-LIP decreased angiographic leakage of laser-induced CNV. Representative late phase (6–8 minutes) fluorescein angiograms 1 week after laser injury of mice treated with IV PBS-LIP before and immediately after (A), with IV Cl2MDP-LIP 2 days before (B), and with IV Cl2MDP-LIP 2 days before and immediately after (C) laser injury.
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