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The Journal of Immunology, 2007, 178: 1783-1790.
Copyright © 2007 by The American Association of Immunologists, Inc.

CD59, a Complement Regulatory Protein, Controls Choroidal Neovascularization in a Mouse Model of Wet-Type Age-Related Macular Degeneration1

Nalini S. Bora2,*, Sankaranarayanan Kaliappan*, Purushottam Jha*, Qin Xu*, Baalasubramanian Sivasankar{dagger}, Claire L. Harris{dagger}, B. Paul Morgan{dagger} and Puran S. Bora2,*

* Department of Ophthalmology, Jones Eye Institute, Pat and Willard Walker Eye Research Center, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and {dagger} Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a–/– mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a–/– mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a–/– mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Age-related macular degeneration (AMD)3 is the leading cause of blindness in the Western world in individuals over 55 years of age. Although the nonexudative (dry) form of AMD is more prevalent, catastrophic vision loss is more frequently associated with the exudative (wet) form, specifically from the complication of neovascularization of the choroid (1, 2, 3, 4, 5, 6, 7, 8, 9). Several risk factors have been implicated in the pathogenesis of new choroidal vessel formation (choroidal neovascularization; CNV) in exudative AMD (1, 2, 3, 4, 5, 6, 7, 8, 9).

Although, several attempts have been made in past to establish an animal model that mimics human wet AMD, the studies of the pathogenesis and treatment of CNV associated with AMD have been hampered by lack of an identical animal model. A reliable way to produce CNV in animals is to rupture Bruch’s membrane (BM) with laser photocoagulation (2, 3, 6). Although, this model does not have some features of human AMD, we and others have found that CNV induced in rodents by laser photocoagulation is useful to gain insights into the pathogenesis of new vessel growth from the choroid (3, 5, 6).

Using murine model of laser-induced CNV, we have shown that presence and activation of complement is essential for the development of CNV (6, 9). The complement system plays a central role in the host defense against microorganism and in the modulation of Ag-specific immune responses (10, 11, 12). It is critical for the body to maintain a balance between complement activation and complement inhibition. In rodents, complement activation is tightly regulated by the complement regulatory proteins (CReg) Crry (5I2 Ag), decay-accelerating factor (CD55), and CD59 (13, 14, 15, 16). Crry and decay-accelerating factor control complement activation at the critical step of C3 and C5 convertase formation (13, 14, 15). CD59 protects autologous tissue from damage caused by complement activation by blocking the formation of membrane attack complex (MAC) (16, 17, 18). In mouse, the CD59 gene is duplicated and the two genes termed CD59a and CD59b show differential tissue expression (17, 19). CD59a is a widely distributed protein, whereas CD59b is expressed only in testis (17). Thus, CD59a is the primary regulator of MAC in mice (17).

Recently, we have shown that MAC formation via the alternative pathway is essential for the development of laser-induced CNV in mice (6, 9). However, the relevance of CD59 in this model remains to be determined. This study was undertaken to investigate the in vivo role of CD59a in the development of murine laser-induced CNV.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Male C57BL/6 mice (6–8 wk old) were purchased from The Jackson Laboratory. B6.129-Cd59atm1Bpm (Cd59a–/–) mice were generated as previously described (16), and male Cd59a–/– mice backcrossed for six generations onto a C57BL/6 background were used in this study. This study was approved by the Institutional Animal Care and Use Committee, University of Arkansas for Medical Sciences.

Induction of CNV in mice

CNV was induced by laser photocoagulation in C57BL/6 and Cd59a–/– mice with an argon laser (50-µm spot size, 0.05 s duration, 250 mW) as previously described by us (6, 7, 8, 9). Three laser spots were placed in each eye close to the optic nerve. Production of a vaporization bubble at the time of laser confirmed the rupture of BM. Animals from each group were sacrificed on days 1, 3, 5, and 7 postlaser.

Animals were perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labeled dextran (FITC-dextran, 2 million average m.w.; Sigma-Aldrich) before they were sacrificed. The eyes were harvested and fixed for 1 h in 10% phosphate-buffered formalin, and retinal pigment epithelium (RPE)-choroid-scleral flat mounts were prepared. Briefly, the cornea and lens were removed and the entire retina was carefully dissected from the eyecup. Five radial cuts were made from the edge to the equator, and the eyecup was flat mounted in Aquamount with the sclera facing down. RPE-choroid-scleral flat mounts were stained for elastin using a mAb specific for elastin (1/200 dilution; Sigma-Aldrich) followed by a Cy-3-labeled secondary Ab (1/2000 dilution; Sigma-Aldrich). The incidence and size of CNV were determined by confocal microscopy. The size of the CNV complex was graded by morphometric analysis of the images (Image-Pro Plus) obtained from confocal microscopy (6, 9). The green color in the laser spots is CNV complex. If the CNV was <3% of the total laser spot area, it was graded as negative, whereas CNV >3% was considered positive. These experiments were repeated three times.

RT-PCR analysis

Laser spots were placed in each eye of C57BL/6 mice as described above. Animals (n = 6/each time point) were sacrificed at days 1, 3, 5, and 7 postlaser treatment and the tissue was prepared as described previously (6, 9). Total RNA was prepared using SV total RNA isolation kit (Promega). Equal amounts of the total RNA (0.1 µg) were used to detect the mRNA levels of beta-actin, CD59, VEGF, beta-fibroblast growth factor (FGF), and TGF-beta2 by semiquantitative RT-PCR using the reagents purchased from Applied Biosystems. The sense and antisense oligonucleotide primers were synthesized at Integrated DNA Technologies. Three different cycles, 25, 35 and 40, were used for PCR, and all three cycles gave similar results. The negative controls consisted of omission of RNA or reverse transcriptase from the reaction mixture. PCR products were analyzed on a 1% agarose gel and were quantitated by densitometry, using Quantity One 4.2.0 (Bio-Rad). These experiments were repeated three times with similar results. RT-PCR was conducted using the following primers: beta-actin (746 bp) forward, 5'-GCC ACC AGT TCG CCA TGG ATG A-3' and reverse, 5'-GTC AGG CAG CTC ATA GCT CTT C-3'; CD59 (CD59a-204 bp and CD59b-237 bp) forward, 5'-GAT TCC TGT CTC TAT GCT GTA-3' and reverse, 5'-CAA AAT GGC CAC CAG AAC- 3'; VEGF (716 and 512 bp) forward, 5'-GCG GGC TGC CTC GCA GTC-3' and reverse, 5'-TCA CCG CCT TGG CTT GTC AC-3'; beta-FGF (298 bp) forward, 5'-AGC GGC TCT ACT GCA AGA AC-3' and reverse, 5'-TCG TTT CAG TGC CAC ATA CC-3'; TGF-beta2 (684 bp) forward, -5'-CCA AAG ACT TAA CAT CTC CCA CC-3' and reverse, -5'-GTT CGA TCT TGG GCG TAT TTC-3'.

ELISA

Laser spots were placed in each eye of C57BL/6 mice as described above. Animals (n = 5/each time point) were sacrificed at days 1, 3, 5, and 7 postlaser treatment and the sample for ELISA was prepared as described previously (6). The samples were assayed (in triplicate) for beta-FGF and VEGF proteins using human beta-FGF and mouse VEGF ELISA kits from R&D Systems. The mouse VEGF assay recognizes both 164 and 120 aa residue forms of mouse VEGF. These experiments were repeated three times with similar results.

Western blot analysis

The protein sample for Western blotting was prepared as described previously (6, 9). After SDS-PAGE on 12% linear slab gel, separated proteins were transferred to a polyvinylidene fluoride membrane using a semidry electrophoretic transfer cell (Trans-Blot; Bio-Rad). Blots were treated with anti-mouse CD59a (mCD59.3) raised in Cd59a–/– mice (20) or monoclonal anti-beta actin (mouse IgG1; Sigma-Aldrich). Control blots were treated with the same dilution of appropriate mouse IgG isotype control. After washing and incubation with HRP-conjugated secondary Ab (1/5000 dilution), blots were developed using the ECL Western blot analysis detection system (ECL Plus; Amersham Biosciences). Experiments were repeated three times with similar results.

Immunohistochemical studies

Flat mounts were stained for MAC. A polyclonal Ab (raised in rabbit; 1.0 mg/ml) reactive with rat/mouse C9 was used to stain for mouse MAC at 1/200 dilution. FITC-conjugated anti-rabbit IgG obtained from Sigma-Aldrich was used as the secondary Ab for MAC staining. Control stains were performed with normal rabbit serum at concentrations similar to those of the primary Abs. Additional controls consisted of staining by omission of the primary or secondary Ab. The flat mounts were examined with a confocal microscope (Zeiss LSM510). Experiments were repeated three times with similar results.

Administration of recombinant soluble CD59a-Fc (rsCD59a-Fc)

rsCD59a-Fc was prepared as previously described (20) with the Fc portion modified to a mouse IgG2a. The most effective dose and route of administration was determined in pilot experiments. Three different routes and two different doses (i.v. (50 and 100 µg), i.p. (50 and 100 µg), and intravitreal (25 and 50 µg)) were used. Intraperitoneal (100 µg) and intravitreal (50 µg) routes gave the best results and were used in future experiments. Animals were divided into four groups (n = 5/group). Animals in group 1 received a single injection of rsCD59a-Fc (100 µg/animal) i.p. 24 h before laser treatment while animals in group 2 received a single intravitreal injection of rsCD59a-Fc (50 µg/animal) at this time point. Animals were sacrificed at day 7 postlaser, and RPE-choroid-scleral flat mounts were examined under confocal microscope to determine the incidence and size of CNV complex as described above. Control animals received a similar injection i.p. (group 3) or intravitreal (group 4) of sterile PBS at the above-mentioned time point and were sacrificed as described above. Experiments were repeated three times with similar results.

In some experiments, rsCD59a-Fc-treated mice (n = 3) were bled 24 h after injection for determination of serum complement hemolytic activity. Hemolytic assay was performed as described previously (6). Serum obtained from C57BL/6 (n = 3) mice injected with sterile PBS was used to determine the 100% value for complement-dependent serum hemolytic activity. Experiment was repeated two times with similar results.

Toxicity studies of i.p. rsCD59a-Fc injection

Various organs such as liver, kidney, lung, and spleen harvested at 24 h and day 6 after i.p. injection of rsCD59a-Fc were analyzed histologically to assess the toxicity and adverse effects of systemic i.p. administration.

Statistical analysis

Data were analyzed and compared using Student’s t test, and differences were considered statistically significant with p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Expression of CD59a in CNV lesions

mRNA levels. Using semiquantitative RT-PCR, we detected CD59a mRNA in eye (RPE-choroid-sclera) of naive mice (n = 18) and at 1, 3, 5, and 7 days postlaser treatment in laser-treated animals (n = 18/each time point). These primers will also amplify CD59b mRNA to give a 237-bp product; no such product was detected in choroid-sclera, demonstrating that CD59b mRNA was not expressed. A decline (compared with naive animals) in the levels of CD59a transcripts was observed at day 1 postlaser (Fig. 1, A and B). CD59a transcripts were further down-regulated at day 3 and remained at low levels at day 5 postlaser (Fig. 1, A and B). A strong band at 746 bp for beta-actin indicated equal amounts of RNA in each lane (Fig. 1). No band was seen in the controls without RNA or reverse transcriptase (data not shown).


Figure 1
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  		FIGURE 1. Expression of CD59a during laser-induced CNV (n = 18). RT-PCR products for CD59a (A) in mouse RPE-choroid-sclera at different time points during laser-induced CNV. A strong band at 746 bp for beta-actin indicated equal amounts of RNA in each lane (A). B represents densitometric analysis of PCR product. The intensity of PCR products was quantitated using Quantity One 4.2.0 image analyzer, and the relative intensity was expressed as ratio of the intensity of the CD59a (A) transcripts to those of beta-actin transcripts. Semiquantitative Western blot (C) and densitometric (D) analysis of CD59a protein in mouse RPE-choroid-sclera. The results of densitometric analysis are expressed as ratio of the intensity of CD59a protein (C) bands to those of beta-actin protein bands. CD59a (mRNA and protein) were down-regulated at day 3 and remained at low levels on day 5. N represents naive animals.

 
Protein levels. The expression of CD59a protein during laser-induced CNV was studied by semiquantitative Western blot analysis. Representative immunoblot for CD59a in CNV lesion is shown in Fig. 1C. CD59a was identified in mouse RPE-choroid-scleral tissue obtained from naive (n = 18) and laser-treated animals (n = 18/each time point) as a protein of ~19 kDa (Fig. 1C). A sharp decrease in CD59a protein levels was detected at days 3 and 5 postlaser (Fig. 1, C and D).

Role of CD59a in the development of laser-induced CNV

The importance of CD59a for the induction of CNV after laser photocoagulation was further investigated by using Cd59a–/– mice as well as rsCD59a-Fc fusion protein.

Cd59a–/– mice

Laser-induced CNV. In view of our previous findings of important roles of MAC in the development of laser-induced CNV (6, 9), we induced CNV in Cd59a–/– mice and their wild-type (WT) controls by laser photocoagulation, and the animals were sacrificed at day 1, 3, 5, and 7 after laser treatment. Confocal analyses of RPE-choroid-scleral flat mounts showed that Cd59a–/– mice were more susceptible to laser-induced CNV than the WT controls (Fig. 2). WT strain-matched male mice sacrificed at day 3 (Table I and Fig. 2F) did not develop CNV and developed only mild CNV at day 5 (Fig. 2G). In contrast, Cd59a–/– mice developed mild CNV at day 3 (p < 0.001; Table I and Fig. 2B) and significantly more severe CNV at days 5 (Fig. 2C) and 7 (Fig. 2D) after laser.


Figure 2
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  		FIGURE 2. Laser-induced subretinal neovascular complex in Cd59a–/– (A–D) and WT control (E–H) mice. Mice were anesthetized and perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labeled dextran. The flat mounts stained for elastin by using elastin Ab and Cy3-conjugated secondary Ab were examined by confocal microscope. Confocal micrograph of the neovascular complex shows the new vessels as green, whereas the damaged BM and RPE were stained red for elastin. Cd59a–/– mice developed mild CNV at day 3 (B) and significantly more severe CNV at days 5 (C) and 7 (D) after laser compared with WT strains. Control mice sacrificed at day 3 (F) did not develop CNV and developed only mild CNV at day 5 (G). A and E represent day 1 postlaser.

 

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  		Table I. Laser-induced CNV in Cd59a knockout (Cd59a–/–) mice on day 3 postlasera

 
Deposition of MAC in CNV complex. Flat mounts of the CNV complex were stained for MAC. At day 1, laser spots stained intensely for MAC in Cd59a–/– mice (n = 9; Fig. 3A). In contrast, very weak staining for MAC was noted at this time point in WT controls (n = 9; Fig. 3B). No staining was observed in the control sections stained without the primary Ab (data not shown).


Figure 3
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  		FIGURE 3. Purified IgG fraction of rabbit antiserum to mouse C9 was used as the primary Ab for MAC staining, FITC-conjugated anti-rabbit IgG served as the secondary Ab. Laser spots showed intense staining (green) for MAC in CD59a–/– mice (A), whereas only weak MAC staining was observed in WT controls (B) at day 1 postlaser.

 
These results established that the lack of CD59a leads to more MAC deposition and exacerbation of laser-induced CNV in mice.

rsCD59a-Fc fusion protein

Laser-induced CNV. We then studied the effect of recombinant soluble CD59a fused to the Fc portion of mouse IgG2a on murine laser-induced CNV. A single i.p. or intravitreal injection of CD59a-Fc was given 24 h before laser. Delivery of rsCD59a-Fc 24 h before laser via the i.p. (Fig. 4B) or intravitreal (Fig. 4D) route inhibited the development of CNV. Similar injection of sterile saline did not have any effect on CNV (Fig. 4, A and C). We observed that the incidence of laser-induced CNV in mice injected i.p. with rsCD59a-Fc (13%) was dramatically reduced compared with PBS-injected controls (94%, p < 0.001; Fig. 4 and Table II). However, the incidence of laser-induced CNV in mice injected intravitreally with rsCD59a-Fc was 30% compared with 93% in PBS-injected controls (Table II).


Figure 4
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  		FIGURE 4. rsCD59a-Fc-mediated inhibition of laser-induced CNV. At day 7 postlaser, CNV complex developed in mice treated with PBS i.p. (A) or intravitreally (C). In contrast, neovascular complex was inhibited in mice treated with rsCD59a-Fc i.p. (B) or intravitreally (D). In this confocal micrograph the new vessels appear green, whereas damaged BM and RPE were stained red for elastin.

 

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  		Table II. Effect of rsCD59a-Fc on laser-induced CNVa

 
Inhibition of complement in mice following i.p. injection of rsCD59a-Fc was confirmed by measurement of complement-dependent serum hemolytic activity. rsCD59a-Fc-injected mice demonstrated a marked decrease (~70%) in total hemolytic activity at 24 h postinjection compared with PBS-injected animals (Fig. 5A).


Figure 5
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  		FIGURE 5. Effect of rsCD59a (injected i.p.) on serum complement hemolytic activity (A) and MAC deposition (B and C). rsCD59a-treated animals had significantly (*, p < 0.0001) reduced (~70%) complement hemolytic activity compared with PBS-injected controls. At day 3 postlaser, the flat mounts stained weakly for MAC in C57BL/6 mice treated i.p. with rsCD59-Fc (B), whereas strong MAC staining was observed in control C57BL/6 mice treated with PBS at this time point (C).

 
Liver, kidney and spleen harvested from mice injected i.p. (n = 6) with rsCD59a-Fc were analyzed histologically for adverse effects associated with systemic delivery of this protein. These organs were well preserved and showed no toxic effect of rsCD59a-Fc i.p. injection (data not shown).

Deposition of MAC in CNV complex. Animals injected with rsCD59a-Fc (n = 5) or PBS (n = 5) were sacrificed at day 3 postlaser, and flat mounts of CNV complex were stained for MAC. Very weak staining for MAC was noted in the laser spots of mice injected i.p. (Fig. 5B) or intravitreally (data not shown) with rsCD59a-Fc 24 h before laser. In contrast, intense MAC staining was observed in the CNV complex of animals injected with PBS via i.p. (Fig. 5C) or intravitreal (data not shown) route at this time point.

Expression of growth factors. We then studied the relationship between inhibition of complement by rsCD59a-Fc and the expression of vascular endothelial growth factor (VEGF), TGF (TGF-beta2), and beta-FGF in the laser spots. RT- PCR (VEGF, TGF-beta2, and beta-FGF) and ELISA (VEGF and beta-FGF) were used in our study. Using RT-PCR, we detected low levels (similar to naive mice) of VEGF (716 and 512 bp), beta-FGF (298 bp), and TGF-beta2 (684 bp) mRNA from day 1 through day 7 postlaser in CD59a-Fc-treated mice (Fig. 6A). In contrast, the levels of VEGF, beta-FGF, and TGF-beta2 transcripts increased on days 3 and 5, and returned to basal levels on day 7 in PBS-injected C57BL/6 mice (Fig. 6B). A strong band at 746 bp for beta-actin indicated equal amounts of RNA in each lane (Fig. 6, A and B). No band was seen in the controls without RNA or reverse transcriptase (data not shown).


Figure 6
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  		FIGURE 6. Effect of rsCD59a (injected i.p.) on the expression of growth factors during laser-induced CNV. VEGF, TGFbeta-2, and beta-FGF mRNA expression during laser-induced CNV in rsCD59a (A) and PBS-treated C57BL/6 mice (B). The figures show ethidium bromide-stained bands for PCR product after UV exposure. In the rsCD59a-treated mice, the VEGF, TGFbeta-2, and beta-FGF transcripts remained at the basal constitutive levels (A). However, in the PBS-treated mice, the VEGF, TGFbeta-2, and beta-FGF transcripts were up-regulated on days 3 and 5 (B). A strong band at 746 bp (A and B) indicated equal amounts of RNA in each lane. Effect of rsCD59a (Figure 6) and PBS ({blacksquare}) treatments on VEGF (C) and beta-FGF (D) protein production during laser-induced CNV in C57BL/6 mice. In rsCD59a-treated mice, VEGF and beta-FGF protein levels remained at the basal constitutive levels (C and D). In contrast, VEGF and beta-FGF protein levels were up-regulated on day 3 and 5 in PBS-injected C57BL/6 mice (C and D). N represents naive animals.

 
We next examined the protein levels of VEGF and beta-FGF by ELISA. The levels of VEGF and beta-FGF proteins remained at low levels (similar to naive) at day 1, 3, 5, and 7 in CD59a-Fc-treated C57BL/6 mice (Fig. 6, C and D). However, in control animals injected with PBS, we observed low levels of VEGF and beta-FGF proteins at day 1 with increased levels of VEGF and beta-FGF (p < 0.001) at day 3 and 5 postlaser. VEGF and beta-FGF returned to basal levels at day 7 (Fig. 6, C and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Choroidal angiogenesis (CNV) causing the wet-type AMD is the leading cause of irreversible visual loss in individuals over age 55 (1, 2, 3, 4, 5, 6, 7, 8, 9). We have recently shown that the formation of MAC, as a result of complement activation via the alternative pathway, is essential for the development of laser-induced CNV in mice (6, 9). Although, MAC formation and deposition has been shown by us to have a central role, the expression and role of CReg CD59 in laser-induced CNV has not been investigated. In this study, we used mice deficient in CD59a and a recombinant soluble form of this protein to define the in vivo role of CD59a in laser-induced CNV in C57BL/6 mice.

The change in the expression pattern of CD59a in laser-induced CNV was studied using semiquantitative RT-PCR and Western blot analysis. We observed that CD59a was expressed constitutively in mouse RPE, choroid, and sclera, and the levels of CD59a were down-regulated during laser-induced CNV. These low levels of CD59a may facilitate complement activation leading to increased MAC deposition and induction of choroidal angiogenesis. We have previously reported increased MAC deposition in CNV complex in C57BL/6 mice after laser-photocoagulation (6, 9). Thus, our results suggest that the levels of CRegs are important in determining the degree of complement activation during the development of CNV. We have previously shown that CRegs are present in normal human (21, 22) and rat (23) eye. The decreased level of CRegs has been reported to increase susceptibility to various diseases in humans and rodents (24).

The importance of CD59a was further confirmed by using gene-deleted mice. These animals have provided valuable information in efforts to understand the role of complement and CRegs in vivo (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). We used gene-deleted mice deficient in CD59a (Cd59a–/–) because CD59a is the primary regulator of MAC formation in mice (16, 17). We examined the disease course and severity in mice deficient in CD59a and observed that these mice with targeted deletion of CD59a gene developed the CNV complex early in the disease process and more severe CNV at later time points compared with genetically matched WT controls. C9/MAC deposition was significantly increased in the Cd59a–/– group compared with Cd59+/+ littermate control. Deficiency of CD59a has been reported to enhance disease severity in experimental allergic encephalomyelitis (32), rheumatoid arthritis (33), ischemia perfusion injury (34), and nephrotoxic nephritis (35) in mice. In this study, we have demonstrated for the first time that deficiency of the CD59a gene exacerbates laser-induced CNV in mice by allowing unregulated MAC deposition.

To further analyze the role of CD59a in CNV, we used rsCD59a-Fc to inhibit choroidal angiogenesis. CD59a coupled to mouse IgG Fc yields an efficient homologous complement inhibitor with a long half-life (20). Complement inhibition in vivo can be accomplished by delivery of recombinant CRegs either systemically or directly into the target tissue (36, 37, 38, 39, 40, 41, 42, 43), and recombinant CRegs have been shown to block diseases in various rodent models of human diseases such as experimental allergic encephalomyelitis (38), collagen-induced arthritis (41), renal disease (42), and experimental autoimmune uveoretinitis (43). Inhibition of complement activation in mice following i.p. injection of rsCD59a-Fc was demonstrated by marked decrease in serum hemolytic activity. Following in vivo injection, rsCD59a-Fc inhibited the development of choroidal angiogenesis in the laser-induced mouse model by blocking MAC deposition as well as the induction/release of angiogenic growth factors such as VEGF, beta-FGF, and TGF-beta2. All three growth factors have been shown to play an important role in the development of CNV associated with wet AMD (6, 9). MAC, the final activation product of the complement cascade has been reported to release growth factors including beta-FGF and VEGF from various nucleated cells (44, 45, 46). Thus, these reports along with our results (6, 9) clearly suggest that MAC-mediated release of growth factors plays a crucial role in the pathogenic mechanism of choroidal angiogenesis.

In conclusion, our study is the first direct demonstration of the in vivo role of CD59a in controlling complement activation and MAC formation during laser-induced CNV in mice. Collectively, our data demonstrate that during laser-induced CNV, the absence of CD59a resulted in more MAC formation (due to increased complement activation), with early onset and exacerbation of laser-induced CNV. The data presented in this study further confirm our previous results (6, 9) that implicate MAC as a major mediator of CNV in this murine model of CNV, and provides a rationale for the inhibition of MAC assembly as a therapeutic strategy for CNV.

There is a substantial body of evidence implicating complement in AMD both in humans and in experimental animals (6, 9, 47, 48, 49, 50, 51, 52, 53, 54). Several studies reported in the literature have indicated a potential role for complement in drusen formation in the nonexudative form of AMD in humans (47, 48, 49). C3a, C5a, MAC along with vitronectin have been localized in drusen in patients with AMD (47, 48, 49, 54). Recently, a specific tyrosine to histidine polymorphism at aa 402 in the factor H gene has been reported to increase the risk of AMD in humans (50, 51, 52). These findings along with the work from our laboratory clearly implicate complement system as a major player in the development of AMD.

Currently, several different approaches are being used to treat wet-AMD in humans. Although photodynamic therapy reduces the rate of vision loss in most patients, it does not lead to significant improvement in vision. Furthermore, repeated photodynamic therapy can cause severe damage to the posterior segment of the eye and is not cost effective. Other treatment regimens require the use of antiangiogenic pharmacological agents such Macugen, Avastin, and Lucentis. These drugs also have many side effects including the risk of infection, hemorrhage of the eye membrane, ocular pain, and floaters. Therefore, alternative therapeutic strategies are needed for the better treatment of AMD patients.

Thus, we propose that rsCD59-Fc may have therapeutic potential in humans. Furthermore, we believe that rsCD59-Fc is a better therapeutic agent than those targeting the activation pathways of complement because it does not have the possible untoward complications of complement inhibition using inhibitors that act at the C3 and C5 convertases (55). However, the side effects as well as the efficacy of this potential new drug in humans are not known. The early complement activation products that are important in host response to infection and immune complex catabolism as well as required to facilitate tissue healing will remain intact and their function will not be hindered by this treatment.


   Acknowledgments
 
We thank Dr. Ammar Safar for the help with the use of argon laser and Brian Shank (Digital and Confocal Microscopy Laboratory, University of Arkansas for Medical Sciences, Little Rock, AR) for his help with the use of confocal microscope.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants EY014623 and EY13335, Research to Prevent Blindness, NY, and grants from University of Arkansas for Medical Sciences (Little Rock, AR). The use of the facilities in the University of Arkansas for Medical Sciences Digital and Confocal Microscopy Laboratory was supported by National Institutes of Health Grant 2P20 RR16460 and National Institutes of Health/National Center for Research Resources Grant 1 S10 RR 19395. Back

2 Address correspondence and reprint requests to Dr. Puran S. Bora, Department of Ophthalmology, Jones Eye Institute, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 523, Little Rock, AR 72205-7199; E-mail address: PBora{at}UAMS.edu or Dr. Nalini S. Bora, Department of Ophthalmology, Jones Eye Institute, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 523, Little Rock, AR 72205-7199; E-mail address: NBora{at}UAMS.edu Back

3 Abbreviations used in this paper: AMD, age-related macular degeneration; CNV, choroidal neovascularization; BM, Bruch’s membrane; CReg, complement regulatory protein; MAC, membrane attack complex; RPE, retinal pigment epithelium; FGF, fibroblast growth factor; WT, wild type; VEGF, vascular endothelial growth factor. Back

Received for publication August 9, 2006. Accepted for publication November 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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