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* Department of Ophthalmology, Jones Eye Institute, Pat and Willard Walker Eye Research Center, University of Arkansas for Medical Sciences, Little Rock, AR 72205; and
Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202
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Abstract |
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2. 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. |
Introduction |
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Complement is a major component of innate immunity. It plays a key role in the body’s defense against infection and in the modulation of various inflammatory responses (7). The complement cascade can be activated through three distinct pathways–the classical, the alternative, and the lectin pathways and complement activation triggers a sequence of biological reactions (8). The classical pathway can be activated by immune complexes or by substances such a C-reactive protein and the complement components involved include C1, C2, C4, and C3 (7, 8). The alternative pathway provides a rapid, Ab-independent route of C activation and amplification. The alternative pathway directly activates C3 when it interacts with certain activating surfaces (e.g., zymosan, LPS) and involves C3, factor B, factor D, and properdin (7, 8). The activation of the lectin pathway is also independent of immune complex generation and can be achieved by interaction of certain serum lectins, such as mannose-binding protein (MBL), with mannose and N-acetyl glucosamine residues present in abundance in bacterial cell walls (7, 8).
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 the Bruch’s membrane with laser photocoagulation (3, 6). Although this model does not have many 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). Our previous studies using this model demonstrated that local complement activation, specifically the formation of the membrane attack complex (MAC) was central to the development of laser-induced CNV in mice (6). However, the underlying mechanism by which complement is activated remains unknown. The present study was undertaken to define the specific pathway by which complement is activated in laser-induced CNV in the mouse.
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Materials and Methods |
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Male C57BL/6 mice (4–6 wk old; The Jackson Laboratory) were used in this study. C4-deficient mice (C4–/–), C5 deficient (C5–/–, B10.D2o), and their sex and age-matched wild-type (WT) controls were purchased from The Jackson Laboratory. C57BL/6 and B10.D2n mice were used as controls for C4–/– and C5–/– mice, respectively. This study was approved by the Institutional Animal Care and Use Committee of University of Louisville and University of Arkansas for Medical Sciences (UAMS).
Induction of CNV in mice
CNV was induced by laser photocoagulation with the krypton red laser (50-µm spot size; 0.05-s duration; 250 mW) as previously described by us and others (9, 10, 11). Three laser spots were placed in each eye close to the optic nerve.
Measurement of CNV and CNV lesions
Seven days after laser treatment, all animals were perfused with 1 ml of PBS containing 50 mg/ml fluorescein-labeled dextran (FITC-dextran; average molecular mass, 2 x 106; Sigma-Aldrich) and sacrificed. The eyes were harvested and fixed in 10% phosphate-buffered formalin, and retinal pigment epithelium (RPE)-choroid-scleral flat mounts were prepared as previously described (9, 10, 11). RPE-choroid-scleral flat mounts were stained for elastin using a mAb specific for elastin (1.0 mg/ml; 1/200 dilution; Sigma-Aldrich) followed by a Cy3-labeled secondary Ab (1.0 mg/ml; 1/200 dilution; Sigma-Aldrich). The incidence and the 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 (10, 11). The green color in the laser spots is the CNV complex. If the CNV was <3% of the total laser spot area, it was graded as negative while CNV >3% was considered positive.
Small interfering RNA (siRNA) synthesis and administration
siRNA was used to silence C1q and factor B genes in vivo. For each gene, three target sequences were identified at different locations on the mRNA and siRNAs were designed corresponding to those sequences using Dharmacon siDESIGN Center, an online siRNA design program. siRNAs (duplexes of sense and antisense strands) were synthesized at Invitrogen Life Technologies and were 25-nt long dsRNA. For C1q, three target sequences for each 
, , and 
polypeptide were used. Sense and antisense strands of siRNAs in 5'-3' direction were: C1q -chain (GenBank Accession no. NM_007572): C1q--1 (sense): 5'-UUG GCA ACG UGG UUA UCU UUG ACA A-3', (antisense): 5'-UUG UCA AAG AUA ACC ACG UUG CCA A-3'; C1q--2 (sense): 5'-ACC AGG AGA GUC CAU ACC AGA ACC A-3', (antisense): 5'-UGG UUC UGG UAU GGA CUC UCC UGG U-3'; C1q--3 (sense): 5'-CGG CUU CUA UUA CUU CAA CUU CCA A-3', (antisense): 5'-UUG GAA GUU GAA GUA AUA GAA GCC G-3'; C1q--control siRNA (sense): 5'-UUG AAG CGU UGA UCU UUG UAC GCA A-3', (antisense): 5' -UUG CGU ACA AAG AUC AAC GCU UCA A-3'.
C1q -chain (GenBank Accession no. NM_009777): C1q--1 (sense): 5'-UGA UCA CCA ACG CGA ACG AGA ACU A-3', (antisense): 5'-UAG UUC UCG UUC GCG UUG GUG AUC A-3'; C1q--2 (sense): 5'-ACC GAA CCA GGU CAU UCG CUU CGA A-3', (antisense): 5'-UUC GAA GCG AAU GAC CUG GUU CGG U-3'; C1q--3 (sense): 5'-AAA AGG UGA UCA CCA ACG CGA ACG A-3', (antisense): 5'-UCG UUC GCG UUG GUG AUC ACC UUU U-3'; C1q--control siRNA (sense): 5'-UGA ACC AAC GCG AAC GAG AAU CCU A-3', (antisense): 5'-UAG GAU UCU CGU UCG CGU UGG UUC A-3'.
C1q -chain (GenBank accession no. NM_007574): C1q--1 (sense): 5'-ACA GAA GCA CCA GUC GGU AUU CAC A-3', (antisense): 5'-UGU GAA UAC CGA CUG GUG CUU CUG U-3'; C1q--2 (sense): 5'-AAG CAC CAG UCG GUA UUC ACA GUC A-3', (antisense): 5'-UGA CUG UGA AUA CCG ACU GGU GCU U-3'; C1q--3 (sense): 5'-UGG AGG GCC GAU ACA AAC AGA AGC A-3', (antisense): 5'-UGC UUC UGU UUG UAU CGG CCC UCC A-3'; C1q--control siRNA (sense): 5'-ACA AGC ACC AGU CGG UAU UCG AAC A-3', (antisense): 5'-UGU UCG AAU ACC GAC UGG UGC UUG U-3'.
Factor B mRNA (GenBank accession no. M57890): factor B-1 (sense): 5'-GGU GCC UCA CCA ACU UGA UUG AGA A-3', (antisense): 5'-UUC UCA AUC AAG UUG GUG AGG CAC C-3'; factor B-2 (sense): 5'-UGA ACA UCA AUG CCU UAG CUU CCA A-3', (antisense): 5'-UUG GAA GCU AAG GCA UUG AUG UUC A-3'; factor B-3 (sense): 5'-UGU UUU CUA CCA AAU GAU UGA UGA A-3', (antisense): 5'-UUC AUC AAU CAU UUG GUA GAA AAC A-3'; factor B-control siRNA (sense): 5'-GGU ACU CAA CCG UUC GUU AAC GGA A-3', (antisense): 5'-UUC CGU UAA CGA ACG GUU GAG UAC C-3'.
C57BL/6 mice were divided into six groups and received four i.v. injections (at 24-h interval) of siRNA (mixture of C1q or factor B), control siRNA or PBS starting from day of laser treatment (day = 0) via the tail vein using a modified "hydrodynamic transfection method". This procedure takes <10 s. Animals in group 1 received C1q siRNA while group 2 received factor B siRNA. Control siRNA for C1q and factor B were injected at above-mentioned time points in groups 3 and 4, respectively. Control siRNA is a randomly scrambled sequence with minimum or no homology with the siRNA as well as target sequence and is generated by the BLOCK-iT RNAi Designer software (Invitrogen Life Technologies). Additional controls (groups 5 and 6) received PBS injections at the above-mentioned time points.
To confirm the suppression of C1q and Factor B mRNA and protein, naive C57BL/6 mice were given four i.v. injections (24 h apart) of C1q siRNA (n = 5) or factor B siRNA (n = 5). The animals were sacrificed 4 h after the last injection. Eyes were harvested and total RNA and total protein were isolated separately. Assessment of C1q and factor B suppression was done by RT-PCR and Western blot analysis (described below). These experiments were repeated three times with similar results.
Toxicity studies of i.v. siRNA therapy
Liver and spleen harvested at 4 h after the fourth i.v. injection of siRNA were analyzed histologically to assess the toxicity and adverse effect of systemic i.v. administration.
Immunohistochemical studies
RPE-choroid-scleral flat mounts and a polyclonal Ab (raised in rabbit) reactive with rat/mouse C9 were used to stain for mouse MAC. This Ab was provided by Dr. B. P. Morgan (University of Wales College of Medicine, Cardiff, U.K.).
FITC-conjugated anti-rabbit IgG (Sigma-Aldrich) was used as the secondary Ab. Control stains were performed with normal rabbit serum at concentration similar to that of primary Ab. Additional controls consisted of staining by omission of the primary or secondary Ab. The flat mounts were examined with a confocal microscope (LSM510; Zeiss).
RT-PCR analysis
Three laser spots were placed in each eye of mice as described above. Animals from each group (n = 20/each time point) were sacrificed at days 1, 3, 5, and 7 postlaser treatment; RPE-choroid-scleral tissues harvested from the enucleated eyes were pooled separately for each time point, and total RNA was prepared using the SV Total RNA Isolation kit (Promega). Equal amounts of the total RNA (0.1 µg) were used to detect the mRNA levels of -actin, VEGF, TGF-2, factor B, and factor H by RT-PCR using the reagents purchased from Applied Biosystems. The sense and antisense oligonucleotide primers were synthesized at Integrated DNA Technologies, and PCR used 30 cycles. 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 examined by using Quantity one (Bio-Rad). These experiments were repeated three times with similar results. RT-PCR was conducted using the following primers: -actin (746 bp) forward: 5'-GCC ACC AGT TCG CCA TGG ATG A-3', reverse: 5'-GTC AGG CAG CTC ATA GCT CTT C-3'; vascular endothelial growth factor (VEGF) (716 and 512 bp) forward: 5'-GCG GGC TGC CTC GCA GTC-3', reverse: 5'-TCA CCG CCT TGG CTT GTC AC-3'; -fibroblast growth factor (684 bp) forward: 5'-AGC GGC TCT ACT GCA AGA AC-3', reverse: 5'-TCG TTT CAG TGC CAC ATA CC-3'; factor B (578 bp) forward: 5'-GAG TAC TTC GTG CTG ACA GCA G-3', reverse: 5'-GCG GCT TCT CTT GTG AAC AAT G-3'; factor H (480 bp) forward: 5'-TTG GAA TTC TCC TGC CAT TC-3', reverse: 5'ACC TTC CAT CTT TGC ACA CC-3'.
Western blot analysis
RPE-choroid-scleral tissues harvested from different animals (as described above) were pooled separately and the pooled tissue was homogenized and solubilized in ice-cold PBS containing protease inhibitors and total protein concentration was determined (6). After SDS-PAGE on 7.5% (for factor B and H) or 10% (for C1q) linear slab gel, separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. Blots were incubated with IgG fraction of rat anti-mouse C1q (Cell Sciences), IgG fraction of goat anti-human factor B (Quidel), IgG fraction of goat anti-mouse Factor H (Santa Cruz Biotechnology), or monoclonal anti- actin (mouse IgG1; Sigma-Aldrich). Control blots were treated with the same dilution of appropriate rat, goat or mouse IgG isotype control. After washing and incubation with HRP-conjugated secondary Ab, blots were developed using the ECL Western blotting detection system "ECL Plus" (Amersham Biosciences).
Serum reconstitution
Serum reconstitution of C5-deficient mice was performed as described (12). Animals were euthanized. Blood was aspirated from the right ventricle of WT complement-sufficient mice and serum was collected. C5-deficient mice (n = 5) were infused i.v. with 0.5 ml of WT complement-sufficient serum at the following two time points: 16 h before laser treatment and at day 3 postlaser treatment. Sixteen hours after serum transfer, blood was collected from these animals and complement hemolysis (CH50) and alternative hemolysis (AH50) hemolytic assays were performed to confirm that normal complement activity has been restored. A single transfer of serum reconstituted the host for over 72 h. This experiment was repeated three times with similar results.
CH50 and AH50 assay
Anticoagulant-free blood was collected from C5–/– mice reconstituted with WT control serum and WT mice. Serum was isolated and CH50 and AH50 assays were performed using the standard methods described in the literature (13, 14, 15).
Statistics
Differences between groups were evaluated by 
2 test.
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Results |
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We have previously shown that activation of complement, specifically the formation of the MAC, is essential for the development of laser-induced choroidal angiogenesis (CNV) in mice (6). To further confirm the role of complement and MAC in laser-induced CNV, we used C5–/– mice. Our results demonstrated that C5 deficiency had a dramatic effect on the induction and development of laser-induced CNV. CNV was significantly (p < 0.001) inhibited in C5–/– mice (Table I, Fig. 1B) compared with sex- and age-matched WT animals (Table I, Fig. 1A). Interestingly, when the serum complement activity was reconstituted in C5–/– mice, animals developed CNV (91% incidence) after laser treatment (Table I, Fig. 1C). In these animals the neovascular complex stained strongly for MAC (Fig. 1D). No MAC staining was observed in C5–/– mice (Fig. 1E). As determined by CH50 and AH50 hemolytic assays, infusion of C5–/– mice with serum obtained from WT complement-sufficient mice restored the normal complement activity in these animals (data not shown).
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The relative importance of the classical, lectin, and alternative pathways of complement activation as trigger for CNV was investigated by using C4–/– mice as well as mice treated with C1q or factor B siRNA.
CNV in C4–/– mice. Using C4–/– mice, we explored whether the development of CNV required activation via the classical and/or lectin pathway. Our data presented in Fig. 2 and Table I demonstrate that laser photocoagulation induced CNV in both C4–/– mice (incidence, 95%) and WT controls (incidence, 97%). This experiment was repeated four times with similar results. These results suggest that classical and/or lectin pathways are not critical for the development of laser-induced CNV. To further dissect the contribution of the classical and lectin pathways in laser-induced CNV, C57BL/6 mice were treated with C1q siRNA to block the activation via the classical pathway.
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-actin mRNA and protein (Fig. 3). These results demonstrated the specificity of message suppression by C1q siRNA and, based on these results, 50 µg of siRNA was used in subsequent experiments.
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CNV in factor B siRNA-treated mice.
C57BL/6 mice were treated with factor B siRNA to explore the contribution of the alternative pathway in the development of laser-induced CNV. We first determined the optimum dose of factor B siRNA required to inhibit expression of gene encoding factor B within the eye, liver, and spleen. Three doses of factor B siRNA–15, 25 and 50 µg–were injected i.v. via the tail vein in three different groups of mice. For each dose, naive C57BL/6 mice (n = 4) were given four injections (24 h apart) of factor B siRNA. Animals were sacrificed 4 h after the fourth injection and their eyes, liver, and spleen were harvested for RT-PCR and Western blot analysis. We found that injection of 50 µg of factor B siRNA resulted in 95–98% inhibition of factor B transcripts and protein within the eye (Fig. 3), liver, and spleen (data not shown). Similar treatment of naive C57BL/6 mice with control siRNA had no effect on factor B mRNA in the eye (Fig. 3), liver, and spleen (data not shown). Furthermore, siRNA targeted to factor B did not affect the expression of -actin (Fig. 3). These results demonstrated the specificity of message suppression by factor B siRNA. Based on these results, 50 µg of factor B siRNA was used in subsequent experiments.
We then investigated the in vivo silencing effect of siRNA targeting the gene for factor B on CNV. Animals (n = 4/experiment) received four injections of factor B siRNA (50 µg/injection) at day 0 (the day of laser) and on days 1, 2, and 3 postlaser. Factor B siRNA-treated mice had a significant (p < 0.001) decrease in the incidence of CNV compared with control siRNA or PBS-treated mice at day 7 postlaser (Table II, Fig. 4). RPE-choroid-sclera flat mount analysis revealed that the incidence of CNV in control siRNA and PBS-treated animals was 89 and 92%, respectively. However, in vivo inhibition of factor B gene using siRNA markedly reduced (p < 0.001) the development of CNV to 8% (Table II).
Thus, these results suggest that in the mouse model of laser-induced CNV complement is activated via the alternative pathway because the absence of factor B, a key component of the alternative pathway significantly inhibited laser-induced CNV.
Deposition of MAC in CNV complex of factor B siRNA-injected mice
Factor B siRNA (n = 3) and control siRNA (n = 3) injected animals were sacrificed on day 3 postlaser and flat mounts of the CNV complex were stained for MAC. The neovascular complex stained very strongly for MAC in control siRNA-injected C57BL/6 mice (Fig. 5B). In contrast, very weak MAC staining was observed in the laser spots of factor B siRNA-treated mice at this time point (Fig. 5A). No staining was observed in the control sections stained without the primary Ab (data not shown).
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We then studied the relationship between inhibition of factor B gene and the expression of VEGF and TGF- 2 in the laser spots because we have previously reported an important role for these factors in the laser-induced CNV (6). Using RT-PCR, we detected low levels (similar to naive mice) of VEGF (716 and 512 bp) and TGF-2 (684 bp) mRNA at day 1 postlaser in control siRNA-treated C57BL/6 mice (Fig. 6A). In these animals, VEGF and TGF-2 transcripts increased on days 3 and 5, and returned to basal levels (similar to naive mice) on day 7 (Fig. 6A). In contrast, VEGF and TGF-2 mRNA did not change and remained at low basal levels (similar to naive mice) through day 7 in factor B siRNA-treated mice (Fig. 6B). A strong band at 746 bp for -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).
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Laser-treated complement-sufficient mice (n = 5/time point) were sacrificed at days 1, 3, 5, and 7 postlaser and RPE-choroidal-scleral tissue was harvested to determine the expression of factor B and H. Using RT-PCR and Western blot analysis, we detected low basal levels of factor B mRNA and protein in non-laser-treated naive mice (Fig. 6). Factor B transcripts (Fig. 6C) and protein (Fig. 6D) increased on day 1 and remained elevated until day 7 postlaser. On day 1 postlaser, factor H mRNA (Fig. 6C) and protein (Fig. 6D) levels were similar to those observed in naive animals. Interestingly, levels of factor H transcripts (Fig. 6C) and protein (Fig. 6D) were markedly reduced at day 3 postlaser but were elevated on days 5 and 7. A strong band at 746 bp (Fig. 6C) and 42 kDa for -actin (Fig. 6D) indicated equal amounts of RNA and protein, respectively, in each lane. This experiment was repeated three times with similar results.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The importance of complement and its downstream mediator, MAC, was further confirmed by using C5-deficient mice (C5–/–). These mice are unable to form MAC and serve as a very important tool in defining the role of MAC in disease pathogenesis (12). We found that laser-induced CNV was significantly (p < 0.001) inhibited in mice with homozygous deficiency of C5 complement component. To further determine that MAC generation is key to the development of CNV, additional C5-deficient mice were reconstituted with WT serum to restore complement activation and MAC generation. These animals with reconstituted complement activity developed laser-induced CNV, thus confirming the importance of complement activation and MAC formation in laser-induced CNV (6). Although MAC is essential for the development of CNV, the pathway of complement activation responsible for MAC formation in this model is not known (13, 14). We next used mice with a genetic deficiency in C4, as well as siRNA directed against C1q and factor B, to determine precisely which complement pathway(s) is important in the development of laser-induced CNV in mice.
C4 molecule is a component of both the classical and lectin pathways (28) and mice with a homozygous deficiency of complement component C4 have been useful in defining the role of classical and lectin pathways in the pathogenesis of various complement-mediated diseases (29, 30). We found that mice with targeted deletion of C4 gene developed CNV following laser, thus suggesting that classical and/or lectin pathways may not play a key role in the development of laser-induced CNV.
We next used C1q siRNA-treated mice to distinguish between the classical and lectin pathways. The absence of C1q did not inhibit laser-induced CNV. C1q is the first component of the classical pathway and the inhibition of C1q will specifically block the activation of the complement system via the classical pathway (28, 29). Thus, by using C4–/– mice and inhibiting C1q by siRNA, we ruled out a possible role for both the classical and lectin pathways in the development of CNV.
Because development of CNV was C1q and C4 independent, siRNA targeted against factor B were used to evaluate potential role of alternative pathway in the development of laser-induced CNV. Factor B is the key component of the alternative pathway and is necessary for the function and activation of the alternative pathway (14, 28, 29). We observed that the inhibition of the alternative pathway with the use of factor B siRNA almost completely abolished CNV in the model. Furthermore, laser spots in factor B siRNA-treated mice stained weakly for MAC and the levels of angiogenic growth factors (VEGF and TGF-2) remained at low constitutive (similar to naive mice) levels. In contrast, control siRNA-treated animals demonstrated increased deposition of MAC as well as increased production of angiogenic growth factors within 3 days of laser photocoagulation confirming our previous observations (6).
The importance of the alternative pathway activation in laser-induced CNV was further supported by our observation that during the course of CNV in complement-sufficient C57BL/6 mice the expression of factor B was up-regulated while the expression of factor H was down-regulated. Factor B is a component of the alternative pathway and factor H is the major regulator of alternative pathway (28, 29, 30). A specific polymorphism in the factor B and H gene has recently been reported to increase the risk of AMD in humans (31, 32, 33, 34).
Collectively, our results suggest that the formation of MAC via the alternative complement pathway is important for the release of angiogenic growth factors that are critical for generation of CNV complex in laser-induced CNV. Our results further indicate that the alternative pathway activation is due to increased production of a key component–factor B as well as reduced expression of regulatory protein–factor H.
Several reports have shown that complement and complement-related proteins may play an important role in the development of AMD (6, 35, 36, 37, 38). However, none of the reports have shown precisely the role played by complement or which complement activation pathway is involved in the development of AMD (34, 35, 36). Our results clearly indicated that the complement activation via the alternative pathway may play an important role in the development of laser-induced CNV.
To the best of our knowledge, this study is the first to demonstrate that alternative pathway activation, but not the classical or lectin pathway, plays a key role in the development of laser-induced CNV in mouse. Furthermore, in the absence of factor B, the classical and lectin pathways of complement activation will not be affected and continue to protect the body from pathogens (14, 28, 29). Thus, specific blockade of the alternative complement pathway may represent a therapeutically relevant strategy for the inhibition of CNV or wet AMD.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported in part by National Institutes of Health Grants EY014623 and EY13335, Commonwealth of Kentucky Research Challenge Trust Fund and Research to Prevent Blindness (NY), and grants from University of Arkansas for Medical Sciences (Little Rock, AR). 
2 S.K. and P.J. contributed equally to this work.
3 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, No. 523, Little Rock, AR 72205. E-mail address: pbora{at}uams.edu
4 Abbreviations used in this paper: AMD, age-related macular degeneration; CNV, choroidal neovascularization; MAC, membrane attack complex; WT, wild type; RPE, retinal pigment epithelium; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor; CH50, complement hemolysis; AH50, alternative hemolysis.
Received for publication December 19, 2005. Accepted for publication April 21, 2006.
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