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• W2104426232 abstract "Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets. Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets. Age-related macular degeneration (AMD) is a disease that progressively damages the photoreceptors and retinal pigment epithelium (RPE) in the macula, resulting in loss of vision in the center of the visual field. AMD is the leading cause of blindness in the elderly population, affecting approximately 15% of the population older than 70 years of age.1Friedman D.S. O'Colmain B.J. Munoz B. Tomany S.C. McCarty C. de Jong P.T. Nemesure B. Mitchell P. Kempen J. Eye Diseases Prevalence Research GroupPrevalence of age-related macular degeneration in the United States.Arch Ophthalmol. 2004; 122: 564-572Crossref PubMed Scopus (78) Google Scholar, 2Klein R. Peto T. Bird A. Vannewkirk M.R. The epidemiology of age-related macular degeneration.Am J Ophthalmol. 2004; 137: 486-495Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar The earliest clinical signs of AMD are pigmentary changes and the appearance of drusen, focal protein- and lipid-rich extracellular deposits that form between the RPE and the Bruch membrane (BrM). Late-stage AMD is characterized by choroidal neovascularization, geographic atrophy, or both, resulting in irreversible central loss of the choriocapillaris, RPE, and photoreceptors.3Bowes Rickman C. Farsiu S. Toth C.A. Klingeborn M. Dry age-related macular degeneration: mechanisms, therapeutic targets, and imaging.Invest Ophthalmol Vis Sci. 2013; 54: ORSF68-ORSF80Crossref PubMed Scopus (200) Google Scholar, 4Jager R.D. Mieler W.F. Miller J.W. Age-related macular degeneration.N Engl J Med. 2008; 358: 2606-2617Crossref PubMed Scopus (1238) Google Scholar, 5de Jong P.T. Age-related macular degeneration.N Engl J Med. 2006; 355: 1474-1485Crossref PubMed Scopus (726) Google Scholar Abnormalities in dark adaptation are often the earliest functional change detected in patients with AMD and reflect impairment of rod photoreceptor function.6Jackson G.R. Owsley C. McGwin Jr., G. Aging and dark adaptation.Vision Res. 1999; 39: 3975-3982Crossref PubMed Scopus (235) Google Scholar, 7Jackson G.R. McGwin Jr., G. Phillips J.M. Klein R. Owsley C. Impact of aging and age-related maculopathy on activation of the a-wave of the rod-mediated electroretinogram.Invest Ophthalmol Vis Sci. 2004; 45: 3271-3278Crossref PubMed Scopus (38) Google Scholar AMD is a complex disease influenced by a variety of risk factors, including age, which is the greatest.8Age-Related Eye Disease Study Research GroupRisk factors associated with age-related macular degeneration. a case-control study in the age-related eye disease study: Age-Related Eye Disease Study Report Number 3.Ophthalmology. 2000; 107: 2224-2232Abstract Full Text Full Text PDF PubMed Scopus (733) Google Scholar Of the genetic risk factors, a common polymorphism (T1277C) in the complement factor H (CFH) gene, resulting in a tyrosine (Y) to histidine (H) substitution at amino acid 402 (H402), has been found to be strongly associated with the development of AMD.9Edwards A.O. Ritter III, R. Abel K.J. Manning A. Panhuysen C. Farrer L.A. Complement factor H polymorphism and age-related macular degeneration.Science. 2005; 308: 421-424Crossref PubMed Scopus (2100) Google Scholar, 10Hageman G.S. Anderson D.H. Johnson L.V. Hancox L.S. Taiber A.J. Hardisty L.I. Hageman J.L. Stockman H.A. Borchardt J.D. Gehrs K.M. Smith R.J. Silvestri G. Russell S.R. Klaver C.C. Barbazetto I. Chang S. Yannuzzi L.A. Barile G.R. Merriam J.C. Smith R.T. Olsh A.K. Bergeron J. Zernant J. Merriam J.E. Gold B. Dean M. Allikmets R. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration.Proc Natl Acad Sci U S A. 2005; 102: 7227-7232Crossref PubMed Scopus (1714) Google Scholar, 11Haines J.L. Hauser M.A. Schmidt S. Scott W.K. Olson L.M. Gallins P. Spencer K.L. Kwan S.Y. Noureddine M. Gilbert J.R. Schnetz-Boutaud N. Agarwal A. Postel E.A. Pericak-Vance M.A. Complement factor H variant increases the risk of age-related macular degeneration.Science. 2005; 308: 419-421Crossref PubMed Scopus (2091) Google Scholar, 12Klein R.J. Zeiss C. Chew E.Y. Tsai J.Y. Sackler R.S. Haynes C. Henning A.K. SanGiovanni J.P. Mane S.M. Mayne S.T. Bracken M.B. Ferris F.L. Ott J. Barnstable C. Hoh J. Complement factor H polymorphism in age-related macular degeneration.Science. 2005; 308: 385-389Crossref PubMed Scopus (3570) Google Scholar The same polymorphism has also been linked to dense deposit disease (DDD, also known as membranoproliferative glomerulonephritis type II).13Abrera-Abeleda M.A. Nishimura C. Smith J.L. Sethi S. McRae J.L. Murphy B.F. Silvestri G. Skerka C. Jozsi M. Zipfel P.F. Hageman G.S. Smith R.J. Variations in the complement regulatory genes factor H (CFH) and factor H related 5 (CFHR5) are associated with membranoproliferative glomerulonephritis type II (dense deposit disease).J Med Genet. 2006; 43: 582-589Crossref PubMed Scopus (182) Google Scholar, 14Pickering M.C. de Jorge E.G. Martinez-Barricarte R. Recalde S. Garcia-Layana A. Rose K.L. Moss J. Walport M.J. Cook H.T. de Cordoba S.R. Botto M. Spontaneous hemolytic uremic syndrome triggered by complement factor H lacking surface recognition domains.J Exp Med. 2007; 204: 1249-1256Crossref PubMed Scopus (222) Google Scholar DDD is a condition that primarily affects kidney function. The symptoms usually appear between ages 5 and 15 years and include hematuria, proteinuria, and nephrotic syndrome.15Appel G.B. Cook H.T. Hageman G. Jennette J.C. Kashgarian M. Kirschfink M. Lambris J.D. Lanning L. Lutz H.U. Meri S. Rose N.R. Salant D.J. Sethi S. Smith R.J. Smoyer W. Tully H.F. Tully S.P. Walker P. Welsh M. Wurzner R. Zipfel P.F. Membranoproliferative glomerulonephritis type II (dense deposit disease): an update.J Am Soc Nephrol. 2005; 16: 1392-1403Crossref PubMed Scopus (319) Google Scholar, 16Walker P.D. Dense deposit disease: new insights.Curr Opin Nephrol Hypertens. 2007; 16: 204-212Crossref PubMed Scopus (31) Google Scholar CFH is a major regulator of the alternative pathway (AP) of the complement system. The AP is initiated by complement component 3 (C3) hydrolysis in which a C3 thioester interacts with water to form C3(H2O). This occurs spontaneously and causes the AP to be continuously active. Uninhibited, C3(H2O) reacts with complement factor B (FB) to generate C3(H2O)B, which in the presence of complement factor D is converted to the C3 convertase C3(H2O)Bb. This conversion allows for further cleavage of C3 to C3b and formation of the C3bBb convertase and initiates the amplification loop. Without negative regulators, this positive feedback loop will quickly deplete all available C3 in the system. CFH is the soluble negative regulator of AP activation. CFH can inhibit formation of the C3 convertase by competing with FB for binding to C3b, accelerate decay of the C3 convertase, and act as a cofactor for factor I–mediated cleavage of C3b.17Morgan B.P. Harris C.L. Complement Regulatory Proteins. Academic Press, San Diego1999Google Scholar The complement system is important in defending an organism against foreign invasion by lyzing alien pathogens via the formation of membrane attack complex and signaling debris clearance. However, if dysregulated, the complement system can also attack host cells, causing local inflammation and tissue damage.18Holers V.M. The spectrum of complement alternative pathway-mediated diseases.Immunol Rev. 2008; 223: 300-316Crossref PubMed Scopus (177) Google Scholar Available data support the idea that in AMD, the AP is dysregulated owing to CFH malfunction, causing local inflammation and tissue damage in the macular region of the retina. However, the cellular/molecular mechanism(s) for the risk association of CFH in AMD pathogenesis remains unclear. Consistent with a role for CFH in AMD pathogenesis, aged mice lacking CFH (Cfh−/−) have an ocular phenotype that shares characteristics of early AMD. These characteristics include decreased electroretinogram (ERG) response, loss of visual acuity, disrupted photoreceptor organization, and structural abnormalities of the BrM.19Coffey P.J. Gias C. McDermott C.J. Lundh P. Pickering M.C. Sethi C. Bird A. Fitzke F.W. Maass A. Chen L.L. Holder G.E. Luthert P.J. Salt T.E. Moss S.E. Greenwood J. Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction.Proc Natl Acad Sci U S A. 2007; 104: 16651-16656Crossref PubMed Scopus (176) Google Scholar, 20Hoh Kam J. Lenassi E. Malik T.H. Pickering M.C. Jeffery G. Complement component C3 plays a critical role in protecting the aging retina in a murine model of age-related macular degeneration.Am J Pathol. 2013; 183: 480-492Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar These mice also spontaneously develop DDD-like pathologic abnormalities.21Pickering M.C. Cook H.T. Warren J. Bygrave A.E. Moss J. Walport M.J. Botto M. Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H.Nat Genet. 2002; 31: 424-428Crossref PubMed Scopus (420) Google Scholar To further elucidate the function of CFH in AMD pathogenesis, we generated mice in which full-length human CFH is expressed instead of mouse CFH. Using this mouse model, we tested whether i) human CFH interacts with the mouse AP of the complement system, ii) human CFH protein can rescue the ocular and renal phenotype in Cfh−/− mice, and iii) there are phenotypic differences between mice expressing the normal Y402 or AMD risk–associated H variant of CFH. Mice were housed under normal lighting conditions with 12-hour on/off cycles. The care and treatment of animals were strictly in accordance with the Institutional Animal Care and Use Committee at Duke University (Durham, NC). The bacterial artificial chromosome (BAC) clone RP11-347L19 contains a 180-kb insert spanning the entire CFH gene and two truncated flanking genes (KCNT2 on the 5′ side and CFHR3 on the 3′ side). We confirmed by sequencing that the CFH gene codes for an H402 form of CFH. To generate the Y402 variant of the humanized CFH mice we used BAC clone CTD-2580H3, which has a 132-kb insert in the region spanned by the H402 BAC (UCSC Genome Browser). In collaboration with the Duke Neurotransgenic Laboratory, we generated founder transgenic mice from these BAC clones containing the full-length Y402 [Tg(CTD-2580H3)402Cbr, or CFH-Y mice for short] and H402 [Tg(RP11-347L19)301Cbr, or CFH-H mice for short] variants of the human CFH gene. The founders were then crossed to C57Bl/6J (C57) mice (The Jackson Laboratory, Bar Harbor, ME). Germline transmission was confirmed by analyzing the genomic DNA of the offspring. Human CFH-positive N1 littermates were bred to Cfh−/− mice21Pickering M.C. Cook H.T. Warren J. Bygrave A.E. Moss J. Walport M.J. Botto M. Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H.Nat Genet. 2002; 31: 424-428Crossref PubMed Scopus (420) Google Scholar to generate the CFH-Y:Cfh−/− and CFH-H:Cfh−/− mouse lines. Cfh−/− mice on a C57 background were obtained from Dr. Glenn Jaffe (Durham, NC) with an material transfer agreement from Imperial College London and permission from Dr. Marina Botto (London, UK). We maintained these two lines by crossing CFH:Cfh−/− mice with Cfh−/− mice. The Cfh−/− littermates served as controls. Animals used in this study were crossed to Cfh−/− for more than six generations. We also mated CFH-H:Cfh−/− mice together to generate a line of homozygous CFH-H transgenic mice (CFH-HH:Cfh−/−), bearing in mind that this might interrupt an endogenous gene important for normal development.22Meisler M.H. Insertional mutation of ‘classical’ and novel genes in transgenic mice.Trends Genet. 1992; 8: 341-344Abstract Full Text PDF PubMed Scopus (105) Google Scholar We confirmed that none of the mice carry the rd8 mutation.23Mattapallil M.J. Wawrousek E.F. Chan C.C. Zhao H. Roychoudhury J. Ferguson T.A. Caspi R.R. The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.Invest Ophthalmol Vis Sci. 2012; 53: 2921-2927Crossref PubMed Scopus (495) Google Scholar The ocular and renal phenotypes of 2-year-old C57, Cfh−/−, CFH-Y:Cfh−/−, and CFH-H:Cfh−/− mice were studied using biochemical, histologic, ultrastructural, and ERG methods. Tissue mRNA and real-time quantitative PCR (qPCR) studies were conducted on 3-month-old CFH-Y:Cfh−/− and CFH-H:Cfh−/− mice. The renal histologic composition was also investigated in 8-month-old mice of these four genotypes and CFH-HH:Cfh−/− mice. The numbers of mice for each class of experiments are summarized in Table 1.Table 1Numbers of Mice Used for Each Class of ExperimentsGenotypeReal-time PCRBiochemistryIHC analysisERGRetina histologyRetina TEMKidney histologyAged 2 yearsAged 8 monthsC576449943Cfh−/−636111345CFH-Y:Cfh−/−3931171054CFH-H:Cfh−/−3931391053CFH-HH:Cfh−/−444ERG, electroretinogram; IHC, immunohistochemical; TEM, transmission electron microscopy. Open table in a new tab ERG, electroretinogram; IHC, immunohistochemical; TEM, transmission electron microscopy. Transgenic mouse lines were identified and maintained by PCR using DNA isolated from the tail. A human CFH gene fragment was amplified using 5′-GCAAACCTTTGTTAGTAACTTTAG-3′ (forward) and 5′-GTATTGTGTTCAAATTCTTTTACTG-3′ (reverse) primers, resulting in a 550-bp amplicon. For the Cfh−/− sequence, there is an absence of the 462-bp product amplified in the normal C57 using 5′-GCTACCTACAAATGCCGCCCTG-3′ (forward) and 5′-TCCAACTGCCAGCCTAAAGGAC-3′ (reverse) primers and the presence of a 200-bp amplicon with 5′-GAGGCTATTCGGCTATGACTG-3′ (forward) and 5′-CCACGATAGCCGCGCTGCCTCG-3′ (reverse). Primers used to determine the presence of the rd8 mutation were 5′-GCCCCTGTTTGCATGGAGGAAACTTGGAAGACAGCTACAGTTCTTCTG-3′ (forward) and 5′-GCCCCATTTGCACACTGATGAC-3′ (reverse), which would produce an amplicon of 244 bp if the mutation was present; using 5′-GTGAAGACAGCTACAGTTCTGATC-3′ (forward) with 5′-GCCCCATTTGCACACTGATGAC-3′ (reverse), no 220-bp amplicon would be seen if the mutation was present. To genotype the CFH-HH:Cfh−/−, CFH-H:Cfh−/−, and Cfh−/− mice obtained through the CFH-H:Cfh−/− and CFH-H:Cfh−/− crosses, we used a qBiomarker copy number variant PCR assay (Qiagen Inc., Valencia, CA) specific for the intron and exon boundary of exon 2 of CFH to determine the relative genomic copy number of CFH. qPCR reactions were run in duplicate (iCycler; Bio-Rad Laboratories, Hercules, CA) at 95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds (qBiomarker SYBR fluor mastermix; Qiagen Inc.). Genomic CFH copies were normalized to mouse Gapdh (RT² PCR primer set Gapdh; Qiagen Inc.) using the 2−ΔΔCT method.24Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (127160) Google Scholar CFH-HH:Cfh−/− mice have approximately twice the number of genomic CFH copies compared with CFH-H:Cfh−/− mice (Supplemental Figure S1). Mice were euthanized with carbon dioxide. Brain, eye, intestine, heart, kidney, liver, lung, gut, and spleen were collected from three CFH-Y:Cfh−/− and three CFH-H:Cfh−/− mice. Total RNA was extracted using an RNeasy lipid tissue mini kit (Qiagen Inc.) according to the manufacturer's instructions. cDNA was synthesized from total RNA (SuperScript VILO cDNA synthesis kit; Invitrogen, Grand Island, NY). qPCR reactions were run in triplicate (iCycler) at 95°C for 3 minutes, followed by 40 cycles at 95°C for 10 seconds and 60°C for 20 seconds, then 72°C for 15 seconds (EXPRESS SYBR GreenER qPCR supermix universal kit; Invitrogen). Each reaction contained 25 ng of cDNA, 200 nmol/L each primer, and 10 μL of qPCR supermix in 25 μL of total volume. Relative CFH mRNA expression was normalized to an endogenous reference gene, GAPDH, for each tissue using the quantitative 2−ΔΔCT method.24Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (127160) Google Scholar The primers used were 5′-TGTGAGGGTGGTTTCAGGAT-3′ (forward) and 5′-CCATGAGAAATCTCAGGTGGA-3′ (reverse) for CFH and 5′-AGGTCGGTGTGAACGGATTTG-3′ (forward) and 5′-TGTAGACCATGTAGTTGAGGTCA-3′ (reverse) for GAPDH. Mice were euthanized with carbon dioxide, the chest was cut open, and blood was collected from the heart into EDTA tubes (BD Microtainer; BD Biosciences, Franklin Lakes, NJ) at 4°C. Tubes were centrifuged for 5 minutes at 1500 rpm, and plasma was collected. RPE/choroid/sclera was dissected from each mouse eye. Radioimmunoprecipitation assay buffer (80 μL) with protease inhibitors (cOmplete, mini, EDTA-free; Roche Diagnostics GmbH, Mannheim, Germany) was added to each eyecup and was homogenized using a battery-operated microgrinder from Argos Technologies (Elgin, IL). After sitting on ice for 30 minutes with vortexing every 10 minutes, the lysates were centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant was transferred to a new tube. Protein estimation was performed using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL), and 5 μg of total protein for CFH and 20 μg of total protein for C3 and FB were used for Western blot analyses. The polyclonal goat anti-human CFH antibody (Quidel Corp., San Diego, CA) recognized human and mouse CFH with significantly different affinity. Therefore, mouse CFH standards were prepared by purifying CFH from C57 plasma, as previously described, and were quantified using human CFH standards (CompTech, Tyler, TX) on Coomassie-stained gels using a standard curve of 100 to 600 ng of CFH per well.25Kelly U. Yu L. Kumar P. Ding J.D. Jiang H. Hageman G.S. Arshavsky V.Y. Frank M.M. Hauser M.A. Bowes Rickman C. Heparan sulfate, including that in Bruch's membrane, inhibits the complement alternative pathway: implications for age-related macular degeneration.J Immunol. 2010; 185: 5486-5494Crossref PubMed Scopus (42) Google Scholar We also established that the antibody identically recognized human CFH-H and CFH-Y purified from human plasma (obtained from homozygous individuals) and mouse plasma of CFH-Y:Cfh−/− and CFH-H:Cfh−/− mice. We quantified the isolated human CFH from these sources on Coomasie-stained gels and compared 8, 32, and 64 pg of each CFH on a Western blot analysis (Supplemental Figure S2). Protein levels of mouse CFH in mouse plasma were measured using mouse CFH standards that were loaded onto the gels with C57 plasma, and human CFH standards were loaded onto the gels measuring the human CFH concentration in the CFH-Y:Cfh−/− and CFH-H:Cfh−/− mouse plasma. Plasma samples were diluted 1:500 in 5% sample buffer, then 1.5 μL of this dilution for CFH and 20 μL for C3, C3b, and FB were run, nonreduced on 10% Bis-Tris Criterion XT gels in a 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, transferred to nitrocellulose, blocked with 10% nonfat milk for C3 or 4% bovine serum albumin for CFH and FB, then probed with goat anti-CFH, anti-FB (Kent Laboratories, Bellingham, WA), or rabbit anti-C3d (Dako, Carpinteria, CA). Subsequent incubation with horseradish peroxidase–conjugated anti-goat or anti-rabbit secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) was followed by detection with ECL Plus reagent (Pierce Biotechnology). The images were scanned and the densities measured by ImageJ software version 1.46 (NIH, Bethesda, MD). Normally, the functional activity of complement is measured by the CH50 hemolytic assay. However, because of the small volumes of mouse blood and the low CH50 values obtained, this assay is difficult to perform on mouse plasma. The assay described herein measures functional, intact mouse C3 based on the ability of mouse plasma to provide C3 to reconstitute C3-depleted human serum in the lysis of antibody-sensitized sheep erythrocytes. To sheep erythrocytes (EAs; Lampire Biological Products, Pipersville, PA) at 1 × 109/mL in GVBS++ (Veronal-buffered saline, 0.1% gelatin, 0.15 mmol/L CaCl2, and 1.0 mmol/L MgCl2) was added an equal volume of diluted rabbit anti-sheep erythrocyte IgG, incubated at 30°C for 15 minutes, then centrifuged and washed in GVBS++. Keeping everything on ice, six doubling dilutions of mouse plasma were made in GVBS++ starting with 1:20 for Cfh−/−, CFH-Y:Cfh−/−, and CFH-H:Cfh−/− mouse plasma and 1:50 for C57 mouse plasma. Two hundred microliters of each dilution was added to EAs (1 × 108) in 200 μL of GVBS++, then 100 μL of a 1:20 dilution of C3-depleted human serum was added. The reaction mixture was incubated at 37°C in a shaking water bath for 1 hour, then 500 μL of ice-cold EDTA-GVBS was added to stop the reaction. The tubes were spun at 3000 rpm for 5 minutes, and OD was measured at 412 nm. The reciprocal of the dilution at which 50% hemolysis was achieved gave the hemolytic units for that sample. One hundred percent hemolysis was determined by EAs (1 × 108) in 200 μL of GVBS++ with 300 μL of H2O minus the hemolysis due to EAs (1 × 108) in 200 μL of GVBS++ plus 100 μL of a 1:20 dilution of C3-depleted human serum plus 200 μL of GVBS++. Mice were deeply anesthetized using a ketamine and xylazine mixture and were perfused transcardially with phosphate-buffered saline briefly, followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer (PB), pH 7.4. Eyes and kidneys were postfixed in the same fixative overnight and were cut using a vibratome into approximately 50-μm-thick sections. Free-floating sections were blocked with 10% normal donkey serum and then were incubated overnight with the following primary antibodies: goat anti-human CFH (dilution 1:100) (A312; Quidel Corp.) or goat anti-mouse C3 (dilution 1:500) (#55444; MP Biomedicals, Santa Ana, CA), followed by Alexa Fluor–conjugated donkey anti-goat secondary antibody (dilution 1:500) (Invitrogen) and Hoechst 33258 (Invitrogen) to counterstain the nuclei. Tyramide signal amplification was used for rat anti-mouse C3b/iC3b/C3c antibody (clone 2/11; Hycult Biotech, Uden, the Netherlands) to enhance the signal. Images were acquired using a Leica SP5 laser scanning confocal microscope (Leica Microsystems Inc., Buffalo Grove, IL). Mice were dark adapted overnight, their pupils were dilated with 0.5% tropicamide and 1.25% phenylephrine, and they were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg). Scotopic ERGs were recorded using an Espion E2 system (Diagnosys LLC, Lowell, MA) at flash intensities of 2.5 × 10−5, 5 × 10−5, 5 × 10−4, 5 × 10−3, 0.05, 0.5, 5, 50, and 500 cd·s/m2, as previously described.26Ding J.D. Johnson L.V. Herrmann R. Farsiu S. Smith S.G. Groelle M. Mace B.E. Sullivan P. Jamison J.A. Kelly U. Harrabi O. Bollini S.S. Dilley J. Kobayashi D. Kuang B. Li W. Pons J. Lin J.C. Bowes Rickman C. Anti-amyloid therapy protects against retinal pigmented epithelium damage and vision loss in a model of age-related macular degeneration.Proc Natl Acad Sci U S A. 2011; 108: E279-E287Crossref PubMed Scopus (176) Google Scholar, 27Ding J.D. Lin J. Mace B.E. Herrmann R. Sullivan P. Bowes Rickman C. Targeting age-related macular degeneration with Alzheimer's disease based immunotherapies: anti-amyloid-beta antibody attenuates pathologies in an age-related macular degeneration mouse model.Vision Res. 2008; 48: 339-345Crossref PubMed Scopus (97) Google Scholar Low-pass frequency filtering of 50 Hz was applied to remove oscillatory potentials and noise. Amplitudes of b-waves were calculated from the bottom of the a-wave response to the peak of the b-wave. The data points from the b-wave stimulus-response curves were fitted according to a previously published equation28Herrmann R. Lobanova E.S. Hammond T. Kessler C. Burns M.E. Frishman L.J. Arshavsky V.Y. Phosducin regulates transmission at the photoreceptor-to-ON-bipolar cell synapse.J Neurosci. 2010; 30: 3239-3253Crossref PubMed Scopus (36) Google Scholar using the least-square fitting procedure in OriginPro 9.0 software (OriginLab, Northampton, MA). Mice were deeply anesthetized using a ketamine and xylazine mixture and were perfused transcardially with phosphate-buffered saline briefly, followed by fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L PB, pH 7.4). Eyes were enucleated and fixed in the same fixative overnight. Posterior eyes were osmicated, dehydrated through graded ethanol, infiltrated, and embedded in a mixture of epoxy and Spurr resins. Semithin sections of 0.5 μm were cut, mounted, and stained with toluidine blue. Sections were viewed and photographed under a light microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany). The outer nuclear layer (ONL) thickness was measured along the superior-inferior meridian of the eye at the level of the optic nerve using ImageJ software. For transmission electron microscopy (TEM), approximately 200-μm-thick vibratome sections in the center of the eye through the optic nerve head were osmicated, stained en bloc with uranyl acetate, dehydrated, and embedded in a mixture of epoxy and Spurr resins. Each embedded section was then divided into two parts on each side of the optic nerve head (Supplemental Figure S3A). Thin sections of 60 to 80 nm were cut from each region spanning 70% to 80% of the total length of the retina from the optic nerve toward the ora serrata, collected onto 400-mesh thin-bar copper grids (T400-Cu; Electron Microscopy Sciences, Hatfield, PA), and stained with uranyl acetate and Sato's lead. Sections were viewed using a Tecnai G2 electron microscope (FEI, Hillsboro, OR). Images containing the basal RPE and BrM were taken on the parts of the section adjacent to the grid's bar throughout the length of the retina. For each image, the thickness of the deposits was measured from the central elastin layer of the BrM to the top of the deposit near the edges of the image (Supplemental Figure S3) using ImageJ software. Cumulative distribution of deposit thickness was plotted using OriginPro 9.0 software. A nonparametric Kolmogorov-Smirnov test was used to compare the differences between the distribution curves. Kidneys from 2% paraformaldehyde– and 2% glutaraldehyde–perfused mice were postfixed overnight in the same fixative before being dehydrated and embedded in paraffin. Ten-micron sections were cut and stained with periodic acid–Schiff reagent. Glomerular histologic appearance was graded using the grading system described previously21Pickering M.C. Cook H.T. Warren J. Bygrave A.E. Moss J. Walport M.J. Botto M. Uncontrolled C3 activation causes membranoproliferative glomerulonephritis in mice deficient in complement factor H.Nat Genet. 2002; 31: 424-42" @default.
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• W2104426232 title "Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice" @default.
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