Interleukin 27 Induces the Expression of Complement Factor H (CFH) in the Retina

Complement factor H (CFH) is a central regulator of the complement system and has been implicated in the etiology of age-related macular degeneration (AMD), a leading cause of blindness in the elderly. In view of previous studies showing that reduced expression of CFH in the retina is a risk factor for developing AMD, there is significant interest in understanding how CFH expression is regulated in the retina. In this study, we have shown that the anti-inflammatory cytokine, IL-27, induced CFH expression in mouse retinal cells and human retinal pigmented epithelial cells (RPE) through STAT1-mediated up-regulation of Interferon Regulatory Factor-1 (IRF-1) and IRF-8. We further show that cells in the ganglion and inner-nuclear layers of the retina constitutively express IRF-1 and IRF-8 and enhanced CFH expression in the retina during ocular inflammation correlated with significant increase in the expression of IRF-1, IRF-8 and IL-27 (IL-27p28 and Ebi3). Our data thus reveal a novel role of IL-27 in regulating complement activation through up-regulation of CFH and suggest that defects in IL-27 signaling or expression may contribute to the reduction of CFH expression in the retina of patients with AMD.     

Cigarette smoking strongly modifies the association of LOC387715 and age-related macular degeneration

We used iterative association mapping to identify a susceptibility gene for age-related macular degeneration (AMD) on chromosome 10q26, which is one of the most consistently implicated linkage regions for this disorder. We employed linkage analysis methods, followed by family-based and case-control association analyses, using two independent data sets. To identify statistically the most likely AMD-susceptibility allele, we used the Genotype-IBD Sharing Test (GIST) and conditional haplotype analysis. To incorporate the two most important known AMD risk factors--smoking and the Y402H variant of the complement factor H gene (CFH)--we used logistic regression modeling to test for gene-gene and gene-environment interactions in the case-control data set and used the ordered-subset analysis to account for genetic linkage heterogeneity in the family-based data set. Our results strongly implicate a coding change (Ala69Ser) in the LOC387715 gene as the second major identified AMD-susceptibility allele, confirming earlier suggestions. This variant's effect on AMD is statistically independent of CFH and is of similar magnitude to the effect of Y402H. The overall effect is driven primarily by a strong association in smokers, since we observed significant evidence for a statistical interaction between the LOC387715 variant and a history of cigarette smoking. This gene-environment interaction is supported by statistically independent family-based and case-control analysis methods. We estimate that CFH, LOC387715, and cigarette smoking together explain 61% of the population-attributable risk (PAR) of AMD. The adjusted PAR percentage estimates are 20% for smoking, 36% for LOC387715, and 43% for CFH. We demonstrate, for the first time, that a genetic susceptibility coupled with a modifiable lifestyle factor such as cigarette smoking confers a significantly higher risk of AMD than either factor alone.     

CFH gene variant, Y402H, and smoking, body mass index, environmental associations with advanced age-related macular degeneration

OBJECTIVES: We tested the hypothesis that modifiable lifestyle factors alter the genetic susceptibility associated with a common coding variant in the complement factor H (CFH) gene, Y402H, for the leading cause of blindness among the elderly, age-related macular degeneration (AMD). METHODS: In this case-control association analysis, Caucasian participants in the multicenter Age-Related Eye Disease Study with advanced AMD (n = 574 cases) or no AMD (n = 280 controls) were evaluated. AMD status was determined by grading of fundus photographs. Risk factors including cigarette smoking and body mass index (BMI) were assessed and DNA specimens were genotyped for the variant in the CFH gene. Unconditional logistic regression analyses were performed. Attributable risks and multivariable AMD risk scores were calculated. RESULTS: The number of risk alleles for Y402H was associated with advanced AMD, with odds ratios (OR) of 2.7 (95% confidence interval (CI) 1.8-3.8) for the CT heterozygous genotype and OR 7.4 (4.7-11.8) for the homozygous CC risk genotype, after controlling for demographic and behavioral risk factors. Current cigarette smoking (OR 5.1) and high BMI > or =30 (OR 2.1) were independently related to AMD, controlling for genotype. The association between AMD and BMI varied dependent on genotype (P interaction = 0.006 for the CT vs. TT genotype). The CC genotype plus higher BMI (OR 5.9) or smoking (OR 10.2) conferred the greatest risks. Gene plus environment risk scores provided an area under the receiver operating characteristic (ROC) curve of 0.70-0.75. CONCLUSIONS: Genetic and environmental factors are independently related to advanced AMD, and modifiable factors alter genetic susceptibility. The AMD risk score identifies a highly susceptible population.     

Biochemical analysis of a common human polymorphism associated with age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in developed countries. A large number of human genetic studies have associated a common variant (Y402H) of complement factor H (CFH) with a highly significant increase in AMD risk. CFH is a modular protein with 20 homologous short consensus repeats (SCRs). The Y402H variant is located in SCR7 of both CFH and factor H-like protein 1 (FHL-1), a splice variant of CFH (containing SCR1-7) with unique biochemical properties. Because SCR7 is known to bind to heparin, C-reactive protein (CRP), and M protein from Streptococcus pyogenes, it has been hypothesized that the AMD-associated polymorphism may affect interactions with these CFH ligands. In this study, we tested this hypothesis in the context of full-length CFH (SCR1-20) and FHL-1. We systematically analyzed the interactions of the Y402 and H402 variants of CFH and FHL-1 with heparin, CRP, and several bacterial ligands: M6 protein of Streptococcus pyogenes, PspC of Streptococcus pneumoniea, and BbCRASP-1 of Borrelia burgdorferi. In comparing the Y and H variants of CFH and FHL-1, we found no significant difference in their protein secretion, cofactor activity, or interactions with heparin, BbCRASP-1, or PspC, but a significant difference in binding to CRP and M6 protein. This study reveals the fundamental properties of a common polymorphism of CFH and lays the groundwork for elucidating the role of CFH in AMD pathogenesis.     

Activation of STAT3 stimulates AHSP expression in K562 cells

Studies on the chaperone proteinα-hemoglobin stabilizing protein(AHSP)reveal that abundant AHSP in erythroid cells enhance the cells’tolerance to oxidative stress imposed by excessα-hemoglobin in pathological conditions.However,the potential intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown.The present study examined the effect and molecular mechanism of STAT3,an oxidative regulator,on the expression of AHSP.AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells.Regulation of AHSP in oxidative circumstance was then examined inα-globin-overloaded K562 cells,and real-time PCR showed strengthened expression of both AHSP and STAT3.ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and uponα-globin overexpression.Dual luciferase reporter assays of the wildtype and mutated SB3 element,an IL-6RE site,in the AHSP promoter in K562 cells highlighted the direct regulatory effect of STAT3 on AHSP gene.Finally,direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA assays.Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway,and provides clues to the therapeutic strategy for AHSP enhancement.     

Echinacoside reverses myocardial remodeling and improves heart function via regulating SIRT1/FOXO3a/MnSOD axis in HF rats induced by isoproterenol

Myocardial remodelling is important pathological basis of HF, mitochondrial oxidative stress is a promoter to myocardial hypertrophy, fibrosis and apoptosis. ECH is the major active component of a traditional Chinese medicine Cistanches Herba, plenty of studies indicate it possesses a strong antioxidant capacity in nerve cells and tumour, it inhibits mitochondrial oxidative stress, protects mitochondrial function, but the specific mechanism is unclear. SIRT1/FOXO3a/MnSOD is an important antioxidant axis, study finds that ECH binds covalently to SIRT1 as a ligand and up-regulates the expression of SIRT1 in brain cells. We hypothesizes that ECH may reverse myocardial remodelling and improve heart function of HF via regulating SIRT1/FOXO3a/MnSOD signalling axis and inhibit mitochondrial oxidative stress in cardiomyocytes. Here, we firstly induce cellular model of oxidative stress by ISO with AC-16 cells and pre-treat with ECH, the level of mitochondrial ROS, mtDNA oxidative injury, MMP, carbonylated protein, lipid peroxidation, intracellular ROS and apoptosis are detected, confirm the effect of ECH in mitochondrial oxidative stress and function in vitro. Then, we establish a HF rat model induced by ISO and pre-treat with ECH. Indexes of heart function, myocardial remodelling, mitochondrial oxidative stress and function, expression of SIRT1/FOXO3a/MnSOD signalling axis are measured, the data indicate that ECH improves heart function, inhibits myocardial hypertrophy, fibrosis and apoptosis, increases the expression of SIRT1/FOXO3a/MnSOD signalling axis, reduces the mitochondrial oxidative damages, protects mitochondrial function. We conclude that ECH reverses myocardial remodelling and improves cardiac function via up-regulating SIRT1/FOXO3a/MnSOD axis and inhibiting mitochondrial oxidative stress in HF rats.     

Interaction of Complement Factor H and Fibulin3 in Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of vision loss. It is associated with development of characteristic plaque-like deposits (soft drusen) in Bruch’s membrane basal to the retinal pigment epithelium (RPE). A sequence variant (Y402H) in short consensus repeat domain 7 (SCR7) of complement factor H (CFH) is associated with risk for “dry” AMD. We asked whether the eye-targeting of this disease might be related to specific interactions of CFH SCR7 with proteins expressed in the aging human RPE/choroid that could contribute to protein deposition in drusen. Yeast 2-hybrid (Y2H) screens of a retinal pigment epithelium/choroid library derived from aged donors using CFH SCR7 baits detected an interaction with EFEMP1/Fibulin 3 (Fib3), which is the locus for an inherited macular degeneration and also accumulates basal to macular RPE in AMD. The CFH/Fib3 interaction was validated by co-immunoprecipitation of native proteins. Quantitative Y2H and ELISA assays with different recombinant protein constructs both demonstrated higher affinity for Fib3 for the disease-related CFH 402H variant. Immuno-labeling revealed colocalization of CFH and Fib3 in globular deposits within cholesterol-rich domains in soft drusen in two AMD donors homozygous for CFH 402H (H/H). This pattern of labeling was quite distinct from those seen in examples of eyes with Y/Y and H/Y genotypes. The CFH 402H/Fib3 interaction could contribute to the development of pathological aggregates in soft drusen in some patients and as such might provide a target for therapeutic intervention in some forms of AMD.     

Complement Factor H Expressed by Retinal Pigment Epithelium Cells Can Suppress Neovascularization of Human Umbilical Vein Endothelial Cells: An in vitro Study

Complement factor H (CFH) is one of the most important soluble complement regulatory proteins and is closely associated with age-related macular degeneration (AMD), the leading cause of irreversible central vision loss in the elderly population in developed countries. Our study searches to investigate whether CFH expression is changed in oxidative damaged retinal pigment epithelium (RPE) cells and the role of CFH in the in vitro neovascularization. First, it was confirmed by immunofluorescence staining that CFH was expressed by ARPE-19 cells. CFH mRNA and protein in oxidative (H₂O₂) damaged ARPE-19 cells were both reduced, as determined by Real-time PCR and Western blotting analysis. Enzyme-linked immunosorbent assay (ELISA) also showed that ARPE-19 cells treated with H₂O₂ caused an increase in C3a content, which indicates complement activation. Then, wound assays were performed to show that CFH expression suppression promoted human umbilical vein endothelial cell (HUVECs) migration. Thereafter, ARPE-19 cells were transfected with CFH-specific siRNA and CFH knockdown was confirmed with the aid of Real-time PCR, immunofluorescence staining and Western blotting. The ELISA results showed that specific CFH knockdown in ARPE-19 cells activated the complement system. Finally, in vitro matrigel tube formation assay was performed to determine whether change of CFH expression in RPE would affect tube formation by HUVECs. More tubes were formed by HUVECs co-cultured with ARPE-19 cells transfected with CFH specific-siRNA when compared with controls. Our results suggested that RPE cells might be the local CFH source, and RPE cell injuries (such as oxidative stress) may cause CFH expression suppression, which in turn may lead to complement activation and promotion of tube formation by HUVECs. This finding is of importance in elucidating the role of complement in the pathogenesis of ocular neovascularization including choroidal neovascularization.     

Haplotypes in the complement factor H (CFH) gene: associations with drusen and advanced age-related macular degeneration

Background

Age-related macular degeneration (AMD), the leading cause of blindness in the Western world, is a complex disease that affects people over 50 years old. The complement factor H (CFH) gene has been repeatedly shown to be a major factor in determining susceptibility to the advanced form of the condition. We aimed to better understand the functional role of this gene in the AMD disease process and assess whether it is associated with earlier forms of the disease.

Methodology/Principal Findings

We genotyped SNPs at the CFH gene locus in three independent populations with AMD: (a) extended families where at least 3 family members had AMD; (b) sporadic cases of advanced AMD and (c) cases from the Age-Related Eye Disease Study (AREDS). We investigated polymorphisms and haplotypes in and around the CFH gene to assess their role in AMD. CFH is associated with early/intermediate and advanced AMD in both familial and sporadic cases. In our populations, the CFH SNP, rs2274700, is most strongly associated with AMD and when incorporated into a haplotype with the Y402H SNP and rs1061147, the strongest association is observed (p<10⁻⁹).

Conclusions/Significance

Our results, reproduced in three populations that represent the spectrum of AMD cases, provide evidence that the CFH gene is associated with drusen as well as with advanced AMD. We also identified novel susceptibility and protective haplotypes in the AMD populations.     

Systemic complement activation in age-related macular degeneration

Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.