Complement factor H binds malondialdehyde epitopes and protects from oxidative stress.

Oxidative stress and enhanced lipid peroxidation are linked to many chronic inflammatory diseases, including age-related macular degeneration (AMD). AMD is the leading cause of blindness in Western societies, but its aetiology remains largely unknown. Malondialdehyde (MDA) is a common lipid peroxidation product that accumulates in many pathophysiological processes, including AMD. Here we identify complement factor H (CFH) as a major MDA-binding protein that can block both the uptake of MDA-modified proteins by macrophages and MDA-induced proinflammatory effects in vivo in mice. The CFH polymorphism H402, which is strongly associated with AMD, markedly reduces the ability of CFH to bind MDA, indicating a causal link to disease aetiology. Our findings provide important mechanistic insights into innate immune responses to oxidative stress, which may be exploited in the prevention of and therapy for AMD and other chronic inflammatory diseases.

Complement regulation at necrotic cell lesions is impaired by the age-related macular degeneration-associated factor-H His402 risk variant.

Age-related macular degeneration is a leading form of blindness in Western countries and is associated with a common SNP (rs 1061170/Y402H) in the Factor H gene, which encodes the two complement inhibitors Factor H and FHL1. However, the functional consequences of this Tyr(402) His exchange in domain 7 are not precisely defined. In this study, we show that the Tyr(402) His sequence variation affects Factor H surface recruitment by monomeric C-reactive protein (mCRP) to specific patches on the surface of necrotic retinal pigment epithelial cells. Enhanced attachment of the protective Tyr(402) variants of both Factor H and FHL1 by mCRP results in more efficient complement control and further provides an anti-inflammatory environment. In addition, we demonstrate that mCRP is generated on the surface of necrotic retinal pigment epithelial cells and that this newly formed mCRP colocalizes with the cell damage marker annexin V. Bound to the cell surface, Factor H-mCRP complexes allow complement inactivation and reduce the release of the proinflammatory cytokine TNF-α. This mCRP-mediated complement inhibitory and anti-inflammatory activity at necrotic membrane lesions is affected by residue 402 of Factor H and defines a new role for mCRP, for Factor H, and also for the mCRP-Factor H complex. The increased protective capacity of the Tyr(402) Factor H variant allows better and more efficient clearance and removal of cellular debris and reduces inflammation and pathology.

Defective complement action and control defines disease pathology for retinal and renal disorders and provides a basis for new therapeutic approaches.

The complement system is a central homeostatic system of the vertebrate organism and part f innate immunity. When activated, complement has multiple functions and drives homeostasis and the elimination of infectious microbes (Walport MJ (2001) N Engl J Med 344:1140-1144; Zipfel PF, Skerka C (2009) Nat Rev Immunol 9:729-740). Several inflammatory disorders are caused by defective complement action, and the growing, detailed understanding of the underlying pathophysiological principles translate into therapy with complement inhibitors. As complement inhibitors have been pproved for treatment of the complement-mediated disorders hemolytic uremic syndrome (HUS) and paroxysmal nocturnal hemoglobinuria (PNH), there is a growing interest to extended and improve the options for other complement-mediated diseases. Here, we summarize the current understanding and concepts how defective complement action at biological surfaces lead to pathology and disease, and how this understanding can be used for the development of surface targeting complement inhibitors.

An imbalance of human complement regulatory proteins CFHR1, CFHR3 and factor H influences risk for age-related macular degeneration (AMD).

A frequent deletion of complement factor H (CFH)-related genes CFHR3 and CFHR1 (ΔCFHR3/CFHR1) is considered to have a protective effect against age-related macular degeneration (AMD), although the underlying mechanism remains elusive. The deletion seems to be linked to one of the two protective CFH haplotypes which are both tagged by the protective allele of single nucleotide polymorphism rs2274700 (CFH:A473A). In a German cohort of 530 AMD patients, we now show that protection against AMD conferred by ΔCFHR3/CFHR1 is independent of the effects of rs2274700 and rs1061170 (CFH:Y402H). This suggests a functional role of CFHR1 and/or CFHR3 in disease pathogenesis. We therefore characterized the CFHR3 function and identified CFHR3 as a novel human complement regulator that inhibits C3 convertase activity. CFHR3 displays anti-inflammatory effects by blocking C5a generation and C5a-mediated chemoattraction of neutrophils. In addition, CFHR3 and CFHR1 compete with factor H for binding to the central complement component C3. Thus, deficiency of CFHR3 and CFHR1 results in a loss of complement control but enhances local regulation by factor H. Our findings allude to a critical balance between the complement regulators CFHR3, CFHR1 and factor H and further emphasize the central role of complement regulation in AMD pathology.

Monomeric C-reactive protein modulates classic complement activation on necrotic cells.

The acute-phase protein C-reactive protein (CRP) recruits C1q to the surface of damaged cells and thereby initiates complement activation. However, CRP also recruits complement inhibitors, such as C4b-binding protein (C4bp) and factor H, which both block complement progression at the level of C3 and inhibits inflammation. To define how CRP modulates the classic complement pathway, we studied the interaction of CRP with the classic pathway inhibitor C4bp. Monomeric CRP (mCRP), but not pentameric CRP (pCRP), binds C4bp and enhances degradation of C4b and C3b. Both C1q, the initiator, and C4bp, the inhibitor of the classic pathway, compete for mCRP binding, and this competition adjusts the local balance of activation and inhibition. After attachment of pCRP to the surface of necrotic rat myocytes, generation of mCRP was demonstrated over a period of 18 h. Similarly, a biological role for mCRP, C1q, and C4bp in the disease setting of acute myocardial infarction was revealed. In this inflamed tissue, mCRP, pCRP, C4bp, C1q, and C4d were detected in acetone-fixed and in unfixed tissue. Protein levels were enhanced 6 h to 5 d after infarction. Thus, mCRP bound to damaged cardiomyocytes recruits C1q to activate and also C4bp to control the classic complement pathway.

Factor H-related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation.

Homozygous deletion of a 84-kb genomic fragment in human chromosome 1 that encompasses the CFHR1 and CFHR3 genes represents a risk factor for hemolytic uremic syndrome (HUS) but has a protective effect in age-related macular degeneration (AMD). Here we identify CFHR1 as a novel inhibitor of the complement pathway that blocks C5 convertase activity and interferes with C5b surface deposition and MAC formation. This activity is distinct from complement factor H, and apparently factor H and CFHR1 control complement activation in a sequential manner. As both proteins bind to the same or similar sites at the cellular surfaces, the gain of CFHR1 activity presumably is at the expense of CFH-mediated function (inhibition of the C3 convertase). In HUS, the absence of CFHR1 may result in reduced inhibition of terminal complex formation and in reduced protection of endothelial cells upon complement attack. These findings provide new insights into complement regulation on the cell surface and biosurfaces and likely define the role of CFHR1 in human diseases.

The role of complement in AMD.

Age related macular degeneration (AMD) is a common form of blindness in the western world and genetic variations of several complement genes, including the complement regulator Factor H, the central complement component C3, Factor B, C2, and also Factor I confer a risk for the disease. However deletion of a chromosomal segment in the Factor H gene cluster on human chromosome 1, which results in the deficiency of the terminal pathway regulator CFHR1, and of the putative complement regulator CFHR3 has a protective effect for development of AMD. The Factor H gene encodes two proteins Factor H and FHL1 which are derived from alternatively processed transcripts. In particular a sequence variation at position 402 of both Factor H and FHL1 is associated with a risk for AMD. A tyrosine residue at position 402 represents the protective and a histidine residue the risk variant. AMD is considered a chronic inflammatory disease, which can be caused by defective and inappropriate regulation of the continuously activated alternative complement pathway. This activation generates complement effector products and inflammatory mediators that stimulate further inflammatory reactions. Defective regulation can lead to formation of immune deposits, drusen and ultimately translate into damage of retinal pigment epithelial cells, rupture of the interface between these epithelial cells and the Bruch's membrane and vision loss. Here we describe the role of complement in the retina and summarize the current concept how defective or inappropriate local complement control contributes to inflammation and the pathophysiology of AMD.

Defective complement control of factor H (Y402H) and FHL-1 in age-related macular degeneration.

The common variant in the human complement Factor H gene (CFH), with Tyr402His, is linked to age-related macular degeneration (AMD), a prevalent disorder leading to visual impairment and irreversible blindness in elderly patients. Here we show that the risk variant CFH 402His displays reduced binding to C reactive protein (CRP), heparin and retinal pigment epithelial cells. This reduced binding can cause inefficient complement regulation at the cell surface, particularly when CRP is recruited to injured sites and tissue. In addition, we identify the Factor H-like protein 1 (FHL-1), an alternative splice product of the CFH gene as an additional protein that includes the risk residue 402, and thus confers risk for AMD. FHL-1 is expressed in the eye and the FHL-1 402His risk variant shows similar reduced cell binding and likely reduced complement regulatory functions on the cell surface. CFH and FHL-1 may act in concert in the eye and the reduced surface binding may result in inappropriate local complement control, which in turn can lead to inflammation, disturbance of local physiological homeostasis and progression to cell damage. As a consequence, these processes may lead to AMD pathogenesis.

Endogenous vascular hydrogen peroxide regulates arteriolar tension in vivo.

BACKGROUND: Although many studies suggested direct vasomotor effects of hydrogen peroxide (H2O2) in vitro, little is known about the vasomotor effects of H2O2 in vivo. METHODS AND RESULTS: We have generated mice overexpressing human catalase driven by the Tie-2 promoter to specifically target this transgene to the vascular tissue. Vessels of these mice (cat++) expressed significantly higher levels of catalase mRNA, protein, and activity. The overexpression was selective for vascular tissue, as evidenced by immunohistochemistry in specimens of aorta, heart, lung, and kidney. Quantification of reactive oxygen species by fluorescence signals in cat++ versus catalase-negative (catn) mice showed a strong decrease in aortic endothelium and left ventricular myocardium but not in leukocytes. Awake male cat++ at 3 to 4 months of age had a significantly lower systolic blood pressure (sBP, 102.7+/-2.2 mm Hg, n=10) compared with their transgene-negative littermates (catn, 115.6+/-2.5 mm Hg, P=0.0211) and C57BL/6 mice (118.4+/-3.06 mm Hg, n=6). Treatment with the catalase inhibitor aminotriazole increased sBP of cat++ to 117.3+/-4.3 mm Hg (P=0.0345), while having no effect in catn (118.4+/-2.4 mm Hg, n=4, P>0.05). In contrast, treatment with the NO-synthase inhibitor nitro-L-arginine methyl ester (100 mg.kg BW(-1).d(-1)) increased sBP in cat++ and C57Bl/6 to a similar extent. Likewise, phosphorylation of vasodilator-stimulated phosphoprotein in skeletal muscle, left ventricular myocardium, and lung was identical in cat++ and catn. Endothelium- and NO-dependent aortic vasodilations were unchanged in cat++. Aortic KCl contractions were significantly lower in cat++ and exogenous H2O2 (10 micromol/L)-induced vasoconstriction. CONCLUSIONS: These data suggest that endogenous H2O2 may act as a vasoconstrictor in resistance vessels and contribute to the regulation of blood pressure.

Critical involvement of hydrogen peroxide in exercise-induced up-regulation of endothelial NO synthase.

OBJECTIVE: Recent studies from our groups have indicated that endothelial nitric oxide synthase (eNOS) expression is increased in cell culture by both shear stress and by hydrogen peroxide (H(2)O(2)). In vivo, exercise training, known to increase both endothelial shear stress and oxidative stress, also increases eNOS expression. It is unclear if H(2)O(2) contributes to an increase in eNOS expression in response to exercise training. METHODS: To address this question, we generated mice overexpressing human catalase (hCat) driven by the murine Tie-2 promoter to specifically target this transgene to the endothelium (cat(++)). RESULTS: Vessels of cat(++) expressed significantly higher levels of catalase mRNA and catalase protein and activity but normal levels of eNOS. Exercise alone had no effect on catalase expression in C57BL/6. Wild-type littermates of cat(++) showed an increase in eNOS expression with 3 weeks of exercise (2.53+/-0.42-fold) comparable to C57BL/6 (2.93+/-0.45-fold). In striking contrast, 3 weeks of exercise had no effect on aortic (1.33+/-0.32-fold) and myocardial (1.1+/-0.2-fold) eNOS expression in catalase transgenic mice. CONCLUSIONS: These data suggest that endogenous H(2)O(2) plays a key role in the endothelial adaptation to exercise training by stimulating an up-regulation of eNOS.

Physical inactivity causes endothelial dysfunction in healthy young mice.

OBJECTIVES: We sought to determine if physical inactivity affects endothelial function in young healthy individuals. BACKGROUND: Recent studies have linked exercise training to increased bioavailability of vascular nitric oxide (NO) and to improved endothelial function in patients with cardiovascular disorders. The effects of physical inactivity on normal vascular endothelial function are not known. METHODS: Healthy young male C57Bl/6 mice living in groups of five in large cages, where they were running, climbing, and fighting during their active cycle, were randomly assigned to stay there or to live alone in small cages where they were predominantly resting. After five and nine weeks citrate synthase activity (a measure of mitochondrial respiratory chain activity), heart weight/body weight ratio, vascular reactivity, and protein expression of endothelial nitric oxide synthase (eNOS) were assessed. RESULTS: Singularized mice showed a reduction of citrate synthase activity (p < 0.05), of endothelium-dependent vasorelaxation (to 65 +/- 5% of control levels; p < 0.001), and of eNOS protein expression (to 53 +/- 8% of control levels; p < 0.01). In striking contrast, vascular responses to potassium chloride, phenylephrine, and the NO-donor racemic S-nitroso-N-acetylpenicillamine were unchanged. The alterations of vascular eNOS-activity were completely reversible when singularized mice underwent exercise. In mice living in groups, exercise showed only a small effect on aortic eNOS expression. CONCLUSIONS: In young healthy individuals physical inactivity induces endothelial dysfunction, which is completely reversible by a short period of moderate exercise training. We suggest that physical inactivity, the so-called sedentary lifestyle, increases cardiovascular risk in young healthy individuals by inducing endothelial dysfunction.

Haemophilus influenzae interacts with the human complement inhibitor factor H.

Pathogenic microbes acquire human complement inhibitors to circumvent the innate immune system. In this study, we identify two novel host-pathogen interactions, factor H (FH) and factor H-like protein 1 (FHL-1), the inhibitors of the alternative pathway that binds to Hib. A collection of clinical Haemophilus influenzae isolates was tested and the majority of encapsulated and unencapsulated bound FH. The isolate Hib 541 with a particularly high FH-binding was selected for detailed analysis. An increased survival in normal human serum was observed with Hib 541 as compared with the low FH-binding Hib 568. Interestingly, two binding domains were identified within FH; one binding site common to both FH and FHL-1 was located in the N-terminal short consensus repeat domains 6-7, whereas the other, specific for FH, was located in the C-terminal short consensus repeat domains 18-20. Importantly, both FH and FHL-1, when bound to the surface of Hib 541, retained cofactor activity as determined by analysis of C3b degradation. Two H. influenzae outer membrane proteins of approximately 32 and 40 kDa were detected with radiolabeled FH in Far Western blot. Taken together, in addition to interactions with the classical, lectin, and terminal pathways, H. influenzae interferes with the alternative complement activation pathway by binding FH and FHL-1, and thereby reducing the complement-mediated bactericidal activity resulting in an increased survival. In contrast to incubation with active complement, H. influenzae had a reduced survival in FH-depleted human serum, thus demonstrating that FH mediates a protective role at the bacterial surface.

Arg64 variant of the beta3-adrenergic receptor is associated with gallstone formation.

OBJECTIVES: The beta3-adrenergic receptor (ADRB3) is a transmembrane receptor highly expressed in adipose tissue and thought to be involved in the regulation of lipolysis. ADRB3 is also highly expressed in gallbladder tissue where it may be involved in gallbladder contraction. Because polymorphisms of ADRB3 are present in populations with a high prevalence of gallstones (e.g., Pima-Indians, obese subjects), we hypothesized that known polymorphisms for ADRB3 (Trp64Arg) may represent an independent risk factor for gallstone disease. METHODS: The EMIL cross-sectional study investigated the health behavior and prevalence of chronic diseases in a small Southwestern German town of 12,475 inhabitants. From 3,893 randomly selected citizens 2,147 subjects were enrolled and screened for gallstones employing ultrasonography. Blood samples were drawn for biochemical analysis and isolation of genomic DNA. ADBR3 genotypes were determined by TaqMan SNP Assay. RESULTS: We identified 171 (8%) gallstone carriers of whom 143 participated (46 male, 97 female), with a mean age of 51.4, and mean BMI of 29.3 kg/m2. For these subjects an age, gender and BMI matched partner without gallstones was recruited from the study population. Genotyping for ADRB3 revealed an Arg64 allele frequency of 5.9 versus 0.7% (HR = 11.9, P < 0.05) compared with controls. CONCLUSIONS: Our results indicate that the ADRB3 Trp64Arg polymorphism is associated with gallstone disease thereby representing a genetic marker that identifies subjects at higher risk for gallstone formation.