Human complement factor H Y402H polymorphism causes an age-related macular degeneration phenotype and lipoprotein dysregulation in mice.

One of the strongest susceptibility genes for age-related macular degeneration (AMD) is complement factor H (CFH); however, its impact on AMD pathobiology remains unresolved. Here, the effect of the principal AMD-risk-associated CFH variant (Y402H) on the development and progression of age-dependent AMD-like pathologies was determined in vivo. Transgenic mice expressing equal amounts of the full-length normal human CFH Y402 (CFH-Y/0) or the AMD-risk associated CFH H402 (CFH-H/H) variant on a Cfh-/- background were aged to 90 weeks and switched from normal diet (ND) to a high fat, cholesterol-enriched (HFC) diet for 8 weeks. The resulting phenotype was compared with age-matched controls maintained on ND. Remarkably, an AMD-like phenotype consisting of vision loss, increased retinal pigmented epithelium (RPE) stress, and increased basal laminar deposits was detected only in aged CFH-H/H mice following the HFC diet. These changes were not observed in aged CFH-Y/0 mice or in younger (36- to 40-week-old) CFH mice of both genotypes fed either diet. Biochemical analyses of aged CFH mice after HFC diet revealed genotype-dependent changes in plasma and eyecup lipoproteins, but not complement activation, which correlated with the AMD-like phenotype in old CFH-H/H mice. Specifically, apolipoproteins B48 and A1 are elevated in the RPE/choroid of the aged CFH-H/H mice compared with age-matched control CFH-Y/0 fed a HFC diet. Hence, we demonstrate a functional consequence of the Y402H polymorphism in vivo, which promotes AMD-like pathology development and affects lipoprotein levels in aged mice. These findings support targeting lipoproteins as a viable therapeutic strategy for treating AMD.

Expression of human complement factor H prevents age-related macular degeneration-like retina damage and kidney abnormalities in aged Cfh knockout mice.

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.

The role of complement dysregulation in AMD mouse models.

Variations in several complement genes are now known to be significant risk factors for the development of age-related macular degeneration (AMD). Despite dramatic effects on disease susceptibility, the underlying mechanisms by which common polymorphisms in complement proteins alter disease risk have remained unclear. Genetically modified mice in which the activity of the complement has been altered are available and can be used to investigate the role of complement in the pathogenesis of AMD. In this mini review, we will discuss some existing complement models of AMD and our efforts to develop and characterize the ocular phenotype in a variety of mice in which complement is either chronically activated or inhibited. A spectrum of complement dysregulation was modeled on the APOE4 AMD mouse model by crossing these mice to complement factor H knockout (cfh-/-) mice to test the impact of excess complement activation, and by crossing them to soluble-complement-receptor-1-related protein y (sCrry) mice, in which sCrry acts as a potent inhibitor of mouse complement acting in a manner similar to CFH. In addition, we have also generated humanized CFH mice expressing normal and risk variants of CFH.

Anti-amyloid therapy protects against retinal pigmented epithelium damage and vision loss in a model of age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual dysfunction worldwide. Amyloid ß (Aß) peptides, Aß1-40 (Aß40) and Aß1-42 (Aß42), have been implicated previously in the AMD disease process. Consistent with a pathogenic role for Aß, we show here that a mouse model of AMD that invokes multiple factors that are known to modify AMD risk (aged human apolipoprotein E 4 targeted replacement mice on a high-fat, cholesterol-enriched diet) presents with Aß-containing deposits basal to the retinal pigmented epithelium (RPE), histopathologic changes in the RPE, and a deficit in scotopic electroretinographic response, which is reflective of impaired visual function. Strikingly, these electroretinographic deficits are abrogated in a dose-dependent manner by systemic administration of an antibody targeting the C termini of Aß40 and Aß42. Concomitant reduction in the levels of Aß and activated complement components in sub-RPE deposits and structural preservation of the RPE are associated with anti-Aß40/42 antibody immunotherapy and visual protection. These observations are consistent with the reduction in amyloid plaques and improvement of cognitive function in mouse models of Alzheimer's disease treated with anti-Aß antibodies. They also implicate Aß in the pathogenesis of AMD and identify Aß as a viable therapeutic target for its treatment.

Beta1-adrenergic receptors maintain fetal heart rate and survival.

Beta-adrenergic receptor (betaAR) activation has been shown to maintain heart rate during hypoxia and to rescue the fetus from the fetal lethality that occurs in the absence of norepinephrine. This study examines whether the same subtype of betaAR is responsible for survival and heart rate regulation. It also investigates which betaARs are located on the early fetal heart and whether they can be directly activated during hypoxia. Cultured E12.5 mouse fetuses were treated with subtype-specific betaAR antagonists to pharmacologically block betaARs during a hypoxic insult. Hypoxia alone reduced heart rate by 35-40% compared to prehypoxic levels. During hypoxia, heart rate was further reduced by 31% in the presence of a beta(1)AR antagonist, CGP20712A, at 100 nM, but not with a beta2 (ICI118551)- or a beta3 (SR59230A)-specific antagonist at 100 nM. Survival in utero was also mediated by beta1ARs. A beta1 partial agonist, xamoterol, rescued 74% of catecholamine-deficient (tyrosine-hydroxylase-null) pups to birth, a survival rate equivalent to that with a nonspecific betaAR agonist, isoproterenol (87%). Receptor autoradiography showed that beta1ARs were only found on the mouse heart at E12.5, while beta2ARs were localized to the liver and vasculature. To determine if the response to hypoxia was intrinsic to the heart, isolated fetal hearts were incubated under hypoxic conditions in the presence of a betaAR agonist. Heart rate was reduced to 25-30% by hypoxia alone, but was restored to 63% of prehypoxic levels with 100 nM isoproterenol. Restoration was completely prevented if beta1ARs were blocked with CGP20712A at 300 nM, a concentration that blocks beta1ARs, but not beta2- or beta3ARs. Our results demonstrate that beta1ARs are located on the heart of early fetal mice and that beta1AR stimulation maintains fetal heart rate during hypoxia and mediates survival in vivo.

Catecholamines act via a beta-adrenergic receptor to maintain fetal heart rate and survival.

Mice lacking catecholamines die before birth, some with cardiovascular abnormalities. To investigate the role of catecholamines in development, embryonic day 12.5 (E12.5) fetuses were cultured and heart rate monitored. Under optimal oxygenation, wild-type and catecholamine-deficient fetuses had the same initial heart rate (200-220 beats/min), which decreased by 15% in wild-type fetuses during 50 min of culture. During the same culture period, catecholamine-deficient fetuses dropped their heart rate by 35%. Hypoxia reduced heart rate of wild-type fetuses by 35-40% in culture and by 20% in utero, assessed by echocardiography. However, catecholamine-deficient fetuses exhibited greater hypoxia-induced bradycardia, reducing their heart rate by 70-75% in culture. Isoproterenol, a beta-adrenergic receptor (beta-AR) agonist, reversed this extreme bradycardia, restoring the rate of catecholamine-deficient fetuses to that of nonmutant siblings. Moreover, isoproterenol rescued 100% of catecholamine-deficient pups to birth in a dose-dependent, stereo-specific manner when administered in the dam's drinking water. An alpha-AR agonist was without effect. When wild-type fetuses were cultured with adrenoreceptor antagonists to create pharmacological nulls, blockade of alpha-ARs with 10 microM phentolamine or beta-ARs with 10 microM bupranolol alone or in combination did not reduce heart rate under optimal oxygenation. However, when combined with hypoxia, beta-AR blockade reduced heart rate by 35%. In contrast, the muscarinic blocker atropine and the alpha-AR antagonist phentolamine had no effect. These data suggest that beta-ARs mediate survival in vivo and regulate heart rate in culture. We hypothesize that norepinephrine, acting through beta-ARs, maintains fetal heart rate during periods of transient hypoxia that occur throughout gestation, and that catecholamine-deficient fetuses die because they cannot withstand hypoxia-induced bradycardia.