Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4^−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4^−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4^−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.     

Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

Treatment with interleukin‐33 is non‐toxic and protects retinal pigment epithelium in an ageing model of outer retinal degeneration

The leading cause of central vision loss, age‐related macular degeneration (AMD), is a degenerative disorder characterized by atrophy of retinal pigment epithelium (RPE) and photoreceptors. For 15% of cases, neovascularization occurs, leading to acute vision loss if left untreated. For the remaining patients, there are currently no treatment options and preventing progressive RPE atrophy remains the main therapeutic goal. Previously, we have shown treatment with interleukin‐33 can reduce choroidal neovascularization and attenuate tissue remodelling. Here, we investigate IL‐33 delivery in aged, high‐fat diet (HFD) fed mice on a wildtype and complement factor H heterozygous knockout background. We characterize the non‐toxic effect following intravitreal injection of IL‐33 and further demonstrate protective effects against RPE cell death with evidence of maintaining metabolic retinal homeostasis of Cfh+/−~HFD mice. Our results further support the potential utility of IL‐33 to prevent AMD progression.     

Conditional Ablation of Retinol Dehydrogenase 10 in the Retinal Pigmented Epithelium Causes Delayed Dark Adaption in Mice

Regeneration of the visual chromophore, 11-cis-retinal, is a crucial step in the visual cycle required to sustain vision. This cycle consists of sequential biochemical reactions that occur in photoreceptor cells and the retinal pigmented epithelium (RPE). Oxidation of 11-cis-retinol to 11-cis-retinal is accomplished by a family of enzymes termed 11-cis-retinol dehydrogenases, including RDH5 and RDH11. Double deletion of Rdh5 and Rdh11 does not limit the production of 11-cis-retinal in mice. Here we describe a third retinol dehydrogenase in the RPE, RDH10, which can produce 11-cis-retinal. Mice with a conditional knock-out of Rdh10 in RPE cells (Rdh10 cKO) displayed delayed 11-cis-retinal regeneration and dark adaption after bright light illumination. Retinal function measured by electroretinogram after light exposure was also delayed in Rdh10 cKO mice as compared with controls. Double deletion of Rdh5 and Rdh10 (cDKO) in mice caused elevated 11/13-cis-retinyl ester content also seen in Rdh5^−/−Rdh11^−/− mice as compared with Rdh5^−/− mice. Normal retinal morphology was observed in 6-month-old Rdh10 cKO and cDKO mice, suggesting that loss of Rdh10 in the RPE does not negatively affect the health of the retina. Compensatory expression of other retinol dehydrogenases was observed in both Rdh5^−/− and Rdh10 cKO mice. These results indicate that RDH10 acts in cooperation with other RDH isoforms to produce the 11-cis-retinal chromophore needed for vision.     

Di-retinoid-pyridinium-ethanolamine (A2E) Accumulation and the Maintenance of the Visual Cycle Are Independent of Atg7-mediated Autophagy in the Retinal Pigmented Epithelium

Autophagy is an evolutionarily conserved catabolic mechanism that relieves cellular stress by removing/recycling damaged organelles and debris through the action of lysosomes. Compromised autophagy has been implicated in many neurodegenerative diseases, including retinal degeneration. Here we examined retinal phenotypes resulting from RPE-specific deletion of the autophagy regulatory gene Atg7 by generating Atg7^flox/flox;VMD2-rtTA-cre+ mice to determine whether autophagy is essential for RPE functions including retinoid recycling. Atg7-deficient RPE displayed abnormal morphology with increased RPE thickness, cellular debris and vacuole formation indicating that autophagy is important in maintaining RPE homeostasis. In contrast, 11-cis-retinal content, ERGs and retinal histology were normal in mice with Atg7-deficient RPE in both fasted and fed states. Because A2E accumulation in the RPE is associated with pathogenesis of both Stargardt disease and age-related macular degeneration (AMD) in humans, deletion of Abca4 was introduced into Atg7^flox/flox;VMD2-rtTA-cre+ mice to investigate the role of autophagy during A2E accumulation. Comparable A2E concentrations were detected in the eyes of 6-month-old mice with and without Atg7 from both Abca4^−/− and Abca4^+/+ backgrounds. To identify other autophagy-related molecules involved in A2E accumulation, we performed gene expression array analysis on A2E-treated human RPE cells and found up-regulation of four autophagy related genes; DRAM1, NPC1, CASP3, and EIF2AK3/PERK. These observations indicate that Atg7-mediated autophagy is dispensable for retinoid recycling and A2E deposition; however, autophagy plays a role in coping with stress caused by A2E accumulation.     

Alternative Complement Pathway Deficiency Ameliorates Chronic Smoke-Induced Functional and Morphological Ocular Injury

Background

Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required.

Methods

Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB^−/−) using molecular, histological, electrophysiological, and behavioral outcomes.

Results

CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch''s membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB^−/− mice were protected from developing these CE-mediated alterations.

Conclusions

Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD.     

The Role of Histamine in the Retina: Studies on the Hdc Knockout Mouse

The role of histamine in the retina is not well understood, despite it regulating a number of functions within the brain, including sleep, feeding, energy balance, and anxiety. In this study we characterized the structure and function of the retina in mice that lacked expression of the rate limiting enzyme in the formation of histamine, histidine decarboxylase (Hdc^−/− mouse). Using laser capture microdissection, Hdc mRNA expression was assessed in the inner and outer nuclear layers of adult C57Bl6J wildtype (WT) and Hdc^−/−-retinae. In adult WT and Hdc^−/−-mice, retinal fundi were imaged, retinal structure was assessed using immunocytochemistry and function was probed by electroretinography. Blood flow velocity was assessed by quantifying temporal changes in the dynamic fluorescein angiography in arterioles and venules. In WT retinae, Hdc gene expression was detected in the outer nuclear layer, but not the inner nuclear layer, while the lack of Hdc expression was confirmed in the Hdc^−/− retina. Preliminary examination of the fundus and retinal structure of the widely used Hdc^−/−mouse strain revealed discrete lesions across the retina that corresponded to areas of photoreceptor abnormality reminiscent of the rd8 (Crb1) mutation. This was confirmed after genotyping and the strain designated Hdc^rd8/rd8. In order to determine the effect of the lack of Hdc-alone on the retina, Hdc^−/− mice free of the Crb1 mutation were bred. Retinal fundi appeared normal in these animals and there was no difference in retinal structure, macrogliosis, nor any change in microglial characteristics in Hdc^−/− compared to wildtype retinae. In addition, retinal function and retinal blood flow dynamics showed no alterations in the Hdc^−/− retina. Overall, these results suggest that histamine plays little role in modulating retinal structure and function.     

Annexin A2 Regulates Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina

The daily phagocytosis of shed photoreceptor outer segments by pigment epithelial cells is critical for the maintenance of the retina. In a subtractive polymerase chain reaction analysis, we found that functional differentiation of human ARPE19 retinal pigment epithelial (RPE) cells is accompanied by up-regulation of annexin (anx) A2, a major Src substrate and regulator of membrane–cytoskeleton dynamics. Here, we show that anx A2 is recruited to the nascent phagocytic cup in vitro and in vivo and that it fully dissociates once the phagosome is internalized. In ARPE19 cells depleted of anx A2 by using small interfering RNA and in ANX A2^−/− mice the phagocytosis of outer segments was impaired, and in ANX A2^−/− mice there was an accumulation of phagocytosed outer segments in the RPE apical processes, indicative of retarded phagosome transport. We show that anx A2 is tyrosine phosphorylated at the onset of phagocytosis and that the synchronized activation of focal adhesion kinase and c-Src is abnormal in ANX A2^−/− mice. These findings reveal that anx A2 is involved in the circadian regulation of outer segment phagocytosis, and they provide new insight into the protein machinery that regulates phagocytic function in RPE cells.     

Sema6B,Sema6C,and Sema6D Expression and Function during Mammalian Retinal Development

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B^−/− or Sema6C^−/−; Sema6D^−/− retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.     

Retinal Pigmented Epithelial Cells Obtained from Human Induced Pluripotent Stem Cells Possess Functional Visual Cycle Enzymes in Vitro and in Vivo

Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat^−/− and Rpe65^−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat^−/− and Rpe65^−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases.     

Programmed necrosis,not apoptosis,is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3^−/− mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3^−/− mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.     

Myosin7a Deficiency Results in Reduced Retinal Activity Which Is Improved by Gene Therapy

Mutations in MYO7A cause autosomal recessive Usher syndrome type IB (USH1B), one of the most frequent conditions that combine severe congenital hearing impairment and retinitis pigmentosa. A promising therapeutic strategy for retinitis pigmentosa is gene therapy, however its pre-clinical development is limited by the mild retinal phenotype of the shaker1 (sh1^−/−) murine model of USH1B which lacks both retinal functional abnormalities and degeneration. Here we report a significant, early-onset delay of sh1^−/− photoreceptor ability to recover from light desensitization as well as a progressive reduction of both b-wave electroretinogram amplitude and light sensitivity, in the absence of significant loss of photoreceptors up to 12 months of age. We additionally show that subretinal delivery to the sh1^−/− retina of AAV vectors encoding the large MYO7A protein results in significant improvement of sh1^−/− photoreceptor and retinal pigment epithelium ultrastructural anomalies which is associated with improvement of recovery from light desensitization. These findings provide new tools to evaluate the efficacy of experimental therapies for USH1B. In addition, although AAV vectors expressing large genes might have limited clinical applications due to their genome heterogeneity, our data show that AAV-mediated MYO7A gene transfer to the sh1^−/− retina is effective.     

Fatp1 Deficiency Affects Retinal Light Response and Dark Adaptation,and Induces Age-Related Alterations

FATP1 is involved in lipid transport into cells and in intracellular lipid metabolism. We showed previously that this protein interacts with and inhibits the limiting-step isomerase of the visual cycle RPE65. Here, we aimed to analyze the effect of Fatp1-deficiency in vivo on the visual cycle, structure and function, and on retinal aging. Among the Fatp family members, we observed that only Fatp1 and 4 are expressed in the control retina, in both the neuroretina and the retinal pigment epithelium. In the neuroretina, Fatp1 is mostly expressed in photoreceptors. In young adult Fatp1^−/− mice, Fatp4 expression was unchanged in retinal pigment epithelium and reduced two-fold in the neuroretina as compared to Fatp1^+/+ mice. The Fatp1^−/− mice had a preserved retinal structure but a decreased electroretinogram response to light. These mice also displayed a delayed recovery of the b-wave amplitude after bleaching, however, visual cycle speed was unchanged, and both retinal pigment epithelium and photoreceptors presented the same fatty acid pattern compared to controls. In 2 year-old Fatp1^−/− mice, transmission electron microscopy studies showed specific abnormalities in the retinas comprising choroid vascularization anomalies and thickening of the Bruch membrane with material deposits, and sometimes local disorganization of the photoreceptor outer segments. These anomalies lead us to speculate that the absence of FATP1 accelerates the aging process.     

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm^Flox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm^Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.     

Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition

Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch''s membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch''s membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4^−/− and Rdh8^−/−/Abca4^−/− mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4^−/− but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4^−/− mice revealed that carbonyl adduct deposition in Bruch''s membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4^−/− mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch''s membrane that can confer risk of age-related macular degeneration.     

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.     

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.     

Melanoregulin, Product of the dsu Locus, Links the BLOC-Pathway and Oa1 in Organelle Biogenesis

Humans with Hermansky-Pudlak Syndrome (HPS) or ocular albinism (OA1) display abnormal aspects of organelle biogenesis. The multigenic disorder HPS displays broad defects in biogenesis of lysosome-related organelles including melanosomes, platelet dense granules, and lysosomes. A phenotype of ocular pigmentation in OA1 is a smaller number of macromelanosomes, in contrast to HPS, where in many cases the melanosomes are smaller than normal. In these studies we define the role of the Mreg^dsu gene, which suppresses the coat color dilution of Myo5a, melanophilin, and Rab27a mutant mice in maintaining melanosome size and distribution. We show that the product of the Mreg^dsu locus, melanoregulin (MREG), interacts both with members of the HPS BLOC-2 complex and with Oa1 in regulating melanosome size. Loss of MREG function facilitates increase in the size of micromelanosomes in the choroid of the HPS BLOC-2 mutants ruby, ruby2, and cocoa, while a transgenic mouse overexpressing melanoregulin corrects the size of retinal pigment epithelium (RPE) macromelanosomes in Oa1^ko/ko mice. Collectively, these results suggest that MREG levels regulate pigment incorporation into melanosomes. Immunohistochemical analysis localizes melanoregulin not to melanosomes, but to small vesicles in the cytoplasm of the RPE, consistent with a role for this protein in regulating membrane interactions during melanosome biogenesis. These results provide the first link between the BLOC pathway and Oa1 in melanosome biogenesis, thus supporting the hypothesis that intracellular G-protein coupled receptors may be involved in the biogenesis of other organelles. Furthermore these studies provide the foundation for therapeutic approaches to correct the pigment defects in the RPE of HPS and OA1.