In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE).Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell ® culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis -retinoic acid, and 22( R )-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD. Age-related macular degeneration (AMD) is the leading cause of severe visual loss in the developed world (1, 2). In the early stages of the disease, before visual loss occurs from choroidal neovascularization, there is progressive accumulation of lipids in Bruch's membrane (3-6). Bruch's membrane lies at the critical juncture between the outer retina and its blood supply, the choriocapillaris. Lipid deposition causes reduced hydraulic conductivity and macromolecular permeability in Bruch's membrane and is thought to impair retinal metabolism (7-9). Interestingly, lipid accumulation in Bruch's membrane similar to that in AMD has been observed in apolipoprotein E (apoE) null mice (10, 11). Because of the additional association between apoE alleles and other age-related degenerations, such as Alzheimer's disease and atherosclerosis, there has been recent investigation into a potential role for apoE in AMD.Several studies of apoE polymorphism in AMD have been conducted (12)(13)(14). In contrast to Alzheimer's disease, the apoE4 allele has been associated with a reduced prevalence of AMD. The apoE2 allele is slightly increased in patients with AMD. Further supporting a role in AMD pathogenesis, apoE has been detected in drusen, the Bruch's membrane deposits that are the hallmark of AMD (13,15). Immunohistochemical studies of postmortem eyes have demonstrated apoE in the basal aspect of the retinal pigment epithelium (RPE) (15). Cultured RPE cells synthesize high levels of apoE mRNA, comparable to the levels found in brain (15).Although the role of apoE in AMD is not established, this apolipoprotein has several functions that may affect the course of this disease. ApoE has anti-angiogenic (16), anti-inflammatory (17), and anti-oxidative (18) effects. These are all considered atheroprotective attributes of apoE, but they may also be important in protecting a...

Aims Excessive lipid accumulation in Bruch’s membrane (BrM) is a hallmark of ageing, the major risk factor for age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells may utilise reverse cholesterol transport (RCT) activity to move lipid into BrM, mediated through ATP-binding cassette A1 (ABCA1) and scavenger receptor BI (SR-BI). Methods ABCA1 expression was assessed by reverse transcription polymerase chain reaction (RT-PCR) and western blotting of human RPE cell extracts. Lipid transport assays were performed using radiolabelled photoreceptor outer segments (POS). ABCA1 and SR-BI expression was examined in normal mouse eyes by immunofluorescence staining. BrMs of ABCA1 and SR-BI heterozygous mice were examined microscopically. Results Human RPE cells expressed ABCA1 mRNA and protein. The ABCA1 and SR-BI inhibitor glyburide (also known as glibenclamide) abolished basal transport of POS-derived lipids in RPE cells in the presence of high-density lipoprotein. Mouse retina and RPE expressed ABCA1 and SR-BI. SR-BI was highly expressed in RPE. BrMs were significantly thickened in SR-BI heterozygous mice, but not in ABCA1 heterozygous mice. Conclusion RPE cells express ABCA1 and SR-BI. This implies a significant role for SR-BI and ABCA1 in lipid transport and RCT in the retina and RPE.

Abstract. Drosophila laminin was isolated from the medium of Drosophila K~ cell cultures. It was purified by velocity sedimentation, gel filtration, and chromatography. Drosophila laminin is a disulfide-linked molecule consisting of three chains with apparent molecular masses of 400, 215, and 185 kD. In electron micrographs, it has the cross-shaped appearance with globular domains characteristic of vertebrate laminin with closely similar dimensions. The amino acid composition and lectin-binding properties of Drosophila laminin are given.Polyclonal antibodies to Drosophila laminin were prepared and their specificity was established. In developing embryos immunofluorescence staining was detected between 6 and 8 h of development; and in sections of 8-9-h and older embryos immunostaining was seen at sites where basement membranes are present surrounding internal organs, muscles, underlying the hypodermal epithelium, and in the nervous system. Basement membrane staining was also seen in larva and adults. Cells from Drosophila embryos dissociated at the cellular blastoderm stage were grown in culture and some specific, differentiated cells synthesized laminin after several hours of culture as shown by immunofluorescence. The significance of the evolutionary conservation of the structure of this basement membrane component is discussed.T HE extracellular protein laminin is a component of vertebrate basement membranes (7,44). It is a multidomain protein which facilitates growth and migration of cultured cells (22,23,42,43) and promotes the extension of growth cones by neurites (4,12,15,19,25). The fly Drosophila melanogaster provides a unique experimental system for investigating the control of expression and functioning of gene products during development. To learn more about the assembly of basement membranes, and the roles of these structures in development, we initiated a search for their component materials in Drosophila (18). Here we report the isolation and characterization of a molecule with properties of a Drosophila laminin, and the production of antibodies which enable us to study the early appearance and distribution of this material during Drosophila embryogenesis.Molecules of vertebrate laminin have a characteristic, unique appearance of a cross in the electron microscope after spraying onto a flat surface and rotary metal shadowing (14). After reduction, three polypeptide chains have been obtained from vertebrate laminin: A, B1, mad B2. The glycosylated chains migrate in SDS-PAGE with apparent molecular masses of 400, 220, and 210 kD, respectively. A model of the molecule has been suggested in which the carboxyl portions of one copy of each chain participate in a coiled-coil a-helix in the long arm of the cross, while each short arm of the cross is made up of only one chain. The model is supported by biophysical measurements and a rapidly progressing knowledge of the amino acid and nucleotide coding sequences of the three chains (3,32,38,48).The primary sources of vertebrate laminin have been extra-embryo...

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Conversion of prorenin to renin results from proteolytic cleavage of a 43-amino-acid prorenin prosegment in renal juxtaglomerular cells. The enzyme that performs this processing is not known. Of several enzymes proposed, cathepsin B is a candidate because it colocalizes with renin in juxtaglomerular cell secretory granules and accurately cleaves the prosegment of human prorenin in vitro. It is not known whether cathepsin B can perform this function in the cell. We examined this using secretory granule-containing rat GH4C1 cells transfected with a human preprorenin expression vector. When treated with secretagogue (KCl 50 mmol/L + forskolin 10 micromol/L), these cells secrete 95% prorenin and 5% active renin into the medium, indicating little prorenin processing activity. In contrast, when the cells are cotransfected with a vector that expresses human preprocathepsin B or mouse prohormone convertase 1, secretagogue-induced secretion of active renin increased to 12% and 16.5%, respectively. With antisera that recognize the prosegment and renin, prorenin and renin were identified as proteins of 47 and 43 kD, respectively, and an antibody specific to the prosegment precipitated only the 47-kD species. These results do not address whether cathepsin B is the authentic renal prorenin processing enzyme. However, the results do demonstrate that cathepsin B can localize to the appropriate subcellular compartment and process prorenin to renin in GH4C1 cells and are consistent with a role for this enzyme in prorenin processing.