Ernesto F. Moreira

7-Ketocholesterol is present in lipid deposits in the primate retina: potential implication in the induction of VEGF and CNV formation.

PURPOSE 7-Ketocholesterol is a highly toxic oxysterol found in abundance in atherosclerotic plaques and is believed to play a critical role in atherosclerosis. The purpose of this study was to identify and localize 7-ketocholesterol (7kCh) in the primate retina and to examine the potential consequences of its presence in oxidized lipid deposits in the retina. METHODS Unsterified 7kCh was identified and quantified by high-performance liquid chromatography-mass spectrometry. Localization of 7kCh was performed by immunohistochemistry. VEGF induction was determined by qRT-PCR. Cell viability was determined by measuring cellular dehydrogenase activity. Analyses were performed using ARPE19 and human vascular endothelial cells (HMVECs). RESULTS 7-Ketocholesterol is localized mainly to deposits in the choriocapillaris and Bruchs membrane and on the surfaces of vascular endothelial cells of the neural retina. RPE/choriocapillaris regions contained approximately four times more 7kCh than the neural retina. In ARPE19 cells and HMVECs, oxidized LDL and 7kCh induced VEGF 8- to 10-fold above controls. Hypoxia inducible factor (HIF)-1alpha levels did not increase as a result of 7kCh treatment, suggesting an HIF-independent induction pathway. Cholesterol sulfate, a liver X receptor (LXR) antagonist, had marked attenuation of the 7kCh-mediated VEGF induction. LXR-specific siRNAs also reduced VEGF induction. Inhibition of NF-kappaB with BAY 11-7082 reduced IL-8 but not VEGF induction. CONCLUSIONS The location of 7-kCh in the retina and its induction of VEGF in cultured RPE cells and HMVECs suggest it may play a critical role in choroidal neovascularization. The pathway for VEGF induction seems to be independent of HIF-1alpha and NF-kappaB but seems to be partially regulated by LXRs.

SPACR, a Novel Interphotoreceptor Matrix Glycoprotein in Human Retina That Interacts with Hyaluronan

SPACR (sialoproteinassociated with cones and rods), is the major 147–150-kDa glycoprotein present in the insoluble interphotoreceptor matrix of the human retina. Immunocytochemistry localizes SPACR to the matrix surrounding rods and cones (Acharya, S., Rayborn, M. E., and Hollyfield, J. G. (1998)Glycobiology 8, 997–1006). From affinity-purified SPACR, we obtained seven peptide sequences showing 100% identity to the deduced sequence of IMPG1, a purported chondroitin 6-sulfate proteoglycan core protein, which binds peanut agglutinin and is localized to the interphotoreceptor matrix. We show here that SPACR is the most prominent 147–150-kDa band present in the interphotoreceptor matrix and is the gene product of IMPG1. SPACR is not a chondroitin sulfate proteoglycan, since it is not a product of chondroitinase ABC digestion and does not react to a specific antibody for chondroitin 6-sulfate proteoglycan. Moreover, the deduced amino acid sequence reveals no established glycosaminoglycan attachment site. One hyaluronan binding motif is present in the predicted sequence of SPACR. We present evidence that SPACR has a functional hyaluronan binding domain, suggesting that interactions between SPACR and hyaluronan may serve to form the basic macromolecular scaffold, which comprises the insoluble interphotoreceptor matrix.

Molecular and Biochemical Characterization of a Novel Oxysterol-binding Protein (OSBP2) Highly Expressed in Retina

We are interested in understanding the possible function(s) of the oxysterol-binding proteins in mediating oxysterol cytotoxicity in the retina. In this study we describe the cloning, localization, and biological activity of a novel oxysterol-binding protein (OSBP2), and complete the molecular characterization of the previously known OSBP1. Both OSBP genes contain 14 exons and have similar exon sizes and splice sites suggesting they may have arisen from a gene duplication event. OSBP1 is located in chromosome 11q12.1, and OSBP2 is located in 22q12. At the protein level they share 63% overall similarity and although they have unique N termini, both have similar pleckstrin homology domains within the N terminus region. Northern blot analyses indicate that OSBP1 is broadly expressed in human and monkey tissues. OSBP2 is detected mainly in retina, testis, and fetal liver. Western blot analysis using peptide antibodies specific to OSBP1 and OSBP2 detected the proteins in different subcellular fractions in the retinal monkey tissue. OSBP1 is detected mainly in the soluble or cytosolic fraction and nuclei whereas OSBP2 is detected exclusively in the detergent soluble fraction suggesting association with membranes. Immunohistochemical localization of OSBP1 and OSBP2 in the monkey retina placed these two proteins in similar but distinct areas of the inner retina. OSBP2 was found to bind 7-ketocholesterol but to have very little affinity for cholesterol or 25-hydroxycholesterol.

Molecular dynamics of photoreceptor synapse formation in the developing chick retina

The cellular and molecular mechanisms underlying photoreceptor synaptogenesis are poorly understood. Furthermore, a detailed picture of the molecular composition of photoreceptor synapses, or their subtypes, is not yet available, nor do we know what differences, if any, exist among those subtypes. To address these questions, we investigated temporal and spatial patterns of expression and assembly of photoreceptor presynaptic components during chick embryo retinal development and early posthatched life by using reverse transcriptase polymerase chain reaction (RT‐PCR), dissociated retinal cells, laser‐capture microdissection (LCM), immunocytochemistry and confocal microscopy. Immunocytochemistry in tissue sections and dissociated cells showed many similarities and few differences in the synaptic composition of rods and cone subtypes, which, however, were found to project to different strata within the outer plexiform layer. A striking finding was the precise timetable of expression of synaptic genes and proteins during synaptogenesis. Although mRNAs for some synaptic molecules appeared as early as embryonic day (ED) 5–8 (the time of inner retina synaptogenesis), others were undetectable before the time of onset of photoreceptor synaptogenesis on ED13, including CAST, rim2, synapsin‐2, syntaxin‐3, synaptotagmin, glutamate receptors ‐1, ‐4, and ‐5, homer‐1 and ‐2, and tenascin‐R. Most synaptic proteins in photoreceptors followed a similar sequence of expression: they were negative or weakly positive before ED13, appeared in inner segments between ED13 and ED15, became subsequently detectable in perinuclear and axonal regions, and by ED18 were assembled into synaptic terminals and became undetectable in the inner segments. The identity of the signals that regulate the coordinated expression of these synaptic components remains to be investigated. J. Comp. Neurol. 506:822–837, 2008.

Extracellular matrix nitration alters growth factor release and activates bioactive complement in human retinal pigment epithelial cells

Purpose We have shown previously that non-enzymatic nitration (NEN) of the extracellular matrix (ECM), which serves as a model of Bruch’s membrane (BM) aging, has a profound effect on the behavior of the overlying retinal pigment epithelial (RPE) cells, including altered phagocytic ability, reduced cell adhesion, and inhibition of proliferation. We know that transplanted RPE monolayers will encounter a hostile sub-RPE environment, including age-related alterations in BM that may compromise cell function and survival. Here we use our previous NEN model of BM aging to determine the effects of NEN of the ECM on growth factor release and complement activation in RPE cells. Methods Human induced-pluripotent stem cells (iPSCs) were differentiated into RPE cells, and confirmed by immunohistochemistry, confocal microscopy, and polymerase chain reaction. IPSC-derived RPE cells were plated onto RPE-derived ECM under untreated or nitrite-modified conditions. Cells were cultured for 7 days and barrier function measured by transepithelial resistance (TER). Vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and complement component C3a were measured using enzyme-linked immunosorbent assay (ELISA). Results On average nitrite-modified ECM increased VEGF release both apically and basally by 0.15 ± 0.014 ng/mL (p <0.0001) and 0.21 ± 0.022 ng/mL (p <0.0001), respectively, in iPSC-derived RPE cells. Nitrite-modified ECM increased PEDF release in iPSC-derived RPE cells apically by 0.16 ± 0.031 ng/mL (p <0.0001), but not basally (0.27 ± 0.015 vs. 0.32 ± 0.029 ng/mL, (p >0.05)). Nitrite-modified ECM increased production of C3a in iPSC-derived RPE cells by 0.52 ± 0.123 ng/mL (p <0.05). Conclusion Nitrite-modified ECM increased VEGF, PEDF release, and C3a production in human iPSC-derived RPE cells. This model demonstrates changes seen in the basement membrane can lead to alterations in the cell biology of the RPE cells that may be related to the development of age-related macular degeneration.