Comprehensive gene-expression profile in murine oxygen-induced retinopathy.

BACKGROUND/AIMS: To investigate the correlation between the clinical course and gene-expression pattern in murine oxygen-induced retinopathy (OIR), a commonly used model of retinopathy of prematurity (ROP). METHODS: OIR was induced in C57BL/6N mice by placing postnatal day 7 (P7) pups in 75% oxygen for 5 days. The clinical course of the OIR was evaluated on retinal flat-mounts after fluorescein isothiocyanate-conjugated dextran perfusion from P12 to P21. The expression values of 94 genes, selected by microarray analyses, were determined daily from P12 through P21 by RT-PCR with TaqMan low-density array (TLDA) and analysed by hierarchical clustering. RESULTS: TLDA cluster analyses showed a homology of gene-expression pattern between P12 and P13 and between P16 and P17. Many genes associated with inflammation were upregulated on P12 and P13 when the degree of both central avascular area and central vasoconstriction were maximal, and the upregulation of the genes continued to P21. At P16 and P17 when extraretinal neovascularisation became most noticeable, several genes associated with angiogenesis, for example, vascular endothelial growth factor-A and angiopoietin-2, were most upregulated. CONCLUSION: The gene-expression pattern was well correlated with the clinical appearance in murine OIR. These findings should contribute to the understanding of the pathological conditions in ROP.

Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1.

In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mug of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mug of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential.

Expression of receptor-activity modifying protein (RAMP) mRNAs in the mouse brain.

Receptor activity modifying proteins (RAMPs) comprise a family of accessory proteins for G protein-coupled receptors (GPCRs). They function as receptor modulators that determine the ligand specificity of receptors for calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (ADM). Here we demonstrate for the first time the characteristic distributions of the RAMP family mRNAs in the brain. Northern blot analysis revealed that mRAMP 1 and 3 mRNAs were intensely expressed in the brain, but mRAMP2 mRNA less abundantly. In situ hybridization studies showed the heterogenous and unique distributions of mRAMP mRNAs; RAMP1 mRNA was widely expressed throughout the brain including the cerebral cortex, caudate putamen, amygdaloid complex, hippocampus, cerebellum and ependyma, mRAMP2 was most abundant in the hippocampus, cerebellum, pia mater and blood vessels, while mRAMP3 was specifically distributed in a variety of thalamic nuclei and the cerebellum. In addition, RAMP1 and -3 genes were also detected in the subfornical organ and area postrema, which are members of circumventricular organs lacking blood-brain barrier. The present results help in understanding the diversification and regulation of receptor functions for calcitonin family peptides, and potentially other GPCRs in the brain.

Characterization of a mouse colonic system B(0+) amino acid transporter related to amino acid absorption in colon.

Previous experiments have shown that an amino acid transport system B(0+) transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B(0+) transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB(0+). Northern blot analysis revealed that mCATB(0+) was selectively expressed in the large intestine. In situ hybridization showed the mCATB(0+) mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB(0+) exhibited a Na(+)-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B(0+). In vivo [(3)H]glycine uptake assay demonstrated that a system B(0+)-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB(0+) may absorb amino acids from the intestinal contents in the colon.

The effect of TGF-beta1 on differential gene expression profiles in human corneal epithelium studied by cDNA expression array.

PURPOSE: TGF-betas regulate cell proliferation and differentiation, and they play important roles in maintenance of corneal epithelium. However, the precise function of TGF-betas in the corneal epithelium remains unclear. In this study, cDNA expression array technology was used to demonstrate the effect of TGF-beta1 on the simultaneous expression of a large number of genes in cultured human corneal epithelial cells (HCECs). The change in protein level expression of the specific genes influenced by TGF-beta1 was also investigated. METHODS: Human cDNA expression array technology was used to study the simultaneous expression of 1176 specific cellular genes in HCECs incubated with TGF-beta1 (10 ng/ml). Moreover, gene-specific semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to confirm the gene expression pattern measured by the cDNA expression array. Western blot analysis was used to examine protein expression of the specific genes in the presence or absence of TGF-beta1. RESULTS: TGF-beta1 significantly upregulated the expression of 19 genes and significantly downregulated ras-related protein, caspase10, and beta4-integrin in the treated HCECs. The expression of 277 genes including alpha3-integrin, PAI-2, transferrin receptor, and cyclin-D1 was studied. Semiquantitative RT-PCR analysis confirmed the TGF-beta1-mediated changes in expression patterns of these genes. Furthermore, Western blot analysis revealed that TGF-beta1 remarkably decreased PAI-2, transferrin receptor, and integrin alpha3, and increased caspase10 on the protein level. CONCLUSIONS: TGF-beta1 regulates the expression of specific types of genes in HCECs. These results strongly suggest that TGF-beta1 is critically involved in the maintenance of the corneal epithelium through the control of a network of various signal-transduction pathways.

Retinal fascin: functional nature, subcellular distribution, and chromosomal localization.

PURPOSE: To investigate the functional properties, subcellular localization, and chromosomal location of retinal fascin. METHODS: Recombinant retinal fascin protein was prepared by using a baculovirus-insect expression system. Actin-binding and -bundling assays were performed with chick actin purified from skeletal muscle. Western blot analysis and immunohistochemistry were performed with a polyclonal antibody raised against bovine retinal fascin. A human retinal cDNA library was screened with an expressed sequence tag cDNA fragment. Chromosomal location was determined with fluorescent in situ hybridization. RESULTS: The actin-binding and actin-bundling activities of retinal fascin were demonstrated by high- and low-speed centrifugation assays. Formation of filamentous (F)-actin bundles by retinal fascin in vitro was also morphologically confirmed by fluorescence microscopy and electron microscopy. Immunohistochemical analysis revealed that retinal fascin protein was localized specifically in the outer and inner segments of the photoreceptor cells in the retina. Two splicing variants of human retinal fascin cDNA were also located. One clone encoded 492 amino acids, and the other encoded 516 amino acids. The gene encoding retinal fascin was localized to human chromosome 17, region q24 -25. CONCLUSIONS: These results suggest that retinal fascin may play a role in formation of unique morphologic structures of the photoreceptor cells and is a candidate gene for retinal degenerative disorders.

Molecular cloning and characterization of rat trp homologues from brain.

Identification of trp (transient receptor potential) gene from Drosophila photoreceptor and subsequent molecular cloning of the human cDNA homologues suggest its participation in capacitative calcium entry (CCE) or so called store-operated Ca2+ channel (SOC). We identified five different trp-related amplifications of reverse-transcription-polymerase chain reaction (RT-PCR) from rat brain; these corresponded to mouse trp homologues, mtrp1,3,4,5,6 and were distributed in various tissues with multiple expression levels. Two cDNAs, homologous to Drosophila trp from rat brain, designated rtrp3 and rtrp6, were isolated and characterized. By RT-PCR analysis, mRNAs of rtrp3 and rtrp6 were found to be expressed differently in brain and other tissues. In situ hybridization analysis revealed that rtrp6 mRNA was preferentially expressed in hippocampal dentate gyrus and cortical layers II and III. Expression of rat TRP3 and TRP6 in COS cells revealed an increase in CCE, as compared to that in the mock-transfected COS cells of the control. Isolation of cDNAs of rat trp gene family provides a useful model for studying mechanism of CCE.

Isolation of a cDNA encoding a photoreceptor cell-specific actin-bundling protein: retinal fascin.

We have isolated a novel retina-specific gene, retinal fascin, encoding a new member of actin-bundling protein gene family, from a bovine retina cDNA library. The cDNA encodes a 492 amino acid protein which shows 36-57% amino acid identity with three vertebrate fascins, echinoid fascin and Drosophila singed gene. Northern blot analysis revealed that retinal fascin mRNA was exclusively expressed in the eye and not seen in other tissues examined. In situ hybridization analysis indicated that retinal fascin mRNA signals were found only in the inner segment of the photoreceptor layer and outer nuclear layer, indicating that retinal fascin was specifically expressed in photoreceptor cells. As fascins are actin-bundling proteins important for constructing several intracellular structures, retinal fascin might play a pivotal role in photoreceptor cell-specific events, such as disk morphogenesis.

The differential osmoregulation and localization of taurine transporter mRNA and Na+/myo-inositol cotransporter mRNA in rat eyes.

We studied the cellular localization and osmotic regulation of taurine transporter (TauT) mRNA in the rat eyes using in situ hybridization. TauT mRNA signals were expressed in the ciliary body, and the outer part of the inner nuclear layer (INL), the outer nuclear layer (ONL) and the inner segment (IS) of the adult rat retina. Chronic hypernatrema, induced by gavaging with 1 ml/100 g body weight of 5% NaCl every other day for 7 days, markedly increased in TauT mRNA in the retina compared with control rats. However, there was little change in TauT mRNA in the eyes in acute hypernatremic state that is induced by single injection of high concentration of NaCl. On the contrary, acute hypernatremic rats displayed markedly elevated Na+/myo-inositol cotransporter (SMIT) mRNA in the retina and the iris-ciliary body and the lens epithelium. Under chronic hypernatremic conditions, there was no significant increase in SMIT mRNA in rat eyes. These findings suggest that TauT mRNA is osmotically regulated in vivo to protect retinal neuronal function, especially against chronic hypernatremic conditions, in contrast to rapid up-regulation of SMIT mRNA in acute hypernatremic rats.