In vivo adenovirus-mediated delivery of a uPA/uPAR antagonist reduces retinal neovascularization in a mouse model of retinopathy.

Diabetic retinopathy and retinopathy of prematurity are among the leading causes of vision impairment throughout the world. Both diseases are characterized by pathological angiogenesis, which severely impairs vision. Extracellular proteinases play important roles in endothelial cell migration during angiogenesis. Amino-terminal fragment (ATF) is an angiostatic molecule that targets the uPA/uPAR system and inhibits endothelial cell migration. The angiostatic effect of ATF has been demonstrated in models of cancer, but has never been assessed in pathological retinal neovascularization. Endostatin also has angiostatic effects on tumor growth and retinal neovascularization. We used an adenoviral vector carrying the murine ATF (AdATFHSA) or endostatin gene coupled to human serum albumin (HSA) (AdEndoHSA) to increase the half-life of the therapeutic protein in the circulation. We induced retinopathy by exposing 7-day-old mice to high levels of oxygen. They were intravitreally injected with the vectors. Local injection of AdATFHSA or AdEndoHSA reduced retinal neovascularization by 78.1 and 79.2%, respectively. Thus, the adenovirus-mediated delivery of ATFHSA or EndoHSA reduces retinal neovascularization in a mouse model of hypoxia-induced neovascularization.

Use of denaturing HPLC and automated sequencing to screen the VMD2 gene for mutations associated with Best's vitelliform macular dystrophy.

We identified three novel VMD2 mutations in patients with Best's macular dystrophy. DHPLC analysis of the 11 VMD2 exons revealed abnormal profiles in exon 8. Direct sequencing showed that these abnormal profiles were due to monoallelic transitions and transversions. We also found three polymorphic sequence changes that have been reported previously and annotated to an online database (http://www.uni-wuerzburg.de/humangenetics/vmd2.html).

Identification of novel VMD2 gene mutations in patients with best vitelliform macular dystrophy.

ABSTRACT We report five novel VMD2 mutations in Best's macular dystrophy patients (S16F, I73N, R92H, V235L, and N296S). An SSCP analysis of the VMD2 11 exons revealed electrophoretic mobility shifts exclusively in exons 2, 3, 4, 6 and 8. Direct sequencing indicated that these shifts are caused by mono-allelic transition in exons 2, 4, 6, 8 and transversion in exons 3 and 6. Five novel "silent" polymorphisms are also reported: 213T>C, 323C>A, 1514A>G, 1661C>T, and 1712T>C. Hum Mutat 17:235, 2001.

Prominent beta-5 gene expression in the cardiovascular system and in the cartilaginous primordiae of the skeleton during mouse development.

The alpha v beta (alpha(v)beta5) heterodimer has been implicated in many biological functions, including angiogenesis. We report the beta5 gene expression pattern in embryonic and foetal mouse tissues as determined by Northern blotting and in situ hybridization. During the earliest stages, beta5 mRNA is widespread in the mesoderm. During later developmental stages, it remains mostly confined to tissues of mesodermal origin, although probable inductive effects trigger shifts of beta5 gene expression from some mesenchymatous to epithelial structures. This was observed in the teeth, skin, kidneys, and gut. Of physiological importance is the beta5 labeling in the developing cardiovascular and respiratory systems and cartilages. Furthermore, early beta5 gene expression was observed within the intra- and extraembryonic sites of hematopoiesis. This suggests a major role for beta5 in the hematopoietic and angiogenic stem cells and thus in the development of the vascular system. Later, the beta5 gene was expressed in endothelial cells of the vessels developing both by angiogenesis and vasculogenesis in the lung, heart, and kidneys. Moreover, the beta5 hybridization signal was detected in developing cartilages but not in ossified or ossifying bones. beta5-Integrin is a key integrin involved in angiogenesis, vasculogenesis, hematopoiesis, and bone formation.

Homozygous deletion in the coding sequence of the c-mer gene in RCS rats unravels general mechanisms of physiological cell adhesion and apoptosis.

The RCS rat presents an autosomal recessive retinal pigment epithelium dystrophy characterized by the outer segments of photoreceptors being phagocytosis-deficient. A systematic genetic study allowed us to restrict the interval containing the rdy locus to that between the markers D3Mit13 and D3Rat256. We report the chromosomal localization of the rat c-mer gene in the cytogenetic bands 3q35-36, based on genetic analysis and radiation hybrid mapping. Using a systematic biocomputing analysis, we identified two strong related candidate genes encoding protein tyrosine kinase receptors of the AXL subfamily. The comparison of their expression patterns in human and mice tissues suggested that the c-mer gene was the best gene to screen for mutations. RCS rdy- and RCS rdy+ cDNAs were sequenced. The RCS rdy- cDNAs carried a significant deletion in the 5' part of the coding sequence of the c-mer gene resulting in a shortened aberrant transcript encoding a 20 amino acid peptide. The c-mer gene contains characteristic motifs of neural cell adhesion. A ligand of the c-mer receptor, Gas6, exhibits antiapoptotic properties.

Prominent neuronal-specific tub gene expression in cellular targets of tubby mice mutation.

The tubby strain of mice exhibits maturity-onset obesity and sensory deficits in vision and hearing. The mutated gene, tub , responsible for this phenotype was identified recently, but the function of the TUB protein has not been deduced from its amino acid sequence. This prompted us to undertake expression mapping studies with the hope that they might help to elucidate the biological role of the TUB protein. We report the tub gene expression pattern in embryonic, fetal and adult mice tissues as determined by northern blots and in situ hybridization, using antisense oligonucleotidic probes. In mouse embryos, tub is expressed selectively in differentiating neurons of the ensemble of central and peripheral nervous systems, starting at 9.5 days after conception. In adult mice, tub is transcribed in several major brain areas, including cerebral cortex, hippocampus, several nuclei of the hypothalamus controlling feeding behavior, in the spiral ganglion of the inner ear and in the photoreceptor cells of the retina. These structures contain potential cellular targets of the tubby mutation-induced pathogenesis. The neuronal-specific tub gene distribution allows the establishment of a genotype-phenotype correlation in the tubby mice. This correlation is reminiscent of that observed in fat/fat mice, whose phenotype, also characterized by obesity, is caused by a null mutation in the carboxypeptidase E (CPE) gene. Our observations highlight similarities between CPE, prohormone convertases, several neuropeptides and tub gene expression patterns during embryogenesis, and may narrow down the avenues to explore in order to determine ultimately the function of the TUB protein.