No abstract

The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316 -324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up- In the adult mammalian organism, the vasculature is normally quiescent. Arterial endothelial cells have an extremely low turnover rate (ϳ1 in every 10 5 cells undergoes cell division 1 ). However, adult endothelial cells are not postmitotic and, in response to appropriate stimuli, they can proliferate and form new blood vessels by a process termed angiogenesis. 2-4 Angiogenesis describes the formation of new capillaries and larger vessels by sprouting or splitting from pre-existing vessels. Typically, the sprouting of vessels involves activation of quiescent endothelial cells, proteolytic degradation of the extracellular matrix, chemotactic migration, invasion into the surrounding stroma, proliferation and differentiation of endothelial cells, and formation of a new lumen and maturation of the endothelium. 2,[5][6][7][8] This angiogenic sprouting process occurs under physiological conditions during the female reproductive cycle (ovulation, implantation, pregnancy) and wound healing, as well as under pathological conditions in solid tumors and metastases, rheumatoid arthritis, retinopathies, hemangiomas, and psoriasis. 2,7,9,10 Several of the key players in both embryonic and adult angiogenesis are vascular-specific growth factor ligands

Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and posttranslational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, antihypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally, in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.

Using retroviral entrapment vectors, we identified a novel mouse gene whose expression is restricted to vascular endothelial cells and their precursors in the yolk sac blood islands. A 3.68-kb cDNA corresponding to the endogenous transcript was isolated using genomic DNA flanking the entrapment vector insertion as a probe. We have named this gene Vezf1 for vascular endothelial zinc finger 1. Vezf1 encodes a protein with a predicted molecular mass of 56 kDa and that contains six putative zinc finger domains and shows high homology to a previously identified human gene, DB1, that is believed to be involved in regulating expression of cytokine genes such as interleukin-3. In situ hybridization analysis revealed the onset of expression in advanced primitive streak-stage embryos being located in the extraembryonic mesodermal component of the visceral yolk sac and in the anteriormost mesoderm of the embryo proper. During head-fold and somite stages, expression was restricted to vascular endothelial cells that arise during both vasculogenesis and angiogenesis. Vezf1-related sequences were found to be highly conserved among higher vertebrate species that have acquired extraembryonic yolk sac membranes during evolution. The Vezf1 locus mapped to the proximal part of mouse chromosome 2, a region which has homology to human chromosome 9q. Vezf1 expression correlates temporally and spatially with the early differentiation of angioblasts into the endothelial cell lineage and the proliferation of endothelial cells of the embryonic vascular system. Thus, Vezf1 may play an important role in the endothelial lineage determination and may have an additional role during later stages of embryonic vasculogenesis and angiogenesis.

Mammalian development is orchestrated by a variety of cellular proteins with expression that is regulated precisely. Although some of the genes encoding these factors have been identified, largely by homology to those of simpler organisms, the majority of them presumably remain unknown. We report here on the results of a large‐scale genetic screen that can potentially lead to the identification of many of these unidentified genes in mice. The method we developed takes advantage of the fact that many of the factors that regulate early development are expressed at highly specific stages of early embryogenesis. We therefore established a method for tagging candidate developmental genes by virtue of their expression in a stage‐specific manner during formation of embryoid bodies without a bias for their expression in undifferentiated embryonic stem (ES) cells. Of 2,400 ES cell clones with random insertions of retroviral vectors carrying a human placental alkaline phosphatase reporter gene (AP), 41 clones exhibited stage‐specific reporter gene expression during embryoid body formation. The majority of these insertions were in genes that are not expressed in undifferentiated ES cells. Eleven ES cell clones with characteristic patterns of AP reporter gene expression in vitro were chosen for further examination in vivo for AP expression in developing embryos. Ten ES cell clones exhibited AP expression between day 7.5 and day 10.5 of development. Clones that showed restricted reporter gene expression in vitro also exhibited similar temporally and spatially restricted AP expression in vivo. Sequence analysis of genomic DNA flanking several vector insertions and corresponding cDNAs suggested that several of the insertions identified a previously unidentified gene. Thus, screening for reporter gene expression during embryoid body formation provides an efficient means of enriching clones that contain vector insertions into potentially novel genes that are important for regulating different stages of early postimplantation development. Dev. Dyn. 1998;212:181–197. © 1998 Wiley‐Liss, Inc.