title: Establishment of heparan sulphate deficient primary endothelial cells from EXT-1(flox/flox) mouse lungs and sprouting aortas.
abstract: Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1(flox/flox) mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field.
authors.affiliation: Department of Oncology, Clinical Sciences, Lund University and Lund University Hospital, Barngatan 2B, SE-22185 Lund, Sweden.
authors.forename: Paulina
authors.initials: P
authors.lastname: Kucharzewska
authors.forename: Johanna E
authors.initials: JE
authors.lastname: Welch
authors.forename: Josefin
authors.initials: J
authors.lastname: Birgersson
authors.forename: Mattias
authors.initials: M
authors.lastname: Belting
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001011:Aorta / Q000254:growth & development*; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006497:Heparan Sulfate / Q000172:deficiency* / Q000378:metabolism; D019426:Integrases / Q000378:metabolism; D008168:Lung / Q000098:blood supply* / Q000201:enzymology; D051379:Mice; D008822:Mice, Transgenic; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics* / Q000378:metabolism; D018919:Neovascularization, Physiologic; D011995:Recombination, Genetic / Q000235:genetics; D000099058:Exostosin 1
chemical_list: D006497:Heparan Sulfate; D019426:Integrases; D017351:N-Acetylglucosaminyltransferases; D000099058:Exostosin 1; C045073:Cre recombinase
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