Establishment of heparan sulphate deficient primary endothelial cells from EXT-1(flox/flox) mouse lungs and sprouting aortas.
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.
Kucharzewska
Paulina
P
Department of Oncology, Clinical Sciences, Lund University and Lund University Hospital, Barngatan 2B, SE-22185 Lund, Sweden.
Welch
Johanna E
JE
Birgersson
Josefin
J
Belting
Mattias
M
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
D006497:Heparan Sulfate; D019426:Integrases; D017351:N-Acetylglucosaminyltransferases; D000099058:Exostosin 1; C045073:Cre recombinase
J Biol Chem. 2009 Nov 20;284(47):32959-67
19783663
Genes Dev. 2003 Aug 1;17(15):1835-40
12897053
J Theor Biol. 2005 Apr 21;233(4):483-99
15748910
Cell. 1994 Dec 16;79(6):1015-24
7528103
Dev Biol. 2000 Aug 15;224(2):299-311
10926768
J Biol Chem. 2006 Jan 20;281(3):1731-40
16258170
Genes Dev. 2002 Oct 15;16(20):2684-98
12381667
J Biol Chem. 1997 Feb 28;272(9):5518-24
9038157
Science. 2003 Nov 7;302(5647):1044-6
14605369
Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):668-73
10639137
Nat Med. 1995 Jan;1(1):27-31
7584949
J Biol Chem. 1992 Mar 25;267(9):6093-8
1556117
Int J Oncol. 2008 Apr;32(4):749-56
18360702
J Biol Chem. 2000 Aug 11;275(32):24653-60
10816596
Science. 1991 Jun 21;252(5013):1705-8
1646484
Cancer Metastasis Rev. 1990 Nov;9(3):203-26
1705486
Annu Rev Biochem. 2002;71:435-71
12045103
Nature. 2007 Apr 26;446(7139):1030-7
17460664
Mol Biol Cell. 2002 Jun;13(6):1893-905
12058057
Cell. 1991 Feb 22;64(4):841-8
1847668
J Immunol Methods. 2000 Oct 20;244(1-2):205-15
11033033
Genes Dev. 2007 Feb 1;21(3):316-31
17289920
Nature. 2000 Sep 14;407(6801):249-57
11001068
J Cell Biol. 1984 Dec;99(6):2034-40
6501412
Trends Biochem Sci. 2003 Mar;28(3):145-51
12633994
J Biol Chem. 2002 Mar 29;277(13):10982-6
11790764