title: Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress.
abstract: Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration.
authors.affiliation: Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin; and.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Hoffmann
authors.forename: Jordan R
authors.initials: JR
authors.lastname: Wagner
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Prisco
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Janiak
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Greene
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism*; D015687:Cell Hypoxia; D002465:Cell Movement / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D013058:Mass Spectrometry / Q000379:methods; D008957:Models, Genetic; D018919:Neovascularization, Physiologic / Q000235:genetics; D010446:Peptide Fragments / Q000494:pharmacology; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D013312:Stress, Physiological / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D042461:Vascular Endothelial Growth Factor A; C511613:vascular endothelial growth factor A (138-165); D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: endothelial progenitor cells; hypoxia; mass spectrometry; signaling; vascular endothelial growth factor
references.citation: Proteomics. 2011 Mar;11(6):1058-63
references.pmid: 21365761
references.citation: Physiol Genomics. 2012 Oct 2;44(19):925-33
references.pmid: 22851760
references.citation: Mol Cell Proteomics. 2011 Jan;10(1):M110.002212
references.pmid: 20736410
references.citation: Asian Cardiovasc Thorac Ann. 2004 Jun;12(2):173-81
references.pmid: 15213090
references.citation: BMC Res Notes. 2011 Dec 29;4:569
references.pmid: 22206604
references.citation: Proc Natl Acad Sci U S A. 1990 Apr;87(7):2628-32
references.pmid: 2320579
references.citation: Expert Rev Proteomics. 2004 Oct;1(3):327-41
references.pmid: 15966829
references.citation: Cell Res. 2008 Jul;18(7):792-9
references.pmid: 18560380
references.citation: Mol Cell Proteomics. 2010 Sep;9(9):1849-65
references.pmid: 20460255
references.citation: Nat Rev Mol Cell Biol. 2006 Mar;7(3):177-87
references.pmid: 16496020
references.citation: Circ Res. 2002 Jul 12;91(1):25-31
references.pmid: 12114318
references.citation: J Clin Invest. 2001 Jul;108(1):39-40
references.pmid: 11435455
references.citation: Curr Opin Pharmacol. 2011 Apr;11(2):162-74
references.pmid: 21296616
references.citation: Leukemia. 2001 Jun;15(6):976-80
references.pmid: 11417486
references.citation: Science. 1997 Feb 14;275(5302):964-7
references.pmid: 9020076
references.citation: Am J Respir Crit Care Med. 2002 Jan 1;165(1):67-70
references.pmid: 11779732
references.citation: Proteome Sci. 2011 Sep 15;9:53
references.pmid: 21917185
references.citation: Biochem J. 2011 Jul 15;437(2):169-83
references.pmid: 21711246
references.citation: Clin Sci (Lond). 2009 Feb;116(4):301-14
references.pmid: 19138171
references.citation: Adv Exp Med Biol. 2012;758:1-5
references.pmid: 23080136
references.citation: Circ Res. 2001 Jul 6;89(1):E1-7
references.pmid: 11440984
references.citation: Nat Rev Mol Cell Biol. 2006 May;7(5):359-71
references.pmid: 16633338
references.citation: Proteomics. 2007 Jul;7(14):2371-4
references.pmid: 17623297
references.citation: J Mol Cell Cardiol. 2007 Jun;42(6):1036-44
references.pmid: 17498737
references.citation: PLoS Comput Biol. 2011 Mar;7(3):e1001099
references.pmid: 21408212
references.citation: Hypertens Res. 2004 Feb;27(2):101-8
references.pmid: 15005273
references.citation: Philos Trans A Math Phys Eng Sci. 2010 Jun 28;368(1921):2937-60
references.pmid: 20478915
references.citation: Am J Physiol Heart Circ Physiol. 2003 May;284(5):H1528-35
references.pmid: 12521947
references.citation: Circ Res. 2012 Feb 17;110(4):624-37
references.pmid: 22343557
references.citation: Am J Physiol Heart Circ Physiol. 2000 Aug;279(2):H817-24
references.pmid: 10924082
references.citation: J Biol Chem. 1994 Oct 14;269(41):25646-54
references.pmid: 7929268
references.citation: PLoS Comput Biol. 2009 Dec;5(12):e1000622
references.pmid: 20041209
references.citation: Cancer Res. 2000 Nov 15;60(22):6248-52
references.pmid: 11103778
references.citation: Am J Physiol Heart Circ Physiol. 2008 Jun;294(6):H2814-21
references.pmid: 18441208
references.citation: Clin Exp Pharmacol Physiol. 2009 Oct;36(10):e47-56
references.pmid: 19558529
references.citation: Sci STKE. 2000 Jun 20;2000(37):pl1
references.pmid: 11752594
references.citation: J Cell Biochem. 2007 Oct 15;102(3):580-92
references.pmid: 17668425
references.citation: Nat Med. 2003 Jul;9(7):936-43
references.pmid: 12796773
references.citation: J Mol Cell Cardiol. 2007 Feb;42(2):441-8
references.pmid: 17187821
references.citation: J Cell Biol. 2010 Feb 22;188(4):595-609
references.pmid: 20176926
references.citation: Annu Rev Biochem. 2001;70:437-73
references.pmid: 11395414
references.citation: FASEB J. 2010 Jun;24(6):1981-8
references.pmid: 20056714
references.citation: Am J Physiol Lung Cell Mol Physiol. 2002 Nov;283(5):L1133-42
references.pmid: 12376368
references.citation: Endocr Rev. 1997 Feb;18(1):4-25
references.pmid: 9034784
references.citation: J Biol Chem. 1997 Sep 19;272(38):23659-67
references.pmid: 9295307
references.citation: Clin Sci (Lond). 2012 Aug 1;123(4):205-23
references.pmid: 22548406
references.citation: Kidney Int. 2005 Feb;67(2):449-57
references.pmid: 15673292
references.citation: Anal Biochem. 2007 Oct 15;369(2):241-7
references.pmid: 17624292
references.citation: Curr Opin Cell Biol. 2012 Apr;24(2):188-93
references.pmid: 22366328
references.citation: Cold Spring Harb Perspect Med. 2012 Jul;2(7):a006502
references.pmid: 22762016
references.citation: J Mol Cell Cardiol. 2011 Feb;50(2):266-72
references.pmid: 20673769
references.citation: Cell. 2008 Jan 25;132(2):299-310
references.pmid: 18243104
references.citation: J Med Chem. 2012 Oct 11;55(19):8260-71
references.pmid: 22970990
references.citation: Clin Sci (Lond). 2009 Feb;116(4):283-99
references.pmid: 19138170
references.citation: Physiol Genomics. 2010 Aug;42(3):437-44
references.pmid: 20501694
references.citation: Am J Physiol Cell Physiol. 2001 Jun;280(6):C1358-66
references.pmid: 11350730
references.citation: Microcirculation. 2010 May;17(4):259-70
references.pmid: 20536739
references.citation: EMBO J. 1989 Dec 1;8(12):3801-6
references.pmid: 2684646
references.citation: Biol Pharm Bull. 2010;33(9):1550-4
references.pmid: 20823573
references.citation: Eur J Pharmacol. 2007 Jul 30;568(1-3):222-30
references.pmid: 17553485
references.citation: Curr Opin Nephrol Hypertens. 1996 Jan;5(1):35-44
references.pmid: 8834160
references.citation: EXS. 1997;79:209-32
references.pmid: 9002234
references.citation: Anal Chem. 2010 Aug 15;82(16):6958-68
references.pmid: 20704385
references.citation: Ann Thorac Surg. 2000 Sep;70(3):829-34
references.pmid: 11016318
references.citation: EMBO J. 1999 Jul 15;18(14):3964-72
references.pmid: 10406801
references.citation: Nature. 1995 Jul 6;376(6535):66-70
references.pmid: 7596436
references.citation: Nat Methods. 2010 Feb;7(2):148-55
references.pmid: 20101245
references.citation: J Clin Invest. 1996 Jan 15;97(2):469-76
references.pmid: 8567969