title: Vascular endothelial growth factor (VEGF) and platelet (PF-4) factor 4 inputs modulate human microvascular endothelial signaling in a three-dimensional matrix migration context.
abstract: The process of angiogenesis is under complex regulation in adult organisms, particularly as it often occurs in an inflammatory post-wound environment. As such, there are many impacting factors that will regulate the generation of new blood vessels which include not only pro-angiogenic growth factors such as vascular endothelial growth factor, but also angiostatic factors. During initial postwound hemostasis, a large initial bolus of platelet factor 4 is released into localized areas of damage before progression of wound healing toward tissue homeostasis. Because of its early presence and high concentration, the angiostatic chemokine platelet factor 4, which can induce endothelial anoikis, can strongly affect angiogenesis. In our work, we explored signaling crosstalk interactions between vascular endothelial growth factor and platelet factor 4 using phosphotyrosine-enriched mass spectrometry methods on human dermal microvascular endothelial cells cultured under conditions facilitating migratory sprouting into collagen gel matrices. We developed new methods to enable mass spectrometry-based phosphorylation analysis of primary cells cultured on collagen gels, and quantified signaling pathways over the first 48 h of treatment with vascular endothelial growth factor in the presence or absence of platelet factor 4. By observing early and late signaling dynamics in tandem with correlation network modeling, we found that platelet factor 4 has significant crosstalk with vascular endothelial growth factor by modulating cell migration and polarization pathways, centered around P38α MAPK, Src family kinases Fyn and Lyn, along with FAK. Interestingly, we found EphA2 correlational topology to strongly involve key migration-related signaling nodes after introduction of platelet factor 4, indicating an influence of the angiostatic factor on this ambiguous but generally angiogenic signal in this complex environment.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;
authors.forename: Ta-Chun
authors.initials: TC
authors.lastname: Hang
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Tedford
authors.forename: Raven J
authors.initials: RJ
authors.lastname: Reddy
authors.forename: Tharathorn
authors.initials: T
authors.lastname: Rimchala
authors.forename: Alan
authors.initials: A
authors.lastname: Wells
authors.forename: Forest M
authors.initials: FM
authors.lastname: White
authors.forename: Roger D
authors.initials: RD
authors.lastname: Kamm
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Lauffenburger
mesh_terms: D002465:Cell Movement; D003094:Collagen / Q000737:chemistry; D020405:Dermis / Q000098:blood supply / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005109:Extracellular Matrix / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism; D005782:Gels; D005786:Gene Expression Regulation; D006801:Humans; D013058:Mass Spectrometry; D058977:Molecular Sequence Annotation; D018919:Neovascularization, Physiologic; D019000:Phosphotyrosine / Q000378:metabolism; D010978:Platelet Factor 4 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D061251:Primary Cell Culture; D011485:Protein Binding; D036104:Receptor, EphA2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D005782:Gels; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019000:Phosphotyrosine; D010978:Platelet Factor 4; D003094:Collagen; D036104:Receptor, EphA2; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases
references.citation: J Biol Chem. 1995 Mar 31;270(13):7420-6
references.pmid: 7535770
references.citation: Thromb Haemost. 2007 May;97(5):755-62
references.pmid: 17479186
references.citation: Cancer Sci. 2009 Oct;100(10):1786-93
references.pmid: 19622105
references.citation: Blood. 2012 Aug 30;120(9):1942-52
references.pmid: 22806890
references.citation: Nature. 2009 Apr 30;458(7242):1131-5
references.pmid: 19339967
references.citation: Nat Methods. 2005 Apr;2(4):277-83
references.pmid: 15782220
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):109-15
references.pmid: 22040911
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12867-72
references.pmid: 17646646
references.citation: Mol Cancer Res. 2007 Mar;5(3):283-93
references.pmid: 17374733
references.citation: Nucleic Acids Res. 2013 Jan;41(Database issue):D1063-9
references.pmid: 23203882
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):51-7
references.pmid: 22044885
references.citation: Adv Drug Deliv Rev. 2011 Apr 30;63(4-5):375-8
references.pmid: 21167237
references.citation: J Biol Chem. 2008 Sep 26;283(39):26591-601
references.pmid: 18669639
references.citation: Semin Cell Dev Biol. 2012 Feb;23(1):16-25
references.pmid: 22040915
references.citation: Int J Biochem Cell Biol. 2008;40(9):1764-74
references.pmid: 18291705
references.citation: Nat Rev Cancer. 2003 Jun;3(6):411-21
references.pmid: 12778131
references.citation: J Biol Chem. 2008 Oct 24;283(43):29461-72
references.pmid: 18728010
references.citation: J Signal Transduct. 2011;2011:354091
references.pmid: 21637375
references.citation: J Leukoc Biol. 2001 Apr;69(4):513-21
references.pmid: 11310836
references.citation: Mol Cell Proteomics. 2006 Jun;5(6):1146-57
references.pmid: 16546994
references.citation: Electrophoresis. 1999 Dec;20(18):3551-67
references.pmid: 10612281
references.citation: J Mater Sci Mater Med. 1999 Oct-Nov;10(10/11):589-94
references.pmid: 15347971
references.citation: Adv Mater. 2009 Dec 18;21(47):4863-7
references.pmid: 21049511
references.citation: Cytokine Growth Factor Rev. 2011 Feb;22(1):1-18
references.pmid: 21111666
references.citation: EMBO J. 1991 Nov;10(11):3321-9
references.pmid: 1655417
references.citation: J Biol Chem. 2008 May 30;283(22):15328-38
references.pmid: 18378677
references.citation: Cell Struct Funct. 2001 Feb;26(1):11-7
references.pmid: 11345499
references.citation: FASEB J. 2007 Apr;21(4):968-75
references.pmid: 17215481
references.citation: J Cell Sci. 2004 Apr 15;117(Pt 10):2037-49
references.pmid: 15054110
references.citation: Genes Cancer. 2012 May;3(5-6):394-401
references.pmid: 23226577
references.citation: Cancer Res. 2000 Jul 1;60(13):3631-7
references.pmid: 10910078
references.citation: J Clin Invest. 2012 Feb;122(2):674-92
references.pmid: 22251701
references.citation: J Cell Biol. 2002 Nov 25;159(4):673-83
references.pmid: 12446743
references.citation: Thromb Haemost. 2010 Nov;104(5):941-8
references.pmid: 20806113
references.citation: Genes Cancer. 2010 Oct;1(10):994-1007
references.pmid: 21442024
references.citation: World J Urol. 2000 Feb;18(1):10-8
references.pmid: 10766038
references.citation: Biochem Biophys Res Commun. 2007 Mar 30;355(1):1-5
references.pmid: 17276388
references.citation: J Biol Chem. 1995 Dec 15;270(50):29773-80
references.pmid: 8530369
references.citation: Structure. 2005 Apr;13(4):541-50
references.pmid: 15837193
references.citation: FEBS Lett. 2003 Apr 10;540(1-3):7-14
references.pmid: 12681475
references.citation: Methods Mol Biol. 2009;492:143-60
references.pmid: 19241031
references.citation: FASEB J. 1999 Dec;13(15):2235-45
references.pmid: 10593871
references.citation: Protein Sci. 2011 Jul;20(7):1172-81
references.pmid: 21538645
references.citation: Rheum Dis Clin North Am. 2004 Feb;30(1):97-114
references.pmid: 15061570
references.citation: Circ Res. 2006 Mar 17;98(5):617-25
references.pmid: 16484616
references.citation: Oncogene. 2001 Oct 1;20(44):6322-30
references.pmid: 11607835
references.citation: Mol Biol Cell. 2009 Mar;20(6):1737-48
references.pmid: 19158392
references.citation: Cancer Lett. 2008 Aug 28;267(2):226-44
references.pmid: 18579287
references.citation: FASEB J. 2000 Oct;14(13):2055-64
references.pmid: 11023990
references.citation: Curr Issues Mol Biol. 2008;10(1-2):61-6
references.pmid: 18525107
references.citation: Nature. 2011 May 19;473(7347):298-307
references.pmid: 21593862
references.citation: Mol Cell Proteomics. 2010 Nov;9(11):2558-70
references.pmid: 20631208
references.citation: J Proteome Res. 2010 Jan;9(1):393-403
references.pmid: 19888749
references.citation: J Biomed Mater Res B Appl Biomater. 2007 Nov;83(2):408-15
references.pmid: 17415768
references.citation: Adv Drug Deliv Rev. 2011 Jul 18;63(8):610-5
references.pmid: 21118706
references.citation: Stem Cells. 2009 Nov;27(11):2804-14
references.pmid: 19750537
references.citation: Inflamm Res. 2012 Mar;61(3):233-44
references.pmid: 22138711
references.citation: Eur J Biochem. 2004 Aug;271(16):3310-8
references.pmid: 15291808
references.citation: Nat Rev Cancer. 2010 Mar;10(3):165-80
references.pmid: 20179713
references.citation: Arterioscler Thromb Vasc Biol. 2003 Jun 1;23(6):953-64
references.pmid: 12689916
references.citation: Dev Cell. 2004 Oct;7(4):465-80
references.pmid: 15469835
references.citation: Methods Mol Biol. 2009;511:185-91
references.pmid: 19347298
references.citation: Brain Res. 2009 Feb 23;1256:149-61
references.pmid: 19103176
references.citation: Methods Mol Biol. 2007;359:203-12
references.pmid: 17484120
references.citation: Wound Repair Regen. 2008 Sep-Oct;16(5):585-601
references.pmid: 19128254
references.citation: Arterioscler Thromb Vasc Biol. 2008 Nov;28(11):1928-36
references.pmid: 18757292
references.citation: Nature. 2005 Dec 15;438(7070):932-6
references.pmid: 16355210
references.citation: J Cell Biol. 2008 Aug 25;182(4):753-63
references.pmid: 18710921
references.citation: Biomaterials. 2002 Dec;23(24):4739-51
references.pmid: 12361612
references.citation: Crit Rev Immunol. 2012;32(2):127-38
references.pmid: 23216611
references.citation: Nat Cell Biol. 2008 May;10(5):527-37
references.pmid: 18425119
references.citation: J Biol Chem. 1996 Nov 8;271(45):28086-96
references.pmid: 8910422
references.citation: FASEB J. 2003 Dec;17(15):2240-9
references.pmid: 14656986
references.citation: Nature. 2000 Sep 14;407(6801):242-8
references.pmid: 11001067
references.citation: Wound Repair Regen. 2004 Nov-Dec;12(6):635-42
references.pmid: 15555055
references.citation: J Cell Biol. 1994 Nov;127(4):1121-7
references.pmid: 7962072
references.citation: Oncogene. 2008 Dec 11;27(58):7260-73
references.pmid: 18794797
references.citation: J Immunol Methods. 2003 Feb;273(1-2):83-101
references.pmid: 12535800
references.citation: Nat Cell Biol. 2000 Feb;2(2):62-9
references.pmid: 10655584
references.citation: Mol Cell Proteomics. 2011 Nov;10(11):M111.008433
references.pmid: 21832255
references.citation: J Cell Sci. 2004 Aug 1;117(Pt 17):3759-68
references.pmid: 15252114
references.citation: Proc Natl Acad Sci U S A. 2010 Jul 27;107(30):13252-7
references.pmid: 20616078
references.citation: Mol Cell Biol. 1995 Feb;15(2):954-63
references.pmid: 7529876
references.citation: APMIS. 2004 Jul-Aug;112(7-8):481-95
references.pmid: 15563311
references.citation: J Cell Sci. 2009 Jun 15;122(Pt 12):2064-77
references.pmid: 19470579
references.citation: FEBS Lett. 2004 Dec 3;578(1-2):95-8
references.pmid: 15581623
references.citation: Mutat Res. 2008 Jul-Aug;659(1-2):15-30
references.pmid: 18485806
references.citation: Lymphology. 2005 Mar;38(1):1-8
references.pmid: 15856679
references.citation: Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17886-91
references.pmid: 19805119
references.citation: J Cell Biol. 2003 Aug 18;162(4):661-71
references.pmid: 12925710
references.citation: Tissue Eng Part B Rev. 2009 Jun;15(2):159-69
references.pmid: 19309238
references.citation: Cancer Biol Ther. 2006 Feb;5(2):228-33
references.pmid: 16481735
references.citation: J Biol Chem. 2008 Jun 6;283(23):16017-26
references.pmid: 18387945
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10762-7
references.pmid: 18669648
references.citation: Eur J Cell Biol. 2011 Jun-Jul;90(6-7):521-6
references.pmid: 21295372
references.citation: Mol Cancer Res. 2002 Nov;1(1):79-87
references.pmid: 12496371
references.citation: J Cell Sci. 2000 Apr;113 ( Pt 8):1319-34
references.pmid: 10725216
references.citation: Br J Pharmacol. 2009 Oct;158(4):936-46
references.pmid: 19845684
references.citation: Genes Cells. 2010 Jun;15(7):711-724
references.pmid: 20545765
references.citation: Biochem J. 2012 Aug 1;445(3):323-32
references.pmid: 22587391
references.citation: J Biol Chem. 2007 Feb 2;282(5):3173-81
references.pmid: 17095507
references.citation: Cancer Cell. 2009 Jul 7;16(1):9-20
references.pmid: 19573808
references.citation: Blood. 2007 Sep 15;110(6):1730-8
references.pmid: 17537990
references.citation: Tissue Eng Part A. 2009 Nov;15(11):3297-308
references.pmid: 19382873
references.citation: J Neurochem. 2009 Apr;109(2):297-302
references.pmid: 19187442
references.citation: Int J Biochem Cell Biol. 2005 Jun;37(6):1162-7
references.pmid: 15778080
references.citation: Microbiol Mol Biol Rev. 2004 Jun;68(2):320-44
references.pmid: 15187187
references.citation: Mol Cancer Res. 2002 Nov;1(1):2-11
references.pmid: 12496364
references.citation: Integr Biol (Camb). 2013 Mar;5(3):510-22
references.pmid: 23303249
references.citation: J Biol Chem. 2002 Oct 18;277(42):39274-9
references.pmid: 12167657
references.citation: Physiol Rev. 2003 Jul;83(3):835-70
references.pmid: 12843410
references.citation: PLoS One. 2012;7(7):e40429
references.pmid: 22792315
references.citation: Angiogenesis. 2005;8(4):315-26
references.pmid: 16400523