Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context
2013; Elsevier BV; Volume: 12; Issue: 12 Linguagem: Inglês
10.1074/mcp.m113.030528
ISSN1535-9484
AutoresTa-Chun Hang, Nathan C. Tedford, Raven J. Reddy, Tharathorn Rimchala, Alan Wells, Forest M. White, Roger D. Kamm, Douglas A. Lauffenburger,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThe 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. 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. Angiogenesis, the formation of blood vessels from pre-existing blood vessels, is a complex process essential for repairing injured tissue or supporting tissue growth. A great deal of work has been done to focus on understanding this phenomenon as it occurs in vivo, in particular with regard to its roles in embryonic development (1Carmeliet P. Angiogenesis in life, disease and medicine.Nature. 2005; 438: 932-936Crossref PubMed Scopus (2591) Google Scholar, 2Saharinen P. Eklund L. Miettinen J. Wirkkala R. Anisimov A. Winderlich M. Nottebaum A. Vestweber D. Deutsch U. Koh G.Y. Olsen B.R. Alitalo K. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts.Nat. Cell Biol. 2008; 10: 527-537Crossref PubMed Scopus (310) Google Scholar, 3Singh R. Kaushik S. Wang Y. Xiang Y. Novak I. Komatsu M. Tanaka K. Cuervo A.M. Czaja M.J. Autophagy regulates lipid metabolism.Nature. 2009; 458: 1131-1135Crossref PubMed Scopus (2109) Google Scholar, 4Adair T.H. Montani J.-P. Angiogenesis. Morgan & Claypool Life Sciences, San Rafael, CA2010Google Scholar, 5Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Vascular-specific growth factors and blood vessel formation.Nature. 2000; 407: 242-248Crossref PubMed Scopus (3120) Google Scholar). In contrast to embryonic development, adult angiogenesis and inflammation are closely related phenomena that occur in vivo in a number of physiologically relevant processes. Inflammation lies at the crux of multiple physiological events in biological systems that precede the induction of angiogenesis: wound healing (6Barrientos S. Stojadinovic O. Golinko M.S. Brem H. Tomic-Canic M. Growth factors and cytokines in wound healing.Wound Repair Regen. 2008; 16: 585-601Crossref PubMed Scopus (1894) Google Scholar, 7Gillitzer R. Goebeler M. Chemokines in cutaneous wound healing.J. Leukoc. Biol. 2001; 69: 513-521Crossref PubMed Google Scholar, 8Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol. Rev. 2003; 83: 835-870Crossref PubMed Google Scholar), chronic wounds (8Werner S. Grose R. Regulation of wound healing by growth factors and cytokines.Physiol. Rev. 2003; 83: 835-870Crossref PubMed Google Scholar), inflammatory disorders (9Kobayashi H. Lin P.C. Angiogenesis links chronic inflammation with cancer.Methods Mol. Biol. 2009; 511: 185-191Crossref PubMed Scopus (28) Google Scholar, 10Szekanecz Z. Koch A.E. Vascular endothelium and immune responses: implications for inflammation and angiogenesis.Rheum. Dis. Clin. North Am. 2004; 30: 97-114Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and cancer (9Kobayashi H. Lin P.C. Angiogenesis links chronic inflammation with cancer.Methods Mol. Biol. 2009; 511: 185-191Crossref PubMed Scopus (28) Google Scholar, 11Kundu J.K. Surh Y.-J. Inflammation: gearing the journey to cancer.Mutat. Res. 2008; 659: 15-30Crossref PubMed Scopus (609) Google Scholar, 12Carmeliet P. Jain R.K. Molecular mechanisms and clinical applications of angiogenesis.Nature. 2011; 473: 298-307Crossref PubMed Scopus (3087) Google Scholar). Inflammatory reactions also confound tissue engineered implantable three-dimensional constructs that provide innovative clinical treatments of various diseases and injuries (13Sahota P.S. Burn J.L. Brown N.J. MacNeil S. Approaches to improve angiogenesis in tissue-engineered skin.Wound Repair Regen. 2004; 12: 635-642Crossref PubMed Scopus (0) Google Scholar, 14Feil G. Daum L. Amend B. Maurer S. Renninger M. Vaegler M. Seibold J. Stenzl A. Sievert K.-D. From tissue engineering to regenerative medicine in urology–the potential and the pitfalls.Adv. Drug Deliv. Rev. 2011; 63: 375-378Crossref PubMed Scopus (19) Google Scholar, 15Sherwood J.K. Riley S.L. Palazzolo R. Brown S.C. Monkhouse D.C. Coates M. Griffith L.G. Landeen L.K. Ratcliffe A. A three-dimensional osteochondral composite scaffold for articular cartilage repair.Biomaterials. 2002; 23: 4739-4751Crossref PubMed Scopus (453) Google Scholar, 16Ban D.-X. Kong X.-H. Feng S.-Q. Ning G.-Z. Chen J.-T. Guo S.-F. Intraspinal cord graft of autologous activated Schwann cells efficiently promotes axonal regeneration and functional recovery after rat's spinal cord injury.Brain Res. 2009; 1256: 149-161Crossref PubMed Scopus (0) Google Scholar, 17Hu X. Huang J. Ye Z. Xia L. Li M. Lv B. Shen X. Luo Z. A novel scaffold with longitudinally oriented microchannels promotes peripheral nerve regeneration.Tissue Eng. 2009; 15: 3297-3308Crossref Scopus (85) Google Scholar). As complex tissues become developed for applications in clinical trials, tissue vascularization for constructs of considerable size and volume is required for their survival (18Kaully T. Kaufman-Francis K. Lesman A. Levenberg S. Vascularization–the conduit to viable engineered tissues.Tissue Eng. 2009; 15: 159-169Crossref Scopus (0) Google Scholar, 19Soker S. Machado M. Atala A. Systems for therapeutic angiogenesis in tissue engineering.World J. Urol. 2000; 18: 10-18Crossref PubMed Google Scholar). Once implanted, these constructs will also experience significant inflammatory responses within their host's local milieu (20Kirkpatrick C.J. Otto M. Van Kooten T. Krump V. Kriegsmann J. Bittinger F. Endothelial cell cultures as a tool in biomaterial research.J. Mater. Sci. Mater. Med. 1999; 10: 589-594Crossref PubMed Google Scholar, 21Mendes J.B. Campos P.P. Ferreira M.A.N.D. Bakhle Y.S. Andrade S.P. Host response to sponge implants differs between subcutaneous and intraperitoneal sites in mice.J. Biomed. Mater. Res. 2007; 83: 408-415Crossref Scopus (31) Google Scholar). These circumstances demonstrate the necessity for understanding the interactions between inflammation and angiogenesis, such as the development of predictive models (22Rimchala T. Kamm R.D. Lauffenburger D.A. Endothelial cell phenotypic behaviors cluster into dynamic state transition programs modulated by angiogenic and angiostatic cytokines.Integr. Biol. 2013; 5: 510-522Crossref Scopus (5) Google Scholar). Elucidating specific intracellular mechanisms can provide insight for novel approaches in treatment of diseases as well as predicting responses to artificially engineered tissues. Recently, studies have shown that chemokines, which play a central role in inflammation, can influence the outcomes of angiogenesis (23Bernardini G. Ribatti D. Spinetti G. Morbidelli L. Ziche M. Santoni A. Capogrossi M.C. Napolitano M. Analysis of the role of chemokines in angiogenesis.J. Immunol. Methods. 2003; 273: 83-101Crossref PubMed Scopus (0) Google Scholar, 24Keeley E.C. Mehrad B. Strieter R.M. Chemokines as mediators of neovascularization.Arteriosclerosis, Thrombosis, Vasc. Biol. 2008; 28: 1928-1936Crossref PubMed Scopus (0) Google Scholar, 25Mehrad B. Keane M.P. Strieter R.M. Chemokines as mediators of angiogenesis.Thromb. Haemost. 2007; 97: 755-762Crossref PubMed Scopus (109) Google Scholar, 26Rosenkilde M.M. Schwartz T.W. The chemokine system – a major regulator of angiogenesis in health and disease.APMIS. 2004; 112: 481-495Crossref PubMed Scopus (0) Google Scholar) by promoting new blood vessel growth (e.g. CXCL1–3, CXCL5–8, CXCL12) or inhibiting its formation altogether (e.g. CXCL4, CXCL9–11, CXCL13) (26Rosenkilde M.M. Schwartz T.W. The chemokine system – a major regulator of angiogenesis in health and disease.APMIS. 2004; 112: 481-495Crossref PubMed Scopus (0) Google Scholar). In particular, a large body of information is available on platelet factor 4 (PF-4/CXCL4) and its ability to inhibit and even induce regression of angiogenesis. PF-4 is found throughout the adult body, at roughly 0.25–1.25 nm (2–10 ng/ml) in blood plasma, but as high as 25 μm in localized areas during wound healing (27Slungaard A. Platelet factor 4: a chemokine enigma.Int. J. Biochem. Cell Biol. 2005; 37: 1162-1167Crossref PubMed Scopus (56) Google Scholar, 28Aidoudi S. Bikfalvi A. Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.Thromb. Haemost. 2010; 104: 941-948Crossref PubMed Scopus (46) Google Scholar). Its ubiquitous presence, implication in cancer and vascular diseases, and use as a potential drug therapy have made PF-4 a key point of interest in influencing angiogenesis in vivo (27Slungaard A. Platelet factor 4: a chemokine enigma.Int. J. Biochem. Cell Biol. 2005; 37: 1162-1167Crossref PubMed Scopus (56) Google Scholar, 28Aidoudi S. Bikfalvi A. Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.Thromb. Haemost. 2010; 104: 941-948Crossref PubMed Scopus (46) Google Scholar, 29Vandercappellen J. Van Damme J. Struyf S. The role of the CXC chemokines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer.Cytokine Growth Factor Rev. 2011; 22: 1-18Crossref PubMed Scopus (104) Google Scholar, 30Vandercappellen J. Van Damme J. Struyf S. The role of CXC chemokines and their receptors in cancer.Cancer Letters. 2008; 267: 226-244Crossref PubMed Scopus (0) Google Scholar). In addition to inducing angiostasis, PF-4 can inhibit cell proliferation by halting S phase progression and reducing endothelial cell migration (25Mehrad B. Keane M.P. Strieter R.M. Chemokines as mediators of angiogenesis.Thromb. Haemost. 2007; 97: 755-762Crossref PubMed Scopus (109) Google Scholar, 28Aidoudi S. Bikfalvi A. Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.Thromb. Haemost. 2010; 104: 941-948Crossref PubMed Scopus (46) Google Scholar, 30Vandercappellen J. Van Damme J. Struyf S. The role of CXC chemokines and their receptors in cancer.Cancer Letters. 2008; 267: 226-244Crossref PubMed Scopus (0) Google Scholar, 31Shao X.J. Xie F.M. Influence of angiogenesis inhibitors, endostatin and PF-4, on lymphangiogenesis.Lymphology. 2005; 38: 1-8PubMed Google Scholar, 32Gupta S.K. Singh J.P. Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression.J. Cell Biol. 1994; 127: 1121-1127Crossref PubMed Scopus (106) Google Scholar). Despite the wealth of information on PF-4 and its mechanistic effects on immune cells, scarce literature exists on the nature of the molecular signaling with endothelial cells to inhibit angiogenesis. Furthermore, the complexity of PF-4 mediated signaling and its potential to interact through multiple binding mechanisms makes it difficult to determine how PF-4 can interfere with angiogenesis (28Aidoudi S. Bikfalvi A. Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.Thromb. Haemost. 2010; 104: 941-948Crossref PubMed Scopus (46) Google Scholar, 29Vandercappellen J. Van Damme J. Struyf S. The role of the CXC chemokines platelet factor-4 (CXCL4/PF-4) and its variant (CXCL4L1/PF-4var) in inflammation, angiogenesis and cancer.Cytokine Growth Factor Rev. 2011; 22: 1-18Crossref PubMed Scopus (104) Google Scholar, 33Sulpice E. Contreres J.-O. Lacour J. Bryckaert M. Tobelem G. Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms.Eur. J. Biochem. 2004; 271: 3310-3318Crossref PubMed Scopus (21) Google Scholar, 34Tabruyn S.P. Griffioen A.W. Molecular pathways of angiogenesis inhibition.Biochem. Biophys. Res. Commun. 2007; 355: 1-5Crossref PubMed Scopus (0) Google Scholar). Possible angiogenic signaling network interference mechanisms for PF-4 include the sequestration of growth factors and proteoglycans, antagonism of integrin-mediated signaling, or direct signaling through its chemokine receptor CXCR3, all of which have supporting evidence in previous literature (28Aidoudi S. Bikfalvi A. Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.Thromb. Haemost. 2010; 104: 941-948Crossref PubMed Scopus (46) Google Scholar). Along with the multiple mechanisms PF-4 may utilize for signaling, only limited studies on direct signaling elicited by PF-4 on endothelial cells have been reported; one of interest found that P38 MAPK can be activated via CXCR3 on endothelial cells cultured on plastic (35Petrai I. Rombouts K. Lasagni L. Annunziato F. Cosmi L. Romanelli R.G. Sagrinati C. Mazzinghi B. Pinzani M. Romagnani S. Romagnani P. Marra F. Activation of p38(MAPK) mediates the angiostatic effect of the chemokine receptor CXCR3-B.Int. J. Biochem. Cell Biol. 2008; 40: 1764-1774Crossref PubMed Scopus (51) Google Scholar), whereas another, more definitive study showed PF-4 acting similarly to other CXCR3 ligands in activating PKA to prevent m-calpain-mediated rear de-adhesion of moving cells (36Bodnar R.J. Yates C.C. Wells A. IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain.Circulation Res. 2006; 98: 617-625Crossref PubMed Scopus (0) Google Scholar, 37Bodnar R.J. Yates C.C. Rodgers M.E. Du X. Wells A. IP-10 induces dissociation of newly formed blood vessels.J. Cell Sci. 2009; 122: 2064-2077Crossref PubMed Scopus (90) Google Scholar). Furthermore, PF-4 could have variable sensitivities in different endothelial cell types because of heterogeneous expression of CXCR3 (38Salcedo R. Resau J.H. Halverson D. Hudson E.A. Dambach M. Powell D. Wasserman K. Oppenheim J.J. Differential expression and responsiveness of chemokine receptors (CXCR1–3) by human microvascular endothelial cells and umbilical vein endothelial cells.FASEB J. 2000; 14: 2055-2064Crossref PubMed Google Scholar). In our study, we sought to develop an approach to assess network-level signaling interactions between PF-4 and the major angiogenic inducer vascular endothelial growth factor (VEGF) 1The abbreviations used are:ANXNA1Annexin A1BCAR-1Breast Cancer Anti-Estrogen Resistance 1Cas1CDK1, Cyclin Dependent Kinase 1CTNND1Catenin Delta 1 Isoform 1A (p120CAS)DYRK1BDual-Specificity Tyrosine-Phosphorylation Regulated Kinase 1BDTTDithiothreitolEBMEndothelial Basal MediaEGMEndothelial Growth MediaEphA2Ephrin receptor EphA2ephA1Ephrin ligand A1FAKFocal Adhesion Kinase/Protein Tyrosine Kinase 2 (PTK2)FBSFetal Bovine SerumHDMVECHuman Dermal Microvascular Endothelial CellLC/MS/MSLiquid chromatography tandem mass spectrometryMAPKMitogen Activating Protein KinaseAkt/PKBProtein Kinase BP38αP38α MAPK/Mitogen-Activated Protein Kinase 14PI3KPhosphatidylinositide-3-kinasePRP4KSerine/Threonine Protein Kinase PRP4KErbB2IPErbB2 Interacting ProteinMPZL1Myelin Protein Zero-Like 1CD31Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM-1)IPImmunoprecipitationPBS-T0.1% Tween in 1X PBSPF-4Platelet Factor 4PKAProtein Kinase APTPRαProtein Tyrosine Phosphatase; Receptor Type AlphaPTPRεProtein Tyrosine Phosphatase; Receptor Type EpsilonPXNPaxillinRTKReceptor Tyrosine KinaseSHC1SHC transforming protein 1SHP2Protein Tyrosine Phosphatase; Non-receptor Type 11 (PTPN11)VEGFVascular Endothelial Growth Factor. 1The abbreviations used are:ANXNA1Annexin A1BCAR-1Breast Cancer Anti-Estrogen Resistance 1Cas1CDK1, Cyclin Dependent Kinase 1CTNND1Catenin Delta 1 Isoform 1A (p120CAS)DYRK1BDual-Specificity Tyrosine-Phosphorylation Regulated Kinase 1BDTTDithiothreitolEBMEndothelial Basal MediaEGMEndothelial Growth MediaEphA2Ephrin receptor EphA2ephA1Ephrin ligand A1FAKFocal Adhesion Kinase/Protein Tyrosine Kinase 2 (PTK2)FBSFetal Bovine SerumHDMVECHuman Dermal Microvascular Endothelial CellLC/MS/MSLiquid chromatography tandem mass spectrometryMAPKMitogen Activating Protein KinaseAkt/PKBProtein Kinase BP38αP38α MAPK/Mitogen-Activated Protein Kinase 14PI3KPhosphatidylinositide-3-kinasePRP4KSerine/Threonine Protein Kinase PRP4KErbB2IPErbB2 Interacting ProteinMPZL1Myelin Protein Zero-Like 1CD31Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM-1)IPImmunoprecipitationPBS-T0.1% Tween in 1X PBSPF-4Platelet Factor 4PKAProtein Kinase APTPRαProtein Tyrosine Phosphatase; Receptor Type AlphaPTPRεProtein Tyrosine Phosphatase; Receptor Type EpsilonPXNPaxillinRTKReceptor Tyrosine KinaseSHC1SHC transforming protein 1SHP2Protein Tyrosine Phosphatase; Non-receptor Type 11 (PTPN11)VEGFVascular Endothelial Growth Factor. within a contextually relevant 3-D angiogenesis platform, in a controlled environment to understand what role these two factors may play. We developed methods to reduce extracellular matrix contamination in our samples and were able to successfully use a two-step lysis method with a MS compatible detergent-based lysis buffer. By taking advantage of iTRAQ-based multiplexed quantitation, we were able to collect quantitative phosphoprotein signaling data from our system with early and late temporal resolution. Using correlation network methods to observe differences in our system, we found that simultaneous treatment with PF-4 and VEGF induced changes in migrational pathway topology when compared with VEGF treatment alone. Most often, these changes appeared as losses in correlations between different migrational signaling proteins. We found that several different signaling pathways involved with migration were affected, including central proteins P38α MAPK, focal adhesion kinase (FAK), and Src family kinases. Furthermore, we found statistically significant differences in tyrosine phosphorylation when HDMVECs were stimulated with VEGF and PF-4, as opposed to only VEGF. In addition, we were able to recapitulate previously reported findings on how PF-4 infers its angiostatic effects on endothelial cells. Surprisingly, our data set revealed EphA2 receptor as a central node for PF-4 signaling, indicating that it may possess a complementary role in the balance of angiogenic and angiostatic effects. Annexin A1 Breast Cancer Anti-Estrogen Resistance 1 CDK1, Cyclin Dependent Kinase 1 Catenin Delta 1 Isoform 1A (p120CAS) Dual-Specificity Tyrosine-Phosphorylation Regulated Kinase 1B Dithiothreitol Endothelial Basal Media Endothelial Growth Media Ephrin receptor EphA2 Ephrin ligand A1 Focal Adhesion Kinase/Protein Tyrosine Kinase 2 (PTK2) Fetal Bovine Serum Human Dermal Microvascular Endothelial Cell Liquid chromatography tandem mass spectrometry Mitogen Activating Protein Kinase Protein Kinase B P38α MAPK/Mitogen-Activated Protein Kinase 14 Phosphatidylinositide-3-kinase Serine/Threonine Protein Kinase PRP4K ErbB2 Interacting Protein Myelin Protein Zero-Like 1 Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM-1) Immunoprecipitation 0.1% Tween in 1X PBS Platelet Factor 4 Protein Kinase A Protein Tyrosine Phosphatase; Receptor Type Alpha Protein Tyrosine Phosphatase; Receptor Type Epsilon Paxillin Receptor Tyrosine Kinase SHC transforming protein 1 Protein Tyrosine Phosphatase; Non-receptor Type 11 (PTPN11) Vascular Endothelial Growth Factor. Annexin A1 Breast Cancer Anti-Estrogen Resistance 1 CDK1, Cyclin Dependent Kinase 1 Catenin Delta 1 Isoform 1A (p120CAS) Dual-Specificity Tyrosine-Phosphorylation Regulated Kinase 1B Dithiothreitol Endothelial Basal Media Endothelial Growth Media Ephrin receptor EphA2 Ephrin ligand A1 Focal Adhesion Kinase/Protein Tyrosine Kinase 2 (PTK2) Fetal Bovine Serum Human Dermal Microvascular Endothelial Cell Liquid chromatography tandem mass spectrometry Mitogen Activating Protein Kinase Protein Kinase B P38α MAPK/Mitogen-Activated Protein Kinase 14 Phosphatidylinositide-3-kinase Serine/Threonine Protein Kinase PRP4K ErbB2 Interacting Protein Myelin Protein Zero-Like 1 Platelet/Endothelial Cell Adhesion Molecule 1 (PECAM-1) Immunoprecipitation 0.1% Tween in 1X PBS Platelet Factor 4 Protein Kinase A Protein Tyrosine Phosphatase; Receptor Type Alpha Protein Tyrosine Phosphatase; Receptor Type Epsilon Paxillin Receptor Tyrosine Kinase SHC transforming protein 1 Protein Tyrosine Phosphatase; Non-receptor Type 11 (PTPN11) Vascular Endothelial Growth Factor. To our knowledge, this is the first attempt at performing MS-based analysis of phosphotyrosine signaling networks within the context of an environment that is amenable to angiogenesis. Our work provides a step forward in applying high throughput and systems-level phosphoproteomics data collection to more physiologically relevant experimental conditions. Adult HDMVECs were purchased at passage five (Lonza, Walkersville, MD). Cells were cultured in EGM-2MV (Lonza) medium until near confluency. Once near confluency, cells were rinsed with 1× PBS (Invitrogen, Grand Island, NY) and detached by incubating with 0.05% Trypsin-EDTA (Invitrogen) for 3–5 min at 37 °C and 5% CO2. Trypsin was neutralized by the addition of EBM-2 (Lonza) with 5% FBS (Thermo Fisher Scientific/Hyclone, Logan, UT) and 50 μg/ml gentamicin (Sigma-Aldrich, St. Louis, MO). Cells were pelleted at 450 × g for 5 min and resuspended in EGM-2MV before being seeded onto 50 μg/ml rat tail collagen I (BD Biosciences, Bedford, MA) coated tissue culture flasks at 5000 cells/cm2. Medium was changed 24 h following seeding and replaced once every 48 h until nearing confluency. HDMVEC were grown up to passage 9 for use. Collagen gels cultures were used to study capillary sprouting, as performed in a parallel study on HDMVEC migrational phenotype (22Rimchala T. Kamm R.D. Lauffenburger D.A. Endothelial cell phenotypic behaviors cluster into dynamic state transition programs modulated by angiogenic and angiostatic cytokines.Integr. Biol. 2013; 5: 510-522Crossref Scopus (5) Google Scholar). Collagen gel solutions were made using 10× PBS (10%), collagen solution stock, and 1N NaOH (Sigma-Aldrich) added at 2.3% of the collagen stock solution volume used. Sterile MilliQ purified water was added to reach the desired total volume and kept on ice until use. One milliliter of collagen gel solution was added to each well in six-well plates (9.6 cm2 per well). Gels were formed at a density of 2.0 mg/ml at approximately a pH of 7.2, and allowed to set at 37 °C for 2 h before being rinsed with 1× PBS. Gels were then preconditioned with EBM-2 with 5% FBS and 50 μg/ml gentamicin for 2 days to minimize background changes and nonspecific ligand binding when dosing conditions were introduced to cells seeded on the gels. HDMVEC were thawed and grown on 50 μg/ml collagen I coated tissue culture flasks up to passage 9, collected as above and counted using Neubauer-improved disposable C-Chip hemocytometers (INCYTO, Seoul, Korea) and seeded onto collagen gels at 50,000 cells/cm2. Cells were allowed to adhere for 4–6 h at 37 °C in 5% CO2 before rinsing with PBS and replacing the media with EBM-2 + 5% FBS and gentamicin. HDMVEC were allowed to incubate overnight. 24 h after seeding, plates of HDMVEC were dosed with 20 ng/ml VEGF-165 (Peprotech, Rocky Hill, NJ) and with or without 500 ng/ml PF-4 (Peprotech) across designated time intervals (0 min, 15 min, 30 min, 60 min, 6 h, 24 h, and 48 h). The concentration of PF-4 was selected based on results from previous work on HDMVEC (36Bodnar R.J. Yates C.C. Wells A. IP-10 blocks vascular endothelial growth factor-induced endothelial cell motility and tube formation via inhibition of calpain.Circulation Res. 2006; 98: 617-625Crossref PubMed Scopus (0) Google Scholar, 37Bodnar R.J. Yates C.C. Rodgers M.E. Du X. Wells A. IP-10 induces dissociation of newly formed blood vessels.J. Cell Sci. 2009; 122: 2064-2077Crossref PubMed Scopus (90) Google Scholar). In addition to significant dilution of precursor strength (see phosphotyrosine mass spectrometry and manual validation of mass spectral fragments in Supplemental Data), high error amplification, and previously reported dependence of the angiostatic behavior of PF-4 on the presence of angiogenic growth factors (28Aidoudi S. Bikfalvi A. Interaction of PF4 (CXCL4) with the vasculature: a role in atherosclerosis and angiogenesis.Thromb. Haemost. 2010; 104: 941-948Crossref PubMed Scopus (46) Google Scholar), the inclusion of a no VEGF (0 ng/ml) condition was omitted following initial testing. Collagen gels were formed on 24 well glass bottom MatTek plates (MatTek Corporation, Ashland, MA) and HDMVEC were seeded following the protocol described above. Four hours after seeding, media were replaced with EGM-2MV + 20 ng/ml VEGF (40 ng/ml total). After 72 h, samples were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 30 min and rinsed gently with PBS before being permeabilized with 0.1% Triton-X (Sigma-Aldrich) in PBS. The fixed samples were incubated with 1:100 Alexa 568-conjugated phalloidin (Invitrogen) for actin filaments and 1:200 rabbit polyclonal anti-vascular endothelial cadherin (VE-cadherin) antibody (Enzo Life Sciences, Farmingdale, NY) in 1× PBS with 0.5% BSA for 1 h before being rinsed with PBS. Samples were then stained with 1:1000 DAPI (Invitrogen) and imaged on an Olympus FV1000 Multiphoton Laser Scanning Confocal Microscope using Imaris 7.0.0 (Bitplane, South Windsor, CT). These follow protocols as described previously (22Rimchala T. Kamm R.D. Lauffenburger D.A. Endothelial cell phenotypic behaviors cluster into dynamic state transition programs modulated by angiogenic and angiostatic cytokines.Integr. Biol. 2013; 5: 510-522Crossref Scopus (5) Google Scholar, 39Chung S. Sudo R. Zervantonakis I.K. Rimchala T. Kamm R.D. Surface-treatment-induced three-dimensional capillary morphogenesis in a microfluidic platform.Adv. Mater. Weinheim. 2009; 21: 4863-4867Crossref PubMed Scopus (67) Google Scholar). For more information, please refer to Supplemental Data and Methods. Detergent-based lysis buffer was made following previously established protocols (40Shults M.D. Janes K.A. Lauffenburger D.A. Imperiali B. A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates.Nat. Methods. 2005; 2: 277-283Crossref PubMed Scopus (168) Google Scholar). Slight modifications were made to the base protocol to maintain compatibility with MS lysate preparation protocols. Cell lysis buffer consisted of 1% Triton X-100, 50 mm β-glycerophosphate, 10 mm sodium pyrophosphate, 30 mm sodium fluoride (Sigma-Aldrich), 50 mm Tris (Roche Applied Science, Indianapolis, IN), 150 mm sodium chloride, 2 mm EGTA, 1% Protease Inhibitor Mixture (Sigma-Al
Referência(s)