Vascular Endothelial Growth Factor (VEGF) and Platelet (PF-4) Factor 4 Inputs Modulate Human Microvascular Endothelial Signaling in a Three-Dimensional Matrix Migration Context

• Published in: Molecular & cellular proteomics 2013-12, Vol.12 (12), p.3704-3718
• Main Authors: Hang, Ta-Chun, Tedford, Nathan C., Reddy, Raven J., Rimchala, Tharathorn, Wells, Alan, White, Forest M., Kamm, Roger D., Lauffenburger, Douglas A.
• Format: Article
• Language: English
• Subjects: Cell Movement; Collagen - chemistry; Dermis - blood supply; Dermis - cytology; Dermis - drug effects; Dermis - metabolism; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Extracellular Matrix - drug effects; Extracellular Matrix - genetics; Extracellular Matrix - metabolism; Focal Adhesion Kinase 1 - genetics; Focal Adhesion Kinase 1 - metabolism; Gels; Gene Expression Regulation; Humans; Mass Spectrometry; Molecular Sequence Annotation; Neovascularization, Physiologic; p38 Mitogen-Activated Protein Kinases - genetics; p38 Mitogen-Activated Protein Kinases - metabolism; Phosphotyrosine - metabolism; Platelet Factor 4 - genetics; Platelet Factor 4 - metabolism; Platelet Factor 4 - pharmacology; Primary Cell Culture; Protein Binding; Receptor, EphA2 - genetics; Receptor, EphA2 - metabolism; Signal Transduction - drug effects; src-Family Kinases - genetics; src-Family Kinases - metabolism; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; Vascular Endothelial Growth Factor A - pharmacology
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M113.030528

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.