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.

Harnessing developmental processes for vascular engineering and regeneration

“…Of these, VEGFs and their receptors (Flk and Flt) are considered to be master determinants of vasculogenesis and angiogenesis, as together they regulate the survival, differentiation, proliferation, morphogenesis and migration of ECs (Gentile et al, 2013;Hang et al, 2013;Nakayama et al, 2013). Given their crucial role in vascular development, genes encoding VEGFs have been widely used as therapeutic pro-angiogenic factors in clinical trials or to regulate vascular differentiation and morphogenesis to create vascular network structures in vitro (Hanjaya-Putra et al, 2010;Yee et al, 2011).…”

Fractalkine induces angiogenic potential in CX3CR1-expressing monocytes

“…However, PF‐4, in combination with VEGF, has also been reported to be a proangiogenic cytokine at the initial phase of vascular sprouting by promoting EC separation from vessels . Simultaneous treatment with PF‐4 and VEGF induced changes in migration pathway, including P38α, MAPK, focal adhesion kinase, and Src family kinases, when compared with VEGF treatment alone . Our findings demonstrate that PF‐4 does not promote angiogenesis by itself but, instead, amplifies the angiogenic activity of VEGF.…”

“…PF‐4 can inhibit cell proliferation by halting S‐phase progression and reducing EC migration . However, PF‐4, in combination with VEGF, has also been reported to be a proangiogenic cytokine at the initial phase of vascular sprouting by promoting EC separation from vessels . Simultaneous treatment with PF‐4 and VEGF induced changes in migration pathway, including P38α, MAPK, focal adhesion kinase, and Src family kinases, when compared with VEGF treatment alone .…”

“…A large body of information is available on PF‐4 and its ability to inhibit and even induce regression of angiogenesis . PF‐4 can inhibit cell proliferation by halting S‐phase progression and reducing EC migration . However, PF‐4, in combination with VEGF, has also been reported to be a proangiogenic cytokine at the initial phase of vascular sprouting by promoting EC separation from vessels .…”

Human Th17 Migration in Three-Dimensional Collagen Involves p38 MAPK

“…Several studies that have been performed with 2D models of cell migration have shown the importance of p38 pathway in cell motility. However, p38 has also been associated with cell migration in 3D collagen as is the case with endothelial cells [Hang et al, 2013] and with the invasion of carcinoma cells [Rider et al, 2013;Naci et al, 2015]. Given the more physiological relevance of 3D models of cell migration and the role of p38 in Th17 migration (this study) and in the development of inflammatory diseases, it is likely that p38 might be of critical importance for in vivo T cell migration.…”