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

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 (1–5). 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 (6–8), chronic wounds (8), inflammatory disorders (9, 10), and cancer (9, 11, 12).Inflammatory reactions also confound tissue engineered implantable three-dimensional constructs that provide innovative clinical treatments of various diseases and injuries (13–17). As complex tissues become developed for applications in clinical trials, tissue vascularization for constructs of considerable size and volume is required for their survival (18, 19). Once implanted, these constructs will also experience significant inflammatory responses within their host''s local milieu (20, 21). These circumstances demonstrate the necessity for understanding the interactions between inflammation and angiogenesis, such as the development of predictive models (22). 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 (23–26) by promoting new blood vessel growth (e.g. CXCL1–3, CXCL5–8, CXCL12) or inhibiting its formation altogether (e.g. CXCL4, CXCL9–11, CXCL13) (26). 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 (27, 28). 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 (27–30). In addition to inducing angiostasis, PF-4 can inhibit cell proliferation by halting S phase progression and reducing endothelial cell migration (25, 28, 30–32). 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 (28, 29, 33, 34). 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 (28). 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 (35), 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 (36, 37). Furthermore, PF-4 could have variable sensitivities in different endothelial cell types because of heterogeneous expression of CXCR3 (38).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)¹ 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.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.     

A microtiter assay for quantifying protein-protein interactions associated with cell-cell adhesion

Cell-cell adhesions are a hallmark of epithelial tissues, and the disruption of these contacts plays a critical role in both the early and late stages of oncogenesis. The interaction between the transmembrane protein E-cadherin and the intracellular protein beta-catenin plays a crucial role in the formation and maintenance of epithelial cell-cell contacts and is known to be downregulated in many cancers. The authors have developed a protein complex enzyme-linked immunosorbent assay (ELISA) that can quantify the amount of beta-catenin bound to E-cadherin in unpurified whole-cell lysates with a Z' factor of 0.74. The quantitative nature of the E-cadherin:beta-catenin ELISA represents a dramatic improvement over the low-throughput assays currently used to characterize endogenous E-cadherin:beta-catenin complexes. In addition, the protein complex ELISA format is compatible with standard sandwich ELISAs for parallel measurements of total levels of endogenous E-cadherin and beta-catenin. In 2 case studies closely related to cancer cell biology, the authors use the protein complex ELISA and traditional sandwich ELISAs to provide a detailed, quantitative picture of the molecular changes occurring within adherens junctions in vivo. Because the E-cadherin: beta-catenin protein complex plays a crucial role in oncogenesis, this protein complex ELISA may prove to be a valuable quantitative prognostic marker of tumor progression.     

Engineering of In Vitro 3D Capillary Beds by Self-Directed Angiogenic Sprouting

In recent years, microfluidic systems have been used to study fundamental aspects of angiogenesis through the patterning of single-layered, linear or geometric vascular channels. In vivo, however, capillaries exist in complex, three-dimensional (3D) networks, and angiogenic sprouting occurs with a degree of unpredictability in all x,y,z planes. The ability to generate capillary beds in vitro that can support thick, biological tissues remains a key challenge to the regeneration of vital organs. Here, we report the engineering of 3D capillary beds in an in vitro microfluidic platform that is comprised of a biocompatible collagen I gel supported by a mechanical framework of alginate beads. The engineered vessels have patent lumens, form robust ∼1.5 mm capillary networks across the devices, and support the perfusion of 1 µm fluorescent beads through them. In addition, the alginate beads offer a modular method to encapsulate and co-culture cells that either promote angiogenesis or require perfusion for cell viability in engineered tissue constructs. This laboratory-constructed vascular supply may be clinically significant for the engineering of capillary beds and higher order biological tissues in a scalable and modular manner.