Pharmacologic Characterization of a Kinetic In Vitro Human Co-Culture Angiogenesis Model Using Clinically Relevant Compounds

Abstract

Angiogenesis, the formation of new vessels from preexisting vessels, involves multiple cell types acting in concert to cause endothelial cell proliferation, migration, and differentiation into microvascular arrays. Under pathologic conditions, microenvironment changes result in altered blood vessel production. Historically, in vitro angiogenesis assays study individual aspects of the process and tend to be variable, difficult to quantify, and limited in clinical relevance. Here, we describe a kinetic, quantitative, co-culture angiogenesis model and demonstrate its relevance to in vivo pharmacology. Similar to in vivo angiogenesis, a co-culture of human umbilical vein endothelial cells with normal human dermal fibroblasts remains sensitive to multiple cytokines, resulting in a concentration-dependent stimulation of tube formation over time. Treatment with axitinib, a selective vascular endothelial growth factor (VEGF) antagonist, inhibited VEGF-mediated tube length and branch point formation and was selective for inhibiting VEGF over basic fibroblast growth factor (bFGF), similar to previous studies. Conversely, an FGFR-1 selective compound, PD-161570, was more potent at inhibiting bFGF-mediated angiogenesis. These results demonstrate the cytokine dynamics, selective pharmacology, and translational application of this model system. Finally, combining quantitative angiogenic biology with kinetic, live-content imaging highlights the importance of using validated in vitro models in drug discovery research.

Single-Cell Receptor Quantification of an In Vitro Coculture Angiogenesis Model Reveals VEGFR, NRP1, Tie2, and PDGFR Regulation and Endothelial Heterogeneity

“…Indeed, Ang-Tie signaling pathways are known to play a role in both promoting angiogenesis and quiescence [72][73][74]. The expression of Tie2 protein on EC plasma membrane aligns with previous studies of ECs in vitro [31,32,75,76] and in vivo [77]. Furthermore, Ang-Tie signaling pathways have been identified as one of the escaping mechanisms tumor vasculature develops in response to anti-VEGF treatment, which made Ang-Tie a novel drug target for antiangiogenesis therapy [78,79].…”

Structure-Based Design of GNE-495, a Potent and Selective MAP4K4 Inhibitor with Efficacy in Retinal Angiogenesis

“…Consistent with the good permeability properties, these compounds exhibited adequate potency preventing migration of HUVEC cells in vitro, an assay that is used to evaluate antiangiogenic behavior. 23 Notably, compound 13 showed the best balance of MAP4K4 inhibition, permeability, microsomal stability, and cellular potency.…”

The effect of crude drugs on the angiogenic property and dynamic viscoelasticity of PEMA-based soft polymer materials

“…In the current study, we used the CellPlayer 96-well kinetic angiogenesis PrimeKit assay produced by ESSEN Bioscience, and based on the HUVEC/NHDF co-culture model 27) . Wolfe et al 28) demonstrated that the HUVEC/NHDF co-culture model is suitable to screen the effect of drugs and natural products on angiogenesis at the early stages. Currently, most liquid forms of commercial soft polymer materials include phthalate esters as plasticizers [16][17][18] .…”

“…The various in vivo methods used include the Mile's assay, chicken embryo chorioallantoic membrane method, and the Matrigel subcutaneous implantation method; however, these methods have various limitations 26) . Therefore, in recent year numerous in vitro methods have been developed to assess angiogenesis [26][27][28] . In the current study, we used the CellPlayer 96-well kinetic angiogenesis PrimeKit assay produced by ESSEN Bioscience, and based on the HUVEC/NHDF co-culture model 27) .…”