Real Time Cell Analysis of VEGF and PlGF-Induced Endothelial Cell Proliferation in the Presence of Heparin and Growth Factor Inhibitors

Abstract

Real-time Cell Analysis (RTCA) has been made possible by new instruments that exploit the ability of cells to impede electric current to determine cell proliferation. Because impedance measurement does not interfere with cell processes or destroy cells, repeated measurements can be made and used to produce time-dependent cell response profiles (TCRPs). These TCRPs can be used to study time-dependent alterations in cell proliferation. Heparin has been reported to modulate the proliferative effects of VEGF165 and PlGF-2 in endothelial cells. Whether heparin produces time-dependent differences in growth factor signaling occur has been difficult to study using endpoint assays. RTCA was used to determine if heparin induces time-dependent changes in proliferation in the presence of heparin-binding growth factors -- VEGF165 and PlGF-2-- and whether heparin alters activity of growth factor inhibitors. Human umbilical vein endothelial cells (HUVECs) were stimulated with VEGF and PlGF in the absence or presence of heparin. RTCA impedance measurement was used to determine the effective concentration and maximum cell number for each growth factor and the combination of VEGF165 and PlGF-2. Unlike endpoint measurements, RTCA measurement was also used to determine kinetic differences by determining the time of maximum cell proliferation and area under the curve. These TCRP-derived measurements were used to determine heparin’s time-dependent effects on VEGF and PlGF proliferation responses, and heparin’s impact on the effectiveness of their respective inhibitors. RTCA was shown to provide accurate measurements of proliferation when compared to Bromodeoxyuridine (BrdU) proliferation data. However, unlike BrdU endpoint proliferation measurement, RTCA impedance measurement was also useful for revealing time-dependent differences in proliferation. The effect of soluble heparin binding to VEGF165 and PlGF-2 was found to be more consistent with models of stabilized ligand receptor binding and increased ligand internalization. Additionally, comparison of TCRP data from PlGF-2 and VEGF165-induced proliferation demonstrated that VEGF165 and PlGF-2 have different time-dependent characteristics. RTCA measurements revealed a significant positive correlation between VEGF concentration and Tmax of VEGF-induced proliferation wherein proliferation is delayed at high concentrations of VEGF, but not for PlGF-2-induced proliferation. Time-dependent growth factor differences were reflected in inhibition of single or multi-target inhibitors as unique inhibitor signatures. Comparison of time-dependent inhibitor profiles of single versus multi-target inhibitors in VEGF165: PlGF-2-stimulated HUVECs confirmed previously observed efficacy differences, but also provided information on the completeness of drug inhibition effects. AUC analysis revealed that uninhibited factors were present when either VEGF or PlGF inhibitors were used, while targeting of both factors demonstrated that no significant endogenous growth factor inhibition was present. Soluble heparin was found only to affect the activity of a multi-targeted inhibitor also reported to bind heparin. RTCA impedance measurement is an accurate and useful tool for revealing time-dependent differences in proliferation responses that can be used to differentiate growth factors and their inhibitors and to study signal modulation.