Epidermal growth factor induces intracellular Ca²⁺ oscillations in microvascular endothelial cells

Abstract

An increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) may play a role in the proliferative effect of several growth factors. In this study, the changes in [Ca(2+)](i) elicited by epidermal growth factor (EGF) in rat cardiac microvascular endothelial cells (CMEC) have been investigated by using fura-2 conventional and confocal microscopy. A large heterogeneity in the latency and in the pattern of the Ca(2+) response was found at each dose of EGF (2.5-100 ng/ml), whereas some cells displayed a non-oscillatory behavior and others exhibited a variable number of Ca(2+) oscillations. On average, the fraction of responsive cells, the total number of oscillations and the duration of the Ca(2+) signal were higher at around 10 ng/ml EGF, while there was no dose-dependence in the lag time and in the amplitude of the [Ca(2+)](i) increase. EGF-induced Ca(2+) spikes were abolished by the tyrosine kinase inhibitor genistein, but not by its inactive analogue daidzein, and by the phospholipase C blocker NCDC. Only 1-2 transients could be elicited in Ca(2+)-free solution, while re-addition of extracellular Ca(2+) recovered the spiking activity. The oscillatory signal was prevented by the SERCA inhibitor thapsigargin and abolished by the calcium entry blockers Ni(2+) and La(3+). Moreover, EGF-induced Ca(2+) transients were abolished by the InsP(3) receptor blocker caffeine, while ryanodine was without effect. Confocal imaging microscopy showed that the Ca(2+) response to EGF was localized both in the cytoplasm and in the nucleus. We suggest that EGF-induced [Ca(2+)](i) increase may play a role in the proliferative action of EGF on endothelial cells.

Vascular Endothelial Growth Factor Stimulates Endothelial Colony Forming Cells Proliferation and Tubulogenesis by Inducing Oscillations in Intracellular Ca2+ Concentration

“…These findings suggest that the onset of the oscillatory signal depends on Ca 2þ mobilization from the intracellular Ca 2þ pool. However, Ca 2þ entry maintains the oscillations in [Ca 2þ ] i overtime likely due to its ability to refill intracellular Ca 2þ stores [29,30]. This notion is corroborated by the experiment depicted in Figure 3E, where removal of extracellular Ca 2þ reversibly inhibited the ongoing train of Ca 2þ spikes in 224 of 241 cells.…”

“…Importantly, it has long been known that repetitive Ca 2þ spikes may be exploited by growth factors to drive proliferation, migration, and differentiation [29,[41][42][43][44], which are the main steps underlying the formation of a novel vascular network by circulating ECFCs. This study provides, for the first time, the evidence that VEGF stimulates ECFCs to proliferate and form tubule-like structures by inducing oscillations in [Ca 2þ ] i and the consequent nuclear translocation of the Ca 2þ -sensitive transcription factor, NF-jB.…”

“…First, VEGFR-2 may phosphorylate and activate PLC-c to synthesize InsP 3 and DAG. Second, U73122, a widely used PLC-c inhibitor, prevents the Ca 2þ response to VEGF [20,29]. Third, repetitive Ca 2þ transients rapidly arise also in 0Ca 2þ , although they rapidly cease unless extracellular Ca 2þ is restored.…”

“…The variability in the Ca 2þ response to VEGF might be due to the position of the cells in the cell cycle, as described for the oligodendroglial cell line CEINGE cl3 [28]. ECFCs were starved by serum deprivation (1% FSC in EGM-2 48 hours before Ca 2þ imaging), a maneuver that may synchronize ECs in G 0 [29]. However, VEGF still triggered a heterogeneous pattern of repetitive Ca 2þ spikes in serum-starved ECFCs (Supporting Information Fig.…”

“…The hypothesis that SOCE refills the InsP 3 -sensitive Ca 2þ store was probed by preventing Ca 2þ reuptake into the ER during the ongoing Ca 2þ oscillations by inhibiting SERCA with cyclopiazonic acid (CPA) (10 lM) [11,19,29,33]. Such a maneuver caused an immediate increase in [Ca 2þ ] i due to Ca 2þ leakage from the intracellular stores and inhibited The contribution of PLC-c in the signal transduction cascade leading to the oscillatory response to VEGF was investigated by preincubating the cells with U73122 (10 lM), a widely used PLC antagonist [20,29]. This treatment abrogated the onset of VEGF-induced repetitive Ca 2þ spikes in 113 of 113 ECFCs (Fig.…”