Store-Operated Ca²⁺ Entry Is Remodelled and Controls <em>In Vitro</em> Angiogenesis in Endothelial Progenitor Cells Isolated from Tumoral Patients

<div><h3>Background</h3><p>Endothelial progenitor cells (EPCs) may be recruited from bone marrow to sustain tumor vascularisation and promote the metastatic switch. Understanding the molecular mechanisms driving EPC proliferation and tubulogenesis could outline novel targets for alternative anti-angiogenic treatments. Store-operated Ca²⁺ entry (SOCE), which is activated by a depletion of the intracellular Ca²⁺ pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca²⁺-sensor, Stim1, and the plasmalemmal Ca²⁺ channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca²⁺ influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients.</p> <h3>Methodology/Principal Findings</h3><p>The present study employed Ca²⁺ imaging, recombinant sub-membranal and mitochondrial aequorin, real-time polymerase chain reaction, gene silencing techniques and western blot analysis to investigate the expression and the role of SOCE in EPCs isolated from peripheral blood of patients affected by renal cellular carcinoma (RCC; RCC-EPCs) as compared to control EPCs (N-EPCs). SOCE, activated by either pharmacological (i.e. cyclopiazonic acid) or physiological (i.e. ATP) stimulation, was significantly higher in RCC-EPCs and was selectively sensitive to BTP-2, and to the trivalent cations, La³⁺ and Gd³⁺. Furthermore, 2-APB enhanced thapsigargin-evoked SOCE at low concentrations, whereas higher doses caused SOCE inhibition. Conversely, the anti-angiogenic drug, carboxyamidotriazole (CAI), blocked both SOCE and the intracellular Ca²⁺ release. SOCE was associated to the over-expression of Orai1, Stim1, and transient receptor potential channel 1 (TRPC1) at both mRNA and protein level The intracellular Ca²⁺ buffer, BAPTA, BTP-2, and CAI inhibited RCC-EPC proliferation and tubulogenesis. The genetic suppression of Stim1, Orai1, and TRPC1 blocked CPA-evoked SOCE in RCC-EPCs.</p> <h3>Conclusions</h3><p>SOCE is remodelled in EPCs from RCC patients and stands out as a novel molecular target to interfere with RCC vascularisation due to its ability to control proliferation and tubulogenesis.</p> </div

Store-dependent Ca²⁺ entry is higher in endothelial progenitor cells isolated from patients suffering from renal cellular carcinoma.

<p>A, during exposure to 0Ca²⁺ PSS, depletion of the intracellular Ca²⁺ stores resulted from addition of 10 µM CPA to the bathing medium. Subsequent replenishment of Ca²⁺ (1.5 mM) to the extracellular solution elicited a rise in [Ca²⁺]_i due to Ca²⁺ influx through open store-operated Ca²⁺ channels. Black and grey tracings depict the representative changes in [Ca²⁺]_i recorded from EPCs isolated from healthy volunteers (N-EPCs) and patients suffering from RCC (RCC-EPCs), respectively. The transient increase in [Ca²⁺]_i evoked by CPA under 0Ca²⁺ conditions decayed to the baseline with slower mono-exponential kinetics in RCC-EPCs as compared to N-EPCs (298.06±0.17 sec, n = 58, <i>vs</i>. 342.67±0.07 sec, n = 62, respectively). B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE recorded from all EPCs isolated from both healthy donors (black bar) and RCC patients (white bar). The asterisk indicates p<0.05. C, cells perfused with ATP (100 µM) responded with a transient rise in cytosolic [Ca²⁺]_i. After continued perfusion with 0Ca²⁺, restoration of external Ca²⁺ caused a sustained rise in cytosolic [Ca²⁺]_i due to SOCE activation. Black and grey tracings depict the changes in [Ca²⁺]_i recorded from representative EPCs isolated from healthy volunteers (N-EPCs) and patients suffering from RCC (RCC-EPCs), respectively. In these and the following figures, agonists and drugs were administered at the time indicated by the horizontal bars. The transient increase in [Ca²⁺]_i evoked by ATP under 0Ca²⁺ conditions decayed to the baseline with slower mono-exponential kinetics in RCC-EPCs as compared to N-EPCs (52.26±0.14 sec, n = 35, <i>vs</i>. 101.73±0.17 sec, n = 25, respectively). D, mean±SE of the amplitude of ATP-induced Ca²⁺ release and ATP-induced SOCE recorded from all EPCs isolated from both healthy donors (black bar) and RCC patients (white bar). The asterisk indicates p<0.05. Please note that the amplitude of SOCE was higher upon CPA, rather than ATP, stimulation in N-EPCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042541#pone.0042541-SnchezHernndez1" target="_blank">[27]</a>). E, N-EPCs and RCC-EPCs were transduced by lentiviral particles expressing AEQ fused with SNAP25 (pm-AEQ). Intracellular stores were first depleted by challenging the cells with ATP (100 µM) in 0Ca²⁺, after which SOCE was triggered by restoring extracellular Ca²⁺ in the absence of the agonist. F, mean±SE of the magnitude of the luminescence emitted by pm-AEQ in both control cells (white bar) and RCC-EPCs (black bar). The asterisk indicates p<0.05. G, the cells were infected with lentiviral vector expressing AEQ targeted to the mitochondrial lumen (mit-AEQ) and the experiment conducted as depicted in Panels C and E. H, mean±SE of the amplitude of ATP-induced Ca²⁺ release and ATP-induced SOCE recorded in both N-EPC (black bar) and RCC-EPCs (white bar). The asterisk indicates p<0.05.</p

Primer sequences used for real time reverse transcription/polymerase chain reaction of Orai1-3, Stim1-2, TRPC1-7.

<p>Primer sequences used for real time reverse transcription/polymerase chain reaction of Orai1-3, Stim1-2, TRPC1-7.</p

The amplitude of store-operated Ca²⁺ entry in endothelial progenitor cells isolated from patients affected by renal cellular carcinoma is still higher in the presence of a high-K⁺ extracellular solution.

<p>Mean±SE of CPA-evoked SOCE in N-EPCs and RCC-EPCs bathed in presence of an extracellular solution containing 100 mM KCl to clamp V_M at the same value in both cell types. CPA was applied at 10 µM. The asterisk indicates p<0.05 (p = 0.039).</p

La³⁺ impairs proliferation and tubulogenesis of endothelial progenitor cells.

<p>La³⁺ (10 µM) suppresses proliferation in both N-EPCs (A) and RCC-EPCs (B). Results are expressed as percentage of growth compared with control (given as 100% growth). Similarly, La³⁺ (10 µM) interferes with the tubule-formation capacity of both N-EPCs (C) and RCC-EPCs (D).</p

VEGF does not induce Ca²⁺ oscillations in endothelial progenitor cells isolated from patients affected by renal cellular carcinoma.

<p>VEGF (10 ng/ml) elicits a burst of intracellular Ca²⁺ waves in N-EPCs (black tracing), but not in RCC-ECFCs (grey tracing). The Ca²⁺ traces are representative of 165 N-EPCs and 148 RCC-EPCs, respectively, harvested from three different donors.</p

Silencing of Orai1, Stim1, and TRPC1 reduces store-operated Ca²⁺ entry in endothelial progenitor cells.

<p>A, SOCE evoked by CPA (10 µM) in RCC-EPCs transfected with a control siRNA (black tracing) or with a specific siRNA sequence devised to knock down either Orai1 (grey tracing) or Stim1 (light gray tracing). B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE under each condition described in A. The asterisk indicates p<0.05. C, SOCE triggered by CPA (10 µM) in N-EPCs transfected with a scrambled shRNA (black tracing) and with a shRNA selectively targeting TRPC1 (grey tracing). D, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE under each condition described in C. E, SOCE triggered by CPA (10 µM) in RCC-EPCs transfected with a scrambled shRNA (black tracing) and with a shRNA selectively targeting TRPC1 (grey tracing). F, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE under each condition described in E.</p

Orai1, Stim1, and TRPC1 proteins are over-expressed in endothelial progenitor cells isolated from patients suffering from renal cellular carcinoma.

<p>Western blot and densitometry depicting the significant elevation in Orai1 (A), Stim1 (B), and TRPC1 (C) proteins in RCC-EPCs as compared to N-EPCs. Conversely, TRPC4 protein (D) is down-regulated in RCC-EPCs. Blots for Orai1, Stim1, TRPC1 and TRPC4 representative of 4 different experiments are shown in the lower panel. Lanes were loaded with 20 μg of proteins. Major bands of the expected molecular weight were observed in both cell types. One additional band of 77 kDa was detected by anti-Stim1 in RCC-EPCs. When both Stim1 bands (77 and 100 kDa) were compared to the single band detected at 100 kDa in N-ECFCs, the expression of Stim1 protein became significantly higher in RCC-EPCs (see text for further explanations). Each bar in the upper panel represents the mean±SE of the densitometric analysis of four different experiments. The asterisk indicates p<0.01 (Student's <i>t</i>-test).</p

BTP-2 inhibits store-dependent Ca²⁺ entry in endothelial progenitor cells isolated from patients suffering from renal cellular carcinoma.

<p>A, CPA-elicited SOCE in the absence (black tracing) and presence (grey tracing) of BTP-2 (20 μM). The cells were pre-incubated with the drug for 20 min before the beginning of the experimental protocol. CPA was administered at 10 μM. B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE in the absence and presence of BTP-2. The asterisk indicates p<0.05. C, ATP-evoked Ca²⁺ mobilization and SOCE in the presence (black tracing) and absence (grey tracing) of BTP-2 (20 μM, 20 min of pre-treatment). ATP was applied at 100 μM. D, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE in the absence and presence of BTP-2. The asterisk indicates p<0.05.</p

La³⁺ and Gd³⁺ suppress store-operated Ca²⁺ entry stimulated by ATP in endothelial progenitor cells.

<p>La³⁺ (10 µM) and Gd³⁺ dampen SOCE triggered by ATP (100 µM) in both N-EPCs (A) and RCC-EPCs (B). The traces are representative of the experiments conducted on EPCs from at least three different donors for each condition. Mean±SE of the amplitude of ATP-induced Ca²⁺ release and ATP-induced SOCE in the absence and presence of each trivalent cation in both N-EPCs (C) and RCC-EPCs (D). The asterisk indicates p<0.05.</p