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

Enhanced Expression of Stim, Orai, and TRPC Transcripts and Proteins in Endothelial Progenitor Cells Isolated from Patients with Primary Myelofibrosis

<div><p>Background</p><p>An increase in the frequency of circulating endothelial colony forming cells (ECFCs), the only subset of endothelial progenitor cells (EPCs) truly belonging to the endothelial phenotype, occurs in patients affected by primary myelofibrosis (PMF). Herein, they might contribute to the enhanced neovascularisation of fibrotic bone marrow and spleen. Store-operated Ca²⁺ entry (SOCE) activated by the depletion of the inositol-1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ store drives proliferation in ECFCs isolated from both healthy donors (N-ECFCs) and subjects suffering from renal cellular carcinoma (RCC-ECFCs). SOCE is up-regulated in RCC-ECFCs due to the over-expression of its underlying molecular components, namely Stim1, Orai1, and TRPC1.</p><p>Methodology/Principal Findings</p><p>We utilized Ca²⁺ imaging, real-time polymerase chain reaction, western blot analysis and functional assays to evaluate molecular structure and the functional role of SOCE in ECFCs derived from PMF patients (PMF-ECFCs). SOCE, induced by either pharmacological (i.e. cyclopiazonic acid or CPA) or physiological (i.e. ATP) stimulation, was significantly higher in PMF-ECFCs. ATP-induced SOCE was inhibited upon blockade of the phospholipase C/InsP₃ signalling pathway with U73111 and 2-APB. The higher amplitude of SOCE was associated to the over-expression of the transcripts encoding for Stim2, Orai2–3, and TRPC1. Conversely, immunoblotting revealed that Stim2 levels remained constant as compared to N-ECFCs, while Stim1, Orai1, Orai3, TRPC1 and TRPC4 proteins were over-expressed in PMF-ECFCs. ATP-induced SOCE was inhibited by BTP-2 and low micromolar La³⁺ and Gd³⁺, while CPA-elicited SOCE was insensitive to Gd³⁺. Finally, BTP-2 and La³⁺ weakly blocked PMF-ECFC proliferation, while Gd³⁺ was ineffective.</p><p>Conclusions</p><p>Two distinct signalling pathways mediate SOCE in PMF-ECFCs; one is activated by passive store depletion and is Gd³⁺-resistant, while the other one is regulated by the InsP₃-sensitive Ca²⁺ pool and is inhibited by Gd³⁺. Unlike N- and RCC-ECFCs, the InsP₃-dependent SOCE does not drive PMF-ECFC proliferation.</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

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

Expression of TRPC1 and TRPC4 proteins in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>Western blot and densitometry representative of four separate experiments were shown. Major bands of the expected molecular weights for TRPC1 (A) and TRPC4 (B) were observed. 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

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

BTP-2 inhibits store-operated Ca²⁺ entry in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>A, BTP-2 (20 µM, 20 min of pre-incubation) selectively suppressed CPA (10 µM)-solicited SOCE, while it did not alter intracellular Ca²⁺ mobilization in PMF-ECFCs. Black and grey tracings illustrate CPA-dependent Ca²⁺ signals in the absence and presence of BTP-2, respectively. B, mean±SE of the amplitude of Ca²⁺ release and SOCE evoked by CPA in the absence (black bar; n = 67) and in the presence of BTP-2 (white bar; n = 131). C, BTP-2 (20 µM, 20 min of pre-incubation) did not influence the intracellular Ca²⁺ response to ATP (100 µM), while it abrogated SOCE activation in PMF-ECFCs. Black and grey tracings illustrate ATP-evoked elevations in [Ca²⁺]_i observed in the absence and presence of BTP-2, respectively. D, mean±SE of the amplitude of ATP-elicited Ca²⁺ release and CPA-elicited SOCE in the absence (black bar; n = 49) and in the presence of BTP-2 (white bar; n = 100). The asterisk denotes a p<0.05.</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

Store-operated Ca²⁺ entry is expressed in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>A, intracellular Ca²⁺ stores were depleted by perfusion with cyclopiazonic acid (CPA; 10 µM) in the absence of external Ca²⁺ (0Ca²⁺), and Ca²⁺ influx through store-operated channels was measured on Ca²⁺ restitution to the bathing medium. Black and grey tracings represent the Ca²⁺ signals induced by CPA in ECFCs isolated from healthy donors (N-ECFCs) and PMF patients (PMF-ECFCs), respectively. B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE recorded from N-ECFCs (black bar; n = 130) and PMF-ECFCs (white bar; n = 125). C, cells perfused with ATP (100 µM) during exposure to 0Ca²⁺ responded with a transient rise in [Ca²⁺]_i. After continued perfusion with the Ca²⁺ solution alone, restoration of extracellular Ca²⁺ caused a sustained elevation in intracellular Ca²⁺ levels. Black and grey tracings illustrate ATP-evoked Ca²⁺ signals in N-ECFCs and PMF-ECFCs, respectively. D, mean±SE of the amplitude of ATP-elicited Ca²⁺ release and ATP-elicited SOCE recorded from N-ECFCs (black bar; n = 140) and PMF-ECFCs (white bar; n = 125). The asterisk denotes a p<0.05. In panels A and C, each trace is representative of at least three independent experiments conducted on cells isolated from three distinct healthy donors and three PMF patients.</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