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

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

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

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

Demographic and clinical data of patients and healthy controls enrolled in the study.

<p>Demographic and clinical data of patients and healthy controls enrolled in the study.</p

The amplitude of store-operated Ca²⁺ entry is not reduced by a high-K⁺ extracellular solution in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>100 mM NaCl in the extracellular solution was replaced with an equimolar amount of K⁺ (HighK) to clamp the membrane potential at 0 mV and observe the consequences on the extent of SOCE activation in PMF-ECFCs. A, HighK did not affect either the amplitude or the kinetics of CPA (10 µM)-induced Ca²⁺ signals in PMF-ECFCs. Black and grey tracings illustrate CPA-dependent Ca²⁺ signals in the absence and presence of HighK, respectively. B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE in the absence (black bar; n = 76) and in the presence of HighK (white bar; n = 77). C, the biphasic Ca²⁺ response to ATP (100 µM) was not impaired by HighK. Black and grey tracings illustrate ATP-dependent Ca²⁺ signals in the absence and presence of HighK, respectively. D, mean±SE of the amplitude of ATP-elicited Ca²⁺ release and ATP-elicited SOCE in the absence (black bar; n = 88) and in the presence of HighK (white bar; n = 95). 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

Gd³⁺ does not inhibit CPA-induced SOCE in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>A, Gd³⁺ (10 µM, 40 min of pre-incubation) did not affect both phases (i.e. intracellular Ca²⁺ mobilization and SOCE) of the Ca²⁺ response to CPA (10 µM) in PMF-ECFCs. Black and grey tracings illustrate CPA-dependent increases in [Ca²⁺]_i in the absence and presence of Gd³⁺, respectively. B, mean±SE of the amplitude of CPA-induced Ca²⁺ release and CPA-induced SOCE in the absence (black bar; n = 129) and in the presence of Gd³⁺ (white bar; n = 89). C, Gd³⁺ (10 µM, 40 min of pre-incubation) inhibited ATP (100 µM)-induced SOCE, while it did not modify intracellular Ca²⁺ mobilization. Black and grey tracings illustrate ATP-evoked Ca²⁺ signals in the absence and presence of Gd³⁺, respectively. D, mean±SE of the amplitude of ATP-elicited Ca²⁺ release and ATP-elicited SOCE in the absence (black bar; n = 101) and in the presence of Gd³⁺ (white bar; n = 90). 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

Expression of Stim1–2, Orai1–3, TRPC1 and TRPC4 transcripts in endothelial colony forming cells isolated from patients affected by primary myelofibrosis.

<p>Quantitative real-time reverse transcription polymerase chain reaction of total RNA was performed using specific primers for Stim1–2 (A), Orai1–3 (B), TRPC1 and TRPC4 (C). Bars represent the mean ± SEM of at least 4 different experiments each from different RNA extracts. *P<0.05 versus N-ECFCs (Student’s <i>t</i> test). The PCR products were of the expected size: Orai1, 257 bp; Orai2, 334 bp; Orai3, 159 bp; Stim1, 347 bp; Stim2, 186 bp; TRPC1, 307 bp and TRPC4, 300 bp <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091099#pone.0091099-Lodola1" target="_blank">[23]</a>.</p

Primer sequences used for real time reverse transcription/polymerase chain reaction of the following genes: TRPC1, TRPC3–7, Stim1–2, Orai1–3.

<p>Primer sequences used for real time reverse transcription/polymerase chain reaction of the following genes: TRPC1, TRPC3–7, Stim1–2, Orai1–3.</p

Effect of BTP-2, La³⁺, and Gd³⁺ on ECFC-derived cell growth <i>in vitro</i>.

<p>Results are expressed as percentage of growth compared to control (given as 100% growth). The drugs were administrated at the following concentrations: BTP-2–20 µM; La³⁺ –10 µM; Gd³⁺ –10 µM.</p><p>*compared to control and after Bonferroni’s correction (<i>t</i>-test for paired samples).</p