Endothelial progenitor cells (EPCs) home from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy (CBT) in regenerative medicine and provide a novel promising target in the fight against cancer. Intracellular Ca2+ signals regulate numerous endothelial functions, such as proliferation and tubulogenesis. The growth of endothelial colony forming cells (ECFCs), which are EPCs capable of acquiring a mature endothelial phenotype, is governed by store-dependent Ca2+ entry (SOCE). This study aimed at investigating the nature and the role of VEGF-elicited Ca2+ signals in ECFCs. VEGF induced asynchronous Ca2+ oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca2+ (0Ca2+) and SOCE inhibition with N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) reduced the duration of the oscillatory signal. Blockade of phospholipase C-γ with U73122, emptying the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools with cyclopiazonic acid (CPA), and inhibition of InsP3 receptors with 2-APB prevented the Ca2+ response to VEGF. VEGF-induced ECFC proliferation and tubulogenesis were inhibited by the Ca2+-chelant, BAPTA, and BTP-2. NF-κB activation by VEGF was impaired by BAPTA, BTP-2, and its selective blocker, thymoquinone. Thymoquinone, in turn, suppressed VEGF-dependent ECFC proliferation and tubulogenesis. These data indicate that VEGF-induced Ca2+ oscillations require the interplay between InsP3-dependent Ca2+ release and SOCE, and promote ECFC growth and tubulogenesis by engaging NF-κB. This novel signaling pathway might be exploited to enhance the outcome of CBT and chemotherapy.

BackgroundEndothelial 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.Methodology/Principal FindingsThe 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.ConclusionsSOCE 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.

Endothelial progenitor cells (EPCs) have recently been employed in cell-based therapy (CBT) to promote neovascularization and regeneration of ischemic organs, such as heart and limbs. Furthermore, EPCs may be recruited from bone marrow by growing tumors to drive the angiogenic switch through physical engrafting into the lumen of nascent vessels or paracrine release of pro-angiogenic factors. CBT is hampered by the paucity of EPCs harvested from peripheral blood and suffered from several pitfalls, including the differentiation outcome of transplanted cells and low percentage of engrafted cells. Therefore, CBT will benefit from a better understanding of the signal transduction pathway(s) which govern(s) EPC homing, proliferation and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to combat tumoral neovascularization. We have recently found that store-operated Ca(2+) entry, a Ca(2+)-permeable membrane pathway that is activated upon depletion of the inositol-1,4,5-trisphosphate-sensitive Ca(2+) pool, is recruited by vascular endothelial growth factor to support proliferation and tubulogenesis in human circulating endothelial colony forming cells (ECFCs). ECFCs are a subgroup of EPCs that circulate in the peripheral blood of adult individuals and are able to proliferate and differentiate into endothelial cells and form capillary networks in vitro and contribute to neovessel formation in vivo. The present review will discuss the relevance of SOCE to ECFC-based cell therapy and will address the pharmacological inhibition of store-dependent Ca(2+) channels as a promising target for anti-angiogenic treatments.

Endothelial colony-forming cells (ECFCs) are the only endothelial progenitor cells (EPCs) that are capable of acquiring a mature endothelial phenotype. ECFCs are mainly mobilized from bone marrow to promote vascularization and represent a promising tool for cell-based therapy of severe ischemic diseases. Vascular endothelial growth factor (VEGF) stimulates the proliferation of peripheral blood-derived ECFCs (PB-ECFCs) through oscillations in intracellular Ca(2+) concentration ([Ca(2+)]i). VEGF-induced Ca(2+) spikes are driven by the interplay between inositol-1,4,5-trisphosphate (InsP3)-dependent Ca(2+) release and store-operated Ca(2+) entry (SOCE). The therapeutic potential of umbilical cord blood-derived ECFCs (UCB-ECFCs) has also been shown in recent studies. However, VEGF-induced proliferation of UCB-ECFCs is faster compared with their peripheral counterpart. Unlike PB-ECFCs, UCB-ECFCs express canonical transient receptor potential channel 3 (TRPC3) that mediates diacylglycerol-dependent Ca(2+) entry. The present study aimed at investigating whether the higher proliferative potential of UCB-ECFCs was associated to any difference in the molecular underpinnings of their Ca(2+) response to VEGF. We found that VEGF induces oscillations in [Ca(2+)]i that are patterned by the interaction between InsP3-dependent Ca(2+) release and SOCE. Unlike PB-ECFCs, VEGF-evoked Ca(2+) oscillations do not arise in the absence of extracellular Ca(2+) entry and after pharmacological (with Pyr3 and flufenamic acid) and genetic (by employing selective small interference RNA) suppression of TRPC3. VEGF-induced UCB-ECFC proliferation is abrogated on inhibition of the intracellular Ca(2+) spikes. Therefore, the Ca(2+) response to VEGF in UCB-ECFCs is shaped by a different Ca(2+) machinery as compared with PB-ECFCs, and TRPC3 stands out as a promising target in EPC-based treatment of ischemic pathologies.

Stim1 and Orai1 are ubiquitous proteins that have long been known to mediate Ca2+ release-activated Ca2+ (CRAC) current (ICRAC) and store-operated Ca2+ entry (SOCE) only in non-excitable cells. SOCE is activated following the depletion of the endogenous Ca2+ stores, which are mainly located within the endoplasmic reticulum (ER), to replete the intracellular Ca2+ reservoir and engage specific Ca2+-dependent processes, such as proliferation, migration, cytoskeletal remodeling, and gene expression. Their paralogs, Stim2, Orai2 and Orai3, support SOCE in heterologous expression systems, but their physiological role is still obscure. Ca2+ inflow in neurons has long been exclusively ascribed to voltage-operated and receptor-operated channels. Nevertheless, recent work has unveiled that Stim1–2 and Orai1-2, but not Orai3, proteins are also expressed and mediate SOCE in neurons. Herein, we survey current knowledge about the neuronal distribution of Stim and Orai proteins in rodent and human brains; we further discuss that Orai2 is the main pore-forming subunit of CRAC channels in central neurons, in which it may be activated by either Stim1 or Stim2 depending on species, brain region and physiological stimuli. We examine the functions regulated by SOCE in neurons, where this pathway is activated under resting conditions to refill the ER, control spinogenesis and regulate gene transcription. Besides, we highlighted the possibility that SOCE also controls neuronal excitation and regulate synaptic plasticity. Finally, we evaluate the involvement of Stim and Orai proteins in severe neurodegenerative and neurological disorders, such as Alzheimer’s disease and epilepsy.

The control of stem and progenitor cell fate is emerging as a compelling urgency for regenerative medicine. Here, we propose a innovative strategy to gain optical control of endothelial colony-forming cell fate, which represents the only known truly endothelial precursor showing robust in vitro proliferation and overwhelming vessel formation in vivo. We combine conjugated polymers, used as photo-actuators, with the advantages offered by optical stimulation over current electromechanical and chemical stimulation approaches. Light modulation provides unprecedented spatial and temporal resolution, permitting at the same time lower invasiveness and higher selectivity. We demonstrate that polymer-mediated optical excitation induces a robust enhancement of proliferation and lumen formation in vitro. We identify the underlying biophysical pathway as due to light-induced activation of TRPV1 channel. Altogether, our results represent an effective way to induce angiogenesis in vitro, which represents the proof of principle to improve the outcome of autologous cell-based therapy in vivo.

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Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca concentration ([Ca]), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca] and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca] raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca signals required both intracellular Ca release and external Ca inflow. AA-induced Ca release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca] and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca- and NO-dependent manner. Therefore, AA-evoked Ca signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.