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Background: 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. Methodology/Principal Findings: 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. Conclus

Hematopoietic progenitor and stem cells (HPSCs) have been employed in cell-based therapy (CBT) to promote neovascularisation and regeneration of ischemic organs, such as heart and limbs. Furthermore, endothelial progenitor cells (EPCs) may favour tumour growth and adverse vascular targeting treatment by incorporating into neovessels. CBT is hampered by the paucity of HPSCs harvested from peripheral blood and suffers from several pitfalls, including the differentiation outcome of transplanted cells and low percentage of engrafted cells. Therefore, CBT will benefit of a better understanding of the signal transduction pathway(s) which drive(s) HPSC homing, proliferation and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to combat tumoral neovascularisation. The elevation in intracellular Ca2+ concentration is the key signal in the regulation of cellular motility, replication, and differentiation. Intracellular Ca2+ waves regulate cytoskeleton re-organisation and disassembly at focal adhesions, thus stimulating migration and substrate adhesion, and induce DNA transcription by recruiting Ca2+-sensitive transcription factors. However, the Ca2+ signalling toolkit which underlies Ca2+ release from intracellular stores and Ca2+ entry across the plasmalemma in HPSCs is still unclear. Our recent work has shown that the so-called store-operated Ca2+ entry stimulates EPC growth. Unravelling the mechanisms guiding HPSC behaviour might supply the biological bases required to improve CBT. For instance, genetic manipulation of the Ca2+ signalling machinery (such as transfer of genes encoding for the Ca2+ channels involved in EPC proliferation) could provide a novel approach to increase the extent of limb neovascularisation and regeneration of damaged hearts.

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 Ca2+ entry, a Ca2+-permeable membrane pathway that is activated upon depletion of the inositol-1,4,5-trisphosphate-sensitive Ca2+ 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 Ca2+ channels as a promising target for anti-angiogenic treatments.

Endothelial progenitor cells could be implicated in the aberrant neoangiogenesis that occurs in bone marrow and spleen in patients with primary myelofibrosis (PMF). However, antivascular endothelial growth factor (VEGF) monotherapy had only a modest and transient effect in these individuals. Recently it was found that VEGF-induced proangiogenic intracellular Ca2+ oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca2+ imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca2+ oscillations are altered in PMF-derived endothelial progenitor cells. We focused on endothelial colony-forming cells (ECFCs), which are the only endothelial progenitor cell subtype capable of forming neovessels both in vivo and in vitro. VEGF triggers repetitive Ca2+ spikes in both normal ECFCs (N-ECFCs) and ECFCs obtained from PMF patients (PMF-ECFCs). However, the spiking response to VEGF is significantly weaker in PMF-ECFCs. VEGF-elicited Ca2+ oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca2+ mobilization and store-operated Ca2+ entry. However, in most PMF-ECFCs, Ca2+ oscillations are triggered by a store-independent Ca2+ entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca2+ spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca2+ content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca2+ oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF.

Bordetella holmesii, an emerging pathogen, can be misidentified as Bordetella pertussis by routine polymerase chain reaction (PCR). In some reports, up to 29% of the patients diagnosed with pertussis have in fact B. holmesii infection and invasive, non-respiratory B. holmesii infections have been reported worldwide. This misdiagnosis undermines the knowledge of pertussis' epidemiology, and may lead to misconceptions on pertussis vaccine's efficacy. Recently, the number of whooping cough cases has increased significantly in several countries. The aim of this retrospective study was to determine whether B. holmesii was contributing to the increase in laboratory-confirmed cases of B. pertussis in Switzerland. A multiplex species-specific quantitative PCR assay was performed on 196 nasopharyngeal samples from Swiss patients with PCR-confirmed Bordetella infection (median age: 6 years-old, minimum 21 days-old, maximum 86 years-old), formerly diagnosed as Bordetella pertussis (IS481+). No B. holmesii (IS481+, IS1001-, hIS1001+) was identified. We discuss whether laboratories should implement specific PCR to recognize different Bordetella species. We conclude that in Switzerland B. holmesii seems to be circulating less than in neighboring countries and that specific diagnostic procedures are not necessary routinely. However, as the epidemiological situation may change rapidly, periodic reevaluation is suggested.