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.

Aquaporin-8 (AQP8) allows the bidirectional transport of water and hydrogen peroxide across biological membranes. Depending on its concentration, H 2 O 2 exerts opposite roles, amplifying growth factor signaling in physiological conditions, but causing severe cell damage when in excess. Thus, H 2 O 2 permeability is likely to be tightly controlled in living cells. Aims: In this study, we investigated whether and how the transport of H 2 O 2 through plasma membrane AQP8 is regulated, particularly during cell stress. Results: We show that diverse cellular stress conditions, including heat, hypoxia, and ER stress, reversibly inhibit the permeability of AQP8 to H 2 O 2 and water. Preventing the accumulation of intracellular reactive oxygen species (ROS) during stress counteracts AQP8 blockade. Once inhibition is established, AQP8-dependent transport can be rescued by reducing agents. Neither H 2 O 2 nor water transport is impaired in stressed cells expressing a mutant AQP8, in which cysteine 53 had been replaced by serine. Cells expressing this mutant are more resistant to stress-, drug-, and radiation-induced growth arrest and death. Innovation and Conclusion: The control of AQP8-mediated H 2 O 2 transport provides a novel mechanism to regulate cell signaling and survival during stress. Antioxid.

Clonal endothelial progenitor cells (EPCs) have been implicated in the aberrant vascular growth that features infantile hemangioma (IH), the most common benign vascular tumor in childhood that may cause ulceration, bleeding, and/or permanent disfigurement. Endothelial colony-forming cells (ECFCs), truly endothelial EPCs endowed with clonal ability and capable of forming patent vessels in vivo, remodel their Ca(2+) toolkit in tumor-derived patients to acquire an adaptive advantage. Particularly, they upregulate the proangiogenic store-operated Ca(2+) entry (SOCE) pathway due to the overexpression of its underlying components, that is, stromal interaction molecule 1 (Stim1), Orai1, and transient receptor potential canonical 1 (TRPC1). The present work was undertaken to assess whether and how the Ca(2+) signalosome is altered in IH-ECFCs by employing Ca(2+) and nitric oxide (NO) imaging, real-time polymerase chain reaction, western blotting, and functional assays. IH-ECFCs display a lower intracellular Ca(2+) release in response to either pharmacological (i.e., cyclopiazonic acid) or physiological (i.e., ATP and vascular endothelial growth factor) stimulation. Conversely, Stim1, Orai1, and TRPC1 transcripts and proteins are normally expressed in these cells and mediate a constitutive SOCE, which is sensitive to BTP-2, La(3+), and Pyr6 and recharges the intracellular Ca(2+) pool. The resting SOCE in IH-ECFCs is also associated to an increase in their proliferation rate and the basal production of NO compared to normal cells. Likewise, the pharmacological blockade of SOCE and NO synthesis block IH-ECFC growth. Collectively, these data indicate that the constitutive SOCE activation enhances IH-ECFC proliferation by augmenting basal NO production and sheds novel light on the molecular mechanisms of IH.

Endothelial progenitor cells (EPCs) have recently been employed in cell-based therapy (CBT) to promote regeneration of ischemic organs, such as heart and limbs. Furthermore, EPCs may sustain tumour vascularisation and provide an additional target for anticancer therapies. CBT is limited by the paucity of cells harvested from peripheral blood and suffers from several pitfalls, including the low rate of engrafted EPCs, whereas classic antiangiogenic treatments manifest a number of side effects and may induce resistance into the patients. CBT will benefit of a better understanding of the signal transduction pathway(s) which drive(s) EPC proliferation, trafficking, and incorporation into injured tissues. At the same time, this information might outline alternative molecular targets to impair tumor neovascularisation and improve the therapeutic outcome of antiangiogenic strategies. An increase in intracellular Ca(2+) concentration is the key signal in the regulation of cellular replication, migration, and differentiation. In particular, Ca(2+) signalling may regulate cellcycle progression, due to the Ca(2+)-sensitivity of a number of cycline-dependent kinases, and gene expression, owing to the Ca(2+)-dependence of several transcription factors. Recent work has outlined the role of the so-called store-operated Ca(2+) entry in driving EPC proliferation and migration. Unravelling the mechanisms guiding EPC engraftment into neovessels might supply the biological bases required to improve CBT and anticancer treatments. For example, genetic manipulation of the Ca(2+) signalling machinery could provide a novel approach to increase the extent of limb regeneration or preventing tumour vascularisation by EPCs.

SMYD3 is a methylase previously linked to cancer cell invasion and migration. Here we show that SMYD3 favors TGFβ-induced epithelial–mesenchymal transition (EMT) in mammary epithelial cells, promoting mesenchymal and EMT transcription factors expression. SMYD3 directly interacts with SMAD3 but it is unnecessary for SMAD2/3 phosphorylation and nuclear translocation. Conversely, SMYD3 is indispensable for SMAD3 direct association to EMT genes regulatory regions. Accordingly, SMYD3 knockdown or its pharmacological blockade with the BCI121 inhibitor dramatically reduce TGFβ-induced SMAD3 association to the chromatin. Remarkably, BCI121 treatment attenuates mesenchymal genes transcription in the mesenchymal-like MDA-MB-231 cell line and reduces their invasive ability in vivo, in a zebrafish xenograft model. In addition, clinical datasets analysis revealed that higher SMYD3 levels are linked to a less favorable prognosis in claudin-low breast cancers and to a reduced metastasis free survival in breast cancer patients. Overall, our data point at SMYD3 as a pivotal SMAD3 cofactor that promotes TGFβ-dependent mesenchymal gene expression and cell migration in breast cancer, and support SMYD3 as a promising pharmacological target for anti-cancer therapy.