Vascular Signalling.

In all vertebrates, closed blood and open lymph circulatory systems are essential for the delivery of nutrients and oxygen to tissues, waste clearance, and immune function [...].

AMP-activated protein kinase is a key regulator of acute neurovascular permeability.

Many neuronal and retinal disorders are associated with pathological hyperpermeability of the microvasculature. We have used explants of rodent retinae to study acute neurovascular permeability, signal transduction and the role of AMP-activated protein kinase (AMPK). Following stimulation with either vascular endothelial growth factor (VEGF-A) or bradykinin (BK), AMPK was rapidly and strongly phosphorylated and acted as a key mediator of permeability downstream of Ca2+. Accordingly, AMPK agonists potently induced acute retinal vascular leakage. AMPK activation led to phosphorylation of endothelial nitric oxide synthase (eNOS, also known as NOS3), which in turn increased VE-cadherin (CDH5) phosphorylation on Y685. In parallel, AMPK also mediated phosphorylation of p38 MAP kinases (hereafter p38) and HSP27 (HSPB1), indicating that it regulated paracellular junctions and cellular contractility, both previously associated with endothelial permeability. Endothelial AMPK provided a missing link in neurovascular permeability, connecting Ca2+ transients to the activation of eNOS and p38, irrespective of the permeability-inducing factor used. Collectively, we find that, due to its compatibility with small molecule antagonists and agonists, as well as siRNA, the ex vivo retina model constitutes a reliable tool to identify and study regulators and mechanisms of acute neurovascular permeability.

Endothelial Protease Activated Receptor 1 (PAR1) Signalling Is Required for Lymphocyte Transmigration across Brain Microvascular Endothelial Cells.

Lymphocyte transendothelial migration (TEM) relies on ICAM-1 engagement on the luminal surface of the endothelial cells (ECs). In blood-brain barrier (BBB) ECs, ICAM-1 triggers TEM signalling, including through JNK MAP kinase and AMP-activated protein kinase (AMPK), which lead to the phosphorylation and internalisation of the adherens junction protein VE-cadherin. In addition to ICAM-1, G protein-coupled receptors (GPCRs) are also required for lymphocytes TEM across BBB ECs. Here, we investigated the role of protease activated GPCRs (PARs) and found a specific role for PAR1 in support of lymphocyte TEM across BBB ECs in vitro. PAR1 requirement for TEM was confirmed using protease inhibitors, specific small molecule and peptide antagonists, function blocking antibodies and siRNA-mediated knockdown. In BBB ECs, PAR1 stimulation led to activation of signalling pathways essential to TEM; notably involving JNK and endothelial nitric oxide synthase (eNOS), with the latter downstream of AMPK. In turn, nitric oxide production through eNOS was essential for TEM by modulating VE-cadherin on Y731. Collectively, our data showed that non-canonical PAR1 activation by a lymphocyte-released serine protease is required for lymphocyte TEM across the BBB in vitro, and that this feeds into previously established ICAM-1-mediated endothelial TEM signalling pathways.

Polarised VEGFA Signalling at Vascular Blood­Neural Barriers.

At blood­neural barriers, endothelial VEGFA signalling is highly polarised, with entirely different responses being triggered by luminal or abluminal stimulation. These recent findings were made in a field which is still in its mechanistic infancy. For a long time, endothelial polarity has intuitively been presumed, and likened to that of epithelial cells, but rarely demonstrated. In the cerebral and the retinal microvasculature, the uneven distribution of VEGF receptors 1 and 2, with the former predominant on the luminal and the latter on the abluminal face of the endothelium, leads to a completely polarised signalling response to VEGFA. Luminal VEGFA activates VEGFR1 homodimers and AKT, leading to a cytoprotective response, whilst abluminal VEGFA induces vascular leakage via VEGFR2 homodimers and p38. Whilst these findings do not provide a complete picture of VEGFA signalling in the microvasculature­there are still unclear roles for heterodimeric receptor complexes as well as co-receptors­they provide essential insight into the adaptation of vascular systems to environmental cues that are naturally different, depending on whether they are present on the blood or tissue side. Importantly, sided responses are not only restricted to VEGFA, but exist for other important vasoactive agents.

Endothelial MAPKs Direct ICAM-1 Signaling to Divergent Inflammatory Functions.

Lymphocyte transendothelial migration (TEM) is critically dependent on intraendothelial signaling triggered by adhesion to ICAM-1. Here we show that endothelial MAPKs ERK, p38, and JNK mediate diapedesis-related and diapedesis-unrelated functions of ICAM-1 in cerebral and dermal microvascular endothelial cells (MVECs). All three MAPKs were activated by ICAM-1 engagement, either through lymphocyte adhesion or Ab-mediated clustering. MAPKs were involved in ICAM-1-dependent expression of TNF-α in cerebral and dermal MVECs, and CXCL8, CCL3, CCL4, VCAM-1, and cyclooxygenase 2 (COX-2) in cerebral MVECs. Endothelial JNK and to a much lesser degree p38 were the principal MAPKs involved in facilitating diapedesis of CD4+ lymphocytes across both types of MVECs, whereas ERK was additionally required for TEM across dermal MVECs. JNK activity was critical for ICAM-1-induced F-actin rearrangements. Furthermore, activation of endothelial ICAM-1/JNK led to phosphorylation of paxillin, its association with VE-cadherin, and internalization of the latter. Importantly ICAM-1-induced phosphorylation of paxillin was required for lymphocyte TEM and converged functionally with VE-cadherin phosphorylation. Taken together we conclude that during lymphocyte TEM, ICAM-1 signaling diverges into pathways regulating lymphocyte diapedesis, and other pathways modulating gene expression thereby contributing to the long-term inflammatory response of the endothelium.

Arachidonic acid-evoked Ca2+ signals promote nitric oxide release and proliferation in human endothelial colony forming cells.

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca2+ concentration ([Ca2+]i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca2+ 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 [Ca2+]i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca2+]i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca2+ signals required both intracellular Ca2+ release and external Ca2+ inflow. AA-induced Ca2+ 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 Ca2+ entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca2+ entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2+]i 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 Ca2+- and NO-dependent manner. Therefore, AA-evoked Ca2+ signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.

Endoplasmic Reticulum Ca(2+) Handling and Apoptotic Resistance in Tumor-Derived Endothelial Colony Forming Cells.

Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc.

Dysregulation of VEGF-induced proangiogenic Ca2+ oscillations in primary myelofibrosis-derived endothelial colony-forming cells.

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 Ca(2+) oscillations could be impaired in endothelial progenitor cells of subjects with malignancies. Therefore, we employed Ca(2+) imaging, wavelet analysis, and functional assays to assess whether and how VEGF-induced Ca(2+) 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 Ca(2+) 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 Ca(2+) oscillations are patterned by the interaction between inositol-1,4,5-trisphosphate-dependent Ca(2+) mobilization and store-operated Ca(2+) entry. However, in most PMF-ECFCs, Ca(2+) oscillations are triggered by a store-independent Ca(2+) entry pathway. We found that diacylglycerol gates transient receptor potential canonical 1 channel to trigger VEGF-dependent Ca(2+) spikes by recruiting the phospholipase C/inositol-1,4,5-trisphosphate signaling pathway, reflected as a decrease in endoplasmic reticulum Ca(2+) content. Finally, we found that, apart from being less robust and dysregulated as compared with N-ECFCs, VEGF-induced Ca(2+) oscillations modestly stimulate PMF-ECFC growth and in vitro angiogenesis. These results may explain the modest effect of anti-VEGF therapies in PMF.

Expression and function of toll-like receptors in human circulating endothelial colony forming cells.

Mature endothelial cells are known to sense microbial products through toll-like receptors (TLRs), a family of membrane proteins which serve as pathogen recognition and signaling elements; however, there are limited data in the literature about the expression and function of TLRs in human circulating endothelial colony forming cells (ECFCs), which are considered the most likely endothelial precursors. We expanded and differentiated in vitro umbilical cord blood (UCB) and adult peripheral blood (PB) ECFCs and studied the expression of TLR1 to TLR10 mRNA by qPCR analysis, and we further characterized TLR function in ECFCs through functional assays including in vitro ECFC growth and differentiation, assessment of cytokine production, and measurement of intracellular Ca(2+) signals. Both UCB- and PB-ECFCs had detectable mRNA levels of all the TLRs from 1 to 10; TLR4, a sensor of Gram-negative bacterial lipopolysaccharide (LPS), had a higher level compared to other TLRs. Exposure to LPS induced cytokine production, although with less efficiency compared to PB-mononuclear cells. However, no effect of LPS was seen on ECFC growth and differentiation, and no increase in intracellular Ca(2+) concentrations, which is essential for ECFC proliferation, was observed after exposure to increasing amounts of LPS. Our data show that all TLRs from 1 to 10 are constitutively expressed in ECFCs, and suggest that TLR4 is functional in ECFCs, but its activation through its ligand LPS does not affect ECFC growth and differentiation.

A functional transient receptor potential vanilloid 4 (TRPV4) channel is expressed in human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca(2+) signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca(2+) -permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca(2+) entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca(2+) signals uniquely in presence of extracellular Ca(2+). GSK- and PMA-induced Ca(2+) elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La(3+) and Gd(3+) dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels.

Store-operated Ca2+ entry does not control proliferation in primary cultures of human metastatic renal cellular carcinoma.

Store-operated Ca(2+) entry (SOCE) is activated following depletion of the inositol-1,4,5-trisphosphate (InsP3)-sensitive Ca(2+) pool to regulate proliferation in immortalized cell lines established from either primary or metastatic lesions. The molecular nature of SOCE may involve both Stim1, which senses Ca(2+) levels within the endoplasmic reticulum (ER) Ca(2+) reservoir, and a number of a Ca(2+)-permeable channels on the plasma membrane, including Orai1, Orai3, and members of the canonical transient receptor (TRPC1-7) family of ion channels. The present study was undertaken to assess whether SOCE is expressed and controls proliferation in primary cultures isolated from secondary lesions of heavily pretreated metastatic renal cell carcinoma (mRCC) patients. SOCE was induced following pharmacological depletion of the ER Ca(2+) store, but not by InsP3-dependent Ca(2+) release. Metastatic RCC cells express Stim1-2, Orai1-3, and TRPC1-7 transcripts and proteins. In these cells, SOCE was insensitive to BTP-2, 10 µM Gd(3+) and Pyr6, while it was inhibited by 100 µM Gd(3+), 2-APB, and carboxyamidotriazole (CAI). Neither Gd(3+) nor 2-APB or CAI impaired mRCC cell proliferation. Consistently, no detectable Ca(2+) signal was elicited by growth factor stimulation. Therefore, a functional SOCE is expressed but does not control proliferation of mRCC cells isolated from patients resistant to multikinase inhibitors.

Enhanced expression of Stim, Orai, and TRPC transcripts and proteins in endothelial progenitor cells isolated from patients with primary myelofibrosis.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

Ca2+ signalling in endothelial progenitor cells: a novel means to improve cell-based therapy and impair tumour vascularisation.

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.

Orai1 and transient receptor potential channels as novel molecular targets to impair tumor neovascularization in renal cell carcinoma and other malignancies.

The term "angiogenic switch" describes one of the earlier events of tumorigenesis, that occurs when the balance between pro-and anti-angiogenic factors shifts towards a pro-angiogenic outcome. This leads to the transition from a microscopic indolent lesion to a macroscopic and vascularised primary tumor, that may eventually metastasize and spread to distant sites. The molecular mechanisms underlying such a critical step in the carcinogenetic process have been extensively investigated. Both local endothelial cells (ECs) and endothelial progenitor cells (EPCs), recruited from bone marrow, have been implicated in the angiogenic switch, which is ultimately triggered by a plethora of growth factors released by cancer cells, pivotal among which is vascular endothelial growth factor (VEGF); indeed, VEGF both activates ECs nearby the growing tumor, and leads to EPC mobilization into the circulation. In kidney, in particular, the frequent mutation of the Von Hippel Lindau tumor suppressor gene leads to an overproduction of pro-angiogenic factors which makes this neoplasm quite sensitive to antiangiogenic drugs. However, it is now evident that the use of VEGF(Rs) inhibitors in everyday clinical practice is not as effective as observed in murine models. The investigation of alternative signaling pathways involved in the angiogenic switch is, therefore, imperative in order to induce tumor regression whereby preventing harmful drawback consequences. Ca(2+) entry across the plasma membrane has long been known to stimulate mature ECs to undergo angiogenesis. Recent work from several groups worldwide has then outlined that members of the Transient Receptor Potential (TRP) super-family of cationic channels and Orai1 provide the pathway for such proangiogenic Ca(2+) signal. In addition, Canonical TRP 1 (TRPC1) and Orai1 channels control proliferation and tubulogenesis in both normal EPCs and EPCs isolated from peripheral blood of tumor patients. As a consequence, TRP channels and Orai1 might serve as novel molecular targets to develop alternative and more effective strategies of angiogenesis inhibition.

How to utilize Ca²âº signals to rejuvenate the repairative phenotype of senescent endothelial progenitor cells in elderly patients affected by cardiovascular diseases: a useful therapeutic support of surgical approach?

Endothelial dysfunction or loss is the early event that leads to a host of severe cardiovascular diseases, such as atherosclerosis, hypertension, brain stroke, myocardial infarction, and peripheral artery disease. Ageing is regarded among the most detrimental risk factor for vascular endothelium and predisposes the subject to atheroscleorosis and inflammatory states even in absence of traditional comorbid conditions. Standard treatment to restore blood perfusion through stenotic arteries are surgical or endovascular revascularization. Unfortunately, ageing patients are not the most amenable candidates for such interventions, due to high operative risk or unfavourable vascular involvement. It has recently been suggested that the transplantation of autologous bone marrow-derived endothelial progenitor cells (EPCs) might constitute an alternative and viable therapeutic option for these individuals. Albeit pre-clinical studies demonstrated the feasibility of EPC-based therapy to recapitulate the diseased vasculature of young and healthy animals, clinical studies provided less impressive results in old ischemic human patients. One hurdle associated to this kind of approach is the senescence of autologous EPCs, which are less abundant in peripheral blood and display a reduced pro-angiogenic activity. Conversely, umbilical cord blood (UCB)-derived EPCs are more suitable for cellular therapeutics due to their higher frequency and sensitivity to growth factors, such as vascular endothelial growth factor (VEGF). An increase in intracellular Ca(2+) concentration is central to EPC activation by VEGF. We have recently demonstrated that the Ca(2+) signalling machinery driving the oscillatory Ca(2+) response to this important growth factor is different in UCB-derived EPCs as compared to their peripheral counterparts. In particular, we focussed on the so-called endothelial colony forming cells (ECFCs), which are the only EPC population belonging to the endothelial lineage and able to form capillary-like structures in vitro and stably integrate with host vasculature in vivo. The present review provides a brief description of how exploiting the Ca(2+) toolkit of juvenile EPCs to restore the repairative phenotype of senescent EPCs to enhance their regenerative outcome in therapeutic settings.

Canonical transient receptor potential 3 channel triggers vascular endothelial growth factor-induced intracellular Ca2+ oscillations in endothelial progenitor cells isolated from umbilical cord blood.

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.

Store-operated Ca2+ entry is remodelled and controls in vitro angiogenesis in endothelial progenitor cells isolated from tumoral patients.

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 Ca(2+) entry (SOCE), which is activated by a depletion of the intracellular Ca(2+) pool, regulates the growth of human EPCs, where is mediated by the interaction between the endoplasmic reticulum Ca(2+)-sensor, Stim1, and the plasmalemmal Ca(2+) channel, Orai1. As oncogenesis may be associated to the capability of tumor cells to grow independently on Ca(2+) influx, it is important to assess whether SOCE regulates EPC-dependent angiogenesis also in tumor patients. METHODOLOGY/PRINCIPAL FINDINGS: The present study employed Ca(2+) 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, La(3+) and Gd(3+). 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 Ca(2+) 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 Ca(2+) 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. CONCLUSIONS: 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.

Vascular endothelial growth factor stimulates endothelial colony forming cells proliferation and tubulogenesis by inducing oscillations in intracellular Ca2+ concentration.

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 Ca(2+) 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 Ca(2+) entry (SOCE). This study aimed at investigating the nature and the role of VEGF-elicited Ca(2+) signals in ECFCs. VEGF induced asynchronous Ca(2+) oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca(2+) (0Ca(2+)) 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 (InsP(3))-sensitive Ca(2+) pools with cyclopiazonic acid (CPA), and inhibition of InsP(3) receptors with 2-APB prevented the Ca(2+) response to VEGF. VEGF-induced ECFC proliferation and tubulogenesis were inhibited by the Ca(2+)-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 Ca(2+) oscillations require the interplay between InsP(3)-dependent Ca(2+) 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.

Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs). Sodium hydrosulphide (NaHS), a H2S donor, caused an elevation in [Ca2+]i, which disappeared in absence of extracellular Ca2+. NaHSinduced Ca2+ inflow was sensitive to high doses of Gd3+, but not BTP-2. Inhibition of the reverse-mode of the Na+-Ca2+ exchanger (NCX), with KB-R7943 or upon removal of extracellular Na+, abrogated the Ca2+ response to NaHS. Moreover, NaHS-elicited Ca2+ entry was significantly reduced by TEA and glybenclamide, which hinted at the involvement of ATP-dependent K+ (KATP) channels. Conversely, NaHS-evoked Ca2+ signal was not affected by the reducing agent, dithiothreitol. Acute addition of NaHS hindered both Ca2+ release and Ca2+ entry induced by ATP, a physiological agonist of ECs. Consistently, inhibition of endogenous H2S synthesis with DL-propargylglycine impaired ATP-induced Ca2+ inflow, whereas it did not affect Ca2+ mobilization. These data provide the first evidence that H2S may stimulate Ca2+ influx into ECs by recruiting the reverse-mode of NCX and KATP channels. In addition, they show that such gasotransmitter may modulate the Ca2+ signals elicited by physiological stimuli in intact endothelium.

Store-operated Ca(2+) entry is expressed in human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) may be recruited from the bone marrow to sites of tissue regeneration to sustain neovascularization and reendothelialization after acute vascular injury. This feature makes them particularly suitable for cell-based therapy. In mature endothelium, store-operated Ca(2+) entry (SOCE) is activated following emptying of inositol-1,4,5-trisphosphate-sensitive stores, and controls a wide number of functions, including proliferation, nitric oxide synthesis, and vascular permeability. The present work aimed at investigating SOCE expression in EPCs harvested from both peripheral blood (PB-EPCs) and umbilical cord blood (UCB-EPCs) by employing both Ca(2+) imaging and molecular biology techniques. SOCE was induced upon either pharmacological (ie, cyclopiazonic acid) or physiological (ie, ATP) depletion of the intracellular Ca(2+) pool. Further, store-dependent Ca(2+) entry was inhibited by the SOCE inhibitor, N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Real-time reverse transcription-polymerase chain reaction and western blot analyses showed that both PB-EPCs and UCB-EPCs express all the molecular candidates to mediate SOCE in differentiated cells, including TRPC1, TRPC4, Orai1, and Stim1. Moreover, pharmacological maneuvers demonstrated that, as well as in differentiated endothelial cells, the signal transduction pathway leading to depletion of the intracellular Ca(2+) pool impinged on the phospholipase C/inositol-1,4,5-trisphosphate pathway. Finally, blockage of SOCE with BTP-2 impaired PB-EPC proliferation. These findings provide the first evidence that EPCs express SOCE, which might thus be regarded as a novel target to enhance the regenerative outcome of cell-based therapy.