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Schizophrenia (SZ) is a complex neuropsychiatric disorder associated with severe cognitive dysfunction. Although research has mainly focused on forebrain abnormalities, emerging results support the involvement of the cerebellum in SZ physiopathology, particularly in Cognitive Impairment Associated with SZ (CIAS). Besides its role in motor learning and control, the cerebellum is implicated in cognition and emotion. Recent research suggests that structural and functional changes in the cerebellum are linked to deficits in various cognitive domains including attention, working memory, and decision-making. Moreover, cerebellar dysfunction is related to altered cerebellar circuit activities and connectivity with brain regions associated with cognitive processing. This review delves into the role of the cerebellum in CIAS. We initially consider the major forebrain alterations in CIAS, addressing impairments in neurotransmitter systems, synaptic plasticity, and connectivity. We then focus on recent findings showing that several mechanisms are also altered in the cerebellum and that cerebellar communication with the forebrain is impaired. This evidence implicates the cerebellum as a key component of circuits underpinning CIAS physiopathology. Further studies addressing cerebellar involvement in SZ and CIAS are warranted and might open new perspectives toward understanding the physiopathology and effective treatment of these disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Faris, Pischedda, Palesi and D’Angelo.)

Among the several mechanisms accounting for endocrine resistance in breast cancer, autophagy has emerged as an important player. Previous reports have evidenced that tamoxifen (Tam) induces autophagy and activates transcription factor EB (TFEB), which regulates the expression of genes controlling autophagy and lysosomal biogenesis. However, the mechanisms by which this occurs have not been elucidated as yet. This investigation aims at dissecting how TFEB is activated and contributes to Tam resistance in luminal A breast cancer cells. TFEB was overexpressed and prominently nuclear in Tam-resistant MCF7 cells (MCF7-TamR) compared with their parental counterpart, and this was not dependent on alterations of its nucleo-cytoplasmic shuttling. Tam promoted the release of lysosomal Ca 2+ through the major transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) and two-pore channels (TPCs), which caused the nuclear translocation and activation of TFEB. Consistently, inhibiting lysosomal calcium release restored the susceptibility of MCF7-TamR cells to Tam. Our findings demonstrate that Tam drives the nuclear relocation and transcriptional activation of TFEB by triggering the release of Ca 2+ from the acidic compartment, and they suggest that lysosomal Ca 2+ channels may represent new druggable targets to counteract the onset of autophagy-mediated endocrine resistance in luminal A breast cancer cells.

Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder that may lead patients to sudden cell death through the occurrence of ventricular arrhythmias. ACM is characterised by the progressive substitution of cardiomyocytes with fibrofatty scar tissue that predisposes the heart to life-threatening arrhythmic events. Cardiac mesenchymal stromal cells (C-MSCs) contribute to the ACM by differentiating into fibroblasts and adipocytes, thereby supporting aberrant remodelling of the cardiac structure. Flecainide is an I c antiarrhythmic drug that can be administered in combination with β-adrenergic blockers to treat ACM due to its ability to target both Na v 1.5 and type 2 ryanodine receptors (RyR2). However, a recent study showed that flecainide may also prevent fibro-adipogenic differentiation by inhibiting store-operated Ca 2+ entry (SOCE) and thereby suppressing spontaneous Ca 2+ oscillations in C-MSCs isolated from human ACM patients (ACM C-hMSCs). Herein, we briefly survey ACM pathogenesis and therapies and then recapitulate the main molecular mechanisms targeted by flecainide to mitigate arrhythmic events, including Na v 1.5 and RyR2. Subsequently, we describe the role of spontaneous Ca 2+ oscillations in determining MSC fate. Next, we discuss recent work showing that spontaneous Ca 2+ oscillations in ACM C-hMSCs are accelerated to stimulate their fibro-adipogenic differentiation. Finally, we describe the evidence that flecainide suppresses spontaneous Ca 2+ oscillations and fibro-adipogenic differentiation in ACM C-hMSCs by inhibiting constitutive SOCE.

Colorectal carcinoma (CRC) represents the fourth most common cancer worldwide and is the third most common cause of malignancy-associated mortality. Distant metastases to the liver and lungs are the main drivers of CRC-dependent death. Pro-oxidant therapies, which halt disease progression by exacerbating oxidative stress, represent an antitumour strategy that is currently exploited by chemotherapy and ionizing radiation. A more selective strategy to therapeutically exploit reactive oxygen species (ROS) signaling would consist in targeting a redox sensor that is up-regulated in metastatic cells and is tightly coupled to the stimulation of cancer cell death programs. The non-selective cation channel, Transient Receptor Potential Ankyrin 1 (TRPA1), serves as a sensor of the cellular redox state, being activated to promote extracellular Ca 2+ entry by an increase in oxidative stress. Recent work demonstrated that TRPA1 channel protein is up-regulated in several cancer types and that TRPA1-mediated Ca 2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca 2+ dysfunction and apoptosis. Herein, we sought to assess for the first time the outcome of TRPA1 activation by ROS on primary cultures of metastatic colorectal carcinoma (mCRC cells). We found that TRPA1 channel protein is up-regulated and mediates enhanced hydrogen peroxide (H 2 O 2 )-induced Ca 2+ entry in mCRC cells as compared to non-neoplastic control cells. The lipid peroxidation product 4-hydroxynonenal (4-HNE) is the main ROS responsible for TRPA1 activation upon mCRC cell exposure to oxidative stress. TRPA1-mediated Ca 2+ entry in response to H 2 O 2 and 4-HNE results in mitochondrial Ca 2+ overload, followed by mitochondrial depolarization and caspase-3/7 activation. Therefore, targeting TRPA1 could represent an alternative strategy to eradicate metastatic CRC by enhancing its sensitivity to oxidative stress., (© 2023. The Author(s).)

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca 2+ signaling activation and the response of EGCs markers ( GFAP , SOX10 , S100β , PLP1 , and CCL2 ) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans., Competing Interests: TM holds a patent for the immortalization technology described in this manuscript and is a shareholder of InSCREENeX GmbH, which commercializes cell lines immortalized by the described technology. TM and KN are employees of InSCREENeX GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zanoletti, Valdata, Nehlsen, Faris, Casali, Cacciatore, Sbarsi, Carriero, Arfini, van Baarle, De Simone, Barbieri, Raimondi, May, Moccia, Bozzola, Matteoli, Comincini and Manai.)

Hydrogen sulfide (H 2 S) is an endogenous gaseous molecule present in all living organisms that has been traditionally studied for its toxicity. Interestingly, increased understanding of H 2 S effects in organ physiology has recently shown its relevance as a signalling molecule, with potentially important implications in variety of clinical disorders, including cancer. H 2 S is primarily produced in mammalian cells under various enzymatic pathways are target of intense research biological mechanisms, and therapeutic effects of H 2 S. Herein, we describe the physiological and biochemical properties of H 2 S, the enzymatic pathways leading to its endogenous production and its catabolic routes. In addition, we discuss the role of currently known H 2 S-releasing agents, or H 2 S donors, including their potential as therapeutic tools. Then we illustrate the mechanisms known to support the pleiotropic effects of H 2 S, with a particular focus on persulfhydration, which plays a key role in H 2 S-mediating signalling pathways. We then address the paradoxical role played by H 2 S in tumour biology and discuss the potential of exploiting H 2 S levels as novel cancer biomarkers and diagnostic tools. Finally, we describe the most recent preclinical applications focused on assessing the anti-cancer impact of most common H 2 S-releasing compounds. While the evidence in favour of H 2 S as an alternative cancer therapy in the field of translational medicine is yet to be clearly provided, application of H 2 S is emerging as a potent anticancer therapy in preclinical trails., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABA A receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABA B receptors is still unknown. Preliminary evidence showed that GABA A receptor stimulation can induce an increase in endothelial Ca 2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca 2+ ] i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca 2+ release from neutral and acidic Ca 2+ stores, respectively, and sustained by store-operated Ca 2+ entry. GABA A and GABA B receptors were both required to trigger the endothelial Ca 2+ response. Unexpectedly, we found that the GABA A receptors signal in a flux-independent manner via the metabotropic GABA B receptors. Likewise, the full Ca 2+ response to GABA B receptors requires functional GABA A receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.

Background: Cardiac mesenchymal stromal cells (C-MSC) were recently shown to differentiate into adipocytes and myofibroblasts to promote the aberrant remodeling of cardiac tissue that characterizes arrhythmogenic cardiomyopathy (ACM). A calcium (Ca 2+ ) signaling dysfunction, mainly demonstrated in mouse models, is recognized as a mechanism impacting arrhythmic risk in ACM cardiomyocytes. Whether similar mechanisms influence ACM C-MSC fate is still unknown. Thus, we aim to ascertain whether intracellular Ca 2+ oscillations and the Ca 2+ toolkit are altered in human C-MSC obtained from ACM patients, and to assess their link with C-MSC-specific ACM phenotypes., Methods and Results: ACM C-MSC show enhanced spontaneous Ca 2+ oscillations and concomitant increased Ca 2+ /Calmodulin dependent kinase II (CaMKII) activation compared to control cells. This is manly linked to a constitutive activation of Store-Operated Ca 2+ Entry (SOCE), which leads to enhanced Ca 2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors. By targeting the Ca 2+ handling machinery or CaMKII activity, we demonstrated a causative link between Ca 2+ oscillations and fibro-adipogenic differentiation of ACM C-MSC. Genetic silencing of the desmosomal gene PKP2 mimics the remodelling of the Ca 2+ signalling machinery occurring in ACM C-MSC. The anti-arrhythmic drug flecainide inhibits intracellular Ca 2+ oscillations and fibro-adipogenic differentiation by selectively targeting SOCE., Conclusions: Altogether, our results extend the knowledge of Ca 2+ dysregulation in ACM to the stromal compartment, as an etiologic mechanism of C-MSC-related ACM phenotypes. A new mode of action of flecainide on a novel mechanistic target is unveiled against the fibro-adipose accumulation in ACM., (© 2022. The Author(s).)

Background: Platelets can support cancer progression via the release of microparticles and microvesicles that enhance the migratory behaviour of recipient cancer cells. We recently showed that platelet-derived extracellular vesicles (PEVs) stimulate migration and invasiveness in highly metastatic MDA-MB-231 cells by stimulating the phosphorylation of p38 MAPK and the myosin light chain 2 (MLC2). Herein, we assessed whether the pro-migratory effect of PEVs involves the remodelling of the Ca 2+ handling machinery, which drives MDA-MB-231 cell motility., Methods: PEVs were isolated from human blood platelets, and Fura-2/AM Ca 2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca 2+ dynamics and Ca 2+ -dependent migratory processes in MDA-MB-231 cells., Results: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca 2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP 3 R1/InsP 3 R2 mRNAs and proteins. The consequent enhancement of ER Ca 2+ depletion led to a significant increase in store-operated Ca 2+ entry. The larger Ca 2+ mobilization from the ER was required to potentiate serum-induced migration by recruiting p38 MAPK and MLC2., Conclusions: PEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca 2+ handling machinery.

The mechanism whereby an increase in neuronal activity (NA) leads to a local elevation in cerebral blood flow to supply the active neurons with oxygen and nutrients and remove the catabolic waste has been termed neurovascular coupling (NVC). Although it has long been thought that the vasoactive mediators involved in NVC are generated by neurons and astrocytes, recent evidence unveiled the crucial role of cerebrovascular endothelial cells in NVC. Brain capillary endothelial cells express a complement of ion channels, including inward-rectifier K + (K ir 2.1) channels, Transient Receptor Potential Ankyrin 1 channels and N-methyl-d-aspartate receptors that enable them to sense NA and thereby initiate the retrograde transmission of both electrical (via endothelium-dependent hyperpolarization) and chemical (via intercellular Ca 2+ waves also sustained by TRP Vanilloid 4 channels and inositol-1,4,5-trisphosphate receptors) signals that induce vasodilation in upstream pial arteries and parenchymal arteries. Notably, a defect in the endothelial ion channel machinery (particularly, K ir 2.1 channels) contributes to vascular cognitive impairment and dementia that features many cerebral disorders, including Alzheimer's disease, cerebral small vessel diseases, and traumatic brain injury. Targeting endothelial ion channels through appropriate pharmacological approaches might represent a hitherto unappreciated strategy to rescue CBF and prevent cognitive impairment and dementia in patients affected by cerebral disorders., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier Inc.)

(1) Background: Store-operated Ca 2+ entry (SOCE) drives the cytotoxic activity of cytotoxic T lymphocytes (CTLs) against cancer cells. However, SOCE can be enhanced in cancer cells due to an increase in the expression and/or function of its underlying molecular components, i.e., STIM1 and Orai1. Herein, we evaluated the SOCE expression and function in tumour-infiltrating lymphocytes (TILs) from metastatic colorectal cancer (mCRC) patients. (2) Methods: Functional studies were conducted in TILs expanded ex vivo from CRC liver metastases. Peripheral blood T cells from healthy donors (hPBTs) and mCRC patients (cPBTs) were used as controls. (3) Results: SOCE amplitude is enhanced in TILs compared to hPBTs and cPBTs, but the STIM1 protein is only up-regulated in TILs. Pharmacological manipulation showed that the increase in SOCE mainly depends on tonic modulation by diacylglycerol kinase, which prevents the protein kinase C-dependent inhibition of SOCE activity. The larger SOCE caused a stronger Ca 2+ response to T-cell receptor stimulation by autologous mCRC cells. Reducing Ca 2+ influx with BTP-2 during target cell killing significantly increases cytotoxic activity at low target:effector ratios. (4) Conclusions: SOCE is enhanced in ex vivo-expanded TILs deriving from mCRC patients but decreasing Ca 2+ influx with BTP-2 increases cytotoxic activity at a low TIL density.

Therapeutic neovascularization represents a promising strategy to rescue the vascular network and restore organ function in cardiovascular disorders (CVDs), including acute myocardial infarction, heart failure, peripheral artery disease, and brain stroke. Endothelial colony forming cells (ECFCs), which are mobilized in circulation upon an ischemic insult, are commonly regarded as the most suitable cellular tool to achieve therapeutic neovascularization. ECFCs can be genetically or pharmacologically manipulated to enhance their vasoreparative potential by boosting specific pro-angiogenic signalling pathways. However, optical stimulation represents the most reliable approach to control cellular activity because of its high selectivity and unprecedented spatio-temporal resolution. Herein, we discuss a novel strategy to drive ECFC angiogenic activity in ischemic tissues by combining geneless optical excitation with photosensitive organic semiconductors. We describe how photoexcitation of the conducting polymer poly(3-hexylthiophene-2,5-diyl), also known as P3HT, stimulates extracellular Ca 2+ entry through Transient Receptor Potential Vanilloid 1 (TRPV1) channels upon the production of hydrogen peroxide (H 2 O 2 ) in the cleft between the nanomaterial and the cell membrane. H 2 O 2 -induced TRPV1-dependent Ca 2+ entry stimulates ECFC proliferation and tube formation, thereby providing the proof-of-concept that photoexcitation of organic semiconductors may offer a reliable strategy to stimulate ECFCs-dependent neovascularization in CVDs., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Hibiscus sabdariffa L. is a tropical plant belonging to the Malvaceae family. In Kurdistan, the Autonomous Region of Iraq, water infusion of H. sabdariffa calyces is recommended for the treatment of hypotension and the common cold. Three distillation techniques: hydrodistillation (HD), steam distillation (SD), and solvent-free microwave-assisted extraction (SFME) have been compared to obtain the essential oils from calyces. The composition of the extracts was investigated by GC-FID and GC-MS. A total of 62 compounds have been identified, from which 55 components were found in HD distillates (95.75%), 37 components in SFME (96.06%), and 29 in SD (99.63%). Chemometric tools were applied to optimise and evidence the relation between distillation techniques and composition of the obtained essential oils as an investigation for the essential oils commercialisation approach of Hibiscus sabdariffa L. that have been done from a long time using conventional hydrodistillation in the local Herbal and Tea markets in Kurdistan.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca 2+ release. NAADP-induced lysosomal Ca 2+ release may be amplified by the endoplasmic reticulum (ER) through the Ca 2+ -induced Ca 2+ release (CICR) mechanism. NAADP-induced intracellular Ca 2+ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca 2+ signal that was abolished by disrupting the lysosomal Ca 2+ store with Gly-Phe β-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca 2+ release resulted in the Ca 2+ -dependent recruitment of ER-embedded InsP 3 Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca 2+ to recruit ER-embedded InsP 3 Rs through CICR. NAADP-induced EL Ca 2+ mobilization via EL TPC was found to trigger the intracellular Ca 2+ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca 2+ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Faris, Casali, Negri, Iengo, Biggiogera, Maione and Moccia.)

Endothelial colony forming cells (ECFCs) represent the most suitable cellular substrate to induce revascularization of ischemic tissues. Recently, optical excitation of the light-sensitive conjugated polymer, regioregular Poly (3-hexyl-thiophene), rr-P3HT, was found to stimulate ECFC proliferation and tube formation by activating the non-selective cation channel, Transient Receptor Potential Vanilloid 1 (TRPV1). Herein, we adopted a multidisciplinary approach, ranging from intracellular Ca 2+ imaging to pharmacological manipulation and genetic suppression of TRPV1 expression, to investigate the effects of photoexcitation on intracellular Ca 2+ concentration ([Ca 2+ ] i ) in circulating ECFCs plated on rr-P3HT thin films. Polymer-mediated optical excitation induced a long-lasting increase in [Ca 2+ ] i that could display an oscillatory pattern at shorter light stimuli. Pharmacological and genetic manipulation revealed that the Ca 2+ response to light was triggered by extracellular Ca 2+ entry through TRPV1, whose activation required the production of reactive oxygen species at the interface between rr-P3HT and the cell membrane. Light-induced TRPV1-mediated Ca 2+ entry was able to evoke intracellular Ca 2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors, followed by store-operated Ca 2+ entry on the plasma membrane. These data show that TRPV1 may serve as a decoder at the interface between rr-P3HT thin films and ECFCs to translate optical excitation in pro-angiogenic Ca 2+ signals., (Copyright © 2021. Published by Elsevier Ltd.)

The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) to mediate extracellular Ca 2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO and trigger neurovascular coupling (NVC). Neuronal and glial NMDARs may also operate in a flux-independent manner, although it is unclear whether their non-ionotropic mode of action is involved in NVC. Recently, endothelial NMDARs were found to trigger Ca 2+ -dependent NO production and induce NVC, but the underlying mode of signaling remains elusive. Herein, we report that GluN1 protein, as well as GluN2C and GluN3B transcripts and proteins, were expressed and that NMDA did not elicit inward currents, but induced a dose-dependent increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the human brain microvascular endothelial cell line, hCMEC/D3. A multidisciplinary approach, including live cell imaging, whole-cell patch-clamp recordings, pharmacological manipulation and gene targeting, revealed that NMDARs increase the [Ca 2+ ] i in a flux-independent manner in hCMEC/D3 cells. The Ca 2+ response to NMDA was triggered by endogenous Ca 2+ release from the endoplasmic reticulum and the lysosomal Ca 2+ stores and sustained by store-operated Ca 2+ entry. Unexpectedly, pharmacological and genetic blockade of mGluR1 and mGluR5 dramatically impaired NMDARs-mediated Ca 2+ signals. These findings indicate that NMDARs may increase the endothelial [Ca 2+ ] i in a flux-independent manner via group 1 mGluRs. However, imaging of DAF-FM fluorescence revealed that NMDARs may also induce Ca 2+ -dependent NO release by signaling in a flux-dependent manner. These findings, therefore, shed novel light on the mechanisms whereby brain microvascular endothelium decodes glutamatergic signaling and regulates NVC., (Copyright © 2021. Published by Elsevier Ltd.)

An increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) controls virtually all endothelial cell functions and is, therefore, crucial to maintain cardiovascular homeostasis. An aberrant elevation in endothelial can indeed lead to severe cardiovascular disorders. Likewise, moderate amounts of reactive oxygen species (ROS) induce intracellular Ca 2+ signals to regulate vascular functions, while excessive ROS production may exploit dysregulated Ca 2+ dynamics to induce endothelial injury. Herein, we survey how ROS induce endothelial Ca 2+ signals to regulate vascular functions and, vice versa, how aberrant ROS generation may exploit the Ca 2+ handling machinery to promote endothelial dysfunction. ROS elicit endothelial Ca 2+ signals by regulating inositol-1,4,5-trisphosphate receptors, sarco-endoplasmic reticulum Ca 2+ -ATPase 2B, two-pore channels, store-operated Ca 2+ entry (SOCE), and multiple isoforms of transient receptor potential (TRP) channels. ROS-induced endothelial Ca 2+ signals regulate endothelial permeability, angiogenesis, and generation of vasorelaxing mediators and can be exploited to induce therapeutic angiogenesis, rescue neurovascular coupling, and induce cancer regression. However, an increase in endothelial [Ca 2+ ] i induced by aberrant ROS formation may result in endothelial dysfunction, inflammatory diseases, metabolic disorders, and pulmonary artery hypertension. This information could pave the way to design alternative treatments to interfere with the life-threatening interconnection between endothelial ROS and Ca 2+ signaling under multiple pathological conditions.

Second trimester foetal human amniotic fluid-derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug-induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS-CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony-forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary-like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS-CM Hypo ) triggers intracellular Ca 2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca 2+ -dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca 2+ oscillations by promoting extracellular Ca 2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4-mediated Ca 2+ entry, in turn, promoted the concerted interplay between inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-induced endogenous Ca 2+ release and store-operated Ca 2+ entry (SOCE). hAFS-CM Hypo -induced intracellular Ca 2+ oscillations resulted in the nuclear translocation of the Ca 2+ -sensitive transcription factor p65 NF-κB. Finally, inhibition of either intracellular Ca 2+ oscillations or NF-κB activity prevented hAFS-CM Hypo -induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS-CM Hypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic-related myocardial injury., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Neurovascular coupling (NVC) represents the mechanisms whereby an increase in neuronal activity (NA) may lead to local vasodilation and increase in regional cerebral blood flow (CBF). It has long been thought that neurons and astrocytes generate the vasoactive mediators regulating local changes in CBF, whereas cerebrovascular endothelial cells are not able to directly sense NA. Unexpectedly, recent evidence demonstrated that brain microvascular endothelial cells may sense NA through inward-rectifier K + (K ir 2.1) channels and may detect synaptic activity via N-methyl-d-aspartate (NMDA) receptors (NMDARs). In the present perspective, therefore, we discuss the hypothesis that endothelial K ir 2.1 channels and NMDARs play a key role in NVC and in CBF regulation, which is crucial to unravel the cellular and molecular underpinnings of blood oxygen level-dependent signals., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca 2+ ] i , in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca 2+ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca 2+ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca 2+ ] i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca 2+ ] i . These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

Emerging evidence hints in favor of a life-threatening link between severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and the cardiovascular system. SARS-CoV-2 may result in dramatic cardiovascular complications, whereas the severity of COronaVIrus Disease 2019 (COVID-19) and the incidence of fatalities tend to increase in patients with pre-existing cardiovascular complications. SARS-CoV-2 is internalized into the host cells by endocytosis and may then escape the endolysosomal system via endosomes. Two-pore channels drive endolysosomal trafficking through the release of endolysosomal Ca 2+ . Recent evidence suggested that the pharmacological inhibition of TPCs prevents Ebola virus and Middle East Respiratory Syndrome COronaVirus (MERS-CoV) entry into host cells. In this perspective, we briefly summarize the biophysical and pharmacological features of TPCs, illustrate their emerging role in the cardiovascular system, and finally present them as a reliable target to treat cardiovascular complications in COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moccia, Negri, Faris, Perna, De Luca, Soda, Romani and Guerra.)

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca 2+ -releasing messenger that increases the intracellular Ca 2+ concentration by mobilizing the lysosomal Ca 2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca 2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca 2+ pool endowed with TPC1 is also present in human endothelial colony-forming cells (ECFCs), which represent the only known truly endothelial precursors. Herein, we sought to explore the role of the lysosomal Ca 2+ store and TPC1 in circulating ECFCs by harnessing Ca 2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca 2+ sequestration by acidic organelles, caused endogenous Ca 2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP 3 )-sensitive endoplasmic reticulum (ER) Ca 2+ pool. Likewise, the amount of ER releasable Ca 2+ was reduced by disrupting lysosomal Ca 2+ content. Liposomal delivery of NAADP induced a transient Ca 2+ signal that was abolished by disrupting the lysosomal Ca 2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca 2+ release also required ER-embedded InsP 3 receptors. Finally, NAADP-induced lysosomal Ca 2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca 2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca 2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca 2+ release can selectively be recruited to induce the Ca 2+ response to specific cues in circulating ECFCs., (© 2020 Wiley Periodicals LLC.)

An increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) regulates a plethora of functions in the cardiovascular (CV) system, including contraction in cardiomyocytes and vascular smooth muscle cells (VSMCs), and angiogenesis in vascular endothelial cells and endothelial colony forming cells. The sarco/endoplasmic reticulum (SR/ER) represents the largest endogenous Ca 2+ store, which releases Ca 2+ through ryanodine receptors (RyRs) and/or inositol-1,4,5-trisphosphate receptors (InsP 3 Rs) upon extracellular stimulation. The acidic vesicles of the endolysosomal (EL) compartment represent an additional endogenous Ca 2+ store, which is targeted by several second messengers, including nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ], and may release intraluminal Ca 2+ through multiple Ca 2+ permeable channels, including two-pore channels 1 and 2 (TPC1-2) and Transient Receptor Potential Mucolipin 1 (TRPML1). Herein, we discuss the emerging, pathophysiological role of EL Ca 2+ signaling in the CV system. We describe the role of cardiac TPCs in β-adrenoceptor stimulation, arrhythmia, hypertrophy, and ischemia-reperfusion injury. We then illustrate the role of EL Ca 2+ signaling in VSMCs, where TPCs promote vasoconstriction and contribute to pulmonary artery hypertension and atherosclerosis, whereas TRPML1 sustains vasodilation and is also involved in atherosclerosis. Subsequently, we describe the mechanisms whereby endothelial TPCs promote vasodilation, contribute to neurovascular coupling in the brain and stimulate angiogenesis and vasculogenesis. Finally, we discuss about the possibility to target TPCs, which are likely to mediate CV cell infection by the Severe Acute Respiratory Disease-Coronavirus-2, with Food and Drug Administration-approved drugs to alleviate the detrimental effects of Coronavirus Disease-19 on the CV system., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Exogenous administration of hydrogen sulfide (H 2 S) is emerging as an alternative anticancer treatment. H 2 S-releasing compounds have been shown to exert a strong anticancer effect by suppressing proliferation and/or inducing apoptosis in several cancer cell types, including colorectal carcinoma (CRC). The mechanism whereby exogenous H 2 S affects CRC cell proliferation is yet to be clearly elucidated, but it could involve an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ). Herein, we sought to assess for the first time whether (and how) sodium hydrosulfide (NaHS), one of the most widely employed H 2 S donors, induced intracellular Ca 2+ signals in primary cultures of human metastatic CRC (mCRC) cells. We provided the evidence that NaHS induced extracellular Ca 2+ entry in mCRC cells by activating the Ca 2+ -permeable channel Transient Receptor Potential Vanilloid 1 (TRPV1) followed by the Na + -dependent recruitment of the reverse-mode of the Na + /Ca 2+ (NCX) exchanger. In agreement with these observations, TRPV1 protein was expressed and capsaicin, a selective TRPV1 agonist, induced Ca 2+ influx by engaging both TRPV1 and NCX in mCRC cells. Finally, NaHS reduced mCRC cell proliferation, but did not promote apoptosis or aberrant mitochondrial depolarization. These data support the notion that exogenous administration of H 2 S may prevent mCRC cell proliferation through an increase in [Ca 2+ ] i , which is triggered by TRPV1.

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca 2+ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD.

Background: While there is a long history of measuring death and disability from injuries, modern research methods must account for the wide spectrum of disability that can occur in an injury, and must provide estimates with sufficient demographic, geographical and temporal detail to be useful for policy makers. The Global Burden of Disease (GBD) 2017 study used methods to provide highly detailed estimates of global injury burden that meet these criteria., Methods: In this study, we report and discuss the methods used in GBD 2017 for injury morbidity and mortality burden estimation. In summary, these methods included estimating cause-specific mortality for every cause of injury, and then estimating incidence for every cause of injury. Non-fatal disability for each cause is then calculated based on the probabilities of suffering from different types of bodily injury experienced., Results: GBD 2017 produced morbidity and mortality estimates for 38 causes of injury. Estimates were produced in terms of incidence, prevalence, years lived with disability, cause-specific mortality, years of life lost and disability-adjusted life-years for a 28-year period for 22 age groups, 195 countries and both sexes., Conclusions: GBD 2017 demonstrated a complex and sophisticated series of analytical steps using the largest known database of morbidity and mortality data on injuries. GBD 2017 results should be used to help inform injury prevention policy making and resource allocation. We also identify important avenues for improving injury burden estimation in the future., Competing Interests: Competing interests: Dr. James reports grants from Sanofi Pasteur, outside the submitted work. Dr. Driscoll reports grants from World Health Organisation, during the conduct of the study. Dr Shariful Islam is funded by a Fellowship from National Heart Foundation of Australia and Institute for Physical Activity and Nutrition, Deakin University. Dr. Ivers reports grants from National Health and Medical Research Council of Australia, during the conduct of the study. Dr. Jozwiak reports personal fees from TEVA, personal fees from ALAB, personal fees from BOEHRINGER INGELHEIM, personal fees from SYNEXUS, non-financial support from SERVIER, non-financial support from MICROLIFE, non-financial support from MEDICOVER, outside the submitted work. Walter Mendoza is currently Program Analyst Population and Development at the Peru Country Office of the United Nations Population Fund-UNFPA, which does not necessarily endorses this study. Dr. Rakovac reports grants from World Health Organization, during the conduct of the study. Dr. Sheikh reports grants from Health Data Research UK, outside the submitted work. Dr. Singh reports personal fees from Crealta/Horizon, Medisys, Fidia, UBM LLC, Trio health, Medscape, WebMD, Clinical Care options, Clearview healthcare partners, Putnam associates, Spherix, Practice Point communications, the National Institutes of Health and the American College of Rheumatology, personal fees from Speaker’s bureau of Simply Speaking. Dr. Singh owns stock options in Amarin pharmaceuticals and Viking pharmaceuticals. Dr. Singh serves on the steering committee of OMERACT, an international organization that develops measures for clinical trials and receives arms-length funding from 12 pharmaceutical companies. Dr. Singh serves on the FDA Arthritis Advisory Committee. Dr. Singh is a member of the Veterans Affairs Rheumatology Field Advisory Committee. Dr. Singh is the editor and the Director of the UAB Cochrane Musculoskeletal Group Satellite Center on Network Meta-analysis, outside the submitted work. Dr. Stein reports personal fees from Lundbeck and Sun, outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Background: Past research in population health trends has shown that injuries form a substantial burden of population health loss. Regular updates to injury burden assessments are critical. We report Global Burden of Disease (GBD) 2017 Study estimates on morbidity and mortality for all injuries., Methods: We reviewed results for injuries from the GBD 2017 study. GBD 2017 measured injury-specific mortality and years of life lost (YLLs) using the Cause of Death Ensemble model. To measure non-fatal injuries, GBD 2017 modelled injury-specific incidence and converted this to prevalence and years lived with disability (YLDs). YLLs and YLDs were summed to calculate disability-adjusted life years (DALYs)., Findings: In 1990, there were 4 260 493 (4 085 700 to 4 396 138) injury deaths, which increased to 4 484 722 (4 332 010 to 4 585 554) deaths in 2017, while age-standardised mortality decreased from 1079 (1073 to 1086) to 738 (730 to 745) per 100 000. In 1990, there were 354 064 302 (95% uncertainty interval: 338 174 876 to 371 610 802) new cases of injury globally, which increased to 520 710 288 (493 430 247 to 547 988 635) new cases in 2017. During this time, age-standardised incidence decreased non-significantly from 6824 (6534 to 7147) to 6763 (6412 to 7118) per 100 000. Between 1990 and 2017, age-standardised DALYs decreased from 4947 (4655 to 5233) per 100 000 to 3267 (3058 to 3505)., Interpretation: Injuries are an important cause of health loss globally, though mortality has declined between 1990 and 2017. Future research in injury burden should focus on prevention in high-burden populations, improving data collection and ensuring access to medical care., Competing Interests: Competing interests: Dr. James reports grants from Sanofi Pasteur, outside the submitted work. Dr. Driscoll reports grants from World Health Organization, during the conduct of the study. Dr. Ivers reports grants from National Health and Medical Research Council of Australia, during the conduct of the study. Dr. Jozwiak reports personal fees from TEVA, personal fees from ALAB, personal fees from BOEHRINGER INGELHEIM, personal fees from SYNEXUS, non-financial support from SERVIER, non-financial support from MICROLIFE, non-financial support from MEDICOVER, outside the submitted work. Dr. Rakovac reports grants from World Health Organization, during the conduct of the study. Dr Shariful Islam is funded by National Heart Foundation of Australia and Institute for Physical Activity and Nutrition, Deakin University. Dr. Sheikh reports grants from Health Data Research UK, outside the submitted work. Dr. Singh reports personal fees from Crealta/Horizon, Medisys, Fidia, UBM LLC, Trio health, Medscape, WebMD, Clinical Care options, Clearview healthcare partners, Putnam associates, Spherix, Practice Point communications, the National Institutes of Health and the American College of Rheumatology, and Speaker’s bureau of Simply Speaking, owns stock options in Amarin pharmaceuticals and Viking pharmaceuticals, serves on the steering committee of OMERACT, an international organization that develops measures for clinical trials and receives arm’s length funding from 12 pharmaceutical companies, serves on the FDA Arthritis Advisory Committee, is a member of the Veterans Affairs Rheumatology Field Advisory Committee, and is the editor and the Director of the UAB Cochrane Musculoskeletal Group Satellite Center on Network Meta-analysis, outside the submitted work. Dr. Stein reports personal fees from Lundbeck, personal fees from Sun, outside the submitted work. Dr. Degenhardt reports grants from Indivior, Seqirus, Reckitt Benckiser, outside the submitted work., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

Systemic lupus erythematosus (SLE) is an autoimmune multisystem disease featured by an increased cardiovascular risk that may lead to premature patient's death. It has been demonstrated that SLE patients suffer from early onset endothelial dysfunction which is due to the impairment of endogenous vascular repair mechanisms. Vascular integrity and homeostasis are maintained by endothelial progenitor cells (EPCs), which are mobilized in response to endothelial injury to replace damaged endothelial cells. Two main EPCs subpopulations exist in peripheral blood: endothelial colony forming cells (ECFCs), which represent truly endothelial precursors and can physically engraft within neovessels, and myeloid angiogenic cells (MACs), which sustain angiogenesis in a paracrine manner. Emerging evidence indicates that ECFCs/MACs are down-regulated and display compromised angiogenic activity in SLE, thereby contributing to the pathogenesis of this disease. Intracellular calcium (Ca 2+ ) signaling plays a crucial role in maintaining vascular integrity by stimulating migration, proliferation and tube formation in both ECFCs and MACs. Herein, we illustrate the evidences that support the role played by EPCs dysfunction in SLE. Subsequently, we discuss about the hypothesis that the Ca 2+ handling machinery is compromised in SLE-derived ECFCs and MACs, thereby resulting in their reduced pro-angiogenic activity. Finally, we speculate about the proposal to exploit intracellular Ca 2+ signaling to improve ECFCs' reparative phenotype and suggest this strategy as a new approach to treat SLE patients., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity causes an increase in local cerebral blood flow (CBF) to ensure local supply of oxygen and nutrients to the activated areas. The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-D-aspartate receptors to mediate extracellular Ca 2+ entry and stimulate neuronal nitric oxide (NO) synthase to release NO, thereby triggering NVC. Recent work suggested that endothelial Ca 2+ signals could underpin NVC by recruiting the endothelial NO synthase. For instance, acetylcholine induced intracellular Ca 2+ signals followed by NO release by activating muscarinic 5 receptors in hCMEC/D3 cells, a widely employed model of human brain microvascular endothelial cells. Herein, we sought to assess whether also glutamate elicits metabotropic Ca 2+ signals and NO release in hCMEC/D3 cells. Glutamate induced a dose-dependent increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that was blocked by α-methyl-4-carboxyphenylglycine and phenocopied by trans-1-amino-1,3-cyclopentanedicarboxylic acid, which, respectively, block and activate group 1 metabotropic glutamate receptors (mGluRs). Accordingly, hCMEC/D3 expressed both mGluR1 and mGluR5 and the Ca 2+ response to glutamate was inhibited by their pharmacological blockade with, respectively, CPCCOEt and MTEP hydrochloride. The Ca 2+ response to glutamate was initiated by endogenous Ca 2+ release from the endoplasmic reticulum and endolysosomal Ca 2+ store through inositol-1,4,5-trisphosphate receptors and two-pore channels, respectively, and sustained by store-operated Ca 2+ entry. In addition, glutamate induced robust NO release that was suppressed by pharmacological blockade of the accompanying increase in [Ca 2+ ] i . These data demonstrate for the first time that glutamate may induce metabotropic Ca 2+ signals in human brain microvascular endothelial cells. The Ca 2+ response to glutamate is likely to support NVC during neuronal activity, thereby reinforcing the emerging role of brain microvascular endothelial cells in the regulation of CBF.

Therapeutic angiogenesis represents an emerging strategy to treat ischemic diseases by stimulating blood vessel growth to rescue local blood perfusion. Therefore, injured microvasculature may be repaired by stimulating resident endothelial cells or circulating endothelial colony forming cells (ECFCs) or by autologous cell-based therapy. Endothelial Ca 2+ signals represent a crucial player in angiogenesis and vasculogenesis; indeed, several angiogenic stimuli induce neovessel formation through an increase in intracellular Ca 2+ concentration. Several members of the Transient Receptor Potential (TRP) channel superfamily are expressed and mediate Ca 2+ -dependent functions in vascular endothelial cells and in ECFCs, the only known truly endothelial precursor. TRP Vanilloid 1 (TRPV1), a polymodal cation channel, is emerging as an important player in endothelial cell migration, proliferation, and tubulogenesis, through the integration of several chemical stimuli. Herein, we first summarize TRPV1 structure and gating mechanisms. Next, we illustrate the physiological roles of TRPV1 in vascular endothelium, focusing our attention on how endothelial TRPV1 promotes angiogenesis. In particular, we describe a recent strategy to stimulate TRPV1-mediated pro-angiogenic activity in ECFCs, in the presence of a photosensitive conjugated polymer. Taken together, these observations suggest that TRPV1 represents a useful target in the treatment of ischemic diseases.

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H 1 Rs) which result in the downstream production of nitric oxide (NO), the most powerful vasodilator transmitter in the brain. Although endothelial Ca 2+ signals drive histamine-induced NO release throughout the peripheral circulation, the mechanism by which histamine evokes NO production in human cerebrovascular endothelial cells is still unknown. Herein, we exploited the human cerebral microvascular endothelial cell line, hCMEC/D3, to assess the role of intracellular Ca 2+ signaling in histamine-induced NO release. To achieve this goal, hCMEC/D3 cells were loaded with the Ca 2+ - and NO-sensitive dyes, Fura-2/AM and DAF-FM/AM, respectively. Histamine elicited repetitive oscillations in intracellular Ca 2+ concentration in hCMEC/D3 cells throughout a concentration range spanning from 1 pM up to 300 μM. The oscillatory Ca 2+ response was suppressed by the inhibition of H 1 Rs with pyrilamine, whereas H 1 R was abundantly expressed at the protein level. We further found that histamine-induced intracellular Ca 2+ oscillations were initiated by endogenous Ca 2+ mobilization through inositol-1,4,5-trisphosphate- and nicotinic acid dinucleotide phosphate-sensitive channels and maintained over time by store-operated Ca 2+ entry. In addition, histamine evoked robust NO release that was prevented by interfering with the accompanying intracellular Ca 2+ oscillations, thereby confirming that the endothelial NO synthase is recruited by Ca 2+ spikes also in hCMEC/D3 cells. These data provide the first evidence that histamine evokes NO production from human cerebrovascular endothelial cells through intracellular Ca 2+ oscillations, thereby shedding novel light on the mechanisms by which this neuromodulator controls cerebral blood flow., (© 2019 Wiley Periodicals, Inc.)

Vasculogenesis, angiogenesis and arteriogenesis represent three crucial mechanisms involved in the formation and maintenance of the vascular network in embryonal and post-natal life. It has long been known that endothelial Ca 2+ signals are key players in vascular remodeling; indeed, multiple pro-angiogenic factors, including vascular endothelial growth factor, regulate endothelial cell fate through an increase in intracellular Ca 2+ concentration. Transient Receptor Potential (TRP) channel consist in a superfamily of non-selective cation channels that are widely expressed within vascular endothelial cells. In addition, TRP channels are present in the two main endothelial progenitor cell (EPC) populations, i.e., myeloid angiogenic cells (MACs) and endothelial colony forming cells (ECFCs). TRP channels are polymodal channels that can assemble in homo- and heteromeric complexes and may be sensitive to both pro-angiogenic cues and subtle changes in local microenvironment. These features render TRP channels the most versatile Ca 2+ entry pathway in vascular endothelial cells and in EPCs. Herein, we describe how endothelial TRP channels stimulate vascular remodeling by promoting angiogenesis, arteriogenesis and vasculogenesis through the integration of multiple environmental, e.g., extracellular growth factors and chemokines, and intracellular, e.g., reactive oxygen species, a decrease in Mg 2+ levels, or hypercholesterolemia, stimuli. In addition, we illustrate how endothelial TRP channels induce neovascularization in response to synthetic agonists and small molecule drugs. We focus the attention on TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPV1, TRPV4, TRPM2, TRPM4, TRPM7, TRPA1, that were shown to be involved in angiogenesis, arteriogenesis and vasculogenesis. Finally, we discuss the role of endothelial TRP channels in aberrant tumor vascularization by focusing on TRPC1, TRPC3, TRPV2, TRPV4, TRPM8, and TRPA1. These observations suggest that endothelial TRP channels represent potential therapeutic targets in multiple disorders featured by abnormal vascularization, including cancer, ischemic disorders, retinal degeneration and neurodegeneration., (Copyright © 2020 Negri, Faris, Berra-Romani, Guerra and Moccia.)

Background: Obesity is a major cardiovascular risk factor which dramatically impairs endothelium- dependent vasodilation and leads to hypertension and vascular damage. The impairment of the vasomotor response to extracellular autacoids, e.g., acetylcholine, mainly depends on the reduced Nitric Oxide (NO) bioavailability, which hampers vasorelaxation in large conduit arteries. In addition, obesity may affect Endothelium-Dependent Hyperpolarization (EDH), which drives vasorelaxation in small resistance arteries and arterioles. Of note, endothelial Ca2+ signals drive NO release and trigger EDH., Methods: A structured search of bibliographic databases was carried out to retrieve the most influential, recent articles on the impairment of vasorelaxation in animal models of obesity, including obese Zucker rats, and on the remodeling of the endothelial Ca2+ toolkit under conditions that mimic obesity. Furthermore, we searched for articles discussing how dietary manipulation could be exploited to rescue Ca2+-dependent vasodilation., Results: We found evidence that the endothelial Ca2+ could be severely affected by obese vessels. This rearrangement could contribute to endothelial damage and is likely to be involved in the disruption of vasorelaxant mechanisms. However, several Ca2+-permeable channels, including Vanilloid Transient Receptor Potential (TRPV) 1, 3 and 4 could be stimulated by several food components to stimulate vasorelaxation in obese individuals., Conclusion: The endothelial Ca2+ toolkit could be targeted to reduce vascular damage and rescue endothelium- dependent vasodilation in obese vessels. This hypothesis remains, however, to be probed on truly obese endothelial cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

It has long been known that endothelial Ca 2+ signals drive angiogenesis by recruiting multiple Ca 2+ -sensitive decoders in response to pro-angiogenic cues, such as vascular endothelial growth factor, basic fibroblast growth factor, stromal derived factor-1α and angiopoietins. Recently, it was shown that intracellular Ca 2+ signaling also drives vasculogenesis by stimulation proliferation, tube formation and neovessel formation in endothelial progenitor cells. Herein, we survey how growth factors, chemokines and angiogenic modulators use endothelial Ca 2+ signaling to regulate angiogenesis and vasculogenesis. The endothelial Ca 2+ response to pro-angiogenic cues may adopt different waveforms, ranging from Ca 2+ transients or biphasic Ca 2+ signals to repetitive Ca 2+ oscillations, and is mainly driven by endogenous Ca 2+ release through inositol-1,4,5-trisphosphate receptors and by store-operated Ca 2+ entry through Orai1 channels. Lysosomal Ca 2+ release through nicotinic acid adenine dinucleotide phosphate-gated two-pore channels is, however, emerging as a crucial pro-angiogenic pathway, which sustains intracellular Ca 2+ mobilization. Understanding how endothelial Ca 2+ signaling regulates angiogenesis and vasculogenesis could shed light on alternative strategies to induce therapeutic angiogenesis or interfere with the aberrant vascularization featuring cancer and intraocular disorders., Competing Interests: The authors declare no conflict of interest.

It has long been known that the conditionally essential polyunsaturated arachidonic acid (AA) regulates cerebral blood flow (CBF) through its metabolites prostaglandin E2 and epoxyeicosatrienoic acid, which act on vascular smooth muscle cells and pericytes to vasorelax cerebral microvessels. However, AA may also elicit endothelial nitric oxide (NO) release through an increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ). Herein, we adopted Ca 2+ and NO imaging, combined with immunoblotting, to assess whether AA induces intracellular Ca 2+ signals and NO release in the human brain microvascular endothelial cell line hCMEC/D3. AA caused a dose-dependent increase in [Ca 2+ ] i that was mimicked by the not-metabolizable analogue, eicosatetraynoic acid. The Ca 2+ response to AA was patterned by endoplasmic reticulum Ca 2+ release through type 3 inositol-1,4,5-trisphosphate receptors, lysosomal Ca 2+ mobilization through two-pore channels 1 and 2 (TPC1-2), and extracellular Ca 2+ influx through transient receptor potential vanilloid 4 (TRPV4). In addition, AA-evoked Ca 2+ signals resulted in robust NO release, but this signal was considerably delayed as compared to the accompanying Ca 2+ wave and was essentially mediated by TPC1-2 and TRPV4. Overall, these data provide the first evidence that AA elicits Ca 2+ -dependent NO release from a human cerebrovascular endothelial cell line, but they seemingly rule out the possibility that this NO signal could acutely modulate neurovascular coupling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) gates two-pore channels 1 and 2 (TPC1 and TPC2) to elicit endo-lysosomal (EL) Ca 2+ release. NAADP-induced EL Ca 2+ signals may be amplified by the endoplasmic reticulum (ER) through the Ca 2+ -induced Ca 2+ release mechanism (CICR). Herein, we aimed at assessing for the first time the role of EL Ca 2+ signaling in primary cultures of human metastatic colorectal carcinoma (mCRC) by exploiting Ca 2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide (GPN), and nigericin, which dissipates the ΔpH which drives Ca 2+ refilling of acidic organelles, caused massive Ca 2+ release in the presence of a functional inositol-1,4,5-trisphosphate (InsP₃)-sensitive ER Ca 2+ store. Liposomal delivery of NAADP induced a transient Ca 2+ release that was reduced by GPN and NED-19, a selective TPC antagonist. Pharmacological and genetic manipulations revealed that the Ca 2+ response to NAADP was triggered by TPC1, the most expressed TPC isoform in mCRC cells, and required ER-embedded InsP₃ receptors. Finally, NED-19 and genetic silencing of TPC1 reduced fetal calf serum-induced Ca 2+ signals, proliferation, and extracellular signal-regulated kinase and Akt phoshorylation in mCRC cells. These data demonstrate that NAADP-gated TPC1 could be regarded as a novel target for alternative therapies to treat mCRC.

Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca 2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cβ (PLCβ) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP 3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca 2+ response to Ach was mediated by InsP 3 R3, TPC1-2, and store-operated Ca 2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca 2+ buffer. Moreover, the pharmacological blockade of the Ca 2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca 2+ signal via M5-mAchRs., (© 2018 Wiley Periodicals, Inc.)

The acidic vesicles of the endolysosomal (EL) system are emerging as an intracellular Ca 2+ store implicated in the regulation of multiple cellular functions. The EL Ca 2+ store releases Ca 2+ through a variety of Ca 2+ -permeable channels, including Transient Receptor Potential (TRP) Mucolipin 1-3 (TRPML1-3) and two-pore channels 1-2 (TPC1-2), whereas EL Ca 2+ refilling is sustained by the proton gradient across the EL membrane and/or by the endoplasmic reticulum (ER). EL Ca 2+ signals may be either spatially restricted to control vesicle trafficking, autophagy and membrane repair or may be amplified into a global Ca 2+ signal through the Ca 2+ -dependent recruitment of ER-embedded channels. Emerging evidence suggested that nicotinic acid adenine dinucleotide phosphate (NAADP)-gated TPCs sustain multiple cancer hallmarks, such as migration, invasiveness and angiogenesis. Herein, we first survey the EL Ca 2+ refilling and release mechanisms and then focus on the oncogenic role of EL Ca 2+ signaling. While the evidence in favor of TRPML1 involvement in neoplastic transformation is yet to be clearly provided, TPCs are emerging as an alternative target for anticancer therapies.

Persistent organic pollutants are a group of chemicals that include polychlorinated biphenyls (PCBs). PCBs exposure during adult life increases incidence and severity of cardiomyopathies, whereas in utero exposure determines congenital heart defects. Being fat-soluble, PCBs are passed to newborns through maternal milk, impairing heart functionality in the adult. It is still unknown how PCBs impair cardiac contraction at cellular/molecular levels. Here, we study the molecular mechanisms by which PCBs cause the observed heart contraction defects, analysing the alterations of Ca 2+ toolkit components that regulate contraction. We investigated the effect that Aroclor 1254 (Aroclor), a mixture of PCBs, has on perinatal-like cardiomyocytes derived from mouse embryonic stem cells. Cardiomyocytes, exposed to 1 or 2 µg/ml Aroclor for 24 h, were analyzed for their kinematics contractile properties and intracellular Ca 2+ dynamics. We observed that Aroclor impairs cardiomyocytes contractile properties by inhibiting spontaneous Ca 2+ oscillations. It disrupts intracellular Ca 2+ homeostasis by reducing the sarcoplasmic reticulum Ca 2+ content and by inhibiting voltage-gated Ca 2+ entry. These findings contribute to the understanding of the molecular underpinnings of PCBs-induced cardiovascular alterations, which are emerging as an additional life-threatening hurdle associated to PCBs pollution. Therefore, PCBs-dependent alteration of intracellular Ca 2+ dynamics is the most likely trigger of developmental cardiac functional alteration.

Store-operated Ca 2+ entry (SOCE) provides a major Ca 2+ entry route in cancer cells. SOCE is mediated by the assembly of Stim and Orai proteins at endoplasmic reticulum (ER)-plasma membrane junctions upon depletion of the ER Ca 2+ store. Additionally, Stim and Orai proteins underpin constitutive Ca 2+ entry in a growing number of cancer cell types due to the partial depletion of their ER Ca 2+ reservoir. Herein, we investigated for the first time the structure and function of SOCE in primary cultures of colorectal carcinoma (CRC) established from primary tumor (pCRC) and metastatic lesions (mCRC) of human subjects. Stim1-2 and Orai1-3 transcripts were equally expressed in pCRC and mCRC cells, although Stim1 and Orai3 proteins were up-regulated in mCRC cells. The Mn 2+ -quenching technique revealed that constitutive Ca2 + entry was significantly enhanced in pCRC cells and was inhibited by the pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3. The larger resting Ca 2+ influx in pCRC was associated to their lower ER Ca 2+ content as compared to mCRC cells. Pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 prevented ER-dependent Ca 2+ release, thereby suggesting that constitutive SOCE maintains ER Ca 2+ levels. Nevertheless, pharmacological and genetic blockade of Stim1, Stim2, Orai1 and Orai3 did not affect CRC cell proliferation and migration. These data provide the first evidence that Stim and Orai proteins mediate constitutive Ca 2+ entry and replenish ER with Ca 2+ in primary cultures of CRC cells. However, SOCE is not a promising target to design alternative therapies for CRC., Competing Interests: CONFLICTS OF INTEREST Nothing to declare.