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Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca2+ changes. The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms (SERCA, PMCA and NCX) during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.

Introduction: Endothelial cells (ECs), being located at the interface between flowing blood and vessel wall, maintain cardiovascular homeostasis by virtue of their ability to integrate chemical and physical cues through a spatio-temporally coordinated increase in their intracellular Ca2+ concentration ([Ca2+]i). Endothelial heterogeneity suggests the existence of spatially distributed functional clusters of ECs that display different patterns of intracellular Ca2+ response to extracellular inputs. Characterizing the overall Ca2+ activity of the endothelial monolayer in situ requires the meticulous analysis of hundreds of ECs. This complex analysis consists in detecting and quantifying the true Ca2+ events associated to extracellular stimulation and classifying their intracellular Ca2+ profiles (ICPs). The injury assay technique allows exploring the Ca2+-dependent molecular mechanisms involved in angiogenesis and endothelial regeneration. However, there are true Ca2+ events of nearly undetectable magnitude that are almost comparable with inherent instrumental noise. Moreover, undesirable artifacts added to the signal by mechanical injury stimulation complicate the analysis of intracellular Ca2+ activity. In general, the study of ICPs lacks uniform criteria and reliable approaches for assessing these highly heterogeneous spatial and temporal events.Methods: Herein, we present an approach to classify ICPs that consists in three stages: 1) identification of Ca2+ candidate events through thresholding of a feature termed left-prominence; 2) identification of non-true events, known as artifacts; and 3) ICP classification based upon event temporal location.Results: The performance assessment of true-events identification showed competitive sensitivity = [0.9995, 0.9831], specificity = [0.9946, 0.7818] and accuracy = [0.9978, 0.9579] improvements of 2x and 14x, respectively, compared with other methods. The ICP classifier enhanced by artifact detection showed 0.9252 average accuracy with the ground-truth sets provided for validation.Discussion: Results indicate that our approach ensures sturdiness to experimental protocol maneuvers, besides it is effective, simple, and configurable for different studies that use unidimensional time dependent signals as data. Furthermore, our approach would also be effective to analyze the ICPs generated by other cell types, other dyes, chemical stimulation or even signals recorded at higher frequency.

Abstract: Hydrogen sulfide (H2S) is an endogenous gaseous molecule present in all living organisms and has been traditionally studied for its toxicity. Interestingly, increased understanding of H2S effects in organ physiology has recently shown its relevance as a signalling molecule, with potentially important implications in variety of clinical disorders, including cancer. H2S is primarily produced in mammalian cells under various enzymatic pathways. A developing focus of H2S is a blooming hotspot that studies chemical, biological mechanisms, and therapeutic effects of H2S. Herein, we describe the physiological and biochemical properties of H2S, the enzymatic pathways leading to its endogenous production and its catabolic routes. In addition, we discuss the role of currently known H2S-releasing agents, or H2S donors, including their potential as therapeutic tools. Then we illustrate the mechanisms known to support the pleiotropic effects of H2S, with a particular focus on persulfhydration, which plays a key role in H2S-mediating signalling pathways. We then address the paradoxical role played by H2S in tumour biology and discuss the potential of exploiting H2S 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 H2S-releasing compounds. While the evidence in favour of H2S as an alternative cancer therapy in the field of translational medicine is yet to be clearly provided, application of H2S is emerging as potent anticancer therapies in preclinical trails.

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 (Ca2+) 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 Ca2+ oscillations and the Ca2+ 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 Ca2+ oscillations and concomitant increased Ca2+/Calmodulin dependent kinase II (CaMKII) activation compared to control cells. This is manly linked to a constitutive activation of Store-Operated Ca2+ Entry (SOCE), which leads to enhanced Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors. By targeting the Ca2+ handling machinery or CaMKII activity, we demonstrated a causative link between Ca2+ oscillations and fibro-adipogenic differentiation of ACM C-MSC. Genetic silencing of the desmosomal gene PKP2 mimics the remodelling of the Ca2+ signalling machinery occurring in ACM C-MSC. The anti-arrhythmic drug flecainide inhibits intracellular Ca2+ oscillations and fibro-adipogenic differentiation by selectively targeting SOCE. Conclusions Altogether, our results extend the knowledge of Ca2+ 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.

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

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‐CMHypo) triggers intracellular Ca2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca2+‐dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca2+ oscillations by promoting extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4‐mediated Ca2+ entry, in turn, promoted the concerted interplay between inositol‐1,4,5‐trisphosphate‐ and nicotinic acid adenine dinucleotide phosphate‐induced endogenous Ca2+ release and store‐operated Ca2+ entry (SOCE). hAFS‐CMHypo‐induced intracellular Ca2+ oscillations resulted in the nuclear translocation of the Ca2+‐sensitive transcription factor p65 NF‐κB. Finally, inhibition of either intracellular Ca2+ oscillations or NF‐κB activity prevented hAFS‐CMHypo‐induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS‐CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic‐related myocardial injury.

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 CaPEVs were isolated from human blood platelets, and Fura-2/AM CaPretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in CaPEVs stimulate migration in the highly metastatic MDA-MB-231 breast cancer cell line by inducing a partial remodelling of the Ca

Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca2+concentration ([Ca2+]i), but the underlying signaling mechanisms are still unknown. Herein, we exploited single-cell Ca2+imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca2+signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca2+-sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca2+release/entry pathways responsible for the onset of the Ca2+response. Histamine elicited complex intracellular Ca2+signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca2+response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca2+signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca2+]ievoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca2+signals were initiated by intracellular Ca2+release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3R) and sustained by store-operated Ca2+channels (SOCs). Conversely, L-type voltage-operated Ca2+channels did not support histamine-induced extracellular Ca2+entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP3R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP3and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.

Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca

Moderate levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), fuel tumor metastasis and invasion in a variety of cancer types. Conversely, excessive ROS levels can impair tumor growth and metastasis by triggering cancer cell death. In order to cope with the oxidative stress imposed by the tumor microenvironment, malignant cells exploit a sophisticated network of antioxidant defense mechanisms. Targeting the antioxidant capacity of cancer cells or enhancing their sensitivity to ROS-dependent cell death represent a promising strategy for alternative anticancer treatments. Transient Receptor Potential Ankyrin 1 (TRPA1) is a redox-sensitive non-selective cation channel that mediates extracellular Ca2+ entry upon an increase in intracellular ROS levels. The ensuing increase in intracellular Ca2+ concentration can in turn engage a non-canonical antioxidant defense program or induce mitochondrial Ca2+ dysfunction and apoptotic cell death depending on the cancer type. Herein, we sought to describe the opposing effects of ROS-dependent TRPA1 activation on cancer cell fate and propose the pharmacological manipulation of TRPA1 as an alternative therapeutic strategy to enhance cancer cell sensitivity to oxidative stress.

In light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca2+) dynamics in cultured human cerebral microvascular endothelial cells (hCMEC/D3), as an in vitro human blood-brain barrier (BBB) model, and in cultured astrocytes. mApoE-PA-LIP pre-treatment actively increased both the duration and the area under the curve (A.U.C) of the ATP-evoked Ca2+ waves in cultured hCMEC/D3 cells as well as in cultured astrocytes. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ response to ATP is mainly due to endogenous Ca2+ release. Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer’s disease (AD).

(1) Background: Store-operated Ca

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder that associates with nucleotide sequence variants in genes encoding sarcomere related proteins, and is recognized as the most common heritable cardiac diseases. Clinically, HCM can be extremely variable and this makes the diagnosis difficult until the development of serious or fatal events. Nevertheless, the main hallmark of HCM is represented by left ventricle hypertrophy that can be occasionally associated to cardiac arrhythmias, chest pain, diastolic dysfunction, obstruction of left ventricular outflow tract. The present review aims to focus on the complex interplay existing between the multifaceted non-genetic molecular mechanisms underlying HCM onset and progression, and the key pathophysiological role of abnormal coronary artery function. As the clinical course of HCM shows a mortality rate per year up to 6% the importance of innovative therapeutic strategies will be discussed, especially in regard to the use of potential endogenous coronary modulators to be enrolled as modifiers of HCM phenotype.

Con l’istituzione delle citta metropolitane in Italia, che conferisce loro il compito di redigere il piano territoriale metropolitano, e utile stabilire su quali conoscenze acquisire. Distinguendo il profilo ambientale e funzionale, ci proponiamo di mettere in luce punti di forza e di debolezza di ciascun criterio presentato in relazione alla scala metropolitana, con l’obiettivo costruire quadri conoscitivi meglio ponderati.

Approximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate–dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.

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

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

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ion channels that are activated by the neurotransmitter glutamate, mediate the slow component of excitatory neurotransmission in the central nervous system (CNS), and induce long-term changes in synaptic plasticity. NMDARs are non-selective cation channels that allow the influx of extracellular Na+ and Ca2+ and control cellular activity via both membrane depolarization and an increase in intracellular Ca2+ concentration. The distribution, structure, and role of neuronal NMDARs have been extensively investigated and it is now known that they also regulate crucial functions in the non-neuronal cellular component of the CNS, i.e., astrocytes and cerebrovascular endothelial cells. In addition, NMDARs are expressed in multiple peripheral organs, including heart and systemic and pulmonary circulations. Herein, we survey the most recent information available regarding the distribution and function of NMDARs within the cardiovascular system. We describe the involvement of NMDARs in the modulation of heart rate and cardiac rhythm, in the regulation of arterial blood pressure, in the regulation of cerebral blood flow, and in the blood–brain barrier (BBB) permeability. In parallel, we describe how enhanced NMDAR activity could promote ventricular arrhythmias, heart failure, pulmonary artery hypertension (PAH), and BBB dysfunction. Targeting NMDARs could represent an unexpected pharmacological strategy to reduce the growing burden of several life-threatening cardiovascular disorders.

Store-operated Ca2+ entry (SOCE) is activated in response to the inositol-1,4,5-trisphosphate (InsP3)-dependent depletion of the endoplasmic reticulum (ER) Ca2+ store and represents a ubiquitous mode of Ca2+ influx. In vascular endothelial cells, SOCE regulates a plethora of functions that maintain cardiovascular homeostasis, such as angiogenesis, vascular tone, vascular permeability, platelet aggregation, and monocyte adhesion. The molecular mechanisms responsible for SOCE activation in vascular endothelial cells have engendered a long-lasting controversy. Traditionally, it has been assumed that the endothelial SOCE is mediated by two distinct ion channel signalplexes, i.e., STIM1/Orai1 and STIM1/Transient Receptor Potential Canonical 1(TRPC1)/TRPC4. However, recent evidence has shown that Orai1 can assemble with TRPC1 and TRPC4 to form a non-selective cation channel with intermediate electrophysiological features. Herein, we aim at bringing order to the distinct mechanisms that mediate endothelial SOCE in the vascular tree from multiple species (e.g., human, mouse, rat, and bovine). We propose that three distinct currents can mediate SOCE in vascular endothelial cells: (1) the Ca2+-selective Ca2+-release activated Ca2+ current (ICRAC), which is mediated by STIM1 and Orai1; (2) the store-operated non-selective current (ISOC), which is mediated by STIM1, TRPC1, and TRPC4; and (3) the moderately Ca2+-selective, ICRAC-like current, which is mediated by STIM1, TRPC1, TRPC4, and Orai1.

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

An increase in intracellular Ca

The neuromodulator histamine is able to vasorelax in human cerebral, meningeal and temporal arteries via endothelial histamine 1 receptors (H1 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.

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 (Ca2+) 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 Ca2+ 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 Ca2+ signaling to improve ECFCs' reparative phenotype and suggest this strategy as a new approach to treat SLE patients.

During myocardial ischemia, timely reperfusion is critical to limit infarct area and the overall loss of cardiac contractile function. However, reperfusion further exacerbates the damage of the ischemic heart. This type of injury is known as ischemia-reperfusion injury (IRI). Ischemic conditioning is a procedure which consists of brief cycles of ischemia and reperfusion in order to protect the myocardium against IRI. Remote ischemic conditioning (RIC), namely transient brief episodes of ischemia at a remote site before a subsequent damaging ischemia/reperfusion procedure of the target organ (e.g., the heart), protects against IRI. However, how the stimulus of RIC is transduced from the remote organ to the ischemic heart is still unknown. Recently, extracellular vesicles (EVs) have been proposed to have a role in the RIC procedure. The endothelium releases EVs and is also one of the tissues mostly exposed to EVs during their journey to the target organ. Moreover, EVs may have important roles in angiogenesis and, therefore, in the remodeling of post-ischemic organs. Here we analyze how EVs may contribute to the overall cardioprotective effect and the implication of the endothelium and its EVs in RIC mediated acute cardioprotection as well as in angiogenesis.

The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) to mediate extracellular Ca

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

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI–related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI–null mouse model (Col6a1−/−), we found that collagen VI–null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1−/− megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1−/− mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1−/− mice. Peripheral blood platelets of patients affected by collagen VI–related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI–related diseases.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca2+ -releasing messenger that increases the intracellular Ca2+ concentration by mobilizing the lysosomal Ca2+ store through two-pore channels 1 (TPC1) and 2 (TPC2). NAADP-induced lysosomal Ca2+ release regulates multiple endothelial functions, including nitric oxide release and proliferation. A sizeable acidic Ca2+ 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 Ca2+ store and TPC1 in circulating ECFCs by harnessing Ca2+ imaging and molecular biology techniques. The lysosomotropic agent, Gly-Phe β-naphthylamide, and nigericin, which dissipates the proton gradient which drives Ca2+ sequestration by acidic organelles, caused endogenous Ca2+ release in the presence of a replete inositol-1,4,5-trisphosphate (InsP3 )-sensitive endoplasmic reticulum (ER) Ca2+ pool. Likewise, the amount of ER releasable Ca2+ was reduced by disrupting lysosomal Ca2+ content. Liposomal delivery of NAADP induced a transient Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store and by pharmacological and genetic blockade of TPC1. Pharmacological manipulation revealed that NAADP-induced Ca2+ release also required ER-embedded InsP3 receptors. Finally, NAADP-induced lysosomal Ca2+ release was found to trigger vascular endothelial growth factor-induced intracellular Ca2+ oscillations and proliferation, while it did not contribute to adenosine-5'-trisphosphate-induced Ca2+ signaling. These findings demonstrated that NAADP-induced TPC1-mediated Ca2+ release can selectively be recruited to induce the Ca2+ response to specific cues in circulating ECFCs.

In-depth characterization of heart-brain communication in critically ill patients with severe acute respiratory failure is attracting significant interest in the COronaVIrus Disease 19 (COVID-19) pandemic era during intensive care unit (ICU) stay and after ICU or hospital discharge. Emerging research has provided new insights into pathogenic role of the deregulation of the heart-brain axis (HBA), a bidirectional flow of information, in leading to severe multiorgan disease syndrome (MODS) in patients with confirmed infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Noteworthy, HBA dysfunction may worsen the outcome of the COVID-19 patients. In this review, we discuss the critical role HBA plays in both promoting and limiting MODS in COVID-19. We also highlight the role of HBA as new target for novel therapeutic strategies in COVID-19 in order to open new translational frontiers of care. This is a translational perspective from the Italian Society of Cardiovascular Researches., Graphical Abstract ga1

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 Ca2+ imaging to pharmacological manipulation and genetic suppression of TRPV1 expression, to investigate the effects of photoexcitation on intracellular Ca2+ concentration ([Ca2+]i) in circulating ECFCs plated on rr-P3HT thin films. Polymer-mediated optical excitation induced a long-lasting increase in [Ca2+]i that could display an oscillatory pattern at shorter light stimuli. Pharmacological and genetic manipulation revealed that the Ca2+ response to light was triggered by extracellular Ca2+ 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 Ca2+ entry was able to evoke intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors, followed by store-operated Ca2+ 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 Ca2+ signals.

An increase in intracellular Ca2+ concentration ([Ca2+]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 Ca2+ store, which releases Ca2+ through ryanodine receptors (RyRs) and/or inositol-1,4,5-trisphosphate receptors (InsP3Rs) upon extracellular stimulation. The acidic vesicles of the endolysosomal (EL) compartment represent an additional endogenous Ca2+ store, which is targeted by several second messengers, including nicotinic acid adenine dinucleotide phosphate (NAADP) and phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], and may release intraluminal Ca2+ through multiple Ca2+ 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 Ca2+ 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 Ca2+ 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.

Cardiovascular diseases are the leading cause of mortality worldwide. Such a widespread diffusion makes the conditions affecting the heart and blood vessels a primary medical and economic burden. It, therefore, becomes mandatory to identify effective treatments that can alleviate this global problem. Among the different solutions brought to the attention of the medical-scientific community, therapeutic angiogenesis is one of the most promising. However, this approach, which aims to treat cardiovascular diseases by generating new blood vessels in ischemic tissues, has so far led to inadequate results due to several issues. In this perspective, we will discuss cutting-edge approaches and future perspectives to alleviate the potentially lethal impact of cardiovascular diseases. We will focus on the consolidated role of resident endothelial progenitor cells, particularly endothelial colony forming cells, as suitable candidates for cell-based therapy demonstrating the importance of targeting intracellular Ca2+ signaling to boost their regenerative outcome. Moreover, we will elucidate the advantages of physical stimuli over traditional approaches. In particular, we will critically discuss recent results obtained by using optical stimulation, as a novel strategy to drive endothelial colony forming cells fate and its potential in the treatment of cardiovascular diseases.

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 Ca2+. 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.

Climate changes produce harmful effects on physical, biological and human system, particularly on human health; the possibility to reduce negative impact of phenomena as the urban heat island, the frequency, intensity and duration of extreme precipitations, the heat waves and cool waves, depends on the capacity to plan urban settlements improving their resilience. The green infrastructure planning and the assessment of urban quality in relation to ecosystem services are affirming as outcomes of studies and researches in urban planning. The Municipal Master Plan of Qualiano is an experimentation of green infrastructure planning according to an ecosystem approach aimed to improve ecosystem services supply at a local scale. Furthermore, it is a significant case of Metropolitan Ecological Network realized by a bottom-up approach: despite the lack of a metropolitan plan, in fact, it is possible to evaluate ecosystem values of landscape, both natural and urban, and to plan them with an integrated approach to increase ecosystem services.

The compressive resistance of concrete in new structures is usually characterized on the basis of tests performed on concrete cylinders or cubes under relatively rapid loading conditions. Although efficient for material characterization, these tests do not acknowledge a number of phenomena potentially influencing the compressive resistance of concrete in actual structures. For this reason, when performing a structural analysis, strength reduction factors are usually considered in codes of practice modifying the uniaxial strength of material tests. In this paper, a detailed investigation of the influence of material brittleness and internal stress redistributions on the structural response of reinforced concrete members is presented. This work is based on a number of theoretical considerations and supported by the experimental results of more than 400 reinforced concrete columns tested with or without eccentricity and gathered from the literature. The results show the pertinence of considering a brittleness factor in the calculation of the structural resistance of reinforced concrete columns and compression zones of beams. The results of this work are eventually formulated in terms of code-like proposals, currently considered in the draft of the new Eurocode 2 (prEN 1992-1-1:2018).

Platelets have been extensively implicated in the progression of cancer and platelet-derived extracellular vesicles (PEVs) are gaining growing attention as potential mediators of the platelet-cancer interplay. PEVs are shed from platelet membrane in response to extracellular stimuli and carry important biological signals for intercellular communication. In this study we demonstrate that PEVs specifically bind to different breast cancer cells and elicit cell-specific functional responses. PEVs were massively internalized by the metastatic cell lines MDA-MB-231 and SKBR3 and the ductal carcinoma cell line BT474, but not by the MCF-7 cell line. In SKBR3 cells, PEVs decreased mitochondrial dehydrogenase activities and altered cell cycle progression without affecting cell viability. Conversely, PEVs potently stimulated migration and invasion of MDA-MB-231, without affecting the distribution in the different phases of the cell cycle. In all the analyzed breast cancer cells, PEVs triggered a sustained increase of intracellular Ca2+, but only in MDA-MB-231 cells, this was associated to the stimulation of selected signaling proteins implicated in migration, including p38MAPK and myosin light chain. Importantly, inhibition of myosin light chain phosphorylation by a Rho kinase inhibitor prevented PEVs-stimulated migration of MDA-MB-231 cells. Our results demonstrate that PEVs are versatile regulators of cancer cell behavior and elicit a variety of different responses depending on the specific breast cancer cell subtype.

The cerebellar granule cells (GrCs) are classically described as a homogeneous neuronal population discharging regularly without adaptation. We show that GrCs in fact generate diverse response patterns to current injection and synaptic activation, ranging from adaptation to acceleration of firing. Adaptation was predicted by parameter optimization in detailed computational models based on available knowledge on GrC ionic channels. The models also predicted that acceleration required additional mechanisms. We found that yet unrecognized TRPM4 currents specifically accounted for firing acceleration and that adapting GrCs outperformed accelerating GrCs in transmitting high-frequency mossy fiber (MF) bursts over a background discharge. This implied that GrC subtypes identified by their electroresponsiveness corresponded to specific neurotransmitter release probability values. Simulations showed that fine-tuning of pre- and post-synaptic parameters generated effective MF-GrC transmission channels, which could enrich the processing of input spike patterns and enhance spatio-temporal recoding at the cerebellar input stage., Stefano Masoli, Marialuisa Tognolina et al. show that cerebellar granule cells do not respond uniformly to electrical stimuli. Using computational models and electrophysiology, they show that this diversity in granule cell response affects neurotransmitter release probabilities and can fine-tune cerebellar synaptic transmission.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most recently discovered Ca

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 Ca2+ 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 Ca2+ 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 Ca2+ 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 Mg2+ 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.

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca2+ concentration ([Ca2+ ]i ) in the target cells, which activates the Ca2+ /Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca2+ ]i and NO production. The current study assessed whether and how glutamate drives Ca2+ -dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca2+ ]i , which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca2+ oscillations were triggered by rhythmic endogenous Ca2+ mobilization and maintained over time by extracellular Ca2+ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca2+ release was mediated by InsP3 -sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca2+ entry mediated Ca2+ entry during ongoing Ca2+ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca2+ signals. Of note, glutamate induced Ca2+ -dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca2+ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.

Glioblastoma is one of the most malignant, angiogenic, and incurable tumors in humans. The aberrant communication between glioblastoma cells and tumor microenvironment represents one of the major factors regulating glioblastoma malignancy and angiogenic properties. Emerging evidence implicates sphingosine-1-phosphate signaling in the pathobiology of glioblastoma and angiogenesis, but its role in glioblastoma-endothelial crosstalk remains largely unknown. In this study, we sought to determine whether the crosstalk between glioblastoma cells and brain endothelial cells regulates sphingosine-1-phosphate signaling in the tumor microenvironment. Using human glioblastoma and brain endothelial cell lines, as well as primary brain endothelial cells derived from human glioblastoma, we report that glioblastoma-co-culture promotes the expression, activity, and plasma membrane enrichment of sphingosine kinase 2 in brain endothelial cells, leading to increased cellular level of sphingosine-1-phosphate, and significant potentiation of its secretion. In turn, extracellular sphingosine-1-phosphate stimulates glioblastoma cell proliferation, and brain endothelial cells migration and angiogenesis. We also show that, after co-culture, glioblastoma cells exhibit enhanced expression of S1P1 and S1P3, the sphingosine-1-phosphate receptors that are of paramount importance for cell growth and invasivity. Collectively, our results envision glioblastoma-endothelial crosstalk as a multi-compartmental strategy to enforce pro-tumoral sphingosine-1-phosphate signaling in the glioblastoma microenvironment.

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 Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]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 (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ 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 Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ 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 Ca2+ signal via M5-mAchRs.

The excitatory neurotransmitter glutamate gates post-synaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) to mediate extracellular Ca2+ 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 Ca2+-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 Ca2+ concentration ([Ca2+]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 [Ca2+]i in a flux-independent manner in hCMEC/D3 cells. The Ca2+ response to NMDA was triggered by endogenous Ca2+ release from the endoplasmic reticulum and the lysosomal Ca2+ stores and sustained by store-operated Ca2+ entry. Unexpectedly, pharmacological and genetic blockade of mGluR1 and mGluR5 dramatically impaired NMDARs-mediated Ca2+ signals. These findings indicate that NMDARs may increase the endothelial [Ca2+]i in a flux-independent manner via group 1 mGluRs. However, imaging of DAF-FM fluorescence revealed that NMDARs may also induce Ca2+-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.

Tumor-associated vessels constitution is the result of angiogenesis, the hallmark of cancer essential for tumor to develop in dimension and to spread throughout the organism. Tumor endothelium is configured as an active functioning organ capable of determine interaction with the immune response and all the other components of the variegate cancer microenvironment, determining reciprocal influence. Angiogenesis is here analyzed in its molecular and cellular mechanisms, multiple mediators and principal players, represented by Endothelial Cells. It is discussed the striking heterogeneity of cancer endothelium, due to morphological and molecular aberrations that it often presents and its multiple origin. Among the cells that participate to the composition of tumor vasculature, Endothelial Progenitor Cells represent an important source for physical sustain and paracrine signaling in the process of angiogenesis. Treatment options are reviewed, with particular focus on novel therapeutic strategies for overcoming tumor resistance to anti-angiogenic agents.