The Molecular Heterogeneity of Store-Operated Ca2+ Entry in Vascular Endothelial Cells: The Different roles of Orai1 and TRPC1/TRPC4 Channels in the Transition from Ca2+-Selective to Non-Selective Cation Currents.

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.

The Emerging Role of N-Methyl-D-Aspartate (NMDA) Receptors in the Cardiovascular System: Physiological Implications, Pathological Consequences, and Therapeutic Perspectives.

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.

Cracking the Endothelial Calcium (Ca2+) Code: A Matter of Timing and Spacing.

A monolayer of endothelial cells lines the innermost surface of all blood vessels, thereby coming into close contact with every region of the body and perceiving signals deriving from both the bloodstream and parenchymal tissues. An increase in intracellular Ca2+ concentration ([Ca2+]i) is the main mechanism whereby vascular endothelial cells integrate the information conveyed by local and circulating cues. Herein, we describe the dynamics and spatial distribution of endothelial Ca2+ signals to understand how an array of spatially restricted (at both the subcellular and cellular levels) Ca2+ signals is exploited by the vascular intima to fulfill this complex task. We then illustrate how local endothelial Ca2+ signals affect the most appropriate vascular function and are integrated to transmit this information to more distant sites to maintain cardiovascular homeostasis. Vasorelaxation and sprouting angiogenesis were selected as an example of functions that are finely tuned by the variable spatio-temporal profile endothelial Ca2+ signals. We further highlighted how distinct Ca2+ signatures regulate the different phases of vasculogenesis, i.e., proliferation and migration, in circulating endothelial precursors.

Higher body mass index and disease duration are associated with increased risk of left ventricular diastolic dysfunction in women with systemic lupus erythematosus.

BACKGROUND: Patients with systemic lupus erythematosus (SLE) have an increased cardiovascular (CV) risk. Insulin resistance (IR), which is higher in patients with SLE, adversely impacts left ventricular (LV) remodeling and function. The aims were to determine LV dysfunction and evaluate the influence of potential risk factors on subclinical LV dysfunction in women with SLE, including IR. METHODS: This cross-sectional study included adult women with SLE without diabetes mellitus (DM), hypertension or severe obesity. Diastolic dysfunction (DD) was verified according to current guidelines. Insulin resistance was estimated using the Quantose score. RESULTS: We included 77 women. The frequency of IR was 65%. All participants had a normal ejection fraction (EF), and 11 (15.7%) had abnormal LV global longitudinal strain (GLS). Twenty-three (32.8%) had DD. The GLS% and global circumferential strain (GCS)% did not differ in patients with and without IR (-20.8 ± 3.1 vs -20.5 ± 2.1; p = 0.61 and -27.9 ± 4.4 vs -27.4 ± 3.7; p = 0.57, respectively). The prevalence of DD was 38.1% in patients with IR versus 25% in those without (p = 0.30). E/e' and E/A ratios did not differ between groups (6.6 ± 1.9 vs 6.6 ± 1.5; p = 0.98 and 1.3 ± 0.3 vs 1.3 ± 0.2; p = 0.27). Higher BMI (OR: 1.2, 95% CI 1.1-1.5) and disease duration (OR: 1.2, 95% CI 1.1-1.4) were associated with DD. CONCLUSIONS: Patients with overweight/obesity may be at higher risk of LV dysfunction. Although IR was high in our patients with SLE was not associated with systolic dysfunction or DD. Body mass index and disease duration were associated with an increased risk of DD.

Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation.

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, endothelial progenitor cells and intracellular Ca2+ signaling: A novel approach for an old disease.

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.

Muscarinic M5 receptors trigger acetylcholine-induced Ca2+ signals and nitric oxide release in human brain microvascular endothelial cells.

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 Cß (PLCß) 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.

Type 2 Diabetes Alters Intracellular Ca2+ Handling in Native Endothelium of Excised Rat Aorta.

An increase in intracellular Ca2+ concentration ([Ca2+]i) plays a key role in controlling endothelial functions; however, it is still unclear whether endothelial Ca2+ handling is altered by type 2 diabetes mellitus, which results in severe endothelial dysfunction. Herein, we analyzed for the first time the Ca2+ response to the physiological autacoid ATP in native aortic endothelium of obese Zucker diabetic fatty (OZDF) rats and their lean controls, which are termed LZDF rats. By loading the endothelial monolayer with the Ca2+-sensitive fluorophore, Fura-2/AM, we found that the endothelial Ca2+ response to 20 µM and 300 µM ATP exhibited a higher plateau, a larger area under the curve and prolonged duration in OZDF rats. The "Ca2+ add-back" protocol revealed no difference in the inositol-1,4,5-trisphosphate-releasable endoplasmic reticulum (ER) Ca2+ pool, while store-operated Ca2+ entry was surprisingly down-regulated in OZDF aortae. Pharmacological manipulation disclosed that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) activity was down-regulated by reactive oxygen species in native aortic endothelium of OZDF rats, thereby exaggerating the Ca2+ response to high agonist concentrations. These findings shed new light on the mechanisms by which type 2 diabetes mellitus may cause endothelial dysfunction by remodeling the intracellular Ca2+ toolkit.

Automated Intracellular Calcium Profiles Extraction from Endothelial Cells Using Digital Fluorescence Images.

Endothelial cells perform a wide variety of fundamental functions for the cardiovascular system, their proliferation and migration being strongly regulated by their intracellular calcium concentration. Hence it is extremely important to carefully measure endothelial calcium signals under different stimuli. A proposal to automate the intracellular calcium profiles extraction from fluorescence image sequences is presented. Digital image processing techniques were combined with a multi-target tracking approach supported by Kalman estimation. The system was tested with image sequences from two different stimuli. The first one was a chemical stimulus, that is, ATP, which caused small movements in the cells trajectories, thereby suggesting that the bath application of the agonist does not generate significant artifacts. The second one was a mechanical stimulus delivered by a glass microelectrode, which caused major changes in cell trajectories. The importance of the tracking block is evidenced since more accurate profiles were extracted, mainly for cells closest to the stimulated area. Two important contributions of this work are the automatic relocation of the region of interest assigned to the cells and the possibility of data extraction from big image sets in efficient and expedite way. The system may adapt to different kind of cell images and may allow the extraction of other useful features.

Phosphatidylethanolamine Induces an Antifibrotic Phenotype in Normal Human Lung Fibroblasts and Ameliorates Bleomycin-Induced Lung Fibrosis in Mice.

Lung surfactant is a complex mixture of phospholipids and specific proteins but its role in the pathogenesis of interstitial lung diseases is not established. Herein, we analyzed the effects of three representative phospholipid components, that is, dipalmitoilphosphatidylcoline (DPPC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), on collagen expression, apoptosis and Ca2+ signaling in normal human lung fibroblasts (NHLF) and probed their effect in an experimental model of lung fibrosis. Collagen expression was measured with RT-PCR, apoptosis was measured by using either the APOPercentage assay kit (Biocolor Ltd., Northern Ireland, UK) or the Caspase-Glo 3/7 assay (Promega, Madison, WI, USA) and Ca2+ signaling by conventional epifluorescence imaging. The effect in vivo was tested in bleomycin-induced lung fibrosis in mice. DPPC and PG did not affect collagen expression, which was downregulated by PE. Furthermore, PE promoted apoptosis and induced a dose-dependent Ca2+ signal. PE-induced Ca2+ signal and apoptosis were both blocked by phospholipase C, endoplasmic reticulum pump and store-operated Ca2+ entry inhibition. PE-induced decrease in collagen expression was attenuated by blocking phospholipase C. Finally, surfactant enriched with PE and PE itself attenuated bleomycin-induced lung fibrosis and decreased the soluble collagen concentration in mice lungs. This study demonstrates that PE strongly contributes to the surfactant-induced inhibition of collagen expression in NHLF through a Ca2+ signal and that early administration of Beractant enriched with PE diminishes lung fibrosis in vivo.

Two Signaling Modes Are Better than One: Flux-Independent Signaling by Ionotropic Glutamate Receptors Is Coming of Age.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Glutamatergic transmission can be mediated by ionotropic glutamate receptors (iGluRs), which mediate rapid synaptic depolarization that can be associated with Ca2+ entry and activity-dependent change in the strength of synaptic transmission, as well as by metabotropic glutamate receptors (mGluRs), which mediate slower postsynaptic responses through the recruitment of second messenger systems. A wealth of evidence reported over the last three decades has shown that this dogmatic subdivision between iGluRs and mGluRs may not reflect the actual physiological signaling mode of the iGluRs, i.e., α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA) receptors (AMPAR), kainate receptors (KARs), and N-methyl-D-aspartate (NMDA) receptors (NMDARs). Herein, we review the evidence available supporting the notion that the canonical iGluRs can recruit flux-independent signaling pathways not only in neurons, but also in brain astrocytes and cerebrovascular endothelial cells. Understanding the signaling versatility of iGluRs can exert a profound impact on our understanding of glutamatergic synapses. Furthermore, it may shed light on novel neuroprotective strategies against brain disorders.

Ca²âº-dependent nitric oxide release in the injured endothelium of excised rat aorta: a promising mechanism applying in vascular prosthetic devices in aging patients.

BACKGROUND: Nitric oxide is key to endothelial regeneration, but it is still unknown whether endothelial cell (EC) loss results in an increase in NO levels at the wound edge. We have already shown that endothelial damage induces a long-lasting Ca²âº entry into surviving cells though connexin hemichannels (CxHcs) uncoupled from their counterparts on ruptured cells. The physiological outcome of injury-induced Ca²âº inflow is, however, unknown. METHODS: In this study, we sought to determine whether and how endothelial scraping induces NO production (NOP) in the endothelium of excised rat aorta by exploiting the NO-sensitive fluorochrome, DAF-FM diacetate and the Ca²âº-sensitive fluorescent dye, Fura-2/AM. RESULTS: We demonstrated that injury-induced NOP at the lesion site is prevented in presence of the endothelial NO synthase inhibitor, L-NAME, and in absence of extracellular Ca²âº. Unlike ATP-dependent NO liberation, the NO response to injury is insensitive to BTP-2, which selectively blocks store-operated Ca²âº inflow. However, injury-induced NOP is significantly reduced by classic gap junction blockers, and by connexin mimetic peptides specifically targeting Cx37Hcs, Cx40HCs, and Cx43Hcs. Moreover, disruption of caveolar integrity prevents injury-elicited NO signaling, but not the accompanying Ca²âº response. CONCLUSIONS: The data presented provide the first evidence that endothelial scraping stimulates NO synthesis at the wound edge, which might both exert an immediate anti-thrombotic and anti-inflammatory action and promote the subsequent re-endothelialization.

An automated method to discover true events and classification of intracellular Ca2+ profiles for endothelium in situ injury assay.

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.

Store-Operated Ca2+ Entry as a Putative Target of Flecainide for the Treatment of Arrhythmogenic Cardiomyopathy.

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 Ic antiarrhythmic drug that can be administered in combination with ß-adrenergic blockers to treat ACM due to its ability to target both Nav1.5 and type 2 ryanodine receptors (RyR2). However, a recent study showed that flecainide may also prevent fibro-adipogenic differentiation by inhibiting store-operated Ca2+ entry (SOCE) and thereby suppressing spontaneous Ca2+ 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 Nav1.5 and RyR2. Subsequently, we describe the role of spontaneous Ca2+ oscillations in determining MSC fate. Next, we discuss recent work showing that spontaneous Ca2+ oscillations in ACM C-hMSCs are accelerated to stimulate their fibro-adipogenic differentiation. Finally, we describe the evidence that flecainide suppresses spontaneous Ca2+ oscillations and fibro-adipogenic differentiation in ACM C-hMSCs by inhibiting constitutive SOCE.

Allyl Isothiocianate Induces Ca2+ Signals and Nitric Oxide Release by Inducing Reactive Oxygen Species Production in the Human Cerebrovascular Endothelial Cell Line hCMEC/D3.

Nitric oxide (NO) represents a crucial mediator to regulate cerebral blood flow (CBF) in the human brain both under basal conditions and in response to somatosensory stimulation. An increase in intracellular Ca2+ concentrations ([Ca2+]i) stimulates the endothelial NO synthase to produce NO in human cerebrovascular endothelial cells. Therefore, targeting the endothelial ion channel machinery could represent a promising strategy to rescue endothelial NO signalling in traumatic brain injury and neurodegenerative disorders. Allyl isothiocyanate (AITC), a major active constituent of cruciferous vegetables, was found to increase CBF in non-human preclinical models, but it is still unknown whether it stimulates NO release in human brain capillary endothelial cells. In the present investigation, we showed that AITC evoked a Ca2+-dependent NO release in the human cerebrovascular endothelial cell line, hCMEC/D3. The Ca2+ response to AITC was shaped by both intra- and extracellular Ca2+ sources, although it was insensitive to the pharmacological blockade of transient receptor potential ankyrin 1, which is regarded to be among the main molecular targets of AITC. In accord, AITC failed to induce transmembrane currents or to elicit membrane hyperpolarization, although NS309, a selective opener of the small- and intermediate-conductance Ca2+-activated K+ channels, induced a significant membrane hyperpolarization. The AITC-evoked Ca2+ signal was triggered by the production of cytosolic, but not mitochondrial, reactive oxygen species (ROS), and was supported by store-operated Ca2+ entry (SOCE). Conversely, the Ca2+ response to AITC did not require Ca2+ mobilization from the endoplasmic reticulum, lysosomes or mitochondria. However, pharmacological manipulation revealed that AITC-dependent ROS generation inhibited plasma membrane Ca2+-ATPase (PMCA) activity, thereby attenuating Ca2+ removal across the plasma membrane and resulting in a sustained increase in [Ca2+]i. In accord, the AITC-evoked NO release was driven by ROS generation and required ROS-dependent inhibition of PMCA activity. These data suggest that AITC could be exploited to restore NO signalling and restore CBF in brain disorders that feature neurovascular dysfunction.

Alterations of the Ca2+ clearing mechanisms by type 2 diabetes in aortic smooth muscle cells of Zucker diabetic fatty rat.

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.

Transient receptor potential ankyrin 1 (TRPA1) mediates reactive oxygen species-induced Ca2+ entry, mitochondrial dysfunction, and caspase-3/7 activation in primary cultures of metastatic colorectal carcinoma cells.

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 Ca2+ 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 Ca2+ signals can either engage an antiapoptotic pro-survival signaling pathway or to promote mitochondrial Ca2+ 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 (H2O2)-induced Ca2+ 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 Ca2+ entry in response to H2O2 and 4-HNE results in mitochondrial Ca2+ 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.

The mechanism of injury-induced intracellular calcium concentration oscillations in the endothelium of excised rat aorta.

Endothelial injury is the primary event that leads to a variety of severe vascular disorders. Mechanical injury elicits a Ca(2+) response in the endothelium of excised rat aorta, which comprises an initial Ca(2+) release from inositol-1,4,5-trisphosphate (InsP(3))-sensitive stores followed by a long-lasting decay phase due to Ca(2+) entry through uncoupled connexons. The Ca(2+) signal may also adopt an oscillatory pattern, the molecular underpinnings of which are unclear. In the light of the role played by Ca(2+) spiking in tissue regeneration, this study aimed to unveil the mechanisms underlying injury-induced Ca(2+) oscillations. The latter reversibly ceased upon removal of extracellular Ca(2+) or addition of the gap junction blockers heptanol, 18 α,ß-glycyrrhetinic acid, La(3+) and Ni(2+), but were insensitive to BTP-2 and SKF 96365. The spiking response was abolished by inhibiting the Ca(2+) entry mode of the Na(+)/Ca(2+) exchanger (NCX). The InsP(3)-producing agonist ATP resumed Ca(2+) oscillations in silent cells, while the phospholipase C inhibitor U73122 suppressed them. Injury-induced Ca(2+) transients were prevented by the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) blockers thapsigargin and cyclopiazonic acid, while they were unaffected by suramin and genistein. These data show for the first time that the coordinated interplay between NCX-mediated Ca(2+) entry and InsP(3)-dependent Ca(2+) release contributes to injury-induced intracellular Ca(2+) concentration oscillations.

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

Endothelial progenitor cells (EPCs) home from the bone marrow to the site of tissue regeneration and sustain neovascularization after acute vascular injury and upon the angiogenic switch in solid tumors. Therefore, they represent a suitable tool for cell-based therapy (CBT) in regenerative medicine and provide a novel promising target in the fight against cancer. Intracellular Ca(2+) signals regulate numerous endothelial functions, such as proliferation and tubulogenesis. The growth of endothelial colony forming cells (ECFCs), which are EPCs capable of acquiring a mature endothelial phenotype, is governed by store-dependent Ca(2+) entry (SOCE). This study aimed at investigating the nature and the role of VEGF-elicited Ca(2+) signals in ECFCs. VEGF induced asynchronous Ca(2+) oscillations, whose latency, amplitude, and frequency were correlated to the growth factor dose. Removal of external Ca(2+) (0Ca(2+)) and SOCE inhibition with N-(4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2) reduced the duration of the oscillatory signal. Blockade of phospholipase C-γ with U73122, emptying the inositol-1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) pools with cyclopiazonic acid (CPA), and inhibition of InsP(3) receptors with 2-APB prevented the Ca(2+) response to VEGF. VEGF-induced ECFC proliferation and tubulogenesis were inhibited by the Ca(2+)-chelant, BAPTA, and BTP-2. NF-κB activation by VEGF was impaired by BAPTA, BTP-2, and its selective blocker, thymoquinone. Thymoquinone, in turn, suppressed VEGF-dependent ECFC proliferation and tubulogenesis. These data indicate that VEGF-induced Ca(2+) oscillations require the interplay between InsP(3)-dependent Ca(2+) release and SOCE, and promote ECFC growth and tubulogenesis by engaging NF-κB. This novel signaling pathway might be exploited to enhance the outcome of CBT and chemotherapy.

GABAA and GABAB Receptors Mediate GABA-Induced Intracellular Ca2+ Signals in Human Brain Microvascular Endothelial Cells.

Numerous studies recently showed that the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), can stimulate cerebral angiogenesis and promote neurovascular coupling by activating the ionotropic GABAA receptors on cerebrovascular endothelial cells, whereas the endothelial role of the metabotropic GABAB receptors is still unknown. Preliminary evidence showed that GABAA receptor stimulation can induce an increase in endothelial Ca2+ levels, but the underlying signaling pathway remains to be fully unraveled. In the present investigation, we found that GABA evoked a biphasic elevation in [Ca2+]i that was initiated by inositol-1,4,5-trisphosphate- and nicotinic acid adenine dinucleotide phosphate-dependent Ca2+ release from neutral and acidic Ca2+ stores, respectively, and sustained by store-operated Ca2+ entry. GABAA and GABAB receptors were both required to trigger the endothelial Ca2+ response. Unexpectedly, we found that the GABAA receptors signal in a flux-independent manner via the metabotropic GABAB receptors. Likewise, the full Ca2+ response to GABAB receptors requires functional GABAA receptors. This study, therefore, sheds novel light on the molecular mechanisms by which GABA controls endothelial signaling at the neurovascular unit.