RESUMEN
OBJECTIVE: This study aimed to investigate the current status and performance of machine learning (ML) approaches in providing reproducible treatment response predictions. METHODS: This systematic review was conducted in accordance with the PRISMA statement and the CHARMS checklist. We searched PubMed, Cochrane Library, Web of Science, Scopus, and EBSCO databases for cohort studies that derived and/or validated ML models focused on predicting rheumatoid arthritis (RA) treatment response. We extracted data and critically appraised studies based on the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis (TRIPOD) and Prediction Model Risk of Bias Assessment Tool (PROBAST) guidelines. RESULTS: From 210 unduplicated records identified by the literature search, we retained 29 eligible studies. Of these studies, 10 developed a predictive model and reported a mean adherence to the TRIPOD guidelines of 45.6 % (95 % CI: 38.3-52.8 %). The remaining 19 studies not only developed a predictive model but also validated it externally, with a mean adherence of 42.9 % (95 % CI: 39.1-46.6 %). Most of the articles had an unclear risk of bias (41.4 %), followed by a high risk of bias, which was present in 37.9 %. CONCLUSIONS: In recent years, ML methods have been increasingly used to predict treatment response in RA. Our critical appraisal revealed unclear and high risk of bias in most of the identified models, suggesting that researchers can do more to address the risk of bias and increase transparency, including the use of calibration measures and reporting methods for handling missing data. FUNDING: None.
Asunto(s)
Antirreumáticos , Artritis Reumatoide , Aprendizaje Automático , Artritis Reumatoide/tratamiento farmacológico , Humanos , Antirreumáticos/uso terapéutico , Resultado del Tratamiento , PronósticoRESUMEN
BACKGROUND: The conventional "whole-cell patch-clamp" recording technique is widely used to measure the resting membrane potential (VM) and to dissect the underlying membrane ionic conductances in isolated vascular endothelial cells. NEW METHOD: Herein, we assessed whether the automated patch-clamp (APC) technology, which replaces the traditional patch-pipette with a planar substrate to permit researchers lacking formal training in electrophysiology to generate large amounts of data in a relatively short time, can be used to characterize the bioelectrical activity of vascular endothelial cells. We assessed whether the Port-a-Patch planar patch-clamp system, which is regarded as the smallest electrophysiological rig available on the market, can be used to measure the VM and resting membrane currents in the human cerebrovascular endothelial cell line, hCMEC/D3. COMPARISON WITH EXISTING METHODS: We demonstrated that the Port-a-Patch planar patch-clamp system provides the same values of the resting VM as those provided by the conventional patch-clamp technique. Furthermore, the APC technology provides preliminary data demonstrating that the resting VM of hCMEC/D3 cells is primarily contributed by Cl- and Na+, as demonstrated with the patch-clamp technique for many other endothelial cell types. CONCLUSIONS: The Port-a-Patch planar patch-clamp system can be successfully used to measure the resting VM and the underlying membrane ionic conductances in hCMEC/D3 cells. We envisage that this easy-to-use APC system could also be extremely useful for the investigation of the membrane currents that can be activated by chemical, thermal, optical, and mechanical stimuli in this cell line as well as in other types of isolated vascular endothelial cells.
Asunto(s)
Células Endoteliales , Potenciales de la Membrana , Técnicas de Placa-Clamp , Humanos , Técnicas de Placa-Clamp/métodos , Técnicas de Placa-Clamp/instrumentación , Células Endoteliales/fisiología , Células Endoteliales/citología , Potenciales de la Membrana/fisiología , Línea CelularRESUMEN
Nitric oxide (NO) is a highly versatile gasotransmitter that has first been shown to regulate cardiovascular function and then to exert tight control over a much broader range of processes, including neurotransmitter release, neuronal excitability, and synaptic plasticity. Endothelial NO synthase (eNOS) is usually far from the mind of synaptic neurophysiologists, who have focused most of their attention on neuronal NO synthase (nNOS) as the primary source of NO at the neurovascular unit (NVU). Nevertheless, the available evidence suggests that eNOS could also contribute to generating the burst of NO that, serving as volume intercellular messenger, is produced in response to neuronal activity in the brain parenchyma. Herein, we review the role of eNOS in both the regulation of cerebral blood flow and of synaptic plasticity and discuss the mechanisms by which cerebrovascular endothelial cells may transduce synaptic inputs into a NO signal. We further suggest that eNOS could play a critical role in vascular-to-neuronal communication by integrating signals converging onto cerebrovascular endothelial cells from both the streaming blood and active neurons.
Asunto(s)
Circulación Cerebrovascular , Óxido Nítrico Sintasa de Tipo III , Óxido Nítrico , Humanos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Circulación Cerebrovascular/fisiología , Animales , Óxido Nítrico/metabolismo , Plasticidad Neuronal , Células Endoteliales/metabolismo , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Neuronas/metabolismo , Acoplamiento Neurovascular/fisiologíaAsunto(s)
Señalización del Calcio , Células Endoteliales , Microvasos , Receptores Acoplados a Proteínas G , Sodio , Humanos , Señalización del Calcio/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/efectos de los fármacos , Sodio/metabolismo , Microvasos/metabolismo , Microvasos/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Calcio/metabolismoRESUMEN
Lysosomal Ca2+ signaling is emerging as a crucial regulator of endothelial Ca2+ dynamics. Ca2+ release from the acidic vesicles in response to extracellular stimulation is usually promoted via Two Pore Channels (TPCs) and is amplified by endoplasmic reticulum (ER)-embedded inositol-1,3,4-trisphosphate (InsP3) receptors and ryanodine receptors. Emerging evidence suggests that sub-cellular Ca2+ signals in vascular endothelial cells can also be generated by the Transient Receptor Potential Mucolipin 1 channel (TRPML1) channel, which controls vesicle trafficking, autophagy and gene expression. Herein, we adopted a multidisciplinary approach, including live cell imaging, pharmacological manipulation, and gene targeting, revealing that TRPML1 protein is expressed and triggers global Ca2+ signals in the human brain microvascular endothelial cell line, hCMEC/D3. The direct stimulation of TRPML1 with both the synthetic agonist, ML-SA1, and the endogenous ligand phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) induced a significant increase in [Ca2+]i, that was reduced by pharmacological blockade and genetic silencing of TRPML1. In addition, TRPML1-mediated lysosomal Ca2+ release was sustained both by lysosomal Ca2+ release and ER Ca2+- release through inositol-1,4,5-trisphophate receptors and store-operated Ca2+ entry. Notably, interfering with TRPML1-mediated lysosomal Ca2+ mobilization led to a decrease in the free ER Ca2+ concentration. Imaging of DAF-FM fluorescence revealed that TRPML1 stimulation could also induce a significant Ca2+-dependent increase in nitric oxide concentration. Finally, the pharmacological and genetic blockade of TRPML1 impaired ATP-induced intracellular Ca2+ release and NO production. These findings, therefore, shed novel light on the mechanisms whereby the lysosomal Ca2+ store can shape endothelial Ca2+ signaling and Ca2+-dependent functions in vascular endothelial cells.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Calcio , Células Endoteliales , Células Endoteliales/metabolismo , Calcio/metabolismo , Señalización del Calcio/fisiología , Endotelio/metabolismo , Calcio de la DietaRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Células Endoteliales , Óxido Nítrico , Humanos , Especies Reactivas de Oxígeno/metabolismo , Células Endoteliales/metabolismo , Óxido Nítrico/metabolismo , Línea CelularRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Canales de Calcio , Células Endoteliales , Animales , Bovinos , Ratones , Ratas , Humanos , Canales de Calcio/metabolismo , Células Endoteliales/metabolismo , Canales Catiónicos TRPC/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Calcio/metabolismo , Proteína ORAI1/metabolismo , Señalización del Calcio/fisiologíaRESUMEN
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.
Asunto(s)
N-Metilaspartato , Receptores de N-Metil-D-Aspartato , Receptores de N-Metil-D-Aspartato/metabolismo , N-Metilaspartato/farmacología , Células Endoteliales/metabolismo , Transmisión Sináptica , Barrera Hematoencefálica/metabolismoRESUMEN
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.
Asunto(s)
Células Endoteliales , Receptores de GABA , Humanos , Receptores de GABA/metabolismo , Células Endoteliales/metabolismo , Ácido gamma-Aminobutírico/farmacología , Ácido gamma-Aminobutírico/metabolismo , Encéfalo/metabolismoRESUMEN
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 Ca2+ handling machinery, which drives MDA-MB-231 cell motility. METHODS: PEVs were isolated from human blood platelets, and Fura-2/AM Ca2+ imaging, RT-qPCR, and immunoblotting were exploited to assess their effect on intracellular Ca2+ dynamics and Ca2+-dependent migratory processes in MDA-MB-231 cells. RESULTS: Pretreating MDA-MB-231 cells with PEVs for 24 h caused an increase in Ca2+ release from the endoplasmic reticulum (ER) due to the up-regulation of SERCA2B and InsP3R1/InsP3R2 mRNAs and proteins. The consequent enhancement of ER Ca2+ depletion led to a significant increase in store-operated Ca2+ entry. The larger Ca2+ 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 Ca2+ handling machinery.
Asunto(s)
Neoplasias de la Mama , Calcio/metabolismo , Vesículas Extracelulares , Plaquetas/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Vesículas Extracelulares/metabolismo , Femenino , Fura-2 , Humanos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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+]i evoked 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 InsP3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.
RESUMEN
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.
Asunto(s)
Resistencia a la Insulina , Lupus Eritematoso Sistémico , Disfunción Ventricular Izquierda , Humanos , Adulto , Femenino , Índice de Masa Corporal , Estudios Transversales , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/epidemiología , Disfunción Ventricular Izquierda/epidemiología , Disfunción Ventricular Izquierda/etiología , Remodelación Ventricular , Función Ventricular Izquierda , Volumen SistólicoRESUMEN
Background: Allergic asthma was considered as an inflammation mediated by specific CD4+ helper lymphocytes (Th2); however, this paradigm changed in 2005, when a third group of helper cells called Th17 cells were identified. Th17 lymphocytes are the main source of interleukin (IL)-17A-F, IL-21, and IL-22; however, their physiological role in children is unclear. This study aimed to determine the percentage of Th17 cells and IL-17A in pediatric patients diagnosed with asthma and to associate it with disease control using a validated questionnaire. Methods: This cross-sectional, prospective, comparative study included 92 asthma-diagnosed children 4-18 years of age. The Asthma Control Test was used as an assessment measure to classify patients as controlled (n = 30), partially controlled (n = 31), and uncontrolled (n = 31). Th17 cells and IL-17A were analyzed by flow cytometry. Patients receiving inhaled steroid therapy as monotherapy or associated with a long-acting bronchodilator were included. Results: The mean percentage of Th17 cells in the participants was 4.55 ± 7.34 (Controlled), 5.50 ± 8.09 (Partially Controlled), and 6.14 ± 7.11 (Uncontrolled). There was no significant difference between the 3 groups (P = 0.71). The mean percentage of IL-17A in all the participants was 9.84 ± 9.4 (Controlled), 10.10 ± 10.5 (Partially Controlled), and 11.42 ± 8.96 (Uncontrolled); no significant difference between the 3 groups (P = 0.79) was observed. Th17 lymphocyte levels were similar among the 3 groups and the same trend was observed with IL-17A. A significant correlation between Th17 or IL-17A and the degree of asthma control (Th17, P = 0.24; IL-17A, P = 0.23) was not found. Conclusions: The percentages of both Th17 lymphocytes and IL-17A found in children with asthma were not significantly different in the 3 groups, which suggests that they do not play an important role in asthma control. Our findings may contribute to the knowledge related to non-Th2 inflammation in children. Clinical-Trials.gov ID: 2015-2102-85.