Evaluation of cardiovascular events in patients with hepatocellular carcinoma treated with sorafenib in the clinical practice. The CARDIO-SOR study.

BACKGROUND AND AIMS: The effectiveness of systemic treatment in advanced hepatocellular carcinoma (HCC) depends on the selection of patients, management of cirrhosis complications and expertise to treat adverse events. The aims of the study are to assess the frequency and management of cardiovascular events in HCC patients treated with sorafenib (SOR) and to create a scale to predict the onset of major adverse cardiovascular events (MACE). METHOD: Observational retrospective study with consecutive HCC patients treated with SOR between 2007 and 2019 in a western centre. In order to classify cardiovascular risk pre-SOR, we designed the CARDIOSOR scale with age, hypertension, diabetes, dyslipidaemia and peripheral vascular disease. Other adverse events, dosing and outcome data were collected during a homogeneous protocolled follow-up. RESULTS: Two hundred ninety-nine patients were included (219 BCLC-C). The median overall survival was 11.1 months (IQR 5.6-20.5), and duration of treatment was 7.4 months (IQR 3.3-14.7). Seventeen patients (6%) stopped SOR due to cardiovascular event. Thirty-three patients suffered MACE (7 heart failure, 11 acute coronary syndrome, 12 cerebrovascular accident and 8 peripheral vascular ischemia); 99 had a minor cardiovascular event, mainly hypertension (n = 81). Age was the only independent factor associated to MACE (HR 1.07; 95% CI 1.03-1.12; P = .002). The CARDIOSOR scale allows to identify the group of patients with higher risk of MACE (sHR 3.4; 95% CI 1.4-6.7; P = .04). CONCLUSION: The incidence of cardiovascular events in HCC patients treated with SOR is higher than expected. Multidisciplinary approach and clinical tools like CARDIOSOR scale could be helpful to manage these patients.

Sigma-1 receptor activation-induced glycolytic ATP production and endothelial barrier enhancement.

OBJECTIVE: We tested the hypothesis that σ1 modulates endothelial barrier function due to its influence on endothelial bioenergetics. METHODS: Cultured HUVEC monolayers were used to model the endothelial barrier. ECIS, Transwell assays, and immunofluorescence labeling of junctional proteins were used to evaluate endothelial barrier function. Endothelial cell bioenergetics was determined using extracellular flux analysis and direct ATP level measurements. The endothelial-specific contribution of σ1 was tested using the σ1-selective agonist, PRE-084, and with targeted knockdown of σ1 expression using siRNA. RESULTS: Activation of σ1 with PRE-084 significantly enhanced endothelial barrier function and decreased permeability to albumin and dextran. Knockdown of σ1 with siRNA reduced barrier function and abolished PRE-084-induced endothelial barrier enhancement. PRE-084 upregulated endothelial glycolysis and glycolytic ATP production, but this response was abolished by siRNA-mediated knockdown of σ1 expression. PRE-084 also reduced the degree of endothelial barrier dysfunction caused by the mitochondrial oxidative phosphorylation uncoupler CCCP. CONCLUSION: Activation of σ1 enhances endothelial barrier function and modulates the ratio of glycolytic versus mitochondrial ATP production. These novel findings suggest that endothelial σ1 may prove beneficial as a novel therapeutic target for reducing microvascular hyperpermeability and counteracting mitochondrial dysfunction.

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

The acetylcholine-activated inward rectifier potassium current ( IKACh) is constitutively active in persistent atrial fibrillation (AF). We tested the hypothesis that the blocking of IKACh with the small molecule chloroquine terminates persistent AF. We used a sheep model of tachypacing-induced, persistent AF, molecular modeling, electrophysiology, and structural biology approaches. The 50% inhibition/inhibitory concentration of IKACh block with chloroquine, measured by patch clamp, was 1 µM. In optical mapping of sheep hearts with persistent AF, 1 µM chloroquine restored sinus rhythm. Molecular modeling suggested that chloroquine blocked the passage of a hydrated potassium ion through the intracellular domain of Kir3.1 (a molecular correlate of IKACh) by interacting with residues D260 and F255, in proximity to I228, Q227, and L299. 1H 15N heteronuclear single-quantum correlation of purified Kir3.1 intracellular domain confirmed the modeling results. F255, I228, Q227, and L299 underwent significant chemical-shift perturbations upon drug binding. We then crystallized and solved a 2.5 Å X-ray structure of Kir3.1 with F255A mutation. Modeling of chloroquine binding to the mutant channel suggested that the drug's binding to the pore becomes off centered, reducing its ability to block a hydrated potassium ion. Patch clamp validated the structural and modeling data, where the F255A and D260A mutations significantly reduced IKACh block by chloroquine. With the use of numerical and structural biology approaches, we elucidated the details of how a small molecule could block an ion channel and exert antiarrhythmic effects. Chloroquine binds the IKACh channel at a site formed by specific amino acids in the ion-permeation pathway, leading to decreased IKACh and the subsequent termination of AF.-Takemoto, Y., Slough, D. P., Meinke, G., Katnik, C., Graziano, Z. A., Chidipi, B., Reiser, M., Alhadidy, M. M., Ramirez, R., Salvador-Montañés, O., Ennis, S., Guerrero-Serna, G., Haburcak, M., Diehl, C., Cuevas, J., Jalife, J., Bohm, A., Lin,Y.-S., Noujaim, S. F. Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule.

Kvß1.1 (AKR6A8) senses pyridine nucleotide changes in the mouse heart and modulates cardiac electrical activity.

The present study investigates the physiological role of Kvß1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvß1.1 knockout (KO) or wild-type (WT) mice and established that Kvß1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvß1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvß1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvß1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvß1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvß1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvß1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart.NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvß1.1 subunit. In the present study, we identify Kvß1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.

Modulation of mesenteric collecting lymphatic contractions by σ1-receptor activation and nitric oxide production.

Recently, it has been reported that a σ-receptor antagonist could reduce inflammation-induced edema. Lymphatic vessels play an essential role in removing excess interstitial fluid. We tested the hypothesis that activation of σ-receptors would reduce or weaken collecting lymphatic contractions. We used isolated, cannulated rat mesenteric collecting lymphatic vessels to study contractions in response to the σ-receptor agonist afobazole in the absence and presence of different σ-receptor antagonists. We used RT-PCR and Western blot analysis to investigate whether these vessels express the σ1-receptor and immunofluorescence confocal microscopy to examine localization of the σ1-receptor in the collecting lymphatic wall. Using N-nitro-l-arginine methyl ester (l-NAME) pretreatment before afobazole in isolated lymphatics, we tested the role of nitric oxide (NO) signaling. Finally, we used 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence as an indicator to test whether afobazole increases NO release in cultured lymphatic endothelial cells. Our results show that afobazole (50-150 µM) elevated end-systolic diameter and generally reduced pump efficiency and that this response could be partially blocked by the σ1-receptor antagonists BD 1047 and BD 1063 but not by the σ2-receptor antagonist SM-21. σ1-Receptor mRNA and protein were detected in lysates from isolated rat mesenteric collecting lymphatics. Confocal images with anti-σ1-receptor antibody labeling suggested localization in the lymphatic endothelium. Blockade of NO synthases with l-NAME inhibited the effects of afobazole. Finally, afobazole elicited increases in NO production from cultured lymphatic endothelial cells. Our findings suggest that the σ1-receptor limits collecting lymphatic pumping through a NO-dependent mechanism.NEW & NOTEWORTHY Relatively little is known about the mechanisms that govern contractions of lymphatic vessels. σ1-Receptor activation has been shown to reduce the fractional pump flow of isolated rat mesenteric collecting lymphatics. The σ1-receptor was localized mainly in the endothelium, and blockade of nitric oxide synthase inhibited the effects of afobazole.

Activation of σ1 and σ2 receptors by afobazole increases glial cell survival and prevents glial cell activation and nitrosative stress after ischemic stroke.

Activation of sigma receptors at delayed time points has been shown to decrease injury following ischemic stroke. The mixed σ1/σ2 receptor agonist, 5-ethoxy-2-[2-(morpholino)-ethylthio]benzimidazole (afobazole), provides superior long-term outcomes compared to other σ ligands in the rat middle cerebral artery occlusion (MCAO) stroke model. Experiments using the MCAO model were carried out to determine the molecular mechanism involved in the beneficial effects of afobazole. Administration of afobazole (3 mg/kg) at delayed time points post-stroke significantly increased the number of microglia and astrocytes detected in the ipsilateral hemisphere at 96 h post-surgery. Morphological analysis of the microglia indicated that a greater number of these cells were found in the ramified resting state in MCAO animals treated with afobazole relative to MCAO vehicle controls. Similarly, fewer reactive astrocytes were detected in the injured hemisphere of afobazole-treated animals. Both the enhanced survival and reduced activation of glial cells were abolished by co-application of either a σ1 (BD-1063) or a σ2 (SM-21) receptor antagonist with afobazole. To gain further insight into the mechanisms by which afobazole lessens stroke injury, we probed the brain sections for markers of neuroinflammation (tumor necrosis factor α) and nitrosative stress (S-nitrosocysteine). Data show that afobazole significantly reduces S-nitrosocysteine levels, but does not alter tumor necrosis factor α expression 96 h after an ischemic stroke. Taken together our data indicate that afobazole acting via both σ1 and σ2 receptors decreases stroke injury by enhancing glial cell survival, blocking ischemia-induced glial cell activation, and decreasing nitrosative stress.

σ-1 Receptor Inhibition of ASIC1a Channels is Dependent on a Pertussis Toxin-Sensitive G-Protein and an AKAP150/Calcineurin Complex.

ASIC1a channels play a major role in various pathophysiological conditions including depression, anxiety, epilepsy, and neurodegeneration following ischemic stroke. Sigma-1 (σ-1) receptor stimulation depresses the activity of ASIC1a channels in cortical neurons, but the mechanism(s) by which σ-1 receptors exert their influence on ASIC1a remains unknown. Experiments were undertaken to elucidate the signaling cascade linking σ-1 receptors to ASIC1a channels. Immunohistochemical studies showed that σ-1 receptors, ASIC1a and A-kinase anchoring peptide 150 colocalize in the plasma membrane of the cell body and processes of cortical neurons. Fluorometric Ca(2+) imaging experiments showed that disruption of the macromolecular complexes containing AKAP150 diminished the effects of the σ-1 on ASIC1a, as did application of the calcineurin inhibitors, cyclosporin A and FK-506. Moreover, whole-cell patch clamp experiments showed that σ-1 receptors were less effective at decreasing ASIC1a-mediated currents in the presence of the VIVIT peptide, which binds to calcineurin and prevents cellular effects dependent on AKAP150/calcineurin interaction. The coupling of σ-1 to ASIC1a was also disrupted by preincubation of the neurons in the G-protein inhibitor, pertussis toxin (PTX). Taken together, our data reveal that σ-1 receptor block of ASIC1a function is dependent on activation of a PTX-sensitive G-protein and stimulation of AKAP150 bound calcineurin.

Non-specific inhibition of ischemia- and acidosis-induced intracellular calcium elevations and membrane currents by α-phenyl-N-tert-butylnitrone, butylated hydroxytoluene and trolox.

Ischemia, and subsequent acidosis, induces neuronal death following brain injury. Oxidative stress is believed to be a key component of this neuronal degeneration. Acute chemical ischemia (azide in the absence of external glucose) and acidosis (external media buffered to pH 6.0) produce increases in intracellular calcium concentration ([Ca2+]i) and inward membrane currents in cultured rat cortical neurons. Two α-tocopherol analogues, trolox and butylated hydroxytoluene (BHT), and the spin trapping molecule α-Phenyl-N-tert-butylnitrone (PBN) were used to determine the role of free radicals in these responses. PBN and BHT inhibited the initial transient increases in [Ca2+]i, produced by ischemia, acidosis and acidic ischemia and increased steady state levels in response to acidosis and the acidic ischemia. BHT and PBN also potentiated the rate at which [Ca2+]i increased after the initial transients during acidic ischemia. Trolox inhibited peak and sustained increases in [Ca2+]i during ischemia. BHT inhibited ischemia induced initial inward currents and trolox inhibited initial inward currents activated by acidosis and acidic ischemia. Given the inconsistent results obtained using these antioxidants, it is unlikely their effects were due to elimination of free radicals. Instead, it appears these compounds have non-specific effects on the ion channels and exchangers responsible for these responses.

In vitro evaluation of guanidine analogs as sigma receptor ligands for potential anti-stroke therapeutics.

Currently, the only Food and Drug Administration-approved treatment of acute stroke is recombinant tissue plasminogen activator, which must be administered within 6 hours after stroke onset. The pan-selective σ-receptor agonist N,N'-di-o-tolyl-guanidine (o-DTG) has been shown to reduce infarct volume in rats after middle cerebral artery occlusion, even when administered 24 hours after stroke. DTG derivatives were synthesized to develop novel compounds with greater potency than o-DTG. Fluorometric Ca(2+) imaging was used in cultured cortical neurons to screen compounds for their capacity to reduce ischemia- and acidosis-evoked cytosolic Ca(2+) overload, which has been linked to stroke-induced neurodegeneration. In both assays, migration of the methyl moiety produced no significant differences, but removal of the group increased potency of the compound for inhibiting acidosis-induced [Ca(2+)](i) elevations. Chloro and bromo substitution of the methyl moiety in the meta and para positions increased potency by ≤160%, but fluoro substitutions had no effect. The most potent DTG derivative tested was N,N'-di-p-bromo-phenyl-guanidine (p-BrDPhG), which had an IC(50) of 2.2 µM in the ischemia assay, compared with 74.7 µM for o-DTG. Microglial migration assays also showed that p-BrDPhG is more potent than o-DTG in this marker for microglial activation, which is also linked to neuronal injury after stroke. Radioligand binding studies showed that p-BrDPhG is a pan-selective σ ligand. Experiments using the σ-1 receptor-selective antagonist 1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine dihydrochloride (BD-1063) demonstrated that p-BrDPhG blocks Ca(2+) overload via σ-1 receptor activation. The study identified four compounds that may be more effective than o-DTG for the treatment of ischemic stroke at delayed time points.

Stimulation of sigma receptors with afobazole blocks activation of microglia and reduces toxicity caused by amyloid-ß25-35.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of senile dementia in the United States. Accumulation of amyloid-ß (Aß) and the effects of this peptide on microglial cells contribute greatly to the etiology of AD. Experiments were carried out to determine whether the pan-selective σ-receptor agonist afobazole can modulate microglial response to the cytotoxic Aß fragment, Aß25-35. Treatment with afobazole decreased microglial activation in response to Aß, as indicated by reduced membrane ruffling and cell migration. The effects of afobazole on Aß25-35-evoked migration were concentration dependent and consistent with σ-receptor activation. When afobazole was coapplied with either BD-1047 [N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine dihydrobromide] or rimcazole, which are σ-1- and σ-2-selective antagonists, respectively, the inhibition of Aß25-35-induced migration by afobazole was reduced. Prolonged exposure of microglia to Aß25-35 resulted in glial cell death that was associated with increased expression of the proapoptotic protein Bax and the death protease caspase-3. Coapplication of afobazole with Aß25-35 decreased the number of cells expressing both Bax and caspase-3 and resulted in a concomitant enhancement in cell survival. Although afobazole inhibited activation of microglia cells by Aß25-35, it preserved normal functional responses in these cells after exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24-hour exposure to Aß25-35. However, coincubation in afobazole returned these responses to near control levels. Therefore, stimulation of σ-1 and σ-2 receptors by afobazole prevents Aß25-35 activation of microglia and inhibits Aß25-35-associated cytotoxicity, suggesting that afobazole may be useful for AD therapeutics.

Afobazole activation of σ-1 receptors modulates neuronal responses to amyloid-ß25-35.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a continual decline of cognitive function. No therapy has been identified that can effectively halt or reverse its progression. One hallmark of AD is accumulation of the amyloid-ß peptide (Aß), which alone induces neuronal injury via various mechanisms. Data presented here demonstrate that prolonged exposure (1-24 hours) of rat cortical neurons to Aß25-35 results in an increase in basal intracellular Ca(2+) concentration ([Ca(2+)]i), and that coincubation with the compound afobazole inhibits these [Ca(2+)]i increases. The effect of afobazole on [Ca(2+)]i is due to activation of σ-1 receptors but could not be mimicked by a second pan-selective σ receptor agonist, 1,3-di-o-tolylguanidine (DTG). Afobazole was also found to lessen nitric oxide (NO) production in response to Aß25-35 application but did not affect elevations in reactive oxygen species elicited by the Aß fragment. The reductions in [Ca(2+)]i and NO perturbation produced by afobazole were associated with a decrease in neuronal cell death, whereas DTG failed to enhance cell survival. Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that afobazole incubation results in lower expression of the proapoptotic protein Bax and the death protease caspase-3, while at the same time increasing expression of the antiapoptotic protein, Bcl-2. Given the importance of Aß neurotoxicity in AD etiology, the findings reported here suggest that afobazole may be an effective AD therapeutic agent. Furthermore, σ-1 receptors may represent a useful target for AD treatment, although not all σ ligands appear to be equally beneficial.

Multispectral optoacoustic tomography (MSOT): Monitoring neurovascular changes in a mouse repetitive traumatic brain injury model.

BACKGROUND: Evidence suggests that mild TBI injuries, which comprise > 75% of all TBIs, can cause chronic post-concussive symptoms, especially when experienced repetitively (rTBI). rTBI is a major cause of cognitive deficit in athletes and military personnel and is associated with neurovascular changes. Current methods to monitor neurovascular changes in detail are prohibitively expensive and invasive for patients with mild injuries. NEW METHOD: We evaluated the potential of multispectral optoacoustic tomography (MSOT) to monitor neurovascular changes and assess therapeutic strategies in a mouse model of rTBI. Mice were subjected to rTBI or sham via controlled cortical impact and administered pioglitazone (PG) or vehicle. Oxygenated and deoxygenated hemoglobin were monitored using MSOT. Indocyanine green clearance was imaged via MSOT to evaluate blood-brain-barrier (BBB) integrity. RESULTS: Mice subjected to rTBI show a transient increase in oxygenated/total hemoglobin ratio which can be mitigated by PG administration. rTBI mice also show BBB disruption shortly after injury and reduction of oxygenated/total hemoglobin in the chronic stage, neither of which were affected by PG intervention. COMPARISON WITH EXISTING METHODS: MSOT imaging has the potential as a noninvasive in vivo imaging method to monitor neurovascular changes and assess therapeutics in mouse models of rTBI. In comparison to standard methods of tracking inflammation and BBB disruption, MSOT can be used multiple times throughout the course of injury without the need for surgery. Thus, MSOT is especially useful in research of rTBI models for screening therapeutics, and with further technological improvements may be extended for use in rTBI patients.

Slow Coronary Blood Flow: Pathogenesis and Clinical Implications.

Coronary slow flow (CSF) phenomenon, also known as cardiac syndrome Y, is defined as the delayed opacification of the coronary vasculature at the distal level. Different hypotheses and theories have been postulated about its substrate and mechanism, such as microvascular and endothelial dysfunction. Several studies have confirmed that CSF is a cause of ischaemia detected by non-invasive testing. Clinically, it can present as angina pectoris, acute coronary syndrome and sudden cardiac death. It has an incidence of 1-5% in patients undergoing coronary angiography and has been most frequently found in young men who are smokers with metabolic syndrome. There are no established treatments for CSF and further studies are still necessary.

Left ventricular noncompaction cardiomyopathy: Recent advances.

From its initial description to the present day, left ventricular noncompaction cardiomyopathy has been the subject of numerous studies and publications. In question as a real cardiomyopathy, left ventricular noncompaction can appear in isolation or in association with other cardiac malformations, genetic syndromes, and neuromuscular disorders. As a genetically heterogeneous disorder, it can be sporadic or familial, with an autosomal dominant pattern with variable penetrance most frequently observed. Different diagnostic criteria have been described through the years, first by using echocardiogram and later on by cardiac magnetic resonance. The lack of universally accepted diagnostic criteria has led to the condition being over-diagnosed in the general population. Differential diagnosis between real cardiomyopathy, epiphenomenon (phenocopy in the setting of loading conditions or even other cardiomyopathies), and physiological hypertrabeculation, like in the athlete's heart must be considered. Clinically it can present as heart failure, ventricular arrhythmias, and even sudden death, but it can also be asymptomatic during familial screening. The main prognosis factors are left ventricular dilatation, dysfunction, and fibrosis. There is no specific treatment. Familial screening is recommended and special recommendations in the case of athletes must be taken into account. In the present article, we review the myth and reality concerning main and more recent aspects of left ventricular noncompaction.

Afobazole modulates microglial function via activation of both sigma-1 and sigma-2 receptors.

Microglial cells play a critical role in the neuroinflammatory response that accompanies various diseases of the central nervous system, such as ischemic stroke, and ATP is a major signaling molecule regulating the response of these cells to these pathophysiological conditions. Experiments were carried out to determine the effects of afobazole on microglial function and to identify the molecular mechanisms by which afobazole affects microglial cells. Afobazole was found to inhibit migration of microglial cells in response to ATP and UTP chemoattraction in a concentration-dependent manner. Inhibition of either σ-1 or σ-2 receptors decreased the effects of afobazole on microglia. In addition to inhibiting microglial cell migration, activation of σ receptors by afobazole decreased intracellular calcium elevation produced by focal application of ATP and UTP in isolated microglial cells. Furthermore, afobazole blocked membrane currents elicited by rapid application of ATP in microglial cells. Taken together, our data indicate that afobazole inhibits microglial response to P2Y and P2X purinergic receptor activation by functioning as a pan-selective σ-receptor agonist. In addition to modulating response to purinergic receptor activation, the effects of afobazole on microglial survival during in vitro ischemia were assessed. Application of afobazole during in vitro ischemia decreased microglial cell death during the ischemic episode and after a 24-h recovery period. Moreover, when afobazole was only applied after the ischemic episode, a significant enhancement in cell survival was still observed. Thus, afobazole acts via σ receptors to decrease microglial response to ATP and provides cytoprotection during and after ischemia.

Afobazole modulates neuronal response to ischemia and acidosis via activation of sigma-1 receptors.

Afobazole is an anxiolytic medication that has been previously shown to be neuroprotective both in vitro and in vivo. However, the mechanism(s) by which afobazole can enhance neuronal survival remain poorly understood. Experiments were carried out to determine whether afobazole can decrease intracellular calcium overload associated with ischemia and acidosis and whether the effects of afobazole are mediated via interaction of the compound with σ receptors. Fluorometric Ca(2+) imaging was used to resolve how application of afobazole affects intracellular Ca(2+) handling in cortical neurons. Application of afobazole significantly depressed, in a concentration-dependent and reversible manner, the intracellular Ca(2+) overload resulting from in vitro ischemia and acidosis. The IC(50) for afobazole inhibition of ischemia-evoked intracellular Ca(2+) overload was considerably less than that for the inhibition of [Ca(2+)](i) increases induced by acidosis. However, afobazole maximally inhibited only 70% of the ischemia-evoked intracellular Ca(2+) overload but effectively abolished intracellular Ca(2+) increases produced by acidosis. The effects of afobazole on ischemia- and acidosis-induced intracellular Ca(2+) dysregulation were inhibited by preincubating the neurons in the irreversible, pan-selective σ-receptor antagonist, metaphit. Moreover, the effects of afobazole on intracellular Ca(2+) increases triggered by acidosis and ischemia were blocked by the selective σ-1-receptor antagonists, BD 1063 and BD 1047, respectively. Experiments examining the effects of afobazole on neuronal survival in response to ischemia showed that afobazole was neuroprotective. Taken together, these data suggest that afobazole regulates intracellular Ca(2+) overload during ischemia and acidosis via activation of σ-1 receptors. This mechanism is probably responsible for afobazole-mediated neuroprotection.

Tipifarnib-induced apoptosis in acute myeloid leukemia and multiple myeloma cells depends on Ca2+ influx through plasma membrane Ca2+ channels.

A major contributing factor to the high mortality rate associated with acute myeloid leukemia and multiple myeloma is the development of resistance to chemotherapy. We have shown that the combination of tipifarnib, a nonpeptidomimetic farnesyltransferase inhibitor (FTI), with bortezomib, a proteosome inhibitor, promotes synergistic death and overcomes de novo drug resistance in acute myeloid leukemia cell lines. Experiments were undertaken to identify the molecular mechanisms by which tipifarnib produces cell death in acute myeloid leukemia and multiple myeloma cell lines (U937 and 8226, respectively). Tipifarnib, but not other FTIs tested [N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) and 2'-methyl-5-((((1-trityl-1H-imidazol-4-yl)methyl)amino)methyl)-[1,1'-biphenyl]-2-carboxylic acid (FTI-2153), promotes elevations in intracellular free-calcium concentrations ([Ca(2+)](i)) in both cell lines. These elevations in [Ca(2+)](i) were accompanied by highly dynamic plasmalemmal blebbing and frequently resulted in membrane lysis. The tipifarnib-induced elevations in [Ca(2+)](i) were not blocked by thapsigargin or ruthenium red, but were inhibited by application of Ca(2+)-free extracellular solution and by the Ca(2+) channel blockers Gd(3+) and La(3+). Conversely, 2-aminoethoxydiphenyl borate (2-APB) potentiated the tipifarnib-evoked [Ca(2+)](i) overload. Preventing Ca(2+) influx diminished tipifarnib-evoked cell death, whereas 2-APB potentiated this effect, demonstrating a link between tipifarnib-induced Ca(2+) influx and apoptosis. These data suggest that tipifarnib exerts its effects by acting on a membrane channel with pharmacological properties consistent with store-operated channels containing the Orai3 subunit. It is noteworthy that Orai3 transcripts were found to be expressed at lower levels in tipifarnib-resistant 8226/R5 cells. Our results indicate tipifarnib causes cell death via a novel mechanism involving activation of a plasma membrane Ca(2+) channel and intracellular Ca(2+) overload.

Loop Diuretics Inhibit Ischemia-Induced Intracellular Ca2+ Overload in Neurons via the Inhibition of Voltage-Gated Ca2+ and Na+ Channels.

One consequence of ischemic stroke is disruption of intracellular ionic homeostasis. Intracellular overload of both Na+ and Ca2+ has been linked to neuronal death in this pathophysiological state. The etiology of ionic imbalances resulting from stroke-induced ischemia and acidosis includes the dysregulation of multiple plasma membrane transport proteins, such as increased activity of sodium-potassium-chloride cotransporter-1 (NKCC-1). Experiments using NKCC1 antagonists, bumetanide (BMN) and ethacrynic acid (EA), were carried out to determine if inhibition of this cotransporter affects Na+ and Ca2+ overload observed following in vitro ischemia-acidosis. Fluorometric Ca2+ and Na+ measurements were performed using cultured cortical neurons, and measurements of whole-cell membrane currents were used to determine target(s) of BMN and EA, other than the electroneutral NKCC-1. Both BMN and EA depressed ischemia-acidosis induced [Ca2+]i overload without appreciably reducing [Na+]i increases. Voltage-gated Ca2+ channels were inhibited by both BMN and EA with half-maximal inhibitory concentration (IC50) values of 4 and 36 µM, respectively. Similarly, voltage-gated Na+ channels were blocked by BMN and EA with IC50 values of 13 and 30 µM, respectively. However, neither BMN nor EA affected currents mediated by acid-sensing ion channels or ionotropic glutamatergic receptors, both of which are known to produce [Ca2+]i overload following ischemia. Data suggest that loop diuretics effectively inhibit voltage-gated Ca2+ and Na+ channels at clinically relevant concentrations, and block of these channels by these compounds likely contributes to their clinical effects. Importantly, inhibition of these channels, and not NKCC1, by loop diuretics reduces [Ca2+]i overload in neurons during ischemia-acidosis, and thus BMN and EA could potentially be used therapeutically to lessen injury following ischemic stroke.

Transcatheter Aortic Valve Implantation and Subclinical and Clinical Leaflet Thrombosis: Multimodality Imaging for Diagnosis and Risk Stratification.

In recent years, the phenomenon of subclinical leaflet thrombosis (SLT) in patients who have undergone transcatheter aortic valve implantation has become increasingly relevant. Hypo-attenuating leaflet thickening and hypo-attenuation affecting motion diagnosed by CT are the hallmarks of SLT, and their incidence varies depending on the intensity of screening. Whether these phenomena are a surrogate for leaflet thrombosis reducing valve durability and increasing the risk of stroke is still a matter of debate. Uncertainty remains over the optimal antithrombotic therapy after TAVI and the best treatment strategy is still not confirmed. Ongoing and future trials will provide more evidence about the best strategy for the prevention and treatment of SLT.