Treating diabetes with combination of phosphodiesterase 5 inhibitors and hydroxychloroquine-a possible prevention strategy for COVID-19?

Type 2 diabetes (T2D) is one of the major risk factors for developing cardiovascular disease and the resultant devastating morbidity and mortality. The key features of T2D are hyperglycemia, hyperlipidemia, insulin resistance, and impaired insulin secretion. Patients with diabetes and myocardial infarction have worse prognosis than those without T2D. Moreover, obesity and T2D are recognized risk factors in developing severe form of COVID-19 with higher mortality rate. The current lines of drug therapy are insufficient to control T2D and its serious cardiovascular complications. Phosphodiesterase 5 (PDE5) is a cGMP specific enzyme, which is the target of erectile dysfunction drugs including sildenafil, vardenafil, and tadalafil. Cardioprotective effects of PDE5 inhibitors against ischemia/reperfusion (I/R) injury were reported in normal and diabetic animals. Hydroxychloroquine (HCQ) is a widely used antimalarial and anti-inflammatory drug and its hyperglycemia-controlling effect in diabetic patients is also under investigation. This review provides our perspective of a potential use of combination therapy of PDE5 inhibitor with HCQ to reduce cardiovascular risk factors and myocardial I/R injury in T2D. We previously observed that diabetic mice treated with tadalafil and HCQ had significantly reduced fasting blood glucose and lipid levels, increased plasma insulin and insulin-like growth factor-1 levels, and improved insulin sensitivity, along with smaller myocardial infarct size following I/R. The combination treatment activated Akt/mTOR cellular survival pathway, which was likely responsible for the salutary effects. Therefore, pretreatment with PDE5 inhibitor and HCQ may be a potentially useful therapy not only for controlling T2D but also reducing the rate and severity of COVID-19 infection in the vulnerable population of diabetics.

Chronic inhibition of phosphodiesterase 5 with tadalafil affords cardioprotection in a mouse model of metabolic syndrome: role of nitric oxide.

Patients with metabolic syndrome (MetS) often exhibit generalized endothelial and cardiac dysfunction with decreased nitric oxide (NO) production and/or bioavailability. Since phosphodiesterase 5 (PDE5) inhibitors restore NO signaling, we hypothesized that chronic treatment with long-acting PDE5 inhibitor tadalafil may enhance plasma NO levels and reduce cardiac dysfunction following ischemia/reperfusion (I/R) injury in C57BL/6NCrl-Leprdb-lb/Crl mice with MetS phenotypes. Adult male MetS mice were randomized to receive vehicle solvent or tadalafil (1 mg/kg,i.p.) daily for 28 days and C57BL/6NCrl mice served as healthy wild-type controls. After 28 days, cardiac function was assessed by echocardiography and hearts from a subset of mice were isolated and subjected to 30 min of global ischemia followed by 60 min of reperfusion (I/R) in ex vivo Langendorff mode. Body weight, blood lipids, and glucose levels were elevated in MetS mice as compared with wild-type controls. The dyslipidemia in MetS was ameliorated following tadalafil treatment. Although left ventricular (LV) systolic function was minimally altered in the MetS mice, there was a significant diastolic dysfunction as indicated by reduction in the ratio of peak velocity of early to late filling of the mitral inflow, which was significantly improved by tadalafil treatment. Post-ischemic cardiac function, heart rate, and coronary flow decreased significantly in MetS mice compared to wild-type controls, but preserved by tadalafil treatment. Myocardial infarct size was significantly smaller following I/R, which was associated with higher plasma levels of nitrate and nitrite in the tadalafil-treated MetS mice. In conclusion, tadalafil induces significant cardioprotective effects as shown by improvement of LV diastolic function, lipid profile, and reduced infarct size following I/R. Tadalafil treatment enhanced NO production, which may have contributed to the cardioprotective effects.

Podocyte NLRP3 Inflammasome Activation and Formation by Adipokine Visfatin.

BACKGROUND/AIMS: NLRP3 inflammasome activation has been reported to be an early mechanism responsible for glomerular inflammation and injury in obese mice. However, the precise mechanism of obesity-induced NLRP3 inflammasome activation remains unknown. The present study explored whether adipokine visfatin mediates obesity-induced NLRP3 inflammasome activation and consequent podocyte injury. METHODS: Inflammasome formation and immunofluorescence expressions were quantified by confocal microscopy. Caspase-activity, IL-1ß production and VEGF concentrations were measured by ELISA. RESULTS: Confocal microscopic analysis showed that visfatin treatment increased the colocalization of Nlrp3 with Asc or Nlrp3 with caspase-1 in podocytes indicating the formation of NLRP3 inflammasomes. This visfatin-induced NLRP3 inflammasome formation was abolished by pretreatment of podocytes with Asc siRNA. Correspondingly, visfatin treatment significantly increased the caspase-1 activity and IL-1ß production in podocytes, which was significantly attenuated by Asc siRNA transfection. Further RT-PCR and confocal microscopic analysis demonstrated that visfatin treatment significantly decreased the podocin expression (podocyte damage). Podocytes pretreatment with Asc siRNA or caspase-1 inhibitor, WEHD attenuated this visfatin-induced podocin reduction. Furthermore, Asc siRNA transfection was found to preserve podocyte morphology by maintaining the distinct arrangement of F-actin fibers normally lost in response to visfatin. It also prevented podocyte dysfunction by restoring visfatin-induced suppression of VEGF production and secretion. CONCLUSION: Visfatin induces NLRP3 inflammasome activation in podocytes and thereby resulting in podocyte injury.

High Mobility Group Box 1 Mediates TMAO-Induced Endothelial Dysfunction.

The intestinal microbe-derived metabolite trimethylamine N-oxide (TMAO) is implicated in the pathogenesis of cardiovascular diseases (CVDs). The molecular mechanisms of how TMAO induces atherosclerosis and CVDs' progression are still unclear. In this regard, high-mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to disrupt cell-cell junctions, resulting in vascular endothelial hyper permeability leading to endothelial dysfunction. The present study tested whether TMAO associated endothelial dysfunction results via HMGB1 activation. Biochemical and RT-PCR analysis showed that TMAO increased the HMGB1 expression in a dose-dependent manner in endothelial cells. However, prior treatment with glycyrrhizin, an HMGB1 binder, abolished the TMAO-induced HMGB1 production in endothelial cells. Furthermore, Western blot and immunofluorescent analysis showed significant decrease in the expression of cell-cell junction proteins ZO-2, Occludin, and VE-cadherin in TMAO treated endothelial cells compared with control cells. However, prior treatment with glycyrrhizin attenuated the TMAO-induced cell-cell junction proteins' disruption. TMAO increased toll-like receptor 4 (TLR4) expression in endothelial cells. Inhibition of TLR4 expression by TLR4 siRNA protected the endothelial cells from TMAO associated tight junction protein disruption via HMGB1. In conclusion, our results demonstrate that HMGB1 is one of the important mediators of TMAO-induced endothelial dysfunction.

Characterization and Activation of NLRP3 Inflammasomes in the Renal Medulla in Mice.

BACKGROUND/AIMS: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. METHODS AND RESULTS: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1ß production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. CONCLUSION: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response.

Mammalian target of rapamycin (mTOR) inhibition with rapamycin improves cardiac function in type 2 diabetic mice: potential role of attenuated oxidative stress and altered contractile protein expression.

Elevated mammalian target of rapamycin (mTOR) signaling contributes to the pathogenesis of diabetes, with increased morbidity and mortality, mainly because of cardiovascular complications. Because mTOR inhibition with rapamycin protects against ischemia/reperfusion injury, we hypothesized that rapamycin would prevent cardiac dysfunction associated with type 2 diabetes (T2D). We also investigated the possible mechanisms and novel protein targets involved in rapamycin-induced preservation of cardiac function in T2D mice. Adult male leptin receptor null, homozygous db/db, or wild type mice were treated daily for 28 days with vehicle (5% DMSO) or rapamycin (0.25 mg/kg, intraperitoneally). Cardiac function was monitored by echocardiography, and protein targets were identified by proteomics analysis. Rapamycin treatment significantly reduced body weight, heart weight, plasma glucose, triglyceride, and insulin levels in db/db mice. Fractional shortening was improved by rapamycin treatment in db/db mice. Oxidative stress as measured by glutathione levels and lipid peroxidation was significantly reduced in rapamycin-treated db/db hearts. Rapamycin blocked the enhanced phosphorylation of mTOR and S6, but not AKT in db/db hearts. Proteomic (by two-dimensional gel and mass spectrometry) and Western blot analyses identified significant changes in several cytoskeletal/contractile proteins (myosin light chain MLY2, myosin heavy chain 6, myosin-binding protein C), glucose metabolism proteins (pyruvate dehydrogenase E1, PYGB, Pgm2), and antioxidant proteins (peroxiredoxin 5, ferritin heavy chain 1) following rapamycin treatment in db/db heart. These results show that chronic rapamycin treatment prevents cardiac dysfunction in T2D mice, possibly through attenuation of oxidative stress and alteration of antioxidants and contractile as well as glucose metabolic protein expression.

Chronic inhibition of phosphodiesterase 5 with tadalafil attenuates mitochondrial dysfunction in type 2 diabetic hearts: potential role of NO/SIRT1/PGC-1α signaling.

Enhanced nitric oxide (NO) production is known to activate silent information regulator 1 (SIRT1), which is a histone deacetylase that regulates PGC-1α, a regulator of mitochondrial biogenesis and coactivator of transcription factors impacting energy homeostasis. Since phosphodiesterase-5 inhibitors potentiate NO signaling, we hypothesized that chronic treatment with phosphodiesterase-5 inhibitor tadalafil would activate SIRT1-PGC-1α signaling and protect against metabolic stress-induced mitochondrial dysfunction in diabetic hearts. Diabetic db/db mice (n = 32/group; 40 wk old) were randomized to receive DMSO (10%, 0.2 ml ip) or tadalafil (1 mg/kg ip in 10% DMSO) for 8 wk. Wild-type C57BL mice served as nondiabetic controls. The hearts were excised and homogenized to study SIRT1 activity and downstream protein targets. Mitochondrial function was determined by measuring oxidative phosphorylation (OXPHOS), and reactive oxygen species generation was studied in isolated mitochondria. Tadalafil-treated diabetic mice demonstrated significantly improved left ventricular function, which is associated with increased cardiac SIRT1 activity. Tadalafil also enhanced plasma NO oxidation levels, myocardial SIRT1, PGC-1α expression, and phosphorylation of eNOS, Akt, and AMPK in the diabetic hearts. OXPHOS with the complex I substrate glutamate was decreased by 50% in diabetic hearts compared with the nondiabetic controls. Tadalafil protected OXPHOS with an improved glutamate state 3 respiration rates. The increased reactive oxygen species production from complex I was significantly decreased by tadalafil treatment. In conclusion, chronic treatment with tadalafil activates NO-induced SIRT1-PGC-1α signaling and attenuates mitochondrial dysfunction in type 2 diabetic hearts.

Liver cell death and anemia in Wilson disease involve acid sphingomyelinase and ceramide.

Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

TACE inhibition: a promising therapeutic intervention against AATF-mediated steatohepatitis to hepatocarcinogenesis.

Metabolic dysfunction-associated steatohepatitis-driven hepatocellular carcinoma (MASH-HCC) is a global clinical challenge for which there is a limited understanding of disease pathogenesis and a subsequent lack of therapeutic interventions. We previously identified that tumor necrosis factor-alpha (TNF-α) upregulated apoptosis antagonizing transcription factor (AATF) in MASH. Here, we investigated the effect of TNF-α converting enzyme (TACE) inhibition as a promising targeted therapy against AATF-mediated steatohepatitis to hepatocarcinogenesis. A preclinical murine model that recapitulates human MASH-HCC was used in the study. C57Bl/6 mice were fed with chow diet normal water (CD) or western diet sugar water (WD) along with a low dose of carbon tetrachloride (CCl4; 0.2 µL·g-1, weekly) for 24 weeks. TACE activity, TNF-α levels, and AATF expression were measured. The mice were treated with the TACE inhibitor Marimastat for 12 weeks, followed by analyses of liver injury, fibrosis, inflammation, and oncogenic signaling. In vitro experiments using stable clones of AATF control and AATF knockdown were also conducted. We found that AATF expression was upregulated in WD/CCl4 mice, which developed severe MASH at 12 weeks and advanced fibrosis with HCC at 24 weeks. WD/CCl4 mice showed increased TACE activity with reduced hepatic expression of sirtuin 1 (Sirt1) and tissue inhibitor of metalloproteinase 3 (Timp3). The involvement of the SIRT1/TIMP3/TACE axis was confirmed by the release of TNF-α, which upregulated AATF, a key molecular driver of MASH-HCC. Interestingly, TACE inhibition by Marimastat reduced liver injury, dyslipidemia, AATF expression, and oncogenic signaling, effectively preventing hepatocarcinogenesis. Furthermore, Marimastat inhibited the activation of JNK, ERK1/2, and AKT, which are key regulators of tumorigenesis in WD/CCl4 mice and in AATF control cells, but had no effect on AATF knockdown cells. This study shows that TACE inhibition prevents AATF-mediated inflammation, fibrosis, and oncogenesis in MASH-HCC, offering a potential target for therapeutic intervention.

Contribution of membrane raft redox signalling to visfatin-induced inflammasome activation and podocyte injury.

Recently we have shown that adipokine visfatin-induced NLRP3 inflammasome activation contributes to podocyte injury. However, the molecular mechanisms of how visfatin-induces the Nlrp3 inflammasome activation and podocyte damage is still unknown. The present study tested whether membrane raft (MR) redox signalling pathway plays a central role in visfatin-induced NLRP3 inflammasomes formation and activation in podocytes. Upon visfatin stimulation an aggregation of NADPH oxidase subunits, gp91phox and p47phox was observed in the membrane raft (MR) clusters, forming a MR redox signalling platform in podocytes. The formation of this signalling platform was blocked by prior treatment with MR disruptor MCD or NADPH oxidase inhibitor DPI. In addition, visfatin stimulation significantly increased the colocalization of Nlrp3 with Asc or Nlrp3 with caspase-1, IL-ß production, cell permeability in podocytes compared to control cells. Pretreatment with MCD, DPI, WEHD significantly abolished the visfatin-induced colocalization of NLRP3 with Asc or NLRP3 with caspase-1, IL-1ß production and cell permeability in podocytes. Furthermore, Immunofluorescence analysis demonstrated that visfatin treatment significantly decreased the podocin and nephrin expression (podocyte damage) and prior treatments with DPI, WEHD, MCD attenuated this visfatin-induced podocin and nephrin reduction. In conclusion, our results suggest that visfatin stimulates membrane raft clustering in the membrane of podocytes to form redox signaling platforms by aggregation and activation of NADPH oxidase subunits enhancing O2·- production and leading to NLRP3 inflammasome activation in podocytes and ultimate podocyte injury.

Chronic treatment with long acting phosphodiesterase-5 inhibitor tadalafil alters proteomic changes associated with cytoskeletal rearrangement and redox regulation in Type 2 diabetic hearts.

Diabetic patients are prone to metabolic perturbations that progressively contribute to structural, functional and proteomic alterations in the myocardium. Phosphodiesterase-5 (PDE-5) inhibitors exhibit cardioprotective effects against ischemic/reperfusion injury, however the effects of chronic administration of PDE-5 inhibitors, particularly under diabetic conditions, remain unknown. Hence, the present study was designed to identify novel protein targets related to long-acting PDE-5 inhibitor tadalafil-induced cardioprotection in diabetes. Using two-dimensional differential in-gel electrophoresis with 3 CyDye labeling and MALDI-TOF/TOF tandem mass spectrometry we identified alterations in the expressions of cardiac proteins in diabetic db/db mice treated with tadalafil. Tadalafil reversed the coordinated alterations of cytoskeletal/contractile proteins such as myosin light chain (MLY) 2 and 4, myosin heavy chain α and myosin-binding protein C which contributes to contractile dysfunction. The expression of intermediate filament protein vimentin and extra-cellular matrix proteins like cysteine and glycine rich protein-3 and collagen type VI α were upregulated in db/db mice indicating cardiac remodeling in diabetes. These detrimental proteomic alterations were reflected in cardiac function which were reversed in tadalafil treated mice. Tadalafil also enhanced antioxidant enzyme glutathione S-transferase Kappa-1 (GSKT-1) and downregulated redox regulatory chaperones like heat shock protein 8 (HSPA8), and 75 kD glucose regulatory protein (75GRP). Furthermore, tadalafil treatment significantly attenuated GSSG/GSH ratio and improved the metabolic status of db/db mice. Chronic treatment with tadalafil in db/db mice modulates proteins involved in cytoskeletal rearrangement and redox signaling of the heart, which may explain the beneficial effects of PDE-5 inhibition in diabetes.

GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak.

Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.

Anemia and splenomegaly in cGKI-deficient mice.

To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119(+) erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119(+) cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca(2+) concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.

Targeting glutathione by dimethylfumarate protects against experimental malaria by enhancing erythrocyte cell membrane scrambling.

The balance between GSH-levels and oxidative stress is critical for cell survival. The GSH-levels of erythrocytes are dramatically decreased during infection with Plasmodium spp. We therefore investigated the consequences of targeting GSH for erythrocyte and Plasmodium survival in vitro and in vivo using dimethylfumarate (DMF) at therapeutically established dosage. We first show that noninfected red blood cells (RBC) exposed to DMF undergo changes typical of apoptosis or eryptosis, such as cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine (PS) exposure. DMF did not induce appreciable hemolysis. DMF-triggered PS exposure was mediated by intracellular GSH depletion and reversed by the antioxidative N-acetyl-l-cysteine. DMF treatment controlled intraerythrocyte DNA amplification and in vitro parasitemia of Plasmodium falciparum-infected RBC. In vivo, DMF treatment had no effect on RBC count or GSH levels in noninfected mice. Consistent with its effects on infected RBC, DMF treatment abrogated parasitemia and enhanced the survival of mice infected with Plasmodium berghei from 0% to 60%. In conclusion, DMF sensitizes the erythrocytes to the effect of Plasmodium infection on PS exposure, thus accelerating the clearance of infected erythrocytes. Accordingly, DMF treatment favorably influences the clinical course of malaria. As DMF targets mechanisms within the host cell, it is not likely to generate resistance of the pathogen.

Functional significance of the intermediate conductance Ca2+-activated K+ channel for the short-term survival of injured erythrocytes.

Increased cytosolic Ca(2+) concentrations activate Gardos K(+) channels in human erythrocytes with membrane hyperpolarization, efflux of K(+), Cl⁻, and osmotically obliged H2O resulting in cell shrinkage, a phenomenon referred to as Gardos effect. We tested whether the Gardos effect delays colloid osmotic hemolysis of injured erythrocytes from mice lacking the Ca(2+)-activated K(+) channel K(Ca)3.1. To this end, we applied patch clamp and flow cytometry and determined in vitro as well as in vivo hemolysis. As a result, erythrocytes from K(Ca)3.1-deficient (K(Ca)3.1(-/-)) mice lacked Gardos channel activity and the Gardos effect. Blood parameters, reticulocyte count, or osmotic erythrocyte resistance, however, did not differ between K(Ca)3.1(-/-) mice and their wild-type littermates, suggesting low or absent Gardos channel activity in unstressed erythrocytes. Oxidative stress-induced Ca(2+) entry and phospholipid scrambling were significantly less pronounced in K(Ca)3.1(-/-) than in wild-type erythrocytes. Moreover, in vitro treatment with α-toxin from Staphylococcus aureus, which forms pores in the cellular membrane, resulted in significantly stronger hemolysis of K(Ca)3.1(-/-) than of wild-type erythrocytes. Intravenous injection of α-toxin induced more profound hemolysis in K(Ca)3.1(-/-) than in wild-type mice. Similarly, intra-peritoneal application of the redox-active substance phenylhydrazine, an agent for the induction of hemolytic anemia, was followed by a significantly stronger decrease of hematocrit in K(Ca)3.1(-/-) than in wild-type mice. Finally, malaria infection triggered the activation of K(Ca)3.1 and transient shrinkage of the infected erythrocytes. In conclusion, K(Ca)3.1 channel activity and Gardos effect counteract hemolysis of injured erythrocytes, thus decreasing hemoglobin release into circulating blood.

Non-selective cation channel-mediated Ca2+-entry and activation of Ca2+/calmodulin-dependent kinase II contribute to G2/M cell cycle arrest and survival of irradiated leukemia cells.

Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.

Long-acting phosphodiesterase-5 inhibitor tadalafil attenuates doxorubicin-induced cardiomyopathy without interfering with chemotherapeutic effect.

Doxorubicin (DOX) is one of the most effective anticancer drugs. However, its cardiotoxicity remains a clinical concern that severely restricts its therapeutic usage. We designed this study to investigate whether tadalafil, a long-acting phosphodiesterase-5 (PDE-5) inhibitor, protects against DOX-induced cardiotoxicity. We also sought to delineate the cellular and molecular mechanisms underlying tadalafil-induced cardioprotection. Male CF-1 outbred mice were randomized into three groups (n = 15-24/group) to receive either saline (0.2 ml i.p.), DOX (15 mg/kg, given by a single intraperitoneal injection), or tadalafil (4 mg/kg p.o. daily for 9 days) plus DOX. Left ventricular function was subsequently assessed by transthoracic echocardiography and Millar conductance catheter. Cardiac contractile function was impaired by DOX, and it was significantly improved by cotreatment with tadalafil. Tadalafil attenuated DOX-induced apoptosis and depletion of prosurvival proteins, including Bcl-2 and GATA-4, in myocardium. Cardiac oxidative stress was attenuated and antioxidant capacity was enhanced by tadalafil possibly via up-regulation of mitochondrial superoxide dismutase (MnSOD). Moreover, the tadalafil-treated group demonstrated increased cardiac cGMP level and protein kinase G (PKG) activity. Tadalafil did not interfere with the efficacy of DOX in killing human osteosarcoma cells in vitro or its antitumor effect in vivo in tumor xenograft model. We conclude that tadalafil improved left ventricular function and prevented cardiomyocyte apoptosis in DOX-induced cardiomyopathy through mechanisms involving up-regulation of cGMP, PKG activity, and MnSOD level without interfering with the chemotherapeutic benefits of DOX.

Regulation of erythrocyte survival by AMP-activated protein kinase.

AMP-activated protein kinase (AMPK), an energy-sensing enzyme, counteracts energy depletion by stimulation of energy production and limitation of energy utilization. On energy depletion, erythrocytes undergo suicidal death or eryptosis, triggered by an increase in cytosolic Ca(2+) activity ([Ca(2+)](i)) and characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. The present study explored whether AMPK participates in the regulation of eryptosis. Western blotting and confocal microscopy disclosed AMPK expression in erythrocytes. [Ca(2+)](i) (Fluo3 fluorescence), cell volume (forward scatter), and PS exposure (annexin V binding) were determined by fluorescence-activated cell sorting (FACS) analysis. Glucose removal increased [Ca(2+)](i), decreased cell volume, and increased PS exposure. The AMPK-inhibitor compound C (20 microM) did not significantly modify eryptosis under glucose-replete conditions but significantly augmented the eryptotic effect of glucose withdrawal. An increase in [Ca(2+)](i) by Ca(2+) ionophore ionomycin triggered eryptosis, an effect blunted by the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mM). As compared with erythrocytes from wild-type littermates (ampk(+/+)), erythrocytes from AMPKalpha1-deficient mice (ampk(-/-)) were significantly more susceptible to the eryptotic effect of energy depletion. The ampk(-/-) mice were anemic despite excessive reticulocytosis, and they suffered from severe splenomegaly, again pointing to enhanced erythrocyte turnover. The observations disclose a critical role of AMPK in the survival of circulating erythrocytes.

Simvastatin improves lysosome function via enhancing lysosome biogenesis in endothelial cells.

Nlrp3 inflammasomes were shown to play a critical role in triggering obesity-associated early onsets of cardiovascular complications such as endothelial barrier dysfunction with endothelial hyperpermeability. Statins prevent endothelial dysfunction and decrease cardiovascular risk in patients with obesity and diabetes. However, it remains unclear whether statin treatment for obesity-induced endothelial barrier dysfunction is in part due to the blockade of Nlrp3 inflammasome signaling axis. The results showed that simvastatin, a clinically and widely used statin, prevented free fatty acid-induced endothelial hyperpermeability and disruption of ZO-1 and VE-cadherin junctions in mouse microvascular endothelial cells (MVECs). This protective effect of simvastatin was largely due to improved lysosome function that attenuated lysosome injury-mediated Nlrp3 inflammasome activation and subsequent release of high mobility group box protein-1 (HMGB1). Mechanistically, simvastatin induces autophagy that promotes removal of damaged lysosomes and also promotes lysosome regeneration that preserves lysosome function. Collectively, simvastatin treatment improves lysosome function via enhancing lysosome biogenesis and its autophagic turnover, which may be an important mechanism to suppress Nlrp3 inflammasome activation and prevents endothelial hyperpermeability in obesity.