Nitric oxide augments signaling for contraction in hypoxic pulmonary arterial smooth muscle-Implications for hypoxic pulmonary hypertension.

Introduction: Hypoxic persistent pulmonary hypertension in the newborn (PPHN) is usually treated with oxygen and inhaled nitric oxide (NO), both pulmonary arterial relaxants. But treatment failure with NO occurs in 25% of cases. We previously demonstrated that 72 h exposure to hypoxia, modeling PPHN, sensitized pulmonary artery smooth muscle cells (PASMC) to the contractile agonist thromboxane and inhibited relaxant adenylyl cyclase (AC) activity. Methods: In this study, we examined the effects of sodium nitroprusside (SNP), as NO donor, on the thromboxane-mediated contraction and NO-independent relaxation pathways and on reactive oxygen species (ROS) accumulation in PASMC. In addition, we examined the effect of the peroxynitrite scavenger 5,10,15,20-Tetrakis (4-sulfonatophenyl)porphyrinato Iron (III) (FeTPPS) on these processes. Results: Exposure of PASMC to 72 h hypoxia increased total intracellular ROS compared to normoxic control cells and this was mitigated by treatment of cells with either SNP or FeTPPS. Total protein nitrosylation was increased in hypoxic PASMC compared to controls. Both normoxic and hypoxic cells treated with SNP exhibited increased total protein nitrosylation and intracellular nitrite; this was reduced by treatment with FeTPPS. While cell viability and mitochondrial number were unchanged by hypoxia, mitochondrial activity was decreased compared to controls; addition of FeTPPS did not alter this. Basal and maximal mitochondrial metabolism and ATP turnover were reduced in hypoxic PASMC compared to controls. Hypoxic PASMC had higher basal Ca2+, and a heightened peak Ca2+ response to thromboxane challenge compared to controls. Addition of SNP further elevated the peak Ca2+ response, while addition of FeTPPS brought peak Ca2+ response down to control levels. AC mediated relaxation was impaired in hypoxic PASMC compared to controls but was normalized following treatment with FeTPPS. Addition of SNP inhibited adenylyl cyclase activity in both normoxic and hypoxic PASMC. Moreover, addition of the Ca2+ chelator BAPTA improved AC activity, but the effect was minimal. Discussion: We conclude that NO independently augments contraction and inhibits relaxation pathways in hypoxic PASMC, in part by a mechanism involving nitrogen radical formation and protein nitrosylation. These observations may partially explain impaired effectiveness of NO when treating hypoxic pulmonary hypertension.

Comparing Flaxseed and Perindopril in the Prevention of Doxorubicin and Trastuzumab-Induced Cardiotoxicity in C57Bl/6 Mice.

BACKGROUND: Two anti-cancer agents, doxorubicin (DOX) and trastuzumab (TRZ), are commonly used in the management of breast cancer in women. Despite their efficacy in reducing the morbidity and mortality of individuals with breast cancer, the use of these agents is limited by adverse cardiotoxic side effects. Both the nutraceutical agent flaxseed (FLX) and the pharmaceutical drug perindopril (PER) have been studied individually in the prevention of chemotherapy-mediated cardiac dysfunction. The objective of this study was to determine whether the prophylactic administration of FLX is comparable and/or synergistic with PER in preventing DOX + TRZ-induced cardiotoxicity. METHODS: Over a six-week period, 81 wild-type C57Bl/6 female mice (8-12 weeks old) were randomized to receive regular chow (RC) or 10% FLX-supplemented diets with or without PER (3 mg/kg/week; oral gavage). Starting at week 4, mice were randomized to receive a weekly injection of saline or DOX (8 mg/kg) + TRZ (3 mg/kg). Serial echocardiography was conducted weekly and histological and biochemical analyses were performed at the end of the study. RESULTS: In mice treated with RC + DOX + TRZ, left ventricular ejection (LVEF) decreased from 75 ± 2% at baseline to 37 ± 3% at week 6. However, prophylactic treatment with either FLX, PER, or FLX + PER partially preserved left ventricular systolic function with LVEF values of 61 ± 2%, 62 ± 2%, and 64 ± 2%, respectively. The administration of FLX, PER, or FLX + PER was also partially cardioprotective in preserving cardiomyocyte integrity and attenuating the expression of the inflammatory biomarker NF-κB due to DOX + TRZ administration. CONCLUSION: FLX was equivalent to PER at preventing DOX + TRZ-induced cardiotoxicity in a chronic in vivo murine model.

Mitochondrial autophagy and cell survival is regulated by the circadian Clock gene in cardiac myocytes during ischemic stress.

Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK.Abbreviations: ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATG14: autophagy related 14; ATG7: autophagy related 7; ATP: adenosine triphosphate; BCA: bovine serum albumin; BECN1: beclin 1, autophagy related; bHLH: basic helix- loop-helix; CLOCK: circadian locomotor output cycles kaput; CMV: cytomegalovirus; COQ5: coenzyme Q5 methyltransferase; CQ: chloroquine; CRY1: cryptochrome 1 (photolyase-like); DNM1L/DRP1: dynamin 1-like; EF: ejection fraction; EM: electron microscopy; FS: fractional shortening; GFP: green fluorescent protein; HPX: hypoxia; i.p.: intraperitoneal; I-R: ischemia-reperfusion; LAD: left anterior descending; LVIDd: left ventricular internal diameter diastolic; LVIDs: left ventricular internal diameter systolic; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; MI: myocardial infarction; mPTP: mitochondrial permeability transition pore; NDUFA4: Ndufa4, mitochondrial complex associated; NDUFA8: NADH: ubiquinone oxidoreductase subunit A8; NMX: normoxia; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PER1: period circadian clock 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; qPCR: quantitative real-time PCR; RAB7A: RAB7, member RAS oncogene family; ROS: reactive oxygen species; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TMRM: tetra-methylrhodamine methyl ester perchlorate; WT: wild -type; ZT: zeitgeber time.

Impact of Wnt/ß-catenin signaling on ethanol-induced changes in brain endothelial cell permeability.

Chronic exposure to ethanol is associated with enhanced leakiness in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). As previous studies suggested Wnt/ß-catenin signaling could improve the BBB phenotype of brain endothelial cells, we examined the extent to which Wnt signaling is altered following ethanol exposure, using both a cell culture model of the BBB and mice exposed to ethanol, and the ability of Wnt activation to reverse the permeability effects of ethanol. The human brain endothelial cells, hCMEC/D3, were exposed to ethanol (17-200 mM) for various periods of time (0-96 hr) and Wnt signaling, as well as expression of downstream genes influencing BBB integrity in the cell monolayers were monitored. Determination of Wnt signaling in both brain homogenates and brain microvessels from mice exposed to ethanol was also performed. The effects of ethanol on the permeability of the hCMEC/D3 monolayers were examined using both small molecular weight (sodium fluorescein) and large molecular weight (IRdye 800CW PEG) fluorescent markers. Exposure of hCMEC/D3 to ethanol (50 mM) caused a down-regulation of Wnt/ß-catenin signaling, a reduction of tight junction protein expression and up-regulation of plasmalemma vesicle associated protein (PLVAP). A similar reduction in Wnt/ß-catenin activity in both cortical brain homogenates and isolated cortical cerebral microvessels were observed in mice. Other areas such as cerebellum and striatum displayed as much as 3-6 fold increases in Dkk-1, an endogenous Wnt inhibitor. Ethanol exposure caused significant changes in both sodium fluorescein and IRdye 800CW PEG permeability (2-fold compared to control). The ethanol-induced increases in permeability were attenuated by treatment with known Wnt activators (i.e. LiCl or Wnt3a). Additional screens of CNS active agents with possible Wnt activity indicated fluoxetine could also prevent the permeability effects of ethanol. These studies suggest that ethanol-induced changes in brain microvessel permeability can be reversed through activation of Wnt signaling.

BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation.

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance.Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3ß: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.

Doxorubicin-loaded iron oxide nanoparticles for glioblastoma therapy: a combinational approach for enhanced delivery of nanoparticles.

Although doxorubicin (DOX) is an effective anti-cancer drug with cytotoxicity in a variety of different tumors, its effectiveness in treating glioblastoma multiforme (GBM) is constrained by insufficient penetration across the blood-brain barrier (BBB). In this study, biocompatible magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a carrier of DOX for GBM chemotherapy. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) released DOX within 4 days with the capability of an accelerated release in acidic microenvironments. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) demonstrated an efficient uptake in mouse brain-derived microvessel endothelial, bEnd.3, Madin-Darby canine kidney transfected with multi-drug resistant protein 1 (MDCK-MDR1), and human U251 GBM cells. The DOX-EDT-IONPs could augment DOX's uptake in U251 cells by 2.8-fold and significantly inhibited U251 cell proliferation. Moreover, the DOX-EDT-IONPs were found to be effective in apoptotic-induced GBM cell death (over 90%) within 48 h of treatment. Gene expression studies revealed a significant downregulation of TOP II and Ku70, crucial enzymes for DNA repair and replication, as well as MiR-155 oncogene, concomitant with an upregulation of caspase 3 and tumor suppressors i.e., p53, MEG3 and GAS5, in U251 cells upon treatment with DOX-EDT-IONPs. An in vitro MDCK-MDR1-GBM co-culture model was used to assess the BBB permeability and anti-tumor activity of the DOX-EDT-IONPs and DOX treatments. While DOX-EDT-IONP showed improved permeability of DOX across MDCK-MDR1 monolayers compared to DOX alone, cytotoxicity in U251 cells was similar in both treatment groups. Using a cadherin binding peptide (ADTC5) to transiently open tight junctions, in combination with an external magnetic field, significantly enhanced both DOX-EDT-IONP permeability and cytotoxicity in the MDCK-MDR1-GBM co-culture model. Therefore, the combination of magnetic enhanced convective diffusion and the cadherin binding peptide for transiently opening the BBB tight junctions are expected to enhance the efficacy of GBM chemotherapy using the DOX-EDT-IONPs. In general, the developed approach enables the chemotherapeutic to overcome both BBB and multidrug resistance (MDR) glioma cells while providing site-specific magnetic targeting.

The Cardioprotective Role of Flaxseed in the Prevention of Doxorubicin- and Trastuzumab-Mediated Cardiotoxicity in C57BL/6 Mice.

BACKGROUND: Although the combination of doxorubicin (DOX) and trastuzumab (TRZ) reduces the progression and recurrence of breast cancer, these anticancer drugs are associated with significant cardiotoxic side effects. OBJECTIVE: We investigated whether prophylactic administration of flaxseed (FLX) and its bioactive components, α-linolenic acid (ALA) and secoisolariciresinol diglucoside (SDG), would be cardioprotective against DOX + TRZ-mediated cardiotoxicity in a chronic in vivo female murine model. METHODS: Wild-type C57BL/6 female mice (10-12 wk old) received daily prophylactic treatment with one of the following diets: 1) regular control (RC) semi-purified diet; 2) 10% FLX diet; 3) 4.4% ALA diet; or 4) 0.44% SDG diet for a total of 6 wks. Within each arm, mice received 3 weekly injections of 0.9% saline or a combination of DOX [8 mg/(kg.wk)] and TRZ [3 mg/(kg.wk)] starting at the end of week 3. The main outcome was to evaluate the effects of FLX, ALA, and SDG on cardiovascular remodeling and markers of apoptosis, inflammation, and mitochondrial dysfunction. Significance between measurements was determined using a 4 (diet) × 2 (chemotherapy) × 2 (time) mixed factorial design with repeated measures. RESULTS: In the RC + DOX + TRZ-treated mice at week 6 of the study, the left ventricular ejection fraction (LVEF) decreased by 50% compared with the baseline LVEF (P < 0.05). However, the prophylactic administration of the FLX, ALA, or SDG diet was partially cardioprotective, with mice in these treatment groups showing an ∼68% increase in LVEF compared with the RC + DOX + TRZ-treated group at week 6 (P < 0.05). Although markers of inflammation (nuclear transcription factor κB), apoptosis [poly (ADP-ribose) polymerase-1 and the ratio of BCL2-associated X protein to B-cell lymphoma-extra large], and mitochondrial dysfunction (BCL2-interacting protein 3) were significantly elevated by approximately 2-fold following treatment with RC + DOX + TRZ compared with treatment with RC + saline at week 6, prophylactic administration of FLX, ALA, or SDG partially downregulated these signaling pathways. CONCLUSION: In a chronic in vivo female C57BL/6 mouse model of DOX + TRZ-mediated cardiotoxicity, FLX, ALA, and SDG were partially cardioprotective.

Salinomycin-Loaded Iron Oxide Nanoparticles for Glioblastoma Therapy.

Salinomycin is an antibiotic introduced recently as a new and effective anticancer drug. In this study, magnetic iron oxide nanoparticles (IONPs) were utilized as a drug carrier for salinomycin for potential use in glioblastoma (GBM) chemotherapy. The biocompatible polyethylenimine (PEI)-polyethylene glycol (PEG)-IONPs (PEI-PEG-IONPs) exhibited an efficient uptake in both mouse brain-derived microvessel endothelial (bEnd.3) and human U251 GBM cell lines. The salinomycin (Sali)-loaded PEI-PEG-IONPs (Sali-PEI-PEG-IONPs) released salinomycin over 4 days, with an initial release of 44% ± 3% that increased to 66% ± 5% in acidic pH. The Sali-IONPs inhibited U251 cell proliferation and decreased their viability (by approximately 70% within 48 h), and the nanoparticles were found to be effective in reactive oxygen species-mediated GBM cell death. Gene studies revealed significant activation of caspases in U251 cells upon treatment with Sali-IONPs. Furthermore, the upregulation of tumor suppressors (i.e., p53, Rbl2, Gas5) was observed, while TopII, Ku70, CyclinD1, and Wnt1 were concomitantly downregulated. When examined in an in vitro blood-brain barrier (BBB)-GBM co-culture model, Sali-IONPs had limited penetration (1.0% ± 0.08%) through the bEnd.3 monolayer and resulted in 60% viability of U251 cells. However, hyperosmotic disruption coupled with an applied external magnetic field significantly enhanced the permeability of Sali-IONPs across bEnd.3 monolayers (3.2% ± 0.1%) and reduced the viability of U251 cells to 38%. These findings suggest that Sali-IONPs combined with penetration enhancers, such as hyperosmotic mannitol and external magnetic fields, can potentially provide effective and site-specific magnetic targeting for GBM chemotherapy.

Simvastatin increases temozolomide-induced cell death by targeting the fusion of autophagosomes and lysosomes.

Temozolomide (TMZ) is a chemotherapy agent used to treat Grade IV astrocytoma, also known as glioblastoma (GBM). TMZ treatment causes DNA damage that results in tumor cell apoptosis and increases the survival rate of GBM patients. However, chemoresistance as a result of TMZ-induced autophagy significantly reduces this anticancer effects over time. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate (MEV) cascade. Statins are best known for their cholesterol (CH)-lowering effect. Long-term consumption of statins, prior to and in parallel with other cancer therapeutic approaches, has been reported to increase the survival rate of patients with various forms of cancers. In this study, we investigated the potentiation of TMZ-induced apoptosis by simvastatin (Simva) in human GBM cell lines and patient GBM cells, using cell monolayers and three-dimensional cell culture systems. The incubation of cells with a combination of Simva and TMZ resulted in a significant increase in apoptotic cells compared to cells treated with TMZ alone. Incubation of cells with CH or MEV cascade intermediates failed to compensate the decrease in cell viability induced by the combined Simva and TMZ treatment. Simva treatment inhibited the autophagy flux induced by TMZ by blocking autophago-lysosome formation. Our results suggest that Simva sensitizes GBM cells to TMZ-induced cell death in a MEV cascade-independent manner and identifies the inhibition of autophagosome-lysosome fusion as a promising therapeutic strategy in the treatment of GBM.

Acute upregulation of bone morphogenetic protein-4 regulates endogenous cell response and promotes cell death in spinal cord injury.

Traumatic spinal cord injury (SCI) elicits a cascade of secondary injury mechanisms that induce profound changes in glia and neurons resulting in their activation, injury or cell death. The resultant imbalanced microenvironment of acute SCI also negatively impacts regenerative processes in the injured spinal cord. Thus, it is imperative to uncover endogenous mechanisms that drive these acute injury events. Here, we demonstrate that the active form of bone morphogenetic protein-4 (BMP4) is robustly and transiently upregulated in acute SCI in rats. BMP4 is a key morphogen in neurodevelopment; however, its role in SCI is not fully defined. Thus, we elucidated the ramification of BMP4 upregulation in a preclinical model of compressive/contusive SCI in the rat by employing noggin, an endogenous antagonist of BMP ligands, and LDN193189, an intracellular inhibitor of BMP signaling. In parallel, we studied cell-specific effects of BMP4 on neural precursor cells (NPCs), oligodendrocyte precursor cells (OPCs), neurons and astrocytes in vitro. We demonstrate that activation of BMP4 inhibits differentiation of spinal cord NPCs and OPCs into mature myelin-expressing oligodendrocytes, and acute blockade of BMPs promotes oligodendrogenesis, oligodendrocyte preservation and remyelination after SCI. Importantly, we report for the first time that BMP4 directly induces caspase-3 mediated apoptosis in neurons and oligodendrocytes in vitro, and noggin and LDN193189 remarkably attenuate caspase-3 activation and lipid peroxidation in acute SCI. BMP4 also enhances the production of inhibitory chondroitin sulfate proteoglycans (CSPGs) in activated astrocytes in vitro and after SCI. Interestingly, our work reveals that despite the beneficial effects of BMP inhibition in acute SCI, neither noggin nor LDN193189 treatment resulted in long-term functional recovery. Collectively, our findings suggest a role for BMP4 in regulating acute secondary injury mechanisms following SCI, and a potential target for combinatorial approaches to improve endogenous cell response and remyelination.

Impaired NF-κB signalling underlies cyclophilin D-mediated mitochondrial permeability transition pore opening in doxorubicin cardiomyopathy.

AIMS: The chemotherapy drug doxorubicin (Dox) is commonly used for treating a variety of human cancers; however, it is highly cardiotoxic and induces heart failure. We previously reported that the Bcl-2 mitochondrial death protein Bcl-2/19kDa interaction protein 3 (Bnip3), is critical for provoking mitochondrial perturbations and necrotic cell death in response to Dox; however, the underlying mechanisms had not been elucidated. Herein, we investigated mechanism that drives Bnip3 gene activation and downstream effectors of Bnip3-mediated mitochondrial perturbations and cell death in cardiac myocytes treated with Dox. METHODS AND RESULTS: Nuclear factor-κB (NF-κB) signalling, which transcriptionally silences Bnip3 activation under basal states in cardiac myocytes was dramatically reduced following Dox treatment. This was accompanied by Bnip3 gene activation, mitochondrial injury including calcium influx, permeability transition pore (mPTP) opening, loss of nuclear high mobility group protein 1, reactive oxygen species production, and cell death. Interestingly, impaired NF-κB signalling in cells treated with Dox was accompanied by protein complexes between Bnip3 and cyclophilin D (CypD). Notably, Bnip3-mediated mPTP opening was suppressed by inhibition of CypD-demonstrating that CypD functionally operates downstream of Bnip3. Moreover, restoring IKKß-NF-κB activity in cardiac myocytes treated with Dox suppressed Bnip3 expression, mitochondrial perturbations, and necrotic cell death. CONCLUSIONS: The findings of the present study reveal a novel signalling pathway that functionally couples NF-κB and Dox cardiomyopathy to a mechanism that is mutually dependent upon and obligatorily linked to the transcriptional control of Bnip3. Our findings further demonstrate that mitochondrial injury and necrotic cell death induced by Bnip3 is contingent upon CypD. Hence, maintaining NF-κB signalling may prove beneficial in reducing mitochondrial dysfunction and heart failure in cancer patients undergoing Dox chemotherapy.

Mechanisms of simvastatin myotoxicity: The role of autophagy flux inhibition.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.

Role of renin-angiotensin system antagonists in the prevention of bevacizumab- and sunitinib-mediated cardiac dysfunction.

Although anticancer systemic therapy agents clearly lead to improved survival in patients with cancer, these can come at the cost of serious complications including cardiotoxicity. Two types of targeted systemic therapies currently in use for colorectal cancer (CRC) and renal cell cancer (RCC), respectively, include the vascular endothelial growth factor inhibitor bevacizumab (BVZ) and the tyrosine kinase inhibitor sunitinib (SNT). Despite the beneficial effects of BVZ and SNT in improving clinical outcomes in the settings of CRC and RCC, there is an increased risk of cardiac dysfunction. The aim of the present study was to determine whether prophylactic administration of renin-angiotensin system (RAS) inhibitors would attenuate the cardiotoxic side effects of BVZ or SNT in a chronic in vivo murine model. A total of 194 wild-type C57Bl/6 male mice received: 1) 0.9% saline, 2) BVZ (10 mg·kg-1·wk-1), or 3) SNT (40 mg·kg-1·day-1) for 4 wk. Within each arm, mice received daily prophylactic treatment with hydralazine (0.05 mg/ml), aliskiren (50 mg/kg), perindopril (4 mg/kg), or valsartan (2 mg/kg). Although hydralazine effectively lowered blood pressure in BVZ- or SNT-treated mice, it did not prevent left ventricular systolic dysfunction. Prophylactic administration of aliskiren, perindopril, or valsartan prevented adverse cardiovascular remodeling in mice treated with either BVZ or SNT. The addition of RAS antagonists also downregulated expression of phosphorylated p38 and Bcl-2-like 19-kDa interacting protein 3 in SNT-treated mice. In our chronic in vivo murine model, RAS antagonists partially attenuated the development of BVZ- or SNT-mediated cardiac dysfunction. Future clinical studies are warranted to investigate the cardioprotective effects of prophylactic treatment with RAS inhibitors in the settings of CRC and RCC. NEW & NOTEWORTHY In the evolving field of cardio-oncology, bevacizumab and sunitinib improve clinical outcomes in the settings of metastatic colorectal cancer and renal cell cancer, respectively. These anticancer drugs, however, are associated with an increased risk of cardiotoxicity. The prophylactic administration of renin-angiotensin system antagonists is partially cardioprotective against bevacizumab- and sunitinib-mediated cardiac dysfunction.

Autophagy modulates temozolomide-induced cell death in alveolar Rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells significantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co-treatment with TMZ significantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not significantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is significantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death.

Statins: a new approach to combat temozolomide chemoresistance in glioblastoma.

Patients with glioblastoma multiforme (GBM) have an average life expectancy of approximately 15 months. Recently, statins have emerged as a potential adjuvant cancer therapy due to their ability to inhibit cell proliferation and induce apoptosis in many types of cancer. The exact mechanisms that mediate the inhibitory actions of statins in cancer cells are largely unknown. The purpose of this proceeding paper is to discuss some of the known anticancer effects of statins, while focusing on GBM therapy that includes adjunct therapy of statins with chemotherapeutic agents.

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells.

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFß1) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFß1 can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFß1 treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFß1 treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFß1-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFß1, in the clinical progression of NSCLC.

Steroids Limit Myocardial Edema During Ex Vivo Perfusion of Hearts Donated After Circulatory Death.

BACKGROUND: Normothermic ex vivo heart perfusion (EVHP) has been shown to improve the preservation of hearts donated after circulatory arrest and to facilitate clinical successful transplantation. Steroids are added to the perfusate solution in current clinical EVHP protocols; however, the impact of this approach on donor heart preservation has not been previously investigated. We sought to determine the impact of steroids on the inflammatory response and development of myocardial edema during EVHP. METHODS: Thirteen pigs were anesthetized, mechanical ventilation was discontinued, and a hypoxemic cardiac arrest ensued. A 15-minute warm-ischemic standoff period was observed, and then hearts were resuscitated with a cardioplegic solution. Donor hearts were then perfused ex vivo in a normothermic beating state for 6 hours with 500 mg of methylprednisolone (steroid: n = 5) or without (control: n = 8). RESULTS: The addition of steroids to the perfusate solution reduced the generation of proinflammatory cytokines (interleukin-6, -8, -1ß, and tumor necrosis factor-α) and the development of myocardial edema during EVHP (percentage of weight gain: control = 26% ± 7% versus steroid = 16% ± 10%, p = 0.049). Electron microscopy suggested less endothelial cell edema in the steroid group (injury score: control = 1.8 ± 0.2 versus steroid = 1.2 ± 0.2, p = 0.06), whereas perfusate troponin-I (control = 11.9 ± 1.9 ng/mL versus steroid = 9.5 ± 2.4 ng/mL, p = 0.448) and myocardial function were comparable between the groups. CONCLUSIONS: The addition of methylprednisolone to the perfusion solution minimizes the generation of proinflammatory cytokines and development of myocardial edema during normothermic ex vivo perfusion of hearts donated after circulatory arrest.

Simple, Hackable, Size-Selective, Amine-Functionalized Fe-Oxide Nanoparticles for Biomedical Applications.

A facile one-pot method for synthesizing amine-functionalized nonspherical Fe3O4 nanoparticles in gram-scale quantities is presented using just a single source of iron (iron(II) chloride) and an amine (triethylamine). The amine not only transforms iron salt to Fe3O4, but also directs the morphology of the nanoparticles along with the temperature of the reaction and functionalizes them, making the synthesis very economical. By modifying the surface further, these nanoparticles promise to offer useful biomedical applications. For example, after biocide coating, the particles are found to be 100% effective in deactivating methicillin-resistant Staphylococcus aureus (MRSA) bacteria in 2 h. Cellular-uptake studies using biocompatible EDTA-Na3 (N-(trimethoxysilyl-propyl)ethylenediaminetriacetate, trisodium salt)-coated nanoparticles in human glioblastoma U-251 cells show that the majority of the particles are internalized by the cells in the presence of a small dc-magnetic field, making these particles a potential candidate as drug carriers for magnetic field-targeted delivery and hyperthermia.

Neuregulin-1 promotes remyelination and fosters a pro-regenerative inflammatory response in focal demyelinating lesions of the spinal cord.

Oligodendroglial cell death and demyelination are hallmarks of neurotrauma and multiple sclerosis that cause axonal damage and functional impairments. Remyelination remains a challenge as the ability of endogenous precursor cells for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating conditions. Here, in a rat model of lysolecithin lysophosphatidyl-choline (LPC)-induced focal demyelination, we report that Neuregulin-1 (Nrg-1), an important factor for oligodendrocytes and myelination, is dysregulated in demyelinating lesions and its bio-availability can promote oligodendrogenesis and remyelination. We delivered recombinant human Nrg-1ß1 (rhNrg-1ß1) intraspinally in the vicinity of LPC demyelinating lesion in a sustained manner using poly lactic-co-glycolic acid microcarriers. Availability of Nrg-1 promoted generation and maturation of new oligodendrocytes, and accelerated endogenous remyelination by both oligodendrocyte and Schwann cell populations in demyelinating foci. Importantly, Nrg-1 enhanced myelin thickness in newly remyelinated spinal cord axons. Our complementary in vitro studies also provided direct evidence that Nrg-1 significantly promotes maturation of new oligodendrocytes and facilitates their transition to a myelinating phenotype. Nrg-1 therapy remarkably attenuated the upregulated expression chondroitin sulfate proteoglycans (CSPGs) specific glycosaminoglycans in the extracellular matrix of demyelinating foci and promoted interleukin-10 (IL-10) production by immune cells. CSPGs and IL-10 are known to negatively and positively regulate remyelination, respectively. We found that Nrg-1 effects are mediated through ErbB2 and ErbB4 receptor activation. Our work provides novel evidence that dysregulated levels of Nrg-1 in demyelinating lesions of the spinal cord pose a challenge to endogenous remyelination, and appear to be an underlying cause of myelin thinning in newly remyelinated axons.

Impact of Reperfusion Calcium and pH on the Resuscitation of Hearts Donated After Circulatory Death.

BACKGROUND: Hearts donated after circulatory death may represent an additional donor source. The influx of sodium and calcium ions across the sarcolemma play a central role in the pathogenesis of ischemia-reperfusion injury; however, this process may be inhibited if the initial reperfusion solution is rendered hypocalcemic and acidic. We sought to determine the calcium concentration and pH of the initial reperfusion solution that yielded optimal functional recovery of hearts donated after circulatory death during ex vivo heart perfusion. METHODS: Pigs were anesthetized, mechanical ventilation was discontinued, and a 15-minute standoff period was observed after circulatory arrest. Hearts were reperfused with a normothermic cardioplegia of varying calcium concentrations (part 1 [50 µmol/L, n = 4; 220 µmol/L, n = 9; 500 µmol/L, n = 4; and 1,250 µmol/L, n = 5]) and pH (part 2 [7.9, n = 5; 7.4, n = 9; 6.9, n = 8; and 6.4, n = 6]). Myocardial function was then assessed in a physiologic working model 1 hour after initiation of normothermic ex vivo heart perfusion. RESULTS: The calcium concentration and pH of the cardioplegic solution affected the development of myocardial edema (part 1: 50 µmol/L = 5.8% ± 0.9%; 220 µmol/L = 4.3% ± 0.4%; 500 µmol/L = 7.0% ± 0.6%; and 1,250 µmol/L = 6.6% ± 0.8% weight gain, p = 0.015; part 2: 7.9 = 3.6% ± 0.4%, 7.4 = 4.3% ± 0.4%, 6.9 = 3.7% ± 0.6%, and 6.4 = 6.4% ± 1.3% weight gain, p = 0.056) and the recovery of myocardial function (cardiac index part 1: 50 µmol/L = 2.6 ± 0.6; 220 µmol/L = 6.0 ± 0.8; 500 µmol/L = 2.3 ± 0.5; and 1,250 µmol/L = 1.9 ± 0.6 mL · m-1 · g-1, p < 0.001; part 2: 7.9 = 1.5 ± 0.7; 7.4 = 6.0 ± 0.8; 6.9 = 8.4 ± 1.8; and 6.4 = 3.1 ± 0.8 mL · m-1 · g-1, p = 0.003) during ex vivo heart perfusion. CONCLUSIONS: Initial reperfusion of hearts donated after circulatory death with a hypocalcemic and moderately acidic cardioplegia minimizes edema and optimizes functional recovery during subsequent ex vivo heart perfusion.