An ERK5-PFKFB3 axis regulates glycolysis and represents a therapeutic vulnerability in pediatric diffuse midline glioma.

Metabolic reprogramming in pediatric diffuse midline glioma is driven by gene expression changes induced by the hallmark histone mutation H3K27M, which results in aberrantly permissive activation of oncogenic signaling pathways. Previous studies of diffuse midline glioma with altered H3K27 (DMG-H3K27a) have shown that the RAS pathway, specifically through its downstream kinase, extracellular-signal-related kinase 5 (ERK5), is critical for tumor growth. Further downstream effectors of ERK5 and their role in DMG-H3K27a metabolic reprogramming have not been explored. We establish that ERK5 is a critical regulator of cell proliferation and glycolysis in DMG-H3K27a. We demonstrate that ERK5 mediates glycolysis through activation of transcription factor MEF2A, which subsequently modulates expression of glycolytic enzyme PFKFB3. We show that in vitro and mouse models of DMG-H3K27a are sensitive to the loss of PFKFB3. Multi-targeted drug therapy against the ERK5-PFKFB3 axis, such as with small-molecule inhibitors, may represent a promising therapeutic approach in patients with pediatric diffuse midline glioma.

Lysosomal mitochondrial interaction promotes tumor growth in squamous cell carcinoma of the head and neck.

Tumor growth and proliferation are regulated by numerous mechanisms. Communication between intracellular organelles has recently been shown to regulate cellular proliferation and fitness. The way lysosomes and mitochondria communicate with each other (lysosomal/mitochondrial interaction) is emerging as a major determinant of tumor proliferation and growth. About 30% of squamous carcinomas (including squamous cell carcinoma of the head and neck, SCCHN) overexpress TMEM16A, a calcium-activated chloride channel, which promotes cellular growth and negatively correlates with patient survival. TMEM16A has recently been shown to drive lysosomal biogenesis, but its impact on mitochondrial function is unclear. Here, we show that (1) patients with high TMEM16A SCCHN display increased mitochondrial content specifically complex I; (2) In vitro and in vivo models uniquely depend on mitochondrial complex I activity for growth and survival; (3) ß-catenin/NRF2 signaling is a critical linchpin that drives mitochondrial biogenesis, and (4) mitochondrial complex I and lysosomal function are codependent for proliferation. Taken together, our data demonstrate that LMI drives tumor proliferation and facilitates a functional interaction between lysosomes and mitochondria. Therefore, inhibition of LMI may serve as a therapeutic strategy for patients with SCCHN.

Release of hepatic xanthine oxidase (XO) to the circulation is protective in intravascular hemolytic crisis.

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 µmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

Mitochondria-containing extracellular vesicles (EV) reduce mouse brain infarct sizes and EV/HSP27 protect ischemic brain endothelial cultures.

Ischemic stroke causes brain endothelial cell (BEC) death and damages tight junction integrity of the blood-brain barrier (BBB). We harnessed the innate mitochondrial load of BEC-derived extracellular vesicles (EVs) and utilized mixtures of EV/exogenous 27 kDa heat shock protein (HSP27) as a one-two punch strategy to increase BEC survival (via EV mitochondria) and preserve their tight junction integrity (via HSP27 effects). We demonstrated that the medium-to-large (m/lEV) but not small EVs (sEV) transferred their mitochondrial load, that subsequently colocalized with the mitochondrial network of the recipient primary human BECs. Recipient BECs treated with m/lEVs showed increased relative ATP levels and mitochondrial function. To determine if the m/lEV-meditated increase in recipient BEC ATP levels was associated with m/lEV mitochondria, we isolated m/lEVs from donor BECs pre-treated with oligomycin A (OGM, mitochondria electron transport complex V inhibitor), referred to as OGM-m/lEVs. BECs treated with naïve m/lEVs showed a significant increase in ATP levels compared to untreated OGD cells, OGM-m/lEVs treated BECs showed a loss of ATP levels suggesting that the m/lEV-mediated increase in ATP levels is likely a function of their innate mitochondrial load. In contrast, sEV-mediated ATP increases were not affected by inhibition of mitochondrial function in the donor BECs. Intravenously administered m/lEVs showed a reduction in brain infarct sizes compared to vehicle-injected mice in a mouse middle cerebral artery occlusion model of ischemic stroke. We formulated binary mixtures of human recombinant HSP27 protein with EVs: EV/HSP27 and ternary mixtures of HSP27 and EVs with a cationic polymer, poly (ethylene glycol)-b-poly (diethyltriamine): (PEG-DET/HSP27)/EV. (PEG-DET/HSP27)/EV and EV/HSP27 mixtures decreased the paracellular permeability of small and large molecular mass fluorescent tracers in oxygen glucose-deprived primary human BECs. This one-two punch approach to increase BEC metabolic function and tight junction integrity may be a promising strategy for BBB protection and prevention of long-term neurological dysfunction post-ischemic stroke.

Aging-Associated Changes in Oxidative Stress Negatively Impacts the Urinary Bladder Urothelium.

PURPOSE: Lower urinary tract symptoms are known to significantly increase with age, negatively impacting quality of life and self-reliance. The urothelium fulfills crucial tasks, serving as a barrier protecting the underlying bladder tissue from the harsh chemical composition of urine, and exhibits signaling properties via the release of mediators within the bladder wall that affect bladder functioning. Aging is associated with detrimental changes in cellular health, in part by increasing oxidative stress in the bladder mucosa, and more specifically the urothelium. This, in turn, may impact urothelial mitochondrial health and bioenergetics. METHODS: We collected mucosal tissue samples from both young (3-4 months old) and aged (25-30 months old) rats. Tissue was evaluated for p21-Arc, nitrotyrosine, and cytochrome C expression by western immunoblotting. Urothelial cells were cultured for single-cell imaging to analyze basal levels of reactive oxygen species and the mitochondrial membrane potential. Mitochondrial bioenergetics and cellular respiration were investigated by the Seahorse assay, and measurements of adenosine triphosphate release were made using the luciferin-luciferase assay. RESULTS: Aging was associated with a significant increase in biomarkers of cellular senescence, oxidative stress, and basal levels of reactive oxygen species. The mitochondrial membrane potential was significantly lower in urothelial cell cultures from aged animals, and cultures from aged animals showed a significant decrease in mitochondrial bioenergetics. CONCLUSION: Aging-related increases in oxidative stress and excessive reactive oxygen species may be contributing factors underlying lower urinary tract symptoms in older adults. The mechanisms outlined in this study could be utilized to identify novel pharmaceutical targets to improve aging-associated bladder dysfunction.

Dysregulation of Lipid and Glucose Homeostasis in Hepatocyte-Specific SLC25A34 Knockout Mice.

Nonalcoholic fatty liver disease (NAFLD) is an epidemic affecting 30% of the US population. It is characterized by insulin resistance, and by defective lipid metabolism and mitochondrial dysfunction in the liver. SLC25A34 is a major repressive target of miR-122, a miR that has a central role in NAFLD and liver cancer. However, little is known about the function of SLC25A34. To investigate SLC25A34 in vitro, mitochondrial respiration and bioenergetics were examined using hepatocytes depleted of Slc25a34 or overexpressing Slc25a34. To test the function of SLC25A34 in vivo, a hepatocyte-specific knockout mouse was generated, and loss of SLC25A34 was assessed in mice maintained on a chow diet and a fast-food diet (FFD), a model for NAFLD. Hepatocytes depleted of Slc25a34 displayed increased mitochondrial biogenesis, lipid synthesis, and ADP/ATP ratio; Slc25a34 overexpression had the opposite effect. In the knockout model on chow diet, SLC25A34 loss modestly affected liver function (altered glucose metabolism was the most pronounced defect). RNA-sequencing revealed changes in metabolic processes, especially fatty acid metabolism. After 2 months on FFD, knockouts had a more severe phenotype, with increased lipid content and impaired glucose tolerance, which was attenuated after longer FFD feeding (6 months). This work thus presents a novel model for studying SLC25A34 in vivo in which SLC25A34 plays a role in mitochondrial respiration and bioenergetics during NAFLD.

Layer-specific Nos3 expression and genotypic distribution in bicuspid aortic valve aortopathy.

OBJECTIVES: We hypothesized that expression and activity of nitric oxide synthase-3 enzyme (Nos3) in bicuspid aortic valve (BAV) aortopathy are related to tissue layer and Nos3 genotype. METHODS: Gene expression of Nos3 and platelet and endothelial cell adhesion molecule-1 (Pecam1) and NOS activity were measured in intima-containing media and adventitial specimens of ascending aortic tissue. The presence of 2 Nos3 single-nucleotide polymorphisms (SNPs; -786T/C and 894G/T) was determined for non-aneurysmal (NA) and aneurysmal patients with BAV (n = 40, 89, respectively); patients with tricuspid aortic valve (TAV) and aneurysm (n = 151); and NA patients with TAV (n = 100). RESULTS: Elevated Nos3 relative to Pecam1 and reduced Pecam1 relative to a housekeeping gene were observed within intima-containing aortic specimens from BAV patients when compared with TAV patients. Lower Nos3 in the adventitia of aneurysmal specimens was noted when compared with specimens of NA aorta, independent of valve morphology. NOS activity was similar among cohorts in media/intima and decreased in the diseased adventitia, relative to control patients. Aneurysmal BAV patients exhibited an under-representation of the wild-type genotype for -786 SNP. No differences in genotype distribution were noted for 894 SNP. Primary intimal endothelial cells from patients with at least 1 C allele at -786 SNP exhibited lower Nos3 when compared with wild-type cells. CONCLUSIONS: These findings of differential Nos3 in media/intima versus adventitia depending on valve morphology or aneurysm reveal new information regarding aneurysmal pathophysiology and support our ongoing assertion that there are distinct mechanisms giving rise to ascending aortopathy in BAV and TAV patients.

Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

Metabolic Syndrome Mediates ROS-miR-193b-NFYA-Dependent Downregulation of Soluble Guanylate Cyclase and Contributes to Exercise-Induced Pulmonary Hypertension in Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Many patients with heart failure with preserved ejection fraction have metabolic syndrome and develop exercise-induced pulmonary hypertension (EIPH). Increases in pulmonary vascular resistance in patients with heart failure with preserved ejection fraction portend a poor prognosis; this phenotype is referred to as combined precapillary and postcapillary pulmonary hypertension (CpcPH). Therapeutic trials for EIPH and CpcPH have been disappointing, suggesting the need for strategies that target upstream mechanisms of disease. This work reports novel rat EIPH models and mechanisms of pulmonary vascular dysfunction centered around the transcriptional repression of the soluble guanylate cyclase (sGC) enzyme in pulmonary artery (PA) smooth muscle cells. METHODS: We used obese ZSF-1 leptin-receptor knockout rats (heart failure with preserved ejection fraction model), obese ZSF-1 rats treated with SU5416 to stimulate resting pulmonary hypertension (obese+sugen, CpcPH model), and lean ZSF-1 rats (controls). Right and left ventricular hemodynamics were evaluated using implanted catheters during treadmill exercise. PA function was evaluated with magnetic resonance imaging and myography. Overexpression of nuclear factor Y α subunit (NFYA), a transcriptional enhancer of sGC ß1 subunit (sGCß1), was performed by PA delivery of adeno-associated virus 6. Treatment groups received the SGLT2 inhibitor empagliflozin in drinking water. PA smooth muscle cells from rats and humans were cultured with palmitic acid, glucose, and insulin to induce metabolic stress. RESULTS: Obese rats showed normal resting right ventricular systolic pressures, which significantly increased during exercise, modeling EIPH. Obese+sugen rats showed anatomic PA remodeling and developed elevated right ventricular systolic pressure at rest, which was exacerbated with exercise, modeling CpcPH. Myography and magnetic resonance imaging during dobutamine challenge revealed PA functional impairment of both obese groups. PAs of obese rats produced reactive oxygen species and decreased sGCß1 expression. Mechanistically, cultured PA smooth muscle cells from obese rats and humans with diabetes or treated with palmitic acid, glucose, and insulin showed increased mitochondrial reactive oxygen species, which enhanced miR-193b-dependent RNA degradation of nuclear factor Y α subunit (NFYA), resulting in decreased sGCß1-cGMP signaling. Forced NYFA expression by adeno-associated virus 6 delivery increased sGCß1 levels and improved exercise pulmonary hypertension in obese+sugen rats. Treatment of obese+sugen rats with empagliflozin improved metabolic syndrome, reduced mitochondrial reactive oxygen species and miR-193b levels, restored NFYA/sGC activity, and prevented EIPH. CONCLUSIONS: In heart failure with preserved ejection fraction and CpcPH models, metabolic syndrome contributes to pulmonary vascular dysfunction and EIPH through enhanced reactive oxygen species and miR-193b expression, which downregulates NFYA-dependent sGCß1 expression. Adeno-associated virus-mediated NFYA overexpression and SGLT2 inhibition restore NFYA-sGCß1-cGMP signaling and ameliorate EIPH.

Myoglobin promotes nitrite-dependent mitochondrial S-nitrosation to mediate cytoprotection after hypoxia/reoxygenation.

It is well established that myoglobin supports mitochondrial respiration through the storage and transport of oxygen as well as through the scavenging of nitric oxide. However, during ischemia/reperfusion (I/R), myoglobin and mitochondria both propagate myocardial injury through the production of oxidants. Nitrite, an endogenous signaling molecule and dietary constituent, mediates potent cardioprotection after I/R and this effect relies on its interaction with both myoglobin and mitochondria. While independent mechanistic studies have demonstrated that nitrite-mediated cardioprotection requires the presence of myoglobin and the post-translational S-nitrosation of critical cysteine residues on mitochondrial complex I, it is unclear whether myoglobin directly catalyzes the S-nitrosation of complex I or whether mitochondrial-dependent nitrite reductase activity contributes to S-nitrosation. Herein, using purified myoglobin and isolated mitochondria, we characterize and directly compare the nitrite reductase activities of mitochondria and myoglobin and assess their contribution to mitochondrial S-nitrosation. We demonstrate that myoglobin is a significantly more efficient nitrite reductase than isolated mitochondria. Further, deoxygenated myoglobin catalyzes the nitrite-dependent S-nitrosation of mitochondrial proteins. This reaction is enhanced in the presence of oxidized (Fe3+) myoglobin and not significantly affected by inhibitors of mitochondrial respiration. Using a Chinese Hamster Ovary cell model stably transfected with human myoglobin, we show that both myoglobin and mitochondrial complex I expression are required for nitrite-dependent attenuation of cell death after anoxia/reoxygenation. These data expand the understanding of myoglobin's role both as a nitrite reductase to a mediator of S-nitrosation and as a regulator of mitochondrial function, and have implications for nitrite-mediated cardioprotection after I/R.

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection.

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.

Toll interacting protein protects bronchial epithelial cells from bleomycin-induced apoptosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by altered epithelial cell phenotypes, which are associated with myofibroblast accumulation in the lung. Atypical alveolar epithelial cells in IPF express molecular markers of airway epithelium. Polymorphisms within and around Toll interacting protein (TOLLIP) are associated with the susceptibility to IPF and mortality. However, the functional role of TOLLIP in IPF is unknown. Using lung tissues from IPF and control subjects, we showed that expression of TOLLIP gene in the lung parenchyma is globally lower in IPF compared to controls. Lung cells expressing significant levels of TOLLIP include macrophages, alveolar type II, and basal cells. TOLLIP protein expression is lower in the parenchyma of IPF lungs but is expressed in the atypical epithelial cells of the distal fibrotic regions. Using overexpression and silencing approaches, we demonstrate that TOLLIP protects cells from bleomycin-induced apoptosis using primary bronchial epithelial cells and BEAS-2B cells. The protective effects are mediated by reducing mitochondrial reactive oxygen species (ROS) levels and upregulating autophagy. Therefore, global downregulation of the TOLLIP gene in IPF lungs may predispose injured lung epithelial cells to apoptosis and to the development of IPF.

Activation of AMP-activated protein kinase during sepsis/inflammation improves survival by preserving cellular metabolic fitness.

The purpose was to determine the role of AMPK activation in the renal metabolic response to sepsis, the development of sepsis-induced acute kidney injury (AKI) and on survival. In a prospective experimental study, 167 10- to 12-week-old C57BL/6 mice underwent cecal ligation and puncture (CLP) and human proximal tubule epithelial cells (TEC; HK2) were exposed to inflammatory mix (IM), a combination of lipopolysaccharide (LPS) and high mobility group box 1 (HMGB1). Renal/TEC metabolic fitness was assessed by monitoring the expression of drivers of oxidative phosphorylation (OXPHOS), the rates of utilization of OXPHOS/glycolysis in response to metabolic stress, and mitochondrial function by measuring O2 consumption rates (OCR) and the membrane potential (Δψm ). Sepsis/IM resulted in AKI, increased mortality, and in renal AMPK activation 6-24 hours after CLP/IM. Pharmacologic activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin during sepsis improved the survival, while AMPK inhibition with Compound C increased mortality, impaired mitochondrial respiration, decreased OCR, and disrupted TEC metabolic fitness. AMPK-driven protection was associated with increased Sirt 3 expression and restoration of metabolic fitness. Renal AMPK activation in response to sepsis/IM is an adaptive mechanism that protects TEC, organs, and the host by preserving mitochondrial function and metabolic fitness likely through Sirt3 signaling.

Impact of triptolide during ex vivo lung perfusion on grafts after transplantation in a rat model.

OBJECTIVE: Ex vivo lung perfusion creates a proinflammatory environment leading to deterioration in graft quality that may contribute to post-transplant graft dysfunction. Triptolide has been shown to have a therapeutic potential in various disease states because of its anti-inflammatory properties. On this basis, we investigated the impact of triptolide on graft preservation during ex vivo lung perfusion and associated post-transplant outcomes in a rat transplant model. METHODS: We performed rat normothermic ex vivo lung perfusion with acellular Steen solution containing 100 nM triptolide for 4 hours and compared the data with untreated lungs. Orthotopic single lung transplantation after ex vivo lung perfusion was performed. RESULTS: Physiologic and functional parameters of lung grafts on ex vivo lung perfusion with triptolide were better than those without treatment. Graft glucose consumption was significantly attenuated on ex vivo lung perfusion with triptolide via inhibition of hypoxia signaling resulting in improved mitochondrial function and reduced oxidative stress. Also, intragraft inflammation was markedly lower in triptolide-treated lungs because of inhibition of nuclear factor-κB signaling. Furthermore, post-transplant graft function and inflammatory events were significantly improved in the triptolide group compared with the untreated group. CONCLUSIONS: Treatment of lung grafts with triptolide during ex vivo lung perfusion may serve to enhance graft preservation and improve graft protection resulting in better post-transplant outcomes.

Nitrite attenuates mitochondrial impairment and vascular permeability induced by ischemia-reperfusion injury in the lung.

Primary graft dysfunction (PGD) is directly related to ischemia-reperfusion (I/R) injury and a major obstacle in lung transplantation (LTx). Nitrite (NO2-), which is reduced in vivo to form nitric oxide (NO), has recently emerged as an intrinsic signaling molecule with a prominent role in cytoprotection against I/R injury. Using a murine model, we provide the evidence that nitrite mitigated I/R-induced injury by diminishing infiltration of immune cells in the alveolar space, reducing pulmonary edema, and improving pulmonary function. Ultrastructural studies support severe mitochondrial impairment in the lung undergoing I/R injury, which was significantly protected by nitrite treatment. Nitrite also abrogated the increased pulmonary vascular permeability caused by I/R. In vitro, hypoxia-reoxygenation (H/R) exacerbated cell death in lung epithelial and microvascular endothelial cells. This contributed to mitochondrial dysfunction as characterized by diminished complex I activity and mitochondrial membrane potential but increased mitochondrial reactive oxygen species (mtROS). Pretreatment of cells with nitrite robustly attenuated mtROS production through modulation of complex I activity. These findings illustrate a potential novel mechanism in which nitrite protects the lung against I/R injury by regulating mitochondrial bioenergetics and vascular permeability.

RNA-seq reveals novel mechanistic targets of withaferin A in prostate cancer cells.

Withaferin A (WA) is a promising phytochemical exhibiting in vitro and in vivo anticancer activities against prostate and other cancers, but the mechanism of its action is not fully understood. In this study, we performed RNA-seq analysis using 22Rv1 human prostate cancer cell line to identify mechanistic targets of WA. Kyoto Encyclopedia of Genes and Genomes pathway analysis of the differentially expressed genes showed most significant enrichment of genes associated with metabolism. These results were validated using LNCaP and 22Rv1 human prostate cancer cells and Hi-Myc transgenic mice as models. The intracellular levels of acetyl-CoA, total free fatty acids and neutral lipids were decreased significantly following WA treatment in both cells, which was accompanied by downregulation of mRNA (confirmed by quantitative reverse transcription-polymerase chain reaction) and protein levels of key fatty acid synthesis enzymes, including ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A. Ectopic expression of c-Myc, but not constitutively active Akt, conferred a marked protection against WA-mediated suppression of acetyl-CoA carboxylase 1 and fatty acid synthase protein expression, and clonogenic cell survival. WA was a superior inhibitor of cell proliferation and fatty acid synthesis in comparison with known modulators of fatty acid metabolism including cerulenin and etomoxir. Intraperitoneal WA administration to Hi-Myc transgenic mice (0.1 mg/mouse, three times/week for 5 weeks) also resulted in a significant decrease in circulating levels of total free fatty acids and phospholipids, and expression of ATP citrate lyase, acetyl-CoA carboxylase 1, fatty acid synthase and carnitine palmitoyltransferase 1A proteins in the prostate in vivo.

Platelet Extracellular Vesicles Drive Inflammasome-IL-1ß-Dependent Lung Injury in Sickle Cell Disease.

Rationale: Intraerythrocytic polymerization of Hb S promotes hemolysis and vasoocclusive events in the microvasculature of patients with sickle cell disease (SCD). Although platelet-neutrophil aggregate-dependent vasoocclusion is known to occur in the lung and contribute to acute chest syndrome, the etiological mechanisms that trigger acute chest syndrome are largely unknown.Objectives: To identify the innate immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vasoocclusion and injury in SCD.Methods:In vivo imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system was performed. SCD mice were systemically challenged with nanogram quantities of LPS to trigger lung vasoocclusion.Measurements and Main Results: Platelet-inflammasome activation led to generation of IL-1ß and caspase-1-carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation, and platelet-neutrophil aggregation were enhanced by the presence of LPS at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1ß pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro.Conclusions: These results are the first to identify that platelet-inflammasome-dependent shedding of IL-1ß and caspase-1-carrying platelet EVs promote lung vasoocclusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome-dependent innate immune pathway to prevent acute chest syndrome.

Bioenergetics and translational metabolism: implications for genetics, physiology and precision medicine.

It is now becoming clear that human metabolism is extremely plastic and varies substantially between healthy individuals. Understanding the biochemistry that underlies this physiology will enable personalized clinical interventions related to metabolism. Mitochondrial quality control and the detailed mechanisms of mitochondrial energy generation are central to understanding susceptibility to pathologies associated with aging including cancer, cardiac and neurodegenerative diseases. A precision medicine approach is also needed to evaluate the impact of exercise or caloric restriction on health. In this review, we discuss how technical advances in assessing mitochondrial genetics, cellular bioenergetics and metabolomics offer new insights into developing metabolism-based clinical tests and metabolotherapies. We discuss informatics approaches, which can define the bioenergetic-metabolite interactome and how this can help define healthy energetics. We propose that a personalized medicine approach that integrates metabolism and bioenergetics with physiologic parameters is central for understanding the pathophysiology of diseases with a metabolic etiology. New approaches that measure energetics and metabolomics from cells isolated from human blood or tissues can be of diagnostic and prognostic value to precision medicine. This is particularly significant with the development of new metabolotherapies, such as mitochondrial transplantation, which could help treat complex metabolic diseases.

Neutrophil Extracellular Traps Drive Mitochondrial Homeostasis in Tumors to Augment Growth.

Neutrophil infiltration and neutrophil extracellular traps (NET) in solid cancers are associated with poorer prognosis, but the mechanisms are incompletely understood. We hypothesized that NETs enhance mitochondrial function in tumor cells, providing extra energy for accelerated growth. Metastatic colorectal cancer tissue showed increased intratumoral NETs and supranormal preoperative serum MPO-DNA, a NET marker. Higher MPO-DNA correlated with shorter survival. In mice, subcutaneous tumor implants and hepatic metastases grew slowly in PAD4-KO mice, genetically incapable of NETosis. In parallel experiments, human cancer cell lines grew slower in nu/nu mice treated with DNAse, which disassembles NETs. PAD4-KO tumors manifested decreased proliferation, increased apoptosis, and increased evidence of oxidative stress. PAD4-KO tumors had decreased mitochondrial density, mitochondrial DNA, a lesser degree of ATP production, along with significantly decreased mitochondrial biogenesis proteins PGC1α, TFAM, and NRF-1. In vitro, cancer cells treated with NETs upregulated mitochondrial biogenesis-associated genes, increased mitochondrial density, increased ATP production, enhanced the percentage of cancer cells with reduced mitochondrial membrane potential, and increased the oxygen consumption rate. Furthermore, NETs increased cancer cells' expression of fission and fusion-associated proteins, DRP-1 and MFN-2, and mitophagy-linked proteins, PINK1 and Parkin. All of which were decreased in PAD4-KO tumors. Mechanistically, neutrophil elastase released from NETs activated TLR4 on cancer cells, leading to PGC1α upregulation, increased mitochondrial biogenesis, and accelerated growth. Taken together, NETs can directly alter the metabolic programming of cancer cells to increase tumor growth. NETs represent a promising therapeutic target to halt cancer progression. SIGNIFICANCE: Neutrophils through the release of NETs facilitate the growth of stressed cancer cells by altering their bioenergetics, the inhibition of which induces cell death.

Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane.

Inhibition of metabolic re-programming represents an attractive approach for prevention of prostate cancer. Studies have implicated increased synthesis of fatty acids or glycolysis in pathogenesis of human prostate cancers. We have shown previously that prostate cancer prevention by sulforaphane (SFN) in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model is associated with inhibition of fatty acid metabolism. This study utilized human prostate cancer cell lines (LNCaP, 22Rv1 and PC-3), two different transgenic mouse models (TRAMP and Hi-Myc) and plasma specimens from a clinical study to explore the glycolysis inhibition potential of SFN. We found that SFN treatment: (i) decreased real-time extracellular acidification rate in LNCaP, but not in PC-3 cell line; (ii) significantly downregulated expression of hexokinase II (HKII), pyruvate kinase M2 and/or lactate dehydrogenase A (LDHA) in vitro in cells and in vivo in neoplastic lesions in the prostate of TRAMP and Hi-Myc mice; and (iii) significantly suppressed glycolysis in prostate of Hi-Myc mice as measured by ex vivo1H magnetic resonance spectroscopy. SFN treatment did not decrease glucose uptake or expression of glucose transporters in cells. Overexpression of c-Myc, but not constitutively active Akt, conferred protection against SFN-mediated downregulation of HKII and LDHA protein expression and suppression of lactate levels. Examination of plasma lactate levels in prostate cancer patients following administration of an SFN-rich broccoli sprout extract failed to show declines in its levels. Additional clinical trials are needed to determine whether SFN treatment can decrease lactate production in human prostate tumors.