Paola Finocchietto

The pan-caspase inhibitor Emricasan (IDN-6556) decreases liver injury and fibrosis in a murine model of non-alcoholic steatohepatitis

Hepatocyte apoptosis, the hallmark of non‐alcoholic steatohepatitis (NASH) contributes to liver injury and fibrosis. Although, both the intrinsic and extrinsic apoptotic pathways are involved in the pathogenesis of NASH, the final common step of apoptosis is executed by a family of cysteine‐proteases termed caspases. Thus, our aim was to ascertain if administration of Emricasan, a pan‐caspase inhibitor, ameliorates liver injury and fibrosis in a murine model of NASH.

Mitochondrial Nitric Oxide Synthase: A Masterpiece of Metabolic Adaptation, Cell Growth, Transformation, and Death

Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)–dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.

Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: implications for the metabolic syndrome.

Background In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO2 and H2O or to drive glucose storage to glycogen. Methodology/Principal Findings We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4–8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (−25 to −60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-14C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37±3 vs 10±1 µmolO2/h.g tissue and 13±1 vs 7.2±1 µmol 3H2O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half. Conclusions/Significance These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.

Defective Leptin–AMP-Dependent Kinase Pathway Induces Nitric Oxide Release and Contributes to Mitochondrial Dysfunction and Obesity in ob/ob Mice

AIMS Obesity arises on defective neuroendocrine pathways that increase energy intake and reduce mitochondrial metabolism. In the metabolic syndrome, mitochondrial dysfunction accomplishes defects in fatty acid oxidation and reciprocal increase in triglyceride content with insulin resistance and hyperglycemia. Mitochondrial inhibition is attributed to reduced biogenesis, excessive fission, and low adipokine-AMP-activated protein kinase (AMPK) level, but lateness of the respiratory chain contributes to perturbations. Considering that nitric oxide (NO) binds cytochrome oxidase and inhibits respiration, we explored NO as a direct effector of mitochondrial dysfunction in the leptin-deficient ob/ob mice. RESULTS A remarkable three- to fourfold increase in neuronal nitric oxide synthase (nNOS) expression and activity was detected by western blot, citrulline assay, electronic and confocal microscopy, flow cytometry, and NO electrode sensor in mitochondria from ob/ob mice. High NO reduced oxygen uptake in ob/ob mitochondria by inhibition of complex IV and nitration of complex I. Low metabolic status restricted β-oxidation in obese mitochondria and displaced acetyl-CoA to fat synthesis; instead, small interference RNA nNOS caused a phenotype change with fat reduction in ob/ob adipocytes. INNOVATION We evidenced that leptin increases mitochondrial respiration and fat utilization by potentially inhibiting NO release. Accordingly, leptin administration to ob/ob mice prevented nNOS overexpression and mitochondrial dysfunction in vivo and rescued leptin-dependent effects by matrix NO reduction, whereas leptin-Ob-Rb disruption increased the formation of mitochondrial NO in control adipocytes. We demonstrated that in ob/ob, hypoleptinemia is associated with critically low mitochondrial p-AMPK and that, oppositely to p-Akt2, p-AMPK is a negative modulator of nNOS. CONCLUSION Thereby, defective leptin-AMPK pathway links mitochondrial NO to obesity with complex I syndrome and dysfunctional mitochondria.

Abnormal mitochondrial fusion–fission balance contributes to the progression of experimental sepsis

Abstract Sepsis-associated multiple organ failure is a major cause of mortality characterized by a massive increase of reactive oxygen and nitrogen species (ROS/RNS) and mitochondrial dysfunction. Despite intensive research, determining events in the progression or reversal of the disease are incompletely understood. Herein, we studied two prototype sepsis models: endotoxemia and cecal ligation and puncture (CLP)—which showed very different lethality rates (2.5% and 67%, respectively)—, evaluated iNOS, ROS and respiratory chain activity, and investigated mitochondrial biogenesis and dynamics, as possible processes involved in sepsis outcome. Endotoxemia and CLP showed different iNOS, ROS/RNS, and complex activities time-courses. Moreover, these alterations reverted after 24-h endotoxemia but not after CLP. Mitochondrial biogenesis was not elicited during the first 24 h in either model but instead, 50% mtDNA depletion was observed. Mitochondrial fusion and fission were evaluated using real-time PCR of mitofusin-2 (Mfn2), dynamin-related protein-1 (Drp1), and using electron microscopy. During endotoxemia, we observed a decrease of Mfn2-mRNA levels at 4–6 h, and an increase of mitochondrial fragmentation at 6 h. These parameters reverted at 24 h. In contrast, CLP showed not only decreased Mfn2-mRNA levels at 12–18 h but also increased Drp1-mRNA levels at 4 h, and enhanced and sustained mitochondrial fragmentation. The in vivo pretreatment with mdivi-1 (Drp1 inhibitor) significantly attenuated mitochondrial dysfunction and apoptosis in CLP. Therefore, abnormal fusion-to-fission balance, probably evoked by ROS/RNS secondary to iNOS induction, contributes to the progression of sepsis. Pharmacological targeting of Drp1 may be a potential novel therapeutic tool for sepsis.

Cardiac-specific overexpression of thioredoxin 1 attenuates mitochondrial and myocardial dysfunction in septic mice.

Sepsis-induced myocardial dysfunction is associated with increased oxidative stress and mitochondrial dysfunction. Current evidence suggests a protective role of thioredoxin-1 (Trx1) in the pathogenesis of cardiovascular diseases. However, it is unknown yet a putative role of Trx1 in sepsis-induced myocardial dysfunction, in which oxidative stress is an underlying cause. Transgenic male mice with Trx1 cardiac-specific overexpression (Trx1-Tg) and its wild-type control (wt) were subjected to cecal ligation and puncture or sham surgery. After 6, 18, and 24h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28h; p=0.0204). Sepsis induced a decrease in left ventricular developed pressure in both groups, while the contractile reserve, estimated as the response to β-adrenergic stimulus, was higher in Trx1-Tg in relation to wt, after 6h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24h. Ultrastructural alterations in mitochondrial cristae were accompanied by reduced optic atrophy 1 (OPA1) fusion protein, and activation of dynamin-related protein 1 (Drp1) (fission protein) in wt mice at 24h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24h, suggesting mitochondrial biogenesis induction. Autophagy, demonstrated by electron microscopy and increased LC3-II/LC3-I ratio, was observed earlier in Trx1-Tg. In conclusion, Trx1 overexpression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover (mitophagy and biogenesis), preserves contractile reserve and prolongs survival during sepsis.

TAFRO Syndrome in a Patient of South-American Descent

A 31-year-old Caucasian woman of South-American descent was diagnosed with a variant of multicentric Castleman disease (MCD) that has been reported in Japan as Castleman-Kojima disease. This is a systemic inflammatory disorder known as TAFRO Syndrome which includes thrombocytopenia, polyserositis (ascites/pleural effusion), microcytic anemia, myelofibrosis, fever, renal dysfunction and organomegaly, with immunologic disorder, polyclonal hypergammaglobulinemia, and elevated levels of interleukin-6 (IL-6) and the vascular endothelial growth factor present in serum and/or effusions. Optimal therapies are not well established. The patient was treated with methylprednisolone and rituximab. Following the start of treatment, the patient has been asymptomatic for over 8 months. This is one of only a few reports of TAFRO Syndrome in a non-Japanese patient.

p66Shc Inactivation Modifies RNS Production, Regulates Sirt3 Activity, and Improves Mitochondrial Homeostasis, Delaying the Aging Process in Mouse Brain

Programmed and damage aging theories have traditionally been conceived as stand-alone schools of thought. However, the p66Shc adaptor protein has demonstrated that aging-regulating genes and reactive oxygen species (ROS) are closely interconnected, since its absence modifies metabolic homeostasis by providing oxidative stress resistance and promoting longevity. p66Shc(−/−) mice are a unique opportunity to further comprehend the bidirectional relationship between redox homeostasis and the imbalance of mitochondrial biogenesis and dynamics during aging. This study shows that brain mitochondria of p66Shc(−/−) aged mice exhibit a reduced alteration of redox balance with a decrease in both ROS generation and its detoxification activity. We also demonstrate a strong link between reactive nitrogen species (RNS) and mitochondrial function, morphology, and biogenesis, where low levels of ONOO− formation present in aged p66Shc(−/−) mouse brain prevent protein nitration, delaying the loss of biological functions characteristic of the aging process. Sirt3 modulates age-associated mitochondrial biology and function via lysine deacetylation of target proteins, and we show that its regulation depends on its nitration status and is benefited by the improved NAD+/NADH ratio in aged p66Shc(−/−) brain mitochondria. Low levels of protein nitration and acetylation could cause the metabolic homeostasis maintenance observed during aging in this group, thus increasing its lifespan.

Takotsubo Myocardiopathy and Hyperthyroidism: A Case Report and Literature Review

Patient: Male, 34 Final Diagnosis: Takotsubo myocardiopathy and hyperthyroidism Symptoms: Chest pain • dyspnea Medication: — Clinical Procedure: — Specialty: Cardiology Objective: Rare co-existance of disease or pathology Background: Takotsubo cardiomyopathy (TM), also called stress myocardiopathy or transient left ventricular apical ballooning syndrome, is characterized by acute left ventricular dysfunction with reversible wall motion abnormalities. TM resembles acute coronary syndrome (ACS) in the absence of coronary artery disease (CAD). In several reports, TM has been described in association with hyperthyroidism, suggesting the potential role of thyrotoxicosis in the pathophysiology. Case Report: We present the case of a 34-year-old man with TM associated with hyperthyroidism caused by Graves’ disease. In this case, TM was also preceded by an emotional trigger. The diagnosis of TM was based on clinical manifestations, electrocardiographic and echocardiographic abnormalities, and the absence of coronary artery disease (CAD) in the angiography. A diagnosis of hyperthyroidism was made based on hormonal and antibody measurements. The patient had a favorable outcome, and the cardiac and thyroid disorders resolved. Conclusions: Our case illustrates that thyroid disease, mainly hyperthyroidism, should be considered in patients with TM with or without previous emotional triggers. As in our patient, the outcome in TM is usually favorable, with reversibility of cardiac abnormalities.

Intravascular Large B Cell Lymphoma Presenting as Fever of Unknown Origin and Diagnosed by Random Skin Biopsies: A Case Report and Literature Review

Patient: Female, 66 Final Diagnosis: Intravascular B-cell lymphoma Symptoms: Fever of unknown origin Medication: — Clinical Procedure: — Specialty: Hematology Objective: Rare disease Background: Intravascular lymphoma (IVL) is a rare lymphoproliferative disorder characterized by the proliferation of large B lymphoma cells within the lumen of small-caliber blood vessels. Clinical features are nonspecific, presenting as a systemic disease with fever and may be life-threatening. Antemortem diagnosis is difficult but may be made with biopsies of affected tissues or with random skin biopsies. Case Report: We report the case of a 66-year-old white woman presenting with fever of unknown origin (FUO) who developed neurologic, pulmonary, and hematologic manifestations. The diagnosis of intravascular large B cell lymphoma (IVLBCL) was made by random skin biopsies. She received treatment with steroids, rituximab, cyclophosphamide, vincristine, and doxorubicin (R-CHOP). Her disease evolution was unfavorable and she died after her first cycle of chemotherapy. Conclusions: Our case illustrates that IVL can present as FUO and should be considered in the differential diagnosis of this syndrome, especially in patients with neurologic compromise and persistently elevated serum lactate dehydrogenase. In this case, the diagnosis was made with cutaneous biopsies of visibly unaffected skin. As in our patient, the course of IVL is usually fatal within a few months.