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 24 h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28 h; 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 6 h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24 h. 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 24 h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24 h, 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.     

The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice

During inflammation, neutrophils infiltrate into the involved site and undergo apoptosis. Early apoptotic neutrophils are then cleared by phagocytes, leading to resolution of the inflammation, whereas if late apoptotic neutrophils are accumulated for some reason, they provoke proinflammatory responses such as TNF-α production. To determine how endogenously produced nitric oxide (NO) regulates neutrophil apoptosis and the resolution of inflammation, we compared peritoneal inflammation induced by Staphylococcus aureus bioparticles in wild type mice with that in inducible NO synthase (iNOS)-deficient ones. In this model, NO production was largely dependent on iNOS, the NO level peaking at 24 h. There were increases in the numbers of neutrophils and late apoptotic ones at 24 h in iNOS-deficient mice as compared with in wild type ones, and consequently TNF-α production at 36 h in iNOS-deficient mice. On the other hand, the administration of a NO donor to iNOS-deficient mice at 12 h decreased the numbers of neutrophils and late apoptotic ones at 24 h, and thereafter TNF-α production at 36 h. In addition, coculturing of macrophages with late apoptotic neutrophils caused TNF-α production and a NO donor inhibited the transmigration of neutrophils in a dose-dependent manner. Collectively, these results suggest a novel mechanism that endogenously produced NO suppresses neutrophil accumulation at a late stage of inflammation, thereby preventing the appearance of late apoptotic neutrophils and subsequent proinflammatory responses.     

Cardiac mitochondrial biogenesis in endotoxemia is not accompanied by mitochondrial function recovery

Mitochondrial biogenesis emerges as a compensatory mechanism involved in the recovery process in endotoxemia and sepsis. The aim of this work was to analyze the time course of the cardiac mitochondrial biogenesis process occurring during endotoxemia, with emphasis on the quantitative analysis of mitochondrial function. Female Sprague-Dawley rats (45 days old) were ip injected with LPS (10 mg/kg). Measurements were performed at 0–24 h after LPS administration. PGC-1α and mtTFA expression for biogenesis and p62 and LC3 expression for autophagy were analyzed by Western blot; mitochondrial DNA levels by qPCR, and mitochondrial morphology by transmission electron microscopy. Mitochondrial function was evaluated as oxygen consumption and respiratory chain complex activity. PGC-1α and mtTFA expression significantly increased in every time point analyzed, and mitochondrial mass was increased by 20% (P<0.05) at 24 h. p62 expression was significantly decreased in a time-dependent manner. LC3-II expression was significantly increased at all time points analyzed. Ultrastructurally, mitochondria displayed several abnormalities (internal vesicles, cristae disruption, and swelling) at 6 and 18 h. Structures compatible with fusion/fission processes were observed at 24 h. A significant decrease in state 3 respiration was observed in every time point analyzed (LPS 6 h: 20%, P<0.05). Mitochondrial complex I activity was found decreased by 30% in LPS-treated animals at 6 and 24 h. Complex II and complex IV showed decreased activity only at 24 h. The present results show that partial restoration of cardiac mitochondrial architecture is not accompanied by improvement of mitochondrial function in acute endotoxemia. The key implication of our study is that cardiac failure due to bioenergetic dysfunction will be overcome by therapeutic interventions aimed to restore cardiac mitochondrial function.     

Liver-targeting of primaquine-(poly-γ-glutamic acid) and its degradation in rat hepatocytes

We have synthesized poly-γ-glutamic acid (PGA) modified with a synthetic trivalent glyco-ligand (TriGalNAc) for the hepatocyte asialoglycoprotein receptor (ASGP-R). We investigated in vivo distribution of unmodified PGA and TriGalNAc-modified PGA (TriGalNAc-PGA) in mice after intravenous injection. Most of unmodified PGA administered was transported to the bladder over 20–80 min, suggesting a rapid excretion of unmodified PGA into urine. In contrast, TriGalNAc-PGA was found exclusively in the liver over the same period of time. We further synthesized TriGalNAc-PGA–primaquine conjugate (TriGalNAc-PGA–PQ), and investigated binding, uptake, and catabolism of the conjugate by rat hepatocytes. Our studies indicated that approximately 250 ng per million cells of the conjugate bound to one million rat hepatocytes at 0 °C, and approximately 2 μg per million cells of the conjugate was taken up over 7 h incubation at 37 °C. Furthermore, our results suggested that TriGalNAc-PGA–PQ was almost completely degraded over 24 h, and small degradation products were secreted into cell culture medium.The results described in this report suggest that the TriGalNAc ligand can serve as an excellent targeting device for delivery of PGA-conjugates to the liver hepatocytes, and rat hepatocytes possess sufficient capacity to digest PGA even modified with other substituents.     

Whole-body basal nitric oxide production is impaired in postprandial endothelial dysfunction in healthy rats

In healthy humans, a high-saturated-fat/high-sucrose meal induces vascular endothelial dysfunction, a hallmark of atherogenesis. This transient dysfunction indicates a loss in nitric oxide (NO) production and/or bioactivity in the vasculature but it remains unknown if this is the local manifestation of a general impairment in NO pathway in the postprandial state. Here, we studied whole-body NO production and systemic NO bioactivity in postprandial endothelial dysfunction, as induced by a high-saturated-fat, high-sucrose meal.We first developed a physiological test of endothelial function on conscious rats, based on the transient fall in blood pressure after iv acetylcholine, and showed that this response was NO-dependent. As assessed with this method in healthy rats, endothelial function decreased during the postprandial state, being 60 ± 7% lower than baseline at 6 h after the meal challenge, associated with important elevations in plasma triglycerides and hydroperoxides. Aortic superoxide anion production, as assessed by oxidative fluorescent detection, was higher 6 h after the meal challenge than after the nutrients vehicle (water). During the postprandial period, plasma cGMP, but not plasma ANP, markedly decreased, indicating a general decrease in NO bioavailability, which was numerically maximal 4 h after the meal challenge. As determined 4 h after ingestion by a tracer-based method using iv [¹⁵N₂-(guanido)]-arginine, the whole-body NO production fell by 27 ± 9% postprandially.This is the first study evidencing that a meal challenge that impairs the stimulated, NO-mediated, vascular response also reduces whole-body basal NO production and bioavailability. Postprandial pathophysiology may build on this general, fundamental alteration in NO production.     

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including Acetazolamide in human Glioblastoma

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24 h at 0.1%, O₂). Aza. application was at a range between 250 and 8000 nM and the HIF-1α inhibitor Chetomin at a concentration range of 150–500 nM. Cell culture plates were incubated for 24 h under hypoxia (0.1% O₂). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24 h of hypoxia (0.1% O₂) at concentrations between 3500 and 8000 nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150–500 nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.     

Time-course correlation of early toxic events in three models of striatal damage: Modulation by proteases inhibition

Metabolic alterations in the nervous system can be produced at early stages of toxicity and are linked with oxidative stress, energy depletion and death signaling. Proteases activation is responsible for triggering deadly cascades during cell damage in toxic models. In this study we evaluated the early time-course of toxic events (oxidative damage to lipids, mitochondrial dysfunction and LDH leakage, all at 1, 3 and 6 h) in rat striatal slices exposed to quinolinic acid (QUIN, 100 μM) as an excitotoxic/pro-oxidant model, 3-nitropropionic acid (3-NP, 1 mM) as an inhibitor of mitochondrial succinate dehydrogenase, and a combined model produced by the co-administration of these two toxins at subtoxic concentrations (21 and 166 μM for QUIN and 3-NP, respectively). In order to further characterize a possible causality of caspases or calpains on the toxic mechanisms produced in these models, the broad calpain inhibitor IC1 (50 μM), and the pan-caspase inhibitor Z-VAD (100 μM) were tested. Lipid peroxidation (LP) was increased at all times and in all models evaluated. Both IC1 and Z-VAD exerted significant protection against LP in all models and at all times evaluated. Mitochondrial dysfunction (MD) was consistently affected by all toxic models at 3 and 6 h, but was mostly affected by 3-NP and QUIN at 1 h. IC1 differentially protected the slices against 3-NP and QUIN at 1 h and against QUIN at 3 h, while Z-VAD exhibited positive actions against QUIN and 3-NP at all times tested, and against their combination at 3 and 6 h. LDH leakage was enhanced at 1 and 3 h in all toxic models, but this effect was evident only for 3-NP + QUIN and 3-NP at 6 h. IC1 protected against LDH leakage at 1 h in 3-NP + QUIN and 3-NP models, at 3 h in all toxic models, and at 6 h in 3-NP + QUIN and 3-NP models. In turn, Z-VAD protected at 1 and 6 h in all models tested, and at 3 h in the combined and QUIN models. Our results suggest differential chronologic and mechanistic patterns, depending on the toxic insult. Although LP, MD and membrane cell rupture are shared by the three models, the occurrence of each event seems to obey to a selective recruitment of damaging signals, including a differential activation of proteases in time. Proteases activation is likely to be an up-stream event influencing oxidative stress and mitochondrial dysfunction in these toxic models.     

Chronic hypoxia alters mitochondrial composition in human macrophages

Hypoxia inducible factors (HIFs) are important mediators of the cellular adaptive response during acute hypoxia. The role of HIF-1 and HIF-2 during prolonged periods of hypoxia, i.e. chronic hypoxia is less defined. Therefore, we used human THP-1 macrophages with a knockdown of either HIF-1α, HIF-2α, or both HIFα-subunits, incubated them for several days under hypoxia (1% O₂), and analyzed responses to hypoxia using 2D-DIGE coupled to MS/MS-analysis. Chronic hypoxia was defined as a time point when the early but transient accumulation of HIFα-subunits and mRNA expression of classical HIF target genes returned towards basal levels, with a new steady state that was constant from 72 h onwards. From roughly 800 spots, that were regulated comparing normoxia to chronic hypoxia, about 100 proteins were unambiguously assigned during MS/MS-analysis. Interestingly, a number of glycolytic proteins were up-regulated, while a number of inner mitochondrial membrane proteins were down-regulated independently of HIF-1α or HIF-2α. Chronic hypoxic conditions depleted the mitochondrial mass by autophagy, which occurred independently of HIF proteins. Macrophages tolerate periods of chronic hypoxia very well and adaptive responses occur, at least in part, independently of HIF-1α and/or HIF-2α and comprise mitophagy as a pathway of particular importance.     

Pre-treatment with isoflurane ameliorates renal ischemic-reperfusion injury in mice

AimsPerioperative renal dysfunction is associated with a high mortality. The aim of this study was to investigate whether isoflurane preconditioning provides a protection against renal ischemic–reperfusion injury and whether hypoxia inducible factor 1α (HIF-1α) is responsible for the protection afforded by isoflurane in mice.Main methodsAdult male C57BL/6 mice received vehicle (PBS), scrambled siRNA or HIF-1α siRNA via hydrodynamic injection through tail vein. Twenty-four hours after injection, they were exposed to 1.5% isoflurane in oxygen enriched air for 2 h while controls without injection were exposed to oxygen enriched air. Twenty-four hours after gas exposure, mice were sacrificed and their kidney were harvested for western blot while other cohorts underwent renal ischemia–reperfusion injury induced by bilateral renal pedicle clamping for 25 min for renal histological or functional analysis 24 h after reperfusion or by unilateral clamping for 40 min for survival rate analysis.Key findingsSurvival rate and the expression of HIF-1α and erythropoietin were significantly increased while apoptosis, renal tubule score, blood plasma creatinine and urea were decreased by isoflurane preconditioning. HIF-1α siRNA but not scrambled siRNA injection abrogated the protective effect of isoflurane preconditioning.SignificanceOur data suggested that isoflurane preconditioning provided a protection against renal ischemic–reperfusion injury which is very likely due to hypoxia inducible factor-1α upregulation.     

Combined effect of bone marrow derived mesenchymal stem cells and nitric oxide inducer on injured gastric mucosa in a rat model

AimTo study the effect of intravenous injection of bone marrow mesenchymal stem cells (BMMSCs), alone and combined with NO inducer in gastric ulcer healing in a rat model.MethodsRats were divided into controls, gastric ulcer, gastric ulcer receiving mesenchymal stem cells (MSCs), gastric ulcer receiving NO inducer (l-Arginine), gastric ulcer receiving MSCs plus NO inducer (l-Arginine) groups. MSCs were given in a dose of (10⁶cells) by intravenous injection. l-Arginine was given 300 mg/kg body weight intraperitoneally. 24 h and 7 days after BMMSCs and NO inducer injection, VEGF, PGE, TNF-α were assessed by ELISA. Gene expression of HGF, caspase-3, eNOS and BAX/Bcl-2 in gastric tissues were studied by real time PCR. Histopathology staining of gastric tissues was performed.ResultsInjection of MSCs or NO inducer or both to the gastric ulcer group significantly decreased caspase-3 and BAX genes expression (apoptotic factors) and increased Bcl-2 gene expression (anti-apoptotic factor) compared to that of the gastric ulcer group after both 24 h and 7 days with more significant results in the gastric group received both MSCs and NO inducer. HGF gene expression was significantly increased in the groups injected with MSCs or NO inducer or both compared with the corresponding gastric ulcer group (p < 0.05, p < 0.05 & p < 0.001 respectively). There was a significant decrease in the mean PGE2 and TNF-α levels in the gastric ulcer group receiving MSCs, the gastric ulcer group receiving NO and the gastric ulcer group receiving both MSCs and  NO compared to the gastric ulcer group after both 24 h and 7 days. Histopathological examination of gastric tissue of groups that received stem cells or NO alone, showed mucosal regenerative changes with increased thickness together with reduced inflammatory cellular infiltrate in the submucosa and decreased congestion. There was complete restoration in gastric mucosa in the group that received both stem cells and NO.ConclusionAdministration of MSCs, NO, or MSCs plus NO may exert a therapeutic effect on the mucosal lesion in gastric ulcer through their anti-inflammatory, angiogenic and antiapoptotic actions.