Metformin Attenuates ox-LDL-Induced Macrophage Senescence and Inflammation via NR4A1-Mediated Mitophagy Regulation.

Caffeine and acetaminophen association: Effects on mitochondrial bioenergetics

Secretion of the proinflammatory cytokines, interleukin (IL)-1beta and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1beta and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines.

Oxidative stress, resulting from increased production of reactive oxygen species (ROS) and/or reduced antioxidant defences, has been implicated in cardiovascular disease pathophysiology for over 2 decades. Based on the concept that this drives both the genesis and progression of conditions such as heart failure, numerous clinical trials of antioxidant therapies were undertaken but were unsuccessful. Nevertheless, experimental data linking oxidative stress and heart disease remain compelling and support continued efforts to develop more effective therapies than antioxidant vitamins.1 In the current issue of Circulation , Zhao et al .2 report that cardiomyocyte-specific high-level overexpression of the ROS-generating enzyme NADPH oxidase-4 (Nox4) aggravated angiotensin II-induced cardiac remodeling and was mitigated by a small molecule Nox inhibitor. The authors propose that Nox4 inhibition may have therapeutic potential to treat cardiac remodeling. Is this proposal reasonable and how should such studies be interpreted within a pathophysiological framework for the roles of ROS in heart failure?

Previous studies proposed that acidic reperfusion may be a protective strategy for myocardial ischemia-reperfusion therapy with potential of clinical transformation. In this study, we investigated whether therapeutic hypercapnia could mimic acidosis postconditioning in isolated hearts with a 30-min left coronary artery ligation-reperfusion model in rats. Therapeutic hypercapnia (inhalation 20% CO2 for 10min) is cardioprotective with a strict therapeutic time window and acidity: it reduced the infarct ratio and serum myocardial enzyme and increased the myocardial ATP content. Furthermore, mitochondrial morphology damage, the loss of mitochondrial membrane potential, and the formation of mitochondrial permeability transition pore were effectively inhibited, indicating the improvements in mitochondrial function. The expression of the mitochondrial biogenesis regulators was upregulated simultaneously. These findings indicated therapeutic hypercapnia in animals can mimic ex vivo acidosis postconditioning to alleviate myocardial ischemia-reperfusion injury. The effect is related to improvement in mitochondrial function and regulation of the mitochondrial biogenesis pathway.

There is still debate about the relationship between fat accumulation and mitochondrial function in nonalcoholic fatty liver disease. It is a critical question as only a small proportion of individuals with steatosis progress to steatohepatitis. In this study, we focused on defining (i) the effects of triglyceride accumulation and reactive oxygen species (ROS) on mitochondrial function (ii) the contributions of triglyceride, ROS and subsequent mitochondrial impairment on the metabolism of energy substrates. Human hepatoblastoma C3A cells, were treated with various combinations of oleate, octanoate, lactate (L), pyruvate (P) and ammonia (N) acutely or for 72 h, before measurements of triglyceride concentration, cell respiration, ROS production, mitochondrial membrane potential, ketogenesis and gluconeogenesis, TCA cycle metabolite analysis and electron microscopy. Acutely, LPON treatment enhanced mitochondrial respiration and ROS formation. After 72 h, despite the similarities in triglyceride accumulation, LPON treatment, but not oleate, dramatically affected mitochondrial function as evidenced by decreased respiration, increased mitochondrial membrane potential and ROS formation with concomitant enhanced ketogenesis. By comparison, respiration and ROS formation remained unperturbed with oleate. Importantly, this was accompanied by an increased gluconeogenesis and ketogenesis. The addition of the antioxidant N-acetyl-L-cysteine prevented mitochondrial dysfunction and reversed metabolic changes seen with LPON, strongly suggesting ROS involvement in mediating mitochondrial impairment. Our data indicate that ROS formation, rather than cellular steatosis per se, impairs mitochondrial function. Thus, reduction in cellular steatosis may not always be the desired outcome without concomitant improvement in mitochondrial function and/or reducing of ROS formation.

Nuclear factor-erythroid 2 related factor 2 (Nrf2)-mediated signaling plays a central role in maintaining cellular redox homeostasis of hepatic cells. Carbon monoxide releasing molecule-A1 (CORM-A1) has been reported to stimulate up-regulation and nuclear translocation of Nrf2 in hepatocytes. However, the role of CORM-A1 in improving lipid metabolism, antioxidant signaling and mitochondrial functions in nonalcoholic steatohepatitis (NASH) is unknown. In this study, we report that CORM-A1 prevents hepatic steatosis in high fat high fructose (HFHF) diet fed C57BL/6J mice, used as model of NASH. The beneficial effects of CORM-A1 in HFHF fed mice was associated with improved lipid homeostasis, Nrf2 activation, upregulation of antioxidant responsive (ARE) genes and increased ATP production. As, mitochondria are intracellular source of reactive oxygen species (ROS) and important sites of lipid metabolism, we further investigated the mechanisms of action of CORM-A1-mediated improvement in mitochondrial function in palmitic acid (PA) treated HepG2 cells. Cellular oxidative stress and cell viability were found to be improved in PA + CORM-A1 treated cells via Nrf2 translocation and activation of cytoprotective genes. Furthermore, in PA treated cells, CORM-A1 improved mitochondrial oxidative stress, membrane potential and rescued mitochondrial biogenesis thru upregulation of Drp1, TFAM, PGC-1α and NRF-1 genes. CORM-A1 treatment improved cellular status by lowering glycolytic respiration and maximizing OCR. Improvement in mitochondrial respiration and increment in ATP production in PA + CORM-A1 treated cells further corroborate our findings. In summary, our data demonstrate for the first time that CORM-A1 ameliorates tissue damage in steatotic liver via Nrf2 activation and improved mitochondrial function, thus, suggesting the anti-NASH potential of CORM-A1.

The evolving role of mitochondria in metabolism

Trauma-induced heterotopic ossification (HO) is a complex disorder after musculoskeletal injury and characterized by aberrant extraskeletal bone formation. Recent studies shed light on critical role of dysregulated osteogenic differentiation in aberrant bone formation. Krupel-like factor 2 (KLF2) and peroxisome proliferator-activated receptor gamma (PPARγ) are master adapter proteins that link cellular responses to osteogenesis; however, their roles and relationships in HO remain elusive. Using a murine burn/tenotomy model in vivo, we identified elevated KLF2 and reduced PPARγ levels in tendon stem/progenitor cells (TSPCs) during trauma-induced HO formation. Both KLF2 inhibition and PPARγ promotion reduced mature HO, whereas the effects of PPARγ promotion were abolished by KLF2 overexpression. Additionally, mitochondrial dysfunction and reactive oxygen species (ROS) production also increased after burn/tenotomy, and improvements in mitochondrial function (ROS scavenger) could alleviate HO formation, but were abolished by KLF2 activation and PPARγ suppression by affecting redox balance. Furthermore, in vitro, we found increased KLF2 and decreased PPARγ levels in osteogenically induced TSPCs. Both KLF2 inhibition and PPARγ promotion relieved osteogenesis by improving mitochondrial function and maintaining redox balance, and effects of PPARγ promotion were abolished by KLF2 overexpression. Our findings suggest that KLF2/PPARγ axis exerts regulatory effects on trauma-induced HO through modulation of mitochondrial dysfunction and ROS production in TSPCs by affecting redox balance. Targeting KLF2/PPARγ axis and mitochondrial dysfunction can represent attractive approaches to therapeutic intervention in trauma-induced HO.

Intervention by picroside II on FFAs induced lipid accumulation and lipotoxicity in HepG2 cells

Cancer treatments in general share various detrimental effects in common, especially upregulation of cardiovascular risk factors. Therefore, the science of onco-cardiology should not be restricted in scope to the side effects of each specific cancer drug. In particular, premature aging induced by cancer treatment may contribute to the chronic health problems of cancer survivors. About 1 660 000 people, including more than 12 000 children below the age of 18 years, are newly diagnosed with a malignancy in the United States every year.1 The American Cancer Society reported that in 2016 there are 15.5 million cancer survivors in the United States (http://www.cancer.org/cancer/news/news/report-number-of-cancer-survivors-continues-to-grow). At present, the 5-year survival rate of patients treated for cancer is 67%. Seventy-five percent of children in whom cancer is diagnosed today will live for at least 10 years; 20% will survive for longer than 35 years.1 Although these numbers are impressive compared with those from decades ago, further improvement of cancer survivors’ life span as well as quality of life and functional status is still necessary. Approximately 75% of cancer survivors have some form of chronic health problem. Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality in this population, particularly after recurrent or second malignancy. The risk of CVD in cancer survivors is 8× higher than that of the general population. The relative risks of coronary artery disease and heart failure in cancer survivors are 10× and 15× higher, respectively, than their siblings without cancer.1 Cancer treatments, including chemotherapy and radiation, can lead to both short- and long-term cardiovascular complications. Evidence of subclinical cardiac and vascular damage was observed in more than 50% of survivors 5 to 10 years after chemotherapy.1 Onco-cardiology is a medical subspecialty concerned with the diagnosis and treatment of CVDs and organ failure mediated by microcirculatory or macrocirculatory …

Context Aging leads to senile osteoporosis (SOP), marked by bone loss and increased fracture risk. Macrophages, as active immune cells in bone tissue, play an important role in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) during aging. Wen-Shen-Tong-Luo-Zhi-Tong Decoction (WSTLZTD), a traditional Chinese herbal formula, has been clinically validated for its efficacy in treating SOP. However, the specific mechanisms by which WSTLZTD exerts its anti-SOP effects—particularly through modulating macrophage senescence—remain unclear. Objective The study aims to elucidate the role of WSTLZTD in macrophage senescence and SOP. Materials and methods Aged mice received low, medium, high-dose WSTLZTD. Bone loss was evaluated via micro-computed tomography, hematoxylin and eosin staining and osteocalcin, tartrate-resistant acid phosphatase marker analysis. Macrophage senescence detection (β-galactosidase staining, p16, p21) and molecular mechanisms by Western blot, immunohistochemistry, immunofluorescence method were investigated. Macrophage-conditioned medium’s effects on BMSC osteogenesis and mitochondrial function were assessed through alkaline phosphatase, Alizarin Red S staining, reactive oxygen species and JC-1 mitochondrial membrane potential (ΔΨm) assays. Results In vivo experiments demonstrated that WSTLZTD effectively ameliorated macrophage senescence and osteoporosis in naturally aged mice. Mechanistically, high-dose WSTLZTD attenuated senescence in bone marrow-derived macrophages by mediating LONP1, concurrently suppressing the cyclic GMP-AMP synthase (cGAS)/STING signaling pathway in BMSCs, thereby enhancing osteogenic differentiation of BMSCs. In vitro studies further confirmed that WSTLZTD-containing serum attenuated the senescent phenotype of macrophages. Notably, the LONP1 inhibitor, LONP1-IN-2, was found to diminish the anti-senescence effects of WSTLZTD on macrophages and BMSC osteogenesis. Discussion and conclusion WSTLZTD potentially modulate macrophage senescence via LONP1, which subsequently suppresses the activation of the cGAS/STING pathway in BMSCs, ultimately promoting their osteogenic differentiation and ameliorating osteoporosis.

Objective To investigate the protective roles of bone marrow-derived mesenchymal stem cells in glucolipotoxicity–induced INS–1 cell damage and the possible mechanisms involved. Methods INS–1 cells were divided into three groups according to different treatment, control group, high glucose group/ PA group and high glucose/PA with BMSCs co–culture group. INS–1 cells in high glucose/PA with BMSCs co-culture group were exposured to the culture medium containing 16.7 mmol/L glucose and 0.4 mmol/L palmitic acid (PA) for 48 hours, and then co–cultured with BMSCs for another 24 hours. The viability of INS- 1 cells was assessed by CCK8 assay. The expression of Cleaved cysteine asparate pro–tease- 3 (Caspase–3) were detected by western blot. The cell apoptosis incidence was measured using Annexin V/propidium iodinate (PI) staining by a flow cytometer. The insulin secretion of INS–1 cells were measured using a Ratio Immunity Assay (RIA) kit. The mitochondrial membrane potential (MMP) was detected with JC–1 probe by a flow cytometer. Intracellular reactive oxygen species (ROS) production was detected using dichlorofluoresceindiacetate (DCFH – DA) and quantified by a flow cytometer. Comparisons between two groups were measured using Student's t – test. Results Compared with control group, there were a significant decrease of INS–1 cell viability((100.0%±0.8%) vs (71.9%±3.2%),t=8.46, P<0.01), upregulated expression of Cleaved Caspase- 3 (0.25±0.03 vs 1.01±0.07,t=10.28, P<0.01) and an increase of apoptosis incidence ((6.9%±0.4%)vs (16.4%±1.2%),t=17.38, P<0.01), combined with reduced basal insulin secretion (BIS) and glucose stimulated insulin secretion (GSIS) (t=5.745,13.559, P<0.01) in high glucose/ PA group. BMSCs co – culture improved the viability of INS – 1 cells, down – regulated the expression of Cleaved Caspase–3, reduced the apoptosis incidence t=3.98,5.16,13.08, P<0.05), and significantly increased the BIS((80.743±0.012) vs (109.67±1.058) ng/mg,t=5.674, P<0.05)and GSIS ((120.0±1.3) vs (231.2±1.6)ng/mg protein,t=10.148, P<0.01), compared with chronic high glucose/PA–treated INS – 1 cells. Additionally, mitochondrial function was impaired according to the glucolipotoxicity–induced mitochondrial depolarization (95.7±8.5 vs 45.9±4.2,t=5.239, P<0.05) and considerable ROS accumulation ((13.3%±0.9%)vs(34.2% ± 0.8%),t=17.38, P<0.01). However, compared with chronic high glucose/PA- treated INS- 1 cells, BMSCs treatment effectively reversed glucolipotoxicity–induced depolarization (46±4 vs 87±14,t=2.822, P<0.05) and reduced the cellular ROS level ((34.3%±0.8%) vs (22.5%±0.4%),t=13.08, P<0.01), indicating that BMSCs could recover the injury of the mitochondrial function. Conclusion BMSCs played an important cyto-protective role in glucolipotoxicity–induced INS–1 cell damage, and the possible mechanism underlying this effect might be associated with the improvement of mitochondrial function. Key words: Mesenchymal stem cells; Glucolipotoxicity; Mitochondria; Insulinoma- 1 cells

Senescence, which is irreversible cell cycle arrest, is induced by various types of DNA damage, including genotoxic stress. Senescent cells show dysregulation of tumor suppressor genes and other regulators of cellular proliferation. Activating transcription factor 3 (ATF3) plays a pleiotropic role in biological processes through genotoxic stress. In this study, we examined the effects of acrylamide (ACR), a genotoxic carcinogen, on cellular senescence and the molecular mechanisms of ATF3 function in macrophages. Treatment of macrophages with ACR at low concentrations (<1.0 mM) resulted in senescence-like morphology and an increase in senescence-associated β-galactosidase (SA-β-gal) activity. Exposure of macrophages to ACR led to stress-induced, telomerase-independent senescence. In addition, ACR treatment for 1, 3, or 5 days showed a concentration-dependent increase in ATF3 expression and G0/G1 phase arrest. To better understand the role of ATF3 in controlling the senescence response to ACR, SA-β-gal activity was examined using ATF3 knockdown and overexpression. ACR-mediated senescence was significantly decreased by knockdown of ATF3, whereas it was increased with ATF3 overexpression. We found that ATF3 regulated p53 and p21 levels. ATF3 also played an important role in regulating intracellular reactive oxygen species (ROS) production in response to ACR treatment. Moreover, phosphorylation of p38 and JNK kinases, which were activated during ATF3-mediated senescence, was observed in ACR-treated macrophages. Taken together, these results suggest that ATF3 contributes to ACR-induced senescence by enhancing ROS production, activating p38 and JNK kinases, and promoting the ATF3-dependent expression of p53, resulting in regulation of cellular senescence in macrophages.

Temperature (T) reduction increases lifespan, but the mechanisms are not understood. Because reactive oxygen species (ROS) contribute to aging, we hypothesized that lowering T might decrease mitochondrial ROS production. We measured respiratory response and ROS production in isolated mitochondria at 32, 35, and 37 °C. Lowering T decreased the rates of resting (state 4) and phosphorylating (state 3) respiration phases. Surprisingly, this respiratory slowdown was associated with an increase of ROS production and hydrogen peroxide release and with elevation of the mitochondrial membrane potential, ΔΨ(m). We also found that at lower T mitochondria produced more carbon-centered lipid radicals, a species known to activate uncoupling proteins. These data indicate that reduced mitochondrial ROS production is not one of the mechanisms mediating lifespan extension at lower T. They suggest instead that increased ROS leakage may mediate mitochondrial responses to hypothermia.

IntroductionRemote ischemic preconditioning (RIPC), repeated cycles of brief limb ischemia/reperfusion, attenuates postoperative troponin release in patients undergoing surgical coronary revascularization and improves clinical outcome in some, but not all studies. Mitochondria are the end‐effectors of cardioprotection by local ischemic conditioning maneuvers in experimental studies. Here, we now analyzed mitochondrial function in response to RIPC in human atrial tissue.MethodsPatients undergoing elective surgical coronary revascularization under isoflurane anesthesia were randomized to RIPC by 3× 5 min/5 min upper arm blood pressure cuff inflation/deflation or placebo (n=30/30), and their right atrial appendages were harvested prior to ischemic cardioplegic arrest. Cardioprotection was reflected by a 14% decrease with RIPC vs. placebo in the area under the curve of serum troponin I/T concentrations over 72 h after surgery. Mitochondria were isolated from the atrial tissue (n=10/10). Mitochondrial function, i.e. mitochondrial respiration, adenosine triphosphate (ATP) production, reactive oxygen species (ROS) production and calcium retention capacity (CRC) to estimate mitochondrial permeability transition pore (mPTP) opening, was measured at physiological temperature of 37°C. Data are given as mean±SEM.ResultsAssociated with the observed cardioprotection in patients, mitochondrial function was improved by RIPC. Basal respiration was not different between RIPC and placebo. ADP‐stimulated complex I respiration was significantly greater with RIPC than with placebo. Mitochondrial complex IV respiration and maximal oxygen uptake of uncoupled mitochondria were not different between RIPC and placebo, reflecting an equal loading of viable mitochondria (Figure 1A). The mitochondrial ATP production (Figure 1B) was greater whereas mitochondrial ROS production (Figure 1C) was less with RIPC than with placebo. CRC of mitochondria (Figure 1D) was improved with RIPC in comparison to placebo, with and without cyclosporine A.ConclusionIn human myocardium, mitochondria are an intracellular target of protection by RIPC.Support or Funding InformationSupported by: German Research Foundation (SFB 1116 B8)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.