Weili Shen

Recent studies have revealed robust metabolic changes during cell differentiation. Mitochondria, the organelles where many vital metabolic reactions occur, may play an important role. Here, we report the involvement of SIRT3-regulated mitochondrial stress in osteoblast differentiation and bone formation. In both the osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts, robust mitochondrial biogenesis and supercomplex formation were observed during differentiation, accompanied by increased ATP production and decreased mitochondrial stress. Inhibition of mitochondrial activity or an increase in mitochondrial superoxide production significantly suppressed osteoblast differentiation. During differentiation, SOD2 was specifically induced to eliminate excess mitochondrial superoxide and protein oxidation, whereas SIRT3 expression was increased to enhance SOD2 activity through deacetylation of K68. Both SOD2 and SIRT3 knockdown resulted in suppression of differentiation. Meanwhile, mice deficient in SIRT3 exhibited obvious osteopenia accompanied by osteoblast dysfunction, whereas overexpression of SOD2 or SIRT3 improved the differentiation capability of primary osteoblasts derived from SIRT3-deficient mice. These results suggest that SIRT3/SOD2 is required for regulating mitochondrial stress and plays a vital role in osteoblast differentiation and bone formation.

Inflammatory response and chemotaxis of vascular wall cells play an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant for monocytes. Besides the induction of monocyte recruitment, it has been suggested that MCP-1 may directly activate smooth muscle cells. We investigated whether MCP-1 affects the proliferation and cytokine production of human vascular smooth muscle cells (VSMCs) and determined the underlying signal transduction pathways. Stimulation of VSMCs with MCP-1 induced proliferation and resulted in a concentration- and time-dependent release of the proinflammatory cytokine interleukin-6 (IL-6). Pretreatment with pertussis toxin, GF109203X, and pyrrolidine dithiocarbamate inhibited MCP-1-dependent IL-6 release, suggesting the involvement of G(i) proteins, protein kinase C, and nuclear factor-kappaB (NF-kappaB). MCP-1 also induced extracellular signal-regulated kinase, which, along with IL-6 release, was inhibited by pertussis toxin. PD98059 prevented MCP-1-induced extracellular signal-regulated kinase activation and cell proliferation. MCP-1 stimulated the binding activity of NF-kappaB and of activator protein-1 (AP-1). As demonstrated by cis element double-stranded (decoy) oligodeoxynucleotides, NF-kappaB was involved in IL-6 release by MCP-1, whereas proliferation was dependent on AP-1. The results clearly demonstrate that MCP-1 induces differential activation of NF-kappaB and AP-1 in VSMCs. Thus, our data propose a new mechanism for the proatherogenic effect of MCP-1.

Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1–10 μmol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1α, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1α by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1α activation signaling pathway demonstrated that HT is an activator of 5′AMP-activated protein kinase and also up-regulates gene expression of PPARα, CPT-1 and PPARγ. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis.

Studies in this laboratory have previously shown that hydroxytyrosol, the major antioxidant polyphenol in olives, protects ARPE-19 human retinal pigment epithelial cells from oxidative damage induced by acrolein, an environmental toxin and endogenous end product of lipid oxidation, that occurs at increased levels in age-related macular degeneration lesions. A proposed mechanism for this is that protection by hydroxytyrosol against oxidative stress is conferred by the simultaneous activation of two critically important pathways, viz., induction of phase II detoxifying enzymes and stimulation of mitochondrial biogenesis. Cultured ARPE-19 cells were pretreated with hydroxytyrosol and challenged with acrolein. The protective effects of hydroxytyrosol on key factors of mitochondrial biogenesis and phase II detoxifying enzyme systems were examined. Hydroxytyrosol treatment simultaneously protected against acrolein-induced inhibition of nuclear factor-E2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor coactivator 1 alpha (PPARGC1α) in ARPE-19 cells. The activation of Nrf2 led to activation of phase II detoxifying enzymes, including γ-glutamyl-cysteinyl-ligase, NADPH (nicotinamide adenine dinucleotide phosphate)-quinone-oxidoreductase 1, heme-oxygenase-1, superoxide dismutase, peroxiredoxin and thioredoxin as well as other antioxidant enzymes, while the activation of PPARGC1α led to increased protein expression of mitochondrial transcription factor A, uncoupling protein 2 and mitochondrial complexes. These results suggest that hydroxytyrosol is a potent inducer of phase II detoxifying enzymes and an enhancer of mitochondrial biogenesis. Dietary supplementation of hydroxytyrosol may contribute to eye health by preventing the degeneration of retinal pigment epithelial cells induced by oxidative stress.

Oxidative stress and mitochondrial dysfunction are known to play important roles in type 2 diabetes mellitus (T2DM) and insulin resistance. However, the pathology of T2DM remains complicated; in particular, the mechanisms of mitochondrial dysfunction in skeletal muscle and other insulin-sensitive tissues are as yet unclear. In the present study, we investigated the underlying mechanisms of oxidative stress and mitochondrial dysfunction by focusing on mitochondrial dynamics, including mitochondrial biogenesis and autophagy, in skeletal muscle of a nonobese diabetic animal model — the Goto–Kakizaki (GK) rat. The results showed that GK rats exhibited impaired glucose metabolism, increased oxidative stress and decreased mitochondrial function. These dysfunctions were found to be associated with induction of LC3B, Beclin1 and DRP1 (key molecules mediating the autophagy pathway), while they appeared not to affect the mitochondrial biogenesis pathway. In addition, (−)-epigallocatechin-3-gallate (EGCG) was tested as a potential autophagy-targeting nutrient, and we found that EGCG treatment improved glucose tolerance and glucose homeostasis in GK rats, and reduced oxidative stress and mitochondrial dysfunction in skeletal muscle. Amelioration of excessive muscle autophagy in GK rats through the down-regulation of the ROS-ERK/JNK-p53 pathway leads to improvement of glucose metabolism, reduction of oxidative stress and inhibition of mitochondrial loss and dysfunction. These results suggest (a) that hyperglycemia-associated oxidative stress may induce autophagy through up-regulation of the ROS-ERK/JNK-p53 pathway, which may contribute to mitochondrial loss in soleus muscle of diabetic GK rats, and (b) that EGCG may be a potential autophagy regulator useful in treatment of insulin resistance.

Emerging evidence indicates that impaired angiogenesis may contribute to hypertension-induced cardiac remodeling. The nicotinamide adenine dinucleotide-dependent deacetylase Sirtuin 3 (SIRT3) has the potential to modulate angiogenesis, but this has not been confirmed. As such, the aim of this study was to examine the relationship between SIRT3-mediated angiogenesis and cardiac remodeling.Our experiments were performed on SIRT3 knockout and age-matched wild-type mice infused with angiotensin II (1400 ng/kg per minute) or saline for 14 days. After angiotensin II infusion, SIRT3 knockout mice developed more severe microvascular rarefaction and functional hypoxia in cardiac tissues compared with wild-type mice. These events were concomitant with mitochondrial dysfunction and enhanced collagen I and collagen III expression, leading to cardiac fibrosis. Silencing SIRT3 facilitated angiotensin II-induced aberrant Pink/Parkin acetylation and impaired mitophagy, while excessive mitochondrial reactive oxygen species generation limited angiogenic capacity in primary mouse cardiac microvascular endothelial cells. Moreover, SIRT3 overexpression in cardiac microvascular endothelial cells enhanced Pink/Parkin-mediated mitophagy, attenuated mitochondrial reactive oxygen species generation, and restored vessel sprouting and tube formation. In parallel, endothelial cell-specific SIRT3 transgenic mice showed decreased fibrosis, as well as improved cardiac function and microvascular network, compared with wild-type mice with similar stimuli.Collectively, these findings suggest that SIRT3 could promote angiogenesis through attenuating mitochondrial dysfunction caused by defective mitophagy.

Defective autophagy of monocytes or macrophages might result in NLRP3 inflammasome activation and cause vascular metabolic inflammation. However, the mechanism underlying the initiation of the autophagy response to hyperlipidaemia remains unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is sensitive to the metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated autophagy in regulating NLRP3 inflammasome activation. We determined that the inhibition of autophagy and the activation of the NLRP3 inflammasome were concomitant with reduced SIRT3 levels both in peripheral blood monocytes from obese humans and in palmitate-treated THP-1 cells. Furthermore, we demonstrated that SIRT3 could form a molecular complex with ATG5, while SIRT3 overexpression altered the acetylation of endogenous ATG5. ATG5 acetylation inhibited autophagosome maturation and induced NLRP3 inflammasome activation. In parallel, SIRT3 overexpression in THP-1 cells decreased the palmitate-induced generation of mitochondrial reactive oxygen species, restored autophagy, and attenuated NLRP3 inflammasome activation. The incubation of human aortic endothelial cells (HAECs) with macrophage-conditioned medium (MCM) induced HAEC expression of vascular cell adhesion molecule-1, intercellular adhesion molecule 1, α-smooth muscle actin, and collagen-1. The effect of MCM could be reversed by the addition of neutralizing anti-IL-1β antibody or the overexpression of SIRT3. Consistent with this, en face analyses displayed a marked increase in α-SMC-positive endothelial cells in SIRT3-/- mice with acute hyperlipidaemia. Taken together, these findings revealed that SIRT3-deficient macrophages displayed impaired autophagy and accelerated NLRP3 inflammasome activation and endothelial dysfunction.

Objective— Recent studies have shown that altered mitochondrial dynamics impairs the function in senescent endothelial cells (ECs). However, the underlying molecular mechanism remains to be elucidated. Herein, we investigated the role and underlying mechanism of mitochondrial fission protein dynamin-related protein 1 (DRP1) in vascular aging. Approach and Results— We found that DRP1 expression is decreased in senescent ECs, accompanied with long interconnected mitochondria and impaired angiogenic function. In addition, there was marked increase of autophagosomes but not of autolysosomes (assessed as punctate dual fluorescent mCherry-GFP (green fluorescent protein) tandem-tagged light chain 3 expression) in senescent ECs, indicating impaired autophagic flux. DRP1 knockdown or pharmacological inhibition in young ECs resulted in elongated mitochondria, suppressed autophagic flux, premature senescence, and impaired angiogenic function. In contrast, adenoviral-mediated overexpression of DRP1 in senescent ECs restored autophagic flux and improved angiogenic function. EC senescence was associated with the increase of mitochondrial reactive oxygen species and antioxidant N-acetyl-cysteine restored autophagosome clearance and improved angiogenic function. Consistently, en face staining of old rat thoracic aorta revealed a decrease of DRP1 expression and increase of autophagosomes accumulation. Furthermore, in vivo knockdown of Drp1 in common carotid arteries significantly impaired the autophagosome clearance. Importantly, downregulation of Drp1 directly abrogated microvessels outgrowth from ex vivo aortic rings. Conclusions— These results suggest that loss of DRP1 during senescence exacerbates ECs dysfunction by increasing mitochondrial reactive oxygen species and subsequently inhibiting autophagic flux.

Overwhelming oxidative stress is implicated as crucial in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Liraglutide, a well-established antidiabetes drug, was recently reported to ameliorate NAFLD with an elusive mechanism. We used a mouse model to examine whether liraglutide could ameliorate NAFLD and explored the possible mechanisms.Twenty C57BL/6J mice were randomly treated with a normal-fat diet or high-fat diet for 16 weeks, then further distributed into four groups and subjected to s.c. injection of liraglutide or saline for 4 weeks. The growth/metabolism, oxidative stress, mitochondrial architecture and autophagy were assessed prospectively at the 20th week.High-fat diet inducement resulted in severe NAFLD while liraglutide treatment significantly reversed the trend, marked by reduced bodyweight, improved glucose tolerance and liver triglyceride composition. Reduced hepatic malondialdehyde level, increased mRNA and protein levels of CATALASE and MNSOD indicated liraglutide affected both the oxidative and antioxidative process to ameliorate oxidative stress. After liraglutide administration, the upregulated mRNA and protein levels of mitochondrial fission and fusion-related DRP1, OPA1 and respiratory chain-related COMPLEX1, UCP2 demonstrated the enhancement of mitochondrial architecture which may attenuate the generation of reactive oxygen species (ROS), while the diminished mRNA and protein level of P62 and increased levels of Beclin1 and LC3II/I ratio indicated the promoting autophagy, which probably contribute to the ROS elimination. Further, restored protein levels of Sirtuin1/Sirtuin3 and the downstream p-FOXO3a reveal the probable pathways of liraglutide acting on autophagy.Liraglutide diminishes oxidative stress by enhancing mitochondrial architecture and promoting autophagy through the SIRT1/SIRT3-FOXO3a-LC3 pathway to ameliorate diet-induced NAFLD.

Whether prediabetes mellitus alone or combined with other disorders means a higher risk for cardiovascular disease (CVD) is still controversial. This study aimed to investigate the association between prediabetes mellitus and CVD and diabetes mellitus and to explore whether prediabetes mellitus alone or combined with other syndromes, such as hypertension, could promote CVD risks significantly. This longitudinal population-based study of 1609 residents from Shanghai in Southern China was conducted between 2002 and 2014. Participants with a history of CVD at baseline were excluded from analysis. Multivariate log-binomial regression models were used to adjust possible coexisting factors. Incidence of CVD during follow-up was 10.1%. After adjusting for age, sex, and other factors, the association between prediabetes mellitus and CVD was not observed. When hypertension was incorporated in stratifying factors, adjusted CVD risk was elevated significantly (odds ratio, 2.41; 95% confidence interval, 1.25-4.64) in prediabetes mellitus and hypertension combined group, and coexistence of diabetes mellitus and hypertension made CVD risk highly significantly increased, reaching 3.43-fold higher than the reference group. Blood glucose level within prediabetic range is significantly associated with elevated risks for diabetes mellitus after multivariable adjustment, but only when it is concurrent with other disorders, such as hypertension, it will significantly increase CVD risk.

Insulin resistance is an important feature of type 2 diabetes and obesity. The underlying mechanisms of insulin resistance are still unclear and may involve pathological changes in multiple tissues. Mitochondrial dysfunction, including mitochondrial loss and over-production of oxidants, has been suggested to be involved in the development of insulin resistance. Increasing evidence suggests that targeting mitochondria to protect mitochondrial function as a unique measure, i.e. mitochondrial medicine, could prevent and ameliorate various diseases associated with mitochondrial dysfunction. In this review, we have summarized recent progress in pharmaceutical and nutritional studies of drugs and nutrients to targeting mitochondria by stimulating mitochondrial metabolism (biogenesis and degradation) to improve mitochondrial function and decrease oxidative stress for preventing and ameliorating insulin resistance. We have focused on nutrients from natural sources to stimulating mitochondrial biogenesis in cellular systems and in animal models. The in vitro and in vivo studies, especially our own work on the effects and mechanisms of mitochondrial targeting nutrients or their combinations, may help us to understand the importance and mechanisms of mitochondrial biogenesis in insulin resistance, and provide hope for developing mitochondria-targeting agents for preventing and treating insulin resistance in type 2 diabetes and obesity.

Physical exercise is considered to exert a positive effect on health, whereas strenuous or excessive exercise (Exe) causes fatigue and damage to muscle and immune functions. The underlying molecular mechanisms are still unclear. We designed a protocol to mimic Exe and explore the ensuing cellular damage and involvement of mitochondrial dynamics. We found that Exe was prone to decrease endurance capacity and induce damage to renal function and the immune system. Muscle atrophy markers atrogin-1 and MuRF1 mRNA were increased by Exe, accompanied by increased autophagy and mitochondrial fission in skeletal muscle. Exe caused a decrease in PGC-1α and complex I expression; it also activated JNK and Erk1/2 pathways and consequently induced p53, p21, and MnSOD expression in skeletal muscle. The involvement of oxidant-induced autophagy and mitochondrial dysfunction was confirmed in C2C12 myoblasts. Hydroxytyrosol (HT), a natural olive polyphenol, efficiently enhanced endurance capacity and prevented Exe-induced renal and immune system damage. Also, HT treatment inhibited both the Exe-induced increase in autophagy and mitochondrial fission and the decrease in PGC-1α expression. In addition, HT enhanced mitochondrial fusion and mitochondrial complex I and II activities in muscle of Exe rats. These results demonstrate that Exe-induced fatigue and damage to muscle and immune functions may be mediated via the regulation of mitochondrial dynamic remodeling, including the downregulation of mitochondrial biogenesis and upregulation of autophagy. HT supplementation may regulate mitochondrial dynamic remodeling and enhance antioxidant defenses and thus improve exercise capacity under Exe conditions.

Endurance exercise causes fatigue due to mitochondrial dysfunction and oxidative stress. In order to find an effective strategy to prevent fatigue or enhance recovery, the effects of a combination of mitochondrial targeting nutrients on physical activity, mitochondrial function and oxidative stress in exercised rats were studied. Rats were subjected to a four-week endurance exercise regimen following four weeks of training. The effects of exercise and nutrient treatment in rat liver were investigated by assaying oxidative stress biomarkers and activities of mitochondrial complexes. Endurance exercise induced an increase in activities of complexes I, IV, and V and an increase in glutathione (GSH) levels in liver mitochondria; however, levels of ROS and malondialdehyde (MDA) and activities of complexes II and III remained unchanged. Exercise also induced a significant increase in MDA and activities of glutathione S-transferase and NADPH-quinone-oxidoreductase 1 (NQO-1) in the liver homogenate. Nutrient treatment caused amelioration of complex V and NQO-1 activities and enhancement of activities of complex I and IV, but had no effect on other parameters. These results show that endurance exercise can cause oxidative and mitochondrial stress in liver and that nutrient treatment can either ameliorate or enhance this effect, suggesting that endurance exercise-induced oxidative and mitochondrial stress may be either damaging by causing injury or beneficial by activating defense systems.

The aim of the study was to address the importance of mitochondrial function in insulin resistance and type 2 diabetes, and also to identify effective agents for ameliorating insulin resistance in type 2 diabetes. We examined the effect of two mitochondrial nutrients, R-alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC), as well as their combined effect, on mitochondrial biogenesis in 3T3-L1 adipocytes.Mitochondrial mass and oxygen consumption were determined in 3T3-L1 adipocytes cultured in the presence of LA and/or ALC for 24 h. Mitochondrial DNA and mRNA from peroxisome proliferator-activated receptor gamma and alpha (Pparg and Ppara) and carnitine palmitoyl transferase 1a (Cpt1a), as well as several transcription factors involved in mitochondrial biogenesis, were evaluated by real-time PCR or electrophoretic mobility shift (EMSA) assay. Mitochondrial complexes proteins were measured by western blot and fatty acid oxidation was measured by quantifying CO2 production from [1-14C]palmitate.Treatments with the combination of LA and ALC at concentrations of 0.1, 1 and 10 micromol/l for 24 h significantly increased mitochondrial mass, expression of mitochondrial DNA, mitochondrial complexes, oxygen consumption and fatty acid oxidation in 3T3L1 adipocytes. These changes were accompanied by an increase in expression of Pparg, Ppara and Cpt1a mRNA, as well as increased expression of peroxisome proliferator-activated receptor (PPAR) gamma coactivator 1 alpha (Ppargc1a), mitochondrial transcription factor A (Tfam) and nuclear respiratory factors 1 and 2 (Nrf1 and Nrf2). However, the treatments with LA or ALC alone at the same concentrations showed little effect on mitochondrial function and biogenesis.We conclude that the combination of LA and ALC may act as PPARG/A dual ligands to complementarily promote mitochondrial synthesis and adipocyte metabolism.

MicroRNAs (miRNAs), which are genomically encoded small RNAs, negatively regulate target gene expression at the post-transcriptional level. Our recent study indicated that microRNA-155 (miR-155) might be negatively correlated with blood pressure, and it has been suggested that miR-155-mediated target genes could be involved in the cardiovascular diseases. Bioinformatic analyses predict that angiotensin II type 1 receptor (AT(1)R) is a miR-155 target gene. The present study investigated the potential role of miR-155 in regulating AT(1)R expression and phenotypic differentiation in rat aortic adventitial fibroblasts (AFs). Luciferase assay demonstrated that miR-155 suppressed AT(1)R 3'-UTR reporter construct activity. miR-155 overexpression in AFs did not reduce target mRNA levels, but significantly reduced target protein expression. In addition, AFs transfected with pSUPER/miR-155 exhibited reduced Ang II-induced ERK1/2 activation. miR-155 overexpression in cells attenuated Ang II-induced α-smooth muscle actin (α-SMA, produces myofibroblast) expression, but did not transform growth factor beta-1 (TGF-β1). This study demonstrated that miR-155 could have an important role in regulating adventitial fibroblast differentiation and contribute to suppression of AT(1)R expression.

Mitochondrial dysfunction and oxidative damage are highly involved in the pathogenesis of Parkinson's disease (PD). Some mitochondrial antioxidants/nutrients that can improve mitochondrial function and/or attenuate oxidative damage have been implicated in PD therapy. However, few studies have evaluated the preventative effects of a combination of mitochondrial antioxidants/nutrients against PD, and even fewer have sought to optimize the doses of the combined agents. The present study examined the preventative effects of two mitochondrial antioxidant/nutrients, R-alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC), in a chronic rotenone-induced cellular model of PD. We demonstrated that 4-week pretreatment with LA and/or ALC effectively protected SK-N-MC human neuroblastoma cells against rotenone-induced mitochondrial dysfunction, oxidative damage and accumulation of alpha-synuclein and ubiquitin. Most notably, we found that when combined, LA and ALC worked at 100-1000-fold lower concentrations than they did individually. We also found that pretreatment with combined LA and ALC increased mitochondrial biogenesis and decreased production of reactive oxygen species through the up-regulation of the peroxisome proliferator-activated receptor-gamma coactivator 1alpha as a possible underlying mechanism. This study provides important evidence that combining mitochondrial antioxidant/nutrients at optimal doses might be an effective and safe prevention strategy for PD.

Chemerin is a novel adipokine associated with obesity and metabolic syndrome. Previous studies indicate that chemerin may also function as a stimulator of angiogenesis. However, the underlying mechanism of its regulatory role in angiogenesis remains largely unknown. In this study, we determined the role of autophagy in chemerin-induced angiogenesis. Treatment of human aorta endothelial cells (HAECs) with chemerin increased the generation of mitochondrial reactive oxygen species (ROS) concurrent with the induced, time-dependent expression of LC3II and upregulation of the autophagy-related genes beclin-1, Atg7, and Atg12–Atg5 . Knockdown of chemerin receptor 23 (ChemR23) by shRNA or treatment with the mitochondria-targeted antioxidant Mito-TEMPO decreased the chemerin-associated ROS generation and abolished the upregulation of autophagy-related genes. Furthermore, chemerin treatment of HAECs augmented AMP-activated protein kinase-α (AMPKα) activity and acetyl-CoA carboxylase phosphorylation and reduced phosphorylation of the mammalian target of rapamycin, ribosomal protein S6 kinase-1, and eukaryotic initiation factor 4E-binding protein 1, which were blocked by coadministration of Mito-TEMPO or shRNA-mediated knockdown of AMPKα. Analysis of the HAECs revealed that inhibition of autophagy by Mito-TEMPO or shRNA against ChemR23, AMPKα, and beclin-1 impaired chemerin-induced tube formation and cell proliferation. These studies show that mitochondrial ROS are important for autophagy in chemerin-induced angiogenesis and that targeting autophagy may provide an important new tool for treating cardiovascular disease.

Endothelial-to-mesenchymal transition (EndoMT) has recently emerged as a potentially important contributor in promoting fibrosis in chronic kidney disease. However, little is known about the role and molecular basis of its involvement in hypertensive renal injury. Here, we aim to determine the role of SIRT (sirtuin) 3 on EndoMT in hypertensive renal injury and to explore its underlying mechanisms. We found that SIRT3 expression was significantly reduced in Ang II (angiotensin II)-induced hypertensive model, accompanied with induction of EndoMT and increased reactive oxygen species and renal fibrosis. In SIRT3-/- (SIRT3 knockout) mice subjected to Ang II infusion, renal dysfunction was aggravated with an increased EndoMT and reactive oxygen species level, whereas in SIRT3-TgEC (SIRT3 endothelial cell-specific transgenic) mice, the Ang II-induced renal fibrosis and EndoMT and oxidative stress were ameliorated. With primary mouse glomerular endothelial cells, we confirmed that Ang II treatment initiated EndoMT and decreased catalase expression, which were suppressed by SIRT3 overexpression. Using immunoprecipitation, luciferase, and chromatin immunoprecipitation assay, we demonstrated that SIRT3-mediated deacetylation and nuclear localization of Foxo3a (forkhead box O3a) resulted in activated Foxo3a-dependent catalase expression. Moreover, Foxo3a knockdown abolished SIRT3-mediated suppression of EndoMT. In conclusion, these results established the SIRT3-Foxo3a-catalase pathway as a critical factor in the maintenance of endothelial homeostasis and point to an important role of EndoMT in the vascular pathology of renal fibrosis, which may provide a new therapeutic target to impede the progression of hypertensive renal injury.

Objective: Infiltrated macrophages actively promote perivascular adipose tissue remodeling and represent a dominant population in the perivascular adipose tissue microenvironment of hypertensive mice. However, the role of macrophages in initiating metabolic inflammation remains uncertain. SIRT3 (sirtuin-3), a NAD-dependent deacetylase, is sensitive to metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated metabolic shift in regulating NLRP3 (Nod-like receptor family pyrin domain-containing 3) inflammasome activation. Approach and Results: Here, we report that Ang II (angiotensin II) accelerates perivascular adipose tissue inflammation and fibrosis, accompanied by NLRP3 inflammasome activation and IL (interleukin)-1β secretion in myeloid SIRT3 knockout (SIRT3 −/ − ) mice. This effect is associated with adipose tissue mitochondrial dysfunction. In vitro studies indicate that the deletion of SIRT3 in bone marrow–derived macrophages induces IL-1β production by shifting the metabolic phenotype from oxidative phosphorylation to glycolysis. Mechanistically, SIRT3 deacetylates and activates PDHA1 (pyruvate dehydrogenase E1 alpha) at lysine 83, and the loss of SIRT3 leads to PDH activity decrease and lactate accumulation. Knocking down LDHA (lactate dehydrogenase A) or using carnosine, a buffer against lactic acid, attenuates IL-1β secretion. Furthermore, the blockade of IL-1β from macrophages into brown adipocytes restores thermogenic markers and mitochondrial oxygen consumption. Moreover, NLRP3 knockout (NLRP3 −/− ) mice exhibited reduced IL-1β production while rescuing the mitochondrial function of brown adipocytes and alleviating perivascular adipose tissue fibrosis. Conclusions: SIRT3 represents a potential therapeutic target to attenuate NLRP3-related inflammation. Pharmacological targeting of glycolytic metabolism may represent an effective therapeutic approach.

The Chinese economy has entered the phase of high-quality development. Urbanization is an important driving factor in promoting the domestic economic cycle, while tourism is an emerging force in the development of urbanization. The convergence of these two factors will contribute to the high-quality development of regional economies. By constructing an evaluation index system of tourism development and urbanization level, 26 cities in the Yangtze River Delta Urban Agglomeration have been identified as the study area. The study has adopted the entropy method and the coupling coordination model to analyze the comprehensive development level of tourism and urbanization and the coupling coordination relationship between them from 2008 to 2018. The results show that the Yangtze River Delta presents a spatial pattern of orderly changes in the development of tourism, forming a spatial structure of “one pole and many centers”, with Shanghai as the core. In terms of spatial distribution, it generally presents the spatial trend characteristics of “high in the east and low in the west” in the east–west direction, and “protruding in the middle and lower at both ends” in the south–north direction. Coordination and interaction are steadily developing to a high level, with significant spatial dependence and spillover effects. Based on the research results, the study applies a new development vision to explore the coupling coordination high-quality development mechanism of tourism and urbanization in the Yangtze River Delta region; the corresponding policy recommendations are discussed.

Phenotypic differentiation of adventitial fibroblasts into myofibroblasts is an essential feature of vascular remodeling. The present study was undertaken to test the hypothesis that reactive oxygen species (ROS) are involved in rat adventitial fibroblast differentiation to myofibroblast. Activation of α-smooth muscle a‘ctin (α-SMA) was used as a marker of myofibroblast. Angiotensin II increased intracellular ROS in adventitial fibroblasts that was completely inhibited by the free radical scavenger NAC, the NAD(P)H oxidase inhibitor DPI, and transfection of antisense gp91phox oligonucleotides. Myofibroblast differentiation was prevented by inhibition of ROS generation with DPI, NAC, and antisense gp91phox as shown by decreased expression of α-SMA. Angiotensin II rapidly induced phosphorylation of p38 MAPK and JNK, both of which were inhibited by DPI, NAC, antisense gp91phox, and the selective AT1 receptor antagonist, losartan. Inhibiting p38MAPK with SB202190 or JNK with SP600125 also reduced angiotensin II-induced α-SMA expression. These findings demonstrate that angiotensin II induces adventitial fibroblast differentiation to myofibroblast via a pathway that involves NADPH oxidase generation of ROS and activation of p38MAPK and JNK pathways.

Abstract The development of type 2 diabetes is accompanied by decreased immune function and the mechanisms are unclear. We hypothesize that oxidative damage and mitochondrial dysfunction may play an important role in the immune dysfunction in diabetes. In the present study, we investigated this hypothesis in diabetic Goto‐Kakizaki rats by treatment with a combination of four mitochondrial‐targeting nutrients, namely, R‐α‐lipoic acid, acetyl‐L‐carnitine, nicotinamide and biotin. We first studied the effects of the combination of these four nutrients on immune function by examining cell proliferation in immune organs (spleen and thymus) and immunomodulating factors in the plasma. We then examined, in the plasma and thymus, oxidative damage biomarkers, including lipid peroxidation, protein oxidation, reactive oxygen species, calcium and antioxidant defence systems, mitochondrial potential and apoptosis‐inducing factors (caspase 3, p53 and p21). We found that immune dysfunction in these animals is associated with increased oxidative damage and mitochondrial dysfunction and that the nutrient treatment effectively elevated immune function, decreased oxidative damage, enhanced mitochondrial function and inhibited the elevation of apoptosis factors. These effects are comparable to, or greater than, those of the anti‐diabetic drug pioglitazone. These data suggest that a rational combination of mitochondrial‐targeting nutrients may be effective in improving immune function in type 2 diabetes through enhancement of mitochondrial function, decreased oxidative damage, and delayed cell death in the immune organs and blood.

Emerging evidence indicates exercise training could mediate mitochondrial quality control through the improvement of mitochondrial dynamics. Ginsenoside Rg3 (Rg3), one of the active ingredients in Panax ginseng, is well known in herbal medicine as a tonic and restorative agent. However, the molecular mechanism underlying the beneficial effects of Rg3 has been elusive. In the present study, we compared the effects of Rg3 administration with aerobic exercise on mitochondrial adaptation in cardiac muscle tissue of Sprague–Dawley (SD) rats. Three groups of SD rats were studied: (1) sedentary control, (2) Rg3-treated and (3) aerobic exercise trained. Both aerobic exercise training and Rg3 supplementation enhanced peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) and nuclear factor-E2-related factor 2 (Nrf2) protein levels in cardiac muscle. The activation of PGC-1α led to increased mRNA levels of mitochondrial transcription factor A (Tfam) and nuclear related factor 1(Nrf1), these changes were accompanied by increases in mitochondrial DNA copy number and complex protein levels, while activation of Nrf2 increased levels of phase II detoxifying enzymes, including nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1(NQO1), superoxide dismutase (MnSOD) and catalase. Aerobic exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of beclin1 and autophagy-related protein 7 (ATG7), these effects of aerobic exercise are comparable to that of Rg3. These results demonstrate that Rg3 mimics improved cardiac adaptations to exercise by regulating mitochondria dynamic remodeling and enhancing the quantity and quality of mitochondria.

The lysosomal degradation pathway, autophagy, is essential for the maintenance of cellular homeostasis. Recently, autophagy has been demonstrated to be required in the process of adipocyte conversion. However, its role in mature adipocytes under physiological and pathological conditions remains unclear. Here, we report a major function of BECN1 in the regulation of basal autophagy in mature adipocytes. We also show that berberine, a natural plant alkaloid, inhibits basal autophagy in adipocytes and adipose tissue of mice fed a high-fat diet via downregulation of BECN1 expression. We further demonstrate that berberine has a pronounced effect on the stability of Becn 1 mRNA through the Mir30 family. These findings explore the potential of BECN1 as a key molecule and a drug target for regulating autophagy in mature adipocytes.

Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto-Kakizaki rats.We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-alpha-lipoic acid, acetyl-L-carnitine, nicotinamide, and biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator-activated receptor-alpha (Ppar alpha), peroxisome proliferator-activated receptor-delta (Ppar delta), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain.These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain.

Chemerin is an adipokine involved in obesity, inflammation, and innate immune system that is highly expressed in the liver. In the present study, we find that chemerin mRNA expression is decreased in the livers of rodents with nonalcoholic fatty liver disease as well as in HepG2 cells after lipid overloading. Moreover, we report that chemerin expression and secretion are induced in HepG2 cells and primary hepatocytes from wild-type mice, but not farnesoid X receptor (FXR)−/− mice, in response to the synthetic FXR ligand GW4064. Hepatic chemerin expression is decreased in FXR−/− mice but up-regulated by GW4064 administration in wild-type mice. Dual-luciferase reporter assay and chromatin immunoprecipitation analyses further identified a functional FXR response element located in the −258-bp /+121-bp region of the chemerin gene. These data demonstrate that chemerin, a novel target gene of FXR, is related to nonalcoholic steatohepatitis.

FoxO1 acts as a pivotal transcription factor in insulin signaling. However, in hyperglycemia induced cardiac complications, whether FoxO1 is involved remains unclear. The goal of this study was to delineate the potential role of FoxO1 under high-glucose condition.We investigated insulin resistance and reactive oxygen species (ROS) generation in H9c2 cardiomyoblasts after high-glucose exposure. A series of autophagy biomarkers were measured and further confirmed by LC3 turnover assay. Using gene silencing and overexpression experiments we dissected the molecular mechanisms of FoxO1 regulated autophagy. We also tested the protective effect of (-)-epigallocatechin-3-gallate (EGCG, a green tea-derived polyphenol) in high-glucose treated H9c2 cardiomyoblasts.High-glucose elicited elevated ROS, autophagy and FoxO1 abundance in cultured H9c2 cardiomyoblasts. Specifically, high-glucose significantly augmented the acetylated FoxO1 in cytosol. In line, compared with 3A-FoxO1 (majorly localized in nuclei with a strong transcriptional activity), overexpression of WT-FoxO1 led to more intense elevated autophagy with enhanced acetylation of FoxO1. In addition, FoxO1 RNAi brought down autophagy induced by high-glucose. Intriguingly, EGCG successfully reversed ROS, autophagy and acetylated FoxO1 in high-glucose treated H9c2 cells.Our findings suggest that FoxO1, especially the acetylated form, regulates high-glucose induced autophagy in H9c2 cardiomyoblasts, which can be prevented by EGCG via a possible ROS-FoxO1 pathway.

BACKGROUND: Efferocytosis is an activity of macrophages that is pivotal for the resolution of inflammation in hypertension. The precise mechanism by which macrophages coordinate efferocytosis and internalize apoptotic cardiomyocytes remains unknown. The aim of this study was to determine whether SIRT3 (sirtuin-3) is required for both apoptotic cardiomyocyte engulfment and anti-inflammatory responses during efferocytosis. METHODS: We generated myeloid SIRT3 knockout mice and FXN (frataxin) knock-in mice carrying an acetylation-defective lysine to arginine K189R mutation (FXN K189R ). The mice were given Ang II (angiotensin II) infusion for 7 days. We analyzed cardiac macrophages’ mitochondrial iron levels, efferocytosis activity, and phenotype both in vivo and in vitro. RESULTS: We showed that SIRT3 deficiency exacerbated Ang II–induced downregulation of the efferocytosis receptor MerTK (c-Mer tyrosine kinase) and proinflammatory cytokine production, accompanied by disrupted mitochondrial iron homeostasis in cardiac macrophages. Quantitative acetylome analysis revealed that SIRT3 deacetylated FXN at lysine 189. Ang II attenuated SIRT3 activity and enhanced the acetylation level of FXN K189 . Acetylated FXN further reduced the synthesis of ISCs (iron-sulfur clusters), resulting in mitochondrial iron accumulation. Phagocytic internalization of apoptotic cardiomyocytes increased myoglobin content, and derived iron ions promoted mitochondrial iron overload and lipid peroxidation. An iron chelator deferoxamine improved the levels of MerTK and efferocytosis, thereby attenuating proinflammatory macrophage activation. FXN K189R mice showed improved macrophage efferocytosis, reduced cardiac inflammation, and suppressed cardiac fibrosis. CONCLUSIONS: The SIRT3-FXN axis has the potential to resolve cardiac inflammation by increasing macrophage efferocytosis and anti-inflammatory activities.

α-Lipoic acid (LA) has been widely studied as an agent for preventing and treating various diseases associated with oxidative disruption of mitochondrial functions. To investigate a related mitochondrial antioxidant, we compared the effects of lipoamide (LM), the neutral amide of LA, with LA for measures of oxidative damage and mitochondrial dysfunction in a human retinal pigment epithelial (RPE) cell line. Acrolein, a major component of cigarette smoke and a product of lipid peroxidation, was used to induce oxidative mitochondrial damage in RPE cells. Overall, using comparable concentrations, LM was more effective than LA at preventing acrolein-induced mitochondrial dysfunction and oxidative stress. Relative to LA, LM improved ATP levels, membrane potentials, and activities of mitochondrial complexes I, II, and V and dehydrogenases that had been decreased by acrolein exposure. LM reduced acrolein-induced oxidant generation, calcium levels, protein oxidation, and DNA damage to a greater degree than LA. And, total antioxidant capacity, glutathione content, glutathione S-transferase, and superoxide dismutase activities and expression of nuclear factor-E2-related factor 2 were increased by LM relative to LA. These results suggest that LM is a more potent mitochondrial-protective agent and antioxidant than LA in protecting RPE from oxidative damage.

Metabolic dysfunction due to loss of mitochondria plays an important role in diabetes, and stimulation of mitochondrial biogenesis by anti-diabetic drugs improves mitochondrial function. In a search for potent stimulators of mitochondrial biogenesis, we examined the effects and mechanisms of lipoamide and α-lipoic acid (LA) in adipocytes.Differentiated 3T3-L1 adipocytes were treated with lipoamide or LA. Mitochondrial biogenesis and possible signalling pathways were examined.Exposure of 3T3-L1 cells to lipoamide or LA for 24 h increased the number and mitochondrial mass per cell. Such treatment also increased mitochondrial DNA copy number, protein levels and expression of transcription factors involved in mitochondrial biogenesis, including PGC-1α, mitochondrial transcription factor A and nuclear respiratory factor 1. Lipoamide produced these effects at concentrations of 1 and 10 µmol·L⁻¹, whereas LA was most effective at 100 µmol·L⁻¹. At 10 µmol·L⁻¹, lipoamide, but not LA, stimulated mRNA expressions of PPAR-γ, PPAR-α and CPT-1α. The potency of lipoamide was 10-100-fold greater than that of LA. Lipoamide dose-dependently stimulated expression of endothelial nitric oxide synthase (eNOS) and formation of cGMP. Knockdown of eNOS (with small interfering RNA) prevented lipoamide-induced mitochondrial biogenesis, which was also blocked by the soluble guanylate cyclase inhibitor, ODQ and the protein kinase G (PKG) inhibitor, KT5823. Thus, stimulation of mitochondrial biogenesis by lipoamide involved signalling via the eNOS-cGMP-PKG pathway.Our data suggest that lipoamide is a potent stimulator of mitochondrial biogenesis in adipocyte, and may have potential therapeutic application in obesity and diabetes.

Obesity-induced vascular dysfunction is related to chronic low-grade systemic inflammation. Recent studies indicate that NLRP3, a multiprotein complex formed by NOD-like receptor (NLR) family members, is a key component mediating internal sterile inflammation, but the role in obesity-related vascular dysfunction is largely unknown. In the present study, we investigate whether NLRP3 activation is involved in vascular inflammation in obese Otsuka Long-Evans Tokushima Fatty rats (OLETF).Male OLETF with their control Long-Evans Tokushima Otsuka rats (LETO) were studied at 3 and 12 months of age. Aortic relaxation in response to acetylcholine decreased gradually with age in both strains, with early and persistent endothelium dysfunction in obese OLETF compared with age-matched LETO controls. These changes are associated with parallel changes of aortic endothelial nitric oxide synthase (eNOS) content, macrophage accumulation and intimal thickening. NLRP3 increased in OLETF rats compared to LETO. Consistent with inflammasome activation, the conversion of procaspase-1 to cleaved and activated forms as well as IL-1β markedly increased in OLETF rats. Additionally, we observed increased expression of dynamin-related protein-1 (Drp1) and decreased fusion-relative protein optic atropy-1(OPA1). Altered mitochondrial dynamics was associated with elevated oxidative stress level in OLETF aortas.These results demonstrate that obesity seems to accelerate endothelial dysfunction in OLETFs via the activation of NLRP3 and mitochondrial dysfunction.

Nonoriented coupling of nanobodies (Nb) to nanomaterials can considerably reduce epitope recognition or induce other dysfunctions, thereby restricting the application of Nb in field detection. Herein, using Avi-tag/streptavidin (Avi/SA)-oriented coupling strategy, we developed a lateral-flow immunochromatographic assay (LFIA) based on Nb-Avi/SA@quantum dots (QDs) probes (Nb-LFIA) for Aflatoxin B1 (AFB1) detection. Compared with nonoriented coupled Nb@QD probes, the detection sensitivity and stability of Nb-Avi/SA@QD probes were greatly retained. Furthermore, Nb showed higher tolerance to the extreme detection environments compared with monoclonal antibodies in traditional enzyme-linked immunosorbent assay and LFIA. After optimization, the limit of detection of Nb-LFIA was 0.095 ng mL−1, half-maximal inhibitory concentration was 0.85 ng mL−1, and the visual cut-off level was 1.25 ng mL−1. Nb-Avi/SA@gold nanoparticle probes were assessed for the wide applicability of the Avi/SA strategy, and they showed excellent performance (coefficient of determination = 0.999). Finally, the analysis performance of probes was assessed in oat samples, revealing a recovery rate of 88.8–116.7%, which was consistent with the results of high-pressure liquid chromatography coupled with tandem mass spectrometry. Altogether, our study provides a convenient and sensitive tool for AFB1 detection, and presents a viable path for efficient Nb application.

The aim of this study was to investigate the effects of a combination of nutrients on physical performance, oxidative stress and mitochondrial biogenesis in rats subjected to exhaustive exercise. Rats were divided into sedentary control (SC), exhaustive exercise (EC) and exhaustive exercise with nutrient supplementation (EN). The nutrients include (mg/kg/day): R ‐α‐lipoic acid 50, acetyl‐ l ‐carnitine 100, biotin 0.1, nicotinamide 15, riboflavin 6, pyridoxine 6, creatine 50, CoQ10 5, resveratrol 5 and taurine 100. Examination of running distances over the 4‐week period revealed that EN rats ran significantly longer throughout the entire duration of the exhaustive exercise period compared with the EC rats. Nutrient supplementation significantly inhibited the increase in activities of alanine transaminase, lactate dehydrogenase and creatine kinase, reversed increases in malondialdehyde, inhibited decreases in glutathione S ‐transferase and total antioxidant capacity in plasma, and suppressed the elevation of reactive oxygen species and apoptosis in splenic lymphocytes. Nutrient supplementation increased the protein expression of mitochondrial complexes I, II and III, mtDNA number and transcription factors involved in mitochondrial biogenesis and fusion in skeletal muscle. These findings suggest that mitochondrial nutrient supplementation can reduce exhaustive exercise‐induced oxidative damage and mitochondrial dysfunction, thus leading to enhancement of physical performance and of fatigue recovery.

Treatment with a combination of four nutrients, i.e. R-α-lipoic acid, acetyl-l-carnitine, nicotinamide and biotin, just as with pioglitazone, significantly improves glucose tolerance, insulin release, plasma NEFA, skeletal muscle mitochondrial biogenesis and oxidative stress in Goto-Kakizaki (GK) rats. However, it is not known whether treatment with these nutrients can improve mitochondrial function and reduce oxidative stress in GK rats. The effects of a combination of these four nutrients on mitochondrial function, oxidative stress and apoptosis in GK rat liver were investigated. Livers of untreated GK rats showed (1) abnormal changes in the activities of mitochondrial complexes (decreases in I, III and IV and increases in II and V), (2) increases in protein oxidation, (3) decreases in antioxidant enzymes (superoxide dismutase, glutathione S-transferase, NADH-quinone oxidoreductase-1), (4) a decrease in total antioxidant capacity but increases in reduced glutathione level and glyceraldehyde 3-phosphate dehydrogenase expression and (5) significant increases in apoptosis biomarkers, including expression of p21 and p53. A 3-month treatment with the four nutrients significantly improved most of these abnormalities in GK rats, and the effects of the nutrient combination were greater than those of pioglitazone for most of these indices. These results suggest that dietary supplementation with nutrients that are thought to influence mitochondrial function may be an effective strategy for improving liver dysfunction in GK diabetic rats.

Chemerin is a novel adipocyte-derived factor that induces insulin resistance in skeletal muscle. However, the effect of chemerin on skeletal muscle mitochondrial function has received little attention. In the present study, we investigated whether mitochondrial dysfunction is involved in the pathogenesis of chemerin-mediated insulin resistance. In this study, we used recombinant adenovirus to express murine chemerin in C57BL/6 mice. The mitochondrial function and structure were evaluated in isolated soleus muscles from mice. The oxidative mechanism of mitochondrial dysfunction in cultured C2C12 myotubes exposed to recombinant chemerin was analysed by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction. The overexpression of chemerin in mice reduced the muscle mitochondrial content and increased mitochondrial autophagy, as determined by the increased conversion of LC3-I to LC3-II and higher expression levels of Beclin1 and autophagy-related protein-5 and 7. The chemerin treatment of C2C12 myotubes increased the generation of mitochondrial reactive oxygen species, concomitant with a reduced mitochondrial membrane potential and increased the occurrence of mitochondrial protein carbonyls and mitochondrial DNA deletions. Knockdown of the expression of chemokine-like receptor 1 or the use of mitochondria-targeting antioxidant Mito-TEMPO restored the mitochondrial dysfunction induced by chemerin. Furthermore, chemerin exposure in C2C12 myotubes not only reduced the insulin-stimulated phosphorylation of protein kinase B (AKT) but also dephosphorylated forkhead box O3α (FoxO3α). Chemerin-induced mitochondrial autophagy likely through an AKT-FoxO3α-dependent signalling pathway. These findings provide direct evidence that chemerin may play an important role in regulating mitochondrial remodelling and function in skeletal muscle.

Regulatory T cells (Treg cells) play important roles in hypertension and organ damages. MicroRNA-31 (miR-31) is a critical regulator for Treg cell generation. However, the role of miR-31 in hypertension has not been elucidated. We aim to study the functionality of miR-31 and the detailed mechanism in Ang II (Angiotensin II)-induced hypertensive mouse model. We found: In vitro, miR-31 expression was higher in T helper 17 cells and lower in Treg cells than that of naïve T cells. The genetic deficiency of miR-31 promoted Treg cell differentiation, whereas no impact on T helper 17 cells differentiation. Ang II-induced hypertension resulted in increased expression of miR-31 in the aorta, splenic CD4+ T cells, and kidney leukocytes. MiR-31 deficiency strikingly decreased systolic blood pressure and diastolic blood pressure and attenuated renal and vascular damage. MiR-31 deletion altered the accumulation of Treg cells and macrophages and expression of inflammatory cytokines in kidneys in Ang II-induced hypertensive mice. Ang II treatment reduced the levels of anti-inflammatory cytokines and increased proinflammatory cytokines in plasma that were blunted by the miR-31 deletion. Ppp6C (protein phosphatase 6c; a direct target of miR-31) specific deletion in Treg cells led to marked impairment of Treg cell induction, increased Ang II-induced blood pressure elevation, and organ damage in mice. In conclusion, we provided novel evidence of miR-31 as an emerging key posttranscriptional regulator of hypertension-associated immunosuppression through targeting ppp6C which is a critical regulator in the differentiation of Treg cells. This study offers new perspectives on miRNA-based therapeutic approaches.

The FASEB JournalVolume 34, Issue 12 p. 16645-16661 RESEARCH ARTICLEFree Access Sirtuin 3 governs autophagy-dependent glycolysis during Angiotensin II-induced endothelial-to-mesenchymal transition Jing Gao, Jing Gao Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaSearch for more papers by this authorTong Wei, Tong Wei Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaSearch for more papers by this authorChenglin Huang, Chenglin Huang Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaSearch for more papers by this authorMengwei Sun, Mengwei Sun Key Laboratory of State General Administration of Sport, Shanghai Research Institute of Sports Science, Shanghai, ChinaSearch for more papers by this authorWeili Shen, Corresponding Author Weili Shen wlshen@sibs.ac.cn weili_shen@hotmail.com Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Correspondence Weili Shen, Department of Hypertension, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. Email: wlshen@sibs.ac.cn, weili_shen@hotmail.comSearch for more papers by this author Jing Gao, Jing Gao Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaSearch for more papers by this authorTong Wei, Tong Wei Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaSearch for more papers by this authorChenglin Huang, Chenglin Huang Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaSearch for more papers by this authorMengwei Sun, Mengwei Sun Key Laboratory of State General Administration of Sport, Shanghai Research Institute of Sports Science, Shanghai, ChinaSearch for more papers by this authorWeili Shen, Corresponding Author Weili Shen wlshen@sibs.ac.cn weili_shen@hotmail.com Department of Cardiovascular Medicine, Department of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Correspondence Weili Shen, Department of Hypertension, Shanghai Key Laboratory of Hypertension, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China. Email: wlshen@sibs.ac.cn, weili_shen@hotmail.comSearch for more papers by this author First published: 01 November 2020 https://doi.org/10.1096/fj.202001494R Jing Gao and Tong Wei contributed equally to this work. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract The impairment of autophagy can cause cellular metabolic perturbations involved in endothelial-to-mesenchymal transition (EndoMT). However, the interplay between the cellular autophagy machinery and endothelial metabolism remains elusive. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is a major cellular sensor of energy metabolism. The aim of this work was to determine the role of SIRT3-mediated autophagy in cellular metabolism and the process of EndoMT. We demonstrated that Angiotensin II (Ang II) led to defective autophagic flux and high levels of glycolysis in endothelial cells (ECs) accompanied by a loss of mitochondrial SIRT3 during EndoMT. The loss of SIRT3 further induced the hyperacetylation of endogenous autophagy-regulated gene 5 (ATG5), which in turn inhibited autophagosome maturation and increased pyruvate kinase M2 (PKM2) dimer expression. The M2 dimer is the less active form of PKM2, which drives glucose through aerobic glycolysis. Additionally, TEPP-46, a selective PKM2 tetramer activator, produced lower concentrations of lactate and led to the reduction of EndoMT both in vitro and in vivo. In parallel, the blockade of lactate influx from ECs into vascular smooth muscle cells (VSMCs) downregulated synthetic VSMC markers. EC-specific SIRT3 transgenic mice exhibited reduced endothelial cell transition but partial rescue of vascular fibrosis and collagen accumulation. Taken together, these findings reveal that SIRT3 regulates EndoMT by improving the autophagic degradation of PKM2. Pharmacological targeting of glycolysis metabolism may, therefore, represent an effective therapeutic strategy for hypertensive vascular remodeling. Abbreviations 2-DG 2-Deoxy-D-glucose Ang II Angiotensin II ATG5 autophagy-regulated gene 5 CHX cycloheximide CM culture medium ECAR extracellular acidification rate EndoMT endothelial-to-mesenchymal transition NAM nicotinamide OCR oxygen consumption rate PKM2 pyruvate kinase M2 SIRT3 Sirtuin 3 TEPP-46 Thieno[3,2-b]Pyrrole[3,2-d]Pyridazinones 1 INTRODUCTION Autophagy is a cellular catabolic process that degrades damaged proteins and organelles via a lysosome-dependent pathway. It is crucial for regulating protein and mitochondrial quality control and ensuring intracellular homeostasis.1 Evidence suggests that defective autophagy in endothelial cells (ECs) contributes to endothelial dysfunction, which is involved in the pathological process of vascular diseases.2, 3 Systemic- or endothelial-specific deletion of core autophagy-regulated genes (ATG), including ATG3, ATG5, and ATG7, in mice attenuates nitric oxide (NO) generation and ischemia-related angiogenesis while accelerating the endothelial-to-mesenchymal transition (EndoMT).4-6 Loss of Beclin 1- induced endothelium senescence.7 In view of these findings, EC-associated autophagy may serve as a potential therapeutic target to improve vessel functions. EndoMT is the hallmark of endothelial plasticity and contributes to pathological fibrosis. During the complex biological process, ECs lose their specific markers, such as VE-cadherin and CD31, and gain mesenchymal cell markers, such as α-smooth muscle actin (α-SMA) and fibroblast-specific protein 1 (FSP-1).8, 9 Functionally, cells acquire myofibroblast-like characteristics with contractile function, enhanced migratory phenotypes, and increased extracellular matrix production.10 Pharmacological inhibition of autophagy or small interfering RNA (siRNA) for ATG5-induced EndoMT.4, 11 Metabolic changes that rely on autophagy impairment drive endothelial phenotypic differentiation. In general, ECs tend to utilize aerobic glycolysis metabolism to survive, even under ample oxygen conditions. When ECs switch from a quiescent to proliferative state, glycolytic flux is increased, and oxygen consumption is reduced.12, 13 Pyruvate kinase (PK) is responsible for the last step in glucose metabolism by converting phosphoenolpyruvate (PEP) to pyruvate and phosphorylating ADP to produce ATP. Among the four PK isoforms, PKM2 is the preferential form expressed by hyperproliferative cells. PKM2 shifts between inactive dimeric and active tetrameric forms. The active PKM2 tetramer produces pyruvate for oxidative phosphorylation, whereas the less active PKM2 dimer drives aerobic glycolysis.14 Feng et al demonstrated that autophagy-related proteins inhibit the Warburg effect by suppressing PKM2 activity.15 Similar reports indicate that HK2 is required for the regulation of glycolysis by autophagy.16 These results revealed that impaired autophagy contributes to the substantial concomitant enhancement of glycolysis; thus, autophagy may provide a novel mechanism of metabolic reprogramming during EndoMT. Nevertheless, the precise mechanisms through which the autophagy pathway facilitates glycolysis remain unclear. SIRT3 (Sirtuin 3) is a mitochondrial NAD+-dependent deacetylase that controls the energy metabolism by deacetylation of key oxidative phosphorylation enzymes. SIRT3 also acts as a positive regulator of autophagy via its deacetylation of crucial autophagic proteins.17-19 Our previous study revealed that ablation of SIRT3 directly impairs EC-associated autophagy, resulting in severe microvascular rarefaction. Moreover, several groups have found that the loss of SIRT3 causes a shift toward glycolytic metabolism.20 This evidence implies that SIRT3 may coordinate autophagy activity and glycolytic metabolism to regulate ECs phenotype; however, their roles in the mechanisms of autophagy defect-induced glycolysis during EndoMT are unknown. Angiotensin II (Ang II) is the main effector peptide of the renin-angiotensin system (RAS) and plays a pivotal role in initiating EndoMT progression.21 Here, we hypothesized that SIRT3 might serve as an important regulator of the balance between autophagy and glycolytic pathways during Ang II-induced EndoMT. We investigated the role of SIRT3-mediated autophagy in regulating the metabolic switch by altering the metabolic fate of glucose using a fully labeled form (13C6-glucose) in ECs. Finally, we evaluated the effects by which lactate accumulation by ECs creates a vascular acidic microenvironment, altering the neighboring VSMC phenotype. 2 MATERIALS AND METHODS 2.1 Materials Ang II, β-actin, myosin, and LC3B antibodies were purchased from Sigma Chemicals (St. Louis, MO, USA). Antibodies against CD31, VE-cadherin, α-SMA, and collagen I were purchased from Abcam (Cambridge, MA, USA). Anti-monocarboxylate transporter 4 (MCT4) was purchased from Santa Cruz (Dallas, TX, USA). Pyruvate kinase muscle isozyme M2 (PKM2), autophagy-related protein 5 (ATG5), P62, vimentin, and SIRT3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-monocarboxylate transporter 1 (MCT1), ki67 and glucose transporter 1 (GLUT1) antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Secondary antibodies for Western blot analysis, including peroxidase-conjugated rabbit anti-goat IgG, rabbit anti-mouse IgG, and goat anti-rabbit IgG, were supplied by Jackson ImmunoResearch (West Grove, PA, USA). Fluorescence-labeled secondary antibodies, including Alexa Fluor 555-labeled anti-mouse IgG and Alexa Fluor 488-labeled anti-rabbit IgG, were purchased from Life Technologies (Carlsbad, CA, USA). Cell culture reagents were purchased from HyClone (Logan, UT, USA). 2-Deoxy-D-glucose (2-DG) was purchased from Profleader (Shanghai, China). TEPP-46 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). 2.2 Cell culture Murine aortic endothelial cells (MAECs) were purchased from Chi Scientific (Jiangsu, China) and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 μg/mL endothelial cell growth factor, 100 μg/mL heparin, and 1% (v/v) penicillin-streptomycin. Cells were maintained in a humidified atmosphere with 95% air and 5% CO2 at 37°C. Cells between passages 4 and 6 were used in this study. Cells were subcultured in 24-well plates and treated with or without 1 × 10−7 mol/L Ang II in 1% (w/v) bovine serum albumin (BSA)/DMEM. The conditioned medium can be divided into the following 10 types: those derived from WT-ECs with or without Ang II stimulation, those derived from SIRT3-knockdown ECs with or without Ang II stimulation, those derived from SIRT3-overexpression ECs with or without Ang II stimulation, those derived from ATG5-knockdown ECs with or without Ang II stimulation, and those derived from ATG5-overexpression ECs with or without Ang II stimulation. A mouse vascular smooth muscle cell line (MOVAS) was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. To detect the effect of lactate on the phenotype in MOVAS, cells were cultured in medium without glucose supplementation with lactate (4 or 8 × 10−6 mol/L) for 24 hours. 2.3 Lentivirus infection To silence SIRT3 or ATG5 expression, the DNA oligonucleotides used to generate short-hairpin RNA (shRNA) against the open reading frame of SIRT3 mRNA were 5′-CTGTACTGGCGTTGTGAAA-3′. The sequence of ATG5 shRNA was 5′-ATCTGAGCTACCCAGATAA-3′. Two shRNA fragments were synthesized and inserted into the lentiviral shRNA GV115 vector (Genechem, Shanghai, China). The viruses containing nonsense shRNA were used as controls. In order to overexpress SIRT3 or ATG5, the subcloned full length cDNA for SIRT3 and ATG5 were synthesized and inserted into lentiviral vector GV365 (Genechem, Shanghai, China). The scramble sequences were used as negative controls. MAECs were subcultured into 6-well plates. The cells were grown in regular media until 50% confluence and infected with recombinant lentivirus at a multiplicity of infection (MOI) of 25. The lentiviruses harbored murine SIRT3 shRNA, LV-SIRT3 , murine ATG5 shRNA, LV-ATG5 or a GFP-expressing control vector. The transduced cells were treated with or without Ang II (1 × 10−7 mol/L) for 24 hours. 2.4 Animal models SIRT3 KO (SIRT3−/−) mice on a 129/Sv background were purchased from Jackson Laboratories (Bar Harbor, ME, USA). SIRT3flox/flox transgenic mice were a gift from Professor Weiliang Xia (Shanghai Jiaotong University). Endothelial-specific SIRT3 overexpression (SIRT3-TgEC) mice were constructed by crossing SIRT3-Tg mice with Tek-Cre transgenic mice (Shanghai Research Centre for Model Organisms). The offspring were identified by tail-snip PCR with the following primers: forward 5′-ACTGCTCATCAACCGGGAC-3′; reverse 5′-CGCACACCGGCCTTATTCCAA-3′; forward 5′-ACTCCAAGGCCACTTATCACC-3′; reverse 5′-ATTGTTACCAACTGGGACGACA-3′.18 Eight-week-old male WT, SIRT3 KO, and SIRT3-TgEC mice were infused with 1400 ng/kg/min Ang II or saline for 2 weeks (n = 12/group) via subcutaneously implanted Alzet mini-osmotic pumps. TEPP-46 (1 mg/kg) was administered to encourage tetramer formation of PKM2 and was intraperitoneally injected 3 d prior to Ang II or saline infusion. The systolic blood pressure was measured by tail-cuff plethysmography (BP-2000, Visitech Systems, Apex, NC, USA) every 2 days. All procedures were approved by the Institutional Animal Care and Use Committee at Shanghai Jiaotong University and performed in conformance with the Guide for the Care and Use of Laboratory Animals (NIH Publication, 8th edition, 2011). The animals were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg). The thoracic aorta was placed in ice-cold, modified Krebs/Hepes buffer (buffer composition (1 × 10−3 mol/L): NaCl 99.01; KCl 4.69; CaCl2 1.87; MgSO4 1.20; K2HPO4 1.03; NaHCO3 25.0; Na-Hepes 20.0, and glucose, 11.1, pH 7.4) and excessive adventitial tissue was removed. After being cut into 2-3 mm ring segments, the thoracic aortas were mounted on isometric force transducers (Danish Myo Technology Model 610M, Denmark) and to assess vessel function. 2.5 Enface analysis After removing peripheral fat, the thoracic aorta was cut open longitudinally with the luminal surface facing up. The tissue sections were stained with CD31 (1:200) and α-SMA (1:200) overnight and washed with phosphate-buffered saline (PBS) thrice. After incubation with secondary antibody for 30 minutes, the fluorescence of the ECs was measured using a Zeiss AxiovertA1 microscope.22 2.6 Autophagy flux To detect the different stages of autophagy, an adenovirus expressing RFP-GFP-LC3B was used to infect mouse aortic ECs. After treatment with or without Ang II for 24 hours, GFP and RFP expression in cells was assessed using confocal fluorescence microscopy. In this assay, as GFP fluorescence is quenched in the acidic environment of the lysosomal compartment, the overlay of green and red fluorescence creates yellow dots and identifies the numbers of autophagosomes relative to the number of autolysosomes (remaining red dots) in the merged images. Nuclei were stained with Hoechst. Chloroquine, a compound that elevates lysosomal pH, efficiently inhibits lysosome functions. To evaluate autophagic flux, the MAECs were pretreated with chloroquine (2 × 10−6 mol/L) for 1 hour and then, stimulated with Ang II (10−7 mol/L) for 24 hours. Images were acquired and analyzed using Zeiss Axiovision software (Carl Zeiss, Göttingen, Germany). Punctate fluorescence-tagged LC3 dots were counted and expressed as the number per cell.23 2.7 Western blot analysis Equal amounts of proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membrane (PVDF) membranes. After blocking with 5% (w/v) nonfat milk in Tris-buffered saline (TBS) supplemented with 0.1% Tween-20 (TBS-T), membranes were incubated with primary antibodies directed against β-actin (1:6000), α-SMA (1:6000), CD31 (1:500), SIRT3 (1:1000), ATG5 (1:1000), LC3B (1:2000), GLUT1 (1:1000), MCT1 (1:2000), MCT4 (1:1000), collagen I (1:1000), vimentin (1:1500), and myosin (1:1000) in 5% (w/v) BSA/TBS-T overnight at 4°C. The membranes were washed thrice with TBST and then, incubated with peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Western blots were developed using electrochemiluminescence and imaged by an Amersham Biosciences 600 Imager. Signals were quantitated by quantity-one software (Bio-Rad Laboratories). 2.8 Blue native polyacrylamide gel electrophoresis to separate the PKM2 oligomer Lysates of MAECs were prepared with 1% (w/v) N-Dodecyl-β-D-maltoside (DDM), 5 × 10−2 mol/L Bis-Tris, 5 × 10−2 mol/L NaCl, 6N HCl, and 10% (v/v) Glycerol. Samples were centrifuged at 10 000 g for 15 minutes at 4°C before the supernatant was collected into new tubes. Three microliter of 5% Coomassie G-250 (Coomassie G-250 dissolved in 5 × 10−1 mol/L 6-aminocaproic acid) was added toper 10 μL lysates. Equal amounts of proteins were separated onto native-gels and transferred to PVDF membranes. 2.9 Isolation of mitochondria Isolation of mitochondria from cells was performed according to the manufacturer's protocol (Beyotime Co., Nantong, China). Briefly, after exposed to Ang II for 24 hours, cells (2 × 107 cells/mL) were harvested and centrifuged at 600 g at 4°C for 10 minutes, the pellets were incubated with 0.5 mL of 2 × 10−2 mol/L Hepes buffer, and cell suspension was transferred to glass grinder and disrupted to homogenates. These homogenates were centrifuged at 600 g at 4°C for 10 minutes, the resulting supernatants transferred to 1.5 mL new tube, and then, centrifuged at 11 000 g at 4°C for 15 minutes. Transfer the supernatant (cytosol fraction) to a new tube. The pellet contains the isolated mitochondria. 2.10 Immunoprecipitation and coimmunoprecipitation Equal amounts of lysates were mixed with protein A/G-agarose bound acetyl lysine antibody overnight at 4°C with agitation. Pellets were washed five times using PBS and then, boiled in sample buffer. The prepared samples were subjected to 10% SDS-PAGE to assess the relative quantity of acetylated ATG5. The Pierce Co-Immunoprecipitation kit (Thermo Scientific, Rockford, MD, USA) was used to determine the interaction between SIRT3 and ATG5 according to the manufacturer's instructions. SIRT3 antibody was immobilized in resin for 90 minutes at room temperature and washed with coupling buffer. Equal amounts of protein (50 μg) were added to the resin and incubated with gentle mixing overnight at 4°C. The following day, the resin was washed thrice using IP lysis buffer, and then, the immunoprecipitation complex was eluted for Western blot analysis to determine ATG5 protein levels. Total mitochondrial protein was used as an input. The specificity of antibodies used for immunoprecipitation was routinely validated by negative controls, including nonimmune IgG. 2.11 Immunofluorescence analysis Cells were fixed in acetone and methanol (1:1) at −20°C and then, washed with PBS thrice followed by permeabilization with 0.01% Triton X-100. Nonspecific binding sites were blocked with 5% BSA in PBS for 30 minutes. Cells were incubated with primary antibodies, including CD31 (1:200) antibody and α-SMA antibody (1:400), in 5% BSA at 4°C overnight. They were washed thrice with PBS and then, incubated with the corresponding secondary fluorescently labeled antibodies for an hour, including Alexa Fluor 555-labeled anti-mouse IgG and Alexa Fluor 488-labeled anti-rabbit IgG. Nuclei were counterstained with DAPI. Samples were examined using a Zeiss AxiovertA1 microscope, and images were analyzed by ImageJ software. 2.12 Seahorse and metabolic assessments An XF96 Extracellular flux (XF) Analyzer (Seahorse Bioscience, Billerica, MA, USA) was used to monitor the metabolism of cells in real time.24 Briefly, 4×104 cells per well were adhered to XF 96-well microplates, treated with or without Ang II as described above. Before measuring the lactic acid release (ECAR) and oxygen consumption rate (OCR), the plates were incubated at 37 °C with XF assay medium for 1 h. For testing ECAR, glucose (10−2 mol/L), oligomycin (10−6 mol/L) and 2-DG (2×10−2 mol/L) were loaded into drug delivery ports. For testing OCR, oligomycin (10−6 mol/L), FCCP (2×10−6 mol/L) and rotenone/antimycin A (5×10−7mol/L) were loaded into drug delivery ports and added sequentially at the indicated time points. The results were analyzed with Seahorse Software and corrected according to the protein concentration per well. 2.13 Glucose uptake Glucose uptake was measured using the glucose fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-il) amino]-2-desoxi-d-glucose (2-NBDG). After treatment with Ang II, MAECs were incubated at 37°C in basic medium containing 1 × 10−4 mol/L 2-NBDG. Following 30 minutes of incubation, the reaction was terminated by washing the cells three times with PBS to remove the residual 2-NBDG. The fluorescence of the cells was then captured by a Zeiss AxiovertA1 microscope. 2.14 Isotope labeling and GC-MS for metabolic flux analysis Glucose labeled with 13C ([U-13C6] glucose) was obtained from ProfLeader Biotech Co. MAECs were grown in 10-cm dishes with regular medium until they reached 80% confluence. Cells were then starved and supplemented with 2.5 × 10−2 mol/L [U-13C6] glucose in glucose-free medium. An equal number of cells from each treatment was collected in 1 mL of 1:1 water/methanol and subjected to processing via five cycles of 1 minute ultrasonication before being mixed with ice-cold chloroform. After centrifugation at 14 000 g for 15 minutes at 4°C, 600 μL supernatant was evaporated to dryness with a nitrogen stream and then, reconstituted with 30 μL of methoxyamine hydrochloride (20 g/L) in anhydrous pyridine. Then, a tert-butyldimethylsilyl (TBDMS) derivatization was initiated by adding 30 μL of MTBSTFA (with 1% TBDMCS, Regis Technologies) and incubating at 55°C for 60 minutes. Samples were injected and analyzed by an Agilent 7890A gas chromatography system coupled to an Agilent 5975C inert MSD system (Agilent Technologies Inc, CA, USA). Steady state metabolic flux was calculated based on 13C mass isotopologue distributions (MIDs) for glucose metabolism intermediates using a package in the R language.25, 26 2.15 Statistical analysis All quantitative data were confirmed in at least three independent experiments. The data are expressed as the means ± SEM. The statistical significance was performed using GraphPad Prism 6 (GraphPad Software, Inc, La Jolla, CA, USA) and determined by one-way ANOVA with Bonferroni's posttest between groups. A P-value <.05 indicates a statistically significant difference. 3 RESULTS 3.1 SIRT3 deficiency accelerates Ang II-induced EndoMT We investigated the mechanism of SIRT3 ablation in hypertensive vascular remodeling in vivo. SIRT3-KO and WT mice were infused with Ang II for 14 days. As expected, Ang II treatment induced a significant increase in systolic blood pressure that was similar in magnitude between the WT and SIRT3-KO mice compared with their saline-treated controls (Figure S1A). SIRT3 deficiency accelerated the impairment of ACh-induced relaxation by Ang II treatment (Figure S1B). In addition, en face staining revealed that in the Ang II-infused SIRT3 KO mice, aortic endothelial CD31 levels decreased, whereas levels of the myofibroblast marker α-SMA significantly increased (Figure 1A,B). For the in vitro analyses, ECs were infected with the shRNA-SIRT3 lentivirus, which resulted in a 72% reduction in SIRT3 protein expression in ECs. In contrast, MAECs infected with the lentivirus-mediated SIRT3 overexpression vector (LV-SIRT3) increased SIRT3 expression 1.5-fold (Figure 1C,D). Western blot and fluorescence imaging results revealed that SIRT3 knockdown accelerated the Ang II-induced increase in α-SMA protein levels but decreased CD31 levels. Conversely, MAEC overexpression of SIRT3 attenuated Ang II-induced EndoMT (Figure 1C-F). In addition, SIRT3 knockdown caused an enhanced migration capacity in both wound healing and transwell chamber assays, whereas SIRT3 overexpression reduced the migratory capacity of cells (Figure 1G-J). These results suggest that SIRT3 plays an important role in regulating Ang II-induced EndoMT. FIGURE 1Open in figure viewer SIRT3 deficiency accelerates Ang II-induced EndoMT. A, Eight-week-old male WT and SIRT3−/− mice were given 1400 ng/kg/min of Ang II or saline for 14 days. Representative fluorescence micrographs of en face staining with CD31 (green) and α-SMA (red) in the aortic endothelia. Scale bar: 100 μm. B, Fluorescence intensity of α-SMA in the aortic endothelia (n = 7). C, MAECs were infected with shRNA-SIRT3, lentivirus encoding SIRT3 (LV-SIRT3), or empty vector. Representative western blots of SIRT3, CD31, and α-SMA in MAECs. D, Quantitative analysis of SIRT3, CD31, and α-SMA levels (n = 6). E, Representative fluorescence of MAECs labeled with α-SMA (red) after 24 hours Ang II treatment. Scale bar: 100 μm. F, Fluorescence intensity of α-SMA (n = 6). G, Transwell assay of MAECs infected with shRNA-SIRT3, LV-SIRT3, or empty vector. Scale bar: 200 μm. H, The number of cells migrating through microporous membrane (n = 6). I, The wound healing test of MAECs infected with shRNA-SIRT3, LV-SIRT3, or empty vector. Scale bar: 200 μm. J, Percentage of wound closure (n = 6). *P < .05, **P < .01 vs control. #P < .05, ##P < .01 vs Ang II treatment group 3.2 Ang II inhibits autophagic flux during EndoMT To assess the effects of Ang II on autophagic flux during EndoMT, en face immunofluorescence staining of endothelial CD31 and LC3B was performed. We observed that the level of the autophagic marker LC3B dramatically decreased in the aortic ECs of mice receiving Ang II infusion and that SIRT3 deficiency resulted in an accelerated decrease in LC3B puncta (Figure 2A,B). Western blotting confirmed that SIRT3 knockdown decreased the level of LC3-II and promoted P62 expression (Figure 2C,D). FIGURE 2Open in figure viewer Ang II inhibited autophagic flux during EndoMT. A, Eight-week-old male WT and SIRT3−/− mice were given 1400 ng/kg/min of Ang II or saline for 14 days. Representative fluorescence micrographs of en face staining with CD31 (green) and LC3B (red) in the aortic endothelia. Scale bar: 100 μm. B, Fluorescence intensity of LC3B (n = 7). C, MAECs were infected with shRNA-SIRT3 or empty vector, followed by stimulation with or without Ang II for 24 hours. Representative western blots of LC3B and P62 in MAECs. D, Quantitative analysis of LC3B and P62 (n = 6). E, MAECs were infected with RFP-GFP-LC3B, and then, treated with Ang II combined with or without chloroquine (CQ, 2 × 10-6mol/L, 24 hours). Representative immunofluorescence images of mCherry-green fluorescent protein (GFP)-LC3B. Scale bar: 100 μm. F, Quantitative analysis autophagosomes (yellow dots) and autolysosomes (red dots) in merged images per cell (n = 6). G, Representative western blots of P62 and LC3B in MAECs treated with Ang II combined with or without chloroquine. H, Quantitative analysis of P62 and LC3B (n = 6). *P < .05, **P < .01 vs control. #P < .05, ##P < .01 vs Ang II treatment group. §P < .05 vs chloroquine treatment group We also used MAECs transfected with the RFP-GFP-LC3 plasmid

Abstract Mitochondrial dysfunction due to oxidative stress and concomitant impaired β‐cell function may play a key role in type 2 diabetes. Preventing and/or ameliorating oxidative mitochondrial dysfunction with mitochondria‐specific nutrients may have preventive or therapeutic potential. In the present study, the oxidative mechanism of mitochondrial dysfunction in pancreatic β‐cells exposed to sublethal levels of oleic acid (OA) and the protective effects of mitochondrial nutrients [ R ‐alpha‐lipoic acid (LA) and acetyl‐ L ‐carnitine (ALC)] were investigated. Chronic exposure (72 h) of insulinoma MIN6 cells to OA (0.2–0.8 mM) increased intracellular oxidant formation, decreased mitochondrial membrane potential (MMP), enhanced uncoupling protein‐2 (UCP‐2) mRNA and protein expression, and consequently, decreased glucose‐induced ATP production and suppressed glucose‐stimulated insulin secretion. Pretreatment with LA and/or ALC reduced oxidant formation, increased MMP, regulated UCP‐2 mRNA and protein expression, increased glucose‐induced ATP production, and restored glucose‐stimulated insulin secretion. The key findings on ATP production and insulin secretion were verified with isolated rat islets. These results suggest that mitochondrial dysfunction is involved in OA‐induced pancreatic β‐cell dysfunction and that pretreatment with mitochondrial protective nutrients could be an effective strategy to prevent β‐cell dysfunction. J. Cell. Biochem. 104: 1232–1243, 2008. © 2008 Wiley‐Liss, Inc.

In this study, we investigated the interaction between exercise-induced mitochondrial adaptation of large vessels and the effects of chronic anabolic androgenic steroids (AASs).Four groups of Sprague-Dawley rats were studied: (i) sedentary, (ii) sedentary + nandrolone-treated, (iii) aerobic exercise trained, and (iv) trained + nandrolone-treated. Aerobic training increased the levels of aortic endothelial nitric oxide synthase (eNOS) and heme oxygenase-1 (HO-1) in accordance with improved acetylcholine-induced vascular relaxation. These beneficial effects were associated with induction of mitochondrial complexes I and V, increased mitochondrial DNA copy number, and greater expression of transcription factors involved in mitochondrial biogenesis/fusion. We also observed enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of beclin1 and autophagy-related protein-7 (ATG7). The levels of thiobarbituric acid-reactive substances and protein carbonyls remained unchanged, whereas significant increases in catalase and mitochondrial manganese superoxide dismutase (MnSOD) levels were observed in the aortas of trained animals, when compared with sedentary controls. Nandrolone increased oxidative stress biomarkers and inhibited exercise-induced increases of eNOS, HO-1, catalase, and MnSOD expression. In addition, it also attenuated elevated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and mitofusin-2 expression, and further up-regulated LC3II conversion, beclin1, ATG7, and dynamin-related protein-1 expression.These results demonstrate that nandrolone attenuates aortic adaptations to exercise by regulating mitochondrial dynamic remodelling, including down-regulation of mitochondrial biogenesis and intensive autophagy.

Abstract Previous study indicated that Sirtuin 3 (SIRT3) is a central regulator of adaptive thermogenesis in brown adipose tissue (BAT). Here we investigate the role of SIRT3 in the modulation of cellular phenotype in BAT under high salt intake (HS). HS downregulated SIRT3 level in BAT, accompanied by decreased oxygen consumption rate, and caused a severe loss of BAT characteristics. Mechanically, SIRT3 interacted with pyruvate dehydrogenase E1α (PDHA1) and deacetylated Lys-83 both in vitro and in vivo under HS. In parallel, HS suppressed salt-induced kinase (Sik) 2 phosphorylation. Silencing Sik2 further diminished SIRT3 activity and enhanced acetylation of PDHA1 K83 level. Reconstruction of SIRT3 restored PDH activity and thermogenic markers expression in differentiated brown adipocytes from SIRT3 knockout (KO) mice. In addition, loss of SIRT3 induced selective remodelling of phospholipids and glycerolipids in BAT exposure to HS. These data indicate that SIRT3 is an essential enzymatic switch that controls brown adipose cell phenotype.

Oxidative stress may be an important cause of erythrocyte senescence. Angiotensin II (Ang II) has recently been shown to promote vascular cell senescence. However, its effects on erythrocytes remain unclear. This study aims at investigating the role of Ang II in regulating erythrocyte lifespan through oxidative stress. Experiments were performed in C57/BL6J mice infused with Ang II (1500 ng/kg per minute) or saline for 7 days. After Ang II infusion, we found that Ang II increased erythrocyte number, hemoglobin, and red blood cell distribution width. These differences were accompanied by a decrease in glutathione (GSH) and an increase in malondialdehyde (MDA) concentration. In vitro, after 24 hours of Ang II treatment, erythrocytes showed reduced surface expression of CD47 and increased phosphatidylserine exposure. In parallel, Ang II reduced the levels of antioxidant enzymes, including Cu/ZnSOD, catalase, and peroxidase 2 (PRDX2). These effects were reversed by the addition of the antioxidant N-acetyl-L-cysteine or the Ang II type 1 (AT1) receptor blocker losartan. In addition, Ang II treatment increased pro-inflammatory oxylipin, including hydroxyeicosatetraenoic acids (HETEs) and dihydroxyoctadecenoic acids (DiHOMEs), in the erythrocyte membranes. Collectively, Ang II induced erythrocyte senescence and susceptibility to eryptosis, partially due to enhanced oxidative stress.

Experimental evidence has suggested that vascular adventitial fibroblasts (AFs) may migrate into the neointima of arteries after balloon injury in various animal models. However, the research on migration of AFs has been limited to the effects of acute vascular injury. The role of AFs in chronic vascular injury and hypertension is not yet known. In this study, the migration of spontaneously hypertensive rat (SHR)-AFs and Wistar-Kyoto rat (WKY)-AFs from the thoracic aorta was determined by a transwell technique. Our results showed that fetal calf serum, angiotensin II (Ang II), phorbol ester, basic fibroblast growth factor and platelet-derived growth factor-BB induced migration in a dose-dependent manner, and the migration of SHR-AFs was always greater than that of WKY-AFs. Ang II-induced migration of AFs was considered to have been mediated by Ang II type 1 receptor (AT1-R), because the AT1-R antagonist losartan (10−7−10−5 mol/l) suppressed Ang II-induced migration. Ang II-induced migration was also blocked by the extracellular-regulated protein kinase 1/2 (ERK1/2) inhibitor PD98059 (10−5 mol/l) and p38 kinase inhibitor SB202190 (10−5 mol/l), indicating that ERK1/2 and p38 kinase were involved in Ang II-induced migration. Ang II (10−7 mol/l)-induced ERK1/2 and p38 kinase phosphorylation, both of which peaked after 5 min, were suppressed by PD98059 and SB202190, respectively. The Ang-II induced phosphorylation of both proteins was suppressed by losartan, whereas no effect was observed with PD123319, a specific inhibitor of Ang II type 2 receptor (AT2-R). Thus, in the present study, various factors stimulated the migration of SHR-AFs and, to a leber extent, WKY-AFs from the thoracic aorta, and the ERK1/2 and p38 kinase pathways are involved in Ang II-stimulated migration of fibroblasts.

We propose that elevation of mitochondrial enzyme cofactors may prevent or ameliorate neurodegenerative diseases by improving mitochondrial function. In the present study, we investigated the effects of high doses of B vitamins, the precursors of mitochondrial enzyme cofactors, on mitochondrial dysfunction, oxidative stress, and Parkinsonism in a 4-week long rotenone treatment-induced cellular model of Parkinson's disease (PD). Pretreatment with B vitamins (also 4 weeks) prevented rotenone-induced: (1) mitochondrial dysfunction, including reduced mitochondrial membrane potential and activities of complex I; (2) oxidative stress, including increase in reactive oxygen species, oxidative DNA damage and protein oxidation, and (3) Parkinsonism parameters, including accumulation of alpha-synuclein and poly-ubiquitin. The optimum doses were found around 2.5- and 5-fold of that in normal MEM medium. The 4-week pretreatment was chosen based on time-dependent experiments that pretreatments longer than 2 weeks resulted in a decrease in oxidants, an increase in oxygen consumption, and up-regulation of complex I activity and PGC-1alpha expression. Individual B vitamins at the same doses did not show a similar effect suggesting that these B vitamins work synergistically. These results suggest that administration of high doses of B vitamins sufficient to elevate mitochondrial enzyme cofactors may be effective in preventing PD by reducing oxidative stress and improving mitochondrial function.

Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-l-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

Chronic exercise training is known to protect the vasculature; however, the underlying mechanisms remain obscure. The present study hypothesized that exercise may improve aortic endothelial and mitochondrial function through an adenosine monophosphate-activated protein kinase α2 (AMPKα2)-dependent manner. Ten-week-old AMPKα2 knockout (AMPKα2-/-) mice and age-matched wild-type (WT) mice were subjected to daily treadmill running for 6 weeks, and the thoracic aorta from these mice were used for further examination. Our results showed that exercise significantly promoted vasodilatation and increased expression and phosphorylation of endothelial nitric oxide synthase (eNOS), concomitant with increased AMPKα2 expression in WT mice. These effects were not observed in AMPKα2-/- mice. Furthermore, exercise training increased thoracic aortic mitochondrial content as indicated by increased Complex I and mitochondrial DNA (mtDNA) in WT mice but not in AMPKα2-/- mice. This may be caused by decreased mitochondrial autophagy since the expression of BH3 domain-containing BCL2 family members BNIP3-like (BNIP3L) and LC3B were decreased in WT mice with exercise. And these changes were absent with AMPKα2 deletion in mice. Importantly, exercise increased the expression of manganous superoxide dismutase (MnSOD) and catalase, suggesting that mitochondrial antioxidative capacity was increased. Notably, the improved antioxidative capacity was lost in AMPKα2-/- mice with exercise. In conclusion, this study illustrated that AMPKα2 plays a critical role in exercise-related vascular protection via increasing endothelial and mitochondrial function in the artery.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is highly expressed in heart and skeletal muscles, and is the major enzyme that metabolizes acetaldehyde and toxic aldehydes. The cardioprotective effects of ALDH2 during cardiac ischemia/reperfusion injury have been recognized. However, less is known about the function of ALDH2 in skeletal muscle. This study was designed to evaluate the effect of ALDH2 on exhaustive exercise-induced skeletal muscle injury. We created transgenic mice expressing ALDH2 in skeletal muscles. Male wild-type C57/BL6 (WT) and ALDH2 transgenic mice (ALDH2-Tg), 8-weeks old, were challenged with exhaustive exercise for 1 week to induce skeletal muscle injury. Animals were sacrificed 24 h post-exercise and muscle tissue was excised. ALDH2-Tg mice displayed significantly increased treadmill exercise capacity compared to WT mice. Exhaustive exercise caused an increase in mRNA levels of the muscle atrophy markers, Atrogin-1 and MuRF1, and reduced mitochondrial biogenesis and fusion in WT skeletal muscles; these effects were attenuated in ALDH2-Tg mice. Exhaustive exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of Beclin1 and Bnip3; the effects of which were mitigated by ALDH2 overexpression. In addition, ALDH2-Tg reversed the increase of an oxidative stress biomarker (4-hydroxynonenal) and decreased levels of mitochondrial antioxidant proteins, including manganese superoxide dismutase and NAD(P)H:quinone oxidoreductase 1, in skeletal muscle induced by exhaustive exercise. ALDH2 may reverse skeletal muscle mitochondrial dysfunction due to exhaustive exercise by regulating mitochondria dynamic remodeling and enhancing the quality of mitochondria.

We investigated the associations between time in target range (TTR) of blood pressure (BP) and cardiovascular outcomes in patients with diabetes.4651 participants from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) BP trial were included in the present study. The diastolic BP target range was defined as 70 to 80 mm Hg, and the systolic as 120 to 140 mm Hg and 110 to 130 mm Hg for the standard and intensive therapy, respectively.After adjusting for covariates, 1-SD increase of diastolic TTR was significantly associated with lower risks of primary outcome (HR 0.82, 95% CI: 0.74-0.91, P < 0.001; HR 0.86, 95% CI: 0.77-0.95, P = 0.0044, as well as nonfatal myocardial infarction (HR 0.79, 95% CI: 0.69-0.91, P < 0.001). Meanwhile, systolic TTR was significantly associated with various cardiovascular outcomes (P ≤ 0.016) in fully-adjusted models. The diastolic TTR sustained significance in myocardial infarction when systolic blood pressure average was higher than 120 mm Hg.In patients with diabetes, TTR of diastolic and systolic BP was independently associated with lower risks of major outcomes. The diastolic BP within the optimal target range was considerably important for reducing the risk of myocardial infarction, even when systolic BP was under stable control.

Molecular diagnostics play an important role in illness detection, prevention, and treatment, and are vital in point-of-care test. In this investigation, a novel CRISPR/Cas12a based small-molecule detection platform was developed using Antibody-Controlled Cas12a Biosensor (ACCBOR), in which antibody would control the trans-cleavage activity of CRISPR/Cas12a. In this system, small-molecule was labelled around the PAM sites of no target sequence(NTS), and antibody would bind on the labelled molecule to prevent the combination of CRISPR/Cas12a, resulting the decrease of trans-cleavage activity. Biotin-, digoxin-, 25-hydroxyvitamin D3 (25-OH-VD3)-labelled NTS and corresponding binding protein were separately used to verify its preformance, showing great universality. Finally, one-pot detection of 25-OH-VD3 was developed, exhibiting high sensitivity and excellent specificity. The limit of detection could be 259.86 pg/mL in serum within 30 min. This assay platform also has the advantages of low cost, easy operation (one-pot method), and fast detection (∼30 min), would be a new possibilities for the highly sensitive detection of other small-molecule targets.

The specificity of antibodies and efficient amplification of nucleic acid molecules play an important role in developing biosensors for monitoring human and environmental health. Herein, integrating their capabilities by site-selective modification of small molecules on DNA, a biosensor based on antibody-controlled strand displacement amplification (SDA) and hybridization chain reaction (HCR) was developed. The SDA–HCR reaction was activated by the competitive binding of antibodies to free small molecules and small molecule-conjugated DNA. As a proof-to-concept, we developed a biosensor capable of detecting tetracycline (TC) with high sensitivity and specificity. First, TC was labeled with azide group through chemical modification, and TC–DNA was prepared by copper-free click chemistry. Further, the antibody-controlled SDA–HCR reaction was developed through serial optimization. By this method, TC could be detected in a linear range of 0.01–100 µM with a detection limit of 0.006 µM, and the recovery of actual samples was 96.62–100.31 % with 1.70–4.00 % RSD, suggesting satisfactory performance by the biosensor. Using this method, TC or any other small molecules can be detected without washing, immobilization, and multiple additional steps, which would simplify detection to a great degree. Consequently, the distinctive strategy proposed in this work may find more applications in the detection of other small molecules.

Industry convergence is a popular term that has been widely referenced in the context of rural tourism development in China. All levels of government (local, regional, national) in China have repeatedly addressed the significance of industry convergence in their tourism plans and related policies. Despite its popularity, limited studies at present have explored this concept in-depth. Using Huai'an as a case, this study applied a path analysis and reported the industry convergence process in a destination. The findings of this study can provide both theoretical and practical implications that are useful for tourism planners and policy makers.

Abstract NLRP3 is involved in obesity-induced cardiac remodeling and dysfunction. In this study, we evaluated whether the cardiac protective effects of nebivolol relied on attenuating NLRP3 activation in a juvenile-adolescent animal model of diet-induced obesity. Weaning male Sprague-Dawley rats were fed with either a standard chow diet (ND) or a high-fat diet (HFD) for 8 weeks. The obese rats were subsequently subdivided into three groups: 1) HFD control group; 2) HFD with low-dose nebivolol (5 mg/kg/d); 3) HFD with high-dose nebivolol (10 mg/kg/d). Treatment with nebivolol prevented HFD-induced obesity associated excess cardiac lipid accumulation as well as myocardial mitochondrial dysfunction. Nebivolol attenuated pro-inflammatory cytokines secretion and NLRP3 inflammasome activation in myocardium of obese rats. In parallel, nebivolol treatment of obese animals increased cardiac β3-AR expression, reversing the reduction of endothelial nitric oxide synthase (eNOS). In vitro, nebivolol treatment of palmitate-incubated H9C2 cells suppressed autophagy, restored mitochondrial biogenesis, leading to decreased mitochondrial reactive oxygen species (mtROS) generation and suppressed NLRP3 inflammasome activation. Meanwhile the presence of shRNA against β3-AR or against eNOS deteriorated the protective effects of nebivolol. These data suggest the beneficial effect of nebivolol on myocardial lipotoxicity contributing to inhibiting NLRP3 inflammasome activation possibly via improved mitochondrial dysfunction.

To investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice. Experiments were performed using irradiated LDL receptor-deficient (LDLR−/−) mice with marrow from either TLR4-deficient (TLR4−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4−/− → LDLR−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4−/− → LDLR−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects. These results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.

Abstract: Nebivolol is a novel β-adrenergic receptor (β-AR) blocker with anti-inflammatory and antioxidant properties. The NLRP3 inflammasome plays a pivotal role in the pathogenesis of obesity-induced vascular dysfunction. Our study aimed to explore the effect of nebivolol on the NLRP3 inflammasome and vascular remodeling in diet-induced obese rats. Eight-week-old Sprague–Dawley male rats were fed with either a standard chow diet or a high-fat diet (HFD) for 8 weeks. Next, the obese rats were subdivided into 3 groups as follows: (1) HFD control group, (2) HFD with low doses of nebivolol (5 mg/kg·d −1 ), and (3) HFD with high doses of nebivolol (10 mg/kg·d −1 ). A 4-week treatment with nebivolol improved acetylcholine-induced vascular relaxation in accordance with an increased aortic endothelial nitric oxide synthase. Nebivolol attenuated NLRP3 inflammasome activation and suppressed autophagy. In parallel, nebivolol enhanced the levels of phase-II detoxifying enzymes, including superoxide dismutase and catalase. These effects were associated with an increased β3-AR level. Moreover, nebivolol treatment significantly increased Adenosine 5′-monophosphate (AMP)-activated protein kinase activity and decreased phosphorylation of the mammalian target of rapamycin. These results demonstrated that nebivolol improves obesity-induced vascular remodeling by attenuating NLRP3 inflammasome activation and restoring the antioxidant defense.

We investigated large vessel function in lean Goto-Kakizaki diabetic rats (GK) and Otsuka Long-Evans Tokushima Fatty diabetic rats (OLETF) with possible roles of hyperglycemia/hyperosmolarity and insulin. Both young and old GK showed marked hyperglycemia with normal insulin level and well-preserved endothelium-dependent and endothelium-independent vasodilation in aorta and carotid artery. There were significant elevations in endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive heme oxygenase (HO-1/HO-2) in GK. The endothelium-dependent vasodilation in GK was inhibited partly by NOS blockade and completely by simultaneous blocking of HO and NOS. In contrast, OLETF showed hyperinsulinemia and mild hyperglycemia but significant endothelium dysfunction beginning at early ages with concomitantly reduced eNOS. Insulin injection corrected hyperglycemia in GK but induced endothelium dysfunction and intima hyperplasia. Hyperglycemia/hyperosmolarity in vitro enhanced vessel eNOS/HO. We suggest that hyperinsulinemia plays a role in endothelium dysfunction in obese diabetic OLETF, while hyperglycemia/hyperosmolarity-induced eNOS/HO upregulation participates in the adaptation of endothelium function in lean diabetic GK.

Peroxisome proliferator-activated receptor (PPAR) γ ligands oppose the effect induced by angiotensin II (Ang II) to reduce oxidative stress and improve antioxidant status. In this study, Ang II inhibited catalase (CAT) and peroxisome proliferator-activated receptor γ (PPAR γ) protein and mRNA expressions. Transfection with PPAR γ small-interfering RNA (siRNA) led to a reduction in CAT expression. PPAR γ ligands enhanced CAT expression and inhibited extracellular signal-regulated kinase 1/2 activation. We further reveal that Ang II type 1 receptor is not involved in the inhibitory effects of PPAR γ ligands on Ang II stimulatory events.

Interleukin-18 (IL-18) was discovered as an interferon-γ-inducing factor and has been regarded as a proinflammatory cytokine. However, IL-18 is ubiquitously expressed both in immune/inflammatory cells and in nonimmune cells, and its biological roles have not been sufficiently elucidated. Here, we demonstrate that IL-18-deficient [IL-18 knockout (KO)] mice have heart abnormalities that may be related to impaired autophagy. In endurance running tests, IL-18KO mice ran significantly shorter distances compared with wild-type (WT) mice. Echocardiographs indicated disability in the systolic and diastolic functions of the IL-18KO mouse heart. Immunostaining of connexin 43 showed heterogeneous localization of gap junctions in the lateral membranes of the IL-18KO cardiac myocytes. Western blotting analysis revealed decreased phosphorylated connexin 43 in the IL-18KO heart. Electron microscopy revealed unusual localization of intercalated disks, swollen or damaged mitochondria, and broad, indistinct Z-lines in the IL-18KO heart. In accordance with the morphological observation, mitochondrial respiratory function, including that of complexes I and IV, was impaired, and production of reactive oxygen species was augmented in IL-18KO hearts. Notably, levels of LC3-II were markedly lower in the IL-18KO hearts than in WT hearts. In the culture of cardiac myocytes of IL-18KO neonates, exogenous IL-18 upregulated LC3-II and increased the number of intact mitochondria with high mitochondrial membrane potential. These results indicated that IL-18 has roles apart from those as a proinflammatory cytokine in cardiac myocytes and suggested that IL-18 contributes to the homeostatic maintenance of mitochondrial function and gap-junction turnover in cardiac myocytes, possibly by upregulating autophagy.

Cardiac fibroblasts (CFs) and cardiomyocytes are the major cell populations in the heart. CFs not only support cardiomyocytes by producing extracellular matrix (ECM) but also assimilate myocardial nutrient metabolism. Recent studies suggest that the classical intercellular lactate shuttle may function in the heart, with lactate transported from CFs to cardiomyocytes. However, the underlying mechanisms regarding the generation and delivery of lactate from CFs to cardiomyocytes have yet to be explored.In this study, we found that angiotensin II (Ang II) induced CFs differentiation into myofibroblasts that, driven by cell metabolism, then underwent a shift from oxidative phosphorylation to aerobic glycolysis. During this metabolic conversion, the expression of amino acid synthesis 5-like 1 (GCN5L1) was upregulated and bound to and acetylated mitochondrial pyruvate carrier 2 (MPC2) at lysine residue 19. Hyperacetylation of MPC2k19 disrupted mitochondrial pyruvate uptake and mitochondrial respiration. GCN5L1 ablation downregulated MPC2K19 acetylation, stimulated mitochondrial pyruvate metabolism, and inhibited glycolysis and lactate accumulation. In addition, myofibroblast-specific GCN5L1-knockout mice (GCN5L1fl/fl: Periostin-Cre) showed reduced myocardial hypertrophy and collagen content in the myocardium. Moreover, cardiomyocyte-specific monocarboxylate transporter 1 (MCT1)-knockout mice (MCT1fl/fl: Myh6-Cre) exhibited blocked shuttling of lactate from CFs to cardiomyocytes and attenuated Ang II-induced cardiac hypertrophy.Our findings suggest that GCN5L1-MPC2 signalling pathway alters metabolic patterns, and blocking MCT1 interrupts the fibroblast-to-cardiomyocyte lactate shuttle, which may attenuate cardiac remodelling in hypertension.

This study investigates the effects on aortic stiffness and vasodilation by arotinolol and the underlying mechanisms in spontaneously hypertensive rats (SHR).The vasodilations of rat aortas, renal and mesenteric arteries were evaluated by isometric force recording. Nitric oxide (NO) was measured in human aortic endothelial cells (HAECs) by fluorescent probes. Sixteen-week old SHRs were treated with metoprolol (200 mg·kg-1·d⁻¹), arotinolol (30 mg·kg-1·d⁻¹) for 8 weeks. Central arterial pressure (CAP) and pulse wave velocity (PWV) were evaluated via catheter pressure transducers. Collagen was assessed by immunohistochemistry and biochemistry assay, while endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation (p-eNOS) of HAECs or aortas were analyzed by western blotting.Arotinolol relaxed vascular rings and the relaxations were attenuated by Nω-nitro-L-arginine methyl ester (L-NAME, NO synthase inhibitor) and the absence of endothelium. Furthermore, arotinolol-induced relaxations were attenuated by 4-aminopyridine (4-AP, Kv channels blocker). Arotinolol produced more nitric oxide compared to metoprolol and increased the expression of p-eNOS in HAECs. These results indicated that arotinolol-induced vasodilation involves endothelium-derived NO and Kv channels. The treatement with arotinolol in 8 weeks, but not metoprolol, markedly decreased CAP and PWV. Biochemistry assay and immunohistochemistry showed that aortic collagen depositions in the arotinolol groups were reduced compared with SHRs with metoprolol. Moreover, eNOS phosphorylation was significantly increased in aortinolol-treated SHR compared with SHRs with metoprolol.Arotinolol improves arterial stiffness in SHR, which involved in increasing NO and decreasing collagen contents in large arteries.

Background: Hyperosmotic solutions have been used successfully in different shock resuscitations with cardioprotection. This study was to examine the effects of hyperosmotic sodium chloride on isolated heart function and heart responses to ischemia/reperfusion in normotensive and hypertensive rats. The roles of hyperosmolarity-induced antioxidants including hyperosmolarity-relevant heat shock proteins as well as vasodilating endothelial nitric oxide synthase (eNOS) and vasoactive catecholamines were investigated.Methods: Hearts of normal rats and stroke-prone spontaneously hypertensive rats were isolated and perfused for 30 min with control Krebs-Henseleit buffer (osmolarity 300 mOsm/L) or hyperosmotic buffer of different sodium chloride concentrations (320, 350 and 400 mOsm/L) before subjected to 40-min global ischemia followed by 10-min hyperosmotic reperfusion and 30-min normal buffer reperfusion. Heart function, creatine phosphokinase leakage and myocardial antioxidants were examined. Myocardial antioxidants after hyperosmotic perfusion with different osmolytes were assayed with Western blotting.Results: Pre-ischemic hyperosmotic sodium chloride perfusion enhanced heart contractility and diastole function and reduced coronary vascular resistance in both normal and hypertensive hearts. Post-ischemic recoveries of heart function were improved in hyperosmotic perfused hearts, associated with lower creatine phosphokinase leakage, higher coronary flow, reduced coronary resistance and lower norepinephrine overflow. At the end of reperfusion, the myocardial activities of total superoxide dismutase and catalase, glutathione content as well as osmosis-relevant heat shock protein 32 and 90 were increased in hyperosmotic hearts. In addition to sodium chloride, in vitro hyperosmotic mannitol, glucose and raffinose also increased protein expressions of antioxidants including superoxide dismutase, catalase, heat shock protein 32 and 90 and vasodilating eNOS.Conclusion: Hyperosmotic perfusion enhanced heart function and preconditioned normal and hypertensive hearts against ischemia/reperfusion injury. The hyperosmolarity-induced up-regulations in myocardial antioxidants including heat shock proteins and eNOS may play an important role in the hyperosmolarity-induced cardioprotection.

Ternary oxides nanomaterials (TONs) are the mostly investigated as a promising candidate for ultraviolet detection due to its excellent photoconductivity. Among them, Zn2SnO4 (ZTO) has received extensive attention, but its advantages cannot be utilized in deep-ultraviolet (DUV) detection due to band gap limitation. Herein, we synthesized ZnSn(OH)6 nanocube/ ZTO nanowires yolk–shell hierarchical structure with tunable band gap between 3.6 and 4.4 eV by the dual laser ablation in liquid plus hydrothermal method. This hierarchical structure is applied to DUV photodetection and the results showed that it could improve the Ilight/ Idark ratio of ZTO DUV photodetection by two orders of magnitude while retaining the advantage of ZTO’s rapid response time (τrise =0.43 ms, τdecay =0.55 ms). Moreover, deep-UV imaging is also realized based on this material. This approach provides a new way of tuning the TONs’ band gap and its application to DUV photodetection or imaging.

Skin mesenchymal stem cells (S-MSCs) revealed an important immunomodulatory activity to markedly suppress the formation of the atherosclerosis (AS) plaque by modulating macrophages, and also inhibit the development of experimental autoimmune encephalomyelitis (EAE) by regulating T helper 17 (Th17) cell differentiation. Macrophages and Th17 cells play important roles in hypertension. However, it remains unclear whether S-MSCs are capable of improving angiotensin (AngII)-induced hypertension by acting on inflammatory cells. Therefore, we studied a direct effect of S-MSC treatment on an AngII-induced hypertensive mouse model. Twenty-seven C57BL/6 (WT) mice were divided into three groups: Control group (WT-NC), AngII-infused group (WT-AngII), and S-MSC treatment group (WT-AngII + S-MSCs). In contrast to WT-AngII group, systolic blood pressure (SBP) and vascular damage were strikingly attenuated after tail-vein injection of S-MSCs. Numbers of Th17 cells in mouse peripheral blood of S-MSC treated group were significantly decreased, and IL-17 mRNA and protein levels were also reduced in the aorta and serum compared with WT-AngII group. Furthermore, macrophages in S-MSC treated group were switched to a regulatory profile characterized by a low ability to produce pro-inflammatory cytokine TNF-α and a high ability to produce anti-inflammatory cytokines Arg1 and IL-10. Mechanistically, we found that S-MSCs inhibited Th17 cell differentiation and induced M2 polarization. Moreover, we found proliferation and migration of S-MSCs were elevated, and expression of CXCR4, the receptor for Stromal derivated factor -1(SDF-1), was markedly increased in lipopolysaccharide (LPS)- stimulated S-MSCs. Given that SDF-1 expression was increased in the serum and aorta in AngII- induced hypertensive mice, the immunomodulatory effects exerted by S-MSCs involved the CXCR4/SDF-1 signaling. Collectively, our data demonstrated that S-MSCs attenuated AngII-induced hypertension by inhibiting Th17 cell differentiation and by modulating macrophage M2 polarization, suggesting that S-MSCs potentially have a role in stem cell based therapy for hypertension.

Low birthweight is known to predict high risk of metabolic diseases in adulthood, while regular endurance exercises are believed sufficient to improve metabolic dysfunction. In this study, we established a mouse model to determine whether long-term exercise training could ameliorate catch-up growth, and we explored the possible underlying mechanisms. By restricting maternal food intake during the last week of gestation, we successfully produced low birthweight pups. Further, normal birthweight mice and low birthweight mice were randomly distributed into one of three groups receiving either a normal fat diet, high fat diet, or high fat diet with exercise training. The growth/metabolism, mitochondrial content and functions were assessed at 6 months of age. Through group comparisons and correlation analyses, the 4th week was demonstrated to be the period of crucial growth and chosen to be the precise point of intervention, as the growth rate at this point is significantly correlated with body weight, intraperitoneal glucose tolerance test (IPGTT), Lee's index and fat mass in adulthood. In addition, regular endurance exercises when started from 4 weeks remarkably ameliorated low birthweight outcomes and induced catch-up growth and glucose intolerance in the 25th week. Furthermore, real-time PCR and western blot results indicated that the effect of long-term exercise on mitochondrial functions alleviated catch-up related metabolic dysfunction. To conclude, long-term exercise training from the 4th week is sufficient to ameliorate catch-up growth and related metabolic disturbances in adulthood by promoting mitochondrial functions in skeletal muscle.

Our previous study demonstrated that TGF-beta1 could induce the differentiation of vascular adventitial fibroblasts (AFs) to myofibroblasts (MFs). The aim of this study was to identify the genes which might be responsible for the cell phenotypic change using genechips. Cultured rat AFs were treated with TGF-beta1 (10 ng/ml) for 0 min, 5 min, 15 min, 2 h, 12 h and 24 h, respectively. Then the cells were gathered to prepare total RNA. We examined TGF-beta1-induced gene expression profiling using Affymetrix oligonucleotide microarrays and analyzed data by GCOS1.2 software. Moreover, expressional similarity was measured by hierarchical clustering. Some of genechip results were confirmed by real-time quantitative RT-PCR. Microarray analysis identified 2121 genes with a 2-fold change or above after TGF-beta1 stimulation. 1318 genes showed a greater than 2-fold increase and 761 genes were reduced 2 folds or more at mRNA levels, whereas a small portion of the total regulated genes (42 genes) displayed dynamically up- and down-regulated pattern. Genes were further segregated for early (peak at 5 min, 15 min and/or 2 h), late (peak at 12 h and/or 24 h), and sustained (2-fold change or above at five time points) temporal response groups according to the time of their peak expression level. Among 1318 up-regulated genes, 333 genes (25.3%) responded rapidly to TGF-beta1 and 159 genes (12.1%) responded in a sustained manner. Most genes (826, 62.6%) were regulated at 12 h or later. For the 761 down-regulated genes, numbers of early and late responsive genes were 335 (44%) and 267 (36.1%), respectively. There were also 159 genes, 19.9% of total down-regulated genes, decreased at five time points treated by TGF-beta1. The results suggested that the gene expressions of secreted phosphoprotein 1 (APP1) and Rho-associated coiled-coil forming kinase 2 (ROCK2) had the same trends as alpha-smooth muscle-actin, a marker of MF differentiation. In addition, the gene expression of potassium voltage-gated channel, Shal-related family and member 2 (KCND2) was up-regulated. Furthermore, it was found that endothelin 1 (EDN1), some complement components, NADPH oxidase 4 (NOX4) and NAD(P)H dehydrogenase, quinone 1 (NQO1) might be involved in MF differentiation. Using microarrary technique, we confirmed some genes that have been identified by other techniques were implicated in MF differentiation and observed new genes involved in this process. Our results suggest that gene expression profiling study is helpful in identifying genes and pathways potentially involved in cell differentiation.

Background: It has been reported that Angiotensin II (Ang II) induced skeletal muscle atrophy. However, the precise mechanisms remain elusive. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, plays a central role in maintaining cellular metabolic homeostasis. This work aims to determine the role of SIRT3-mediated cellular metabolism in skeletal muscle wasting.Methods and Results: Eight-week-old male wild-type (WT) and SIRT3 knockout (SIRT3 KO) mice were infused with Ang II or saline for 4 weeks. Ang II induces skeletal muscle atrophy by inducing expression of the muscle-enriched E3 ubiquitin ligase muscle RING-finger-1 (MuRF1) and atrogin-1, accompanied by a reduction in SIRT3 in skeletal muscle. SIRT3 deficiency accelerated Ang II-induced loss of lean mass and protein hyper-acetylation, while the activities of mitochondrial oxidative enzymes, such as complex I and complex V, were significantly decreased. Furthermore, SIRT3 deficiency accelerated the Ang II-induced shift from slow-twitch towards fast-twitch fibres. Similarly, the three major rate-limiting enzymes in the glycolytic pathway, hexokinase 2 (HK2), phosphofructokinase-1(PFK) and pyruvate kinase (PK), were upregulated in Ang II-treated SIRT3 KO mice.Conclusion: These studies indicate that SIRT3 deficiency augmented Ang II-induced fibre-type shifting and metabolic reprogramming.

Abstract:

Postnatal catch-up growth may be related to reduce mitochondrial content and oxidation capacity in skeletal muscle.The aim of this study is to explore the effect and mechanism of antioxidant MitoQuinone mesylate beta cyclodextrin complex (MMCC) ameliorates catch-up growth related metabolic disorders.Catch-up growth mice were created by restricting maternal food intake during the last week of gestation and providing high fat diet after weaning.Low birthweight mice and normal birthweight controls were randomly subjected to normal fat diet, high fat diet and high fat diet with MMCC drinking from the 4th week.MMCC treatment for 21 weeks slowed down the catch up growth and ameliorated catch-up growth related obesity, glucose intolerance and insulin resistance.MMCC administration significantly inhibited the peroxidation of the membrane lipid and up-regulated the antioxidant enzymes Catalase and MnSOD.In addition, MMCC treatment effectively enhanced mitochondrial functions in skeletal muscle through the up-regulation of the ATP generation, and the promotion of mitochondrial replication and remodeling.To conclude, this study demonstrates that antioxidant MMCC effectively ameliorates catch-up growth related metabolic dysfunctions by increasing mitochondrial functions in skeletal muscle.

Hypertension can cause structural and functional abnormalities in the cardiovascular system, which can be attributed to both hemodynamic and nonhemodynamic factors. These alterations are linked with metabolic changes and are induced by pathological stressors. Sirtuins are enzymes that act as stress sensors and regulate metabolic adaptation by deacetylating proteins. Among them, mitochondrial SIRT3 performs a crucial role in maintaining metabolic homeostasis. Evidence from experimental and clinical studies has shown that hypertension-induced decreases in SIRT3 activity can lead to cellular metabolism reprogramming and, subsequently, increased susceptibility to endothelial dysfunction, myocardial hypertrophy, myocardial fibrosis, and heart failure. This review presents recent research advances in SIRT3-mediated metabolic adaptation in hypertensive cardiovascular remodeling.

Biomarkers are the most sensitive reactants and early indicators of many kinds of diseases. The development of highly sensitive and simple techniques to quantify them is challenging. In this study, based on rolling cycle amplification (RCA) and the Nicked PAM/CRISPR-Cas12a system (RNPC) as a signal reporter, a sandwich-type method was developed using antibody@magnetic beads and aptamer for the high-sensitive detection of the C-reactive protein (CRP). The antibody-antigen (target)-aptamer sandwich-like reaction was coupled to RCA, which can produce hundreds of similar binding sites and are discriminated by CRISPR/Cas12a for signal amplification. The ultrasensitivity is achieved based on the dual-signal enhancing strategy, which involves the special recognition of aptamers, RCA, and trans-cleavage of CRISPR/Cas12a. By incorporating the CRISPR/Cas12a system with cleaved PAM, the nonspecific amplification of the RCA reaction alone was greatly reduced, and the dual signal output of RCA and Cas12a improved the detection sensitivity. Our assay can be performed only in two steps. The first step takes only 20 min of target capture, followed by a one-pot reaction, where the target concentration can be obtained by fluorescence values as long as there are 37 °C reaction conditions. Under optimal conditions, this system detected CRP with high sensitivity. The fabricated biosensor showed detection limits of 0.40 pg/mL in phosphate-buffered saline and 0.73 pg/mL in diluted human serum and a broad linear dynamic range of 1.28 pg/mL to 100 ng/mL within a total readout time of 90 min. The method could be used to perform multi-step signal amplification, which can help in the ultrasensitive detection of other proteins. Overall, the proposed biosensor might be used as an immunosensor biosensor platform.

Objectives: Defective autophagy of monocytes or macrophages might result in NLRP3 inflammasome activation and cause vascular metabolic inflammation. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is sensitive to the metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated autophagy in regulating NLRP3 inflammasome activation. Methods: 20 patients with a BMI above 25 kg/m2 and 20 healthy controls were enrolled. 8-week male WT and SIRT3 KO mice were injected intraperitoneally with 0.5 g/kg of poloxamer 407 or saline. The expression of autophagy-related proteins and inflammatory mediators were analyzed using western blot. Results: The inhibition of autophagy and the activation of the NLRP3 inflammasome were concomitant with reduced SIRT3 levels both in peripheral blood monocytes from obese humans and in palmitate-treated THP-1 cells. SIRT3 could form a molecular complex with ATG5, while SIRT3 overexpression altered the acetylation of endogenous ATG5. ATG5 acetylation inhibited autophagosome maturation and induced NLRP3 inflammasome activation. In parallel, SIRT3 overexpression in THP-1 cells decreased the palmitate-induced generation of mitochondrial ROS, restored autophagy, and attenuated NLRP3 inflammasome activation. The incubation of HAECs with macrophage-conditioned medium (MCM) induced HAEC expression of VCAM-1, ICAM-1, α-SMA and collagen-1. The effect of MCM could be reversed by the addition of neutralizing anti-IL-1β antibody or the overexpression of SIRT3. En face analyses displayed a marked increase in α-SMC-positive endothelial cells in SIRT3−/− mice with acute hyperlipidaemia. Conclusion: These findings revealed that SIRT3-deficient macrophages displayed impaired autophagy and accelerated NLRP3 inflammasome activation and endothelial dysfunction.

Objective: Infiltrated macrophages are a dominant population in the perivascular adipose tissue (PVAT) microenvironment and actively promote PVAT remodeling in hypertensive mice. However, the molecular mechanism underlying the role of macrophages in initiating metabolic inflammation remains uncertain. Here, we investigate whether Situin-3(SIRT3) is determinant in macrophage activation. Design and method: 10-week-old male WT, SIRT3 KO, and NLRP3 KO mice were infused with Angiotension II (Ang II, 1000ng/kg/min) or normal saline (NS) for 7 days. The morphological and functional changes of thoracic aorta and PVAT were determined by histological analysis and immunofluorescence staining. In vitro studies were performed on bone marrow-derived macrophages (BMDMs) and brown adipocytes isolated from WT or SIRT3 KO mice. Results: We report that Ang II accelerates PVAT inflammation and fibrosis in SIRT3-deficient mice. This effect is associated with attenuating browning markers and lipid droplet-associated protein expression. Immunofluorescence analysis confirmed that there is an increase in NLRP3 inflammasome and secretion of interleukin-1β (IL-1β). In vitro studies indicate that BMDMs from SIRT3 KO mice induce interleukin-IL-1β production by shifting metabolic phenotype from oxidative phosphorylation towards glycolysis. Mechanically, SIRT3 deacetylates and activates pyruvate dehydrogenase E1 alpha (PDHE1a) at lysine 83, loss of SIRT3 lead to decreased PDH activity, and accumulation of pyruvate and lactate metabolites. The PDHK inhibitor dichloroacetate (DCA) suppresses both pro-IL-1β maturation and caspase-1 activation in macrophages in response to Ang II. The blockade of IL-1β from macrophage into brown adipocytes restores expression of brown adipocyte specific markers. Coincidentally, NLRP3 KO mice exhibited a reduction in infiltrated macrophages in PVAT, while partially rescuing brown adipocytes mitochondrial function and collagen accumulation. Conclusions: Taken together, these findings reveal that SIRT3-mediated activity of PDHE1a inhibits macrophage activation. Pharmacological targeting of glycolysis metabolism may therefore represent an effective approach.

Objective: This study aims to investigate different patterns of aortic stiffness and vasodilation in spontaneously hypertensive rats treated with β-blockers, with or without vasodilatory properties. Methods: Spontaneously hypertensive rats (SHRs) were treated with metoprolol (200 mg·kg−1·d−1), arotinolol (30 mg·kg−1·d−1), and nebivolol (15 mg·kg−1·d−1) for 8 weeks. Age-matched Wistar-Kyoto rats and untreated SHRs were used as controls. Central arterial pressure and pulse wave velocity were evaluated via catheter pressure transducers. The rings of rat aorta, and renal and mesenteric arteries were evaluated by isometric force recording. Collagen was assessed by immunohistochemistry, while endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation (p-eNOS) were analyzed by western blotting. Nitric oxide and reactive oxygen species were measured in human aortic endothelial cells by using fluorescent probes. Results: Data showed that nebivolol and arotinolol, rather than metoprolol, markedly decreased central arterial pressure and pulse wave velocity at week 8, and compared to metoprolol, both nebivolol and arotinolol obviously increased vasorelaxation—aortic vasorelaxation to acetylcholine and p-eNOS/eNOS, and the effects of nebivolol were more pronounced. In addition, aortic collagen depositions in the nebivolol and arotinolol groups were reduced compared with those in the metoprolol group or untreated SHRs. Nebivolol and arotinolol produced more nitric oxide and had higher oxygen radical-scavenging capacities compared to metoprolol in the endothelial cells. Intriguingly, a potassium channel inhibitor (4-aminopyridine) caused a significant reduction in artinolol-induced vasorelaxation, but not nebivolol. Conclusions: These findings suggested that vasodilating β-blockers with nitric oxide production may cause increased reduction of arterial stiffness via different mechanisms.

Objectives: Whether reactive oxygen species (ROS) involves in the arterial stiffness has not been reported. Here we observe the relationship between ROS and the structural and functional changes of large arteries, and the influence of apocynin and losartan in arterial stiffness in this model. Methods: DOCA salt hypertensive rats were generated as previous described. For apocynin treatment groups, three concentrations of apocynin or losartan were administered orally in a tap water solution. At the end of the sixth week, tail blood pressure, central blood pressure, pulse wave velocity (PWV) were measured. The structure and composition and the ROS level in throat aorta also been analyzed at the end of the study. Results: DOCA salt hypertensive rats are characterized by the increased collagen content and stiffness in aorta accompanied by the enhanced level of ROS. The higher dosages of apocynin reduced blood pressure(159.83 ± 7.99, 156.83 ± 11.47 vs 183.89 ± 9.87mmHg, P < 0.05), and PWV(5.17 ± 0.33, 5.31 ± 0.28, vs 6.21 ± 0.24m/s, P < 0.05). But only 1.5 mM Apocynin treatment reduced the collagen content in arterial wall(0.84 ± 0.04 vs 1.03 ± 0.09 mg/cm, P < 0.05)and ROS level markedly (436.50 ± 61.92 vs 781.65 ± 108.68 RLU/min/mg, P < 0.05). Losartan treatment also significantly decreased PWV (5.75 ± 0.20 vs. 6.33 ± 0.20 m/s, P < 0.05) but without altering tail blood pressure, central blood pressure and collagen content in arterial wall in DSH rats. The heightened ROS production in the aortic ring also dropped after losartan treatment. Conclusion: These results suggest that blockade of NADPH oxidase or local AT1R signaling exerts a therapeutic effect on increased arterial stiffness by reducing oxidative stress in DSH rats.

Objective To study the difference of the incidence of cerebral micro-bleeding(CMBs)in patients with stroke and the normal controls using MR susceptibility weighted imaging(SWI),and to analyze the relationship between CMBs and severity of lacunar infarction and cerebral white matter changes.Methods Conventional MR sequences and SWI were performed in 28 patients with ischemic stroke,19 patients with cerebral hemorrhage and 26 cases of control group.The frequency,location,number of CMBs and stroke location,leukoaraiosis of patients were recorded.The severity of CMBs was classified as absent,mild,moderate and severe.All data were statistically analyzed by using SPSS 13.0.Results The incidence of CMBs in cerebral hemorrhage group,cerebral infarction group and control group was 63.2%,32.1%and 7.7%respectively.The most common locations of CMBs were deep location of cerebral,followed by cortico-subcortical region,and infratentorial.There was significant correlation between the presence of CMBs and the severity of lacunar infarction and the severity of white matter lesions(P 0.01).Conclusions CMBs are closely related with cerebral microangiopathy and occur frequently in patients with stroke.SWI sequence has a marked advantage on detection of CMBs.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is highly expressed in heart and skeletal muscles, and is the major enzyme that metabolizes acetaldehyde and toxic aldehydes. The cardioprotective effects of ALDH2 during cardiac ischemia/reperfusion injury have been recognized. However, less is known about the function of ALDH2 in skeletal muscle. PURPOSE: This study was designed to evaluate the effect of ALDH2 on exhaustive exercise-induced skeletal muscle injury. METHODS: We created transgenic mice expressing ALDH2 in skeletal muscles. Male wild-type C57/BL6 (WT) and ALDH2 transgenic mice (ALDH2-Tg), 8-weeks old, were challenged with exhaustive exercise for 1 week to induce skeletal muscle injury. Animals were sacrificed 24 h post-exercise and muscle tissue was excised. RESULTS: ALDH2-Tg mice displayed significantly increased treadmill exercise capacity compared to WT mice. Exhaustive exercise caused an increase in mRNA levels of the muscle atrophy markers, Atrogin-1 and MuRF1, and reduced mitochondrial biogenesis and fusion in WT skeletal muscles; these effects were attenuated in ALDH2-Tg mice. Exhaustive exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of Beclin1 and Bnip3; the effects of which were mitigated by ALDH2 overexpression. In addition, ALDH2-Tg reversed the increase of an oxidative stress biomarker (4-hydroxynonenal) and decreased levels of mitochondrial antioxidant proteins, including manganese superoxide dismutase and NAD(P)H:quinone oxidoreductase 1, in skeletal muscle induced by exhaustive exercise. CONCLUSIONS: ALDH2 may reverse skeletal muscle mitochondrial dysfunction due to exhaustive exercise by regulating mitochondria dynamic remodeling and enhancing the quality of mitochondria.

Objectives: Enhanced glycolysis has been recognized as a critical role in initiating endothelial to mesenchymal transition (EndMT) progression. The underlying regulators of the metabolic program in Angiotension II (Ang II)-induced EndMT remain unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is a major cellular sensor of energy metabolism and mediates adaptation responses. The present study is designed to investigate the role of SIRT3-medated glycolysis enhancing in regulating the processes of EndMT. Methods: Experiments were preformed with murine aortic endothelial cells (MAECs) which were infected with lentivirus SIRT3 shRNA, ATG5 shRNA or GFP-expressing vector. EndMT was induced by Ang II (1 μM for 24 h) and the expression of endothelial and mesenchymal markers (CD31, FSP1 and α-SMA) were detected by Western blot analysis. Results: AngII leads to impaired mitophagy flux in SIRT3-deficient ECs, accompanying marked changes in EC architecture, endothelial loss and gain of mesenchymal markers. The accumulation of damaged mitochondria during EndMT is coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes, which correlates with the upregulation of acetylation of endogenous ATG5. ATG5 acetylation inhibits autophagosome maturation andinduces metabolic reprogramming. Consistent with this, en face analyses displays a marked increase in α-SMC-positive endothelial cells in SIRT3−/− mice challenged with Ang II. Further, we find that SIRT3 overexpression represses glycolysis and transition in endothelial cells. Pharmacological or genetic inhibition of glycolysis significantly attenuates α-SMA expression. Conclusion: Taken together, these findings reveal that SIRT3-deficient ECs display impaired autophagy and accelerate glycolysis and the processes of EndMT.

Objectives: Endothelial to mesenchymal transition (EndoMT) has recently emerged as a potentially important contributor in promoting fibrosis in chronic kidney disease. However, little is known about the role and molecular basis of its involvement in hypertensive renal injury. Here, we aim to determine the role of SIRT3 on EndoMT in hypertensive renal injury and to explore its underlying mechanisms. Methods: Adult wild-type, SIRT3 knockout (SIRT3−/−), non transgenic and SIRT3 endothelial cell-specific transgenic (SIRT3-TgEC) mice were infused with Ang II for 4 weeks. Results: We found that SIRT3 expression was significantly reduced in Ang II-induced hypertensive model, accompanied with induction of EndoMT and increased reactive oxygen species (ROS) and renal fibrosis. In SIRT3−/− mice subjected to Ang II infusion, renal dysfunction was aggravated with an increased EndoMT and ROS level, while in SIRT3-TgEC mice, the Ang II-induced renal fibrosis and EndoMT and oxidative stress were ameliorated. With primary mouse glomerular endothelial cells we confirmed that Ang II treatment initiated EndoMT and decreased catalase expression, which were suppressed by SIRT3 overexpression. Using immunoprecipitation, luciferase and chromatin immunoprecipitation assay we demonstrated that SIRT3-mediated deacetylation and nuclear localization of forkhead box O3a (Foxo3a) resulted in activated Foxo3a-dependent catalase expression. Moreover, Foxo3a knockdown abolished SIRT3-mediated suppression of EndoMT. Conclusion: In conclusion, these results established the SIRT3-Foxo3a-catalase pathway as a critical factor in the maintenance of endothelial homeostasis and point to an important role of EndoMT in the vascular pathology of renal fibrosis, which may provide a new therapeutic target to impede the progression of hypertensive renal injury.

Objective: Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is a major cellular sensor of energy metabolism. The aim of this work was therefore to determine the role of SIRT3-mediated autophagy in cellular metabolism and the processes of endothelial-to-mesenchymal transition (EndoMT). Design and method: Experiments were preformed on widetype (WT), SIRT3 knockout (SIRT3 KO) and SIRT3 transgenic (SIRT3 Tg) mice infused with Angiotension II (Ang II) or saline for 2 weeks. Murine aortic endothelial cells (MAECs) were cultured with Ang II, while the protein levels of mesenchymal markers, autophagy related protein and glycolytic enzymes were evaluated by Western blot analysis or immunofluorescence. Glycolysis was determined by measuring lactic acid release (ECAR) using an XF24 XF analyser. Results: We demonstrated that Ang II led to defective autophagy flux and high levels of glycolysis during EndoMT. The loss of SIRT3 further induced the hyperacetylation of endogenous ATG5, which in turn inhibited autophagosome maturation and increased pyruvate kinase M2 (PKM2) dimer expression. The M2 dimer is the less active form of PKM2, which drives glucose through the route of aerobic glycolysis. Additionally, TEPP-46, a selective PKM2 tetramer activator, had lower concentrations of lactate and led to the reduction of EndoMT both in vitro and in vivo. In parallel, the blockade of lactate influx from ECs into vascular smooth muscle cells (VSMCs) downregulated synthetic VSMC markers. EC-specific SIRT3-Tg mice exhibited a reduction in their endothelial cell transition, while partially rescuing vascular fibrosis and collagen accumulation. Conclusions: Taken together, these findings reveal that SIRT3 regulates EndoMT by improving the autophagic degradation of PKM2. Pharmacological targeting of glycolysis metabolism may therefore represent an effective therapeutic strategy for hypertensive vascular remodelling.

Industry convergence is a popular term that has been widely referenced in the context of rural tourism development in China. All levels of government (local, regional, national) in China have repea...

To test whether P38 MAPK is involved in angiotensin II (Ang II)-enhanced migration potential of adventitial fibroblasts (AFs) from spontaneously hypertensive rat (SHR).Migratory potential was estimated by transwell chamber in vitro. Activation of P38 MAPK pathway was determined with phosphospecific antibodies by immunoblotting.Ang II induced migration of SHR-AFs was markedly increased in a dose-dependent manner when compared with WKY-AFs. Addition of the Ang II receptor type-1 (AT1-R) antagonist Losartan and P38 MAPK inhibitor SB202190 suppressed Ang II-induced migration of SHR-AFs. Ang II could induce P38 MAPK phosphorylation in SHR-AFs in a time-and dose-dependent manner. Phosphorylation of P38 MAPK was suppressed by Losartan and SB202190.This study indicated that Ang II-induced migration involves P38 MAPK pathway via AT1 receptor in aortic adventitial fibroblasts from SHR.

A high performance liquid chromatography was used for the determination of gardenia yellow pigment according to the standard (GB/T5009.149-2003) was studied. The contents of gardenia yellow pigment were analyzed and compared by spectrophotometry. The results showed that it was not reasonable to assay the gardenia yellow pigment with the standard control in food by high performance liquid chromatography. All these other works could establish a foundation for a better qualitative and quantitative analysis method of gardenia yellow pigment.

Aim: To investigate and study the sensibility of leukemic cells to arsenic trioxide. Methods: Histochemistry, propidium iodile and flow cytometry were used in the experiments. Results: More than 0. 5μmol/L of arsenic trioxide for 48 h could produce the apoptosis to NB4 leukemic cells; however, 2μmol/L of arsenic trioxide for 72 h could produce the apoptosis to K562 leukemic cells. WTHZConclusion: WTBZIt was discovered that there exists the different sensibility of leukemic cells to arsenic trioxide.