ß-Hydroxybutyrate Prevents Vascular Senescence through hnRNP A1-Mediated Upregulation of Oct4.

ß-hydroxybutyrate (ß-HB) elevation during fasting or caloric restriction is believed to induce anti-aging effects and alleviate aging-related neurodegeneration. However, whether ß-HB alters the senescence pathway in vascular cells remains unknown. Here we report that ß-HB promotes vascular cell quiescence, which significantly inhibits both stress-induced premature senescence and replicative senescence through p53-independent mechanisms. Further, we identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) as a direct binding target of ß-HB. ß-HB binding to hnRNP A1 markedly enhances hnRNP A1 binding with Octamer-binding transcriptional factor (Oct) 4 mRNA, which stabilizes Oct4 mRNA and Oct4 expression. Oct4 increases Lamin B1, a key factor against DNA damage-induced senescence. Finally, fasting and intraperitoneal injection of ß-HB upregulate Oct4 and Lamin B1 in both vascular smooth muscle and endothelial cells in mice in vivo. We conclude that ß-HB exerts anti-aging effects in vascular cells by upregulating an hnRNP A1-induced Oct4-mediated Lamin B1 pathway.

Mitochondria-derived Vesicle Packaging as a Novel Therapeutic Mechanism in Pulmonary Hypertension.

Increasing evidence suggests that mitochondrial dysfunction in pulmonary endothelial cells (ECs) plays a causative role in the initiation and progression of pulmonary hypertension (PH); how mitochondria become dysfunctional in PH remains elusive. Mitochondria-derived vesicles (MDVs) are small subcellular vesicles that excise from mitochondria. Whether MDV deregulation causes mitochondrial dysfunction in PH is unknown. The aim of this study was to determine MDV regulation in ECs and to elucidate how MDV deregulation in ECs leads to PH. MDV formation and mitochondrial morphology/dynamics were examined in ECs of EC-specific liver kinase B1 (LKB1) knockout mice (LKB1ec-/-), in monocrotaline-induced PH rats, and in lungs of patients with PH. Pulmonary ECs of patients with PH and hypoxia-treated pulmonary ECs exhibited increased mitochondrial fragmentation and disorganized mitochondrial ultrastructure characterized by electron lucent-swelling matrix compartments and concentric layering of the cristae network, together with defective MDV shedding. MDVs actively regulated mitochondrial membrane dynamics and mitochondrial ultrastructure via removing mitofission-related cargoes. The shedding of MDVs from parental mitochondria required LKB1-mediated mitochondrial recruitment of Rab9 GTPase. LKB1ec-/- mice spontaneously developed PH with decreased mitochondrial pools of Rab9 GTPase, defective MDV shedding, and disequilibrium of the mitochondrial fusion-fission cycle in pulmonary ECs. Aerosol intratracheal delivery of adeno-associated virus LKB1 reversed PH, together with improved MDV shedding and mitochondrial function in rats in vivo. We conclude that LKB1 regulates MDV shedding and mitochondrial dynamics in pulmonary ECs by enhancing mitochondrial recruitment of Rab9 GTPase. Defects of LKB1-mediated MDV shedding from parental mitochondria instigate EC dysfunction and PH.

SMYD2 regulates vascular smooth muscle cell phenotypic switching and intimal hyperplasia via interaction with myocardin.

The SET and MYND domain-containing protein 2 (SMYD2) is a histone lysine methyltransferase that has been reported to regulate carcinogenesis and inflammation. However, its role in vascular smooth muscle cell (VSMC) homeostasis and vascular diseases has not been determined. Here, we investigated the role of SMYD2 in VSMC phenotypic modulation and vascular intimal hyperplasia and elucidated the underlying mechanism. We observed that SMYD2 expression was downregulated in injured carotid arteries in mice and phenotypically modulated VSMCs in vitro. Using an SMC-specific SMYD2 knockout mouse model, we found that SMYD2 ablation in VSMCs exacerbated neointima formation after vascular injury in vivo. Conversely, SMYD2 overexpression inhibited VSMC proliferation and migration in vitro and attenuated arterial narrowing in injured vessels in mice. SMYD2 downregulation promoted VSMC phenotypic switching accompanied with enhanced proliferation and migration. Mechanistically, genome-wide transcriptome analysis and loss/gain-of-function studies revealed that SMYD2 up-regulated VSMC contractile gene expression and suppressed VSMC proliferation and migration, in part, by promoting expression and transactivation of the master transcription cofactor myocardin. In addition, myocardin directly interacted with SMYD2, thereby facilitating SMYD2 recruitment to the CArG regions of SMC contractile gene promoters and leading to an open chromatin status around SMC contractile gene promoters via SMYD2-mediated H3K4 methylation. Hence, we conclude that SMYD2 is a novel regulator of VSMC contractile phenotype and intimal hyperplasia via a myocardin-dependent epigenetic regulatory mechanism.

Indoleamine 2,3-Dioxygenase 1 Deletion-Mediated Kynurenine Insufficiency in Vascular Smooth Muscle Cells Exacerbates Arterial Calcification.

BACKGROUND: IDO1 (indoleamine 2,3-dioxygenase 1) is the rate-limiting enzyme for tryptophan metabolism. IDO1 malfunction is involved in the pathogenesis of atherosclerosis. Vascular smooth muscle cells (VSMCs) with an osteogenic phenotype promote calcification and features of plaque instability. However, it remains unclear whether aberrant IDO1-regulated tryptophan metabolism causes VSMCs osteogenic reprogramming and calcification. METHODS: We generated global Apoe (apolipoprotein E) and Ido1 double knockout mice, and Apoe knockout mice with specific deletion of IDO1 in VSMCs or macrophages. Arterial intimal calcification was evaluated by a Western diet-induced atherosclerotic calcification model. RESULTS: Global deficiency of IDO1 boosted calcific lesion formation without sex bias in vivo. Conditional IDO1 loss of function in VSMCs rather than macrophages promoted calcific lesion development and the abundance of RUNX2 (runt-related transcription factor 2). In contrast, administration of kynurenine via intraperitoneal injection markedly delayed the progression of intimal calcification in parallel with decreased RUNX2 expression in both Apoe-/- and Apoe-/-Ido1-/- mice. We found that IDO1 deletion restrained RUNX2 from proteasomal degradation, which resulted in enhanced osteogenic reprogramming of VSMCs. Kynurenine administration downregulated RUNX2 in an aryl hydrocarbon receptor-dependent manner. Kynurenine acted as the endogenous ligand of aryl hydrocarbon receptor, controlled resultant interactions between cullin 4B and aryl hydrocarbon receptor to form an E3 ubiquitin ligase that bound with RUNX2, and subsequently promoted ubiquitin-mediated instability of RUNX2 in VSMCs. Serum samples from patients with coronary artery calcification had impaired IDO1 activity and decreased kynurenine catabolites compared with those without calcification. CONCLUSIONS: Kynurenine, an IDO1-mediated tryptophan metabolism main product, promotes RUNX2 ubiquitination and subsequently leads to its proteasomal degradation via an aryl hydrocarbon receptor-dependent nongenomic pathway. Insufficient kynurenine exerts the deleterious role of IDO1 ablation in promoting RUNX2-mediated VSMCs osteogenic reprogramming and calcification in vivo.

Perioperative EEG background and discharge abnormalities in children undergoing cardiac surgery: a prospective single-centre observational study.

BACKGROUND: We analysed the characteristics of abnormal electroencephalogram (EEG) patterns before, during, and 48 h after cardiac surgery in patients with heterogeneous congenital heart disease to assess their relationship to demographic and perioperative variables and to early patient outcomes. METHODS: In 437 patients enrolled in a single centre, EEG was evaluated for background (including sleep-wake cycle) and discharge (seizures, spikes/sharp waves, pathological delta brushes) abnormalities. Clinical data (arterial blood pressure, doses of inotropic drugs, and serum lactate concentrations) were recorded every 3 h. Postoperative brain MRI was performed before discharge. RESULTS: Preoperative, intraoperative, and postoperative EEG was monitored in 139, 215, and 437 patients, respectively. Patients with a degree of preoperative background abnormalities (n=40) had more severe intraoperative and postoperative EEG abnormalities (P<0.0001). Intraoperatively, 106/215 (49.3%) patients progressed into an isoelectric EEG. Longer durations of isoelectric EEG were associated with more severe postoperative EEG abnormalities and brain injury on MRI (Ps≤0.003). Postoperative background abnormalities occurred in 218/437 (49.9%) patients, and 119 (54.6%) of them had not recovered after surgery. Seizures occurred in 36/437 (8.2%) patients, spikes/sharp waves in 359/437 (82.2%), and pathological delta brushes in 9/437 (2.0%). Postoperative EEG abnormalities correlated with degree of brain injury on MRI (Ps≤0.02). Demographic and perioperative variables were significantly correlated with postoperative EEG abnormalities, which in turn correlated with adverse clinical outcomes. CONCLUSIONS: Perioperative EEG abnormalities occurred frequently and correlated with numerous demographic and perioperative variables and adversely correlated with postoperative EEG abnormalities and early outcomes. The relation of EEG background and discharge abnormalities with long-term neurodevelopmental outcomes remains to be explored.

Acaricidal activities of paeonol from Moutan Cortex, dried bark of Paeonia × suffruticosa, against the grain pest mite Aleuroglyphus ovatus (Acari: Acaridae).

Aleuroglyphus ovatus (Acari: Acaridae) is a major pest mite of stored grains that is distributed worldwide. Paeonol, a phenolic component of the essential oil extracted from the Chinese herb Paeonia moutan, possesses a range of biological activities, including antiviral, antifungal and acaricidal activity. This study investigated the bioactivity of paeonol against A. ovatus and its effect on the activity of detoxification enzymes. The bioactivity of paeonol against A. ovatus was determined by contact, fumigation and repellency bioassays, and the mechanism was preliminarily explored via morphological observation of the color changes of mite epidermis and determination of the changing trend of some important enzymes associated with acaricidal efficacy in the mites. The results showed that the median lethal concentration (LC50) in the contact and fumigation bioassays was 9.832 µg/cm2 and 14.827 µg/cm3, respectively, and the acaricidal activity of paeonol was higher under direct contact than under fumigation. Dynamic symptomatology studies registered typical neurotoxicity symptoms including excitation, convulsion and paralysis in A. ovatus treated with paeonol. The enzyme activity of catalase (CAT), nitric oxide synthase (NOS) and glutathione-S-transferase (GST) was higher, whereas the activity of superoxide dismutase (SOD) and acetylcholinesterase (AChE) was lower, compared to the control group. CAT, NOS and GST were activated, whereas SOD and AChE activities were inhibited after paeonol intervention. Our findings suggest paeonol has potent acaricidal activity against A. ovatus and thus may be used as an agent to control the stored-product mite A. ovatus.

New insight into the role of lipid metabolism-related proteins in rheumatic heart valve disease.

PURPOSE: The aim of this study was to determine the expression of lipid metabolism-related proteins in rheumatic heart valve disease (RHVD). METHODS: This retrospective study involved a total of 20 cases of moderate or severe rheumatic mitral valve stenosis and 4 cases of mitral regurgitation due to secondary causes from September 2018 to September 2021. The patients enrolled included 12 males and 12 females who underwent surgical excision of the mitral valve at the cardiac surgery department of Hainan General Hospital. The samples of mitral valve were collected during surgery treatment as the study group, and mitral valves collected from patients with ischemic heart disease were allocated into the control group. Hematoxylin-eosin (HE), oil red staining and immunohistochemical (IHC) staining were conducted to compare the expression of lipid metabolism-related proteins (ATP-binding cassette transporter A1 and acyl-coenzyme A: cholesterol acyltransferase-1), and real-time polymerase chain reaction (RT-PCR) was applied to compare the mRNA levels of ABCA1, ACAT1, and the inflammatory cytokines TNF-α, IL-10, and MCP-1. RESULTS: In general, the rheumatic mitral valve showed leaflet thickening along with border adhesions and visible yellow fats. Oil red O staining also revealed the abovementioned results as well as fat cells. Both ABCA1 and ACAT1 were expressed in the rheumatic mitral valve via IHC, whereas only ACAT1 showed a faint level of expression in the ischemic mitral valve with no expression of ABCA1. In addition, compared with the ischemic mitral valve, RT-PCT showed increased mRNA expression levels of ABCA1, ACAT1, and the inflammatory cytokines TNF-α, IL-10, and MCP-1 (P < 0.05). After dividing the RMs into two groups for RT-PCR, we found that the higher the expression of ABCA1 and ACAT1 was, the lower the relative expression of inflammatory factors. CONCLUSION: This study showed that adipose tissue, adipose cells, and lipid transport-related proteins were expressed strongly in the rheumatic mitral valve, suggesting that adipose tissue formation might be one of the important pathways in the pathology of rheumatic heart disease. In addition, adipose tissue and adipocytes were also involved in the inflammatory process. These data provide new insight into pathological mechanisms in rheumatic heart disease.

Features of Lipid Metabolism in Humanized ApoE Knockin Rat Models.

Apolipoprotein E (ApoE), an essential plasma apolipoprotein, has three isoforms (E2, E3, and E4) in humans. E2 is associated with type III hyperlipoproteinemia. E4 is the major susceptibility gene to Alzheimer's disease (AD) and coronary heart disease (CHD). We investigated lipid metabolism and atherosclerotic lesions of novel humanized ApoE knockin (hApoE KI) rats in comparison to wide-type (WT) and ApoE knockout (ApoE KO) rats. The hApoE2 rats showed the lowest bodyweight and white fat mass. hApoE2 rats developed higher serum total cholesterol (TC), total triglyceride (TG), and low- and very low density lipoprotein (LDL-C&VLDL-C). ApoE KO rats also exhibited elevated TC and LDL-C&VLDL-C. Only mild atherosclerotic lesions were detected in hApoE2 and ApoE KO aortic roots. Half of the hApoE2 rats developed hepatic nodular cirrhosis. A short period of the Paigen diet (PD) treatment led to the premature death of the hApoE2 and ApoE KO rats. Severe vascular wall thickening of the coronary and pulmonary arteries was observed in 4-month PD-treated hApoE4 rats. In conclusion, hApoE2 rats develop spontaneous hyperlipidemia and might be suitable for studies of lipid metabolism-related diseases. With the PD challenge, hApoE4 KI rats could be a novel model for the analysis of vascular remodeling.

BRD4 inhibition by JQ1 prevents high-fat diet-induced diabetic cardiomyopathy by activating PINK1/Parkin-mediated mitophagy in vivo.

BRD4 is a member of the BET family of epigenetic regulators. Inhibition of BRD4 by the selective bromodomain inhibitor JQ1, alleviates thoracic aortic constriction-induced cardiac hypertrophy and heart failure. However, whether BRD4 inhibition by JQ1 has therapeutic effect on diabetic cardiomyopathy, a major cause of heart failure in patients with Type 2 diabetes, remains unknown. Here, we discover a novel link between BRD4 and PINK1/Parkin-mediated mitophagy during diabetic cardiomyopathy. Upregulation of BRD4 in diabetic mouse hearts inhibits PINK1/Parkin-mediated mitophagy, resulting in accumulation of damaged mitochondria and subsequent impairment of cardiac structure and function. BRD4 inhibition by JQ1 improves mitochondrial function, and repairs the cardiac structure and function of the diabetic heart. These effects depended on rewiring of the BRD4-driven transcription and repression of PINK1. Deletion of Pink1 suppresses mitophagy, exacerbates cardiomyopathy, and abrogates the therapeutic effect of JQ1 on diabetic cardiomyopathy. Our results illustrate a valid therapeutic strategy for treating diabetic cardiomyopathy by inhibition of BRD4.

Hyperglycemia-Driven Inhibition of AMP-Activated Protein Kinase α2 Induces Diabetic Cardiomyopathy by Promoting Mitochondria-Associated Endoplasmic Reticulum Membranes In Vivo.

BACKGROUND: Fundc1 (FUN14 domain containing 1), an outer mitochondrial membrane protein, is important for mitophagy and mitochondria-associated endoplasmic reticulum membranes (MAMs). The roles of Fundc1 and MAMs in diabetic hearts remain unknown. The aims of this study, therefore, were to determine whether the diabetes mellitus-induced Fundc1 expression could increase MAM formation, and whether disruption of MAM formation improves diabetic cardiac function. METHODS: Levels of FUNDC1 were examined in the hearts from diabetic patients and nondiabetic donors. Levels of Fundc1-induced MAMs and mitochondrial and heart function were examined in mouse neonatal cardiomyocytes exposed to high glucose (HG, 30 mmol/L d-glucose for 48 hours), and in streptozotocin-treated cardiac-specific Fundc1 knockout mice and cardiac-specific Fundc1 knockout diabetic Akita mice, as well. RESULTS: FUNDC1 levels were significantly elevated in cardiac tissues from diabetic patients in comparison with those from nondiabetic donors. In cultured mouse neonatal cardiomyocytes, HG conditions increased levels of Fundc1, the inositol 1,4,5-trisphosphate type 2 receptor (Ip3r2), and MAMs. Genetic downregulation of either Fundc1 or Ip3r2 inhibited MAM formation, reduced endoplasmic reticulum-mitochondrial Ca2+ flux, and improved mitochondrial function in HG-treated cardiomyocytes. Consistently, adenoviral overexpression of Fundc1 promoted MAM formation, mitochondrial Ca2+ increase, and mitochondrial dysfunction in cardiomyocytes exposed to normal glucose (5.5 mmol/L d-glucose). In comparison with nondiabetic controls, levels of Fundc1, Ip3r2, and MAMs were significantly increased in hearts from streptozotocin-treated mice and Akita mice. Furthermore, in comparison with control hearts, diabetes mellitus markedly increased coimmunoprecipitation of Fundc1 and Ip3r2. The binding of Fundc1 to Ip3r2 inhibits Ip3r2 ubiquitination and proteasome-mediated degradation. Cardiomyocyte-specific Fundc1 deletion ablated diabetes mellitus-induced MAM formation, prevented mitochondrial Ca2+ increase, mitochondrial fragmentation, and apoptosis with improved mitochondrial functional capacity and cardiac function. In mouse neonatal cardiomyocytes, HG suppressed AMP-activated protein kinase activity. Furthermore, in cardiomyocytes of Prkaa2 knockout mice, expression of Fundc1, MAM formation, and mitochondrial Ca2+ levels were significantly increased. Finally, adenoviral overexpression of a constitutively active mutant AMP-activated protein kinase ablated HG-induced MAM formation and mitochondrial dysfunction. CONCLUSIONS: We conclude that diabetes mellitus suppresses AMP-activated protein kinase, initiating Fundc1-mediated MAM formation, mitochondrial dysfunction, and cardiomyopathy, suggesting that AMP-activated protein kinase-induced Fundc1 suppression is a valid target to treat diabetic cardiomyopathy.

Comprehensive analysis of differential immunocyte infiltration and the potential ceRNA networks during epicardial adipose tissue development in congenital heart disease.

BACKGROUND: To detect the development, function and therapeutic potential of epicardial adipose tissue (EAT); analyze a related gene expression dataset, including data from neonates, infants, and children with congenital heart disease (CHD); compare the data to identify the codifferentially expressed (DE) mRNAs and lncRNAs and the corresponding miRNAs; generate a potential competitive endogenous RNA (ceRNA) network; and assess the involvement of immunocyte infiltration in the development of the EAT. METHODS: Multiple algorithms for linear models for microarray data algorithms (LIMMA), CIBERSORT, gene-set enrichment analysis (GSEA), and gene set variation analysis (GSVA) were used. The miRcode, miRDB, miRTarBase, and TargetScan database were used to construct the ceRNA network. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DE mRNAs were performed. RESULTS: Thirteen co-DE mRNAs and 47 co-DE lncRNAs were subsequently identified. The related categories included negative regulation of myoblast differentiation, regulation of ion transmembrane transport, and heart development, which were primarily identified for further pathway enrichment analysis. Additionally, the hub ceRNA network in EAT development involving MIR210HG, hsa-miR-449c-5p, and CACNA2D4 was generated and shown to target monocyte infiltration. CONCLUSION: These findings suggest that the pathways of myoblast differentiation and ion transmembrane transport may be potential hub pathways involved in EAT development in CHD patients. In addition, the network includes monocytes, MIR210HG, and CACNA2D4, which were shown to target the RIG-I-like receptor signaling pathway and PPAR signaling pathway, indicating that these factors may be novel regulators and therapeutic targets in EAT development.

AMPKα2 Protects Against the Development of Heart Failure by Enhancing Mitophagy via PINK1 Phosphorylation.

RATIONALE: Mitochondrial dysfunction plays an important role in heart failure (HF). However, the molecular mechanisms regulating mitochondrial functions via selective mitochondrial autophagy (mitophagy) are poorly understood. OBJECTIVE: We sought to determine the role of AMPK (AMP-activated protein kinase) in selective mitophagy during HF. METHODS AND RESULTS: An isoform shift from AMPKα2 to AMPKα1 was observed in failing heart samples from HF patients and transverse aortic constriction-induced mice, accompanied by decreased mitophagy and mitochondrial function. The recombinant adeno-associated virus Serotype 9-mediated overexpression of AMPKα2 in mouse hearts prevented the development of transverse aortic constriction-induced chronic HF by increasing mitophagy and improving mitochondrial function. In contrast, AMPKα2-/- mutant mice exhibited an exacerbation of the early progression of transverse aortic constriction-induced HF via decreases in cardiac mitophagy. In isolated adult mouse cardiomyocytes, AMPKα2 overexpression mechanistically rescued the impairment of mitophagy after phenylephrine stimulation for 24 hours. Genetic knockdown of AMPKα2, but not AMPKα1, by short interfering RNA suppressed the early phase (6 hours) of phenylephrine-induced compensatory increases in mitophagy. Furthermore, AMPKα2 specifically interacted with phosphorylated PINK1 (PTEN-induced putative kinase 1) at Ser495 after phenylephrine stimulation. Subsequently, phosphorylated PINK1 recruited the E3 ubiquitin ligase, Parkin, to depolarized mitochondria, and then enhanced the role of the PINK1-Parkin-SQSTM1 (sequestosome-1) pathway involved in cardiac mitophagy. This increase in cardiac mitophagy was accompanied by the elimination of damaged mitochondria, improvement in mitochondrial function, decrease in reactive oxygen species production, and apoptosis of cardiomyocytes. Finally, Ala mutation of PINK1 at Ser495 partially suppressed AMPKα2 overexpression-induced mitophagy and improvement of mitochondrial function in phenylephrine-stimulated cardiomyocytes, whereas Asp (phosphorylation mimic) mutation promoted mitophagy after phenylephrine stimulation. CONCLUSIONS: In failing hearts, the dominant AMPKα isoform switched from AMPKα2 to AMPKα1, which accelerated HF. The results show that phosphorylation of Ser495 in PINK1 by AMPKα2 was essential for efficient mitophagy to prevent the progression of HF.

Yes-Associated Protein Promotes Angiogenesis via Signal Transducer and Activator of Transcription 3 in Endothelial Cells.

RATIONALE: Angiogenesis is a complex process regulating endothelial cell (EC) functions. Emerging lines of evidence support that YAP (Yes-associated protein) plays an important role in regulating the angiogenic activity of ECs. OBJECTIVE: The objective of this study was to specify the effect of EC YAP on angiogenesis and its underlying mechanisms. METHOD AND RESULTS: In ECs, vascular endothelial growth factor reduced YAP phosphorylation time and dose dependently and increased its nuclear accumulation. Using Tie2Cre-mediated YAP transgenic mice, we found that YAP promoted angiogenesis in the postnatal retina and tumor tissues. Mass spectrometry revealed signal transducer and activator of transcription 3 (STAT3) as a potential binding partner of YAP in ECs. Western blot and immunoprecipitation assays indicated that binding with YAP prolonged interleukin 6-induced STAT3 nuclear accumulation by blocking chromosomal maintenance 1-mediated STAT3 nuclear export without affecting its phosphorylation. Moreover, angiopoietin-2 expression induced by STAT3 was enhanced by YAP overexpression in ECs. Finally, a selective STAT3 inhibitor or angiopoietin-2 blockage partly attenuated retinal angiogenesis in Tie2Cre-mediated YAP transgenic mice. CONCLUSIONS: YAP binding sustained STAT3 in the nucleus to enhance the latter's transcriptional activity and promote angiogenesis via regulation of angiopoietin-2.

Peroxynitrite-Mediated SIRT (Sirtuin)-1 Inactivation Contributes to Nicotine-Induced Arterial Stiffness in Mice.

Objective- Inhibition of SIRT (sirtuin)-1, a nicotinamide adenine dinucleotide-dependent protein deacetylase, is linked to cigarette smoking-induced arterial stiffness, but the underlying mechanisms remain largely unknown. The aim of the present study was to determine the effects and mechanisms of nicotine, a major component of cigarette smoke, on SIRT1 activity and arterial stiffness. Approach and Results- Arterial stiffness, peroxynitrite (ONOO-) formation, SIRT1 expression and activity were monitored in mouse aortas of 8-week-old C57BL/6 mice (wild-type) or Sirt1-overexpressing ( Sirt1 Super) mice with or without nicotine for 4 weeks. In aortas of wild-type mice, nicotine reduced SIRT1 protein and activity by ≈50% without affecting its mRNA levels. In those from Sirt1 Super mice, nicotine also markedly reduced SIRT1 protein and activity to the levels that were comparable to those in wild-type mice. Nicotine infusion significantly induced collagen I, fibronectin, and arterial stiffness in wild-type but not Sirt1 Super mice. Nicotine increased the levels of iNOS (inducible nitric oxide synthase) and the co-staining of SIRT1 and 3-nitrotyrosine, a footprint of ONOO- in aortas. Tempol, which ablated ONOO- by scavenging superoxide anion, reduced the effects of nicotine on SIRT1 and collagen. Mutation of zinc-binding cysteine 395 or 398 in SIRT1 into serine (C395S) or (C398S) abolished SIRT1 activity. Furthermore, ONOO- dose-dependently inhibited the enzyme and increased zinc release in recombinant SIRT1. Finally, we found SIRT1 inactivation by ONOO- activated the YAP (Yes-associated protein) resulting in abnormal ECM (extracellular matrix) remodeling. Conclusions- Nicotine induces ONOO-, which selectively inhibits SIRT1 resulting in a YAP-mediated ECM remodeling. Visual Overview- An online visual overview is available for this article.

AMPK, Mitochondrial Function, and Cardiovascular Disease.

Adenosine monophosphate-activated protein kinase (AMPK) is in charge of numerous catabolic and anabolic signaling pathways to sustain appropriate intracellular adenosine triphosphate levels in response to energetic and/or cellular stress. In addition to its conventional roles as an intracellular energy switch or fuel gauge, emerging research has shown that AMPK is also a redox sensor and modulator, playing pivotal roles in maintaining cardiovascular processes and inhibiting disease progression. Pharmacological reagents, including statins, metformin, berberine, polyphenol, and resveratrol, all of which are widely used therapeutics for cardiovascular disorders, appear to deliver their protective/therapeutic effects partially via AMPK signaling modulation. The functions of AMPK during health and disease are far from clear. Accumulating studies have demonstrated crosstalk between AMPK and mitochondria, such as AMPK regulation of mitochondrial homeostasis and mitochondrial dysfunction causing abnormal AMPK activity. In this review, we begin with the description of AMPK structure and regulation, and then focus on the recent advances toward understanding how mitochondrial dysfunction controls AMPK and how AMPK, as a central mediator of the cellular response to energetic stress, maintains mitochondrial homeostasis. Finally, we systemically review how dysfunctional AMPK contributes to the initiation and progression of cardiovascular diseases via the impact on mitochondrial function.

Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts.

Recent evidence indicates that activation of adenosine monophosphate-activated protein kinase (AMPK), a highly conserved sensor and modulator of cellular energy and redox, regulates cell mitosis. However, the underlying molecular mechanisms for AMPKα subunit regulation of chromosome segregation remain poorly understood. This study aimed to ascertain if AMPKα1 deletion contributes to chromosome missegregation by elevating Polo-like kinase 4 (PLK4) expression. Centrosome proteins and aneuploidy were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J) or AMPKα1 homozygous deficient (AMPKα1-/-) mice by Western blotting and metaphase chromosome spread. Deletion of AMPKα1, the predominant AMPKα isoform in immortalized MEFs, led to centrosome amplification and chromosome missegregation, as well as the consequent aneuploidy (34-66%) and micronucleus. Furthermore, AMPKα1 null cells exhibited a significant induction of PLK4. Knockdown of nuclear factor kappa B2/p52 ameliorated the PLK4 elevation in AMPKα1-deleted MEFs. Finally, PLK4 inhibition by Centrinone reversed centrosome amplification of AMPKα1-deleted MEFs. Taken together, our results suggest that AMPKα1 plays a fundamental role in the maintenance of chromosomal integrity through the control of p52-mediated transcription of PLK4, a trigger of centriole biogenesis.

Macrophage Liver Kinase B1 Inhibits Foam Cell Formation and Atherosclerosis.

RATIONALE: LKB1 (liver kinase B1) is a serine/threonine kinase and tumor suppressor, which regulates the homeostasis of hematopoietic cells and immune responses. Macrophages transform into foam cells upon taking-in lipids. No role for LKB1 in foam cell formation has previously been reported. OBJECTIVE: We sought to establish the role of LKB1 in atherosclerotic foam cell formation. METHODS AND RESULTS: LKB1 expression was examined in human carotid atherosclerotic plaques and in western diet-fed atherosclerosis-prone Ldlr-/- and ApoE-/- mice. LKB1 expression was markedly reduced in human plaques when compared with nonatherosclerotic vessels. Consistently, time-dependent reduction of LKB1 levels occurred in atherosclerotic lesions in western diet-fed Ldlr-/- and ApoE-/- mice. Exposure of macrophages to oxidized low-density lipoprotein downregulated LKB1 in vitro. Furthermore, LKB1 deficiency in macrophages significantly increased the expression of SRA (scavenger receptor A), modified low-density lipoprotein uptake and foam cell formation, all of which were abolished by blocking SRA. Further, we found LKB1 phosphorylates SRA resulting in its lysosome degradation. To further investigate the role of macrophage LKB1 in vivo, ApoE-/-LKB1fl/flLysMcre and ApoE-/-LKB1fl/fl mice were fed with western diet for 16 weeks. Compared with ApoE-/-LKB1fl/fl wild-type control, ApoE-/-LKB1fl/flLysMcre mice developed more atherosclerotic lesions in whole aorta and aortic root area, with markedly increased SRA expression in aortic root lesions. CONCLUSIONS: We conclude that macrophage LKB1 reduction caused by oxidized low-density lipoprotein promotes foam cell formation and the progression of atherosclerosis.

SNRK (Sucrose Nonfermenting 1-Related Kinase) Promotes Angiogenesis In Vivo.

OBJECTIVE: SNRK (sucrose nonfermenting 1-related kinase) is a novel member of the AMPK (adenosine monophosphate-activated protein kinase)-related superfamily that is activated in the process of angiogenesis. Currently, little is known about the function of SNRK in angiogenesis in the physiological and pathological conditions. APPROACH AND RESULTS: In this study, in Snrk global heterozygous knockout mice, retina angiogenesis and neovessel formation after hindlimb ischemia were suppressed. Consistently, mice with endothelial cell (EC)-specific Snrk deletion exhibited impaired retina angiogenesis, and delayed perfusion recovery and exacerbated muscle apoptosis in ischemic hindlimbs, compared with those of littermate wide-type mice. Endothelial SNRK expression was increased in the extremity vessel samples from nonischemic human. In ECs cultured in hypoxic conditions, HIF1α (hypoxia inducible factor 1α) bound to the SNRK promoter to upregulate SNRK expression. In the nuclei of hypoxic ECs, SNRK complexed with SP1 (specificity protein 1), and together, they bound to an SP1-binding motif in the ITGB1 (ß1 integrin) promoter, resulting in enhanced ITGB1 expression and promoted EC migration. Furthermore, SNRK or SP1 deficiency in ECs ameliorated hypoxia-induced ITGB1 expression and, consequently, inhibited EC migration and angiogenesis. CONCLUSIONS: Taken together, our data have revealed that SNRK/SP1-ITGB1 signaling axis promotes angiogenesis in vivo.

AMP-activated protein kinase α1 promotes atherogenesis by increasing monocyte-to-macrophage differentiation.

Monocyte-to-macrophage differentiation, which can be initiated by physiological or atherogenic factors, is a pivotal process in atherogenesis, a disorder in which monocytes adhere to endothelial cells and subsequently migrate into the subendothelial spaces, where they differentiate into macrophages and macrophage-derived foam cells and cause atherosclerotic lesions. However, the monocyte-differentiation signaling pathways that are activated by atherogenic factors are poorly defined. Here we report that the AMP-activated protein kinase α1 (AMPKα1) in monocytes promotes atherosclerosis by increasing monocyte differentiation and survival. Exposure of monocytes to oxidized low-density lipoprotein, 7-ketocholesterol, phorbol 12-myristate 13-acetate, or macrophage colony-stimulated factor (M-CSF) significantly activated AMPK and promoted monocyte-to-macrophage differentiation. M-CSF-activated AMPK is via M-CSF receptor-dependent reactive oxygen species production. Consistently, genetic deletion of AMPKα1 or pharmacological inhibition of AMPK blunted monocyte-to-macrophage differentiation and promoted monocyte/macrophage apoptosis. Compared with apolipoprotein E knock-out (ApoE-/-) mice, which show impaired clearing of plasma lipoproteins and spontaneously develop atherosclerosis, ApoE-/-/AMPKα1-/- mice showed reduced sizes of atherosclerotic lesions and lesser numbers of macrophages in the lesions. Furthermore, aortic lesions were decreased in ApoE-/- mice transplanted with ApoE-/-/AMPKα1-/- bone marrow and in myeloid-specific AMPKα1-deficient ApoE-/- mice. Finally, rapamycin treatment, which abolished delayed monocyte differentiation in ApoE-/-/AMPKα1-/- mice, lost its atherosclerosis-lowering effects in these mice. Mechanistically, we found that AMPKα1 regulates FoxO3-dependent expression of both LC3 and ULK1, which are two important autophagy-related markers. Rapamycin treatment increased FoxO3 activity as well as LC3 and ULK1 expressions in macrophages from AMPKα1-/- mice. Our results reveal that AMPKα1 deficiency impairs autophagy-mediated monocyte differentiation and decreases monocyte/macrophage survival, which attenuates atherosclerosis in ApoE-/- mice in vivo.