Vitamin B-6 Prevents Heart Failure with Preserved Ejection Fraction Through Downstream of Kinase 3 in a Mouse Model.

BACKGROUND: There is an urgent need to develop an efficient therapeutic strategy for heart failure with preserved ejection fraction (HFpEF), which is mediated by phenotypic changes in cardiac macrophages. We previously reported that vitamin B-6 inhibits macrophage-mediated inflammasome activation. OBJECTIVES: We sought to examine whether the prophylactic use of vitamin B-6 prevents HFpEF. METHODS: HFpEF model was elicited by a combination of high-fat diet and Nω-nitro-l-arginine methyl ester supplement in mice. Cardiac function was assessed using conventional echocardiography and Doppler imaging. Immunohistochemistry and immunoblotting were used to detect changes in the macrophage phenotype and myocardial remodeling-related molecules. RESULTS: Co-administration of vitamin B-6 with HFpEF mice mitigated HFpEF phenotypes, including diastolic dysfunction, cardiac macrophage phenotypic shifts, fibrosis, and hypertrophy. Echocardiographic improvements were observed, with the E/E' ratio decreasing from 42.0 to 21.6 and the E/A ratio improving from 2.13 to 1.17. The exercise capacity also increased from 295.3 to 657.7 min. However, these beneficial effects were negated in downstream of kinase (DOK) 3-deficient mice. Mechanistically, vitamin B-6 increased DOK3 protein concentrations and inhibited macrophage phenotypic changes, which were abrogated by an AMP-activated protein kinase inhibitor. CONCLUSIONS: Vitamin B-6 increases DOK3 signaling to lower risk of HFpEF by inhibiting phenotypic changes in cardiac macrophages.

Statins improve cardiac endothelial function to prevent heart failure with preserved ejection fraction through upregulating circRNA-RBCK1.

Heart failure with preserved ejection fraction (HFpEF) is associated with endothelial dysfunction. We have previously reported that statins prevent endothelial dysfunction through inhibition of microRNA-133a (miR-133a). This study is to investigate the effects and the underlying mechanisms of statins on HFpEF. Here, we show that statins upregulate the expression of a circular RNA (circRNA-RBCK1) which is co-transcripted with the ring-B-box-coiled-coil protein interacting with protein kinase C-1 (RBCK1) gene. Simultaneously, statins increase activator protein 2 alpha (AP-2α) transcriptional activity and the interaction between circRNA-RBCK1 and miR-133a. Furthermore, AP-2α directly interacts with RBCK1 gene promoter in endothelial cells. In vivo, lovastatin improves diastolic function in male mice under HFpEF, which is abolished by loss function of endothelial AP-2α or circRNA-RBCK1. This study suggests that statins upregulate the AP-2α/circRNA-RBCK1 signaling to suppress miR-133a in cardiac endothelial cells and prevent diastolic dysfunction in HFpEF.

S-nitrosylation of AMPKγ impairs coronary collateral circulation and disrupts VSMC reprogramming.

Collateral circulation is essential for blood resupply to the ischemic heart, which is dictated by the contractile phenotypic restoration of vascular smooth muscle cells (VSMC). Here we investigate whether S-nitrosylation of AMP-activated protein kinase (AMPK), a key regulator of the VSMC phenotype, impairs collateral circulation. In rats with collateral growth and development, nitroglycerin decreases coronary collateral blood flow (CCBF), inhibits vascular contractile phenotypic restoration, and increases myocardial infarct size, accompanied by reduced AMPK activity in the collateral zone. Nitric oxide (NO) S-nitrosylates human recombinant AMPKγ1 at cysteine 131 and decreases AMP sensitivity of AMPK. In VSMCs, exogenous expression of S-nitrosylation-resistant AMPKγ1 or deficient NO synthase (iNOS) prevents the disruption of VSMC reprogramming. Finally, hyperhomocysteinemia or hyperglycemia increases AMPKγ1 S-nitrosylation, prevents vascular contractile phenotypic restoration, reduces CCBF, and increases the infarct size of the heart in Apoe-/- mice, all of which is rescued in Apoe-/-/iNOSsm-/- mice or Apoe-/- mice with enforced expression of the AMPKγ1-C130A mutant following RI/MI. We conclude that nitrosative stress disrupts coronary collateral circulation during hyperhomocysteinemia or hyperglycemia through AMPK S-nitrosylation.

Prognostic significance of SPARC expression in non-small cell lung cancer: A meta-analysis and bioinformatics analysis.

The aim of the present study was to elucidate the significance of secreted protein acidic and cysteine rich (SPARC) expression in non-small cell lung cancer (NSCLC) in terms of clinicopathology, immune-cell infiltration and survival prognosis. A meta-analysis and bioinformatics analysis were performed using studies retrieved with PubMed, Web of Science, Wanfang Data and the Chinese National Knowledge Infrastructure databases. The meta-analysis suggested that, compared with normal tissues, SPARC expression was elevated in NSCLC tissues. The expression of SPARC was not significantly associated with TNM stage and lymph-node metastasis, and was positively associated with patient gender. Regarding the differential expression of SPARC and the relationship between expression levels and survival, the Oncomine database was consulted and Kaplan-Meier curves were drawn. It was indicated that SPARC mRNA expression levels were higher in NSCLC tissues than in normal tissues. Low expression of SPARC mRNA was negatively associated with overall survival, first progression survival and post-progression survival of patients. Further exploration of the relationship between SPARC expression and survival by univariate analysis indicated that TNM stage, lymph node metastasis, distant metastasis and depth of infiltration of lung cancer were negatively associated with patient prognosis. Cox multifactorial analysis suggested that SPARC expression levels and TNM stage were risk factors significantly affecting the prognosis of patients with NSCLC. Analysis with the GEPIA and UALCAN databases further indicated that the mRNA expression level of SPARC in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) was higher than that in normal lung tissue, and the SPARC expression levels were affected by factors such as the TNM stage of lung cancer. A lower the level of SPARC mRNA expression was associated with a better relative survival prognosis of patients. In the Human Protein Atlas database, the expression level of SPARC protein was higher in LUAD and LUSC than in normal lung tissue. In the Timer database, the expression level of SPARC was closely linked to immune cells related to the occurrence of lung cancer, and the degree of immune-cell infiltration and SPARC protein expression were closely related to the prognosis of patients with lung cancer. Immune cells were indicated to exhibit significant inhibition of DNA proliferation mutation mechanisms in lung cancer (P<0.05). In summary, SPARC expression may be used as a valuable indicator of prognosis in patients with NSCLC, which may provide new approaches for preventative treatment.

Continuous Infusion of Angiotensin IV Protects against Acute Myocardial Infarction via the Inhibition of Inflammation and Autophagy.

Acute myocardial infarction (AMI) is a major cause of morbidity and mortality worldwide. Angiotensin (Ang) IV possesses many biological properties that are not yet completely understood. Therefore, we investigated the function and mechanism of Ang IV in AMI in in vivo and in vitro conditions. AMI was performed by ligation of the left anterior descending coronary artery (LAD) in male C57 mice. Ang IV was continuously infused by a minipump 3 d before AMI for 33 d. The neonatal rat ventricular myocytes (NRVCs) were stimulated with Ang IV and cultured under hypoxic conditions. In vivo, Ang IV infusion significantly reduced the mortality after AMI. By the 7th day after AMI, compared with the AMI group, Ang IV reduced the inflammatory cytokine expression. Moreover, terminal deoxyribonucleotidyl transferase- (TDT-) mediated dUTP nick-end labeling (TUNEL) assay showed that Ang IV infusion reduced AMI-induced cardiomyocyte apoptosis. Compared with AMI, Ang IV reduced autophagosomes in cardiomyocytes and improved mitochondrial swelling and disarrangement, as assessed by transmission electron microscopy. By 30th day after AMI, Ang IV significantly reduced the ratio of heart weight to body weight. Echocardiography showed that Ang IV improved impaired cardiac function. Hematoxylin and eosin (H&E) and Masson staining showed that Ang IV infusion reduced the infarction size and myocardial fibrosis. In vitro, dihydroethidium (DHE) staining and comet assay showed that, compared with the hypoxia group, Ang IV reduced oxidative stress and DNA damage. Enzyme-linked immunosorbent assay (ELISA) showed that Ang IV reduced hypoxia-induced secretion of the tumor necrosis factor- (TNF-) ɑ and interleukin- (IL-) 1ß. In addition, compared with the hypoxia group, Ang IV reduced the transformation of light chain 3- (LC3-) I to LC3-II but increased p62 expression and decreased cardiomyocyte apoptosis. Overall, the present study showed that Ang IV reduced the inflammatory response, autophagy, and fibrosis after AMI, leading to reduced infarction size and improved cardiac function. Therefore, administration of Ang IV may be a feasible strategy for the treatment of AMI.

Krüppel-Like Factor 15 Reduces Ischemia-Induced Apoptosis Involving Regulation of p38/MAPK Signaling.

Cardiomyocyte apoptosis is a characteristic of a variety of cardiac diseases, including myocardial infarction (MI). Krüppel-like factor 15 (KLF15) is a transcription factor of Krüppel family that plays an important part in cardiovascular diseases. However, the function and the underlying mechanism of KLF15 in MI remain unknown. The expression of KLF15 was downregulated both in ischemic myocardium of MI mice model and hypoxia-treated neonatal rat ventricular myocytes (NRVCs). KLF15 overexpression mediated by adeno-associated virus significantly abrogated the ischemia-induced cardiac dysfunction, increased the survival rate, and reduced infarct size after MI. Meanwhile, KLF15 overexpression dramatically reduced the myocardial apoptosis, regulated apoptosis-related genes, such as Bcl2 and Bax, diminished the activities of caspase-9/3, and inactivated p38/MAPK signaling in the border zone. Similar results were observed in NRVCs exposed to hypoxia. We demonstrated for the first time that KLF15 overexpression could reduce cardiomyocyte apoptosis and improve cardiac dysfunction in MI mice at least partially by inhibiting p38/MAPK signaling pathway.

Qindan Capsule Attenuates Myocardial Hypertrophy and Fibrosis in Pressure Overload-Induced Mice Involving mTOR and TGF-ß1/Smad Signaling Pathway Inhibition.

Qindan capsule (QC), a traditional Chinese medicine compound, has been used to treat hypertension in the clinic for over 30 years. It is still not known about the effects of QC on pressure overload-induced cardiac remodeling. Hence, this study aims to investigate the effects of QC on pressure overload-induced cardiac hypertrophy, fibrosis, and heart failure in mice and to determine the possible mechanisms. Transverse aortic constriction (TAC) surgery was used to induce cardiac hypertrophy and heart failure in C57BL/6 mice. Mice were treated with QC or losartan for 8 weeks after TAC surgery. Cardiac function indexes were evaluated with transthoracic echocardiography. Cardiac pathology was detected using HE and Masson's trichrome staining. Cardiomyocyte ultrastructure was detected using transmission electron microscopy. Hypertrophy-related fetal gene expression was investigated using real-time RT-PCR. The expression of 8-OHdG and the concentration of MDA and Ang-II were assessed by immunohistochemistry stain and ELISA assay, respectively. The total and phosphorylated protein levels of mTOR, p70S6K, 4EBP1, Smad2, and Smad3 and the expression of TGF-ß1 and collagen I were measured using western blot. The results showed that low- and high-dose QC improved pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction. QC inhibited ANP, BNP, and ß-MHC mRNA expression in failing hearts. QC improved myocardial ultrastructure after TAC surgery. Furthermore, QC downregulated the expression of 8-OHdG and the concentration of MDA, 15-F2t-IsoP, and Ang-II in heart tissues after TAC surgery. We also found that QC inhibited the phosphorylation of mTOR, p70S6K, and 4EBP1 and the expression of TGF-ß1, p-Smad2, p-Smad3, and collagen I in pressure overload-induced failing hearts. These data indicate that QC has direct benefic effects on pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction. The protective effects of QC involve prevention of increased oxidative stress injury and Ang-II levels and inhibition of mTOR and TGF-ß1/Smad pathways in failing hearts.

Excessive expression of miR-1a by statin causes skeletal injury through targeting mitogen-activated protein kinase kinase kinase 1.

BACKGROUNDS: A major side effect of statin, a widely used drug to treat hyperlipidemia, is skeletal myopathy through cell apoptosis. The aim of this study is to investigate the roles of microRNA in statin-induced injury. METHODS: Apolipoprotein E knockout (ApoE-/-) mice were administered with simvastatin (20 mg/kg/day) for 8 weeks. Exercise capacity was evaluated by hanging grid test, forelimb grip strength, and running tolerance test. RESULTS: In cultured skeletal muscle cells, statin increased the levels of miR-1a but decreased the levels of mitogen-activated protein kinase kinase kinase 1 (MAP3K1) in a time or dose dependent manner. Both computational target-scan analysis and luciferase gene reporter assay indicated that MAP3K1 is the target gene of miR-1a. Statin induced cell apoptosis of skeletal muscle cells, but abolished by downregulating of miR-1a or upregulation of MAP3K1. Further, the effects of miR-1a inhibition on statin-induced cell apoptosis were ablated by MAP3K1 siRNA. In ApoE-/- mice, statin induced cell apoptosis of skeletal muscle cells and decreased exercise capacity in mice infected with vector, but not in mice with lentivirus-mediated miR-1a gene silence. CONCLUSION: Statin causes skeletal injury through induction of miR-1a excessive expression to decrease MAP3K1 gene expression.

Clinical Course and Risk Factors of Disease Deterioration in Critically Ill Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began in December 2019 and rapidly spread to other provinces in China as well as other countries. In this study, 262 patients diagnosed with moderate to severe SARS-CoV-2 pneumonia in Wuhan, China, were analyzed. Data were compared between survivors and nonsurvivors. Of all the 262 patients, 23 (8.8%) patients died and 239 (91.2%) were discharged. The median age was 63.5 years and 46.9% of patients were male. The main complaints were fever (83.6%), cough (63.4%), and fatigue (49.2%) in the surviving group, while there were more complaints of dyspnea (39.1%) and shortness of breath (56.5%) in the nonsurviving group. The main comorbidities were hypertension (35.5%), diabetes mellitus (16.4%), and coronary artery disease (9.9%). Morbidity is higher in elderly patients with more comorbidities. Patients were mainly treated with nasal cannula (93.9%), while the nonsurviving group received more invasive mechanical ventilation (39.1%). Arbidol (80.9%), ribavirin (36.6%), oseltamivir (38.9%), interferon (16.4%), and ganciclovir (14.5%) were used for the antiviral treatment. In the nonsurviving group, the number of white blood cells (WBC) was significantly increased and lymphocytes were decreased, and lymphopenia was more common. The levels of aspartate transaminase (AST), brain natriuretic peptide (BNP), creatine kinase isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and C-reactive protein (CRP) were also significantly increased in the nonsurviving group. The adjusted hazard ratios (HRs) for association of known variables for all-cause mortality due to the coronavirus disease 2019 were 2.467 (95% confidence interval [CI], 1.007-6.044; p = 0.048) for shortness of breath and 1.025 (95% CI, 1.001-1.049; p = 0.042) for AST. Elderly patients with more comorbidities and complaints of dyspnea and shortness of breath had increased risk of death. Patients with lymphopenia and high levels of WBC, AST, BNP, CK-MB, LDH, and CRP may be more likely to deteriorate.

Vitamin B6 inhibits macrophage activation to prevent lipopolysaccharide-induced acute pneumonia in mice.

Macrophage activation participates in the pathogenesis of pulmonary inflammation. As a coenzyme, vitamin B6 (VitB6) is mainly involved in the metabolism of amino acids, nucleic acids, glycogen and lipids. We have previously reported that activation of AMP-activated protein kinase (AMPK) produces anti-inflammatory effects both in vitro and in vivo. Whether VitB6 via AMPK activation prevents pulmonary inflammation remains unknown. The model of acute pneumonia was induced by injecting mice with lipopolysaccharide (LPS). The inflammation was determined by measuring the levels of interleukin-1 beta (IL-1ß), IL-6 and tumour necrosis factor alpha (TNF-α) using real time PCR, ELISA and immunohistochemistry. Exposure of cultured primary macrophages to VitB6 increased AMP-activated protein kinase (AMPK) Thr172 phosphorylation in a time/dose-dependent manner, which was inhibited by compound C. VitB6 downregulated the inflammatory gene expressions including IL-1ß, IL-6 and TNF-α in macrophages challenged with LPS. These effects of VitB6 were mirrored by AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). However, VitB6 was unable to inhibit LPS-induced macrophage activation if AMPK was in deficient through siRNA-mediated approaches. Further, the anti-inflammatory effects produced by VitB6 or AICAR in LPS-treated macrophages were abolished in DOK3 gene knockout (DOK3-/- ) macrophages, but were enhanced in macrophages if DOK3 was overexpressed. In vivo studies indicated that administration of VitB6 remarkably inhibited LPS-induced both systemic inflammation and acute pneumonia in wild-type mice, but not in DOK3-/- mice. VitB6 prevents LPS-induced acute pulmonary inflammation in mice via the inhibition of macrophage activation.

Up-regulation of paired-related homeobox 2 promotes cardiac fibrosis in mice following myocardial infarction by targeting of Wnt5a.

Cardiac fibrosis is a key factor to determine the prognosis in patient with myocardial infarction (MI). The aim of this study is to investigate whether the transcriptional factor paired-related homeobox 2 (Prrx2) regulates Wnt5a gene expression and the role in myocardial fibrosis following MI. The MI surgery was performed by ligation of left anterior descending coronary artery. Cardiac remodelling was assessed by measuring interstitial fibrosis performed with Masson staining. Cell differentiation was examined by analysis the expression of alpha-smooth muscle actin (α-SMA). Both Prrx2 and Wnt5a gene expressions were up-regulated in mice following MI, accompanied with increased mRNA and protein levels of α-SMA, collagen I and collagen III, compared to mice with sham surgery. Adenovirus-mediated gene knock down of Prrx2 increased survival rate, alleviated cardiac fibrosis, decreased infarction sizes and improved cardiac functions in mice with MI. Importantly, inhibition of Prrx2 suppressed ischaemia-induced Wnt5a gene expression and Wnt5a signalling. In cultured cardiac fibroblasts, TGF-ß increased gene expressions of Prrx2 and Wnt5a, and induced cell differentiations, which were abolished by gene silence of either Prrx2 or Wnt5a. Further, overexpression of Prrx2 or Wnt5a mirrored the effects of TGF-ß on cell differentiations of cardiac fibroblasts. Gene silence of Wnt5a also ablated cell differentiations induced by Prrx2 overexpression in cardiac fibroblasts. Mechanically, Prrx2 was able to bind with Wnt5a gene promoter to up-regulate Wnt5a gene expression. In conclusions, targeting Prrx2-Wnt5a signalling should be considered to improve cardiac remodelling in patients with ischaemic heart diseases.

Correction: Tongxinluo protects against pressure overload-induced heart failure in mice involving VEGF/Akt/eNOS pathway activation.

[This corrects the article DOI: 10.1371/journal.pone.0098047.].

Berberine suppresses the ectopic expression of miR-133a in endothelial cells to improve vascular dementia in diabetic rats.

Objective: Vascular dementia is the second leading cause of dementia, which is strongly associated with diabetes. Ectopic expression of miR-133a in endothelial cells is involved in endothelial dysfunction in diabetes. Whether berberine, as a natural product in Coptis chinensis, improves vascular dementia induced by diabetes remains unknown.Methods: Diabetes and subsequent vascular dementia were induced in rats by injecting streptozotocin (50 mg/kg/day) for five consecutive days. The expression of miR-133a was determined by fluorescence in situ hybridization. The learning and memory were evaluated by step-down, step-through, and morris water maze (MWM) tests.Results: In streptozotocin-injected rats, hyperglycemia dramatically induced miR-133a ectopic expressions in vascular endothelium, reduced GTPCH1 gene expressions and BH4 levels, which were reversed by berberine administration (1.0 g/kg/day, 8 weeks). Hyperglycemia also inhibited acetylcholine-induced vasorelaxation in middle cerebral artery and reduced blood supply to the brain, which were bypassed by berberine. Ex vivo studies indicated that miR-133a agomirs abolished these beneficial effects of berberine on acetylcholine-induced vasorelaxation, while supplement of L-sepiapterin prevented endothelial dysfunction in middle cerebral artery isolated from rats. By performing step-down, step-through, and MWM tests, we observed that hyperglycemia significantly caused the impairments of learning and memory in streptozotocin-injected rats. Importantly, these aberrant phenotypes in diabetic rats were normalized by berberine therapy. Finally, berberine reduced miR-133a expression, and increased both BH4 levels and NO production in cultured endothelial cells treated with high glucose.Conclusion: Berberine improves vascular dementia in diabetes, which is possibly related to the suppression of miR-133a ectopic expression in endothelial cells.

PARP-1 (Poly[ADP-Ribose] Polymerase 1) Inhibition Protects From Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysm in Mice.

Abdominal aortic aneurysm (AAA) is a common vascular degenerative disease. PARP-1 (poly[ADP-ribose] polymerase 1) is a nuclear enzyme, which plays a critical role in vascular diseases. We hypothesized that PARP-1 inhibition might have protective effects on AAA. In vivo, Ang II (angiotensin II) was continuously infused by a micropump for 28 days to induce AAA in mice. In vitro, aortic endothelial cells and smooth muscle cells were stimulated by Ang II for 24 hours. Ang II infusion increased PARP-1 expression and activity and successfully induced AAA formation partly with a hemorrhage in ApoE-/- mice. Genetic deletion of PARP-1 markedly reduced the AAA incidence, abdominal aortic diameter, macrophage infiltration, ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular adhesion molecule 1) expression, and MMP (matrix metalloproteinase) expression, as well as MMP activity; but increased smooth muscle cells content and collagens expression in AAA. PARP-1 inhibition by PJ-34 also exerted a protective effect on AAA in mice. In aortic endothelial cells, Ang II-induced oxidative stress and DNA damage, resulting in increased PARP-1 expression and activity. Compared with the control, Ang II increased TNF-α (tumor necrosis factor α) and IL-6 (interleukin-6) secretions, ICAM-1 expression and THP-1 (human acute monocytic leukemia cell line) cells adhesion, while PARP-1 inhibition by siRNA reduced the inflammatory response probably through inhibition of the phosphorylation of ERK (extracellular signal-regulated kinase), NF-κB (nuclear factor-κB), and Akt signaling pathways. In smooth muscle cells, Ang II promoted cell migration, proliferation, and apoptosis, reduced collagens expression, but increased MMPs expression, while PARP-1 deletion alleviated these effects partly by reducing NF-κB-targeted MMP-9 expression. PARP-1 inhibition might be a feasible strategy for the treatment of AAA.

Peptidyl-prolyl isomerase Pin1 deficiency attenuates angiotensin II-induced abdominal aortic aneurysm formation in ApoE-/- mice.

Peptidyl-prolyl isomerase Pin1 has been reported to be associated with endothelial dysfunction. However, the role of smooth muscle Pin1 in the vascular system remains unclear. Here, we examined the potential function of Pin1 in smooth muscle cells (SMCs) and its contribution to abdominal aortic aneurysm (AAA) pathogenesis. The level of Pin1 expression was found to be elevated in human AAA tissues and mainly localized to SMCs. We constructed smooth muscle-specific Pin1 knockout mice to explore the role of this protein in AAA formation and to elucidate the underlying mechanisms. AAA formation and elastin degradation were hindered by Pin1 depletion in the angiotensin II-induced mouse model. Pin1 depletion reversed the angiotensin II-induced pro-inflammatory and synthetic SMC phenotype switching via the nuclear factor (NF)-κB p65/Klf4 axis. Moreover, Pin1 depletion inhibited the angiotensin II-induced matrix metalloprotease activities. Mechanically, Pin1 deficiency destabilized NF-κB p65 by promoting its polyubiquitylation. Further, we found STAT1/3 bound to the Pin1 promoter, revealing that activation of STAT1/3 was responsible for the increased expression of Pin1 under angiotensin II stimulation. Thus, these results suggest that Pin1 regulates pro-inflammatory and synthetic SMC phenotype switching and could be a novel therapeutic target to limit AAA pathogenesis.

Period2 deficiency blunts hypoxia-induced mobilization and function of endothelial progenitor cells.

BACKGROUND: In the clinic, variations in circadian rhythm are evident in patients with cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. In this study, we focused on the role of the circadian gene period2 (per2) in mobilization and function of endothelial progenitor cells (EPCs) in vitro and in vivo after myocardial infarction (MI) in mice. METHODS AND RESULTS: MI was produced by surgical ligation of the left anterior descending coronary artery in mice with and without per2 deficiency. Trans-thoracic echocardiography was used to evaluate cardiac function in mice. Per2-/- mice with MI showed decreased cardiac function and increased infarct size. The number of CD34+ cells and capillary density were decreased in the myocardium of per2-/- mice on immunohistochemistry. Flow cytometry revealed decreased number of circulating EPCs in per2-/- mice after MI. In vitro, per2-/- EPCs showed decreased migration and tube formation capacity under hypoxia. Western blot analysis revealed inhibited activation of extracellular signal-regulated kinase and Akt signaling in the bone marrow of per2-/- mice and inhibited PI3K/Akt expression in per2-/- EPCs under hypoxia. CONCLUSIONS: Per2 modulates EPC mobilization and function after MI, which is important to recovery after MI in mice.

Resveratrol-enhanced autophagic flux ameliorates myocardial oxidative stress injury in diabetic mice.

Autophagic dysfunction is observed in diabetes mellitus. Resveratrol has a beneficial effect on diabetic cardiomyopathy. Whether the resveratrol-induced improvement in cardiac function in diabetes is via regulating autophagy remains unclear. We investigated the mechanisms underlying resveratrol-mediated protection against heart failure in diabetic mice, with a focus on the role of sirtuin 1 (SIRT1) in regulating autophagic flux. Diabetic cardiomyopathy in mice was induced by streptozotocin (STZ). Long-term resveratrol treatment improved cardiac function, ameliorated oxidative injury and reduced apoptosis in the diabetic mouse heart. Western blot analysis revealed that resveratrol decreased p62 protein expression and promoted SIRT1 activity and Rab7 expression. Inhibiting autophagic flux with bafilomycin A1 increased diabetic mouse mortality and attenuated resveratrol-induced down-regulation of p62, but not SIRT1 activity or Rab7 expression in diabetic mouse hearts. In cultured H9C2 cells, redundant or overactive H2O2 increased p62 and cleaved caspase 3 expression as well as acetylated forkhead box protein O1 (FOXO1) and inhibited SIRT1 expression. Sirtinol, SIRT1 and Rab7 siRNA impaired the resveratrol amelioration of dysfunctional autophagic flux and reduced apoptosis under oxidative conditions. Furthermore, resveratrol enhanced FOXO1 DNA binding at the Rab7 promoter region through a SIRT1-dependent pathway. These results highlight the role of the SIRT1/FOXO1/Rab7 axis in the effect of resveratrol on autophagic flux in vivo and in vitro, which suggests a therapeutic strategy for diabetic cardiomyopathy.

Tongxinluo protects against pressure overload-induced heart failure in mice involving VEGF/Akt/eNOS pathway activation.

BACKGROUND: It has been demonstrated that Tongxinluo (TXL), a traditional Chinese medicine compound, improves ischemic heart disease in animal models via vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). The present study aimed to investigate whether TXL protects against pressure overload-induced heart failure in mice and explore the possible mechanism of action. METHODS AND RESULTS: Transverse aortic constriction (TAC) surgery was performed in mice to induce heart failure. Cardiac function was evaluated by echocardiography. Myocardial pathology was detected using hematoxylin and eosin or Masson trichrome staining. We investigated cardiomyocyte ultrastructure using transmission electron microscopy. Angiogenesis and oxidative stress levels were determined using CD31 and 8-hydroxydeoxyguanosine immunostaining and malondialdehyde assay, respectively. Fetal gene expression was measured using real-time PCR. Protein expression of VEGF, phosphorylated (p)-VEGF receptor 2 (VEGFR2), p-phosphatidylinositol 3-kinase (PI3K), p-Akt, p-eNOS, heme oxygenase-1 (HO-1), and NADPH oxidase 4 (Nox4) were measured with western blotting. Twelve-week low- and high-dose TXL treatment following TAC improved cardiac systolic and diastolic function and ameliorated left ventricular hypertrophy, fibrosis, and myocardial ultrastructure derangement. Importantly, TXL increased myocardial capillary density significantly and attenuated oxidative stress injury in failing hearts. Moreover, TXL upregulated cardiac nitrite content and the protein expression of VEGF, p-VEGFR2, p-PI3K, p-Akt, p-eNOS, and HO-1, but decreased Nox4 expression in mouse heart following TAC. CONCLUSION: Our findings indicate that TXL protects against pressure overload-induced heart failure in mice. Activation of the VEGF/Akt/eNOS signaling pathway might be involved in TXL improvement of the failing heart.

Period 2 is essential to maintain early endothelial progenitor cell function in vitro and angiogenesis after myocardial infarction in mice.

Cellular therapeutic neovascularization has been successfully performed in clinical trials for patients with ischaemia diseases. Despite the vast knowledge of cardiovascular disease and circadian biology, the role of the circadian clock in regulating angiogenesis in myocardial infarction (MI) remains poorly understood. In this study, we aimed to investigate the role and underlying mechanisms of Period 2 (Per2) in endothelial progenitor cell (EPC) function. Flow cytometry revealed lower circulating EPC proportion in per2(-/-) than in wild-type (WT) mice. PER2 was abundantly expressed in early EPCs in mice. In vitro, EPCs from per2(-/-) mice showed impaired proliferation, migration, tube formation and adhesion. Western blot analysis demonstrated inhibited PI3k/Akt/FoxO signalling and reduced C-X-C chemokine receptor type 4 (CXCR4) protein level in EPCs of per2(-/-) mice. The impaired proliferation was blocked by activated PI3K/Akt/FoxO signalling. Direct interaction of CXCR4 and PER2 was detected in WT EPCs. To further study the effect of per2 on in vivo EPC survival and angiogenesis, we injected saline or DiI-labelled WT or per2(-/-) EPC intramyocardially into mice with induced MI. Per2(-/-) reduced the retention of transplanted EPCs in the myocardium, which was associated with significantly reduced DiI expression in the myocardium of MI mice. Decreased angiogenesis in the myocardium of per2(-/-) EPC-treated mice coincided with decreased LV function and increased infarct size in the myocardium. Per2 may be a key factor in maintaining EPC function in vitro and in therapeutic angiogenesis in vivo.

Tongxinluo Improves Cardiac Function and Ameliorates Ventricular Remodeling in Mice Model of Myocardial Infarction through Enhancing Angiogenesis.

Background. Myocardial infarction (MI) is a major cause of morbidity and mortality in the world. Tongxinluo (TXL) is a traditional Chinese compound prescription which has cardioprotective functions. The present study was aimed to determine the effect of TXL on postischemic cardiac dysfunction and cardiac remodeling and to elucidate the underlying mechanisms. Methods and Results. MI was performed by ligation of left anterior descending coronary artery (LAD) in male adult mice. Mice were randomly divided into four groups: (1) sham group (Sham); (2) MI-control group (Control); (3) MI-low dose TXL group (TXL-L); and (4) MI-high dose TXL (TXL-H) group. Compared with the control group, TXL treatment restored cardiac function, increased revascularization, attenuated cardiomyocyte apoptosis, and reduced interstitial fibrosis. TXL treatment increased the phosphorylation of Akt, extracellular signal regulated kinase (ERK), and endothelial nitric oxide synthase (eNOS); the expression of phosphatidylinositol3-kinase (PI3K), hypoxia-inducible factors 1 α (HIF-1 α ), and vascular endothelial growth factor (VEGF); and the DNA binding activity of HIF-1 α after MI. Conclusion. TXL may improve cardiac function and ameliorate cardiac remodeling by increasing neovascularization through enhancing the phosphorylation of Akt and ERK, the expression and activity of HIF-1 α , and the protein level of VEGF and p-eNOS.