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1.
J Cell Mol Med ; 24(5): 3139-3148, 2020 03.
Article in English | MEDLINE | ID: mdl-31970902

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Interleukin-1beta/genetics , Pneumonia/drug therapy , Tumor Necrosis Factor-alpha/genetics , Vitamin B 6/pharmacology , AMP-Activated Protein Kinases/genetics , Animals , Disease Models, Animal , Humans , Inflammation/chemically induced , Inflammation/drug therapy , Inflammation/genetics , Inflammation/pathology , Lipopolysaccharides/toxicity , Macrophage Activation/drug effects , Mice , Phosphorylation/drug effects , Pneumonia/chemically induced , Pneumonia/genetics , Pneumonia/pathology , Signal Transduction
2.
Clin Pharmacol Ther ; 105(1): 201-209, 2019 01.
Article in English | MEDLINE | ID: mdl-29672839

ABSTRACT

Development of nitrate tolerance is a major drawback to nitrate therapy. Prostacyclin (PGI2) is a powerful vasodilator produced from prostaglandin (PGH2) by prostacyclin synthase (PGIS) in endothelial cells. This study aimed to determine the role of PGIS S-nitrosylation in nitrate tolerance induced by nitroglycerin (GTN). In endothelial cells, GTN increased PGIS S-nitrosylation and disturbed PGH2 metabolism, which were normalized by mutants of PGIS cysteine 231/441 to alanine (C231/441A). Clearance of nitric oxide by carboxy-PTIO or inhibition of S-nitrosylation by N-acetyl-cysteine decreased GTN-induced PGIS S-nitrosylation. Enforced expression of mutated PGIS with C231/441A markedly abolished GTN-induced PGIS S-nitrosylation and nitrate cross-tolerance in Apoe-/- mice. Inhibition of cyclooxygenase 1 by aspirin, supplementation of PGI2 by beraprost, and inhibition of PGIS S-nitrosylation by N-acetyl-cysteine improved GTN-induced nitrate cross-tolerance in rats. In patients, increased PGIS S-nitrosylation was associated with nitrate tolerance. In conclusion, GTN induces nitrate cross-tolerance through PGIS S-nitrosylation at cysteine 231/441.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Drug Tolerance/physiology , Intramolecular Oxidoreductases/metabolism , Nitrates/metabolism , Nitric Oxide/metabolism , Nitroglycerin/pharmacology , Aged , Aged, 80 and over , Amino Acid Sequence , Animals , Cattle , Cricetinae , Cytochrome P-450 Enzyme System/genetics , Dose-Response Relationship, Drug , Female , Human Umbilical Vein Endothelial Cells , Humans , Intramolecular Oxidoreductases/genetics , Male , Mice , Mice, Knockout , Middle Aged , Rats , Rats, Sprague-Dawley , Vasodilator Agents/pharmacology
3.
J Mol Med (Berl) ; 96(5): 403-412, 2018 05.
Article in English | MEDLINE | ID: mdl-29502204

ABSTRACT

Diabetes mellitus is one of risk factors of cardiovascular diseases including atherosclerosis. Whether and how diabetes promotes the formation of unstable atherosclerotic plaque is not fully understood. Here, we show that streptozotocin-induced type 1 diabetes reduced collagen synthesis, leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout (Apoe-/-) mice. These detrimental effects of hyperglycemia on plaque stability were reversed by metformin in vivo without altering the levels of blood glucose and lipids. Mechanistically, we found that high glucose reduced the phosphorylated level of AMP-activated protein kinase alpha (AMPKα) and the transcriptional activity of activator protein 2 alpha (AP-2α), increased the expression of miR-124 expression, and downregulated prolyl-4-hydroxylase alpha 1 (P4Hα1) protein expression and collagen biosynthesis in cultured vascular smooth muscle cells. Importantly, these in vitro effects produced by high glucose were abolished by AMPKα pharmacological activation or adenovirus-mediated AMPKα overexpression. Further, adenovirus-mediated AMPKα gain of function remitted the process of diabetes-induced plaque destabilization in Apoe-/- mice injected with streptozotocin. Administration of metformin enhanced pAP-2α level, reduced miR-124 expression, and increased P4Hα1 and collagens in carotid atherosclerotic plaque in diabetic Apoe-/- mice. We conclude that streptozotocin-induced toxic diabetes promotes the formation of unstable atherosclerotic plaques based on the vulnerability index in Apoe-/- mice, which is related to the inactivation of AMPKα/AP-2α/miRNA-124/P4Hα1 axis. Clinically, targeting AMPKα/AP-2α/miRNA-124/P4Hα1 signaling should be considered to increase the plaque stability in patients with atherosclerosis. KEY MESSAGES: Hyperglycemia reduced collagen synthesis, leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout mice. Hyperglycemia destabilizes atherosclerotic plaque in vivo through an AMPKα/AP-2α/miRNA-124/P4Hα1-dependent collagen synthesis. Metformin functions as a stabilizer of atherosclerotic plaque to reduce acute coronary accent.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , MicroRNAs/metabolism , Plaque, Atherosclerotic/metabolism , Procollagen-Proline Dioxygenase/metabolism , Transcription Factor AP-2/metabolism , Animals , Collagen Type I/metabolism , Collagen Type II/metabolism , Hypoglycemic Agents/pharmacology , Male , Metformin/pharmacology , Mice, Knockout, ApoE , Myocytes, Smooth Muscle/metabolism
4.
Oncotarget ; 8(9): 14294-14305, 2017 Feb 28.
Article in English | MEDLINE | ID: mdl-28179583

ABSTRACT

We have previously reported that activation of AMP-activated kinase alpha 2 (AMPKα2) by nicotine or angiotensin II (AngII) instigates formation of abdominal aortic aneurysms (AAA) in Apoe-/- mice. Statins, used to treat hyperlipidemia widely, activate AMPK in vascular cells. We sought to examine the effects of pravastatin on AAA formation and uncover the molecular mechanism. The AAA model was induced by AngII and evaluated by incidence, elastin degradation, and maximal abdominal aortic diameter in Apoe-/- mice. The phosphorylated levels of AMPKα2 and activator protein 2 alpha (AP-2α) were examined in cultured vascular smooth muscle cells (VSMCs) or in mice. We observed that pravastatin (50 mg/kg/day, 8 weeks) remarkably increased the AngII-induced AAA incidence in mice. In VSMCs, pravastatin increased the levels of pAMPK, pAP-2α, and MMP2 in both basal and AngII-stressed conditions, which were abolished by tempol and compound C. Pravastatin-upregulated MMP2 was abrogated by AMPKα2 or AP-2α siRNA. Lentivirus-mediated gene silence of AMPKα2 or AP-2α abolished pravastatin-worsened AAA formations in AngII-infused Apoe-/- mice. Clinical investigations demonstrated that both AMPKα2 and AP-2α phosphorylations were increased in AAA patients or human subjects taking pravastatin. In conclusion, pravastatin promotes AAA formation through AMPKα2-dependent AP-2α activations.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Angiotensin II/adverse effects , Aortic Aneurysm, Abdominal/etiology , Apolipoproteins E/physiology , Gene Expression Regulation/drug effects , Pravastatin/adverse effects , Transcription Factor AP-2/metabolism , Animals , Anticholesteremic Agents/pharmacology , Aortic Aneurysm, Abdominal/metabolism , Aortic Aneurysm, Abdominal/pathology , Blotting, Western , Cells, Cultured , Disease Models, Animal , Humans , Male , Mice , Mice, Knockout , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Phosphorylation , Signal Transduction
5.
Oncotarget ; 8(6): 9021-9034, 2017 Feb 07.
Article in English | MEDLINE | ID: mdl-28061433

ABSTRACT

AIMS: Proteasome-linked oxidative stress is believed to cause endothelial dysfunction, an early event in cardiovascular diseases (CVD). Statin, as HMG-CoA reductase inhibitor, prevents endothelial dysfunction in CVD. However, the molecular mechanism of statin-mediated normalization of endothelial function is not completely elucidated. METHODS AND RESULTS: Lovastatin time/dose-dependently increased miR-29b expression and decreased proteasome activity in cultured human umbilical vein endothelial cells (HUVECs). Anti-miR-29b or overexpression of PA200 abolished lovastatin-induced inhibition of proteasome activity in HUVECs. In contrast, pre-miR-29b or PA200 siRNA mimics these effects of lovastatin on proteasome activity. Lovastatin inhibited oxidative stress induced by multiple oxidants including ox-LDL, H2O2, TNFα, homocysteine thiolactone (HTL), and high glucose (HG), which were reversed by inhibition of miR-29b in HUVECs. Ex vivo analysis indicated that lovastatin normalized the acetylcholine-induced endothelium-dependent relaxation and the redox status in isolated rat aortic arteries exposure to multiple cardiovascular risk factors. In vivo analysis revealed that administration of lovastatin remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats with hyperglycemia, dyslipidemia, and hyperhomocysteinemia, as well as increased miR-29b expressions, reduced PA200 protein levels, and suppression of proteasome activity in aortic tissues. CONCLUSION: Upregulation of miR-29b expression is a common mechanism contributing to endothelial dysfunction induced by multiple cardiovascular risk factors through PA200-dependent proteasome-mediated oxidative stress, which is prevented by lovastatin.


Subject(s)
Antioxidants/pharmacology , Cardiovascular Diseases/prevention & control , Diabetes Mellitus, Experimental/drug therapy , Dyslipidemias/prevention & control , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hyperhomocysteinemia/drug therapy , Lovastatin/pharmacology , MicroRNAs/metabolism , Oxidative Stress/drug effects , AMP-Activated Protein Kinases/metabolism , Animals , Aorta/drug effects , Aorta/metabolism , Aorta/physiopathology , Cardiovascular Diseases/genetics , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/pathology , Cells, Cultured , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Dose-Response Relationship, Drug , Dyslipidemias/genetics , Dyslipidemias/metabolism , Dyslipidemias/pathology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Hyperhomocysteinemia/genetics , Hyperhomocysteinemia/metabolism , Hyperhomocysteinemia/pathology , MicroRNAs/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , RNA Interference , Signal Transduction/drug effects , Time Factors , Transfection , Up-Regulation , Vasodilation/drug effects
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