Far-Infrared Irradiation Decreases Proliferation in Basal and PDGF-Stimulated VSMCs Through AMPK-Mediated Inhibition of mTOR/p70S6K Signaling Axis.

BACKGROUND: Far-infrared (FIR) irradiation has been reported to improve diverse cardiovascular diseases, including heart failure, hypertension, and atherosclerosis. The dysregulated proliferation of vascular smooth muscle cells (VSMCs) is well established to contribute to developing occlusive vascular diseases such as atherosclerosis and in-stent restenosis. However, the effects of FIR irradiation on VSMC proliferation and the underlying mechanism are unclear. This study investigated the molecular mechanism through which FIR irradiation inhibited VSMC proliferation. METHODS: We performed cell proliferation and cell death assay, adenosine 5'-triphosphate (ATP) assay, inhibitor studies, transfection of dominant negative (dn)-AMP-activated protein kinase (AMPK) α1 gene, and western blot analyses. We also conducted confocal microscopic image analyses and ex vivo studies using isolated rat aortas. RESULTS: FIR irradiation for 30 minutes decreased VSMC proliferation without altering the cell death. Furthermore, FIR irradiation accompanied decreases in phosphorylation of the mammalian target of rapamycin (mTOR) at Ser2448 (p-mTOR-Ser2448) and p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr389). The phosphorylation of AMPK at Thr172 (p-AMPK-Thr172) was increased in FIR-irradiated VSMCs, which was accompanied by a decreased cellular ATP level. Similar to in vitro results, FIR irradiation increased p-AMPK-Thr172 and decreased p-mTOR-Ser2448 and p-p70S6K-Thr389 in isolated rat aortas. Pre-treatment with compound C, a specific AMPK inhibitor, or ectopic expression of dn-AMPKα1 gene, significantly reversed FIR irradiation-decreased VSMC proliferation, p-mTOR-Ser2448, and p-p70S6K-Thr389. On the other hand, hyperthermal stimulus (39°C) did not alter VSMC proliferation, cellular ATP level, and AMPK/mTOR/p70S6K phosphorylation. Finally, FIR irradiation attenuated platelet-derived growth factor (PDGF)-stimulated VSMC proliferation by increasing p-AMPK-Thr172, and decreasing p-mTOR-Ser2448 and p-p70S6K-Thr389 in PDGF-induced in vitro atherosclerosis model. CONCLUSION: These results show that FIR irradiation decreases the basal and PDGF-stimulated VSMC proliferation, at least in part, by the AMPK-mediated inhibition of mTOR/p70S6K signaling axis irrespective of its hyperthermal effect. These observations suggest that FIR therapy can be used to treat arterial narrowing diseases, including atherosclerosis and in-stent restenosis.

Nucleus-targeted delivery of nitric oxide in human mesenchymal stem cells enhances osteogenic differentiation.

Nitric oxide (NO) is an important gaseous signaling molecule in various physiological processes, which functions through interactions with its acceptor molecules located in organelles. NO has an extremely short half-life, making it challenging to experimentally achieve effective NO levels in organelles to study these interactions. Here we developed an organelle-specific, peptide-based NO delivery material that targets the nucleus. NO was attached to the SH group of a cysteine residue inserted into the N-terminus of a cell-penetrating peptide (CPP) conjugated to varying repeats of the nuclear localization signal (NLS), which we denoted NO-CysCPP-NLS, through S-nitrosylation. NO-CysCPP-NLS strongly induced osteogenic differentiation of mesenchymal stem cells. This delivery concept can be extended to cells other than stem cells to elucidate the effects of NO release in the nucleus. Furthermore, conjugation of NO to CysCPP fused to mitochondria- or lysosome-targeting signals can be used to deliver NO to other organelles such as mitochondria and lysosomes, respectively.

Valproic acid decreases vascular smooth muscle cell proliferation via protein phosphatase 2A-mediated p70 S6 kinase inhibition.

Valproic acid (VPA) has been used to treat epilepsy and bipolar disorder. Although the abnormal proliferation of vascular smooth muscle cells (VSMCs) is a well-established contributor to the development of various vascular diseases including atherosclerosis, the effect of VPA on VSMC proliferation and its mechanism of action have not been fully revealed. Herein, we investigated the molecular mechanism by which VPA inhibits rat VSMC proliferation. VPA dose-dependently decreased VSMC proliferation, which was accompanied by the dose-dependent decrease in phosphorylation of p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr389), and overexpression of the p70S6K-T389E mutant gene significantly reversed VPA-inhibited VSMC proliferation. Co-treatment with okadaic acid, a specific protein phosphatase 2A (PP2A) inhibitor, significantly restored p-p70S6K-Thr389. Furthermore, knockdown of PP2Ac gene expression by siRNA significantly reversed VPA-inhibited p-p70S6K-Thr389 and VSMC proliferation. Confocal microscopic analyses and co-immunoprecipitation results clearly showed that the physical binding of p70S6K and PP2Ac was promoted by VPA. Valpromide, a VPA's structural derivative with no histone deacetylase (HDAC) inhibition activity, as well as VPA and sodium butyrate, an HDAC inhibitor similar to VPA, decreased VSMC proliferation and p-p70S6K-Thr389, indicating that HDAC is not involved in VPA-inhibited VSMC proliferation. Finally, the inhibitory effects of VPA on p-p70S6K-Thr389 and VSMC proliferation were reiterated in a platelet-derived growth factor (PDGF)-induced in vitro atherosclerosis model. In conclusion, our results demonstrate that VPA decreased cell proliferation via PP2A-mediated inhibition of p-p70S6K-Thr389 in basal and PDGF-stimulated VSMCs. The results suggest that VPA could be used in the treatment and prevention of atherosclerosis and in-stent restenosis.

Far-infrared irradiation inhibits breast cancer cell proliferation independently of DNA damage through increased nuclear Ca2+/calmodulin binding modulated-activation of checkpoint kinase 2.

Far-infrared (FIR) irradiation is reported to inhibit cell proliferation in various types of cancer cells; the underlying mechanism, however, remains unclear. We explored the molecular mechanisms using MDA-MB-231 human breast cancer cells. FIR irradiation significantly inhibited cell proliferation and colony formation compared to hyperthermal stimulus, with no alteration in cell viability. No increase in DNA fragmentation or phosphorylation of DNA damage kinases including ataxia-telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase, and DNA-dependent protein kinase indicated no DNA damage. FIR irradiation increased the phosphorylation of checkpoint kinase 2 (Chk2) at Thr68 (p-Chk2-Thr68) but not that of checkpoint kinase 1 at Ser345. Increased nuclear p-Chk2-Thr68 and Ca2+/CaM accumulations were found in FIR-irradiated cells, as observed in confocal microscopic analyses and cell fractionation assays. In silico analysis predicted that Chk2 possesses a Ca2+/calmodulin (CaM) binding motif ahead of its kinase domain. Indeed, Chk2 physically interacted with CaM in the presence of Ca2+, with their binding markedly pronounced in FIR-irradiated cells. Pre-treatment with a Ca2+ chelator significantly reversed FIR irradiation-increased p-Chk2-Thr68 expression. In addition, a CaM antagonist or small interfering RNA-mediated knockdown of the CaM gene expression significantly attenuated FIR irradiation-increased p-Chk2-Thr68 expression. Finally, pre-treatment with a potent Chk2 inhibitor significantly reversed both FIR irradiation-stimulated p-Chk2-Thr68 expression and irradiation-repressed cell proliferation. In conclusion, our results demonstrate that FIR irradiation inhibited breast cancer cell proliferation, independently of DNA damage, by activating the Ca2+/CaM/Chk2 signaling pathway in the nucleus. These results demonstrate a novel Chk2 activation mechanism that functions irrespective of DNA damage.

Nuclear localization of endothelial nitric oxide synthase and nitric oxide production attenuates aphidicolin-induced endothelial cell death.

Aphidicolin represses DNA replication by inhibiting DNA polymerase α and δ, which leads to cell cycle arrest and cell damage. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) plays an essential role in maintenance of endothelial integrity including endothelial cell (EC) survival. Previously, we reported that aphidicolin increases NO production in bovine aortic ECs (BAECs). However, the role of aphidicolin-induced NO on EC viability and its molecular mechanism remain to be elucidated. Treatment with 20 µM aphidicolin for 24 h reduced BAEC viability by ~40%, which was accompanied by increased NO production, phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179), and eNOS protein expression. The aphidicolin-increased eNOS expression and p-eNOS-Ser1179 were not altered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), a cell permeable and specific intracellular Ca2+ chelator. Co-treatment with 2-phenyl-4, 4, 5, 5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), an NO scavenger, or Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), a NOS inhibitor, exacerbated aphidicolin-stimulated BAEC death. Knockdown of eNOS gene expression using siRNA aggravated aphidicolin-induced BAEC death. However, exogenous NO donors including S-nitroso-l-glutathione (GSNO) or diethylenetriamine NONOate (DETA NO) had no effect on aphidicolin-decreased BAEC viability and aggravated BAEC viability at higher doses. Interestingly, aphidicolin accumulated eNOS protein in the active form, p-eNOS-Ser1179, in the nucleus. When cells were ectopically transfected with a wild-type (WT)-eNOS gene, aphidicolin induced significant localization of the protein product in the nucleus. Additionally, aphidicolin-elicited cell death was significantly reversed in WT-eNOS gene-transfected BAECs. Furthermore, overexpression of the eNOS gene containing nuclear localization signal (NLS) but not nuclear export signal (NES) significantly attenuated aphidicolin-induced BAEC death. When G2A-eNOS mutant lacking myristoylation at Gly2 was transfected, its intracellular distribution became diffuse and included the nucleus. Finally, expression of N-myristoyltransferase 2 (NMT2) but not NMT1 significantly decreased in aphidicolin-treated BAECs. Taken together, our results suggest that aphidicolin attenuates BAEC death in part by increasing nuclear eNOS localization and NO production.

Activation of AMPK by Telmisartan Decreases Basal and PDGF-stimulated VSMC Proliferation via Inhibiting the mTOR/p70S6K Signaling Axis.

BACKGROUND: Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is widely used to treat hypertension by blocking the renin-angiotensin-aldosterone system. Although abnormal proliferation of vascular smooth muscle cells (VSMCs) is a well-established contributor to the development of various vascular diseases, such as atherosclerosis, the effect of telmisartan on VSMC proliferation and its mechanism of action have not been fully revealed. Herein, we investigated the molecular mechanism whereby telmisartan inhibits rat VSMC proliferation. METHODS: We measured VSMC proliferation by MTT assay, and performed inhibitor studies and western blot analyses using basal and platelet-derived growth factor (PDGF)-stimulated rat VSMCs. To elucidate the role of AMP-activated protein kinase (AMPK), we introduced dominant-negative (dn)-AMPKα1 gene into VSMCs. RESULTS: Telmisartan decreased VSMC proliferation, which was accompanied by decreased phosphorylations of mammalian target of rapamycin (mTOR) at Ser2448 (p-mTOR-Ser2448) and p70 S6 kinase (p70S6K) at Thr389 (p-p70S6K-Thr389) in dose- and time-dependent manners. Telmisartan dose- and time-dependently increased phosphorylation of AMPK at Thr172 (p-AMPK-Thr172). Co-treatment with compound C, a specific AMPK inhibitor, or ectopic expression of the dn-AMPKα1 gene, significantly reversed telmisartan-inhibited VSMC proliferation, p-mTOR-Ser2448 and p-p70S6K-Thr389 levels. Among the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr172 and decreased p-mTOR-Ser2448, p-p70S6K-Thr389, and VSMC proliferation. Furthermore, GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, did not affect any of the telmisartan-induced changes. Finally, telmisartan also exhibited inhibitory effects on VSMC proliferation by increasing p-AMPK-Thr172 and decreasing p-mTOR-Ser2448 and p-p70S6K-Thr389 in a PDGF-induced in vitro atherosclerosis model. CONCLUSION: These results demonstrated that telmisartan-activated AMPK inhibited basal and PDGF-stimulated VSMC proliferation, at least in part, by downregulating the mTOR/p70S6K signaling axis in a PPARγ-independent manner. These observations suggest that telmisartan could be used to treat arterial narrowing diseases such as atherosclerosis and restenosis.

Zearalenone-Induced Interaction between PXR and Sp1 Increases Binding of Sp1 to a Promoter Site of the eNOS, Decreasing Its Transcription and NO Production in BAECs.

Zearalenone (ZEN) is a non-steroidal mycotoxin that has various toxicological impacts on mammalian health. Here, we found that ZEN significantly affected the production of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS) of bovine aortic endothelial cells (BAECs). A promoter analysis using 5'-serially deleted human eNOS promoter revealed that the proximal region (-135 to +22) was responsible for ZEN-mediated reduction of the human eNOS promoter activity. This effect was reversed by mutation of two specificity protein 1 (Sp1) binding elements in the human eNOS promoter. A chromatin immunoprecipitation assay revealed that ZEN increased Sp1 binding to the bovine eNOS promoter region (-113 to -12), which is homologous to -135 to +22 of the human eNOS promoter region. We also found that ZEN promoted the binding of the pregnane X receptor (PXR) to Sp1 of the bovine eNOS, consequently decreasing eNOS expression. This reduction of eNOS could have contributed to the decreased acetylcholine-induced vessel relaxation upon ZEN treatment in our ex vivo study using mouse aortas. In conclusion, our data demonstrate that ZEN decreases eNOS expression by enhancing the binding of PXR-Sp1 to the eNOS promoter, thereby decreasing NO production and potentially causing vessel dysfunction.

Activation of ATM/Akt/CREB/eNOS Signaling Axis by Aphidicolin Increases NO Production and Vessel Relaxation in Endothelial Cells and Rat Aortas.

Although DNA damage responses (DDRs) are reported to be involved in nitric oxide (NO) production in response to genotoxic stresses, the precise mechanism of DDR-mediated NO production has not been fully understood. Using a genotoxic agent aphidicolin, we investigated how DDRs regulate NO production in bovine aortic endothelial cells. Prolonged (over 24 h) treatment with aphidicolin increased NO production and endothelial NO synthase (eNOS) protein expression, which was accompanied by increased eNOS dimer/monomer ratio, tetrahydrobiopterin levels, and eNOS mRNA expression. A promoter assay using 5'-serially deleted eNOS promoters revealed that Tax-responsive element site, located at -962 to -873 of the eNOS promoter, was responsible for aphidicolin-stimulated eNOS gene expression. Aphidicolin increased CREB activity and ectopic expression of dominantnegative inhibitor of CREB, A-CREB, repressed the stimulatory effects of aphidicolin on eNOS gene expression and its promoter activity. Co-treatment with LY294002 decreased the aphidicolin-stimulated increase in p-CREB-Ser133 level, eNOS expression, and NO production. Furthermore, ectopic expression of dominant-negative Akt construct attenuated aphidicolin-stimulated NO production. Aphidicolin increased p-ATM-Ser1981 and the knockdown of ATM using siRNA attenuated all stimulatory effects of aphidicolin on p-Akt-Ser473, p-CREB-Ser133, eNOS expression, and NO production. Additionally, these stimulatory effects of aphidicolin were similarly observed in human umbilical vein endothelial cells. Lastly, aphidicolin increased acetylcholine-induced vessel relaxation in rat aortas, which was accompanied by increased p-ATM-Ser1981, p-Akt-Ser473, p-CREB-Ser133, and eNOS expression. In conclusion, our results demonstrate that in response to aphidicolin, activation of ATM/Akt/CREB/eNOS signaling cascade mediates increase of NO production and vessel relaxation in endothelial cells and rat aortas.

SRT1720-induced activation of SIRT1 alleviates vascular smooth muscle cell senescence through PKA-dependent phosphorylation of AMPKα at Ser485.

Aging is a major risk factor for hypertension and atherosclerosis, and vascular smooth muscle cell (VSMC) senescence can promote aging-related vascular diseases. Sirtuin-1 (SIRT1) and AMP-activated protein kinase (AMPK) were previously reported to modulate vascular senescence; however, its effects have not been well characterized. To determine the nature of the interaction between SIRT1 and AMPK in VSMC senescence, we investigated the effects of SRT1720 on its downstream targets of SIRT1 and the phosphorylation of AMPKα at Ser485. During Adriamycin-induced VSMC senescence, SRT1720 increased the activity of SIRT1 and AMPKα phosphorylation at Ser485 via the cAMP-protein kinase A (PKA) pathway. Telomere length and telomerase reverse transcriptase expression were increased by SIRT1 activation with SRT1720. Taken together, these data show that activation of the SIRT1/cAMP-PKA/p-AMPKα (Ser485) pathway may be an effective antisenescence mechanism for VSMCs.

Activation of AMPK/proteasome/MLCK degradation signaling axis by telmisartan inhibits VSMC contractility and vessel contraction.

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is widely used to treat hypertension. Dysfunction of vascular smooth muscle cells (VSMCs) is well-established to contribute to the pathogenesis of various vascular diseases. A growing body of evidence indicates that increased VSMC contractility plays a primary role in the development of pathological artery spasms. Nevertheless, effect of telmisartan on VSMC contractility, and its mechanism of action remain unknown. Here, we investigated the mechanism by which telmisartan inhibits VSMC contractility and vessel contraction in rat VSMCs and endothelium-deprived aortas. Telmisartan inhibited phenylephrine-induced vessel contraction in endothelium-deprived aortas, and decreased myosin light chain kinase (MLCK) levels (without altering corresponding mRNA levels) and myosin light chain (MLC) phosphorylation at Ser19 (p-MLC-Ser19) in VSMCs. MG-132 but not doxycycline significantly restored telmisartan-inhibited MLCK expression and p-MLC-Ser19. Telmisartan induced AMP-activated protein kinase (AMPK) phosphorylation at Thr172 (p-AMPK-Thr172), and compound C or ectopic expression of the dominant negative (dn)-AMPKα1 gene significantly reversed telmisartan-inhibited MLCK expression and p-MLC-Ser19. Of the ARBs tested (including losartan and fimasartan), only telmisartan increased p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19. GW9662 had no effects on telmisartan-induced changes. Similar to the in vitro results, telmisartan enhanced p-AMPK-Thr172, and inhibited MLCK expression and p-MLC-Ser19 in endothelium-deprived aortas. Furthermore, the telmisartan-inhibited vessel contraction in the aortas was significantly reversed by MG-132 or compound C. In conclusion, we demonstrated that telmisartan inhibits VSMC contractility and vessel contraction by activating AMPK/proteasome/MLCK degradation signaling axis. These results suggest that telmisartan can be used to treat pathological vasospasms.

Metformin ameliorates bile duct ligation-induced acute hepatic injury via regulation of ER stress.

Cholestasis is a condition in which the bile duct becomes narrowed or clogged by a variety of factors and bile acid is not released smoothly. Bile acid-induced liver injury is facilitated by necrotic cell death, neutrophil infiltration, and inflammation. Metformin, the first-line treatment for type 2 diabetes, is known to reduce not only blood glucose but also inflammatory responses. In this study, we investigated the effects of metformin on liver injury caused by cholestasis with bile acid-induced hepatocyte injury. Static bile acid-induced liver injury is thought to be related to endoplasmic reticulum (ER) stress, inflammatory response, and chemokine expression. Metformin treatment reduced liver injury caused by bile acid, and it suppressed ER stress, inflammation, chemokine expression, and neutrophil infiltration. Similar results were obtained in mouse primary hepatocytes exposed to bile acid. Hepatocytes treated with tauroursodeoxycholic acid, an ER stress inhibitor, showed inhibition of ER stress, as well as reduced levels of inflammation and cell death. These results suggest that metformin may protect against liver injury by suppressing ER stress and inflammation and reducing chemokine expression. [BMB Reports 2020; 53(6): 311-316].

Telmisartan Inhibits Nitric Oxide Production and Vessel Relaxation via Protein Phosphatase 2A-mediated Endothelial NO Synthase-Ser1179 Dephosphorylation.

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser1179 levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser1179 levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser1179 levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser1179 dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.

Telmisartan increases hepatic glucose production via protein kinase C ζ-dependent insulin receptor substrate-1 phosphorylation in HepG2 cells and mouse liver.

Background: Dysregulation of hepatic glucose production (HGP) contributes to the development of type 2 diabetes mellitus. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), has various ancillary effects in addition to common blood pressure-lowering effects. The effects and mechanism of telmisartan on HGP have not been fully elucidated and, therefore, we investigated these phenomena in hyperglycemic HepG2 cells and high-fat diet (HFD)-fed mice. Methods: Glucose production and glucose uptake were measured in HepG2 cells. Expression levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase α (G6Pase-α), and phosphorylation levels of insulin receptor substrate-1 (IRS-1) and protein kinase C ζ (PKCζ) were assessed by western blot analysis. Animal studies were performed using HFD-fed mice. Results: Telmisartan dose-dependently increased HGP, and PEPCK expression was minimally increased at a 40 µM concentration without a change in G6Pase-α expression. In contrast, telmisartan increased phosphorylation of IRS-1 at Ser302 (p-IRS-1-Ser302) and decreased p-IRS-1-Tyr632 dose-dependently. Telmisartan dose-dependently increased p-PKCζ-Thr410 which is known to reduce insulin action by inducing IRS-1 serine phosphorylation. Ectopic expression of dominant-negative PKCζ significantly attenuated telmisartan-induced HGP and p-IRS-1-Ser302 and -inhibited p-IRS-1-Tyr632. Among ARBs, including losartan and fimasartan, only telmisartan changed IRS-1 phosphorylation and pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, did not alter this effect. Finally, in the livers from HFD-fed mice, telmisartan increased p-IRS-1-Ser302 and decreased p-IRS-1-Tyr632, which was accompanied by an increase in p-PKCζ-Thr410. Conclusion: These results suggest that telmisartan increases HGP by inducing p-PKCζ-Thr410 that increases p-IRS-1-Ser302 and decreases p-IRS-1-Tyr632 in a PPARγ-independent manner.

Anti-malarial Drugs Reduce Vascular Smooth Muscle Cell Proliferation via Activation of AMPK and Inhibition of Smad3 Signaling.

OBJECTIVE: The aim of this study was to investigate the effects of 2 anti-malarial drugs, chloroquine (CQ) and hydroxychloroquine (HCQ), on inhibition of vascular smooth muscle cell (VSMC) proliferation both in vivo and in vitro via Adenosine monophosphate-activated protein kinase (AMPK) activation. METHODS: Protein and mRNA levels were determined by western blot analysis and real-time reverse transcription-polymerase chain reaction in primary rat VSMCs treated with CQ and HCQ, respectively. Cell proliferation was measured by flow cytometry and cell counting. Mice carotid arteries were ligated and treated with CQ or HCQ every other day for 3 weeks. Pathological changes of carotid arteries were visualized by both microscopy and fluorescence microscopy. RESULTS: CQ and HCQ increase AMPK phosphorylation in VSMCs. Both CQ and HCQ decrease platelet-derived growth factor-induced VSMC proliferation and cell cycle progression in an AMPK-dependent manner. In addition, CQ and HCQ inhibit Smad3 phosphorylation and VSMC proliferation induced by transforming growth factor-ß1. Moreover, CQ and HCQ diminished neointimal proliferation in a mouse model of carotid artery ligation-induced neointima formation. CONCLUSION: The results demonstrated that CQ and HCQ inhibit cell proliferation and cell cycle progression in VSMCs via the AMPK-dependent signaling pathway. Carotid artery ligation-induced intima thickness was reduced in mouse arteries treated with CQ or HCQ, suggesting a role for antimalarial drugs in treating atherosclerosis and restenosis.

Telmisartan mitigates hyperglycemia-induced vascular inflammation by increasing GSK3ß-Ser9 phosphorylation in endothelial cells and mouse aortas.

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), attenuates hyperglycemia-aggravated vascular inflammation by decreasing IκB kinase ß (IKKß) expression in endothelial cells. Because glycogen synthase 3ß (GSK3ß) is involved in inflammatory process by regulating nuclear factor-κB (NF-κB) activity, we investigated whether GSK3ß mediates telmisartan-ameliorated vascular inflammation in hyperglycemia-treated endothelial cells and high-fat diet (HFD)-fed mice. Telmisartan remarkably induced GSK3ß-Ser9 phosphorylation in hyperglycemia-treated endothelial cells that accompanied a decrease in hyperglycemia-induced NF-κB p65-Ser536 phosphorylation, vascular cell adhesion molecule-1 (VCAM-1) expression, and THP-1 monocyte adhesion. Ectopic expression of GSK3ß-S9A, a constitutively active mutant of GSK3ß, significantly restored complete telmisartan-inhibited NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and THP-1 monocyte adhesion. In addition, it reversed telmisartan-repressed IKKß expression. Among the ARB, including losartan and fimasartan, only telmisartan increased GSK3ß-Ser9 phosphorylation, and telmisartan-induced GSK3ß-Ser9 phosphorylation remained unchanged by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Finally, in the aortas of HFD-fed mice, telmisartan treatment significantly attenuated HFD-induced upregulation of NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and IKKß expression and downregulation of GSK3ß-Ser9 phosphorylation. Taken together, our findings demonstrated that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by inducing GSK3ß-Ser9 phosphorylation, which consequently inhibits IKKß expression, NF-κB p65-Ser536 phosphorylation, and VCAM-1 expression in a PPARγ-independent manner.

Disruption of neuronal nitric oxide synthase dimerization contributes to the development of Alzheimer's disease: Involvement of cyclin-dependent kinase 5-mediated phosphorylation of neuronal nitric oxide synthase at Ser(293).

Although previous studies have suggested that neuronal nitric oxide synthase (nNOS)-derived NO has neuroprotective effects on the development of Alzheimer's disease (AD), the underlying molecular mechanisms are not fully elucidated. Here, we investigated whether and how disruption of nNOS dimerization contributes to the development of AD. No differences in synaptic number or expression of synaptic markers, including synaptophysin and postsynaptic density 95, were found in the cortex of 5 × FAD mice, which possess 5 familial AD mutations, at 6 months of age compared with control littermates. nNOS dimerization was disrupted in the 5 × FAD cortex, accompanied by an increase in reactive oxygen species (ROS) production. The subcellular distribution of cyclin-dependent kinase 5 (CDK5) shifted more diffusely toward a cytosolic compartment, but there was no change in total expression. Furthermore, the levels of p25, a CDK5 activator, increased significantly and it colocalized with nNOS in the 5 × FAD cortex. In silico analysis revealed that a new nNOS-specific GSP (glycine-serine-proline) motif was well-conserved across species at nNOS-Ser(293), which is located ahead of the N-terminal hook. This motif was not present in the closely related isoform, endothelial NOS. Motif scan analysis also predicted that CDK5 can phosphorylate nNOS-Ser(293) with a high likelihood. An in vitro phosphorylation assay clearly showed that CDK5/p25 does indeed phosphorylate nNOS-Ser(293). Finally, nNOS-S293D mutant, a phosphomimetic form of nNOS-Ser(293), and nNOS-S293A mutant, a neutral form of nNOS-Ser(293), significantly decreased nNOS dimerization and NO production. Taken together, our results demonstrate that nNOS dimers are disrupted in the 5 × FAD cortex, and nNOS-Ser(293), a potential site of CDK5 phosphorylation, may be involved in the decrease in nNOS dimerization and NO production, and the development of AD.

Citron Rho-interacting kinase mediates arsenite-induced decrease in endothelial nitric oxide synthase activity by increasing phosphorylation at threonine 497: Mechanism underlying arsenite-induced vascular dysfunction.

We reported that arsenite causes an acute decrease in nitric oxide (NO) production by increasing phosphorylation of endothelial NO synthase at threonine 497 (eNOS-Thr(497)); however, the detailed mechanism has not yet been clarified. Here, we investigated the kinase involving in arsenite-stimulated eNOS-Thr(497) phosphorylation. Although treatment with H-89, a known protein kinase A (PKA) inhibitor, inhibited arsenite-stimulated eNOS-Thr(497) phosphorylation, no inhibition was found in cells treated with other PKA inhibitors, including Rp-8-Br-cAMPS or PKI. Based on previous reports, we also tested whether RhoA mediates arsenite-stimulated eNOS-Thr(497) phosphorylation and found that arsenite causes an acute increase in RhoA activity. Ectopic expression of dominant negative (DN)-RhoA significantly reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. An in vitro phosphorylation assay also revealed that the well-known Rho effectors, Rho-associated protein kinase 1/2 (ROCK1/2), directly phosphorylate eNOS-Thr(497). Y27632, a selective ROCK inhibitor, reversed arsenite-stimulated eNOS-Thr(497) phosphorylation. However, overexpression of a small interfering RNA (siRNA) against ROCK1/2 or DN-ROCK did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation, thereby providing no conclusive evidence of a role for ROCK1/2. Knockdown of PKC-related protein kinase 1/2, another Rho effector, also did not reverse arsenite-stimulated eNOS-Thr(497) phosphorylation. In contrast, we found that transfection with an siRNA against citron Rho-interacting kinase (CRIK), the other downstream effector of Rho, significantly reversed the arsenite-induced eNOS-Thr(497) phosphorylation that was accompanied by restoration of eNOS enzymatic activity repressed by arsenite. Moreover, CRIK directly phosphorylated eNOS-Thr(497)in vitro. Finally, we also found that arsenite increased eNOS-Thr(497) phosphorylation and decreased acetylcholine-induced vessel relaxation in rat aortas. In conclusion, we demonstrate that arsenite acutely inhibits eNOS enzymatic activity and vessel relaxation in part by increasing the RhoA/CRIK/eNOS-Thr(497) phosphorylation signaling axis, which provides a molecular mechanism underlying arsenite-induced impaired vascular diseases.

Telmisartan attenuates hyperglycemia-exacerbated VCAM-1 expression and monocytes adhesion in TNFα-stimulated endothelial cells by inhibiting IKKß expression.

Uncontrolled hyperglycemia accelerates endothelial damage and vascular inflammation caused by proinflammatory cytokines including tumor necrosis factor α (TNFα), which leads to arteriosclerotic cardiovascular diseases such as myocardial infarction. Telmisartan, an angiotensin II type 1 receptor blocker (ARB), is prescribed for treatment of hypertensive patients with concurrent diabetes mellitus (DM). Although a few clinical trials have suggested that telmisartan decreases cardiovascular complications in diabetic patients, the molecular mechanism for the beneficial effects remains elusive. Here, we investigated a molecular mechanism and effects of telmisartan on the expression of vascular cell adhesion molecule-1 (VCAM-1) and attachment of monocytes onto endothelial cells induced by TNFα in hyperglycemia-treated bovine aortic endothelial cells (BAEC). Telmisartan dose-dependently decreased hyperglycemia-aggravated IκB kinase ß (IKKß) expression and nuclear factor-κB (NF-κB) p65-Ser(536) phosphorylation, which accompanied a decrease in VCAM-1 expression and THP-1 monocytes adhesion. Among ARBs, including losartan and fimasartan, only telmisartan showed the inhibitory effects on expression of VCAM-1 and IKKß, and phosphorylation of NF-κB p65-Ser(536). The telmisartan's beneficial effects were not changed by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist, although GW9662 clearly inhibited rosiglitazone-induced CD36 expression. Finally, ectopic expression of wild type (WT)-IKKß significantly restored telmisartan-attenuated VCAM-1 expression, NF-κB p65-Ser(536) phosphorylation, and THP-1 monocytes adhesion. Taken together, our findings demonstrate that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by decreasing expression of IKKß and VCAM-1 independently of PPARγ. Telmisartan may be useful for the treatment of DM-associated vascular inflammation and cardiovascular diseases.

The Ameliorating Effect of Myrrh on Scopolamine-Induced Memory Impairments in Mice.

Myrrh has been used since ancient times for the treatment of various diseases such as inflammatory diseases, gynecological diseases, and hemiplegia. In the present study, we investigated the effects of aqueous extracts of myrrh resin (AEM) on scopolamine-induced memory impairments in mice. AEM was estimated with (2E,5E)-6-hydroxy-2,6-dimethylhepta-2,4-dienal as a representative constituent by HPLC. The oral administration of AEM for 7 days significantly reversed scopolamine-induced reduction of spontaneous alternation in the Y-maze test. In the passive avoidance task, AEM also restored the decreased latency time of the retention trial by scopolamine treatment. In addition, Western blot analysis and Immunohistochemistry revealed that AEM reversed scopolamine-decreased phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Our study demonstrates for the first time that AEM ameliorates the scopolamine-induced memory impairments in mice and increases the phosphorylation of Akt and ERK in the hippocampus of mice brain. These results suggest that AEM has the therapeutic potential in memory impairments.

B56δ subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production.

DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser1179 phosphorylation, aphidicolin decreased eNOS-Ser116 phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116 residue was identified as the substrate site of the regulatory subunit B56δ of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser116 dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56δ-Ser566 phosphorylation, although expression of neither the catalytic subunit Cα (PP2A Cα) nor B56δ was altered. Ectopic expression of dominant negative (dn)-B56δ reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser116 and B56δ-Ser566. Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56δ. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser116, at least in part by activating PP2A B56δ, resulting in NO release in BAEC.