Objective To explore the role and mechanism of mammalian target of rapamycin (mTOR) in oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in vascular smooth muscle cells (VSMCs). Methods A model of oxLDL-induced VSMC ferroptosis was established. VSMCs were co-treated with either the mTOR inhibitor rapamycin or the autophagy inducer carbonyl cyanide m-chlorophenylhydrazone (CCCP), followed by detection of autophagy and ferroptosis-related indexes. Quantitative real-time PCR and Western blot were used respectively to analyze the expression of mTOR, glutathione peroxidase 4 (GPX4), sequestosome 1 (p62), and microtubule-associated protein 1 light chain 3 (LC3). Flow cytometry was employed to assess VSMC death. C11 BODIPY fluorescent staining was used to measure cellular lipid peroxidation levels. Colorimetric assays were performed to determine the contents of malondialdehyde (MDA), ferrous ion (Fe²⁺) and glutathione (GSH). Results oxLDL significantly upregulated mTOR expression in VSMCs, while increasing p62 expression and reducing LC3 expression, thereby suppressing VSMC autophagy. Compared with oxLDL treatment alone, rapamycin co-treatment reversed oxLDL-induced VSMC ferroptosis, as characterized by reduced VSMC death, increased GPX4 expression and GSH contents, along with decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Similarly, CCCP co-treatment activated autophagy characterized by reduced p62 expression and elevated LC3 expression, which subsequently alleviated oxLDL-induced ferroptosis, showing reduced VSMC death, increased GPX4 expressions and GSH contents, and decreased MDA content, Fe²⁺ content and lipid peroxidation levels. Moreover, mTOR inhibition by rapamycin significantly reversed the oxLDL-induced upregulation of p62 and downregulation of LC3. Conclusion mTOR may promote oxLDL-induced VSMC ferroptosis by suppressing autophagy.
Ferroptosis/drug effects* ; Lipoproteins, LDL/metabolism* ; TOR Serine-Threonine Kinases/physiology* ; Autophagy/drug effects* ; Muscle, Smooth, Vascular/metabolism* ; Animals ; Rats ; Myocytes, Smooth Muscle/cytology* ; Cells, Cultured ; Lipid Peroxidation/drug effects* ; Sequestosome-1 Protein/genetics* ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism* ; Microtubule-Associated Proteins/genetics* ; Sirolimus/pharmacology*

The present study attempted to test a novel hypothesis that Ca(2+) sparks play an important role in arterial relaxation induced by tacrolimus. Recorded with confocal laser scanning microscopy, tacrolimus (10 µmol/L) increased the frequency of Ca(2+) sparks, which could be reversed by ryanodine (10 µmol/L). Electrophysiological experiments revealed that tacrolimus (10 µmol/L) increased the large-conductance Ca(2+)-activated K(+) currents (BKCa) in rat aortic vascular smooth muscle cells (AVSMCs), which could be blocked by ryanodine (10 µmol/L). Furthermore, tacrolimus (10 and 50 µmol/L) reduced the contractile force induced by norepinephrine (NE) or KCl in aortic vascular smooth muscle in a concentration-dependent manner, which could be also significantly attenuated by iberiotoxin (100 nmol/L) and ryanodine (10 µmol/L) respectively. In conclusion, tacrolimus could indirectly activate BKCa currents by increasing Ca(2+) sparks released from ryanodine receptors, which inhibited the NE- or KCl-induced contraction in rat aorta.
Animals ; Aorta ; cytology ; metabolism ; physiology ; Calcium Signaling ; Cells, Cultured ; Large-Conductance Calcium-Activated Potassium Channels ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; metabolism ; physiology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; pharmacology ; Tacrolimus ; pharmacology ; Vasoconstriction

The aim of this study was to investigate the inhibitory effect of heparin/fibronectin (Hep/Fn) complexes on neointimal hyperplasia following endovascular intervention. Hep/Fn complexes were immobilized onto titanium (Ti) surfaces, with subsequent X-ray photoelectron spectroscopy (XPS), Toluidine Blue O (TBO) and immunohistochemistry methods were used to characterize surface properties. Smooth muscle cell (SMC) cultures were used to evaluate the effect of Hep/Fn complexes on SMC proliferation. Results showed that Hep/Fn complexes successfully immobilized onto Ti surfaces and resulted in an inhibition of SMC proliferation. This study suggests that Hep/Fn surface-immobilized biomaterials develop as a new generation of biomaterials to prevent neointimal hyperplasia, particularly for use in cardiovascular implants.
Biocompatible Materials ; Cell Proliferation ; drug effects ; physiology ; Cells, Cultured ; Fibronectins ; chemistry ; pharmacology ; Heparin ; chemistry ; pharmacology ; Humans ; Immobilized Proteins ; chemistry ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Surface Properties ; Titanium ; chemistry ; Umbilical Arteries

OBJECTIVETo explore the effects of NF-κB on proliferation of rat pulmonary artery smooth muscle cells (PASMC) inhibited by simvastatin.

METHODSPASMC isolated from rats and cultured in vitro were randomly divided into four groups (n=6 each): control, platelet-derived growth factor (PDGF) treatment, PDGF+simvastatin treatment, and PDGF+simvastatin+parthenolide (NF-κB inhibitor) treatment. MTT colorimetric assay and flow cytometry were performed to detect cell proliferation and cell cycle distribution. Immunohistochemistry was performed to detect the expression of NF-κB protein. Real-Time PCR was performed to detect NF-κB mRNA expression.

RESULTSCompared with the control group, MTT values of PASMC at all time points, cell proportion at the S phase and G2+M phase, NF-κB protein and mRNA expression increased significantly in the PDGF group (P<0.05). With the intervention of simvastatin, the levels of above indexes decreased compared with the PDGF group (P<0.05). With the intervention of simvastatin and parthenolide, the levels of above indexes decreased more obviously, but were not significantly different from those in the simvastatin intervention group.

CONCLUSIONSSimvastatin can inhibit proliferation of PASMC and cell cycle process. NF-κB may play an important role in the inhibitory effect of simvastatin on the proliferation of PASMC.

Animals ; Cell Proliferation ; drug effects ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; NF-kappa B ; analysis ; genetics ; physiology ; Pulmonary Artery ; cytology ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Simvastatin ; pharmacology

The purpose of the present study is to investigate the effect of isoliquiritigenin (ISL) on the cerebral basilar artery in spontaneously hypertensive rats (SHR). The change of SHR systolic pressure was measured by tail artery pressure measurement instrument before and after ISL intervention. After perfusion with 1 × 10(-5) mol/L phenylephrine (PE), 1 × 10(-5) mol/L PE + 1 × 10(-4) mol/L ISL and 1 × 10(-5) mol/L PE, the diameter of the cerebral basilar artery separated from SHR was measured by pressure myograph. The current of large-conductance calcium-activated potassium (BKCa) channel of SHR single vascular smooth muscle cell (VSMC) was recorded by whole-cell patch-clamp technique and the cGMP levels of basilar artery was evaluated by ELISA. The results showed that 1) after intervention with ISL for 14 days, the systolic pressure of SHR was decreased from (218.3 ± 1.6) mmHg to (119.2 ± 1.9) mmHg (P < 0.01), but there was no difference in systolic pressure between ISL-treated SHR and Wistar-Kyoto (WKY) rat; 2) 1 × 10(-4) mol/L ISL relaxed the SHR cerebral basilar artery (P < 0.01); 3) ISL significantly increased the outward current density of VSMC from SHR cerebral basilar artery (P < 0.01, n = 6), and the effect could be reversed by 1 × 10(-3) mol/L TEA (a BKCa channel inhibitor), but 3 × 10(-4) mol/L 4-AP (a Kv channel inhibitor) had no effect on the enhanced current density induced by ISL in VSMC; 4) 1 × 10(-5) mol/L Methylene blue (a sGC inhibitor) significantly inhibited the ISL-enhanced current density in VSMC (P < 0.05, n = 6); 5) ISL significantly increased the cGMP level of SHR basilar artery (P < 0.05, n = 6). The results suggest that the role of the ISL in relaxing the SHR cerebral basilar artery may be related to its effect in enhancing BKCa current by increasing the levels of cGMP in the VSMC.
Animals ; Basilar Artery ; drug effects ; Blood Pressure ; Cerebral Arteries ; drug effects ; Chalcones ; pharmacology ; Cyclic GMP ; physiology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; physiology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Systole

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca2+-activated K+, inward rectifier K+ and ATP-sensitive K+ channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca2+ channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, alpha-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.
4-Aminopyridine/pharmacology ; Action Potentials ; Animals ; Calcium Channel Blockers/pharmacology ; Calcium Channels/metabolism ; Cells, Cultured ; Ketanserin/pharmacology ; Male ; Mesenteric Arteries/drug effects/*metabolism/physiology ; Muscle Contraction ; Muscle, Smooth, Vascular/cytology/drug effects/metabolism/physiology ; Myocytes, Smooth Muscle/drug effects/metabolism/physiology ; Nifedipine/pharmacology ; Potassium Channel Blockers/pharmacology ; Potassium Channels, Voltage-Gated/antagonists & inhibitors/*metabolism ; Protein Kinase Inhibitors/pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptor, Serotonin, 5-HT2A/*metabolism ; Serotonin/*pharmacology ; Serotonin 5-HT2 Receptor Antagonists/pharmacology ; Spiperone/pharmacology ; *Vasoconstriction ; src-Family Kinases/antagonists & inhibitors/*metabolism

OBJECTIVETo investigate effects of dahuang zhechong pill ( DHZCP) on the cell cycle and the related signal pathways in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF) with the method of serum pharmacology.

METHODSDNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. The cycle of VSMCs was evaluated with flow cytometry. Expressions of cyclin D1, p27, protein kinase Cα (PKCα), and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) were quantified by Western blot method.

RESULTSDHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs, arrested the cells in G G(1) phase, modulated the protein expressions of cyclin D D(1) and p27, and suppressed the activation of PKCα and ERK1/2.

CONCLUSIONDHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G G(1) phase, which is attributed to, at least in part, suppressing PKCα-ERK1/2 signaling in VSMCs.

Animals ; Aorta, Thoracic ; cytology ; Blood Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; G1 Phase ; drug effects ; physiology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; enzymology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley

PURPOSE: Dexmedetomidine, a full agonist of alpha2B-adrenoceptors, is used for analgesia and sedation in the intensive care units. Dexmedetomidine produces an initial transient hypertension due to the activation of post-junctional alpha2B-adrenoceptors on vascular smooth muscle cells (SMCs). The aims of this in vitro study were to identify mitogen-activated protein kinase (MAPK) isoforms that are primarily involved in full, alpha2B-adrenoceptor agonist, dexmedetomidine-induced contraction of isolated rat aortic SMCs. MATERIALS AND METHODS: Rat thoracic aortic rings without endothelium were isolated and suspended for isometric tension recording. Cumulative dexmedetomidine (10(-9) to 10(-6) M) dose-response curves were generated in the presence or absence of extracellular signal-regulated kinase (ERK) inhibitor PD 98059, p38 MAPK inhibitor SB 203580, c-Jun NH2-terminal kinase (JNK) inhibitor SP 600125, L-type calcium channel blocker (verapamil and nifedipine), and alpha2-adrenoceptor inhibitor atipamezole. Dexmedetomidine-induced phosphorylation of ERK, JNK, and p38 MAPK in rat aortic SMCs was detected using Western blotting. RESULTS: SP 600125 (10(-6) to 10(-5) M) attenuated dexmedetomidine-evoked contraction in a concentration-dependent manner, whereas PD 98059 had no effect on dexmedetomidine-induced contraction. SB 203580 (10(-5) M) attenuated dexmedetomidine-induced contraction. Dexmedetomidine-evoked contractions were both abolished by atipamezole and attenuated by verapamil and nifedipine. Dexmedetomidine induced phosphorylation of JNK and p38 MAPK in rat aortic SMCs, but did not induce phosphorylation of ERK. CONCLUSION: Dexmedetomidine-induced contraction involves a JNK- and p38 MAPK-mediated pathway downstream of alpha2-adrenoceptor stimulation in rat aortic SMCs. In addition, dexmedetomidine-induced contractions are primarily dependent on calcium influx via L-type calcium channels.
Adrenergic alpha-2 Receptor Agonists/*pharmacology ; Animals ; Anthracenes/pharmacology ; Aorta/cytology ; Dexmedetomidine/*pharmacology ; Enzyme Inhibitors/pharmacology ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/physiology ; Flavonoids/pharmacology ; Imidazoles/pharmacology ; JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors/*physiology ; Male ; *Muscle Contraction ; Muscle, Smooth, Vascular/drug effects/enzymology/*physiology ; Protein Isoforms/antagonists & inhibitors/physiology ; Pyridines/pharmacology ; Rats ; Rats, Sprague-Dawley ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/physiology

PURPOSE: Stimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration. MATERIALS AND METHODS: nLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H2O2 generation was determined by microscopic analysis using 2',7'-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates. RESULTS: nLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H2O2 generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H2O2. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody. CONCLUSION: PTX-sensitive G-protein coupled receptor-dependent H2O2 generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.
Cell Movement/*physiology ; Cells, Cultured ; Humans ; Hydrogen Peroxide/*metabolism ; Interleukin-8/*biosynthesis ; Lipoproteins, LDL/*pharmacology ; Muscle, Smooth, Vascular/cytology/*metabolism ; Myocytes, Smooth Muscle/cytology/*metabolism ; Pertussis Toxin/pharmacology ; Phosphorylation/drug effects ; Reactive Oxygen Species/metabolism ; Receptors, G-Protein-Coupled/*physiology ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo investigate the contribution of mitochondrial pathway in the apoptosis induced by puerarin (PUE) in pulmonary artery smooth muscle cells.

METHODCultured rat pulmonary artery smooth muscle cells (PASMC) were intervened by high, middle and low dose of puerarin (1.5 x 10(-3), 1.5 x 10(-4), 1.5 x 10(-5) mol x L(-1)). The change of mitochondrial membrane potential was observed. Western blot detected the expression of apoptosis-related gene Caspase-9, Bax and Bcl-2 protein.

RESULTCompared with the control group, mitochondrial membrane potential significantly decreased in puerarin groups. Puerarin can enhance the expression of Caspase-9 and Bax protein, decrease the expression of Bcl-2 protein. Puerarin also has a concentration-dependent on the induction of PASMC.

CONCLUSIONPuerarin can induce PASMC apoptosis through mitochondrial pathway.

Animals ; Apoptosis ; drug effects ; Caspase 9 ; metabolism ; Cells, Cultured ; Isoflavones ; pharmacology ; Male ; Mitochondria ; drug effects ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Pulmonary Artery ; cytology ; drug effects ; Rats ; Rats, Wistar