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

OBJECTIVETo explore the effect of ouabain on intracellular Ca(2+) concentration ([Ca(2+)]i) in thoracic aorta vascular smooth muscle cells (VSMCs) in vitro.

METHODSPrimary SD rat thoracic aorta VSMCs were cultured by tissue adherent method and identified by immunochemistry. The binding ability between ouabain and VSMCs was detected by autoradiography, and fluo 3-AM (a Ca(2+) fluorescent probe) was employed to investigate whether ouabain affected VSMCs within a short period of time. The effect of a truncated fragment of the sodium pump α2 subunit was assayed in antagonizing the effect of ouabain on [Ca(2+)]i in the VSMCs.

RESULTSWithin the concentration range of 0.1-100 nmol/L, ouabain was found to dose-dependently bind to the VSMCs. Different concentrations of ouabain (0-3200 nmol/L) caused a transient, dose-dependent increase in [Ca(2+)]i in the VSMCs, which was antagonized by the application of the truncated fragment of sodium pump α2 subunit.

CONCLUSIONSElevations in [Ca(2+)]i in the VSMCs can be the cytological basis of high ouabain-induced hypertension. The truncated fragment of the sodium pump α2 subunit can antagonize ouabain-induced increase of [Ca(2+)]i in the VSMCs, which provides a clue for understanding the pathogenesis of and devising a therapeutic strategy for high ouabain-induced hypertension.

Animals ; Aorta, Thoracic ; cytology ; Calcium ; metabolism ; Cells, Cultured ; Cytoplasm ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Ouabain ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase

Leptin is a peptide hormone, which has a central role in the regulation of body weight; it also exerts many potentially atherogenic effects. Ferulic acid ethyl ester (FAEE) has been approved for antioxidant properties. The aim of this study was to investigate whether FAEE can inhibit the atherogenic effects of leptin and the possible molecular mechanism of its action. Both of cell proliferation and migration were measured when the aortic smooth muscle cell (A10 cell) treated with leptin and/or FAEE. Phosphorylated p44/42MAPK, cell cycle-regulatory protein (for example, cyclin D1, p21, p27), beta-catenin and matrix metalloproteinase-9 (MMP-9) proteins levels were also measured. Results demonstrated that leptin (10, 100 ng ml-1) significantly increased the proliferation of cells and the phosphorylation of p44/42MAPK in A10 cells. The proliferative effect of leptin was significantly reduced by the pretreatment of U0126 (0.5 muM), a MEK inhibitor, in A10 cells. Meanwhile, leptin significantly increased the protein expression of cyclin D1, p21, beta-catenin and decreased the expression of p27 in A10 cells. In addition, leptin (10 ng ml-1) significantly increased the migration of A10 cells and the expression of MMP-9 protein. Above effects of leptin were significantly reduced by the pretreatment of FAEE (1 and 10 muM) in A10 cells. In conclusion, FAEE exerts multiple effects on leptin-induced cell proliferation and migration, including the inhibition of p44/42MAPK phosphorylation, cell cycle-regulatory proteins and MMP-9, thereby suggesting that FAEE may be a possible therapeutic approach to the inhibition of obese vascular disease.
Animals ; Antioxidants/*pharmacology ; Aorta/cytology/*drug effects ; Caffeic Acids/*pharmacology ; Cell Line ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Leptin/*metabolism ; Matrix Metalloproteinase 9/metabolism ; Muscle, Smooth, Vascular/cytology/drug effects ; Myocytes, Smooth Muscle/cytology/*drug effects ; Rats ; beta Catenin/metabolism

Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; pathology ; Cell Differentiation ; Coculture Techniques ; Gene Expression Regulation ; Isoenzymes ; genetics ; metabolism ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Vascular Calcification ; genetics ; metabolism ; pathology

OBJECTIVETo investigate the effects and mechanisms of high glucose on the phenotype transformation of rat vascular smooth muscle cells (VSMCs).

METHODSVSMCs ere isolated from rat thoracic aorta and the 3rd-5th VSMCs were incubated with normal glucose (5.5 mmol/L), high glucose (25 mmol/L), or high glucose (25 mmol/L) + P38 inhibitor (25 mmol/L +SB203580) for another 24 hours. Then the gene expression of osteopontin (OPN), alpha smooth-actin (alpha-SMA), matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9(MMP-9) were assayed by real time RT-PCR, the protein expression of P38 were assayed by Western blot.

RESULTS(1) High glucose promoted the phenotype transformation of VSMCs and up-regulated the expression of MMP-2 and MMP-9. (2) High glucose promoted the phosphorylation of P38. (3) SB203580, the inhibitor of P38/MAPK signal pathway, inhibited the effects of high glucose on phenotype transformation and expression of MMP-2 and MMP-9.

CONCLUSIONHigh glucose may promote phenotype transformation of VSMCs via the signal pathway of P38/MAPK.

Actins ; metabolism ; Animals ; Aorta, Thoracic ; cytology ; Blotting, Western ; Cells, Cultured ; Glucose ; pharmacology ; Imidazoles ; pharmacology ; MAP Kinase Signaling System ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Osteopontin ; metabolism ; Phenotype ; Pyridines ; pharmacology ; Rats ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDUrotensin II (UII), a potent vasoconstrictive peptide, is able to stimulate phenotypic differentiation of adventitial fibroblasts. This study aimed to determine the effect of UII on monocyte chemoattractant protein-1 (MCP-1) expression in rat aortic adventitial fibroblasts, so as to explore possible mechanisms in the development of vascular inflammation.

METHODSGrowth-arrested adventitial fibroblasts were incubated in serum-free medium with UII (10(-10)-10(-7) mol/L) and inhibitors of signal transduction pathways for 1 to 24 hours. MCP-1 mRNA and protein expression and secretion were determined by RT-PCR, Western blotting analysis and enzyme-linked immunosorbent assay (ELISA), respectively.

RESULTSUII dose- and time-dependently promoted MCP-1 mRNA and protein expression and secretion in cells, with maximal effect at 10(-8) mol/L at 3 hours for mRNA expression, 24 hours for protein expression in the cells, and 12 hours for protein secretion from the cells. Furthermore, the UII effects were significantly inhibited by treatment with its receptor antagonist SB710411, Rho kinase inhibitor Y27632, protein kinase C (PKC) inhibitor H7, mitogen-activated protein kinase inhibitor PD98059, calcineurin inhibitor cyclosporine A, and the Ca(2+)channel blocker nicardipine.

CONCLUSIONUII may stimulate MCP-1 expression in rat aortic adventitial fibroblasts through its receptor and Rho kinase, PKC, mitogen-activated protein kinase, calcineurin and Ca(2+) channel signal transduction, thus contributing to adventitial inflammation.

Adventitia ; cytology ; Animals ; Aorta ; cytology ; Cells, Cultured ; Chemokine CCL2 ; genetics ; metabolism ; Fibroblasts ; drug effects ; metabolism ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Urotensins ; pharmacology

OBJECTIVETo investigate the effects of non-neuronal muscarinic receptors (NNMR) stimulation on atherosclerosis and endothelial cells activation.

METHODSAtherosclerosis model was established in ApoE-/- mice by a high fat diet for 7 weeks. During the experimental periods, animals were received a low (7 mg/kg/d) or a high (21 mg/kg/d) dose of arecoline by gavage. At the termination of the treatments, serum total cholesterol and NO levels were measured, and the aorta morphology was analyzed by hematoxylin and eosin staining. The gene expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in the thoracic aortas was determined by RT-PCR, and the MCP-1 protein expression and NF-κB activity were detected by Western blot analysis. NO production, MCP-1 secretion in cultured rat aortic endothelial cells (RAECs), and monocyte-endothelium adhesion assay were also performed after arecoline treatments.

RESULTSArecoline efficiently decreased atherosclerotic plaque areas, increased serum nitric oxide (NO) content, suppressed the mRNA and protein expression of MCP-1, and modulated the IκB-α degradation and P65 phosphorylation in the aortae of ApoE-/- mice. Furthermore, arecoline promoted NO production and suppressed MCP-1 secretion in cultured RAECs after ox-LDL exposure, and either atropine or NG-nitro-L-arginine methylester could abrogate these effects. Arecoline also significantly inhibited the adherence of U937 monocytes to the ox-LDL injured human umbilical vein endothelial cells, which could be abolished by atropine.

CONCLUSIONOur results indicate that arecoline attenuates the progression of atherosclerosis and inhibits endothelial cells activation and adherence by stimulating endothelial NNMR. These effects, at least in part, are due to its modulation on NF-κB activity.

Animals ; Aorta ; cytology ; Apolipoproteins E ; Arecoline ; pharmacology ; Atherosclerosis ; physiopathology ; prevention & control ; Cell Adhesion Molecules ; metabolism ; Chemokine CCL2 ; metabolism ; Cholesterol ; blood ; Disease Progression ; Endothelial Cells ; cytology ; drug effects ; Endothelium, Vascular ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; I-kappa B Proteins ; metabolism ; Lipoproteins, LDL ; Mice ; Mice, Knockout ; Monocytes ; cytology ; NF-KappaB Inhibitor alpha ; Nitric Oxide ; blood ; Nitroarginine ; pharmacology ; Rats ; Receptors, Muscarinic ; physiology ; Transcription Factor RelA ; metabolism

The present study was aimed to investigate the effect of pretreatment with Danshensu (DSS) on rat aortic endothelial cells (RAECs) senescence and the underlying mechanisms. Cultured RAECs at fourth and twelfth passages were taken as young and old groups, respectively. DSS and DSS+nicotinamide (DSS+N) groups were incubated with DSS and DSS in combination with nicotinamide, an inhibitor of silent information regulator 1 (SIRT1), from the fourth to twelfth passage, respectively. The cell status of senescence was determined by the senescence-associated β-galactosidase (SA β-gal) staining, and 4,6-diamino-2-phenyl indole (DAPI) fluorescent dye was used to detect senescence associated heterochromatin foci (SAHF) formation; Thiobarbituric acid (TBA) and colorimetric methods were used to evaluate malondialdehyde (MDA) and H₂O₂contents; Western blot was employed to analysis the expressions of xanthine oxidase (XOD), SIRT1 and superoxide dismutase 2 (SOD₂) in the RAECs. The results showed that, in comparison with young group, the old group exhibited higher SA β-gal positive and SAHF formation rates, as well as higher MDA and H₂O₂levels (P < 0.05 or P < 0.01), whereas DSS pretreatment reduced SA β-gal positive and SAHF formation rates, decreased MDA and H2O2 contents (P < 0.05 or P < 0.01). The protection of DSS was reversed by nicotinamide. Compared with the young group, the old group showed higher expression levels of XOD, but lower SIRT1 and SOD₂expression levels (P < 0.05 or P < 0.01). With the pretreatment of DSS, the expression of XOD was declined, and the expression levels of SIRT1 and SOD₂were elevated, while nicotinamide reversed the effects of DSS. These results suggest that DSS delays senescence of RAECs via up-regulation of SIRT1.
Animals ; Aorta ; cytology ; Cells, Cultured ; Cellular Senescence ; drug effects ; Endothelial Cells ; cytology ; Hydrogen Peroxide ; metabolism ; Lactates ; pharmacology ; Niacinamide ; pharmacology ; Rats ; Sirtuin 1 ; metabolism ; Superoxide Dismutase ; metabolism ; Up-Regulation

AIM: The compound B19 (C21H22O5) is a newly synthesized, mono-carbonyl analog of curcumin that has exhibited potential antitumor effects. This present study was performed to identify the anti-angiogenic activity of this compound. METHODS AND RESULTS: B19 inhibited migration and tube formation of human umbilical vein endothelial cells, and arrested microvessel outgrowth from rat aortic rings. In addition, B19 suppressed the neovascularization of chicken chorioallantoic membrane. Mechanistic studies revealed that B19 suppressed the downstream protein kinase activation of vascular endothelial growth factor (VEGF) by decreasing phosphorylated forms of serine/threonine kinase Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase, with or without stimulating vascular endothelial growth factor (VEGF). CONCLUSIONS B19 exerted anti-angiogenic activity in vitro and ex vivo, which suggests that it merits further investigation as a promising anticancer angiogenesis compound.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Animals ; Aorta ; drug effects ; metabolism ; Cell Movement ; drug effects ; Curcumin ; analogs & derivatives ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; In Vitro Techniques ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo observe the effects of ginkgo flavone aglycone (GA) on oxidized low-density lipoprotein (ox-LDL) induced oxidative injury of human aortic endothelial cells (HAECs) and its mechanisms.

METHODSHAECs were in vitro cultured. Then they were divided into 6 groups, i.e., the vehicle control group, the ox-LDL group, the GA 30 mg/L group, the GA 60 mg/L group, the GA 90 mg/L group, and the Vit E group. The oxidative injury model was duplicated in the rest 5 groups by adding 150 mg/L ox-LDL except the vehicle control group. GA was added as intervention at corresponding dose to the GA 30 mg/L group, the GA 60 mg/L group, and the GA 90 mg/L group. Vit E at 200 micromol/L was administered to those in the Vit E group. The survival rate of HAECs was detected by MTT. The contents of reactive oxygen species (ROS) in HAECs were determined by CM-H2DCFDA fluorescent probe. The contents of NADPH oxidase were detected by ELISA. The levels of malondialdehyde (MDA) were measured by thiobarbituric acid (TBA) test. The contents of nitric oxide (NO) were determined by Griess reagent method. The contents of superoxide dismutase (SOD) were detected by xanthine oxidase method.

RESULTSCompared with the vehicle control group (100.00%), the cell survival rate in the ox-LDL group (70.68%) obviously decreased (P <0.05). The cell survival rate was 88. 95% in the VitE group, 83.25% in the GA 30 mg/L group, and 94.93% in the GA 60 mg/L group, obviously higher than that of the ox-LDL group (70.68%, P <0.05). The optimal effects were shown in the GA 60 mg/L group. Compared with the vehicle control group, the contents of ROS, MDA, and NADPH oxidase increased, the contents of NO and the SOD activity decreased in the ox-LDL group, showing statistical difference (P <0.05). Compared with the ox-LDL group, the contents of ROS, MDA, and NADPH oxidase decreased, the NO content and the SOD activity increased in the GA 30 mg/L group, the GA 60 mg/L group, and the Vit E group, showing statistical difference (P <0.05). The optimal effects were shown in the GA 60 mg/L group.

CONCLUSIONSGA could obviously inhibit ox-LDL induced synthesis of ROS, lower the contents of MDA, and elevate the levels of NO. Its mechanisms might be associated with increasing the activity of SOD and lowering the activity of NADPH oxidase.

Aorta ; cytology ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Ginkgo biloba ; chemistry ; Humans ; Isoflavones ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Malondialdehyde ; metabolism ; Nitric Oxide ; metabolism ; Oxidation-Reduction ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Vitamin E ; pharmacology