OBJECTIVE: To investigate the effect of rosuvastatin on homocysteine (Hcy) induced mousevascular smooth muscle cells(VSMCs) dedifferentiation and endoplasmic reticulum stress(ERS). METHODS: VSMCs were co-cultured with Hcy and different concentration of rosuvastatin (0.1, 1.0 and 10 μmol/L). Cytoskeleton remodeling, VSMCs phenotype markers (smooth muscle actin-α, calponin and osteopontin) and ERS marker mRNAs (Herpud1, XBP1s and GRP78) were detected at predicted time. Tunicamycin was used to induce, respectively 4-phenylbutyrate(4-PBA) inhibition, ERS in VSMCs and cellular migration, proliferation and expression of phenotype proteins were analyzed. Mammalian target of rapamycin(mTOR)-P70S6 kinase (P70S6K) signaling agonist phosphatidic acid and inhibitor rapamycin were used in Rsv treated VSMCs. And then mTOR signaling and ERS associated mRNAs were detected. RESULTS: Compared with Hcy group, Hcy+ Rsv group (1.0 and 10 μmol/L) showed enhanced α-SMA and calponin expression (<0.01), suppressed ERS mRNA levels (<0.01) and promoted polarity of cytoskeleton. Compared with Hcy group, Hcy+Rsv group and Hcy+4-PBA group showed suppressed proliferation, migration and enhanced contractile protein expression (<0.01); while tunicamycin could reverse the effect of Rsv on Hcy treated cells. Furthermore, alleviated mTOR-P70S6K phosphorylation and ERS (<0.01)were observed in Hcy+Rsv group and Hcy+rapamycin group, compared with Hcy group; while phosphatidic acid inhibited the effect of Rsv on mTOR signaling activation and ERS mRNA levels (<0.01). CONCLUSIONS Rosuvastatin could inhibit Hcy induced VSMCs dedifferentiation suppressing ERS, which might be regulated by mTOR-P70S6K signaling.
Actins ; metabolism ; Animals ; Calcium-Binding Proteins ; metabolism ; Cell Dedifferentiation ; drug effects ; Cells, Cultured ; Endoplasmic Reticulum Stress ; drug effects ; Heat-Shock Proteins ; metabolism ; Homocysteine ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Ribosomal Protein S6 Kinases, 70-kDa ; metabolism ; Rosuvastatin Calcium ; pharmacology ; TOR Serine-Threonine Kinases ; metabolism ; X-Box Binding Protein 1 ; metabolism

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

PURPOSE: Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. MATERIALS AND METHODS: Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-kappaB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. RESULTS: We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-kappaB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. CONCLUSION: Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-kappaB pathways. These effects were inhibited by the antioxidant rutin.
Animals ; Caspase 3/metabolism ; Cell Movement/*drug effects ; Cell Proliferation/*drug effects ; Flavonoids/*pharmacology ; Glucose/*metabolism/pharmacology ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Kinase 1 ; Male ; Mitogen-Activated Protein Kinase 3 ; Muscle, Smooth, Vascular/cytology/*drug effects/enzymology ; Myocytes, Smooth Muscle/metabolism ; NF-kappa B/metabolism ; Phosphatidylinositol 3-Kinases ; Protein Kinase Inhibitors/*pharmacology ; Rats ; Rats, Inbred OLETF ; Rats, Long-Evans ; Reactive Oxygen Species/metabolism ; Rutin/*pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo observe the effect of Pinggan Qianyang Recipe (PQR) on inhibiting angiotensin II (Ang II) induced proliferation and migration of vascular smooth muscle cells (VSMCs) and changes of DNA methylation.

METHODSVSMCs were cultured using tissue explant method, and PQR containing serum was prepared. Primarily cultured VSMCs were divided into four groups, the normal group, the model group, the folate group (folic acid intervention) , and the PQR group. The proliferation and migration of VSMCs was duplicated by Ang II. After 24-h Ang II induced culture, 40 microg/mL folic acid was added to the folate group for 48 h, while 5% PQR containing serum was added to the PQR group for 48 h. The cell growth curve of VSMCs was drawn by using Cell Counting Kit (CCK-8). The proliferative activity of VSMC was determined by MTT assay. The migration of VSMCs was measured by Millicell chamber. The general level of cytosine methylation in cell nucleus was detected via 5-mC antibodies immunofluorescence, and mRNA expression levels of DNA methyltransferase 1 (DNMT1) were measured by Real-time q-polymerase chain reaction (q-PCR).

RESULTSVSMCs were promoted by Ang II at 10(-6) mol/L for 24 h. Compared with the normal group, the proliferative activity and migration quantity of VSMCs obviously increased, and DNA methylation level obviously decreased (P < 0.05, P < 0.01). Compared with the model group, the cell growth, proliferative activity and migration quantity of VSMCs obviously decreased and the general DNA methylation level increased in the folate group and the PQR group (P < 0.05, P < 0.01). Compared with the normal group, the mRNA expression of DNMT1 decreased in the model group (P < 0.01). Compared with the model group, mRNA expression of DNMT1 in Ang II induced VSMCs was obviously enhanced in the folate group and the PQR group (P < 0.01).

CONCLUSIONSPQR could inhibit Ang II induced proliferation and migration of VSMCs, and cause high genomic DNA methylation level. Changes of DNA methylation might be associated with DNMT1 expression.

Angiotensin II ; pharmacology ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; DNA Methylation ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects

BACKGROUNDClarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2has not been fully elucidated. This study was designed to investigate if SO2inhibited VSMC proliferation in mice with hypertension induced by AngII.

METHODSThirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2groups. Mice in AngII group and AngII + SO2group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg-1·min-1. In addition, mice in AngII + SO2received intraperitoneal injections of SO2donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting.

RESULTSIn animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P-ERK induced by AngII (P < 0.05). In cell experiment, PD98059, an ERK phosphorylation inhibitor, blocked the inhibitory effect of SO2on VSMC proliferation (P < 0.05).

CONCLUSIONSERK signaling is involved in the mechanisms by which SO2inhibits VSMC proliferation in AngII-induced hypertensive mice via ERK signaling.

Angiotensin II ; pharmacology ; Animals ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hypertension ; chemically induced ; drug therapy ; Male ; Mice ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Signal Transduction ; drug effects ; Sulfur Dioxide ; therapeutic use

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

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

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

Scoparone, which is a major constituent of Artemisia capillaries, has been identified as an anticoagulant, hypolipidemic, vasorelaxant, anti-oxidant and anti-inflammatory drug, and it is used for the traditional treatment of neonatal jaundice. Therefore, we hypothesized that scoparone could suppress the proliferation of VSMCs by interfering with STAT3 signaling. We found that the proliferation of these cells was significantly attenuated by scoparone in a dose-dependent manner. Scoparone markedly reduced the serum-stimulated accumulation of cells in the S phase and concomitantly increased the proportion of cells in the G0/G1 phase, which was consistent with the reduced expression of cyclin D1, phosphorylated Rb and survivin in the VSMCs. Cell adhesion markers, such as MCP-1 and ICAM-1, were significantly reduced by scoparone. Interestingly, this compound attenuated the increase in cyclin D promoter activity by inhibiting the activities of both the WT and active forms of STAT3. Similarly, the expression of a cell proliferation marker induced by PDGF was decreased by scoparone with no change in the phosphorylation of JAK2 or Src. On the basis of the immunofluorescence staining results, STAT3 proteins phosphorylated by PDGF were predominantly localized to the nucleus and were markedly reduced in the scoparone-treated cells. In summary, scoparone blocks the accumulation of STAT3 transported from the cytosol to the nucleus, leading to the suppression of VSMC proliferation through G1 phase arrest and the inhibition of Rb phosphorylation. This activity occurs independent of the form of STAT3 and upstream of kinases, such as Jak and Src, which are correlated with abnormal vascular remodeling due to the presence of an excess of growth factors following vascular injury. These data provide convincing evidence that scoparone may be a new preventative agent for the treatment of cardiovascular diseases.
Active Transport, Cell Nucleus ; Animals ; Biomarkers ; Cell Cycle Proteins/genetics/metabolism ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Coumarins/*pharmacology ; Gene Expression Regulation/drug effects ; Hep G2 Cells ; Humans ; Muscle, Smooth, Vascular/*cytology ; Myocytes, Smooth Muscle/*metabolism ; Proto-Oncogene Proteins c-sis/metabolism ; Rats ; STAT3 Transcription Factor/genetics/*metabolism ; Signal Transduction/drug effects ; Transcription, Genetic

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