Animals ; Cell Proliferation ; drug effects ; Female ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tamoxifen ; analogs & derivatives ; pharmacology

OBJECTIVETo study the action mechanism of tetramethylpyrazine (TMP) on the proliferation of vascular smooth muscle cells (VSMCs), thus providing experimental evidence for Chinese medicine to effectively prevent restenosis.

METHODSRats' thoracic aorta VSMCs in vitro cultured (cell line A7r5) were divided into five groups, i.e., the negative control group, the angiotensin II (Ang II, 10(-6) mol/L) group, the low dose TMP (20 micromol/L) plus Ang II group, the middle dose TMP (40 micromol/L) plus Ang II group, the high dose TMP (80 micromol/L) plus Ang II group. The proliferation ratio was detected by MTT. Gene and protein expressions of Wnt4, Dvl-1, beta-catenin, CyclinD1, and collagen I and III were detected with real-time fluorescent quantitative PCR and Western blot respectively.

RESULTSCompared with the negative control group, the proliferation ratio of VSMCs obviously increased in the Ang II group (P < 0.05). Compared with the Ang II group, the proliferation ratio of VSMCs obviously decreased in the middle dose TMP plus Ang II group and the high dose TMP plus Ang II group (P < 0.05). Compared with the negative control group, gene and protein expressions of Wnt4, Dvl-1, beta-catenin, CyclinD1, Col I, and Col III were obviously up-regulated in the Ang II group (P < 0.05). Compared with the Ang II group, mRNA and protein expressions of Wnt4, Dvl-1, beta-catenin, CyclinD1, Col I, and Col III were obviously down-regulated in the middle dose TMP plus Ang II group and the high dose TMP plus Ang II group (P < 0.05). The aforesaid indices were dose-dependent in the low, middle, and high dose TMP plus Ang II groups.

CONCLUSIONTMP inhibited Ang II induced proliferation and collagen secretion of VSMCs through down-regulating Wnt signal pathway.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen ; biosynthesis ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Pyrazines ; pharmacology ; RNA, Messenger ; genetics ; Rats

AIMAccumulated evidence suggest that the development of vascular calcification is similar to osteogenesis. Here we want to elucidate the effect of the common used osteo-regulatory factor 1,25(OH)2D3 on vascular calcification.

METHODS AND RESULTSAdding 10(-9) mol/L to the culture media 1,25(OH)2D3 time dependently increased the calcium deposition on the in vitro calcification of bovine vascular smooth muscle cells (BVSMCs) induced by beta-GP. It also increased cellular alkaline phosphatase activity by 301.1% during the calcified process. Osteocalcin, one of the osteogenic specific metric proteins, was dramatically elevated by 58.3% during the calcified processes, which indicate the transformation of BVSMCs to osteoblastic cell. 1,25(OH)2D3 had no such effect on non-calcified BVSMCs.

CONCLUSIONThese data suggest that 1,25(OH)2D3 exerts a stimulatory effect on vascular calcification through increasing the synthesis of ALP. This effect shares the same character as osteoblast cells. This effect is limited to the calcified prone vascular cell.

Animals ; Calcitriol ; metabolism ; Cattle ; Cells, Cultured ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteocalcin ; metabolism ; Vascular Calcification ; metabolism ; pathology ; Vitamin D ; analogs & derivatives ; pharmacology

Animals ; Cells, Cultured ; Interleukin-1 ; pharmacology ; Matrix Metalloproteinase 1 ; secretion ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Platelet-Derived Growth Factor ; pharmacology ; Rabbits

OBJECTIVETo explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).

METHODSVSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.

RESULTSThe resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).

CONCLUSIONTestosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.

Animals ; Calcium ; metabolism ; Cells, Cultured ; Dinoprost ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism ; physiology

OBJECTIVETo investigate the effects of 17β-estrogen on expressions of C-reactive protein (CRP) and its mRNA in vascular smooth muscle cells(VSMCs).

METHODSImmunocytochemistry was used to detect CRP level in normal VSMCs. The expressions of C-reactive protein and p-ERK1/2 in Ang-II-stimulated VSMCs were evaluated with Western blot. C-reactive protein mRNA was examined with RT-PCR.

RESULTS17β-estrogen had no effect on cell morphology and C-reactive protein expression in normal VSMCs; however, C-reactive protein and mRNA, as well as p-ERK1/2 were decreased in Ang-II-stimulated VSMCs after 17β-estrogen treatment in a concentration-dependent manner.

CONCLUSION17β-estrogen may inhibit the expression of C-reactive protein and its mRNA in Ang-II-stimulated VSMCs via ERK1/2 signal transduction pathway in a concentration-dependent way.

Animals ; C-Reactive Protein ; genetics ; metabolism ; Cells, Cultured ; Estrogens ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect of transforming growth factor beta (TGF-beta) on ectomesenchymal stem cells differentiating to smooth muscle cells.

METHODS60 pmol/L TGF-beta was added to the ectomesenchymal stem cells of embryonic facial processes. Immunohistochemistry assay and image analysis were used to value the expression extent of a smooth muscle actin (alpha-SMA) and quantitative RT-PCR was used to value the quantity of alpha-SMA.

RESULTS2 days later, about 95% cells in TGF-beta group and 65% cells in control group without differentiation inhibitor expressed alpha-SMA. Expression of alpha-SMA in TGF-beta group was stronger than that of control group after one and two days. Quantitative RT-PCR showed the quantity of alpha-SMA mRNA in treated group cells was more than that of in control group.

CONCLUSIONQuantity of alpha-SMA in TGF-beta group is more than that of spontaneous differentiation group. TGF-beta has positive effect on ectomesenchymal stem cells differentiating to smooth muscle cells.

Actins ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Transforming Growth Factor beta ; pharmacology

AIMTo explore the effects of sinomenine(Sin) on intracellular free calcium ([Ca2+]i) and the activity of PKC (protein kinase C) of the cultured aortic vascular smooth muscle cells (VSMC) during ischemia and hypoxia.

METHODSThe effect of Sin on changes in [Ca2+]i were determined in cultured rabbit VSMC after exposure to high K+, norepinephrine (NE) and caffeine (Caf). Fluorescent Ca2+ -indicater fura-2/AM was used. The effects of Sin were compared with that of verapamil (Ver). The hypoxia model was made, then the activity of PKC was measured by y scintillation counting instrument.

RESULTSSin (10 x 10(-6) mol x L(-1), 3 x 10(-5) mol x L(-1) 10(-4) mol x L(-1)) inhibited the elevation of [Ca2+]i induced by high K+ -depolarization in a concentration dependent manner. In addition, Sin inhibited the elevation of [Ca2+]i induced by NE in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, Sin (3 x 10(-5) mol.L(-1)) also had no blocking effect on the NE-induced [Ca2+]i increase. It was found that the activity of PKC treated with Sin in VSMC cytoplasm and cell membrane in normal condition increased, the activity of PKC in cytoplasm in ischemia and hypoxia situation increased, but the activity of PKC in cell membrane decreased. When VSMC was treated with Sin, the activity of PKC in cytoplasm decreased and that of cell membrane increased.

CONCLUSIONThe results suggest that Sin might decrease the[Ca2+] i of VSMC by blocking both VDC and ROC, could regulate the PKC activities induced by ischemia and hypoxia.

Animals ; Aorta ; cytology ; drug effects ; Calcium ; metabolism ; Cell Hypoxia ; Cells, Cultured ; Cytoplasm ; metabolism ; Morphinans ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; physiology ; Protein Kinase C ; metabolism ; Rabbits

OBJECTIVETo investigate the effect of allicin in inhibiting insulin-induced vascular smooth muscle cell (VSMC) proliferation and migration.

METHODThe tissue explant method was adopted to culture rat's aVSMCs, and the immunofluorescence method was used to identify α-SMA. Cells from the third to fifth generations were selected in the experiment The insulin-induced VSMC model was established. The experiment was carried out in five groups: the control group, the insulin group, the allicin group, the ERK inhibitors PD98059 group (20 μmol · L(-1)) and the PD98059 + allicin group. VSMCs' proliferation was determined by CCK8 colorimetric method, while its migration was detected by cell counting; The western blotting was used to detect total ERK, Phospho-ERK, PCNA protein's expression.

RESULTPrimary cultured VSMCs grew well in the spindle shape under the lightmicroscope, with peak and valley. α-SMA immunofluorescence results showed that the cultured cells had typical VSMCs' features. Insulin could stimulate VSMCs' proliferation and migration, with the best effect at the concentration of 100 nmol · L(-1). The pretreatment with allicin could significantly inhibit VSMCs' proliferation and migration induced by insulin in a dose-dependent manner. The pretreatment with PD98059 and allicin + PD98059 could inhibit VSMCs' proliferation and migration induced by insulin remarkably as well. Insulin could significantly accelerate VSMCs' expression of such proteins as p-ERK, PCNA. Contrarily, allicin could notably inhibit VSMCs' expression of such proteins as p-ERK, PCNA in a dose-dependent manner.

CONCLUSIONAllicin could significantly inhibit VSMCs' proliferation and migration induced by insulin, which may be related to the inhibition of the activation of ERK signal path.

Animals ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Insulin ; metabolism ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; cytology ; drug effects ; Plant Extracts ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfinic Acids ; pharmacology

OBJECTIVE: To determine the effect of human corticotrophin-releasing hormone (CRH) on the expression of connexin-43 phosphate (P-Cx43) in human myometrial smooth muscle cells (SMCs) and the function of cell gap junction intercellular communication in SMCs. METHODS: Human non-conceive myometial SMCs were cultured with different concentrations of CRH (0, 5.85, 58.5, 585 and 5850 pmol/L). Western blot was used to test P-Cx43 and Cx43 non-phosphate (NP-Cx43) of protein expression. Cell scratch was used to test cell gap junction intercellular communication opening status in human myometrial SMCs. RESULTS: Compared with the control group, the expression of P-Cx43 was higher in the CRH groups (P<0.01), and was concentration-dependent. There was no significant difference in NPCx43 between the control group and the CRH groups (P>0.05). The transmission of cell layers in the CRH groups was higher than that in the control group (P<0.01), and as the concentration of CRH increased, the time was concentration-dependent (P<0.01). CONCLUSION CRH can enhance the expression of P-Cx43 and the function of gap junction intercellular communication in the primary cultured myometrial SMCs.
Cell Communication ; drug effects ; Cells, Cultured ; Connexin 43 ; metabolism ; Corticotropin-Releasing Hormone ; pharmacology ; Female ; Gap Junctions ; drug effects ; Humans ; Myocytes, Smooth Muscle ; metabolism ; Myometrium ; cytology ; Phosphorylation ; drug effects