AIMThe process of vascular calcification involves various genetic alterations which may play a very important role in the vascular calcification. Vascular smooth muscle cells undoubtedly composed the main part of vascular cells, and are involved in vascular calcification. So bovine artery smooth muscle cell (BASMC) was used to investigate the gene changes during BASMC's calcification.

METHODSBovine artery smooth muscle cells cultured in vitro was induced calcified by beta-Glycerophosphate (beta-GP). Using DD-PCR technique to screening differentially expressed genes and those differentially expressed bands were reexamined by reverse Northern blot. All the ESTs were sequenced and BLAST with GenBank.

RESULTSTotal 65 cDNAs were isolated as differentially expressed genes and 40 of them were successfully reamplified. Using reverse-Northern blot, seven of these 40 cDNAs were reproducibly expressed differentially between the two cells. Three of them are new bands and have not been reported before.

CONCLUSIONThis is the first time using DD-PCR to screen differentially expressed genes of BASMC calcification. Seven related ESTs were identified relating to BASMC calcification.

Animals ; Arteriosclerosis ; genetics ; metabolism ; pathology ; Cattle ; Cells, Cultured ; Expressed Sequence Tags ; Genetic Variation ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism ; pathology ; Vascular Calcification ; genetics ; metabolism ; pathology

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

AIM AND METHODSMTT colorimetric assay, in situ hybridization and immunocytochemistry were performed to investigate the effect of endogenous and exogenous CO on the proliferation of PASMCs and the expression of PDGF-B and protooncogene bcl-2, P53 (mutant type) in PASMCs, in order to elucidate the mechanism by which CO suppressed the proliferation of PASMC in hypoxic environment.

RESULTSThe results of in situ hybridization of PDGF-B mRNA and immunocytochemical staining of PDGF-B were negative. Hypoxia could upregulate the expression of Bcl-2, mutant P53 protein in comparison with the control group (P < 0.01). Compared with the hypoxic group, the expression of Bcl-2 and mutant P53 were decreased after treated with hemin or CO, but increased after treated with hemoglobin (P < 0.01).

CONCLUSIONCO could suppress the expression of oncogene bcl-2 and mutant P53. This partially explained how CO suppressed the proliferation of PASMCs in hypoxic environment.

Animals ; Carbon Monoxide ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Muscle, Smooth, Vascular ; cytology ; metabolism ; pathology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Pulmonary Artery ; metabolism ; Rats ; Rats, Wistar

Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are the major cause of in-stent restenosis (ISR). Intervention proliferation and migration of VSMCs is an important strategy for antirestenotic therapy. Roscovitine, a second-generation cyclin-dependent kinase inhibitor, can inhibit cell cycle of multiple cell types. We studied the effects of roscovitine on cell cycle distribution, proliferation and migration of VSMCs in vitro by flow cytometry, BrdU incorporation and wound healing assay, respectively. Our results showed that roscovitine increased the proportion of G0/G1 phase cells after 12 h (69.57±3.65 vs. 92.50±1.68, P=0.000), 24 h (80.87±2.24 vs. 90.25±0.79, P=0.000) and 48 h (88.08±3.86 vs. 88.87±2.43, P=0.427) as compared with control group. Roscovitine inhibited proliferation and migration of VSMCs in a concentration-dependent way. With the increase of concentration, roscovitine showed increased capacity for growth and migration inhibition. Roscovitine (30 μmol/L) led to an almost complete VSMCs growth and migration arrest. Combined with its low toxicity and selective inhibition to ISR-VSMCs, roscovitine may be a potential drug in the treatment of vascular stenosis diseases and particularly useful in the prevention and treatment of ISR.
Animals ; Cell Cycle ; drug effects ; Cell Line ; Cell Movement ; drug effects ; Graft Occlusion, Vascular ; drug therapy ; metabolism ; pathology ; Muscle, Smooth, Vascular ; metabolism ; pathology ; Myocytes, Smooth Muscle ; metabolism ; pathology ; Protein Kinase Inhibitors ; pharmacology ; Purines ; pharmacology ; Rats

OBJECTIVETo investigate the novel hyperplasia suppressor gene (HSG) expression in vascular smooth muscle cells derived from normotensive and hypertensive patients underwent bypass surgery.

METHODSCoronary heart disease patients underwent coronary artery bypass graft (CABG) operation in BEIJING ANZHEN hospital from 4 - 9, 2006 were enrolled in this study and divided into hypertensive group (n = 28) and normotensive group (n = 26). The preoperative venous blood samples were taken for serum biochemical and vasoactive peptides measurements. Total RNA was extracted from WBC, explanted-vessels and cultured VSMCs using TRIZOL and HSG expression was determined by Semi-Quantitative RT-PCR.

RESULTSBody mass index (BMI) was significantly higher in hypertensive group compared to normotensive group (P < 0.01) while other biochemic parameters and vasoactive peptides were similar between the groups. BMI and GLU, BMI and SBP, BMI and DBP, GLU and TG, SBP and DBP were positively correlated (all P < 0.05). HSG expression in WBC, VSMCs and vessel tissue were significantly lower in hypertensive group than those in normotensive group (all P < 0.05). HSG expression in tissue was negatively correlated to BMI, SBP and DBP (all P < 0.05).

CONCLUSIONSReduced HSG expression and the negative correlation on vascular tissue HSG expression to BMI, SBP and DBP suggested a possible inhibitory role of HSG on VSMC proliferation and blood pressure.

Aged ; Body Mass Index ; Female ; Gene Expression ; Genes, Suppressor ; Humans ; Hyperplasia ; genetics ; Hypertension ; genetics ; pathology ; Male ; Middle Aged ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; metabolism

AIM AND METHODSTo determine the relationship between the nuclear envelope nucleoside triphosphatase (EC 3. 6. 1. 15, NTPase) activity and the phenotypic modulation of vascular smooth muscle cell (VSMC), the NTPase activity was detected during restenosis after de-endothelialization in vascular wall. The activities of three enzymes involved in carbohydrate and nucleic acid metabolism were also investigated by spectrophotometry.

RESULTSThe activity of NTPase increased continuously and associated with the process of intimal thickening. Western blotting showed that expression of SMalpha-actin, as the marker of contractile phenotype of VSMC, decreased continuously. Osteopontin (OPN), the marker of synthetic phenotype of VSMC, was up-regulated during the process. These suggested that intimal injury induced phenotypic modulation of VSMC. The activities of 5'-nucleotidase, adenosine deaminase and succinate dehydrogenase increased and reached their peaks on 7 days after de-endothelialization. The changes of three enzymes were associated with proliferation in VSMC.

CONCLUSIONThe efflux of mRNA and the changes of enzyme activity involved in carbohydrate or nucleic acid metabolism may be the biochemical basis in the development and progression of restenosis.

Animals ; Constriction, Pathologic ; Endothelium, Vascular ; pathology ; Female ; Hyperplasia ; enzymology ; pathology ; Male ; Muscle, Smooth, Vascular ; pathology ; Nucleoside-Triphosphatase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tunica Intima ; enzymology ; pathology

The objective of this paper was to investigate the contribution of mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) and mitochondrial membrane potential (Deltapsim) to the distribution of cytochrome C in human pulmonary arterial smooth muscle cells (HPASMCs) and to the proliferation of HPASMCs induced by hypoxia. HPASMCs were divided into 6 groups, as following: (1) control group: cultured under normoxia; (2) diazoxide group: cultured in normoxia with diazoxide, an opener of mitoK(ATP); (3) 5-HD group: cultured in normoxia with 5-hydroxydecanoate (5-HD), an antagonist of mitoK(ATP); (4) 24-hour hypoxia group: cultured in hypoxia for 24 h; (5) 24-hour hypoxia + diazoxide group: cultured in hypoxia with diazoxide for 24 h; (6) 24-hour hypoxia + 5-HD group: cultured in hypoxia with 5-HD for 24 h. The relative changes in mitochondrial potential were tested with rhodamine fluorescence (R-123) technique. Western blot was used to detect the expression of cytochrome C protein in cell plasma and mitochondria,respectively. The expression of cell caspase-9 protein was determined with Western blot. The proliferation of HPASMCs was examined by cell cycle analysis and MTT colorimetric assay. The results were as following: after exposure to diazoxide for 24 h, the intensity of R-123 fluorescence in normoxic HPASMCs was significantly increased compared with that in the control group (P<0.05), but there was no significant change of the intensity of R-123 fluorescence after the HPASMCs had been exposed to 5-HD for 24 h; 24-hour hypoxia or 24-hour hypoxia + diazoxide could markedly increase the intensity of R-123 fluorescence in HPASMCs compared with normoxia (P<0.05), and the change was more significant in 24-hour hypoxia + diazoxide group than that in 24-hour hypoxia group (P<0.05); 5-HD could weaken the effect of 24-hour hypoxia on the intensity of R-123 fluorescence. After exposure to diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein was significantly decreased compared with that in the control group (P<0.05), and the expression of caspase-9 protein was significantly decreased compared with that in the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased compared with those in the control group (P<0.05). But there were no significant changes in these tests after HPASMCs had been exposed to 5-HD for 24 h (P>0.05). After exposure to hypoxia or hypoxia + diazoxide for 24 h, the ratio of the expression of cytosolic cytochrome C protein to that of mitochondrial cytochrome C protein and the expression of caspase-9 protein were significantly decreased compared with those in the control group (P<0.05). The percentage of S phase and A value of MTT were significantly increased compared with those in the control group (P<0.05). These changes were more significant in 24-hour hypoxia + diazoxide group than those in 24-hour hypoxia group (P<0.05). 5-HD could weaken the effect of hypoxia on the changes of the distribution of cytochrome C, the expression of caspase-9 in HPASMCs and the proliferation of HPASMCs induced by hypoxia (P<0.05). All these results suggest that the opening of mitoK(ATP) followed by a depolarization of Deltapsim induced by hypoxia might contribute to the inhibition of the release of cytochrome C from mitochondria to plasma in HPASMCs. This might be a mechanism of the development of hypoxic pulmonary hypertension. The signal transduction pathway of mitochondria might play an important role in the relationship between Deltapsim and apoptosis of HPASMCs.
Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Cytochromes c ; metabolism ; Humans ; Hypertension, Pulmonary ; physiopathology ; Membrane Potential, Mitochondrial ; physiology ; Muscle, Smooth, Vascular ; cytology ; pathology ; Myocytes, Smooth Muscle ; metabolism ; pathology ; Potassium Channels ; physiology ; Pulmonary Artery ; cytology ; metabolism ; pathology ; Signal Transduction

OBJECTIVETo investigate whether extracellular signal-regulated kinase (ERK1/2) was involved in changes of vascular smooth muscle cell (VSMC) under hypertension.

METHODSTwo-kidney one clip Wistar hypertensive rats (WHR) were sacrificed and their right kidneys were harvested 4 weeks after surgery. The spontaneously hypertensive rats (SHR) were divided into 4, 8, and 16 weeks old groups (SHR4w, SHR8w, and SHR16w), respectively. The control group were sham operated age-matched Wistar rats. Immunohistochemical technique and Western blotting were applied to study ERK1/2 protein expression in VSMC of the renal vascular trees in WHR, SHR, and control rats.

RESULTSBlood pressure in two-kidney one clip WHR obviously increased at one week after surgery, and reached to 198. 00 +/- 33. 00 mm Hg at the end of experiment, significantly higher than that in the control rats (P < 0.01). Blood pressure in SHR4w (108.00 +/- 11.25 mm Hg) was similar to that in the controls. However, it rose to 122.25 +/- 21.75 mm Hg in SHR8w, and even up to 201.75 +/- 18.00 mm Hg in SHR16w, which were significantly higher than that of both the SHR4w and the controls (P < 0.01). The rate and degree of glomerular fibrosis in WHR were significantly higher than controls (P < 0.05). Hyaline degeneration of the afferent arterioles was found in WHR. In contrast, either fibrosis of glomerulus or hyaline degeneration of the arterioles or protein casts was not observed in SHR4w, SHR8w, and SHR16w. Immunohistochemical staining results showed expression of ERK1 was similar to that of ERK2. The positive rates of ERK2 staining in VSMC of afferent arterioles, interlobular, interlobar, and arcuate arteries in two-kidney one clip WHR were significantly higher (7.09% +/- 1.75%, 14.57% +/- 4.58%, 29.44% +/- 7.35%, and 13.63% +/- 3. 85%, respectively) than that of the controls(P < 0.01). The positive rates of ERK2 staining in VSMC at afferent arterioles, interlobular, interlobar, and arcuate arteries in SHR16w were significantly higher (12.09% +/- 1.40%, 24.17% +/- 6.92%, 32.44% +/- 4.05%, and 18.61% +/- 3.35%, respectively) than that of the controls (P < 0.01), too. The expression of ERK1/2 protein of kidney in WHR and SHR16w was significantly higher than that in the controls by Western blotting assay (P < 0.01).

CONCLUSIONExtracellular signal transduction system are highly expressed in kidney VSMC of two-kidney one clip WHR and SHR. Phospho-ERKI/2 may play an important role in VSMC hypertrophy and hyperplasia under hypertension.

Animals ; Arterioles ; enzymology ; Fibrosis ; Hypertension ; metabolism ; pathology ; Kidney Glomerulus ; blood supply ; pathology ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; enzymology ; Rats ; Rats, Inbred SHR ; Rats, Wistar

Animals ; Cell Differentiation ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Endothelial Cells ; pathology ; physiology ; Humans ; Muscle, Smooth, Vascular ; pathology ; physiology ; Receptors, CXCR4 ; metabolism ; Stem Cells ; metabolism ; pathology ; physiology ; Vascular Diseases ; metabolism ; pathology ; physiopathology

OBJECTIVE: To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC). METHODS: Mouse models of VC were established in ApoE-deficient (ApoE^-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with N^ε-carboxymethyl-lysine for 16 weeks. ApoE^-/- mice (control group), ApoE^-/- diabetic mice (VC group), ApoE^-/- diabetic mice with BI-1 overexpression (VC + BI-1^TG group), and ApoE^-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1^TG+OPA1^-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L β-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining. RESULTS: ApoE^-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007). CONCLUSION BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.
Animals ; Apolipoproteins E/metabolism* ; Calcium/metabolism* ; Caspase 3/metabolism* ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Diabetes Mellitus, Experimental/pathology* ; GTP Phosphohydrolases/metabolism* ; Membrane Proteins/metabolism* ; Mice ; Mice, Knockout ; Muscle, Smooth, Vascular/pathology* ; Myocytes, Smooth Muscle/pathology* ; Optic Atrophy, Autosomal Dominant/pathology* ; Osteogenesis ; Vascular Calcification/pathology* ; bcl-2-Associated X Protein/metabolism*