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

Atherosclerosis is an important pathological basis for coronary artery disease. ANRIL is an antisense non-coding RNA located in Chr9p21 locus, which was identified as the most significant risk locus associated with atherosclerosis. ANRIL can produce multiple transcripts including linear and circular transcripts after various transcript splicing. It has been illustrated that ANRIL plays important roles in the pathology of atherosclerosis by regulating the proliferation and apoptosis of vascular cells. Linear ANRIL can regulate the proliferation of vascular smooth muscle cells (VSMCs) in plaques by chromatin modification, as well as influence the proliferation and the apoptosis of macrophages in post transcription; circular ANRIL can affect the proliferation and apoptosis of VSMCs by chromatin modification as well as interfering with rRNA maturation. In this review, we describe the ANRIL evolution, different transcripts characteristics, and their roles in the proliferation and apoptosis of vascular cells to participate in the process of atherosclerosis, for further understanding the pathogenesis of atherosclerosis and finding potential targets for diagnosis and treatment of atherosclerosis.
Apoptosis ; genetics ; Atherosclerosis ; genetics ; Cell Proliferation ; genetics ; Humans ; Myocytes, Smooth Muscle ; pathology ; RNA, Long Noncoding ; metabolism

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

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

This study investigated the potential role of ERK1/2-cyclinE1 signaling pathway in rat pulmonary artery smooth muscle cells (rPASMCs) proliferation and pulmonary vascular remodeling induced by cigarette smoke exposure. A total of 24 male Wistar rats were randomly divided into 4 groups: control group (C group), S-1M, S-3M and S-6M groups (animals in the groups were exposed to smoke for 1, 3, and 6 months, respectively). HE staining and anti-α-smooth muscle actin antibody staining were performed to observe the degree of pulmonary vascular remodeling. Immunohistochemistry and Western blotting were performed to evaluate ERK1/2 and cyclinE1 expression in pulmonary vessels. Primary cultured rat pulmonary artery smooth muscle cells (rPASMCs) were exposed to cigarette smoke extract (CSE). ERK inhibitor (PD98059) and cyclinE1 siRNA were used to verify the role of ERK1/2 and cyclinE1 in CSE-induced rPASMCs proliferation. Cell proliferation was assessed by cell counting and 5-bromo-2-deoxyuridine (BrdU) incorporation. Our results showed that abnormal pulmonary vascular remodeling was found in cigarette smoked rats. Compared to C group, activated ERK1/2 and cyclinE1 expression was significantly increased in smoke-exposure groups. This up-regulated expression was positively correlated with the severity of pulmonary vascular remodeling, and there was positive correlation between the expression of ERK1/2 and cyclinE1. PD98059 and cyclinE1 siRNA inhibited the proliferation of rPASMCs. The expression of cyclinE1 could be down-regulated by PD98059. Our data demonstrated that increased expression of ERK1/2 and cyclinE1 might be involved in the pathogenesis of abnormal rPASMCs proliferation and rat pulmonary vascular remodelling induced by cigarette smoke exposure.
Animals ; Cells, Cultured ; Cyclins ; metabolism ; MAP Kinase Signaling System ; Male ; Myocytes, Smooth Muscle ; metabolism ; pathology ; Pulmonary Artery ; metabolism ; pathology ; Rats ; Rats, Wistar ; Smoking ; metabolism ; pathology ; Up-Regulation

OBJECTIVETo observe the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) in the unstable plaque of patients with acute coronary syndrome (ACS), and the impact of leukotriene B4 (LTB4) on the EMMPRIN expression in macrophages.

METHODSThe EMMPRIN expression was detected by immunohistochemistry in 11 unstable plaques from patients with ACS. Protein expression of EMMPRIN was evaluated by Western blot on macrophages differentiated from THP-1 which were stimulated with LTB4 in the absence or presence of LTB4 antagonist U75302. There are 8 study groups: 1-THP-1, 2-8-the macrophages derived from THP-1, 2-6-macrophages were stimulated by LTB4 (0, 10(-10), 10(-9), 10(-8) and 10(-7) mol/L) for 24 h, 7-8-the macrophages were pretreated by 10(-6) mol/L or 10(-7) mol/L U75302 2 h before the LTB4 (10(-7) mol/L) stimulation.

RESULTSAbundant EMMPRIN expression was detected in macrophages and smooth muscle cells of unstable plaques from ACS patients. As to the THP-1 derived macrophages, EMMPRIN expression was significantly upregulated in a concentration-dependent manner in LTB4 stimulated groups, which was significantly higher in group 3-6 than in the THP-1 group (group 1) and macrophages group (group 2) (all P < 0.05) and pretreatment with U75302 significantly reduced the LTB4 induced upregulation of EMMPRIN in a dose-dependent manner (P < 0.05).

CONCLUSIONEMMPRIN expression is enhanced in macrophages and smooth muscle cells on unstable coronary artery plaques from ACS patients. LTB4 could stimulate EMMPRIN expression on THP-1 derived macrophages suggesting that LTB4 and EMMPRIN might be both involved in the formation and progression of unstable plaques, future studies are warranted to explore if LTB4 and EMMPRIN antagonists are effective or not for treating patients with ACS.

Acute Coronary Syndrome ; metabolism ; pathology ; Basigin ; metabolism ; Cell Line ; Humans ; Leukotriene B4 ; metabolism ; pharmacology ; Macrophages ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Plaque, Atherosclerotic ; metabolism

Primary leiomyosarcoma of the nipple-areola complex is extremely rare. Less than ten such cases have been reported in English literature so far. Herein we describe a 52-year-old female presenting with a 1.5 cmx1.1 cmx0.7 cm nodular lesion over her left nipple, and leiomyosarcoma was proved by pathological examination of the excised specimen. Positron emitted tomogram (PET) revealed no abnormal signal other than the primary site. Microscopically, this poorly circumscribed tumor was composed of interlacing bundles of smooth muscle cells with bizarre and pleomorphic nuclei, as well as prominent nucleoli. Its mitotic count was up to 7 mitoses per 10 high power fields (HPF). Immunohistochemical study of tumor cells revealed positive stain for alpha-smooth muscle actin and vimentin; and negative for cytokeratin, CD34 and S-100. Left simple mastectomy was undertaken and no residual mass lesion was noted on the resected specimen. Related literatures about the diagnosis and treatment for breast leiomyosarcoma will be presented here.
Antigens, CD34 ; biosynthesis ; Breast ; pathology ; Breast Neoplasms ; diagnosis ; pathology ; Cell Nucleus ; metabolism ; Female ; Humans ; Immunohistochemistry ; methods ; Leiomyosarcoma ; diagnosis ; pathology ; Mastectomy ; Middle Aged ; Myocytes, Smooth Muscle ; pathology ; Nipples ; pathology ; S100 Proteins ; biosynthesis ; Smooth Muscle Tumor ; diagnosis ; pathology ; Treatment Outcome

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

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