Human Muscle Cell Proliferation Research Articles

EPO-receptor is present in mouse C2C12 and human primary skeletal muscle cells but EPO does not influence myogenesis

The role and regulation of the pleiotropic cytokine erythropoietin (EPO) in skeletal muscle are controversial. EPO exerts its effects by binding its specific receptor (EPO‐R), which activates intracellular signaling and gene transcription in response to internal and external stress signals. EPO is suggested to play a direct role in myogenesis via the EPO‐R, but several studies have questioned the effect of EPO treatment in muscle in vitro and in vivo. The lack of certainty surrounding the use of nonspecific EPO‐R antibodies contributes to the ambiguity of the field. Our study demonstrates that the EPO‐R gene and protein are expressed at each stage of mouse C2C12 and human skeletal muscle cell proliferation and differentiation and validates a specific antibody for the detection of the EPO‐R protein. However, in our experimental conditions, EPO treatment had no effect on mouse C2C12 and human muscle cell proliferation, differentiation, protein synthesis or EPO‐R expression. While an increase in Akt and MAPK phosphorylation was observed, we demonstrate that this effect resulted from the stress caused by changing medium and not from EPO treatment. We therefore suggest that skeletal muscle EPO‐R might be present in a nonfunctional form, or too lowly expressed to play a role in muscle cell function.

Cyclooxygenase-2 expression in atherosclerosis tissues and the effect of celecoxib on proliferation and apoptosis of human vascular smooth muscle cells

Objective To investigate the expression of cyclooxygenase-2 (COX-2) in atherosclerosis tissues and the effect of selective COX-2 inhibitor celeboxib on proliferation and apoptosis of human vascular smooth muscle cells (VSMCs).Methods Immunohistochemistry for COX-2 was performed on 22 cases of formalin-fixed,paraffin-embedded human atherosclerosis tissues.Cell growth rate was assessed by methyl thiazol tetrazolium (MTT) assay,and apoptosis was examined by flow cytometry.Results COX-2 protein was detected in 21 of 22 (95％) human atherosclerosis tissues.COX-2 was decreased in VSMCs when treated with celecoxib in vitro.After cells were treated with increasing concentrations of celecoxib (0,10,20,40 and 80 μmol/L) for 24 h,the cell growth rate was 100.00％,90.06％,81.38％,75.41％ and 68.90％ respectively,and the early apoptosis ratio was 0.02％,0.62％,0.92％,1.17％ and 1.63％ respectively.Conclusion COX-2 is highly expressed in human atherosclerosis tissues,and the celecoxib inhibited proliferation and induced apoptosis of VSMCs in a dose-dependent manner. Key words: Cyclooxygenase-2 ; Celecoxib ; Atherosclerosis; Vascular smooth muscle cells; Proliferation ; Apoptosis

Wood smoke condensate had weak proliferation and strong necrotic effects on human airway smooth muscle cells

To investigate the effect of wood smoke condensate (WSC) on proliferation and necrosis of human airway smooth muscle cells (HASMCs). Primary cultured HASMCs between passage 2 and 8 were divided into 3 groups: a control group, a WSC group and a cigarette smoke condensate (CSC) group. The viability of cells was examined by the CCK8 assays. The ratio of cellular proliferative stage (S phase) and cell cycle index were examined by fluorescent-labeled thymidine analogue uptake assays and flow cytometry. The expression of cyclin D1 was detected by quantitative reverse transcriptase polymerase chain reaction (Q-PCR) and Western blot. Cell apoptosis and necrosis were observed by the annexin-V and PI staining. Statistical analysis was performed by using the One-way ANOVA and LSD-t test. Cell viability reached peak at WSC 1 mg/L[(126 ± 12)%] and at CSC 10 mg/L exposure level [(142 ± 11) %] respectively. While at WSC 10 mg/L and CSC 60 mg/L exposure levels, cell viability decreased significantly to 86% and 76%, respectively, as compared with that of the blank control group[(100 ± 0)%] (q = 3.63- 9.33, P < 0.05). In the WSC 1 mg/L group, the cell proliferation ratio and the expression of cyclin D1 protein were (124 ± 20)% and 1.31 ± 0.64, respectively, the differences being significant as compared with the blank control group [(100 ± 0)%, 1.0 ± 0.0] (q = 5.85, 5.91, P < 0.05), while the expression of cyclin D1 mRNA and the percentage of S+G2M phase were 1.18 ± 0.21 and (103 ± 4)%, respectively, not significantly different as compared to the control group [(100 ± 0)%, 1.0 ± 0.0], (q = 1.16, 2.05, P > 0.05). In the CSC 10 mg/L group, the above-mentioned values were (204 ± 45)%, 1.80 ± 0.25, (140 ± 6)%, 1.48 ± 0.2, respectively, which were significantly higher than those in the blank control group (q = 5.38-16.51, P < 0.05) and in the WSC group (q = 3.33-15.35, P < 0.05). However, when HASMCs were exposed to WSC 10 mg/L, the cell death ratio was (13.39 ± 0.15)%, higher than that of the blank control group [(1.57 ± 0.41)%] and the CSC group [(6.61 ± 1.91)%] (q = 18.03, 10.34, P < 0.05). Apoptosis ratio in the CSC 40 mg/L group was [(61.8 ± 10.6)%], higher than that of the blank control group [(0.0 ± 0.0)%] and the WSC group [(1.7 ± 0.4)%] (q = 17.44, 16.95, P < 0.05). Exposure to WSC caused a weak proliferation of HASMCs, but resulted in cell necrosis instead of apoptosis at high doses. There was a slight difference in cell effects between the WSC group and the CSC group.

The effect of cigarette smoke extract on the proliferation of human airway smooth muscle cells sensitized by serum from bronchial asthmatic patients

To study the effect of cigarette smoke extract (CSE) on the proliferation of human airway smooth muscle cells (HASMCs) sensitized by serum from asthmatic patients and the underlying mechanisms. HASMCs were cultured from primary generation. Cells between passage 4 and 8 were used in the study. HASMCs were sensitized by 10% serum from asthmatic patients and were divided into an asthmatic serum group, an asthmatic serum + CSE group, an asthmatic serum + GW8510 (inhibitor of cyclin-dependent kinase-4) group and an asthmatic serum + CSE + GW8510 group. Non-asthmatic human serum treated HASMCs served as the control. The proliferation of HASMCs was examined by cell cycle analysis, MTT colorimetric assay and [(3)H] thymidine incorporation. The expression of cyclinD(1) was detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The percentage of S + G(2)/M phase, the absorbance (A) value and the DNA synthesis value in asthmatic serum group were significantly increased compared with those of the control group (q = 6.25, 5.61, 6.82, respectively, all P < 0.01). The percentage of S + G(2)/M phase, the absorbance (A) value and DNA synthesis value in the asthmatic serum group were (21.4 ± 1.1)%, 0.392 ± 0.124 and 2669 ± 138, respectively. Their value in the asthmatic serum + CSE group were (33.3 ± 1.3)%, 0.612 ± 0.201 and 3552 ± 303, respectively, which were significantly increased compared with those of the asthmatic serum group (q = 5.67, 6.32, 5.56, respectively, all P < 0.01). Their value in the asthmatic serum + GW8510 group were (14.7 ± 1.4)%, 0.301 ± 0.097 and 1812 ± 109, respectively, which were significantly decreased compared with those of the asthmatic serum group (q = 6.02, 5.53, 5.79, respectively, all P < 0.01). The ratios of A value of cyclinD(1) mRNA and the expression of cyclinD(1) protein in the asthmatic serum group were 0.291 ± 0.112 and 0.186 ± 0.002, respectively. The ratios of A value in the asthmatic serum + CSE group were 0.521 ± 0.102 and 0.312 ± 0.002, respectively, which were significantly increased compared with those of the asthmatic serum group (q = 12.09, 9.26, respectively, all P < 0.01). The ratios of A value in the asthmatic serum + GW8510 group were 0.223 ± 0.038 and 0.150 ± 0.002, respectively, which were significantly decreased compared with those of the asthmatic serum group (q = 6.86, 5.60, respectively, all P < 0.01). HASMCs sensitized by serum from asthmatic patients showed accelerated proliferation after intervention by CSE, with increased expression of cyclinD(1). CSE may increase the proliferation of HASMCs sensitized by serum from asthmatic patients via regulating cyclinD expression.

Gene transfer of human endothelial nitric oxide synthase inhibits proliferation of human vascular smooth muscle cells and the mechanism

Objectiye To invesigate the effect of human endothelial nitric oxide synthase (heNOS ) gene transfer on the proliferation of in vitro cultured human vascular smooth muscle cells (HVSMCs)and the mechanism. Methods The HVSMCs were transfeced with multiplicity of infection (MOI) of 50,150,250, 300,450. cGMP was measured by radioimmunoassay in HVSMCs. The expression levels of p21 and p27 were detected by Western blotting. Cell cycle and apoptosis were assayed by flow cytometry. Results (1) At 120th h, the A570values were respectively 1.410±0.081, 1.357 ±-0. 150, 1.303±0.311,0. 995 ±0. 248 and 0. 731 ±0. 101 at MOI of 50, 150,250,300,450. The proliferation of HVSMCs was significantly and stably inhibited with MOI 300 of AdCMV-heNOS; (2) Af72nd h after the gene transfer,cGMP levels were increased in heNOS-transduced ( 12. 89 ±2. 06) compared to LacZ- (8.36 ±0. 34) and non-tranduced (7. 91 ± 0. 39) cells ( P ＜ 0. 01 ); (3) At 48th h after the gene transfer, the expression of heNOS in HVSMCs up-regulated p21 and p27 (P＜0. 05); (4) After 48 h tronsfected with sersuln-depriveol and for 24 h serum stimulation, the cell cycle was significantly arrested in G0/G1 phase in heNOStransduced group, and G0/G1 was respectively ( 64. 23 ± 1.58 ) %, ( 64. 96 ± 1.36 ) %, ( 76. 03 ±2. 27 ) % in non-, LacZ- and heNOS-transduced cells; ( 5 ) At first and third day afer the gene transfer,there was no increase in apoptosis at the first day in all transduced cells ( P ＞ 0. 05 ). Conclusion Adenovirus-mediated heNOS gene transfer to HVSMCs inhibits cell proliferation via up-regulation of p21 and p27 resulting in a delay in cell progression not apoptosis. Key words: Endothelial nitric oxide synthase; Cell cycle; p27

Construction of the recombinant human endothelial nitric oxide synthase gene adenovirus and its expression in human vascular smooth muscle cells

Objective To construct recombinant adenovirus vector carrying the human endothelial nitric oxide synthase gene (heNOS) and detect its expression in human vascular smooth muscle cells in vitro. Methods heNOS was cloned by polymerase chain reaction (PCR) using plasmid PMSCV-heNOS,heNOS cDNA was inserted into adenovirus shuttle plasmid-PSUCMV to generate a recombinant plasmid PSUCMV-heNOS, transfected into E. coli DH5α, and positive clones were correctly constructed and confirmed by DNA sequencing analysis and restriction endonucleas analysis. The plasmid PSUCMV-heNOS was co-transfected with pBHGE3 in 293 cells to generate replication-defective recombinant adenovirus vector carrying heNOS gene. Human vascular smooth muscle cells were transfected by Ad-heNOS. The effect on the proliferation of human vascular smooth muscle cells was investigated by MTT assay. The expression of heNOS was detected by immunohistochemistry staining and Western blotting. Results ( 1 ) PCR, restrictive digestion, and sequencing revealed the successful construction of the recombinant adenovirus vector carrying heNOS gene and its titer was 3.5 × 1010 PFU/ml; (2) It was confirmed that the human vascular smooth muscle cells cultivated in vitro were characterized by morphology and immunohistochemistry strain of α-actin; (3) At 120 h, the A570 values in multiplicity of infection (MOI) 50, 150,250,300,450 were respectively 1. 410 ±0. 081, 1. 357 ±0. 150, 1.303 ±0. 311,0. 995 ±0. 248 and 0. 731 ±0. 101. The proliferation of human vascular smooth muscle cells was significantly and stably inhibited with MOI 300 of AdCMVheNOS ( P ＜ 0. 01 ). Immunostaining and Western blotting with heNOS in engineered cells were positive.Conclusion The successful construction of AdCMV-heNOS and expression may provide an opportunity for the gene therapy of vascular intimal hyperplasia. Key words: Nitric oxide synthase; Vascular smooth muscle cells; Gene transfection