Expression of tropomyosin 2 in aortic dissection tissue / 中华心血管病杂志

Objective: To investigate the expression pattern of tropomyosin 2(TPM2) in aorta of patients with aortic dissection and explore its clinical implication. Methods: Thirteen cases with acute type A aortic dissection(TAAD) diagnosed by transabdominal aortic angiography from 2015 in Tongji Hospital were included. During the operation, the aortic wall tissues of these patients were collected. Ten patients with heart transplantation were selected as control group, and normal aortic wall tissues were taken. The hematoxylin-eosin (HE) and Verhoeff's Van Gieson (EVG) staining were performed to observe the morphological changes of aorta. The mRNA expression level of TPM2 was measured by real-time fluorescent quantitative-PCR, and the protein levels of TPM2 were detected by Western blot and immunohistochemical staining. Image The J software was used to collect the optical density values of each point on the image, obtain the integrated optical density(IOD) value, and calculate the average density(%, IOD/area of the target distribution area). Results: HE and EVG staining revealed medial degeneration and broken elastic fiber in aorta of TAAD patients. The mRNA expression levels of TPM2 were significantly upregulated in aorta of TAAD patients as compared to the control group (P<0.05), so as the TPM2 protein expression levels ((9.73±1.20)% vs. (0.11±0.04)%, P<0.05). And TPM2 was mainly expressed in cytoplasm. Conclusion: The increased expression of TPM2 in TAAD patients hints that TPM2 might be involved in the pathogenesis of aortic dissection.

Effect of ghrelin on proliferation and mitofusin-2 expression of human aortic smooth muscle cells / 重庆医学

Objective To investigate the effects of ghrelin on proliferation of vascular smooth muscle cells(VSMC)and the expression of mitochondrial fusion 2(Mfn-2)in cultured human aortic smooth muscle cells(HASMCs).Methods HASMCs were cultured in vitro,treated with different concentrations(10-9,10-8,10-7,10-6,10-5 mol/L)ghrelin or 10-6 mol/L ghrelin for different time(0,6,12,18,24 h).Subconfluent HASMCs at passage 4-6 were used in experiments.MTT essay was used to investigate the effect on proliferation of HASMCs.RT-PCR and Western blot were used to analyse the expression of Mfn-2.Results 10-7-10-5 mol/L ghrelin inhibited the proliferation of HASMCs,and the inhibitory effect of concentration of 10-6 mol/L was the most obvious(P<0.01).Ghrelin inhibited the proliferation of HASMCs in 6-24 h,and it reached the peak at 24 h(P<0.01).10-6 mol/L ghrelin significantly increased the expression of Mfn-2 mRNA and protein(P<0.01).The up-regulation of 10-6 mol/L ghrelin on Mfn-2 mRNA and protein expression reached the peak at 18 h(P<0.01).Conclusion Ghrelin might inhibit the proliferation of HASMC by up-regulating the expression of Mfn-2.

Expression and biological functions of Myc associated factor X in human aortic dissection tissue / 第二军医大学学报

Objective To observe the expression of Myc associated factor X (MAX) in aortic dissection tissue, and to discuss its biological functions. Methods MAX expression level was evaluated by qRT-PCR and Western blotting analysis in 15 dissected aorta samples. The adenovirus vector was used to transfect human aortic smooth muscle cells (HASMCs) for overexpression of MAX. The effects of MAX overexpression on proliferation and apoptosis of HASMCs were analyzed by Cell Counting Kit-8 and flow cytometry, respectively. Results MAX mRNA and protein expression levels were significantly higher in the aortic dissection tissue compared with that in the healthy controls. Overexpression of MAX significantly inhibited the proliferation of HASMCs and promoted its apoptosis (P<0.05). Conclusion MAX might induce the loss of HASMCs via regulating their proliferation and apoptosis process, thus play an important role in the development and progression of aortic dissection.

Effect of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells / 中华肾脏病杂志

Objective To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs).Methods Uremic serum was incubated at 37℃ for 3 days.Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by uhracentrifugation.Scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX) were performed for analysis of morphological and chemical characteristics of the crystals.HASMCs were treated in vitro with control medium,uremic serummedium,calcium phosphate crystals-medium and uremic supernatant-medium.Calcification was visualizcd by Alizarin red staining.Calcium loads in cells were quantified by o-cresolphthalein complexone method.Gene expression of bone morphogenetic protein-2 (BMP-2),osteopontin (OPN) and core-binding factor α1 (Cbfα1),alkaline phosphate (ALP) and matrix gamma carboxyglutamic acid protein (MGP) were quantified by real-time PCR.Cbfα1,OPN and BMP-2 protein levels were determined by Western blotting or ELISA.Results Calcium phosphate crystals which induced by uremic serum displayed laminated shapes containing crystallized needle-like projections and ranged from 30-500 nm,with Ca/P ratios of 1.41 ±0.05.Compared with the cells in control group,uremic serum induced HASMCs calcification,increased calcium loads (P ＜ 0.05),up-regulated BMP-2,OPN,Cbfα1 mRNA and protein expression (all P＜ 0.01).Similar to uremic serum,calcium phosphate crystals also induced HASMCs calcification,increased calcium loads (P＜0.05),and up-regulated BMP-2,OPN,Cbfα1 mRNA and protein expression (all P ＜ 0.01).However,there was no significant difference between HASMCs growing in uremic supernatant and control medium in calcium loads or the expression levels of these osteogenic proteins (P ＞ 0.05).Conclusions Calcium phosphate crystals induced by uremic serum promote HASMCs calcification,which might be one of the mechanisms of uremic vascular calcification.

Native Low-Density Lipoprotein-Dependent Interleukin-8 Production Through Pertussis Toxin-Sensitive G-Protein Coupled Receptors and Hydrogen Peroxide Generation Contributes to Migration of Human Aortic Smooth Muscle Cells

PURPOSE: Stimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration. MATERIALS AND METHODS: nLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H2O2 generation was determined by microscopic analysis using 2',7'-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates. RESULTS: nLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H2O2 generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H2O2. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody. CONCLUSION: PTX-sensitive G-protein coupled receptor-dependent H2O2 generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.

Tetrahydrobiopterin Inhibits PDGF-stimulated Migration and Proliferation in Rat Aortic Smooth Muscle Cells via the Nitric Oxide Synthase-independent Pathway

Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) activity, is known to play important roles in modulating both NO and superoxide production during vascular diseases such as atherosclerosis. However, the role of BH4 in functions of vascular smooth muscle cells is not fully known. In this study, we tested the effects of BH4 and dihydrobiopterin (BH2), a BH4 precursor, on migration and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) in rat aortic smooth muscle cells (RASMCs). Cell migration and proliferation were measured using a Boyden chamber and a 5-bromo-2'-deoxyuridine incorporation assay, respectively, and these results were confirmed with an ex vivo aortic sprout assay. Cell viability was examined by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assays. BH4 and BH2 decreased PDGF-BB-induced cell migration and proliferation in a dose-dependent manner. The inhibition of cell migration and proliferation by BH4 and BH2 was not affected by pretreatment with N G-nitro-L-arginine methyl ester, a NOS inhibitor. Moreover, the sprout outgrowth formation of aortic rings induced by PDGF-BB was inhibited by BH4 and BH2. Cell viability was not inhibited by BH4 and BH2 treatment. The present results suggest that BH4 and BH2 may inhibit PDGF-stimulated RASMC migration and proliferation via the NOS-independent pathway. Therefore, BH4 and its derivative could be useful for the development of a candidate molecule with an NO-independent anti-atherosclerotic function.

Native low-density lipoprotein-induced superoxide anion contributes to proliferation of human aortic smooth muscle cells / 대한마취과학회지

BACKGROUND: Native low-density lipoprotein (nLDL) was one of the modifiable risk factors contributed directly to cardiovascular diseases development. We investigated that nLDL stimulation induced NADPH oxidase activation and superoxide production that was an important factor on human aortic smooth muscle cells (hAoSMC) proliferation. METHODS: Superoxide generation was recorded with fluorescent-staining of dihydroethidine or by measuring lucigenin-induced chemiluminescence for 5 minutes. We examined cell proliferation with 4[-3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) reagent and analyzed the change of gene expression by northern blot analysis. RESULTS: nLDL stimulation increased superoxide anion production in hAoSMC that confirmed through dihydroethidine staining and lucigenin-induced chemiluminescence methods. nLDL-induced proliferation abolished with preincubation of superoxide scavengers or NADPH oxidase inhibitor. NADPH as a substrate of NADPH oxidase increased superoxide generation in both nLDL-stimulated and unstimulated cell homogenate, which was completely blocked at the diphenylene iodinium (DPI)- or apocynin-pretreated hAoSMC homogenates. Furthermore, superoxide generation was only observed at the fraction of cellular precipitate, but not in soluble fraction. Expression of p22phox in mRNA level increased with nLDL treatment as early as 30 minutes and transfection of anti-sense oligonucleotide of p22phox completely abolished nLDL-induced proliferation of hAoSMC. CONCLUSIONS: The above results have shown that nLDL-induced proliferation in hAoSMC depends on superoxide production through NADPH oxidase activation.

Olibanum Extract Inhibits Vascular Smooth Muscle Cell Migration and Proliferation in Response to Platelet-Derived Growth Factor

Olibanum (Boswellia serrata) has been shown to have anti-inflammatory, anti-arthritic and anti- cancer effects. This study determined the role of a water extract of olibanum in platelet-derived growth factor (PDGF)-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). PDGF-BB induced the migration and proliferation of RASMCs that were inhibited by olibanum extract in a dose-dependent manner. The PDGF-BB-increased phosphorylation of p38 mitogen-activated protein kinase (MAPK); the heat shock protein (Hsp) 27 was significantly inhibited by the olibanum extract. The effects of PDGF-BB-induced extracellular signal-regulated kinase1/2 was not altered by the olibanum extract. Treatment with olibanum extract inhibited PDGF-BB-stimulated sprout out growth of aortic rings. These results suggest that the water extract of olibanum inhibits PDGF-BB-stimulated migration and proliferation in RASMCs as well as sprout out growth, which may be mediated by the inhibition of the p38 MAPK and Hsp27 pathways.

Inhibitory effect of panaxynol on the proliferation of rat aortic smooth muscle cell and its mechanisms / 中国药理学通报

Aim To evaluate the inhibitory effect of panaxynol(PNN) on the proliferation of rat aortic smooth muscle cell(RASMC) and its mechanisms.Methods Cell proliferation was determined using cell count and -TdR incorporation test.Fura-3/AM and confocal were used to measure intracelluar free Ca~(2+) concentration.Expression of mitochondrial transcription factor 1(mtTF1) mRNA was tested by using RT-PR.Results PNN inhibited the RASMC proliferation and DNA synthesis induced by serum and PDGF-BB in a dose-dependent manner.9 ?mol?L~(-1) of PNN inhibited the increase of intracelluar free Ca~(2+) concentration induced by PDGF-BB.PDGF-BB upregulated the expression of mtTF1 mRNA,which could be suppressed by 3,9 ?mol?L~(-1) of PNN significantly.Conclusions PNN can inhibit RASMC proliferation significantly,which might be related to the decrease of intracellular free Ca~(2+) concentration and mtTF1 mRNA expression.