Influence of influenza A and B virus infections on human aortic smooth muscle cells and the expressions of cytokines / 医学研究生学报

Objective　Vascular smooth muscle cells are the main cells in atherosclerosis. Reports are rarely seen on influenza virus infection on human aortic smooth muscle cells (HASMC) and its influence on the expressions of the related cytokines. This study was to investigate the impact of influenza A virus (IAV) and influenza B virus (IBV) infection on HASMCs and the expressions of cytokines. Methods　HASMCs were stimulated with IAV or IBV or not stimulated with virus (the control). The nucleoprotein of the influenza virus in the cells was detected by immunofluorescence assay, the proliferation of the cells determined with CCK8, and the level of influenza virus RNA in the supernatant measured by qPCR. The collected supernatant was used to infect Madin-Darby canine kidney (MDCK) cells and detect the influenza virus nucleoprotein. The expressions of the cytokines of the influenza virus after 24 hours of infection were determined by qPCR.　Results　At 3 and 4 days after infected with influenza virus, the proliferation of the HASMCs was significantly inhibited in the IAV and IBV groups as compared with the control (P<0.05). The expression of virus RNA in the supernatant of the IBV group at 3 days was 5.75 times as high as that at 2 days (P<0.05), dropped at 4 days but still higher than that at 2 days (P<0.01). Compared with the normal culture medium, the medium with virus growth fluid significantly elevated the RNA level of IAV (0.842±0.148 vs 15.182±1.932, P< 0.01) and IBV (0.962±0.033 vs 4.029±0.681, P<0.01). After infection, the expression of MCP-1 was remarkably up-regulated in the IAV and IBV groups (4.364±0.193 and 3.348±0.507) in comparison with that in the control group (1.001±0.001) (P<0.05), and so were the expressions of IL-6 and TNF-α (P<0.05).　Conclusion　Both IAV and IBV can infect HASMCs and increase the expressions of the cytokines MCP-1, IL-6 , and TNF-α.

Propofol Inhibits Platelet-derived Growth Factor-stimulated Migration in Rat Aortic Smooth Muscle Cells / 대한마취과학회지

BACKGROUND: Propofol is the extensively used general anesthetic-sedative agent.Although propofol is known to be involved in migration of various cells, migration response to it in vascular smooth muscle cells is not investigated. This study was carried out to determine the role of propofol in migration of rat aortic smooth muscle cells (RASMCs). METHODS: A7r5 RASMCs were used.Cell migration was examined by the analysis of 5 ng/ml of platelet-derived growth factor (PDGF)-induced RASMC response after treatment of cells with propofol (1-100micrometer) in the Boyden chamber.The activity of cofilin by propofol in RASMCs was measured by the Western blot analysis for the change of cofilin dephosphorylaton in cells treated with 10micrometer propofol for 5, 10, 15 and 20 min, for the effect of propofol (1, 10 and 100micrometer) on cofilin phosphorylation, and for the effects of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (2 mM; EGTA), Na3VO4 (200micrometer), and calyculin A (10 nM) on 10micrometer propofol-induced cofilin dephosphorylation. RESULTS: PDGF increased RASMC migration and this response was dose-dependently inhibited by treatment with propofol. Propofol attenuated the cofilin phosphorylation in RASMCs in a dose- and time-dependent manner.Propofol-induced dephosphorylation of cofilin in RASMCs was abolished by calyculin A, a protein phosphatase 2A inhibitor, but not by EGTA, a Ca2+ chelating agent, or Na3VO4, a protein tyrosine phosphatase inhibitor. CONCLUSIONS: The present results suggest that propofol induces the diminution of PDGF-stimulated RASMC migration and this response may be associated with dephosphorylation of cofilin mediated by the protein phosphatase 2A-dependent pathway.

The effect of anti-hypertensive drugs on DNA synthesis and proliferation of ultured rat aortic smooth muscle cells

The aim of this study was to elucidate the effects of anti-hypertensive drugs, nifedipine, furosemide, hydrochlorothiazide, captopril, and atenolol on DNA synthesis and proliferation of cultured rat aortic smooth muscle cells induced by fetal calf serum. Aortic smooth muscle cells from Sprague-Dawley rats were isolated, cultured, and seeded in multi-well plates. When confluent, cells were cultured in a conditioned medium without fetal calf serum. After 72 hours, cells were cultured in the medium retaining 10% fetal calf serum with or without anti-hypertensive drugs by increasing the concentration between 10(-8) and 10(-4) M. DNA synthesis was assessed by [3H]-thymidine uptake and proliferation by cell numbers using a hemocytometer. Nifedipine at a concentration of 10(-5) M and 5 x 10(-5) M inhibited serum-induced DNA synthesis significantly by 50.8% and 86.6%, respectively (p < 0.05). The results of cell numbers paralleled those of 3H-thymidine incorporation. Serum-induced DNA synthesis was also reduced by 32.6% at the highest dose of furosemide (10(-4) M), but there was no statistical significance. Hydrochlorothiazide, captopril, and atenolol did not show anti-proliferative effect throughout any of the doses. In conclusion, among the various anti-hypertensive drugs, nifedipine seems to be most beneficial in view of its direct inhibitory effect on DNA synthesis and proliferation of smooth muscle cells, as well as for its anti-hypertensive effect.

The effect of lovastatin on proliferation of cultured rat mesangial and aortic smooth muscle cells

In order to investigate the anti-proliferative effect of 3-hydroxy-3-methylglutaryl coenzyme. A reductase inhibitor, we evaluated the effects of lovastatin on DNA replication and the proliferation of rat mesangial and aortic smooth muscle cells, both of which were mesenchymal origin cells. Proliferations were determined by measuring [3H]thymidine uptake, and counting the number of cells. Growth-arrested mesangial and aortic smooth muscle cells were exposed to platelet-derived growth factor (PDGF), endothelin (ET) and angiotensin II (Ang II) to stimulate mitogenesis. All agents exhibited dose-dependent stimulation of [3H] thymidine uptake. PDGF was more potent than the others. Ang II increased [3H] thymidine uptake without demonstrable mitogenic activity. Lovastatin inhibited PDGF (10 ng/ml in mesangial cell, 25 ng/ml in smooth muscle cell)-, ET (10(-7)M)- and Ang II (10(-7)M)-induced [3H] thymidine uptake significantly in a dose-dependent manner in both cells. The increase of cell number in response to PDGF and ET treatment were also inhibited at 10 microM of lovastatin. The inhibitory effect of lovastatin was largely overcome in the presence of exogenous mevalonate at 200 microM, with 75.5% restoration from lovastatin-induced inhibition on PDGF-induced [3H] thymidine uptake in mesangial cells (77.8% in aortic smooth muscle cells). However, the addition of cholesterol did not prevent inhibition by lovastatin. In conclusion, lovastatin had an inhibitory effect on mesangial and aortic smooth muscle cell proliferation, and mevalonate was essential for DNA replication in both types of cells. Lovastatin may reduce glomerular and atherosclerotic injury through an anti-proliferative effect on mesangial and vascular smooth muscle cells, in addition to lowering circulating lipids.

Experimental study of atherosclerosis ⅣChanges of aortic smooth cell proliferation, platelet free calcium and aggregation in atherosclerotic rabbits and the effects of 8501 on these changes / 中国药理学通报

Smooth muscle cell (SMC) proliferation, platelet free calcium level and aggregation of experimental atherosclerotic rabbits were investigated in this study. Aortic SMC ofhyperlipidemic rabbits in vitro showed higher growth activity than did normal rabbit SMC. And also hyperlipidemic serum stimulated SMC to proliferate at a significantly greater rate than control serum. Moreover, the level of platelet free calcium and the platelet aggregation was also higher in hyperlipidemic rabbits, indicating that activitated platelets possibly release more PDGF to act as a stimulator to SMC proliferation and calcium is an important factor to activate platelets. Furthermore, SMC from 8501-treated rabbits appeared lower proliferative rate than thecells from hyperlipidemic rabbits. And serum from those rabbits inhibited SMC proliferation compared with hyperlipidemic serum, the inhibitory effect was even stronger than that of normal serum. It may be relevant to the favorable effects of 8501 to TXA2/PGI2 balance.

AORTIC SMOOTH MUSCLE CELLS PRODUCED ELASTIC FIBERS IN THREE-DIMENSIONAL CULTURE / 解剖学报

Objective To Study if aortic smooth muscle cells(ASMC) can produce elastic fibers in three-dimensional culture imitating the dermis substitute. Methods Primary culture of ASMCs were obtained by explanting of thoracic aortae from one-week old SD rats. ?-actin immunocytochemistry was used to approve the smooth muscle cells. After subculture, ASMCs were mixed with gels composed of collagen, chitin and glucosaminoglycans, then, three-dimensional culture was carried out. After 1 week and 2 weeks, Gomori aldehyde fuchsin staining and elastin immunocytochemistry were applied to detect the elastic fibers in the ASMC-collagen gels. Results More than 98% of the cultured cells were approved as smooth muscle cells by ?-actin immunocytochemistry. After both 1 and 2 weeks of three-dimentional culture, elastic fibers were detected in the ASMC-collagen gels by both stainings.Conclusion ASMCs can synthesize elastin and produce elastic fiber in three-dimentional culture imitating the dermis substitute.

THE STUDY ON TRANSPLANTATION OF THREE-DIMENTIONAL CULTURED ASMC-COLLAGEN GEL TO SUBCUTANEOUS / 解剖学报

Objective To investigate if aortic smooth muscle cells (ASMCs) cultured three-dimentionally with collagen gel can still produce elastic fibers and the acceptance of hosts after transplantated in subcutaneous of rats. Methods AMSC-collagen gels that had been cultured for 2 weeks by three-dimentionally imitating the dermis substitute were transplanted to rats subscutaneous. After 4,7,10,14 and 28 days, the transplantations were examined via HE staining, Gomori aldehyde fuchsin staining, ?-actin immunohistochemistry and elastin immunohistochemistry. Results The amount of elastic fibers increased significantly in the week, whereas on the 28th day, no elastic fibers were detected.There was no remarkable leucocyte infiltration around the ASMC-collagen gel during 28 days.Conclusion Further studies are needed to explore the ASMC-collagen gels as a kind of elastic dermis substitute.