• Journal of Life Science
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• v.26 no.2
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• pp.226-233
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• 2016

Vascular smooth muscle cell (VSMC) apoptosis has been identified in various vascular diseases, including atherosclerosis and restenosis after angioplasty, and has been known to precipitate atherosclerotic plaque instability and rupture. Oxysterols are known as inducers of apoptosis in VSMC, and 7-ketocholesterol (7KC) is the major nonenzymically formed oxysterol in atherosclerotic lesions. The precise mechanism underlying VSMC apoptosis is still poorly understood. In this study, we investigated whether 7KC causes apoptosis, and characterized its apoptotic mechanisms in primary cultured rat aortic VSMC. Cell viability was assessed by MTT assay and trypan blue assay. Apoptosis was assessed by flow cytometry, immunofluorescence, immunoprecipitation, and Western blot analyses. 7KC markedly decreased the VSMC viability in a time- and concentration-dependent manner, and increased the production of 4-hydroxynonenal (HNE), a major end-product of lipid peroxidation, which also decreased the VSMC viability. Pretreatment with 2,4-dinitrophenylhydrazine, a well-known reagent of lipid peroxidation-derived aldehydes, significantly restored the 7KC-decreased viability of VSMC. Furthermore, HNE, as well as 7KC, reduced the level of total Akt, a major mediator of cell survival. The 7KC-decreased level of total Akt was significantly restored by pretreatments with 2,4-dinitrophenylhydrazine and N-acetylcysteine. Lactacystin, a proteasome inhibitor, protected VSMC against apoptosis and Akt degradation, but did not inhibit HNE production. In the immunoprecipitation assay, 7KC increased HNE-modified Akt. From the results, it seems that, in atherosclerotic lesions, 7KC induces HNE production in VSMC, and this HNE binds to Akt, proceeding to proteasomal degradation of Akt, through which mechanism the atherosclerotic plaque instability may be facilitated.

• Journal of Life Science
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• v.22 no.12
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• pp.1595-1599
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• 2012

Cell cycle activation and progression in vascular proliferative disease represent potent therapeutic targets. Luteolin, which occurs as glycosylated forms in celery, green pepper, perilla leaf, and camomile tea, has demonstrated antimutagenic, antitumorigenic, antioxidant, and antiinflammatory properties. In this study, we investigated the effect of luteolin on the proliferation of primary cultured rat aortic vascular smooth muscle cells induced by 5% fetal bovine serum. Luteolin at concentrations of 5, 20, and $50{\mu}M$ significantly inhibited this proliferation by 29.6, 50.8, and 83.1%, respectively. The incorporation of $[^3H]$-thymidine into DNA was also inhibited by 25.8, 57.6, and 81.0%, respectively. Flow cytometry analysis of DNA content revealed that FBS-inducible cell cycle progression was blocked by luteolin. Luteolin showed no cytotoxicity in VSMCs in this experimental condition according to WST-1 assays. Luteolin may represent a potential anti-proliferative agent for treatment of angioplasty restenosis and atherosclerosis.

• Journal of Life Science
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• v.28 no.12
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• pp.1516-1522
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• 2018

Atherosclerosis is an obstructive vessel disease mainly caused by chronic arterial inflammation to which the proliferation and migration of vascular smooth muscle cells (VSMCs) is the main pathological response. In the present study, the primary responsible inflammatory cytokine and its signaling pathway was investigated. The proliferation and migration of VSMCs was significantly enhanced by the prostaglandin $F_{2{\alpha}}$ ($PGF_{2{\alpha}}$), while neither was affected by tumor necrosis factor ${\alpha}$. Prostacyclin $I_2$ was seen to enhance the proliferation of VSMCs while simultaneously suppressing their migration. Both prostaglandin $D_2$ and prostaglandin $E_2$ significantly enhanced the migration of VSMCs, however, proliferation was not affected by either of them. The proliferation and migration of VSMCs stimulated by $PGF_{2{\alpha}}$ progressed in a dose-dependent manner; the $EC_{50}$ value of both proliferation and migration was $0.1{\mu}M$. VSMCs highly expressed the phospholipase isoform $C-{\beta}3$ ($PLC-{\beta}3$) while others such as $PLC-{\beta}1$, $PLC-{\beta}2$, and $PLC-{\beta}4$ were not expressed. Inhibition of the PLCs by U73122 completely blocked the $PGF_{2{\alpha}}$-induced migration of VSMCs, and, in addition, silencing $PLC-{\beta}3$ significantly diminished the $PGF_{2{\alpha}}$-induced proliferation and migration of VSMCs. Given these results, we suggest that $PGF_{2{\alpha}}$ plays a crucial role in the proliferation and migration of VSMCs, and activation of $PLC-{\beta}3$ could be involved in their $PGF_{2{\alpha}}$-dependent migration.

• Journal of Chest Surgery
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• v.40 no.4 s.273
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• pp.256-263
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• 2007

Background: Intimal hyperpiasia is characterized by a proliferation of vascular smooth muscle cells in the intimal layer Epigallocatechin-3-gallate (EGCG) is known to suppress smooth muscle cell proliferation. We propose that EGCG may have a protective effect against the development of intimal hyperplasia through the suppression of smooth muscle cell proliferation. Material and Method: Human umbilical vein endothelial cells (HUVEC) and rat aortic smooth muscle cells (RASMC) were cultured with different concentrations of EGCG, and proliferation and migration speed were measured. In 20 dogs, the autologous jugular veins were interposed into the carotid arteries. For the study group (n=10), the graft was stored for 30 minutes in EGCG solution and 300mM EGCG was applied to the perivascular space after grafting. After 6 weeks, the intimal and medial thickness was measured. Result: The proliferation of RASMC and HUVEC was suppressed with EGCG. The migration of RASMC was suppressed with EGCG, but that of HUVEC was not affected. In the in vivo study, the intimal thickness was thinner in EGCG group than in the control group (p<0.05), but the medial thickness did not show any difference. The intimal/medial thickness ratio was lower in the EGCG group (p<0.05). Conclusion: EGCG suppresses intimal hyperplasia after vascular grafting, and this may be mediated by prevention of migration and proliferation of vascular smooth muscle cells. The use of EGCG may offer new therapeutic modality to prevent intimal hyperplasia.

• Journal of Chest Surgery
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• v.40 no.4 s.273
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• pp.264-272
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• 2007

Background: Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis, including stimulating the proliferation and migration of vascular smooth muscle cells (VSMCs). It has been known that diabetes is associated with accelerated cellular proliferation via VEGF, as compared to that under a normal glucose concentration. We investigated the effects of selective blockade of a VEGF receptor by using anti-Flt-1 peptide on the formation and hyperplasia of the neointima in balloon injured-carotid arteries of OLETF rats and also on the in vitro VSMCS' migration under high glucose conditions. Material and Method: The balloon-injury method was employed to induce neointima formation by VEGF. For f4 days beginning 2 days before the ballon injury, placebo or vascular endothelial growth factor receptor-1 (VEGFR-1) specific peptide (anti-Flt-1 peptide), was injected at a dose of 0.5mg/kg daily into the OLETF rats. At 14 days after balloon injury, the neointimal proliferation and vascular luminal stenosis were measured, and cellular proliferation was assessed by counting the proliferative cell nuclear antigen (PCNA) stained cells. To analyze the effect of VEGF and anti-Flt-1 peptide on the migration of VSMCs under a high glucose condition, transwell assay with a matrigel filter was performed. And finally, to determine the underlying mechanism of the effect of anti-Flt-1 peptide on the VEGF-induced VSMC migration in vitro, the expression of matrix metalloproteinase (MMP) was observed by performing reverse transcription-polymerase chain reaction (RT-PCR). Result: Both the neointimal area and luminal stenosis associated with neointimal proliferation were significantly decreased in the anti-Flt-1 peptide injected rats, ($0.15{\pm}0.04 mm^2$ and $ 36.03{\pm}3.78%$ compared to $0.24{\pm}0.03mm^2\;and\;61.85{\pm}5.11%$, respectively, in the placebo-injected rats (p<0.01, respectively). The ratio of PCNA(+) cells to the entire neointimal cells was also significantly decreased from $52.82{\pm}4.20%\;to\;38.11{\pm}6.89%$, by the injected anti-Flt-1 peptide (p<0.05). On the VSMC migration assay, anti-Flt-1 peptide significantly reduced the VEGF-induced VMSC migration by about 40% (p<0.01). Consistent with the effect of anti-Flt-1 peptide on VSMC migration, it also obviously attenuated the induction of the MMP-3 and MMP-9 mRNA expressions via VEGF in the VSMCS. Conclusion: Anti-Flt-1 peptide inhibits the formation and hyperplasia of the neointima in a balloon-injured carotid artery model of OLETF rats. Anti-Flt-1 peptide also inhibits the VSMCs' migration and the expressions of MMP-3 and MMP-9 mRNA induced by VEGF under a high glucose condition. Therefore, these results suggest that specific blockade of VEGFR-1 by anti-Flt-1 peptide may have therapeutic potential against the arterial stenosis of diabetes mellitus patients or that occurring under a high glucose condition.

• Journal of Life Science
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• v.21 no.5
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• pp.664-670
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• 2011

Oxidative stress results in sustained release of heat shock protein 90 (HSP90) from vascular smooth muscle cells (VSMCs). We investigated whether extracellular HSP90 predisposed VSMCs to pro-inflammatory phenotype. Exposure of human aortic smooth muscle cells to HSP90 not only significantly enhanced CXCL10 secretion but also increased CXCL10 transcription. HSP90-mediated CXCL10 secretion was attenuated by OxPAPC, a TLR-2/4 inhibitor, and curcumin, a TLR-4 dimerization inhibitor. Inhibitors of diphenyleneiodium chloride and the Akt pathway also attenuated CXCL10 secretion in response to HSP90. The gene delivery of I${\kappa}$B using recombinant adenoviruses and treatment with resveratrol, which inhibit NF-${\kappa}$B activity, significantly attenuated HSP90-induced CXCL10 secretion from VSMCs. We propose that extracellular HSP90 contributes to an inflammatory reaction in the stressed vasculature by inducing CXCL10 expression of VSMCs, and that TLR-4, Akt, and NF-${\kappa}$B play active roles in the process.

• Journal of the Korean Society of Food Science and Nutrition
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• v.44 no.8
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• pp.1180-1185
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• 2015

This study was performed to investigate the biological activity of yangha flower buds as well as to manufacture of yanggeng prepared with various levels (0 g, 3 g, 6 g, 9 g, and 12 g) of yangha flower buds. DPPH and ABTS scavenging activities of yangha flower buds were 96% and 57% compared to levels of vitamin C, respectively. In the oxygen radical antioxidant capacity assay, antioxidant activity increased dose dependently up to $500{\mu}g/mL$ of yangha flower buds. There was no toxicity up to $1,000{\mu}g/mL$ in vascular smooth muscle cells, and yanggeng significantly reduced migration and proliferation by platelet-derived growth factor-BB-stimulated rat aortic smooth muscle cell migration and proliferation. In the sensory evaluation, the optimal sample was YY9, which was prepared with 9 g of yangha flower buds. It can be concluded that yangha flower buds show antioxidant and vascular protective activities. The optimal sample (YY9) is expected to contribute as a new functional food.

• Journal of Life Science
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• v.25 no.2
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• pp.231-236
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• 2015

Angiotensin II (AngII) is an essential hormone that affects vascular physiology. For example, stimulation of vascular smooth muscle cells (VSMCs) rapidly induces vasoconstriction and results in the up-regulation of blood pressure. Chronic stimulation of VSMCs with AngII also results in hypertrophy. In this study, we confirmed an involvement of phosphatidylinositol 3-kinase (PI3K)-dependent calcium mobilization in AngII-induced generation of reactive oxygen species (ROS). Stimulation of rat aortic smooth muscle cells (RASMCs) with AngII significantly induced the generation of ROS in a dose- and time-dependent manner. AngII-induced generation of ROS was completely abolished by pharmacological inhibition of PI3K (with LY294002), but inhibition of the ERK signaling pathway had no effect. AngII-induced calcium mobilization was completely blocked by inhibition of PI3K, whereas inhibition of the ERK signaling pathway by PD98059 was ineffective. Depletion of extracellular calcium or inhibition of the L-type calcium channel by nifedipine completely blocked AngII-induced calcium mobilization. Depletion of extracellular calcium by EGTA and incubation of RASMCs with calcium-free medium both significantly blocked AngII-induced ROS generation. Inhibition of the L-type calcium channel also significantly blocked AngII-induced ROS generation. These results suggest that AngII-induced ROS generation is regulated by calcium mobilization, which, in turn, is modulated by a PI3K/L-type calcium channel signaling pathway.

• Journal of Life Science
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• v.21 no.1
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• pp.36-43
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• 2011

Diabetes mellitus is associated with vascular complications. Diabetic patients exhibit high levels of glycated adducts in serum compared to non-diabetic individuals. The aim of this study was to investigate whether extracellular glycated albumin (GA) predisposes vascular smooth muscle cells (VSMCs) to pro-inflammatory phenotype. Exposure of rat aortic smooth muscle cells (AoSMCs) to GA not only enhanced interleukin-6 (IL-6) release but also activated promoter activity of the IL-6 gene. GA-induced IL-6 promoter activation was suppressed by dominant-negative forms of Toll-like receptor (TLR)-4 and myeloid differentiation factor 88 (MyD88), but not by dominant-negative-forms of TLR-2 and TIR-domain-containing adapter-inducing interferon-$\beta$ (TRIF). Extracellular signal-regulated kinase (ERK) inhibition and diphenyleneiodium (DPI) also attenuated IL-6 induction by GA. Mutation at the nuclear factor-${\kappa}B$ (NF-${\kappa}B$)-binding site in the IL-6 promoter region suppressed promoter activation in response to GA. The present study proposes that GA would contribute to inflammatory reaction in the stressed vasculature by inducing IL-6 in VSMCs, and that TLR-4, EKR, and NF-${\kappa}B$ play active roles in the process.

• Applied Microscopy
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• v.29 no.1
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• pp.83-94
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• 1999

The ultrastructure of small arterioles and capillaries in the lumbrical muscles of rat digits were studied by electron microscopy and following results were obtained. 1. The diameter of small arterioles of rat digits were $12\sim20{\mu}m$, and it was relatively smaller than human $(30\sim35{\mu}m)$. 2. All the endothelial cells of small arterioles and capillaries in the lumbrical muscles of rat digits were continuous type, and they had typical morphological characteristics of continuous endothelial cells : numerous cytoplasmic pinocytic vesicles and many tight junctions between the endothelial cells. 3. In the small arterioles subendothelial layers were irregularly spaced beneath the basal lamina, and membrane to membrane contacts were found between the endothelial cells and smooth muscle cells. 4. Pericytes were often found nearby capillary endothelium, and their cytoplasmic processes surrounded part of endothelial cells. They were enclosed by basal lamina. 5. Smooth muscle cells in tunica media of small arterioles were only one layered, that is, they were terminal arterioles. Sarcoplasm of smooth muscle cell was divided to 2 areas; homogeneous or filamentous area and non-homogeneous perinuclear area. 6. The tunica adventitia contained fibroblasts with extremely attenuated cytoplasmic processes and collagen fibirls.