• Journal of Yeungnam Medical Science
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• v.23 no.2
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• pp.182-192
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• 2006

Background: Atherosclerosis has emerged as the leading cause of death in developed countries. At present, human umbilical vein endothelial cells (HUVEC) are most commonly used for the investigation of Endothelial cells (EC). However, HUVEC are not found in arteries but only in veins. Currently there are many reports on methods used to isolate EC;, most of these methods require special equipment to remove contaminating smooth muscle cells (SMC). Materials and Methods: The method described here may be used to isolate not only ECs but also SMCs;,the approach presented here did not require special equipment. Rat aorta was treated with 2 mg/ml of type II collagenase solution for 45 minutes. The isolated cells from the aorta were incubated in medium G for a week;, only ECs could be separated. After the collagenase treatment, the rest of aorta was cut lengthwise, and left undisturbed to obtain SMCs in the culture dish for 10 days. To verify the purity of the isolated cells, we performed immunofluorescence and evaluated the results with transmission electron microscopy analysis. Results: The immunofluorescence study demonstrated specific expression of CD31 and ${\alpha}$-smooth muscle actin in the isolated ECs and SMCs, respectively. Cultured ECs and SMCs showed their own fine structure characteristics. Conclusion: These results suggest that this method for isolating ECs and SMCs may be especially useful for the study of atherosclerosis.

• Animal cells and systems
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• v.5 no.3
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• pp.205-209
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• 2001

Mesenchymal cells from the leg buds of stage 24-chick embryos differentiated into chondrocytes when plated at high density. Treatment of high density micromass culture of chick mesenchymal cells with cytochalasin D(CD, 2 $\mu$M for 24 h) resulted in inhibition of chondrogenesis. CD treatment was found to affect the expression of the contractile protein $\alpha$-smooth muscle actin ($\alpha$-SM actin). In control cultures, $\alpha$-SM actin uniformly expressed from culture day 2, but the CD-treated cells induced expression of $\alpha$-SM actin from the first day of culture followed by a continuous increase. Expression of pan-cadherin (P-cadherin) decreased as chondrogenesis proceeded in the control culture, whereas the CD-treated cells showed sustained expression. These results propose a close connection of chondrogenic differentiation with expression of $\alpha$-SM actin and P-cadherin.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.2
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• pp.139-147
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• 2013

Lysolipids such as LPA, S1P and SPC have diverse biological activities including cell proliferation, differentiation, and migration. We investigated signaling pathways of LPA-induced contraction in feline esophageal smooth muscle cells. We used freshly isolated smooth muscle cells and permeabilized cells from cat esophagus to measure the length of cells. Maximal contraction occurred at $10^{-6}M$ and the response peaked at 30s. To identify LPA receptor subtypes in cells, western blot analysis was performed with antibodies to LPA receptor subtypes. LPA1 and LPA3 receptor were detected at 50 kDa and 44 kDa. LPA-induced contraction was almost completely blocked by LPA receptor (1/3) antagonist KI16425. Pertussis toxin (PTX) inhibited the contraction induced by LPA, suggesting that the contraction is mediated by a PTX-sensitive G protein. Phospholipase C (PLC) inhibitors U73122 and neomycin, and protein kinase C (PKC) inhibitor GF109203X also reduced the contraction. The PKC-mediated contraction may be isozyme-specific since only $PKC{\varepsilon}$ antibody inhibited the contraction. MEK inhibitor PD98059 and JNK inhibitor SP600125 blocked the contraction. However, there is no synergistic effect of PKC and MAPK on the LPA-induced contraction. In addition, RhoA inhibitor C3 exoenzyme and ROCK inhibitor Y27632 significantly, but not completely, reduced the contraction. The present study demonstrated that LPA-induced contraction seems to be mediated by LPA receptors (1/3), coupled to PTX-sensitive G protein, resulting in activation of PLC, PKC-${\varepsilon}$ pathway, which subsequently mediates activation of ERK and JNK. The data also suggest that RhoA/ROCK are involved in the LPA-induced contraction.

• The Journal of Pediatrics of Korean Medicine
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• v.21 no.1
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• pp.139-154
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• 2007

Objectives : The author intended to investigate Seonbangpaedoktang (SBPT) significantly affect in vivo and in vitro mucin secretion from airway epithelial cells. Methods : In vivo experiment, the author induced hypersecretion of airway mucin, hyperplasia of tracheal goblet cells and the increase in intraepithelial mucosubstances. Effects of orally-administered SBPT during 1 week on in vivo mucin secretion and hyperplasia of tracheal goblet cells were assessed. For in vitro experiment, confluent hamster tracheal surface epithelial (HTSE) cells were metabolically radiolabeled and chased in the presence of SBPT to assess the effect of the agent on 3H-mucin secretion. Total elution profiles of control spent media and treatment sample through Sepharose CL-4B column were analysed. Possible cytotoxicity of the agent was assessed by measuring LDH release. Also, the effect of SBPT on contractility of isolated tracheal smooth muscle was investigated. Results : SBPT inhibited hypersecretion of in vivo mucin and inhibited the increase of number of goblet cells ; SBPT did not affect in vitro mucin secretion and the secretion of the other releasable glycoproteins with less molecular weight than mucin from cultured HTSE cells, without significant effect on LDH release; SBPT did not affect Ach-induced contraction of isolated tracheal smooth muscle. Conclusions : SBPT can inihibit hypersecretion of in vivo mucin and the author suggest that the effect SBPT with their components should investigate further.

• Natural Product Sciences
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• v.26 no.1
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• pp.71-78
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• 2020

The plant Dendropanax morbifera Léveille is effective folk medicines for the treatment of several conditions, such as infectious diseases, skin diseases, and other illnesses. Although the inhibitory effects of D. morbifera on the proliferation and migration of vascular smooth muscle cells (VSMCs) have been shown in our previous study, its effects in vivo remain to be elucidated. In this study, we aimed to investigate the protective effects of the extracts from D. morbifera (EDM) on neointimal hyperplasia of rat carotid artery and explore the underlying mechanisms. We observed that the ratio of intima to media thickness (I/M) was significantly decreased in the EDM-treated groups by ~80% compared to that of the control. The expression of Ki-67 and proliferating cell nuclear antigen was decreased by ~70% in the EDM-treated groups compared to that of the control. In addition, matrix metalloproteinase (MMP)2 and MMP9 significantly reduced in the neointimal layer of the EDM-treated groups. Moreover, the decreased levels of contractile phenotypic markers of VSMCs, such as α-smooth muscle actin, myocardin, and smooth muscle-myosin heavy chain, were successfully restored by EDM treatment. Furthermore, the levels of synthetic phenotypic markers, cellular retinal binding protein 1 and connexin 43 were also restored to normal levels. These results suggest that EDM inhibits vascular neointimal hyperplasia induced by balloon injury in rats via phenotypic modulation of VSMCs. Therefore, EDM may be a potential drug candidate for the prevention of restenosis.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.5
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• pp.471-479
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• 1999

The Kv channel activity in vascular smooth muscle cell plays an important role in the regulation of membrane potential and blood vessel tone. It was postulated that increased blood vessel tone in hypertension was associated with alteration of Kv channel and membrane potential. Therefore, using whole cell mode of patch-clamp technique, the membrane potential and the 4-AP-sensitive Kv current in cerebral arterial smooth muscle cells were compared between normotensive rat and one-kidney, one-clip Goldblatt hypertensive rat (lK,lC-GBH rat). Cell capacitance of hypertensive rat was similar to that of normotensive rat. Cell capacitance of normotensive rat and 1K,lC-GBH rat were $20.8{\pm}2.3$ and $19.5{\pm}1.4$ pF, respectively. The resting membrane potentials measured in current clamp mode from normotensive rat and 1K,lC-GBH rat were $-45.9{\pm}1.7$ and $-38.5{\pm}1.6$ mV, respectively. 4-AP (5 mM) caused the resting membrane potential hypopolarize but charybdotoxin $(0.1\;{\mu}M)$ did not cause any change of membrane potential. Component of 4-AP-sensitive Kv current was smaller in 1K,lC-GBH rat than in normotensive rat. The voltage dependence of steady-state activation and inactivation of Kv channel determined by using double-pulse protocol showed no significant difference. These results suggest that 4-AP-sensitive Kv channels playa major role in the regulation of membrane potential in cerebral arterial smooth muscle cells and alterations of 4-AP-sensitive Kv channels would contribute to hypopolarization of membrane potential in 1K,lC-GBH rat.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.25-28
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• 2003

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance $Ca^{2+}$-activated $K^+$ channel ($BK_{Ca}$) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of $BK_{Ca}$. The $BK_{Ca}$ currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single $BK_{Ca}$ currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit $BK_{Ca}$ currentsin the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent $K^+$ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.

• Journal of Nutrition and Health
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• v.53 no.6
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• pp.563-569
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• 2020

Purpose: The vascular smooth muscle cells (VSMCs) in mature animals have implicated to play a major role in the progression of cardiovascular diseases such as atherosclerosis. This study aimed at optimizing the protocol in culturing primary VSMCs (pVSMCs) from rat thoracic aorta and investigating the effect of cellular zinc (Zn) deficiency on cell proliferation of the isolated pVSMCs. Methods: The thoracic aorta from 7-month-old Sprague Dawley rats was isolated, minced and digested by the enzymatic process of collagenase I and elastase, and then inoculated with the culture Dulbecco Modified Eagle Medium (DMEM) at 37℃ in an incubator. The primary cell culture morphology was observed using phase-contrast microscopy and cellular Zn was depleted using Chelex-100 resin (extracellular zinc depletion only) or 3 µM N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) (extracellular and intracellular zinc depletion). Western blot analysis was used for the detection of SM22α and calponin as smooth muscle cell marker proteins and von Willebrand factor as endothelial cell marker protein to detect the culture purity. Cell proliferation by Zn depletion (1 day) was measured by MTT assay. Results: A primary culture protocol for pVSMCs from rat thoracic aorta was developed and optimized. Isolated cultures exhibited hill and valley morphology as the major characteristics of pVSMCs and expressed the smooth muscle cell protein markers, SM22α and calponin, while the endothelial marker von Willebrand factor was hardly detected. Zn deprivation for 1 day culture decreased rat primary vascular smooth muscle cell proliferation and this pattern was more prominent under severe Zn depletion (3 µM TPEN), while less prominent under mild Zn depletion (Chelexing). Conclusion: Our results suggest that cellular Zn deprivation decreased pVSMC proliferation and this may be involved in phenotypic modulation of pVSMC in the aorta.

• The Journal of Internal Korean Medicine
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• v.27 no.1
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• pp.92-101
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• 2006

Objectives : This study was done with intend to investigate whether two oriental medical prescriptions, daecheongryong-tang (DCRT) and prescription A (P-A) significantly affect mucin release from cultured hamster tracheal surface epithelial (HTSE) cells. Methods : Confluent HTSE cells were metabolically radiolabeled with $^3H$-glucosamine for 24 hrs and chased for 30 min in the presence of DCRT or P-A to assess the effect of each agent on $^3H$-mucin release. Possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase (LDH) release. Also, the effects of DCRT and P-A on contractility of isolated tracheal smooth muscle were investigated. Results were as follows : 1. DCRT significantly inhibited mucin release from cultured HTSE cells, with significant cytotoxicity. 2. P-A significantly increased mucin release from cultured HTSE cells, with significant cytotoxicity. 3. DCRT inhibited Ach-induced contraction of isolated tracheal smooth muscle. 4. P-A also inhibited Ach-induced contraction of isolated tracheal smooth muscle. Conclusion: Results suggest that DCRT and P-A have regulating effects on mucin secretion from goblet cells. Further investigation is needed, because of the value in finding novel agents to this purpose, and these oriental medical prescriptions have potential for this role.

• The Journal of Internal Korean Medicine
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• v.27 no.4
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• pp.895-904
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• 2006

Objectives : In the present study, the author intended to investigate whether two oriental medical prescriptions named Socheongryongtang-ga-seoggo (SCTS) and Prescription D (P-D) significantly affect mucin release from cultured hamster tracheal surface epithelial (HTSE) cells. Materials and Methods : Confluent HTSE cells were metabolically radiolabeled with 3H-glucosamine for 24 hrs and chased for 30 min in the presence of SCTS or P-D to assess the effect of each agent on 3H-mucin release. Possible cytotoxicities of each agent were assessed by measuring lactate dehydrogenase (LDH) release. Also, the effects of SCTS and P-D on contractility of isolated tracheal smooth muscle were investigated. Results : SCTS did not affect mucin release from cultured HTSE cells, without cytotoxicity. However, P-D significantly increased mucin release from cultured HTSE cells. with significant cytotoxicity. SCTS inhibited Ach-induced contraction of isolated tracheal smooth muscle. P-D also inhibited Ach-induced contraction of isolated tracheal smooth muscle. Conclusions : The author suggests that the effects of SCTS and P-D with their components should be further investigated and it is valuable to find, from oriental medical prescriptions, novel agents which might regulate mucin secretion from airway goblet cells.