• Clinical and Experimental Pediatrics
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• v.46 no.2
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• pp.167-172
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• 2003

Purpose : Pulmonary vascular hypertension is a common problem in congenital heart disease, the most common cardiac condition in childhood. However, the mechanisms responsible for this pathologic change, treatment, and prevention are poorly understood. Therefore, we studied the gene expression of vascular endothelial growth factor(VEGF) by using a hypoxic model of the pulmonary artery smooth muscle cells. Methods : The main pulmonary artery and its proximal branches of a 6 wk old Fischer rat were excised. They were cut into multiple small pieces and suspended in DMEM medium supplemented with 20% fetal bovine serum and incubated in 5% $CO_2$-95% air atmosphere. The smooth muscle cells were confirmed by immunostaining with smooth muscle myosin and ${\alpha}$-smooth muscle actin antibodies. The VEGF gene expression in the hypoxic group was compared with the one in control the group as well as the one in the starved group by RT-PCR and Northern blot hybridization. Results : There was no statistically significant difference among the control, hypoxic and starved groups. Conclusion : There are few studies of pulmonary vascular hypertension at the molecular level in Korea. Therefore, we studied the expression of VEGF gene in hypoxic pulmonary vascular smooth muscle cells. Further studies will be needed to find the difference between newly born and adult rats, or human and rat pulmonary vascular smooth muscle cells in gene expression. We hope that the study will lead to a better understanding of pulmonary vascular hypertension.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.6
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• pp.431-436
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• 2011

Vascular smooth muscle cells can obtain a proliferative function in environments such as atherosclerosis in vivo or primary culture in vitro. Proliferation of vascular smooth muscle cells is accompanied by changes in ryanodine receptors (RyRs). In several studies, the cytosolic $Ca^{2+}$ response to caffeine is decreased during smooth muscle cell culture. Although caffeine is commonly used to investigate RyR function because it is difficult to measure $Ca^{2+}$ release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated $Ca^{2+}$ entry. Using freshly dissociated rat aortic smooth muscle cells (RASMCs) and cultured RASMCs, we sought to provide direct evidence for the operation of RyRs through the $Ca^{2+}$- induced $Ca^{2+}$ -release pathway by directly measuring $Ca^{2+}$ release from SR in permeabilized cells. An additional goal was to elucidate alterations of RyRs that occurred during culture. Perfusion of permeabilized, freshly dissociated RASMCs with $Ca^{2+}$ stimulated $Ca^{2+}$ release from the SR. Caffeine and ryanodine also induced $Ca^{2+}$ release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and $Ca^{2+}$ failed to trigger $Ca^{2+}$ release in cultured RASMCs. These results are consistent with results obtained by immunocytochemistry, which showed that RyRs were expressed in dissociated RASMCs, but not in cultured RASMCs. This study is the first to demonstrate $Ca^{2+}$ release from the SR by cytosolic $Ca^{2+}$ elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.40-43
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• 2000

Pyrrolidinedithiocarbamate (PDTC) and N-Acetylcysteine (NAC) are metal and nonmetal-chelating antioxidant which can induce rat and human smooth muscle cell death. When the smooth muscle cells from mouse aorta (MASMC) that we successfully cultured recently was exposed to PDTC and NAC in a normal serum state, the cells were induced to death by these compounds. However, PDTC did not induce the cell death in a serum depleted medium. This data suggests that certain factors in the serum may mediate the cytotoxic effect of PDTC. The metal chelator, Ca-EDTA blocked PDTC-induced cell death, but Cu-, Fe-, and Zn-EDTA did not block the PDTC-induced cell death. This data indicated that copper, iron, and zinc in the serum may lead to the cytotoxic effect of PDTC. Investigation of the intracellular zinc level in PDTC-induced smooth muscle cell death using the zinc probe dye N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide shows that only the muscle-containing layers of the arteries have higher level of zinc. As expected, PDTC increased the intracellular fluorescence level of the zinc. In agreement with these results, the addition of an exogenous metal, zinc, induced the vascular aortic smooth muscle cell death which led to an increased intracellular zinc level. We concluded that PDTC induced mouse aortic smooth muscle cell death required not only zinc level but also intracellular copper and iron level. The mechanism of this antioxidant to induce vascular smooth muscle cell death may provide a new strategy to prevent their proliferation in arteriosclerotic lesions.

• BMB Reports
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• v.46 no.11
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• pp.550-554
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• 2013

The platelet-derived growth factor (PDGF) signaling pathway is essential for inducing a dedifferentiated state of vascular smooth muscle cells (VSMCs). Activation of PDGF inhibits smooth muscle cell (SMC)-specific gene expression and increases the rate of proliferation and migration, leading to dedifferentiation of VSMCs. Recently, microRNAs have been shown to play a critical role in the modulation of the VSMC phenotype in response to extracellular signals. However, little is known about microRNAs regulated by PDGF in VSMCs. Herein, we identify microRNA- 15b (miR-15b) as a mediator of VSMC phenotype regulation upon PDGF signaling. We demonstrate that miR-15b is induced by PDGF in pulmonary artery smooth muscle cells and is critical for PDGF-mediated repression of SMC-specific genes. In addition, we show that miR-15b promotes cell proliferation. These results indicate that PDGF signaling regulates SMC-specific gene expression and cell proliferation by modulating the expression of miR-15b to induce a dedifferentiated state in the VSMCs.

• Journal of Nutrition and Health
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• v.49 no.1
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• pp.59-62
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• 2016

Purpose: Zinc, a biomineral present within and outside cells, manages various cellular mechanisms. In this study, we examined whether zinc was involved in vascular smooth muscle cell (VSMC) calcification via regulation of calcification inhibitor protein, osteopontin (OPN). Methods: Rat aorta cell line (A7r5 cells) and primary vascular smooth muscle cells (pVSMCs) from rat aorta were cultured with phosphate (1-5 mM) and zinc ($0-15{\mu}M$) as appropriate, along with osteoblasts (MC3T3-E1) as control. The cells were then stained for Ca and P deposition for calcification examination as well as osteopontin expression as calcification inhibitor protein was measured. Results: Both Ca and phosphate deposition increased as the addition of phosphate increased. In the same manner, the expression of osteopontin was upregulated as the addition of phosphate increased in both cell types. When zinc was added, Ca and P deposition decreased in VSMCs, while it increased in osteoblasts. Conclusion: The results imply that zinc may prevent VSMC calcification by stimulating calcification inhibitor protein OPN synthesis in VSMCs.

• Molecules and Cells
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• v.41 no.3
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• pp.198-206
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• 2018

Aortic dissection (AD) is a catastrophic disease with high mortality and morbidity, characterized with fragmentation of elastin and loss of smooth muscle cells. Although AD has been largely attributable to polymorphisms defect in the elastin-coding gene, tropoelastin (TE), other undermined factors also appear to play roles in AD onset. Here, we investigated the effects of post-transcriptional control of TE by microRNAs (miRNAs) on elastin levels in aortic smooth muscle cells (ASMC). We found that miR-144-3p is a miRNA that targets TE mRNA in both human and mouse. Bioinformatics analyses and dual luciferase reporter assay showed that miR-144-3p inhibited protein translation of TE, through binding to the 3'-UTR of the TE mRNA. Interestingly, higher miR-144-3p levels and lower TE were detected in the ASMC obtained from AD patients, compared to those from non-AD controls. In a mouse model for human AD, infusion of adeno-associated viruses (serotype 6) carrying antisense for miR-144-3p (asmiR-144-3p) under CAG promoter significantly reduced the incidence and severity of AD, seemingly through enhancement of TE levels in ASMC. Thus, our data suggest an essential role of miR-144-3p on the pathogenesis of AD.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.274.1-274.1
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• 2013

We report the circumferential alignment of human aortic smooth muscle cells (HASMCs) in an orthogonally micropatterned circular microfluidic channel to form an in vivo-like smooth muscle cell layer. To realize a biomimetic smooth muscle cell layer which is aligned perpendicular to the axis of blood vessel, we first fabricated a half-circular polydimethylsiloxane (PDMS) microchannel by soft lithography using a convex PDMS mold. The orthogonally micro wrinkle patterns were generated inside the half-circular microchannel by stretching-releasing operation under UV irradiation. Upon UV treatment with uniaxial 40 % stretch of a PDMS substrate and releasing process, the microwrinkle patterns perpendicular to the axial direction of the circular microchannel were generated, which could guide the circumferential alignment of HASMCs successfully during cultivation. The analysis of orientation angle, shape index, and contractile protein marker expression indicates that the cultured HASMCs revealed the in vivo-like cell phenotype. Finally, we produced circular microchannels by bonding two half-circular microchannels, and cultured the HASMCs circumferentially with high alignment and viability for 5 days. These results are the first demonstration for constructing an in vivo-like 3D smooth muscle cell layer in the circular microfluidic channel which can provide novel bioassay platforms for in-depth study of HASMC biology and vascular function.

• Journal of Veterinary Science
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• v.24 no.5
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• pp.69.1-69.11
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• 2023

Background: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Objectives: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. Methods: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Results: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca⁺⁺-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runtrelated transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca²⁺-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. Conclusions: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.2
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• pp.123-129
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• 2009

Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-$l\beta$ plus TNF-$\alpha$), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-l induction in rat VSMCs. Aprotinin induced HO-l protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-l inhibitor, tin protoporphyrin IX (SnPPIX). HO-l is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.