1.Role of non-coding RNAs in vascular smooth muscle cell calcification.
Acta Physiologica Sinica 2022;74(6):885-893
Vascular calcification, the deposition of calcium in the arterial wall, is often linked to increased stiffness of the vascular wall. Vascular calcification is one of the important factors for high morbidity and mortality of cardiovascular and cerebrovascular diseases, as well as an important biomarker in atherosclerotic cardiovascular events, stroke and peripheral vascular diseases. The mechanism of vascular calcification has not been fully elucidated. Recently, non-coding RNAs have been found to play an important role in the process of vascular calcification. In this paper, the main types of non-coding RNAs and their roles involved in vascular smooth muscle cell calcification are reviewed, including the changes of osteoblast-related proteins, calcification signaling pathways and intracellular Ca2+.
Humans
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Muscle, Smooth, Vascular/metabolism*
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Vascular Calcification/metabolism*
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Myocytes, Smooth Muscle/metabolism*
2.Bioinformatics analysis identifies aging/senescence-induced genes in calcified plaques.
Hai-Peng YAO ; Yong-Jiang QIAN ; Zhong-Qun WANG
Acta Physiologica Sinica 2022;74(6):939-948
Vascular calcification is an important pathophysiological basis of cardiovascular disease with its underlying mechanism unclear. In recent years, studies have shown that aging is one of the risk factors for vascular calcification. The purpose of this study was to investigate the microenvironmental characteristics of vascular calcification, identify aging/senescence-induced genes (ASIGs) closely related to calcified plaques, and explore the evolution trajectory of vascular calcification cell subsets. Based on the bioinformatics method, the single cell transcriptome sequencing data (Gene Expression Omnibus: GSE159677) of carotid artery samples from 3 patients undergoing carotid endarterectomy were grouped and annotated. Vascular calcification-related aging genes were identified by ASIGs data set. The pseudotime trend of ASIGs in cell subsets was analyzed by Monocle 3, and the evolution of vascular calcification cells was revealed. After quality control, all cells were divided into 8 cell types, including B cells, T cells, smooth muscle cells, macrophages, endothelial cells, fibroblasts, mast cells, and progenitor cells. Ten ASIGs related to vascular calcification were screened from the data set of ASIGs, which include genes encoding complement C1qA (C1QA), superoxide dismutase 3 (SOD3), lysozyme (LYZ), insulin-like growth factor binding protein-7 (IGFBP7), complement C1qB (C1QB), complement C1qC (C1QC), Caveolin 1 (CAV1), von Willebrand factor (vWF), clusterin (CLU), and αB-crystallin (CRYAB). Pseudotime analysis showed that all cell subsets were involved in the progression of vascular calcification, and these ASIGs may play an important role in cell evolution. In summary, AGIS plays an important role in the progression of vascular calcification, and these high expression genes may provide ideas for early diagnosis and treatment of vascular calcification.
Humans
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Endothelial Cells
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Muscle, Smooth, Vascular
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Aging
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Vascular Calcification/metabolism*
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Computational Biology
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Myocytes, Smooth Muscle/metabolism*
3.Inhibitory mechanism of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells.
Xiao-Jun BAI ; Yan LIU ; Shan-Shan GAO ; Wei-Ping ZHANG
China Journal of Chinese Materia Medica 2021;46(17):4497-4503
This study aimed to observe the inhibitory effect of icariin against oxidative stress-induced calcification in aortic vascular smooth muscle cells(VSMCs) and elucidate the molecular mechanism of icariin in inhibiting endoplasmic reticulum stress(ERS)-mediated atherosclerotic calcification, so as to provide new ideas for exploring the anti-atherosclerotic mechanism of Epimedii Folium. The VSMCs in rat thoracic aorta were subjected to adherent culture and then treated with the complete calcification DMEM containing high glucose and hydrogen peroxide(H_2O_2) for three weeks. The resulting calcified VSMCs were divided into different treatment groups. Icariin was added one week after calcification induction for protecting the VSMCs, whose viability was then detected using cell counting kit-8(CCK-8). Alizarin red-S staining was conducted to observe the calcification degree. The activity of alkaline phosphatase(ALP) in VSMCs was measured using the disodium phenyl phosphate substrate and the calcium content was measured by arsenazo Ⅲ method. The mRNA expression levels of ossification-related factors including osteocalcin(OC), osteopontin(OPN), Runt-related transcription factor 2(Runx2), and type Ⅰ collagen(Col Ⅰa) were detected by real-time PCR. Western blot was carried out to determine the protein expression levels of α-smooth muscle actin(α-SMA), Runx2, activating transcription factor 4(ATF4), and eukaryotic translation initiation factor(eIF)-2α. The results showed that H_2O_2 significantly induced the calcification of VSMCs, increased the ALP activity and calcium content in VSMCs, promoted OC, OPN, Runx2, and Col Ⅰa mRNA expression and Runx2 protein expression, and reduced α-SMA protein expression. The ATF4 protein expression and eIF2α phosphorylation were also elevated significantly. Icariin reversed the calcification of VSMCs induced by H_2O_2, inhibited ALP activity and calcium content in VSMCs, down-regulated the mRNA expression levels of OC, OPN, Runx2 and Col Ⅰa and Runx2 protein expression, and relatively up-regulated the expression of α-SMA. The expression of ATF4 and phosphorylation of eIF2α also declined significantly. All these have demonstrated that icariin inhibited VSMCs calcification by down-regulating the ossification-related factors and lowering ALP activity and calcium content in VSMCs. Besides, the down-regulation of Runx2 expression and the inhibition of ATF4 and eIF2α-mediated cellular calcification pathway in ERS might also be involved in such calcification-suppressing process.
Animals
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Cells, Cultured
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Flavonoids/pharmacology*
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Muscle, Smooth, Vascular/metabolism*
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Myocytes, Smooth Muscle
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Oxidative Stress
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Rats
4.Effects of Neostigmine on Tracheal Smooth Muscle Contraction in Rabbits.
Tae Hyun KIM ; Jae Young SHIM ; Hue Jung PARK ; Ou Kyoung KWON
Korean Journal of Anesthesiology 2001;41(1):71-76
BACKGROUND: Neostigmine, a cholinesterase inhibitor, is known to reverse the neuromuscular blocking action induced by nondepolarizing muscle relaxants at the end of general anesthesia. Some authors, however, reported that neostigmine has the properties of a neuromuscular block in skeletal muscles while others reported that neostigmine caused the smooth muscles such as the diaphragm to relax rather than to contract. The purpose of this study was to evaluate the effect of neostigmine at different doses on the tracheal smooth muscle in rabbits. METHODS: Isolated tracheal ring preparation in rabbits was used. Groups were divided into 7 groups; acetylcholine group (acetylcholine cumulative administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), neostigmine group (neostigmine cumulative administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), acetylcholine 10 6 M + neostigmine group (acetylcholine 10 6 M prior to neostigmine administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), acetylcholine 10 4 M + neostigmine group (acetylcholine 10 4 M prior to neostigmine administered at doses of 10 8, 10 7, 10 6, 10 5, 10 4 and 10 3 M), neostigmine 10 5, 10 4 and 10 3 M groups (neostigmine administered at doses of 10 5, 10 4 and 10 3 M). Smooth muscle contraction was evaluated in isometric tension per gram of tissue. RESULTS: In the acetylcholine group, the contractions increased as the dosage increased (10 8 10 3 M). In the neostigmine group, the contractions increased as the dosage increased (10 8 10 4 M), but at 10 3 M of neostigmine, contractions suddenly decreased. In addition when acetylcholine 10 6 M was given as a pretreatment, there was a sudden decrease in muscle contractions induced by neostigmine at 10 3 M. Also the contractions induced by 10 3 M neostigmine were less than that of 10 4 and 10 5 M. CONCLUSIONS: We concluded that neostigmine caused smooth muscle contraction at low concentrations by blocking acetylcholine metabolism, but at high concentrations, smooth muscle contractions were decreased and this might be due to direct action at the acetylcholine receptor.
Acetylcholine
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Anesthesia, General
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Cholinesterases
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Diaphragm
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Metabolism
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Muscle Contraction
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Muscle, Skeletal
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Muscle, Smooth*
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Neostigmine*
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Neuromuscular Blockade
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Rabbits*
5.Advance in study of vascular endothelial cell and smooth muscle cell co-culture system.
Yujie LI ; Qing YANG ; Xiaogang WENG ; Ying CHEN ; Congxiao RUAN ; Dan LI ; Xiaoxing ZHU
China Journal of Chinese Materia Medica 2012;37(3):265-268
The interactions between endothelial cells (EC) and smooth muscle cells (SMC) contribute to vascular physiological functions and also cause the occurrence and development of different kinds of diseases. Currently, EC-SMC co-culture model is the best way to study the interactions between the two kinds of cells. This article summarizes existing EC-SMC co-culture models and their effects on the structure and functions of the two kinds of cells. Microscopically speaking, it provides a basis for in-depth studies on their interactions as well as a reference for the establishment of in vitro EC-SMC co-culture system that is closer to organic physiology or pathology state.
Animals
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Coculture Techniques
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methods
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Endothelial Cells
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cytology
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metabolism
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Humans
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Muscle, Smooth, Vascular
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cytology
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Myocytes, Smooth Muscle
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cytology
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metabolism
6.Chronic psychological stress exacerbates aortic medial calcification via glucocorticoids.
Yan-Qing LI ; Pan-Na HUANG ; Hao-Zhe ZHANG ; Lu-Yu HAN ; Ruo-Xiang MIAO ; Wan-Yun FENG ; Hua PAN ; Lin FENG ; Xin-Hua WU ; Juan HE ; Xu TENG ; Xiao-Ning WANG
Acta Physiologica Sinica 2022;74(6):927-938
Chronic psychological stress can promote vascular diseases, such as hypertension and atherosclerosis. This study aims to explore the effects and mechanism of chronic psychological stress on aortic medial calcification (AMC). Rat arterial calcification model was established by nicotine gavage in combination with vitamin D3 (VitD3) intramuscular injection, and rat model of chronic psychological stress was induced by humid environment. Aortic calcification in rats was evaluated by using Alizarin red staining, aortic calcium content detection, and alkaline phosphatase (ALP) activity assay. The expression levels of the related proteins, including vascular smooth muscle cells (VSMCs) contractile phenotype marker SM22α, osteoblast-like phenotype marker RUNX2, and endoplasmic reticulum stress (ERS) markers (GRP78 and CHOP), were determined by Western blot. The results showed that chronic psychological stress alone induced AMC in rats, further aggravated AMC induced by nicotine in combination with VitD3, promoted the osteoblast-like phenotype transformation of VSMCs and aortic ERS activation, and significantly increased the plasma cortisol levels. The 11β-hydroxylase inhibitor metyrapone effectively reduced chronic psychological stress-induced plasma cortisol levels and ameliorated AMC and aortic ERS in chronic psychological stress model rats. Conversely, the glucocorticoid receptor agonist dexamethasone induced AMC, promoted AMC induced by nicotine combined with VitD3, and further activated aortic ERS. The above effects of dexamethasone could be inhibited by ERS inhibitor 4-phenylbutyrate. These results suggest that chronic psychological stress can lead to the occurrence and development of AMC by promoting glucocorticoid synthesis, which may provide new strategies and targets for the prevention and control of AMC.
Rats
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Animals
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Glucocorticoids/metabolism*
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Rats, Sprague-Dawley
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Nicotine/metabolism*
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Hydrocortisone/metabolism*
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Muscle, Smooth, Vascular
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Dexamethasone/metabolism*
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Vascular Calcification/metabolism*
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Myocytes, Smooth Muscle/metabolism*
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Cells, Cultured
7.Effects of oxidative stress on the expression of aldose reductase in vascular smooth muscle cells.
Hyo Jung KIM ; Ki Churl CHANG ; Han Geuk SEO
The Korean Journal of Physiology and Pharmacology 2001;5(3):271-278
Oxidative stress and methylglyoxal (MG), a reactive dicarbonyl metabolites produced by enzymatic and non-enzymatic reaction of normal metabolism, induced aldose reductase (AR) expression in rat aortic smooth muscle cells (SMC). AR expression was induced in a time-dependent manner and reached at a maximum of 4.5-fold in 12 h of MG treatment. This effect of MG was completely abolished by cyclohemide and actinomycin D treatment suggesting AR was synthesized by de novo pathway. Pretreatment of the SMC with N-acetyl-L-cysteine significantly down-regulated the MG-induced AR mRNA. Furthermore, DL-Buthionine-(S,R)-sulfoximine, a reagent which depletes intracellular glutathione levels, increased the levels of MG-induced AR mRNA. These results indicated that MG induces AR mRNA by increasing the intracellular peroxide levels. Aminoguanidine, a scanvenger of dicarbonyl, significantly down-regulated the MG-induced AR mRNA. In addition, the inhibition of AR activities with statil, an AR inhibitor, enhanced the cytotoxic effect of MG on SMC under normal glucose, suggesting a protective role of AR against MG-induced cell damages. These results imply that the induction of AR by MG may contribute to an important cellular detoxification of reactive aldehyde compounds generated under oxidative stress in extrahepatic tissues.
Acetylcysteine
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Aldehyde Reductase*
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Animals
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Dactinomycin
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Glucose
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Glutathione
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Metabolism
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Muscle, Smooth, Vascular*
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Myocytes, Smooth Muscle
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Oxidative Stress*
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Pyruvaldehyde
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Rats
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RNA, Messenger
8.1, 25-Dihydroxyvitamin D3 increases in vitro vascular calcification through calcified blood vessel cells.
Yu-Mei WANG ; Shi-Wen WANG ; Wan-Ren TONG
Chinese Journal of Applied Physiology 2002;18(2):162-165
AIMAccumulated evidence suggest that the development of vascular calcification is similar to osteogenesis. Here we want to elucidate the effect of the common used osteo-regulatory factor 1,25(OH)2D3 on vascular calcification.
METHODS AND RESULTSAdding 10(-9) mol/L to the culture media 1,25(OH)2D3 time dependently increased the calcium deposition on the in vitro calcification of bovine vascular smooth muscle cells (BVSMCs) induced by beta-GP. It also increased cellular alkaline phosphatase activity by 301.1% during the calcified process. Osteocalcin, one of the osteogenic specific metric proteins, was dramatically elevated by 58.3% during the calcified processes, which indicate the transformation of BVSMCs to osteoblastic cell. 1,25(OH)2D3 had no such effect on non-calcified BVSMCs.
CONCLUSIONThese data suggest that 1,25(OH)2D3 exerts a stimulatory effect on vascular calcification through increasing the synthesis of ALP. This effect shares the same character as osteoblast cells. This effect is limited to the calcified prone vascular cell.
Animals ; Calcitriol ; metabolism ; Cattle ; Cells, Cultured ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteocalcin ; metabolism ; Vascular Calcification ; metabolism ; pathology ; Vitamin D ; analogs & derivatives ; pharmacology
9.Testosterone at physiological level inhibits PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells.
Rui MA ; Shi-Sen JIANG ; Xun-Min CHENG ; Jian-Bin GONG ; Qi-Gao ZHANG ; Qi-Shui LIN
National Journal of Andrology 2009;15(4):326-330
OBJECTIVETo explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).
METHODSVSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.
RESULTSThe resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).
CONCLUSIONTestosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.
Animals ; Calcium ; metabolism ; Cells, Cultured ; Dinoprost ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Myocytes, Smooth Muscle ; metabolism ; Rats ; Rats, Sprague-Dawley ; Testosterone ; metabolism ; physiology
10.Effect of homocysteine on NF-kappaB activity in cultured rat vascular smooth muscle cells.
Liang-rong ZHENG ; Jian-hua ZHU ; Li ZHANG
Journal of Zhejiang University. Medical sciences 2004;33(3):255-268
OBJECTIVETo investigate the effect of homocysteine (Hcy) on nuclear factor-kappaB (NF-kappaB) activity in cultured rat vascular smooth muscle cells (VSMCs).
METHODSRat VSMCs were stimulated with 0.25 mmol/L Hcy. Cells were collected and nuclear protein was extracted at 30 min, 1 h and 2 h following stimulation. NF-kappaB activity was examined by electrophoretic mobility shift assay (EMSA).
RESULTBaseline NF-kappaB nuclear binding in VSMCs increased more than 1.76-fold at 30 min (P<0.01), 1.91-fold at 1h (P<0.01) and 1.84-fold at 2 h (P<0.01) following stimulation of Hcy.
CONCLUSIONHcy can enhance transient mobilization of N-kappaB in VSMCs, which suggests that some regulating effect of Hcy on VSMCs might be exerted through NF-kappaB activation pathway.
Animals ; Arteriosclerosis ; etiology ; Electrophoretic Mobility Shift Assay ; Female ; Homocysteine ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; NF-kappa B ; metabolism ; Rats ; Rats, Sprague-Dawley