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1.
Summary The interactions of vascular smooth muscle cells with growth modulators and extracellular matrix molecules may play a role in the proliferation and migration of these cells after vascular injury and during the development of atherosclerosis. Time-lapse cinematographic techniques have been used to study cell division and migration of bovine carotid artery smooth muscle cells in response to matrix molecules consisting of solubilized basement membrane (Matrigel) and type I collagen. When cells were grown adjacent to Matrigel, both migration and cell proliferation were increased and interdivision time was shortened. Cells grown in Matrigel or in type I collagen had markedly reduced migration rates but interdivision time was not altered. Further, diffusible components of the Matrigel were found to stimulate proliferation of the smooth muscle cells. This work was supported by grants HL35684 and SCOR HL14212 from the National Institutes of Health, Bethesda, MD. 相似文献
2.
Mary P. McMahon Barbara Faris B. Leslie Wolfe Karen E. Brown Curtis A. Pratt Paul Toselli Carl Franzblau 《In vitro cellular & developmental biology. Plant》1985,21(12):674-680
Summary Elastin accumulation in the extracellular matrix of cultured rat aortic smooth muscle cells was monitored as a function of age. The effect of the animal donor age and time in culture in single or consecutive passages on the cells’ ability to accumulate total protein as well as elastin was evaluated. Smooth muscle cells were obtained from animals ranging in age from 2 d to 36 mo. Protein accumulation by the cells based on DNA content was similar regardless of which of the above aging parameters was examined. Although there were significant amounts of elastin present in the extracellular matrix of those cells originating from the younger animals (2 d and 6 wk old), little or none was detected in cell cultures derived from the oldest animals. A soluble elastin-like fraction which was isolated from the cultures of the 2-d-old rats seemed to be lacking in the cultures of cells from the 36-mo-old animals. This observation may, in part, explain the absence of insoluble elastin in the matrix of some cultures obtained from older animals. The data strongly suggest that the age of the donor animal from which the cells originate has the greatest influence on in vitro elastin accumulation. This study was supported by National Institutes of Health Grants HL 19717 and HL 13262. 相似文献
3.
Shulian Chen Banghua Liao Xi Jin Tangqiang Wei Qing He Yifei Lin Jianzhong Ai Lina Gong Hong Li Kunjie Wang 《Journal of cellular biochemistry》2020,121(11):4496-4504
Extracellular matrix (ECM) accumulation plays a key role in the progression of bladder outlet obstruction (BOO). Muscarinic receptors have been widely reported to serve as pivotal regulators in lung tissue remodeling. However, the influence of them on human bladder smooth muscle cells (HBSMCs) and the underlying molecular mechanisms have not yet been evaluated. The purposes of the present study are to investigate the effect of muscarinic receptors on the synthesis of ECM in HBSMCs and the involvement of intracellular signal transducers. The results indicated that M1-M5 muscarinic receptors were all encoded in HBSMCs. The expression rank order was M2 > M1 > M5 > M3 > M4. The gene and protein expression of collagen I (COL1), TIMP-1, and TIMP-2 was carbachol (CCH) concentration-dependently enhanced. The synthesis of COL1 in the supernatant of cell culture medium was significantly elevated by exposure to CCH. The CCH-induced protein expression of COL1, TIMP-1, and TIMP-2, however, was obviously reduced by the pretreatment of muscarinic receptor antagonists, atropine, and M3-preferring antagonist (1,1-dimethyl-4-diphenyl-acetoxypiperidinium iodide [4-DAMP]). Furthermore, ERK1/2 was activated by 100 µM CCH when compared with the control group and the pretreatment of ERK1/2 inhibitor significantly suppressed the synthesis of COL1 induced by 100 µM CCH. Besides, CCH-induced phosphorylation of ERK1/2 was remarkably restrained by the pretreatment of 4-DAMP. All in all, these findings demonstrated that M3 receptor can modulate extracellular matrix synthesis via the ERK1/2 signaling pathway, which may provide potential novel therapeutic targets for BOO. 相似文献
4.
Joseph A. Skrivanek Elaine Schwartz Olga O. Blumenfeld Robert W. Ledeen 《In vitro cellular & developmental biology. Plant》1990,26(5):502-506
Summary The ganglioside composition of calf aortic smooth muscle cells, cultured in the presence and absence of ascorbate, was analyzed.
Previous work has shown that ascorbate supplementation leads to the formation of an extracellular matrix consisting primarily
of collagen and that this matrix influences the biosynthetic capabilities of the cell. Cell cultures were supplemented with
ascorbate for 3 wk and labeled with [14C]glucosamine for 3 d before harvesting. Ascorbate supplementation resulted in increased ganglioside sialic acid levels and
a change in chromatographic profile involving both absolute and relative increases in GD1a. The latter, along with polysialo
species, showed increased incorporation of [14C]glucosamine. These findings are interpreted in relation to the proposed role of gangliosides as mediators in the interaction
of various cells with extracellular matrix.
This work was supported by grants 1-P01-AG05554 and 2-R01-NS-04834 from the Public Health Service, Washington, DC, as well
as a Presidential Junior Faculty Development Award (JAS) from the Purchase College Foundation. 相似文献
5.
Lysyl oxidase (LO) plays a critical role in the stabilization and insolubilization of fibrous structural proteins of the extracellular matrix and has been implicated in the suppression of Ras-induced tumorigenesis. Several prior reports demonstrate that the expression of this catalyst is strongly influenced by a variety of effectors of cell function and is responsive to the growth state of fibrogenic cells. Using specific inhibitors of components of signal transduction pathways, the present study reveals that a PKC-MEK-MAPK-dependent pathway is critical to the enhanced expression of the LO gene in response to variations in the levels of the serum component of the growth medium and in response to platelet-derived growth factor (PDGF). PDGF is shown to be the major component of fetal bovine serum, which stimulates the activity of a LO promoter construct. 相似文献
6.
Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta 总被引:1,自引:0,他引:1
Jan-Kan Chen Hiroyoshi Hoshi Wallace L. McKeehan 《In vitro cellular & developmental biology. Animal》1991,27(1):6-12
Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis
in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan
sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some
extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan.
Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory
effect on either class of [35S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate
of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized
little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans
into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of
TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation
of [35S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented
indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by
promoting the core protein synthesis.
Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA
37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291). 相似文献
7.
Roy J Tran PK Religa P Kazi M Henderson B Lundmark K Hedin U 《Experimental cell research》2002,273(2):169-177
Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G(0)/G(1) phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27(KIP1) in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27(KIP1) levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors. 相似文献
8.
Al-Fakhri N Wilhelm J Hahn M Heidt M Hehrlein FW Endisch AM Hupp T Cherian SM Bobryshev YV Lord RS Katz N 《Journal of cellular biochemistry》2003,89(4):808-823
Regulation of alphavbeta3 and alpha5beta1 integrin function plays a crucial role in atherosclerosis. Possible regulators of integrin-matrix interactions are integrin-binding ADAMs (proteins with a disintegrin- and metalloproteinase-domain), like ADAM-15 and ADAM-9. Molecular interactions between ADAM-15, alpha5beta1, and alphavbeta3 have been demonstrated. ADAM-9 and ADAM-15 were found to be interdependently regulated. This study, therefore, investigated whether the upregulation of integrins alpha5beta1 and alphavbeta3 was correlated with the expression of integrin-binding ADAMs in atherosclerotic processes. Human arterial and venous vascular smooth muscle cells (VSMCs) were incubated with PDGF over different time intervals up to a 3-day culture period. mRNA concentrations, quantified by real-time RT-PCR and normalized to PBGD, of integrins alphavbeta3 and alpha5beta1 were strongly increased after a 12-h PDGF-incubation in arterial and venous VSMC. ADAM-15 and ADAM-9 mRNA production showed a corresponding increase following integrin upregulation after a 24-h incubation period. Western blot anaylsis revealed an increased protein expression of integrins and ADAMs in PDGF-stimulated VSMC. Additionally, mRNA concentrations of atherosclerotic and normal human specimens were quantified by real-time RT-PCR. mRNA of ADAMs and integrins was significantly increased in atherosclerotic arteries compared to normal arteries. Immunohistochemistry of these specimens showed an increased expression and codistribution of both ADAMs and integrins in atherosclerosis. In conclusion, upregulation of ADAM-15 and ADAM-9 in atherosclerosis appears to follow an increase in alpha5beta1 and alphavbeta3 integrins. Since alpha5beta1 and alphavbeta3 are known to promote smooth muscle cell migration and proliferation, upregulation of ADAM-15 and ADAM-9 could balance integrin-matrix interactions and cell migration, thus modulating neointima progression. 相似文献
9.
Fawzy A. Saad Marie Torres Lila Graham 《Biochemical and biophysical research communications》2010,396(4):944-949
LOX, the principal enzyme involved in crosslinking of collagen, was the first of several lysyl oxidase isotypes to be characterized. Its active form was believed to be exclusively extracellular. Active LOX was later reported to be present in cell nuclei; its function there is unknown. LOX expression opposes the effect of mutationally activated Ras, which is present in about 30% of human cancers. The mechanism of LOX in countering the action of Ras is also unknown. In the present work, assessment of nuclear protein for possible effects of lysyl oxidase activity led to the discovery that proliferating cells dramatically increase their nuclear protein content when exposed to BAPN (β-aminopropionitrile), a highly specific lysyl oxidase inhibitor that reportedly blocks LOX inhibition of Ras-induced oocyte maturation. In three cell types (PC12 cells, A7r5 smooth muscle cells, and NIH 3T3 fibroblasts), BAPN caused a 1.8-, 1.7-, and 2.1-fold increase in total nuclear protein per cell, respectively, affecting all major components in both nuclear matrix and chromatin fractions. Since nuclear size is correlated with proliferative status, enzyme activity restricting nuclear growth may be involved in the lysyl oxidase tumor suppressive effect. Evidence is also presented for the presence of apparent lysyl oxidase isotype(s) containing a highly conserved LOX active site sequence in the nuclei of PC12 cells, which do not manufacture extracellular lysyl oxidase substrates. Results reported here support the hypothesis that nuclear lysyl oxidase regulates nuclear growth, and thereby modulates cell proliferation. 相似文献
10.
Phillip J. Stone Mary P. McMahon Shirley M. Morris James D. Calore Carl Franzblau 《In vitro cellular & developmental biology. Plant》1987,23(10):663-676
Summary A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model
substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the
presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up
to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and
lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer,
HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the
purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained
as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59%
that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding
cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein
for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture
system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently
into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory
fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that
little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture
system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema.
Supported by National Heart, Lung and Blood Institute, Bethesda, MD, grants NIH-HL-25229, HL-19717, and HL-33522. Presented
in part at the April 1985 meeting of the Federation of American Societies for Experimental Biology. 相似文献
11.
目的:建立改进大鼠气道平滑肌细胞(ASMC)的体外培养方法,为相关研究提供实验材料。方法:将组织块连续贴壁进行细胞原代培养,胰酶消化传代培养,差速贴壁进行细胞纯化,形态学及免疫细胞化学染色法进行细胞鉴定。MTT法检测PDGF-BB诱导的ASMC增殖。结果:成功培养大鼠ASMC,以改良组织块消化法最为理想。第四代平滑肌细胞纯度可达95%以上。相差显微镜下培养细胞呈典型“峰谷状”生长。免疫荧光化学染色显示特异性平滑肌肌动蛋白阳性表达。随着PDGF浓度的升高(2-80ng/ml),MTT比色A490值呈上升趋势。与对照组相比较,80ng/ml、20ng/ml PDGF—BB组有统计学意义(P〈0.01)。结论:改良组织块消化法可缩短培养周期,在充分利用标本的基础上获得大量气道平滑肌细胞。 相似文献
12.
Kanie K Narita Y Zhao Y Kuwabara F Satake M Honda S Kaneko H Yoshioka T Okochi M Honda H Kato R 《Biotechnology and bioengineering》2012,109(7):1808-1816
Controlling the balance of endothelial cells (ECs) and smooth muscle cells (SMCs) in blood vessels is critically important to minimize the risk associated with vascular implants. Extracellular matrix (ECM) plays a key role in controlling the cellular balance, suggesting a promising source of cell-selective peptides. To obtain EC- or SMC-selective peptides, we start by highlighting sequence differences found among ECM molecules as enriched targets for cell-selective peptides. We explored the EC- or SMC-selective performance of tripeptides that are specifically enriched only in collagen type IV, but not in types I, II, III, and V. Collagen type IV was chosen since it is the major ECM in the basement membrane of blood vessels, which separates ECs and SMCs. Among 114 collagen type IV-derived tripeptides pre-screened from in silico analysis, 22 peptides (19%) were found to promote cell-selective adhesion, as determined by peptide array. One of the best performing EC-selective peptides (Cys-Ala-Gly (CAG)) was mixed into an electrospun fine-fiber, a vascular graft material, for practical application. Compared to unmodified fiber, the CAG containing fiber surface was found to enhance adhesion of ECs (+190%) while limiting SMCs (-20%). These results are not only consistent with the hypothesis of ECM as a source of cell selective peptides, but also suggest a new genre of EC- or SMC-selective peptides for tissue engineering applications. Collectively, these findings favorably support the screening approach used to discover new peptides for these purposes. 相似文献
13.
Indrani Sinha‐Hikim Ruoqing Shen Ekaterina Kovacheva Albert Crum Nosratola D. Vaziri Keith C. Norris 《Cell biology international》2010,34(5):503-511
CKD (chronic kidney disease) is a public health problem, mediated by haemodynamic and non‐haemodynamic events including oxidative stress. We investigated the effect of two GSH (glutathione) precursors, NAC (N‐acetylcysteine) and cystine as the physiological carrier of cysteine in GSH with added selenomethionine (F1) in preventing spermine (uraemic toxin)‐induced apoptosis in cultured human aortic VSMC (vascular smooth muscle cells). VSMCs exposed to spermine (15 μM) with or without antioxidants (doses 50, 100, 200 and 500 μg/ml) were assessed for apoptosis, JNK (c‐Jun‐NH2‐terminal kinase) activation and iNOS (inducible nitric oxide synthase) induction and activation of intrinsic pathway signalling. Spermine exposure resulted in activation of JNK and iNOS induction and apoptosis. NAC and F1 (dose range 50–500 μg/ml) attenuated spermine‐induced acceleration of VSMC apoptosis but only F1 (at 200 and 500 μg/ml) maintained spermine‐induced apoptosis at control levels. Spermine‐induced JNK activation was prevented by 200 μg/ml of both NAC and F1, while iNOS induction was blocked only by F1. Notably, the adverse effects of spermine on BAX/BCL‐2 ratio, cytochrome c release and caspase activation was fully attenuated by F1. In conclusion, F1 was more effective than NAC in preventing spermine‐induced apoptosis and downstream changes in related signal transduction pathways in VSMCs. Further studies are needed to examine the effect of these compounds in preventing CKD‐associated vascular disease. 相似文献
14.
Aortic stiffening is an independent risk factor that underlies cardiovascular morbidity in the elderly. We have previously shown that intrinsic mechanical properties of vascular smooth muscle cells (VSMCs) play a key role in aortic stiffening in both aging and hypertension. Here, we test the hypothesis that VSMCs also contribute to aortic stiffening through their extracellular effects. Aortic stiffening was confirmed in spontaneously hypertensive rats (SHRs) vs. Wistar‐Kyoto (WKY) rats in vivo by echocardiography and ex vivo by isometric force measurements in isolated de‐endothelized aortic vessel segments. Vascular smooth muscle cells were isolated from thoracic aorta and embedded in a collagen I matrix in an in vitro 3D model to form reconstituted vessels. Reconstituted vessel segments made with SHR VSMCs were significantly stiffer than vessels made with WKY VSMCs. SHR VSMCs in the reconstituted vessels exhibited different morphologies and diminished adaptability to stretch compared to WKY VSMCs, implying dual effects on both static and dynamic stiffness. SHR VSMCs increased the synthesis of collagen and induced collagen fibril disorganization in reconstituted vessels. Mechanistically, compared to WKY VSMCs, SHR VSMCs exhibited an increase in the levels of active integrin β1‐ and bone morphogenetic protein 1 (BMP1)‐mediated proteolytic cleavage of lysyl oxidase (LOX). These VSMC‐induced alterations in the SHR were attenuated by an inhibitor of serum response factor (SRF)/myocardin. Therefore, SHR VSMCs exhibit extracellular dysregulation through modulating integrin β1 and BMP1/LOX via SRF/myocardin signaling in aortic stiffening. 相似文献
15.
In this short review we describe the observations which have led us to conclude that one of the most important components involved in modulating cell proliferation in vitro, and probably in vivo as well, may be the extrac-cellular matrix upon which cells rest. 相似文献
16.
Jones SA Patterson JL Chao JT Ramos KS Wilson E 《Journal of cellular biochemistry》2004,91(6):1248-1259
Chronic oxidative injury by allylamine (AAM) induces proliferative vascular smooth muscle cell (vSMC) phenotypes in the rat aorta similar to those seen in rodent and human atherosclerotic lesions. The proliferative advantage of AAM vSMC compared to control cells is maintained with serial passage of the cells and the advantage is nullified when AAM cells are seeded on a collagen substrate. In this study, we evaluate the potential role of cyclin dependent kinase inhibitors, p27 and p21, and mitogen activated protein (MAP) kinases, ERK1/2, in mediating the proliferative advantage of AAM stressed vSMC over control cells on plastic or collagen substrates. p27 levels in randomly cycling cells were comparable in both cell types irrespective of the substrate. In contrast, basal levels of p21 were 1.9 +/- 0.3 (P < 0.05)-fold higher in randomly cycling AAM cells seeded on plastic compared to controls, a difference that was lost on a collagen substrate. Following G0 synchronization, basal levels of both p27 and p21 were higher in AAM cells seeded on plastic compared to controls (1.7 +/- 0.2 and 2.0 +/- 0.3-fold, respectively, P < 0.05), but these differences were lost upon mitogenic stimulation. Pyrrolidine dithiocarbamate (PDTC) decreased p27 and p21 levels in cycling AAM cells relative to controls in a substrate-dependent manner. AAM cells seeded on plastic exhibited enhanced ERK1/2 activation upon mitogenic stimulation; seeding on collagen nullified this advantage. The duration of ERK1/2 activation was prolonged in AAM cells independently of the seeding substrate. We conclude that substrate-dependent acquisition of proliferative phenotypes following repeated cycles of AAM injury correlates with modulation of the cyclin dependent kinase inhibitors, p27 and p21. 相似文献
17.
《Developmental cell》2022,57(20):2426-2443.e6
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18.
大鼠细小肺动脉平滑肌细胞原代培养和鉴定方法的研究 总被引:2,自引:0,他引:2
目的:建立一种重复性好、培养周期短及传代次数多的大鼠细小肺动脉平滑肌细胞(PASMCs)培养方法。方法:在无菌条件下,分离雄性SD大鼠肺细小动脉,剥离外膜和剔除内皮细胞,经胶原酶I消化,培养PASMCs。0.4%台盼蓝染色测定细胞活力;倒置相差显微镜观察;免疫细胞化学法和免疫荧光染色法,进行平滑肌α-肌动蛋白(α-SMactin)鉴定。结果:形态学观察、免疫细胞化学法及免疫荧光染色法鉴定表明培养细胞为PASMCs;细胞存活率在96.5%以上;原代培养后4~7d即可传代,并且生长特点、细胞形态不易发生改变。结论:采用胶原酶I消化法培养PASMCs,方法简单、酶消化时间易控制、培养周期短、重复性好,培养的原代PASMCs具有数量多和生长迅速的特点。 相似文献
19.
目的:建立一种操作简单、实验仪器要求低的大鼠肺细小动脉平滑肌细胞(PASMCs)分离和培养的方法,并且探索血小板衍生因子(PDGF)介导的增殖、迁移的情况。方法:向右心注射铁及琼脂糖,利用琼脂糖能同时粘附血管内皮细胞、平滑肌细胞及铁粉,再结合胶原酶I的消化,通过磁力架吸引铁,特异性地分选出带血管的肺组织,经过3~4周左右的培养及纯化,得到肺细小动脉平滑肌细胞。用倒置相差显微镜观察细胞形态,免疫细胞化学法和免疫荧光染色法进行α-平滑肌肌动蛋白鉴定。MTT实验和划痕实验检测PDGF诱导的肺动脉细小平滑肌细胞的增殖和迁移。结果:分离后第14天、第21天及传代后进行鉴定,均表明分离培养的细胞为PASMCs。MTT结果表明,与不加PDGF组相比,PDGF增殖明显增加(P<0.05)。划痕实验结果显示PDGF刺激组比不刺激组迁移显著增多。结论:本方法分离培养大鼠的PASMCs,操作方便,实验仪器要求低。PDGF能够促进肺细小动脉平滑肌细胞的增殖、迁移。 相似文献
20.
Christian Freise Uwe Querfeld Antje Ludwig Bernd Hamm Jörg Schnorr Matthias Taupitz 《Journal of cellular and molecular medicine》2021,25(12):5602-5614
Extracellular vesicles (EV) function as messengers between endothelial cells (EC) and vascular smooth muscle cells (VSMC). Since chronic kidney disease (CKD) increases the risk for vascular calcifications, we investigated whether EV derived from uraemic milieu-stimulated EC and derived from uraemic rats impact the osteogenic transdifferentiation/calcification of VSMC. For that purpose, human EC were treated with urea and indoxyl sulphate or left untreated. Experimental uraemia in rats was induced by adenine feeding. ‘Uraemic’ and control EV (EVUR; EVCTRL) were isolated from supernatants and plasma by using an exosome isolation reagent. Rat VSMC were treated with a pro-calcifying medium (CM) with or without EV supplementation. Gene expressions, miRNA contents and protein expressions were determined by qPCR and Western blots, respectively. Calcifications were determined by colorimetric assays. Delivery of miRNA inhibitors/mimics to EV and siRNA to VSMC was achieved via transfection. EVCTRL and EVUR differed in size and miRNA contents. Contrary to EVCTRL, EC- and plasma-derived EVUR significantly increased the pro-calcifying effects of CM, including altered gene expressions of osterix, runx2, osteocalcin and SM22α. Further, EVUR enhanced the protein expression of the phosphate transporter PiT-1 in VSMC and induced a phosphorylation of AKT and ERK. Knock down of PiT-1 and individual inhibition of AKT and ERK signalling in VSMC blocked the pro-calcifying effects of EVUR. Similar effects were achieved by inhibition of miR-221/-222 and mimicking of miR-143/-145 in EVUR. In conclusion, EVUR might represent an additional puzzle piece of the complex pathophysiology of vascular calcifications in CKD. 相似文献