scholarly journals miR-200c is aberrantly expressed in leiomyomas in an ethnic-dependent manner and targets ZEBs, VEGFA, TIMP2, and FBLN5

2012 ◽  
Vol 19 (4) ◽  
pp. 541-556 ◽  
Author(s):  
Tsai-Der Chuang ◽  
Harekrushna Panda ◽  
Xiaoping Luo ◽  
Nasser Chegini
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Target Genes ◽  
Abnormal Uterine Bleeding ◽  
Muscle Cells ◽  
Matrix Remodeling ◽  
Dependent Manner ◽  
Vimentin Expression ◽  
Altered Expression ◽  
Uterine Tumors

MicroRNA-200c (miR-200c) through repression of specific target genes has been associated with cellular transition, tumorigenesis, and tissue fibrosis. We explored the expression and functional aspects of miR-200c in genesis of leiomyomas (LYO), benign uterine tumors with fibrotic characteristic. Using LYO and matched myometrium (MYO;n=76) from untreated and from patients exposed to hormonal therapies (GNRH agonist (GNRHa), Depo-Provera, and oral contraceptives), we found that miR-200c was expressed at significantly lower levels (P<0.05) in LYO as compared with MYO. These levels were lower in LYO from African Americans as compared with Caucasians, patients experiencing abnormal uterine bleeding and those exposed to GNRHa therapy. Gain-of-function of miR-200c in isolated leiomyoma smooth muscle cells (LSMCs), myometrial smooth muscle cells (MSMCs), and leiomyosarcoma cell line (SKLM-S1) repressedZEB1/ZEB2mRNAs and proteins, with concurrent increase in E-cadherin (CDH1) and reduction in vimentin expression, phenotypic alteration, and inhibition of MSMC and LSMC proliferations. We further validatedTIMP2, FBLN5, andVEGFAas direct targets of miR-200c through interaction with their respective 3′ UTRs, and other genes as determined by microarray analysis. At tissue levels, LYO expressed lower levels ofTIMP2andFBLN5mRNAs but increased protein expressions, which to some extent altered due to hormonal exposure. Given the regulatory functions ofZEBs, VEGFA, FBLN5, andTIMP2on cellular activities that promote cellular transition, angiogenesis, and matrix remodeling, we concluded that altered expression of miR-200c may have a significant impact on the outcome of LYO growth, maintenance of their mesenchymal and fibrotic characteristics, and possibly their associated symptoms.

scholarly journals MEF2 is regulated by CaMKIIδ2 and a HDAC4–HDAC5 heterodimer in vascular smooth muscle cells

2012 ◽  
Vol 444 (1) ◽  
pp. 105-114 ◽  
Author(s):  
Roman Ginnan ◽  
Li Yan Sun ◽  
John J. Schwarz ◽  
Harold A. Singer
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Target Genes ◽  
Balloon Catheter ◽  
Vascular Diseases ◽  
Muscle Cells ◽  
Binding Activity ◽  
Dependent Manner

VSMCs (vascular smooth muscle cells) dedifferentiate from the contractile to the synthetic phenotype in response to acute vascular diseases such as restenosis and chronic vascular diseases such as atherosclerosis, and contribute to growth of the neointima. We demonstrated previously that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKII (Ca2+/calmodulin-dependent protein kinase) IIδ2 in the medial wall and the expanding neointima [House and Singer (2008) Arterioscler. Thromb. Vasc. Biol. 28, 441–447]. These findings led us to hypothesize that increased expression of CaMKIIδ2 is a positive mediator of synthetic VSMCs. HDAC (histone deacetylase) 4 and HDAC5 function as transcriptional co-repressors and are regulated in a CaMKII-dependent manner. In the present paper, we report that endogenous HDAC4 and HDAC5 in VSMCs are activated in a Ca2+- and CaMKIIδ2-dependent manner. We show further that AngII (angiotensin II)- and PDGF (platelet-derived growth factor)-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ2 expression is suppressed or CaMKIIδ2 activity is attenuated. The transcriptional activator MEF2 (myocyte-enhancer factor 2) is an important determinant of VSMC phenotype and is regulated in an HDAC-dependent manner. In the present paper, we report that stimulation of VSMCs with ionomycin or AngII potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 (nuclear receptor 77) (NR4A1) and MCP1 (monocyte chemotactic protein 1) (CCL2). Suppression of CaMKIIδ2 attenuates increased MEF2 DNA-binding activity and up-regulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSMCs. Suppression of HDAC4 expression and activity prevents AngII- and PDGF-dependent phosphorylation of HDAC5. Taken together, these results illustrate a mechanism by which CaMKIIδ2 mediates MEF2-dependent gene transcription in VSMCs through regulation of HDAC4 and HDAC5.

Interleukin 6 stimulates growth of vascular smooth muscle cells in a PDGF-dependent manner

1991 ◽  
Vol 260 (5) ◽  
pp. H1713-H1717 ◽  
Author(s):  
U. Ikeda ◽  
M. Ikeda ◽  
T. Oohara ◽  
A. Oguchi ◽  
T. Kamitani ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Interleukin 6 ◽  
Thymidine Incorporation ◽  
Tritiated Thymidine ◽  
Muscle Cells ◽  
Thymidine Uptake ◽  
Dependent Manner

We have investigated the effect of interleukin 6 (IL-6) on the growth of vascular smooth muscle cells (VSMC) isolated from rat aortas. Murine recombinant IL-6 significantly increased the number of VSMC and stimulated tritiated thymidine incorporation into VSMC in a dose-dependent manner. The IL-6-induced thymidine incorporation into VSMC was totally inhibited by the Ca2+ channel blocker verapamil; however, IL-6 showed no effects on the intracellular Ca2+ level ([Ca2+]i) in VSMC. Antibody against platelet-derived growth factor (PDGF) also totally inhibited the IL-6-induced thymidine uptake. PDGF caused a significant increase in the [Ca2+]i, which was totally inhibited by verapamil. IL-6 mRNA was not detected in unstimulated “quiescent” VSMC, but its expression was stimulated by exposure of VSMC to 10% fetal bovine serum. Immunohistochemical study using anti-PDGF antibody showed that IL-6 stimulated PDGF production in VSMC. These results support the premise that IL-6 is released by VSMC in an autocrine manner and promotes the growth of VSMC via induction of endogenous PDGF production.

Binding, degradation, and biological activity of insulin in vascular smooth muscle cells

1985 ◽  
Vol 249 (3) ◽  
pp. E292-E298
Author(s):  
N. Kaiser ◽  
A. Tur-Sinai ◽  
M. Hasin ◽  
E. Cerasi
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Insulin Binding ◽  
Maximal Response ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Igf I

The interaction of insulin with the vascular smooth muscle was studied using cultures derived from the bovine aortic arch. The cultured cells exhibited specific binding of 125I-insulin that was reversible and dependent on pH. Both insulin and insulinlike growth factor (IGF) I competed for 125I-insulin binding; IGF I, however, was less effective than insulin by at least an order of magnitude. Insulin binding was accompanied by internalization and degradation of the hormone in a temperature- and time-dependent manner. Chloroquine and other lysosomotropic agents elevated the internalized insulin and reduced its degradation. Pre-exposure of cell cultures to insulin resulted in downregulation of cell surface receptors. Insulin stimulated alpha-aminoisobutyric acid transport in confluent smooth muscle cells. The maximal response was observed at 100 ng/ml insulin with a half-maximal effect at 10 ng/ml. Sparse, serum-starved smooth muscle cells responded to insulin with a dose-dependent increase in [3H]-thymidine incorporation into DNA. Although the effect was already apparent at 1 ng/ml insulin, it reached near maximal level only at 10,000 ng/ml. IGF I also stimulated DNA synthesis in smooth muscle cells; however, at low concentrations insulin was more efficient in this respect. Human growth hormone was inactive. The data indicate the presence of specific receptors for insulin in bovine aortic smooth muscle cells. These receptors appear to mediate the metabolic activity as well as part of the mitogenic effect of insulin in these cells.

Beta-adrenergic actions on membrane electrical properties of dissociated smooth muscle cells

1988 ◽  
Vol 254 (3) ◽  
pp. C423-C431 ◽  
Author(s):  
H. Yamaguchi ◽  
T. W. Honeyman ◽  
F. S. Fay
Keyword(s):  
Smooth Muscle ◽  
Membrane Potential ◽  
Electrical Properties ◽  
Smooth Muscle Cells ◽  
Input Resistance ◽  
Muscle Cells ◽  
Resting Potential ◽  
Equilibrium Potential ◽  
Dependent Manner ◽  
Beta Adrenergic

Studies were carried out to determine the effects of the beta-adrenergic agent, isoproterenol (ISO), on membrane electrical properties in single smooth muscle cells enzymatically dispersed from toad stomach. In cells bathed in buffer of physiological composition, the average resting potential was -56.4 +/- 1.4 mV (mean +/- SE, n = 35). The dominant effect of exposure to ISO was hyperpolarization. The hyperpolarization was apparent in all cells studied and averaged 11.6 +/- 1.2 mV (n = 27). In the majority of the cells, hyperpolarization was accompanied by a decreased input resistance (Rin). Often the change in resistance appeared to lag behind the change in membrane potential. The lack of coincident changes in membrane potential and resistance may reflect a superposition of the outward rectification properties of the membrane on beta-adrenergic-induced increases in ionic conductance. In about half of the cells, an initial small depolarization (3.1 +/- 0.3 mV, n = 14) was accompanied by a small but distinct increase in Rin (12 +/- 2.5%). When membrane potential was made more negative than the estimated equilibrium potential for K+ (EK) by injection of current, ISO also produced biphasic effects, an initial hyperpolarization which reversed to a sustained depolarization to a value (-90 mV) near the estimated EK. The hyperpolarization by ISO could be diminished in a time-dependent manner by previous exposure to ouabain. The inhibition by ouabain, however, appeared to be a fortuitous result of glycoside-induced positive shifts in EK. These observations indicate that the dominant electrophysiological effect of beta-adrenergic stimuli is to hyperpolarize the cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effect of Paclitaxel+Hirudin on the TLR4-MyD88 Signaling Pathway During Inflammatory Activation of Human Coronary Artery Smooth Muscle Cells and Mechanistic Analysis

2018 ◽  
Vol 50 (4) ◽  
pp. 1301-1317 ◽  
Author(s):  
Hongmei Li ◽  
Xian Wang ◽  
Anlong Xu
Keyword(s):  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Human Coronary Artery ◽  
Post Intervention ◽  
Inflammatory Activation ◽  
Myd88 Pathway

Background/Aims: Approximately 10%-20% of patients with acute cardiovascular disease who have received coronary intervention suffer restenosis and high inflammation. The stent compound paclitaxel+hirudin was prepared for the treatment of post-intervention restenosis. This study aimed to explore the anti-inflammatory and anti-restenosis mechanisms of paclitaxel+hirudin with regard to the TLR4/MyD88/NF-κB pathway. Methods: Human coronary artery smooth muscle cells (HCASMCs) at 4-6 generations after in vitro culture were used as a model. Lipopolysaccharide (LPS) was used as an inducer to maximally activate the TLR4/MyD88/NF-κB inflammation pathway. After MyD88 knockdown and selective blocking of MyD88 degradation with epoxomicin, the effects of paclitaxel+hirudin stenting on key sites of the TLR4/MyD88/NF-κB pathway were detected using ELISA, Q-PCR, and western blot analysis. Results: LPS at 1 μg/mL for 48 h was the optimal modeling condition for inflammatory activation of HCASMCs. Paclitaxel+hirudin inhibited the levels of key proteins and the gene expression, except for that of the MyD88 gene, of the TLR4-MyD88 pathway. The trend of the effect of paclitaxel+hirudin on the pathway proteins was similar to that of MyD88 knockdown. After epoxomicin intervention, the inhibitory effects of paclitaxel+hirudin on the key genes and proteins of the TLR4-MyD88 pathway were significantly weakened, which even reached pre-intervention levels. Paclitaxel+hirudin affected the MyD88 protein in a dosage-dependent manner. Conclusion: The paclitaxel+hirudin compound promotes MyD88 degradation in the TLR4/MyD88/NF-κB pathway to reduce the activity of TLR4 and NF-κB p65 and to weaken the LPS-initiated inflammatory reactions of IL-1β, IL-6, and TNF-α.

scholarly journals N-terminal pyroglutamate formation in CX3CL1 is essential for its full biologic activity

2017 ◽  
Vol 37 (4) ◽  
Author(s):  
Astrid Kehlen ◽  
Monique Haegele ◽  
Livia Böhme ◽  
Holger Cynis ◽  
Torsten Hoffmann ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Adhesion Molecule ◽  
Muscle Cells ◽  
Mitogen Activated Protein Kinase ◽  
Intercellular Adhesion Molecule ◽  
Dependent Manner ◽  
Glutaminyl Cyclase ◽  
N Terminus

CX3CL1 (fractalkine) is a unique member of the CX3C chemokine family and mediates both adhesion and cell migration in inflammatory processes. Frequently, the activity of chemokines depends on a modified N-terminus as described for the N-terminus of CCL2 modified to a pGlu- (pyroglutamate) residue by QC (glutaminyl cyclase) activity. Here, we assess the role of the pGlu-modified residue of the CX3CL1 chemokine domain in human endothelial and smooth muscle cells. For the first time, we demonstrated using MS that QC (QPCT, gene name of QC) or its isoenzyme isoQC (iso-glutaminyl cyclase) (QPCTL, gene name of isoQC) catalyse the formation of N-terminal-modified pGlu-CX3CL1. Expression of QPCT is co-regulated with its substrates CCL2 and CX3CL1 in HUVECs (human umbilical vein endothelial cells) and HCASMCs (human coronary artery smooth muscle cells) upon stimulation with TNF-α and IL-1β whereas QPCTL expression is not affected. By contrast, inhibition of the NF-κB pathway using an IKK2 inhibitor decreased the expression of the co-regulated targets QPCT, CCL2, and CX3CL1. Furthermore, RNAi-mediated inhibition of QPCT expression resulted in a reduction in CCL2 and CX3CL1 mRNA. In HCASMCs, N-terminal-modified pGlu1-CX3CL1 induced a significant stronger effect on phosphorylation of ERK (extracellular signal regulated kinase) 1/2, Akt (protein kinase B), and p38 (p38 mitogen-activated protein kinase) kinases than the immature Gln1-CX3CL1 in a time- and concentration-dependent manner. Furthermore, pGlu1-CX3CL1 affected the expression of CCL2, CX3CL1, and the adhesion molecule ICAM1/CD54 (intercellular adhesion molecule-1) inducing in higher expression level compared with its Gln1-variant in both HCASMCs and HUVECs. These results strongly suggest that QC-catalysed N-terminal pGlu formation of CX3CL1 is important for the stability or the interaction with its receptor and opens new insights into the function of QC in inflammation.

Abstract 361: Cyclophilin A Mediates Angiotensin II--Stimulated NADPH Oxidase Activation in Vascular Smooth Muscle Cells

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Nwe Nwe Soe ◽  
Mark Sowden ◽  
Patrizia Nigro ◽  
Bradford C Berk
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Smooth Muscle Cells ◽  
Fold Increase ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Ang Ii ◽  
Membrane Translocation ◽  
Ppiase Activity

Objective: Cyclophilin A (CyPA) is a ubiquitously expressed cytosolic protein that possesses PPIase activity and scaffold function. CyPA regulates Angiotensin II (Ang II) induced reactive oxygen species (ROS) production in vascular smooth muscle cells. However, the mechanism of this CyPA regulation remains unclear. We hypothesized that CyPA regulates plasma membrane translocation of NADPH oxidase cytosolic subunit, p47phox, which is required for NADPH oxidase structural organization and activity. Methods and results: Immunofluorescence studies in rat aortic smooth muscle cells revealed that CyPA translocated from the cytosol to the plasma membrane in response to Ang II in a time dependent manner with a peak at 10min (46.4±5.4 fold increase). Mouse Aortic Smooth Muscle Cells (MASM) were isolated from mice lacking CyPA (CyPA-/-) and wild type controls (WT), treated with Ang II (100nM) and immunofluorescence analysis was performed. Ang II induced p47phox plasma membrane translocation at 10min in WT mice. However, p47 phox translocation was significantly inhibited in CyPA -/- MASM. CyPA and p47phox colocalized at the plasma membrane in response to Ang II. Further analysis using subcellular fractionation studies confirmed that Ang II induced p47phox plasma membrane translocation was inhibited in CyPA -/- MASM compared to WT (1.2±2.7 vs 4.3±3.4 fold increase). Coimmunoprecipitation analyses confirmed that Ang II increased CyPA association with p47phox in a time dependent manner (2.5±3.4 fold increase at 10min). Finally, pretreatment with the PPIase activity inhibitor, cyclosporine A (1uM), could not inhibit CyPA association with p47phox and CyPA mediated p47phox translocation to the plasma membrane. Conclusion: These data suggest that Ang II promotes an association between CyPA and p47phox that enhances plasma membrane translocation of p47phox. This is proposed to increase the NADPH oxidase activity thereby increasing cellular ROS production. This process is independent of the PPIase activity of CyPA. Therefore, inhibition of the CyPA and p47phox association could be a future therapeutic target for Ang II induced ROS regulated cardiovascular diseases such as atherosclerosis and abdominal aortic aneurysm formation.

ANG II increases TIMP-1 expression in rat aortic smooth muscle cells in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. H635-H643 ◽  
Author(s):  
Giovanna Castoldi ◽  
Cira R. T. di Gioia ◽  
Federico Pieruzzi ◽  
Cristina D'Orlando ◽  
Willy M. M. van de Greef ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Vascular Wall ◽  
Dependent Manner ◽  
Ang Ii ◽  
Maximal Increase ◽  
Aortic Smooth Muscle Cells ◽  
Aortic Smooth Muscle

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tissue remodeling processes. TIMP-1 is the main native inhibitor of MMPs and it contributes to the development of tissue fibrosis. It is known that ANG II plays a fundamental role in vascular remodeling. In this study, we investigated whether ANG II modulates TIMP-1 expression in rat aortic smooth muscle cells. In vitro, ANG II induces TIMP-1 mRNA expression in a dose-dependent manner. The maximal increase in TIMP-1 expression was present after 3 h of ANG II stimulation. The ANG II increase in TIMP-1 expression was mediated by the ANG type 1 receptors because it was blocked by losartan. The increase in TIMP-1 expression was present after the first ANG II treatment, whereas repeated treatments (3 and 5 times) did not modify TIMP-1 expression. In vivo, exogenous ANG II was administered to Sprague-Dawley rats (200 ng · kg−1· min−1sc) for 6 and 25 days. Control rats received physiological saline. After treatment, systolic blood pressure was significantly higher ( P < 0.01), whereas plasma renin activity was suppressed ( P < 0.01), in ANG II-treated rats. ANG II increased TIMP-1 expression in the aorta of ANG II-treated rats both at the mRNA ( P < 0.05) and protein levels as evaluated by Western blotting ( P < 0.05) and/or immunohistochemistry. Neither histological modifications at the vascular wall nor differences in collagen content in the tunica media were present in both the ANG II- and saline-treated groups. Our data demonstrate that ANG II increases TIMP-1 expression in rat aortic smooth muscle cells. In vivo, both short- and long-term chronic ANG II treatments increase TIMP-1 expression in the rat aorta. TIMP-1 induction by ANG II in aortic smooth muscle cells occurs in the absence of histological changes at the vascular wall.

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