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Sample records for mir-143 regulate smooth

  1. MiR-143-3p controls TGF-β1-induced cell proliferation and extracellular matrix production in airway smooth muscle via negative regulation of the nuclear factor of activated T cells 1.

    PubMed

    Cheng, Wei; Yan, Kun; Xie, Li-Yi; Chen, Feng; Yu, Hong-Chuan; Huang, Yan-Xia; Dang, Cheng-Xue

    2016-10-01

    MicroRNAs (miRNAs) are small noncoding RNAs that function in diverse biological processes. However, little is known about the precise role of microRNAs in the functioning of airway smooth muscle cells (ASMCs). Here, we investigated the potential role and mechanisms of the miR-143 -3p on proliferation and the extracellular matrix (ECM) protein production of ASMCs. We demonstrated that miR-143-3p was aberrantly lower in ASMCs isolated from individuals with asthma than in individuals without asthma. Meanwhile, TGF-β1 caused a marked decrease in a time-dependent manner in miR-143-3p expression in ASMCs from asthmatics. Additionally, the overexpression of miR- 143-3p robustly reduced TGF-β1-induced ASMCs proliferation and downregulated CDK and cyclin expression, whereas the inhibition of miR-143-3p significantly enhanced ASMCs proliferation and upregulated the level of CDKs and cyclins. Re-expression of miR-143-3p attenuated ECM protein deposition reflected as a marked decrease in the expression of type I collagen and fibronectin, whereas miR-143-3p downregulation caused an opposite effect on the expression of type I collagen and fibronectin. Moreover, qRT-PCR and western blot analysis indicated that miR-143-3p negatively regulated the expression of nuclear factor of activated T cells 1 (NFATc1). Subsequent analyses demonstrated that NFATc1 was a direct and functional target of miR-143-3p, which was validated by the dual luciferase reporter assay. Most importantly, the overexpression of NFATc1 effectively reversed the inhibition of miR-143-3p on TGF-β1-induced proliferation, and strikingly abrogated the effect of miR-143-3p on the expression of CDK4 and Cyclin D1. Together, miR-143-3p may function as an inhibitor of asthma airway remodeling by suppressing proliferation and ECM protein deposition in TGF-β1-mediated ASMCs via the negative regulation of NFATc1 signaling, suggesting miR-143-3p as a potential therapeutic target for asthma.

  2. miR-143 regulates proliferation and differentiation of bovine skeletal muscle satellite cells by targeting IGFBP5.

    PubMed

    Zhang, Wei Ran; Zhang, Hui Na; Wang, Yi Min; Dai, Yang; Liu, Xin Feng; Li, Xin; Ding, Xiang Bin; Guo, Hong

    2017-03-01

    Development of skeletal muscle is a complicated biological process regulated by various regulation factors and signal pathways. MicroRNAs (miRNAs) are novel gene regulators that control muscle cell development. microRNA-143 (miR-143) is highly expressed in skeletal muscle, and we found that miR-143 level is significantly increased during bovine skeletal muscle satellite cells (MSCs) differentiation process through microarray analysis and qRT-PCR detection. However, the function of miR-143 in bovine muscle development remained unclear. In our work, the functions of miR-143 in bovine MSCs myogenic differentiation were investigated. We discovered that IGFBP5 is directly regulated by miR-143 using a dual-luciferase reporter assay. Overexpression of miR-143 led to decreased level of IGFBP5 protein and restrained cell proliferation and differentiation, while downregulation of miR-143 resulted in increased levels of IGFBP5 protein and restrained cell proliferation but improved differentiation. IGFBP5, an important component of IGF signaling pathway, contributes greatly to bovine muscle cell development. A mechanism that miR-143 can regulate the proliferation and differentiation of bovine MSCs through changing expression of IGFBP5 was elucidated by our study.

  3. MiR-143 and MiR-145 Regulate IGF1R to Suppress Cell Proliferation in Colorectal Cancer

    PubMed Central

    Wang, Nan; Zhang, Suyang; Yan, Xin; Feng, Hui; Pang, Wenjing; Wang, Yanbo; Wang, Xueliang; Fu, Zhen; Liu, Yanqing; Zhao, Chihao; Zhang, Junfeng; Zhang, Chen-Yu; Zen, Ke; Chen, Xi; Wang, Yalei

    2014-01-01

    Insulin-like growth factor 1 receptor (IGF1R) is a transmembrane receptor that is activated by insulin-like growth factor 1 (IGF-1) and by a related hormone called IGF-2. It belongs to the large class of tyrosine kinase receptors and plays an important role in colorectal cancer etiology and progression. In this study, we used bioinformatic analyses to search for miRNAs that potentially target IGF1R. We identified specific target sites for miR-143 and miR-145 (miR-143/145) in the 3′-untranslated region (3′-UTR) of the IGF1R gene. These miRNAs are members of a cluster of miRNAs that have been reported to exhibit tumor suppressor activity. Consistent with the bioinformatic analyses, we identified an inverse correlation between miR-143/145 levels and IGF1R protein levels in colorectal cancer tissues. By overexpressing miR-143/145 in Caco2, HT29 and SW480 colorectal cancer cells, we experimentally validated that miR-143/145 directly recognizes the 3′-UTR of the IGF1R transcript and regulates IGF1R expression. Furthermore, the biological consequences of the targeting of IGF1R by miR-143/145 were examined by cell proliferation assays in vitro. We demonstrated that the repression of IGF1R by miR-143/145 suppressed the proliferation of Caco2 cells. Taken together, our findings provide evidence for a role of the miR-143/145 cluster as a tumor suppressor in colorectal cancer through the inhibition of IGF1R translation. PMID:25474488

  4. Cholesterol loading re-programs the miR-143/145-myocardin axis to convert aortic smooth muscle cells to a dysfunctional macrophage-like phenotype

    PubMed Central

    Vengrenyuk, Yuliya; Nishi, Hitoo; Long, Xiaochun; Ouimet, Mireille; Savji, Nazir; Martinez, Fernando O.; Cassella, Courtney P.; Moore, Kathryn J.; Ramsey, Stephen A.; Miano, Joseph M.; Fisher, Edward A.

    2015-01-01

    Objective We previously showed that cholesterol loading in vitro converts mouse aortic vascular smooth muscle cells (VSMC) from a contractile state to one resembling macrophages. In human and mouse atherosclerotic plaques it has become appreciated that ~40% of cells classified as macrophages by histological markers may be of VSMC origin. We therefore sought to gain insight into the molecular regulation of this clinically relevant process. Approach and Results VSMC of mouse (or human) origin were incubated with cyclodextrin-cholesterol complexes for 72 hours, at which time the expression at the protein and mRNA levels of contractile-related proteins were reduced and of macrophage markers increased. Concurrent was down regulation of miR-143/145, which positively regulate the master VSMC-differentiation transcription factor myocardin (MYOCD). Mechanisms were further probed in mouse VSMC. Maintaining the expression of MYOCD or miR-143/145 prevented and reversed phenotypic changes caused by cholesterol loading. Reversal was also seen when cholesterol efflux was stimulated after loading. Notably, despite expression of macrophage markers, bioinformatic analyses showed that cholesterol-loaded cells remained closer to the VSMC state, consistent with impairment in classical macrophage functions of phagocytosis and efferocytosis. In apoE-deficient atherosclerotic plaques, cells positive for VSMC and macrophage markers were found lining the cholesterol-rich necrotic core. Conclusions Cholesterol loading of VSMC converts them to a macrophage–appearing state by downregulating the miR-143/145-myocardin axis. Though these cells would be classified by immunohistochemistry as macrophages in human and mouse plaques, their transcriptome and functional properties imply that their contributions to atherogenesis would not be those of classical macrophages. PMID:25573853

  5. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    PubMed

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples (n=5) and cervical intraepithelial neoplasia tissue samples (n=5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P<0.05). The MTT assay revealed that the cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group (P<0.05), and the cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group (P<0.05) but higher than the miR-143 mimic group significantly (P<0.05). The expression levels of K-ras protein in the miR-143 mimic group, the negative control group and the

  6. miR-143 suppresses epithelial-mesenchymal transition and inhibits tumor growth of breast cancer through down-regulation of ERK5.

    PubMed

    Zhai, Limin; Ma, Chuanxiang; Li, Wentong; Yang, Shuo; Liu, Zhijun

    2016-12-01

    Epithelial-mesenchymal transition (EMT) plays a pivotal role in the development of cancer invasion and metastasis. Many studies have significantly enhanced the knowledge on EMT through the characterization of microRNAs (miRNAs) influencing the signaling pathways and downstream events that define EMT on a molecular level. In this study, we found that miR-143 suppressed EMT. Up-regulating miR-143 enhanced E-cadherin-mediated cell-cell adhesion ability, reduced mesenchymal markers, and decreased cell proliferation, migration, and invasion in vitro. In vivo, the xenograft mouse model also unveiled the suppressive effects of miR-143 on tumor growth. Additionally, we demonstrated that up-regulating extracellular signal regulated kinase 5 (ERK5) was associated with poor prognosis of breast cancer patients. Moreover, we observed an inverse correlation between miR-143 and ERK5 in breast cancer tissues. miR-143 directly targeted seed sequences in the 3'-untranslated regions of ERK5. Furthermore, we revealed that the downstream molecules of glycogen synthase kinase 3 beta (GSK-3β)/Snail signaling were involved in EMT and modulated by ERK5. In summary, our findings demonstrated that miR-143 down-regulated its target ERK5, leading to the suppression of EMT induced by GSK-3β/Snail signaling of breast cancer. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  7. DDX6 post-transcriptionally down-regulates miR-143/145 expression through host gene NCR143/145 in cancer cells.

    PubMed

    Iio, Akio; Takagi, Takeshi; Miki, Kohei; Naoe, Tomoki; Nakayama, Atsuo; Akao, Yukihiro

    2013-10-01

    In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells.

  8. The Cluster of miR-143 and miR-145 Affects the Risk for Esophageal Squamous Cell Carcinoma through Co-Regulating Fascin Homolog 1

    PubMed Central

    Liu, Ran; Liao, Juan; Yang, Miao; Sheng, Jingyi; Yang, Hao; Wang, Yi; Pan, Enchun; Guo, Wei; Pu, Yuepu; Kim, Sun Jung; Yin, Lihong

    2012-01-01

    MicroRNAs (miRNAs), 18–24 nt non-coding RNAs, are thought to play important roles in cell proliferation, differentiation, apoptosis, and development. Recent studies suggest that some of the known microRNAs map to a single genomic locale within a single polycistronic transcript. But the roles of the cluster remain to be known. In order to understand the role and mechanism of a cluster of miR-143 and miR-145 in esophageal squamous cell carcinoma (ESCC), the association of mature miR-143 and miR-145 expression with the risk for esophageal cancer was evaluated in ESCC patients with a case-control study, and target protein regulated by mature miRNA was analyzed in ESCC cell lines with 3′UTR luciferase reporter assay. The expression levels of miR-143 and miR-145 were determined in 110 pairs of esophageal cancer tissues and adjacent normal tissues using real-time reverse transcription PCR. The relative expression of miR-143 and miR-145 were statistically different between cancer tissues and matched controls. The combined expression of miR-143 and miR-145 was significantly associated with the risk for esophageal cancer. Meanwhile, the reduced expression of two miRNAs in tumor patient was supposed to have a trend of lymph node metastases. The co-expression pattern of miR-143 and miR-145 was analyzed with Pearson correlation. It showed a significant correlation between these two miRNAs expression both in tissues and tumor cell lines. 3′UTR luciferase reporter assay indicated that Fascin Homolog 1 (FSCN1) could be co-regulated by miR-143 and miR-145. The protein level of FSCN1 showed no significant linear correlation with miR-143 and miR-145 expression in ESCC cell lines with Western blotting analysis. In conclusion, since miR-143 and miR-145 could regulate oncogenic FSCN1 and take part in the modulation of metastases, the result suggested the combination variable of miR-143 and miR-145 as a potential biomarker for earlier diagnosis and prognosis of esophageal cancer

  9. Regulation signature of miR-143 and miR-26 in porcine Salmonella infection identified by binding site enrichment analysis.

    PubMed

    Yao, Min; Gao, Weihua; Tao, Hengxun; Yang, Jun; Liu, Guoping; Huang, Tinghua

    2016-04-01

    Salmonella infects many vertebrate species, and pigs colonized with Salmonella are typically Salmonella carriers. Transcriptomic analysis of the response to Salmonella infection in whole blood has been reported for the pig. The objective of this study is to identify the important miRNAs involved in Salmonella infection using binding site enrichment analysis. We predicted porcine microRNA (miRNA) binding sites in the 3' UTR of protein-coding genes for all miRNA families. Based on those predictions, we analyzed miRNA-binding sites for mRNAs expressed in peripheral blood to investigate the functional importance of miRNAs in Salmonella infection in pig. Enrichment analysis revealed that binding sites of five miRNAs (including miR-143, -9839, -26, -2483, and -4335) were significantly over represented for the differentially expressed gene sets. Real-time PCR results indicated that selected members of this miRNA group (miR-143, -26, and -4335) were differentially expressed in whole blood after Salmonella inoculation. The luciferase reporter assay showed that ATP6V1A and IL13RA1 were targets of miR-143 and that miR-26 regulates BINP3L and ARL6IP6. The results strongly suggest that miR-143 and miR-26 play important regulatory roles in the development of Salmonella infection in pig.

  10. PAI-1, a target gene of miR-143, regulates invasion and metastasis by upregulating MMP-13 expression of human osteosarcoma.

    PubMed

    Hirahata, Mio; Osaki, Mitsuhiko; Kanda, Yusuke; Sugimoto, Yui; Yoshioka, Yusuke; Kosaka, Nobuyoshi; Takeshita, Fumitaka; Fujiwara, Tomohiro; Kawai, Akira; Ito, Hisao; Ochiya, Takahiro; Okada, Futoshi

    2016-05-01

    Despite recent improvements in the therapy for osteosarcoma, 30-40% of osteosarcoma patients die of this disease, mainly due to its lung metastasis. We have previously reported that intravenous injection of miR-143 significantly suppresses lung metastasis of human osteosarcoma cells (143B) in a mouse model. In this study, we examined the biological role and mechanism of miR-143 in the metastasis of human osteosarcoma cells. We identified plasminogen activator inhibitor-1 (PAI-1) as a direct target gene of miR-143. To determine the role of PAI-1 in human osteosarcoma cells, siRNA was transfected into 143B cells for knockdown of PAI-1 expression. An in vitro study showed that downregulation of PAI-1 suppressed cell invasion activity, but not proliferation. Moreover, injection of PAI-1 siRNA into a primary lesion in the osteosarcoma mouse model inhibited lung metastasis compared to control siRNA-injected mice, without influencing the proliferative activity of the tumor cells. Subsequent examination using 143B cells revealed that knockdown of PAI-1 expression resulted in downregulation of the expression and secretion of matrix metalloproteinase-13 (MMP-13), which is also a target gene of miR-143 and a proteolytic enzyme that regulates tumor-induced osteolysis. Immunohistochemical analysis using clinical samples showed that higher miR-143 expressing cases showed poor expression of PAI-1 in the primary tumor cells. All such cases belonged to the lung metastasis-negative group. Moreover, the frequency of lung metastasis-positive cases was significantly higher in PAI-1 and MMP-13 double-positive cases than in PAI-1 or MMP-13 single-positive or double-negative cases (P < 0.05). These results indicated that PAI-1, a target gene of miR-143, regulates invasion and lung metastasis via enhancement of MMP-13 expression and secretion in human osteosarcoma cells, suggesting that these molecules could be potential therapeutic target genes for preventing lung metastasis in

  11. RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever

    PubMed Central

    Wong, Justin J.-L.; Au, Amy Y.M.; Gao, Dadi; Pinello, Natalia; Kwok, Chau-To; Thoeng, Annora; Lau, Katherine A.; Gordon, Jane E.A.; Schmitz, Ulf; Feng, Yue; Nguyen, Trung V.; Middleton, Robert; Bailey, Charles G.; Holst, Jeff; Rasko, John E.J.; Ritchie, William

    2016-01-01

    Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40°C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142–5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40°C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142–5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142–5p and miR-143. PMID:26825461

  12. RBM3 regulates temperature sensitive miR-142-5p and miR-143 (thermomiRs), which target immune genes and control fever.

    PubMed

    Wong, Justin J-L; Au, Amy Y M; Gao, Dadi; Pinello, Natalia; Kwok, Chau-To; Thoeng, Annora; Lau, Katherine A; Gordon, Jane E A; Schmitz, Ulf; Feng, Yue; Nguyen, Trung V; Middleton, Robert; Bailey, Charles G; Holst, Jeff; Rasko, John E J; Ritchie, William

    2016-04-07

    Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

  13. Curcumin inhibits prostate cancer by targeting PGK1 in the FOXD3/miR-143 axis.

    PubMed

    Cao, Hongwen; Yu, Hongjie; Feng, Yigeng; Chen, Lei; Liang, Fang

    2017-05-01

    Curcumin is a potent antitumor agent. The objective of this study was to explore the interaction between curcumin and PGK1, an oncogene in the FOXD3/miR-143 axis, in prostate cancer therapy. MiRNA microarray analysis was used to identify miRNAs upregulated by curcumin treatment. MiR-143 was dramatically upregulated by curcumin. Cells were treated with antimiR-143 in combination to curcumin, followed by examining cell viability and migration. Bioinformatics analysis was used to investigate target genes of miR-143. The interaction between miR-143 and PGK1 was evaluated with dual-luciferase assay. Since FOXD3 is important in the regulation of miR-143, we explored whether curcumin regulated FOXD3 expression. FOXD3 was also ectopically overexpressed to investigate its effects on curcumin's regulation of miR-143. Curcumin treatment significantly upregulated miR-143 and decreased prostate cancer cell proliferation and migration. Those effects were attenuated by anti-miR-143 transfection. Both miR-143 overexpression and curcumin treatment inhibited PGK1 expression and ectopic expression of PGK1 antagonized curcumin's antitumor effects. FOXD3 was upregulated by miR-143. Ectopic expression of FOXD3 synergized with curcumin in upregulating miR-143 expression. Curcumin inhibits prostate cancer by upregulating miR-143. PGK1 is downregulated by miR-143, and FOXD3 upregulation is essential for the antitumor effect of curcumin.

  14. miR-143 Interferes with ERK5 Signaling, and Abrogates Prostate Cancer Progression in Mice

    PubMed Central

    Clapé, Cyrielle; Fritz, Vanessa; Henriquet, Corinne; Apparailly, Florence; Fernandez, Pedro Luis; Iborra, François; Avancès, Christophe; Villalba, Martin; Culine, Stéphane; Fajas, Lluis

    2009-01-01

    Background Micro RNAs are small, non-coding, single-stranded RNAs that negatively regulate gene expression at the post-transcriptional level. Since miR-143 was found to be down-regulated in prostate cancer cells, we wanted to analyze its expression in human prostate cancer, and test the ability of miR-43 to arrest prostate cancer cell growth in vitro and in vivo. Results Expression of miR-143 was analyzed in human prostate cancers by quantitative PCR, and by in situ hybridization. miR-143 was introduced in cancer cells in vivo by electroporation. Bioinformatics analysis and luciferase-based assays were used to determine miR-143 targets. We show in this study that miR-143 levels are inversely correlated with advanced stages of prostate cancer. Rescue of miR-143 expression in cancer cells results in the arrest of cell proliferation and the abrogation of tumor growth in mice. Furthermore, we show that the effects of miR-143 are mediated, at least in part by the inhibition of extracellular signal-regulated kinase-5 (ERK5) activity. We show here that ERK5 is a miR-143 target in prostate cancer. Conclusions miR-143 is as a new target for prostate cancer treatment. PMID:19855844

  15. Genetic versus Non-Genetic Regulation of miR-103, miR-143 and miR-483-3p Expression in Adipose Tissue and Their Metabolic Implications—A Twin Study

    PubMed Central

    Bork-Jensen, Jette; Thuesen, Anne Cathrine Baun; Bang-Bertelsen, Claus Heiner; Grunnet, Louise Groth; Pociot, Flemming; Beck-Nielsen, Henning; Ozanne, Susan E.; Poulsen, Pernille; Vaag, Allan

    2014-01-01

    Murine models suggest that the microRNAs miR-103 and miR-143 may play central roles in the regulation of subcutaneous adipose tissue (SAT) and development of type 2 diabetes (T2D). The microRNA miR-483-3p may reduce adipose tissue expandability and cause ectopic lipid accumulation, insulin resistance and T2D. We aimed to explore the genetic and non-genetic factors that regulate these microRNAs in human SAT, and to investigate their impact on metabolism in humans. Levels of miR-103, miR-143 and miR-483-3p were measured in SAT biopsies from 244 elderly monozygotic and dizygotic twins using real-time PCR. Heritability estimates were calculated and multiple regression analyses were performed to study associations between these microRNAs and measures of metabolism, as well as between these microRNAs and possible regulating factors. We found that increased BMI was associated with increased miR-103 expression levels. In addition, the miR-103 levels were positively associated with 2 h plasma glucose levels and hemoglobin A1c independently of BMI. Heritability estimates for all three microRNAs were low. In conclusion, the expression levels of miR-103, miR-143 and miR-483-3p in adipose tissue are primarily influenced by non-genetic factors, and miR-103 may be involved in the development of adiposity and control of glucose metabolism in humans. PMID:25010252

  16. MiR-143/145 deficiency attenuates the progression of atherosclerosis in Ldlr-/-mice.

    PubMed

    Sala, Federica; Aranda, Juan F; Rotllan, Noemi; Ramírez, Cristina M; Aryal, Binod; Elia, Leonardo; Condorelli, Gianluigi; Catapano, Alberico Luigi; Fernández-Hernando, Carlos; Norata, Giuseppe Danilo

    2014-10-01

    The miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background. Ldlr-/- and Ldlr-/--miR-143/145-/- (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. We further confirmed the direct regulation of miR-145 on ABCA1 expression by qRT-PCR, Western blotting and 3'UTR-luciferase reporter assays. In summary, miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease.

  17. Mitigation of arsenic-induced acquired cancer phenotype in prostate cancer stem cells by miR-143 restoration.

    PubMed

    Ngalame, Ntube N O; Makia, Ngome L; Waalkes, Michael P; Tokar, Erik J

    2016-12-01

    Inorganic arsenic, an environmental contaminant and a human carcinogen is associated with prostate cancer. Emerging evidence suggests that cancer stem cells (CSCs) are the driving force of carcinogenesis. Chronic arsenic exposure malignantly transforms the human normal prostate stem/progenitor cell (SC) line, WPE-stem to arsenic-cancer SCs (As-CSCs), through unknown mechanisms. MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression at the posttranscriptional level. In prior work, miR-143 was markedly downregulated in As-CSCs, suggesting a role in arsenic-induced malignant transformation. In the present study, we investigated whether loss of miR-143 expression is important in arsenic-induced transformation of prostate SCs. Restoration of miR-143 in As-CSCs was achieved by lentivirus-mediated miR-143 overexpression. Cells were assessed bi-weekly for up to 30weeks to examine mitigation of cancer phenotype. Secreted matrix metalloproteinase (MMP) activity was increased by arsenic-induced malignant transformation, but miR-143 restoration decreased secreted MMP-2 and MMP-9 enzyme activities compared with scramble controls. Increased cell proliferation and apoptotic resistance, two hallmarks of cancer, were decreased upon miR-143 restoration. Increased apoptosis was associated with decreased BCL2 and BCL-XL expression. miR-143 restoration dysregulated the expression of SC/CSC self-renewal genes including NOTCH-1, BMI-1, OCT4 and ABCG2. The anticancer effects of miR-143 overexpression appeared to be mediated by targeting and inhibiting LIMK1 protein, and the phosphorylation of cofilin, a LIMK1 substrate. These findings clearly show that miR-143 restoration mitigated multiple cancer characteristics in the As-CSCs, suggesting a potential role in arsenic-induced transformation of prostate SCs. Thus, miR-143 is a potential biomarker and therapeutic target for arsenic-induced prostate cancer. Published by Elsevier Inc.

  18. Restoration of miR-143 expression could inhibit migration and growth of MDA-MB-468 cells through down-regulating the expression of invasion-related factors.

    PubMed

    Tavanafar, Fatemeh; Safaralizadeh, Reza; Hosseinpour-Feizi, Mohammad Ali; Mansoori, Behzad; Shanehbandi, Dariush; Mohammadi, Ali; Baradaran, Behzad

    2017-07-01

    Breast adenocarcinoma is the second common cancer in women the incidence of which is increasing in many countries, especially in developing companies. In this study, miRNA143 has been replaced by vector based microRNA-143 in breast adenocarcinoma cells (MDA-MB-468) and its anti-cancer effects on breast adenocarcinoma cells have been evaluated. The pCMV-MIR-143 vector was transfected into MDA-MB-468 cells via JetPEI transfection reagent. The transfected cells were selected by IC50 concentration of Geneticin antibiotic (G418) after a 2-week treatment. To evaluate the effect of miR-143 on the inhibition of migration, scratch wound healing assay was performed. Then, the expression level of miR-143, Kras, Vimentin, CXCR4, MMP9 and E-Cadherin were measured by the qRT-PCR method. Results of MTT and wound healing assays showed that miR-143 inhibited cell growth and cell migration in miR-143 induced cell line compared with control group. The result of gene expression showed that miR-143 reduced Kras, Vimentin, CXCR4 and MMP9 expression, and increased E-Cadherin expression in miR-143 replaced cells compared to control cells. The results showed that miRNA-143 plays an important role in cell growth and migration during breast cancer development and metastasis and it can be a candidate as a therapeutic molecule in microRNA replacement therapy of breast adenocarcinoma. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  19. miR-143 is involved in endothelial cell dysfunction through suppression of glycolysis and correlated with atherosclerotic plaques formation.

    PubMed

    Xu, R-H; Liu, B; Wu, J-D; Yan, Y-Y; Wang, J-N

    2016-10-01

    Atherosclerosis is recognized as a chronic inflammatory disease leading to hardening of the vessel wall and narrowing of arteries. Endothelial cells (ECs) exhibit highly active glycolysis, the dysfunction of which leads to accumulation of lipids in the arterial wall and formation of atherosclerotic plaque. qRT-PCR was performed to compare the deregulated miR-143 between atherosclerotic plaque and normal vessel tissues. The direct target of miR-143 was verified by Western blot and luciferase assay. The metabolic enzymes in atherosclerotic plaque and normal vessel tissues were measured. HUVECs were transfected with miR-143 precursor or control microRNAs, and glucose uptake, lactate production, intracellular ATP, and oxygen consumption were measured. In this study, we report a correlation between up-regulated miR-143, EC dysfunction, and atherosclerotic plaque formation. The glycolysis rate was significantly elevated in ECs, which show relatively low levels of miR-143. Importantly, miR-143 was upregulated in clinical atherosclerotic plaque samples compared with healthy arteries, suggesting that miR-143 might play important roles in the atherosclerotic plaque formation. Moreover, mRNA levels of key enzymes of glycolysis, such as HK2, LDHA, and PKM2 are significantly down-regulated in the atherosclerotic plaque samples. Overexpression of miR-143 in HUVECs suppresses glycolysis through direct targeting of HK2, leading to EC dysfunction. Restoration of HK2 expression rescues glycolysis in miR-143-overexpressing HUVECs. This study provides further insight into the metabolic mechanisms involved in atherosclerotic plaque formation due to microRNAs.

  20. Novel MIR143-NOTCH Fusions in Benign and Malignant Glomus Tumors

    PubMed Central

    Mosquera, Juan-Miguel; Sboner, Andrea; Zhang, Lei; Chen, Chun-Liang; Sung, Yun-Shao; Chen, Hsiao-Wei; Agaram, Narasimhan P.; Briskin, Daniel; Basha, Basma M.; Singer, Samuel; Rubin, Mark A.; Tuschl, Thomas; Antonescu, Cristina R.

    2013-01-01

    Glomus tumors (GT) have been classified among tumors of perivascular smooth muscle differentiation, together with myopericytoma, myofibroma/tosis, and angioleiomyoma, based on their morphologic overlap. However, no molecular studies have been carried out to date to investigate their genetic phenotype and to confirm their shared pathogenesis. RNA sequencing was performed in three index cases (GT1, malignant GT; GT2, benign GT and M1, multifocal myopericytoma), followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired-end RNA-seq data. A gene fusion involving MIR143 in band 5q32 was identified in both GTs with either NOTCH2 in 1p13 in GT1 or NOTCH1 in 9q34 in GT2, but none in M1. After being validated by FISH and RT-PCR, these abnormalities were screened on 33 GTs, 6 myopericytomas, 9 myofibroma/toses, 18 angioleiomyomas and in a control group of 5 sino-nasal hemangiopericytomas. Overall NOTCH2 gene rearrangements were identified in 52% of GT, including all malignant cases and one NF1-related GT. No additional cases showed NOTCH1 rearrangement. As NOTCH3 shares similar functions with NOTCH2 in regulating vascular smooth muscle development, the study group was also investigated for abnormalities in this gene by FISH. Indeed, NOTCH3 rearrangements were identified in 9% of GTs, all present in benign soft tissue GT, one case being fused to MIR143. Only 1/18 angioleiomyomas showed NOTCH2 gene rearrangement, while all the myopericytomas and myofibroma/toses were negative. In summary we describe novel NOTCH1-3 rearrangements in benign and malignant, visceral and soft tissue GTs. PMID:23999936

  1. miR-143 Induces the Apoptosis of Prostate Cancer LNCap Cells by Suppressing Bcl-2 Expression

    PubMed Central

    Ma, Zhiwei; Luo, Yizhao; Qiu, Mingxing

    2017-01-01

    Background Prostate cancer has become a serious threat to the life of patients. microRNAs are small non-coding RNA molecules that regulate the growth and apoptosis of cells. We aimed to investigate the regulation and mechanism of microRNA (miR-143) in the proliferation and apoptosis of prostate cancer LNCap cells. Material/Methods miR-143 and control scramble miRNA were synthesized and respectively transfected into LNCap cells. The proliferation and apoptosis were detected by MTT assay, flow cytometry, and caspase-3 activity assay. The intracellular expression of Bcl-2 was determined by Western blot. Further, LNCap cells were transfected with small interfering RNA (siRNA) targeting Bcl-2 (siBcl-2) or plasmid expressing Bcl-2, followed by transfection of miR-143 or control miRNA. Bcl-2 expression was detected by Western blot, and cell apoptosis was measured by caspase-3 activity assay. Results Transfection of miR-143 significantly inhibited the proliferation of LNCap cells (P=0.0073), increased the percentage of externalized phosphatidylserine (P=0.0042), activated the caspase-3 (P=0.0012), and decreased the expression of Bcl-2 (P=0.012) when compared with the control miRNA group. The expression of Bcl-2 was significantly reduced after siBcl-2 transfection. The apoptosis in the siBcl-2+miR-143 group was significantly increased compared with that in the miR-143 group (P=0.036), whereas there was no significant difference in the apoptosis between the siBcl-2+miRNA and miRNA groups. The expression of Bcl-2 was obviously higher after the transfection of Bcl-2-expressing plasmid. The apoptosis in Bcl-2+miR-143 group was significantly reduced compared with the miR-143 group (P=0.031), whereas no significant difference in the apoptosis was detected between the miRNA and Bcl-2+miRNA groups. Conclusions Transfection of miR-143 induces the apoptosis of prostate cancer LNCap cells by down-regulating Bcl-2 expression, suggesting that Bcl-2 might be a potential therapeutic

  2. miR-143 inhibits bladder cancer cell proliferation and enhances their sensitivity to gemcitabine by repressing IGF-1R signaling

    PubMed Central

    Wang, Hengbing; Li, Qi; Niu, Xiaobing; Wang, Gongcheng; Zheng, Sinian; Fu, Guangbo; Wang, Zengjun

    2017-01-01

    microRNAs (miRNAs) are a class of small RNAs that regulate gene expression. It has been demonstrated that aberrant miRNA expression is associated with cancer development and carcinogenesis. Altered miRNA expression has been suggested to occur in bladder cancer. In other cancer systems, studies have indicated that miR-143, as a tumor suppressor gene, plays essential roles in cancer progression. However, its role in bladder cancer has yet to be elucidated. In the present study, we observed that miR-143 expression was downregulated in human bladder cancer tissues and cells, and that its levels were negatively correlated with bladder cancer clinical stages. We further demonstrated that insulin-like growth factor-1 receptor (IGF-1R) is a functional target of miR-143. Their expression levels were inversely correlated in bladder cancer samples. Overexpression of miR-143 inhibited cell proliferation and promoted chemosensitivity of bladder cancer 5637 cells to gemcitabine. Consistently, small interfering RNA-mediated knockdown of IGF-1R phenocopied miR-143 overexpression. Notably, the expression of IGF-1R is a predictor of patient prognosis. Collectively, our findings indicate that miR-143 is a valuable biomarker for bladder cancer. The miR-143/IGF-1R axis is associated with bladder cancer drug resistance and patient survival. PMID:28123579

  3. MiR-143/145 deficiency protects against progression of atherosclerosis in Ldlr−/− mice

    PubMed Central

    Sala, Federica; Aranda, Juan F.; Rotllan, Noemi; Ramírez, Cristina M.; Aryal, Binod; Elia, Leonardo; Condorelli, Gianluigi; Catapano, Alberico Luigi; Fernández-Hernando, Carlos; Norata, Giuseppe Danilo

    2014-01-01

    Background and Objective The miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background. Methods and Results Ldlr−/− and Ldlr−/−-miR-143/145−/− (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. Experiments with the luciferase coding sequence fused to the ABCA1 3’UTR, Western blotting, qRT-PCR and mimicMiR confirmed the direct regulation of ABCA1 expression by miR-145. Conclusions miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease. PMID:25008143

  4. Serum Response Factor Regulates Expression of PTEN through a Micro-RNA Network in Vascular Smooth Muscle Cells

    PubMed Central

    Horita, Henrick N; Simpson, Peter A.; Ostriker, Allison; Furgeson, Seth; Van Putten, Vicki; Weiser-Evans, Mary C.M.; Nemenoff, Raphael A.

    2011-01-01

    Objective Serum response factor (SRF) is a critical transcription factor in smooth muscle cells (SMC) controlling differentiation and proliferation. Our previous work demonstrated that depleting SRF in cultured SMC decreased expression of SMC markers, but increased proliferation and inflammatory mediators. A similar phenotype has been observed in SMC silenced for PTEN, suggesting that SRF and PTEN may lie on a common pathway. Our goal was to determine the effect of SRF depletion on PTEN levels, and define mechanisms mediating this effect. Methods and Results In SRF-silenced SMC, PTEN protein, but not mRNA levels were decreased, suggesting post-transcriptional regulation. Re-introduction of PTEN into SRF-depleted SMC reversed increases in proliferation and cytokine/chemokine production, but had no effect on SMC marker expression. SRF-depleted cells showed decreased levels of miR-143, and increased miR-21, which was sufficient to suppress PTEN. Increased miR-21 expression was dependent on induction of FRA-1, which is a direct target of miR-143. Introducing miR-143 into SRF-depleted SMC reduced FRA-1 expression and miR-21 levels and restored PTEN expression. Conclusions SRF regulates PTEN expression in SMC through a miR network involving miR-143, targeting FRA-1, which regulates miR-21. Cross talk between SRF and PTEN likely represents a critical axis in phenotypic remodeling of SMC. PMID:21940949

  5. miR-143 inhibits cell proliferation by targeting autophagy-related 2B in non-small cell lung cancer H1299 cells.

    PubMed

    Wei, Jiali; Ma, Zhongliang; Li, Yanli; Zhao, Botao; Wang, Detao; Jin, Yan; Jin, Youxin

    2015-01-01

    microRNAs (miRNAs) are small, non‑coding RNAs involved in multiple biological pathways by regulating post-transcriptional gene expression. Previously, autophagy has been reported to suppress the progression of non-small cell lung cancer (NSCLC). However, how miRNAs regulate autophagy in NSCLC remains to be elucidated. In the present study, the autophagy gene, autophagy-related 2B (ATG2B), was identified as a novel target of miR-143. The overexpression of miR-143 was able to downregulate the expression of atg2b at the transcriptional and translational levels by direct binding to its 3' untranslated region. Cell proliferation was significantly inhibited by the ectopic expression of miR-143 in H1299 cells. Knockdown of ATG2B resulted in a similar phenotype, with the overexpression of miR-143 in NSCLC cells. Furthermore, knockdown of ATG2B and hexokinase 2, a key enzyme in glycolysis and another target of miR-143, co-ordinated to inhibit the proliferation of H1299 cells. The results of the present study demonstrated that miR-143 was a novel and important regulator of autophagy by targeting ATG2B and repression of gene expression in autophagy and high glycolysis had a coordinate effect in H1299 cells. These results suggested that ATG2B may be a new potential therapeutic target for NSCLC. Furthermore, it was implied that interrupting autophagy and glycolysis improves NSCLC therapy.

  6. miR-143 decreases COX-2 mRNA stability and expression in pancreatic cancer cells

    SciTech Connect

    Pham, Hung; Ekaterina Rodriguez, C.; Donald, Graham W.; Hertzer, Kathleen M.; Jung, Xiaoman S.; Chang, Hui-Hua; Moro, Aune; Reber, Howard A.; Hines, O. Joe; Eibl, Guido

    2013-09-13

    Highlights: •Pancreatic cancer cells express low miR-143 levels and elevated p-MEK, p-MAPK and RREB1. •MEK inhibitors U0126 and PD98059 increase miR-143 expression. •miR-143 decreases COX-2 mRNA stability and expression and PGE{sub 2}. •miR-143 decreases p-p38MAPK, p-MEK, p-MAPK and RREB1 expression. -- Abstract: Small non-coding RNAs, microRNAs (miRNA), inhibit the translation or accelerate the degradation of message RNA (mRNA) by targeting the 3′-untranslated region (3′-UTR) in regulating growth and survival through gene suppression. Deregulated miRNA expression contributes to disease progression in several cancers types, including pancreatic cancers (PaCa). PaCa tissues and cells exhibit decreased miRNA, elevated cyclooxygenase (COX)-2 and increased prostaglandin E{sub 2} (PGE{sub 2}) resulting in increased cancer growth and metastases. Human PaCa cell lines were used to demonstrate that restoration of miRNA-143 (miR-143) regulates COX-2 and inhibits cell proliferation. miR-143 were detected at fold levels of 0.41 ± 0.06 in AsPC-1, 0.20 ± 0.05 in Capan-2 and 0.10 ± 0.02 in MIA PaCa-2. miR-143 was not detected in BxPC-3, HPAF-II and Panc-1 which correlated with elevated mitogen-activated kinase (MAPK) and MAPK kinase (MEK) activation. Treatment with 10 μM of MEK inhibitor U0126 or PD98059 increased miR-143, respectively, by 187 ± 18 and 152 ± 26-fold in BxPC-3 and 182 ± 7 and 136 ± 9-fold in HPAF-II. miR-143 transfection diminished COX-2 mRNA stability at 60 min by 2.6 ± 0.3-fold in BxPC-3 and 2.5 ± 0.2-fold in HPAF-II. COX-2 expression and cellular proliferation in BxPC-3 and HPAF-II inversely correlated with increasing miR-143. PGE{sub 2} levels decreased by 39.3 ± 5.0% in BxPC-3 and 48.0 ± 3.0% in HPAF-II transfected with miR-143. Restoration of miR-143 in PaCa cells suppressed of COX-2, PGE{sub 2}, cellular proliferation and MEK/MAPK activation, implicating this pathway in regulating miR-143 expression.

  7. TGF-β signaling controls FSHR signaling-reduced ovarian granulosa cell apoptosis through the SMAD4/miR-143 axis

    PubMed Central

    Du, Xing; Zhang, Lifan; Li, Xinyu; Pan, Zengxiang; Liu, Honglin; Li, Qifa

    2016-01-01

    Follicle-stimulating hormone receptor (FSHR) and its intracellular signaling control mammalian follicular development and female infertility. Our previous study showed that FSHR is downregulated during follicular atresia of porcine ovaries. However, its role and regulation in follicular atresia remain unclear. Here, we showed that FSHR knockdown induced porcine granulosa cell (pGC) apoptosis and follicular atresia, and attenuated the levels of intracellular signaling molecules such as PKA, AKT and p-AKT. FSHR was identified as a target of miR-143, a microRNA that was upregulated during porcine follicular atresia. miR-143 enhanced pGC apoptosis by targeting FSHR, and reduced the levels of intracellular signaling molecules. SMAD4, the final molecule in transforming growth factor (TGF)-β signaling, bound to the promoter and induced significant downregulation of miR-143 in vitro and in vivo. Activated TGF-β signaling rescued miR-143-reduced FSHR and intracellular signaling molecules, and miR-143-induced pGC apoptosis. Overall, our findings offer evidence to explain how TGF-β signaling influences and FSHR signaling for regulation of pGC apoptosis and follicular atresia by a specific microRNA, miR-143. PMID:27882941

  8. Inhibition of miR-143 during ischemia cerebral injury protects neurones through recovery of the hexokinase 2-mediated glucose uptake.

    PubMed

    Zeng, Xianzhu; Liu, Na; Zhang, Jing; Wang, Lei; Zhang, Zhecheng; Zhu, Ju; Li, Qian; Wang, Yuwen

    2017-08-31

    Ischemic stroke, a major cause of death, is caused by occlusion of a blood vessel, resulting in significant reduction in regional cerebral blood flow. MiRNAs are a family of short noncoding RNAs (18-22 nts) and bind the 3'-UTR of their target genes to suppress the gene expression post-transcriptionally. In the present study, we report that miR-143 is down-regulated in rat neurones but highly expressed in astrocytes. In vivo middle cerebral artery occlusion (MCAO) and ex vivo oxygen-glucose deprivation (OGD) results showed that miR-143 was significantly induced by ischemia injury. Meanwhile, we observed suppression of glucose uptake and lactate product of rat brain and primary neurones after MCAO or OGD. The glycolysis enzymes hexokinase 2 (HK2), PKM2, and LDHA were inhibited by MCAO or OGD at protein and mRNA levels. In addition, overexpression of miR-143 significantly inhibited HK2 expression, glucose uptake, and lactate product. We report that HK2 is a direct target of miR-143. Importantly, restoration of HK2 in miR-143 overexpressing rat neurones recovered glucose uptake and lactate product. Our results demonstrated inhibition of miR-143 during OGD could protect rat neuronal cells from ischemic brain injury (IBI). In summary, the present study reveals a miRNA-mediated neuron protection during IBI, providing a new strategy for the development of therapeutic agents against IBI. © 2017 The Author(s).

  9. MiR-143 targets CTGF and exerts tumor-suppressing functions in epithelial ovarian cancer

    PubMed Central

    Wang, Lufei; He, Jin; Xu, Hongmei; Xu, Longjie; Li, Na

    2016-01-01

    A series of recent studies suggested that miR-143 might involve in the tumorigenesis and metastasis of various cancer types. However, the biological function and underlying mechanisms of miR-143 in human epithelial ovarian carcinoma (EOC) remain unknown. Therefore, this study aimed to investigate the miR-143 expression and its clinical diagnosis significance in patients suffering EOC and to analyze its role and underlying molecular mechanism in EOC. Our result showed that the expression levels of miR-143 were downregulated in EOC tissues and cell lines, was associated with International Federation of Gynaecology and Obstetrics (FIGO) stage, pathological grade and lymph node metastasis (all P < 0.01) . Overexpression of miR-143 significantly inhibited EOC cell proliferation, migration, and invasion. Furthermore, computational algorithm combined with luciferase reporter assays identified connective tissue growth factor (CTGF) as the direct target of miR-143 in EOC cells. The expression level of CTGF was significantly increased in EOC tissues, was inversely correlated with miR-143 expression in clinical EOC tissues. Knockdown of CTGF mimicked the suppression effect induced by miR-143 overexpression. Restoration of CTGF expression partially reversed the suppression effect induced by miR-143 overexpression. These results suggested that miR-143 inhibited EOC cell proliferation, migration, and invasion, at least in part, via suppressing CTGF expression. PMID:27398154

  10. Stromal expression of miR-143/145 promotes neoangiogenesis in lung cancer development

    PubMed Central

    Dimitrova, Nadya; Gocheva, Vasilena; Bhutkar, Arjun; Resnick, Rebecca; Jong, Robyn M.; Miller, Kathryn M.; Bendor, Jordan; Jacks, Tyler

    2015-01-01

    The two unrelated miRNAs, miR-143 and miR-145, co-expressed from the miR-143/145 cluster have been proposed to act as tumor suppressors in human cancer and therapeutic benefits of delivering miR-143 and miR-145 to tumors have been reported. In contrast, we found that tumor-specific deletion of miR-143/145 in an autochthonous mouse model of lung adenocarcinoma did not affect tumor development. This was consistent with the lack of endogenous miR-143/145 expression in normal and transformed lung epithelium. Surprisingly, miR-143/145 in the tumor microenvironment dramatically promoted tumor growth by stimulating the proliferation of endothelial cells. Loss of miR-143/145 in vivo led to derepression of the miR-145 target Camk1d, an inhibitory kinase, which when overexpressed prevents mitotic entry of endothelial cells. As a consequence, tumors in miR-143/145-deficient animals exhibited diminished neoangiogenesis, increased apoptosis and their expansion was limited by the tumor’s ability to co-opt the alveolar vasculature. These findings demonstrate that stromal miR-143/145 promotes tumorigenesis and cautions against the use of these miRNAs as agents in cancer therapeutics. PMID:26586766

  11. miR-143 Overexpression Impairs Growth of Human Colon Carcinoma Xenografts in Mice with Induction of Apoptosis and Inhibition of Proliferation

    PubMed Central

    Borralho, Pedro M.; Simões, André E. S.; Gomes, Sofia E.; Lima, Raquel T.; Carvalho, Tânia; Ferreira, Duarte M. S.; Vasconcelos, Maria H.; Castro, Rui E.; Rodrigues, Cecília M. P.

    2011-01-01

    Background MicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu). Methodology/Principal Findings HCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm3, respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01). Conclusions Our results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel

  12. Circulating miR-92a, miR-143 and miR-342 in Plasma are Novel Potential Biomarkers for Acute Myeloid Leukemia

    PubMed Central

    Elhamamsy, Amr Rafat; El Sharkawy, Muhammad Suleiman; Zanaty, Ahmed Farouk; Mahrous, Mohammed Ahmed; Mohamed, Ahmed Ezzat; Abushaaban, Eslam Ahmed

    2017-01-01

    MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional gene expression regulators. The expression profiling of miRNAs has already entered into cancer clinics as diagnostic and prognostic biomarkers to assess tumor initiation, progression and response to treatment in cancer patients. Recent studies have opened the way for the use of circulating miRNAs as non-invasive diagnosis and prognosis of Acute myeloid leukemia (AML). The aim of this study was to identify plasma miR-92a, miR-143 and miR-342 expression signatures in AML patients to introduce new markers for establishing AML diagnosis and prognosis. Blood samples were collected from 65 AML patients and 50 controls. The expression of three target miRNAs (miR-92a, miR-143 and miR-342) was measured using quantitative real-time PCR method. Plasma levels of miR-92a, miR-143 and miR-342 were significantly lower in AML patients in comparison with control group. Receiver operator characteristic (ROC) analysis revealed that the sensitivity and specificity values of miR-92a were 81.5% and 94%, respectively, with a cut-off value of 0.704. The sensitivity and specificity values of miR-143 were 87.7% and 80%, respectively, with a cut-off value of 0.65. The sensitivity and specificity values of miR-342 were 75.4% and 90%, respectively, with a cut-off value of 0.479. Our findings suggest that plasma miR-92a, miR-143 and miR-342 could be promising novel circulating biomarkers in clinical detection of AML. PMID:28890884

  13. MicroRNA Regulation of Smooth Muscle Phenotype

    PubMed Central

    Joshi, Sachindra R.; Comer, Brian S.; McLendon, Jared M.; Gerthoffer, William T.

    2014-01-01

    Advances in studies of microRNA (miRNA) expression and function in smooth muscles illustrate important effects of small noncoding RNAs on cell proliferation, hypertrophy and differentiation. An emerging theme in miRNA research in a variety of cell types including smooth muscles is that miRNAs regulate protein expression networks to fine tune phenotype. Some widely expressed miRNAs have been described in smooth muscles that regulate important processes in many cell types, such as miR-21 control of proliferation and cell survival. Other miRNAs that are prominent regulators of smooth muscle-restricted gene expression also have targets that control pluripotent cell differentiation. The miR-143~145 cluster which targets myocardin and Kruppel-like factor 4 (KLF4) is arguably the best-described miRNA family in smooth muscles with profound effects on gene expression networks that promote serum response factor (SRF)-dependent contractile and cytoskeletal protein expression and the mature contractile phenotype. Kruppel-family members KLF4 and KLF5 have multiple effects on cell differentiation and are targets for multiple miRNAs in smooth muscles (miR-145, miR-146a, miR-25). The feedback and feedforward loops being defined appear to contribute significantly to vascular and airway remodeling in cardiovascular and respiratory diseases. RNA interference approaches applied to animal models of vascular and respiratory diseases prove that miRNAs and RNA-induced silencing are valid targets for novel anti-remodeling therapies that alter pathological smooth muscle hyperplasia and hypertrophy. PMID:25309675

  14. Honokiol inhibits bladder tumor growth by suppressing EZH2/miR-143 axis.

    PubMed

    Zhang, Qing; Zhao, Wei; Ye, Changxiao; Zhuang, Junlong; Chang, Cunjie; Li, Yuying; Huang, Xiaojing; Shen, Lan; Li, Yan; Cui, Yangyan; Song, Jiannan; Shen, Bing; Eliaz, Isaac; Huang, Ruimin; Ying, Hao; Guo, Hongqian; Yan, Jun

    2015-11-10

    The oncoprotein EZH2, as a histone H3K27 methyltransferase, is frequently overexpressed in various cancer types. However, the mechanisms underlying its role in urinary bladder cancer (UBC) cells have not yet fully understood. Herein, we reported that honokiol, a biologically active biphenolic compound isolated from the Magnolia officinalis inhibited human UBC cell proliferation, survival, cancer stemness, migration, and invasion, through downregulation of EZH2 expression level, along with the reductions of MMP9, CD44, Sox2 and the induction of tumor suppressor miR-143. Either EZH2 overexpression or miR-143 inhibition could partially reverse honokiol-induced cell growth arrest and impaired clonogenicity. Importantly, it was first revealed that EZH2 could directly bind to the transcriptional regulatory region of miR-143 and repress its expression. Furthermore, honokiol treatment on T24 tumor xenografts confirmed its anticancer effects in vivo, including suppression tumor growth and tumor stemness, accompanied by the dysregulation of EZH2 and miR-143 expressions. Our data suggest a promising therapeutic option to develop drugs targeting EZH2/miR-143 axis, such as honokiol, for bladder cancer treatment.

  15. Honokiol inhibits bladder tumor growth by suppressing EZH2/miR-143 axis

    PubMed Central

    Chang, Cunjie; Li, Yuying; Huang, Xiaojing; Shen, Lan; Li, Yan; Cui, Yangyan; Song, Jiannan; Shen, Bing; Eliaz, Isaac; Huang, Ruimin; Ying, Hao; Guo, Hongqian; Yan, Jun

    2015-01-01

    The oncoprotein EZH2, as a histone H3K27 methyltransferase, is frequently overexpressed in various cancer types. However, the mechanisms underlying its role in urinary bladder cancer (UBC) cells have not yet fully understood. Herein, we reported that honokiol, a biologically active biphenolic compound isolated from the Magnolia officinalis inhibited human UBC cell proliferation, survival, cancer stemness, migration, and invasion, through downregulation of EZH2 expression level, along with the reductions of MMP9, CD44, Sox2 and the induction of tumor suppressor miR-143. Either EZH2 overexpression or miR-143 inhibition could partially reverse honokiol-induced cell growth arrest and impaired clonogenicity. Importantly, it was first revealed that EZH2 could directly bind to the transcriptional regulatory region of miR-143 and repress its expression. Furthermore, honokiol treatment on T24 tumor xenografts confirmed its anticancer effects in vivo, including suppression tumor growth and tumor stemness, accompanied by the dysregulation of EZH2 and miR-143 expressions. Our data suggest a promising therapeutic option to develop drugs targeting EZH2/miR-143 axis, such as honokiol, for bladder cancer treatment. PMID:26484567

  16. Evaluation of miR-9 and miR-143 expression in urine specimens of sulfur mustard exposed patients.

    PubMed

    Khafaei, Mostafa; Samie, Shahram; Mowla, Seyed Javad; Alvanegh, Akbar Ghorbani; Mirzaei, Behnaz; Chavoshei, Somaye; Dorraj, Ghamar Soltan; Esmailnejad, Mostafa; Tavallaie, Mahmood; Nourani, Mohammadreza

    2015-12-01

    Sulfur mustard (SM) or mustard gas is a chemical alkylating agent that causes blisters in the skin (blister gas), burns the eyes and causes lung injury. Some major cellular pathways are involved in the damage caused by mustard gas such as NF-κb signaling, TGF-β signaling, WNT pathway, inflammation, DNA repair and apoptosis. MicroRNAs are non-coding small RNAs (19-25 nucleotides) that are involved in the regulation of gene expression and are found in two forms, extracellular and intracellular. Changes in the levels of extracellular microRNAs are directly associated with many diseases, it is thus common to study the level of extracellular microRNAs as a biomarker to determine the pathophysiologic status. In this study, 32 mustard gas injured patients and 32healthy subjects participated. Comparative evaluation of miR-9 and miR-143 expression in urine samples was performed by Real Time PCR and Graph Pad software. The Mann Whitney t-test analysis of data showed that the expression level of miR-143 and miR-9 had a significant decrease in sulfur mustard individuals with the respective p-value of 0.0480 and 0.0272 compared to normal samples, with an imbalance of several above mentioned pathways. It seems that reducing the expression level of these genes has a very important role in the pathogenicity of mustard gas injured patients.

  17. Low miR-143/miR-145 Cluster Levels Induce Activin A Overexpression in Oral Squamous Cell Carcinomas, Which Contributes to Poor Prognosis

    PubMed Central

    Bufalino, Andreia; Cervigne, Nilva K.; de Oliveira, Carine Ervolino; Fonseca, Felipe Paiva; Rodrigues, Priscila Campioni; Macedo, Carolina Carneiro Soares; Sobral, Lays Martin; Miguel, Marcia Costa; Lopes, Marcio Ajudarte; Leme, Adriana Franco Paes; Lambert, Daniel W.; Salo, Tuula A.; Kowalski, Luiz Paulo; Graner, Edgard; Coletta, Ricardo D.

    2015-01-01

    Deregulated expression of activin A is reported in several tumors, but its biological functions in oral squamous cell carcinoma (OSCC) are unknown. Here, we investigate whether activin A can play a causal role in OSCCs. Activin A expression was assessed by qPCR and immunohistochemistry in OSCC tissues. Low activin A-expressing cells were treated with recombinant activin A and assessed for apoptosis, proliferation, adhesion, migration, invasion and epithelial-mesenchymal transition (EMT). Those phenotypes were also evaluated in high activin A-expressing cells treated with follistatin (an activin A antagonist) or stably expressing shRNA targeting activin A. Transfections of microRNA mimics were performed to determine whether the overexpression of activin A is regulated by miR-143/miR-145 cluster. Activin A was overexpressed in OSCCs in comparison with normal oral mucosa, and high activin A levels were significantly associated with lymph node metastasis, tumor differentiation and poor survival. High activin A levels promoted multiple properties associated with malignant transformation, including decreased apoptosis and increased proliferation, migration, invasion and EMT. Both miR-143 and miR-145 were markedly downregulated in OSCC cell lines and in clinical specimens, and inversely correlated to activin A levels. Forced expression of miR-143 and miR-145 in OSCC cells significantly decreased the expression of activin A. Overexpression of activin A in OSCCs, which is controlled by downregulation of miR-143/miR-145 cluster, regulates apoptosis, proliferation and invasiveness, and it is clinically correlated with lymph node metastasis and poor survival. PMID:26317418

  18. Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon.

    PubMed

    Pekow, Joel; Meckel, Katherine; Dougherty, Urszula; Butun, Fatma; Mustafi, Reba; Lim, John; Crofton, Charis; Chen, Xindi; Joseph, Loren; Bissonnette, Marc

    2015-02-01

    The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.

  19. Tumor suppressors miR-143 and miR-145 and predicted target proteins API5, ERK5, K-RAS, and IRS-1 are differentially expressed in proximal and distal colon

    PubMed Central

    Meckel, Katherine; Dougherty, Urszula; Butun, Fatma; Mustafi, Reba; Lim, John; Crofton, Charis; Chen, Xindi; Joseph, Loren; Bissonnette, Marc

    2014-01-01

    The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition. PMID:25477374

  20. RETRACTED: MiR-143 inhibits tumor cell proliferation and invasion by targeting STAT3 in esophageal squamous cell carcinoma.

    PubMed

    Liu, Jia; Mao, Yu; Zhang, Dakai; Hao, Shengnan; Zhang, Zicheng; Li, Zhenjiang; Li, Baosheng

    2016-04-01

    This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor in Chief. The term miR-143 has been used instead of the term miR-143* in the introduction and discussion which is misleading. With the misuse of this term subsequent errors and misleading statements appear throughout the paper. The authors apologize for this mistake. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. microRNAs Distinctively Regulate Vascular Smooth Muscle and Endothelial Cells: Functional Implications in Angiogenesis, Atherosclerosis, and In-Stent Restenosis

    PubMed Central

    Santulli, Gaetano

    2016-01-01

    Endothelial cells (EC) and vascular smooth muscle cells (VSMC) are the main cell types within the vasculature. We describe here how microRNAs (miRs)—noncoding RNAs that can regulate gene expression via translational repression and/or post-transcriptional degradation—distinctively modulate EC and VSMC function in physiology and disease. In particular, the specific roles of miR-126 and miR-143/145, master regulators of EC and VSMC function, respectively, are deeply explored. We also describe the mechanistic role of miRs in the regulation of the pathophysiology of key cardiovascular processes including angiogenesis, atherosclerosis, and in-stent restenosis post-angioplasty. Drawbacks of currently available therapeutic options are discussed, pointing at the challenges and potential clinical opportunities provided by miR-based treatments. PMID:26662986

  2. 18F-FDG PET/CT for Monitoring the Response of Breast Cancer to miR-143-Based Therapeutics by Targeting Tumor Glycolysis

    PubMed Central

    Miao, Ying; Zhang, Ling-fei; Guo, Rui; Liang, Sheng; Zhang, Min; Shi, Shuo; Shang-Guan, Cheng-fang; Liu, Mo-fang; Li, Biao

    2016-01-01

    Increased glucose utilization is a hallmark of cancer, and tumor metabolism is emerging as anticancer target for therapeutic intervention. Triple-negative breast cancers TNBC are highly glycolytic and show poor clinical outcomes. We previously identified hexokinase 2, the major glycolytic enzyme, as a target gene of miR-143 in TNBC. Here, we developed a therapeutic formulation using cholesterol-modified miR-143 agomir encapsulated in a neutral lipid-based delivery agent that blocked tumor growth and glucose metabolism in TNBC tumor-bearing mice when administered systemically. The antioncogenic effects were accompanied by a reduction in the direct target hexokinase 2 and [18F]-fluorodeoxyglucose (18F-FDG) uptake based on positron emission tomography/computed tomography. Treatment with miR-143 formulation has minimal toxic effects and mice tolerated it well. Thus, we demonstrated that miR-143 is a robust inhibitor of the Warburg effect and an effective therapeutic target for TNBC. In addition, 18F-FDG positron emission tomography/computed tomography can be used to specifically monitor the response of TNBC to miR-143-based therapeutics by targeting tumor glycolysis. PMID:27574783

  3. (18)F-FDG PET/CT for Monitoring the Response of Breast Cancer to miR-143-Based Therapeutics by Targeting Tumor Glycolysis.

    PubMed

    Miao, Ying; Zhang, Ling-Fei; Guo, Rui; Liang, Sheng; Zhang, Min; Shi, Shuo; Shang-Guan, Cheng-Fang; Liu, Mo-Fang; Li, Biao

    2016-01-01

    Increased glucose utilization is a hallmark of cancer, and tumor metabolism is emerging as anticancer target for therapeutic intervention. Triple-negative breast cancers TNBC are highly glycolytic and show poor clinical outcomes. We previously identified hexokinase 2, the major glycolytic enzyme, as a target gene of miR-143 in TNBC. Here, we developed a therapeutic formulation using cholesterol-modified miR-143 agomir encapsulated in a neutral lipid-based delivery agent that blocked tumor growth and glucose metabolism in TNBC tumor-bearing mice when administered systemically. The antioncogenic effects were accompanied by a reduction in the direct target hexokinase 2 and [(18)F]-fluorodeoxyglucose ((18)F-FDG) uptake based on positron emission tomography/computed tomography. Treatment with miR-143 formulation has minimal toxic effects and mice tolerated it well. Thus, we demonstrated that miR-143 is a robust inhibitor of the Warburg effect and an effective therapeutic target for TNBC. In addition, (18)F-FDG positron emission tomography/computed tomography can be used to specifically monitor the response of TNBC to miR-143-based therapeutics by targeting tumor glycolysis.

  4. (18)F-FDG PET/CT for Monitoring the Response of Breast Cancer to miR-143-Based Therapeutics by Targeting Tumor Glycolysis.

    PubMed

    Miao, Ying; Zhang, Ling-Fei; Guo, Rui; Liang, Sheng; Zhang, Min; Shi, Shuo; Shang-Guan, Cheng-Fang; Liu, Mo-Fang; Li, Biao

    2016-08-30

    Increased glucose utilization is a hallmark of cancer, and tumor metabolism is emerging as anticancer target for therapeutic intervention. Triple-negative breast cancers TNBC are highly glycolytic and show poor clinical outcomes. We previously identified hexokinase 2, the major glycolytic enzyme, as a target gene of miR-143 in TNBC. Here, we developed a therapeutic formulation using cholesterol-modified miR-143 agomir encapsulated in a neutral lipid-based delivery agent that blocked tumor growth and glucose metabolism in TNBC tumor-bearing mice when administered systemically. The antioncogenic effects were accompanied by a reduction in the direct target hexokinase 2 and [(18)F]-fluorodeoxyglucose ((18)F-FDG) uptake based on positron emission tomography/computed tomography. Treatment with miR-143 formulation has minimal toxic effects and mice tolerated it well. Thus, we demonstrated that miR-143 is a robust inhibitor of the Warburg effect and an effective therapeutic target for TNBC. In addition, (18)F-FDG positron emission tomography/computed tomography can be used to specifically monitor the response of TNBC to miR-143-based therapeutics by targeting tumor glycolysis.

  5. Circular RNA hsa_circ_0001982 Promotes Breast Cancer Cell Carcinogenesis Through Decreasing miR-143.

    PubMed

    Tang, Yi-Yin; Zhao, Ping; Zou, Tian-Ning; Duan, Jia-Jun; Zhi, Rong; Yang, Si-Yuan; Yang, De-Chun; Wang, Xiao-Li

    2017-09-21

    Circular RNAs (circRNAs) are a type of noncoding RNAs generated from back-splicing, which have been verified to mediate multiple tumorigenesis. With the development of high-throughput sequencing, massive circRNAs are discovered in tumorous tissue. However, the potential physiological effect of circRNAs in breast cancer is still unknown. The purpose of this study is to investigate the expression profile of circRNA in breast cancer tissue and explore the in-depth regulatory mechanism in breast cancer tumorigenesis. In the present study, we screened the circRNA expression profiles in breast cancer tissue using circRNA microarray analysis. Totally 1705 circRNAs were identified to be significantly aberrant. Among these dysregulated circRNAs, hsa_circ_0001982 was markedly overexpressed in breast cancer tissue and cell lines. Bioinformatics analysis predicted that miR-143 acted as target of hsa_circ_0001982, which was confirmed by Dual-luciferase reporter assay. Loss-of-function and rescue experiments revealed that hsa_circ_0001982 knockdown suppressed breast cancer cell proliferation and invasion and induced apoptosis by targeting miR-143. In summary, our study preliminarily investigates the circRNA expression in breast cancer tissue and explores the role of competing endogenous RNA (ceRNA) mechanism in the progression, providing a novel insight for breast cancer tumorigenesis.

  6. Association between functional polymorphisms in the promoter of the miR-143/145 cluster and risk of intracranial aneurysm

    PubMed Central

    Sima, Xiutian; Sun, Hong; Zhou, Peizhi; You, Chao; Cai, Bowen

    2017-01-01

    MicroRNAs (miRs)-143/145 are involved in various biological processes related to aneurysm formation and are downregulated in patients with intracranial aneurysm (IA). We aimed to determine whether two functional polymorphisms (i.e. rs4705342 and rs4705343) in the promoter of miR-143/145 are related to IA risk. A case-control study was undertaken to examine the association of rs4705342 and rs4705343 with IA risk, including 565 patients with IA and 622 age- and gender-matched controls. rs4705342 was analysed by TaqMan Assay, and rs4705343 was genotyped using polymerase chain reaction-restriction fragment length polymorphism. miR-143/145 expression was quantified using RT-PCR. rs4705342 was associated with a significantly lower risk of IA, with adjusted ORs of 0.74 (95% CI: 0.58–0.95) for TC genotype carriers and 0.74 (95% CI: 0.59–0.94) for TC/CC genotypes carriers. Individuals carrying the rs4705342 C allele had a reduced risk of IA (adjusted OR = 0.82; 95% CI: 0.68–0.98). Haplotype of the two loci of rs4705342 and rs4705343 showed that the CT haplotype carried a lower IA risk and higher miR-143 level. Moreover, the rs4705342 CC/CT genotypes were associated with higher miR-143 levels. Thus, the rs4705342C-rs4705343T haplotype in the promoter of miR-143/145 cluster may be related to IA development. PMID:28272526

  7. Association between functional polymorphisms in the promoter of the miR-143/145 cluster and risk of intracranial aneurysm.

    PubMed

    Sima, Xiutian; Sun, Hong; Zhou, Peizhi; You, Chao; Cai, Bowen

    2017-03-08

    MicroRNAs (miRs)-143/145 are involved in various biological processes related to aneurysm formation and are downregulated in patients with intracranial aneurysm (IA). We aimed to determine whether two functional polymorphisms (i.e. rs4705342 and rs4705343) in the promoter of miR-143/145 are related to IA risk. A case-control study was undertaken to examine the association of rs4705342 and rs4705343 with IA risk, including 565 patients with IA and 622 age- and gender-matched controls. rs4705342 was analysed by TaqMan Assay, and rs4705343 was genotyped using polymerase chain reaction-restriction fragment length polymorphism. miR-143/145 expression was quantified using RT-PCR. rs4705342 was associated with a significantly lower risk of IA, with adjusted ORs of 0.74 (95% CI: 0.58-0.95) for TC genotype carriers and 0.74 (95% CI: 0.59-0.94) for TC/CC genotypes carriers. Individuals carrying the rs4705342 C allele had a reduced risk of IA (adjusted OR = 0.82; 95% CI: 0.68-0.98). Haplotype of the two loci of rs4705342 and rs4705343 showed that the CT haplotype carried a lower IA risk and higher miR-143 level. Moreover, the rs4705342 CC/CT genotypes were associated with higher miR-143 levels. Thus, the rs4705342C-rs4705343T haplotype in the promoter of miR-143/145 cluster may be related to IA development.

  8. Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization.

    PubMed

    Alajbegovic, Azra; Turczyńska, Karolina M; Hien, Tran Thi; Cidad, Pilar; Swärd, Karl; Hellstrand, Per; Della Corte, Alessandro; Forte, Amalia; Albinsson, Sebastian

    2017-06-01

    The dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells. This analysis was performed using cardiovascular disease-focused miRNA arrays in both mouse and human cells. The potential clinical importance of actin polymerization in aortic aneurysm was evaluated using biopsies from mildly dilated human thoracic aorta in patients with stenotic tricuspid or bicuspid aortic valve. By integrating information from multiple qPCR based miRNA arrays we identified a group of five miRNAs (miR-1, miR-22, miR-143, miR-145 and miR-378a) that were sensitive to actin polymerization and MRTF-A overexpression in both mouse and human vascular smooth muscle. With the exception of miR-22, these miRNAs were also relatively enriched in striated and/or smooth muscle containing tissues. Actin polymerization was found to be dramatically reduced in the aorta from patients with mild aortic dilations. This was associated with a decrease in actin/MRTF-regulated miRNAs. In conclusion, the transcriptional co-activator MRTF-A and actin polymerization regulated a subset of miRNAs in vascular smooth muscle. Identification of novel miRNAs regulated by actin/MRTF-A may provide further insight into the mechanisms underlying

  9. Downregulation of anti-oncomirs miR-143/145 cluster occurs before APC gene aberration in the development of colorectal tumors.

    PubMed

    Kamatani, Akemi; Nakagawa, Yoshihito; Akao, Yukihiro; Maruyama, Naoko; Nagasaka, Mitsuo; Shibata, Tomoyuki; Tahara, Tomomitsu; Hirata, Ichiro

    2013-09-01

    Accumulating data indicate that some microRNAs (miRNAs or miRs) can function as tumor suppressors or oncogenes and as such are important in cancer development. We previously reported that miR-143 and -145 are frequently downregulated in colon adenomas and cancers, acting as tumor suppressors. In this present study, we investigated the relationship between the downregulation of the miR-143/145 cluster and genetic aberrations of adenomatous polyposis coli (APC), which are early genetic events in the development of colorectal tumors. The expression levels of both miRs were determined by performing real-time PCR on tissue samples of familial adenomatous polyposis (FAP), colorectal adenoma, colorectal cancer, and paired non-tumorous tissues. Also, the expression of C- or N-terminus of the APC protein and that of the p53 protein in these tissues were examined immunohistochemically. Our data clearly indicated that the decreased expression of miR-143 and -145 frequently occurred before APC gene aberrations. The downregulation of miR-143 and -145 is thus an important genetic event for the initiation step in colorectal tumor development.

  10. Expression of oncogenic miR-17-92 and tumor suppressive miR-143-145 clusters in basal cell carcinoma and cutaneous squamous cell carcinoma.

    PubMed

    Sand, Michael; Hessam, Schapoor; Amur, Susanne; Skrygan, Marina; Bromba, Michael; Stockfleth, Eggert; Gambichler, Thilo; Bechara, Falk G

    2017-05-01

    A variety of cancers are associated with the expression of the oncogenic miR-17-92 cluster (Oncomir-1) and tumor suppressor miR-143-5p/miR-145-5p. Epidermal skin cancer has not been investigated for the expression of miR-17-92 and miR-143-145 clusters, despite being extensively studied regarding global microRNA profiles. The goal of this study was to investigate the expression and possible correlation of expression of miR17-92 and miR-143-145 cluster members in epidermal skin cancer. We evaluated punch biopsies from patients with cutaneous squamous cell carcinoma (cSCC, n=15) and basal cell carcinoma (BCC, n=16), along with control specimens from non-lesional epidermal skin (n=16). Expression levels of the miR17-92 cluster (including miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-3p, miR-19b-1-5p, miR-20a-3p, miR-20a-5p, miR-92a-3p, and miR-92a-5p) and the tumor-suppressive cluster miR-143-145 (including miR-143-5p and miR-145-5p) were detected by quantitative real-time reverse transcriptase polymerase chain reaction. We noted a highly significant increased expression of the miR-17-92 members miR-17-5p, miR-18a-5p, miR19a-3p, and miR-19b-3p and tumor suppressor miR-143-5p (p<0.01) in cSCC. miR-145-5p had a significantly decreased expression (p<0.05) for in BCC. A correlation analysis revealed multiple correlating miRNA-pairs within and between the investigated clusters. This study marks the first evidence for the participation of members of the miR-17-92 cluster in cSCC and miR-143-145 cluster in BCC. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  11. miR-17, miR-21, and miR-143 Enhance Adipogenic Differentiation from Porcine Bone Marrow-Derived Mesenchymal Stem Cells.

    PubMed

    An, Xinglan; Ma, Kuiying; Zhang, Zhiren; Zhao, Tianchuang; Zhang, Xueming; Tang, Bo; Li, Ziyi

    2016-08-01

    Bone marrow-derived mesenchymal stem cells (BMSCs) have multilineage differentiation abilities toward adipocytes and osteoblasts. Recently, numerous studies have focused on the roles of microRNAs (miRNAs) in the process of adipogenic differentiation of human and mouse cells. However, the role of miRNAs in adipogenic differentiation process of porcine BMSCs (pBMSCs) remains unclear. In this study, pBMSCs were induced to differentiate into adipocytes using a chemical approach, and the roles of miR-17, miR-21, and miR-143 in this process were investigated. Our results showed that pBMSCs could be chemically induced to differentiate into adipocytes and that the expression of miR-17, miR-21, and miR-143 increased during differentiation. Then, overexpression of mimics of miR-17, miR-21, and miR-143 increased the number of oil red O-positive cells of adipocyte differentiation. The expression levels of CCAAT/enhancer-binding protein alpha (C/EBPα) mRNA showed increases of 1.8-, 1.5-, and 1.2-fold in the groups expressing mimics of miR-21, miR-17, and miR-143, respectively, at day 20. These results demonstrate that miR-17, miR-21, and miR-143 are involved in and promote the adipogenic differentiation of pBMSCs. This study provides an experimental basis for establishing a stable and efficient adipogenic differentiation model for applications in cell therapy and tissue engineering.

  12. MicroRNA Transcriptome Profile Analysis in Porcine Muscle and the Effect of miR-143 on the MYH7 Gene and Protein.

    PubMed

    Zuo, Jianjun; Wu, Fan; Liu, Yihua; Xiao, Juan; Xu, Mei; Yu, Qinping; Xia, Minhao; He, Xiaojun; Zou, Shigeng; Tan, Huize; Feng, Dingyuan

    2015-01-01

    Porcine skeletal muscle fibres are classified based on their different physiological and biochemical properties. Muscle fibre phenotype is regulated by several independent signalling pathways, including the mitogen-activated protein kinase (MAPK), nuclear factor of activated T cells (NFAT), myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) signalling pathways. MicroRNAs are non-coding small RNAs that regulate many biological processes. However, their function in muscle fibre type regulation remains unclear. The aim of our study was to identify miRNAs that regulate muscle fibre type during porcine growth to help understand the miRNA regulation mechanism of fibre differentiation. We performed Solexa/Illumina deep sequencing for the microRNAome during 3 muscle growth stages (63, 98 and 161 d). In this study, 271 mature miRNAs and 243 pre-miRNAs were identified. We detected 472 novel miRNAs in the muscle samples. Among the mature miRNAs, there are 23 highest expression miRNAs (over 10,000 RPM), account for 85.3% of the total counts of mature miRNAs., including 10 (43.5%) muscle-related miRNAs (ssc-miR-133a-3p, ssc-miR-486, ssc-miR-1, ssc-miR-143-3p, ssc-miR-30a-5p, ssc-miR-181a, ssc-miR-148a-3p, ssc-miR-92a, ssc-miR-21, ssc-miR-126-5p). Particularly, both ssc-miR-1 and ssc-miR-133 belong to the MyomiRs, which control muscle myosin content, myofibre identity and muscle performance. The involvement of these miRNAs in muscle fibre phenotype provides new insight into the mechanism of muscle fibre regulation underlying muscle development. Furthermore, we performed cell transfection experiment. Overexpression/inhibition of ssc-miR-143-3p in porcine skeletal muscle satellite cell induced an/a increase/reduction of the slow muscle fibre gene and protein (MYH7), indicating that miR-143 activity regulated muscle fibre differentiate in skeletal muscle. And it regulate MYH7 through the HDAC4-MEF2 pathway.

  13. A polymorphism rs4705341 in the flanking region of miR-143/145 predicts risk and prognosis of colorectal cancer

    PubMed Central

    Li, Lijuan; Sun, Hong; Nie, Xinwen; Liang, Yundan; Yuan, Fang; Pu, Yan; Bai, Peng; Zhang, Lin; Gao, Linbo

    2016-01-01

    The aim of this study was to investigate the effect of a polymorphism rs4705341 in the flanking region of miR-143/145 on the risk of colorectal cancer (CRC). The rs4705341 polymorphism was analyzed in 1002 cases and 1062 controls using a polymerase chain reaction-restriction fragment length polymorphism method. We found a significantly reduced CRC susceptibility with miR-143/145 rs4705341 in homozygote comparison (adjusted OR = 0.66, 95%CI, 0.50-0.88, P = 0.004), dominant genetic model (adjusted OR = 0.80, 95%CI, 0.67-0.96, P = 0.015), recessive genetic model (adjusted OR = 0.73, 95%CI, 0.56-0.94, P = 0.016), and allele comparison (adjusted OR = 0.83, 95%CI, 0.73-0.94, P = 0.004). Stratification analysis showed that the rs4705341 was related to differentiated status, clinical stage I-II, and patients without lymph node metastasis. Moreover, patients with rs4705341GG had a longer overall survival (adjusted HR = 5.57, 95%CI, 0.95-32.68). These findings indicate that the miR-143/145 rs4705341 may be used as a potential biomarker for the development and prognosis of CRC. PMID:27556691

  14. miR-143 inhibits oncogenic traits by degrading NUAK2 in glioblastoma.

    PubMed

    Fu, Ting-Gang; Wang, Ling; Li, Wei; Li, Jian-Zhong; Li, Jian

    2016-06-01

    Despite evidence that the crucial role of NUAK family, SNF1-like kinase, 2 [NUAK2; also known as sucrose non-fermenting 1 (SNF1)-like kinase (SNARK)], has been highlighted in cancer development and in tumor progression, to the best of our knowledge, there are no studies available to date on the role of NUAK2 in glioblastoma. Thus, in this study, in order to determine the role of NUAK2 in glioblastoma, we performed western blot analysis to detect its expression in glioma. The results demonstrated that NUAK2 expression was upregulated in glioma tissues and that its expression was associated with the advanced stages of the disease. In vitro, NUAK2 overexpression promoted the proliferation, migration and invasion of A172 glioblastoma cells, whereas the silencing of NUAK2 with a plasmid carrying shRNA targeting NUAK2 inhibited the proliferation of A172 glioblastoma cells. Moreover, NUAK2 regulated cancer stem cell (CSC)-related gene expression in the glioblastoma cells. We performed an analysis of potential microRNA (miR or miRNA) target sites using 3 commonly used prediction algorithms, miRanda, TargetScan and PicTar. All 3 algorithms predicted that miR‑143 targeted the 3'-untranslated region (3'UTR) of NUAK2. Subsequent experiments confirmed this prediction. Finally, we found that miR‑143 inhibited the proliferation, migration and invasion of the glioblastoma cells. Thus, the findings of the present study demonstrate that miR‑143 inhibits oncogenic traits by degrading NUAK2 in glioblastoma.

  15. Autophagic regulation of smooth muscle cell biology.

    PubMed

    Salabei, Joshua K; Hill, Bradford G

    2015-01-01

    Autophagy regulates the metabolism, survival, and function of numerous cell types, including those comprising the cardiovascular system. In the vasculature, changes in autophagy have been documented in atherosclerotic and restenotic lesions and in hypertensive vessels. The biology of vascular smooth muscle cells appears particularly sensitive to changes in the autophagic program. Recent evidence indicates that stimuli or stressors evoked during the course of vascular disease can regulate autophagic activity, resulting in modulation of VSMC phenotype and viability. In particular, certain growth factors and cytokines, oxygen tension, and pharmacological drugs have been shown to trigger autophagy in smooth muscle cells. Importantly, each of these stimuli has a redox component, typically associated with changes in the abundance of reactive oxygen, nitrogen, or lipid species. Collective findings support the hypothesis that autophagy plays a critical role in vascular remodeling by regulating smooth muscle cell phenotype transitions and by influencing the cellular response to stress. In this graphical review, we summarize current knowledge on the role of autophagy in the biology of the smooth muscle cell in (patho)physiology. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  16. Dysregulated miR-103 and miR-143 expression in peripheral blood mononuclear cells from induced prediabetes and type 2 diabetes rats.

    PubMed

    Vatandoost, Nasimeh; Amini, Masoud; Iraj, Bijan; Momenzadeh, Sedigheh; Salehi, Rasoul

    2015-11-01

    The progression from normal glucose tolerance (NGT) to type 2 diabetes (T2D) occurs through an intermediate state of glucose intolerance known as pre-diabetes. This transition is usually a gradual phenomenon that occurs over 5-10 years. Among the routinely practiced T2D screening criteria, like, FPG, IFG, IGT or HbA1c, still the issue of a preferable one is debated. The newly emerged microRNAs are created much hope to act as a class of suitable diabetes gene signature detectable at an early stage of the disease development. Although T2D related miRNA fluctuations are reported from the main insulin target organs, sampling of these organs for the sake of screening due to its invasive nature is not practicable. Peripheral blood mononuclear cells (PBMCs) are in constant touch with all body organs hence may exhibit the trace of miRNA changes which take place in insulin target organs. In this study we have evaluated miR-103 and miR-143 expression in three groups of rats namely; normal control, high fat diet (HFD) which is corresponding to prediabetes state, and high fat diet/streptozotocin (STZ) induced T2D. Quantitative real time PCR was used for profiling the selected miRNA expression at various time intervals of the three defined groups of rats. In prediabetes and overt diabetes stages, miR-103 showed significantly elevated expression in PBMC specimens compared to the normal healthy control group. Overexpression pattern of mir-143 was statistically significant in T2D compared to non-diabetic controls. However in HFD (prediabetic) rats also we observed an increasing pattern of miR-143 compared to the normal controls but it was not statistically significant.

  17. RNA-binding protein RBM3 prevents NO-induced apoptosis in human neuroblastoma cells by modulating p38 signaling and miR-143

    PubMed Central

    Yang, Hai-Jie; Ju, Fei; Guo, Xin-Xin; Ma, Shuang-Ping; Wang, Lei; Cheng, Bin-Feng; Zhuang, Rui-Juan; Zhang, Bin-Bin; Shi, Xiang; Feng, Zhi-Wei; Wang, Mian

    2017-01-01

    Nitric oxide (NO)-induced apoptosis in neurons is an important cause of neurodegenerative disease in humans. The cold-inducible protein RBM3 mediates the protective effects of cooling on apoptosis induced by various insults. However, whether RBM3 protects neural cells from NO-induced apoptosis is unclear. This study aimed to investigate the neuroprotective effect of RBM3 on NO-induced apoptosis in human SH-SY5Y neuroblastoma cells. Firstly, we demonstrated that mild hypothermia (32 °C) induces RBM3 expression and confers a potent neuroprotective effect on NO-induced apoptosis, which was substantially diminished when RBM3 was silenced by siRNA. Moreover, overexpression of RBM3 exhibited a strong protective effect against NO-induced apoptosis. Signaling pathway screening demonstrated that only p38 inhibition by RBM3 provided neuroprotective effect, although RBM3 overexpression could affect the activation of p38, JNK, ERK, and AKT signaling in response to NO stimuli. Notably, RBM3 overexpression also blocked the activation of p38 signaling induced by transforming growth factor-β1. Furthermore, both RBM3 overexpression and mild hypothermia abolished the induction of miR-143 by NO, which was shown to mediate the cytotoxicity of NO in a p38-dependent way. These findings suggest that RBM3 protects neuroblastoma cells from NO-induced apoptosis by suppressing p38 signaling, which mediates apoptosis through miR-143 induction. PMID:28134320

  18. RNA-binding protein RBM3 prevents NO-induced apoptosis in human neuroblastoma cells by modulating p38 signaling and miR-143.

    PubMed

    Yang, Hai-Jie; Ju, Fei; Guo, Xin-Xin; Ma, Shuang-Ping; Wang, Lei; Cheng, Bin-Feng; Zhuang, Rui-Juan; Zhang, Bin-Bin; Shi, Xiang; Feng, Zhi-Wei; Wang, Mian

    2017-01-30

    Nitric oxide (NO)-induced apoptosis in neurons is an important cause of neurodegenerative disease in humans. The cold-inducible protein RBM3 mediates the protective effects of cooling on apoptosis induced by various insults. However, whether RBM3 protects neural cells from NO-induced apoptosis is unclear. This study aimed to investigate the neuroprotective effect of RBM3 on NO-induced apoptosis in human SH-SY5Y neuroblastoma cells. Firstly, we demonstrated that mild hypothermia (32 °C) induces RBM3 expression and confers a potent neuroprotective effect on NO-induced apoptosis, which was substantially diminished when RBM3 was silenced by siRNA. Moreover, overexpression of RBM3 exhibited a strong protective effect against NO-induced apoptosis. Signaling pathway screening demonstrated that only p38 inhibition by RBM3 provided neuroprotective effect, although RBM3 overexpression could affect the activation of p38, JNK, ERK, and AKT signaling in response to NO stimuli. Notably, RBM3 overexpression also blocked the activation of p38 signaling induced by transforming growth factor-β1. Furthermore, both RBM3 overexpression and mild hypothermia abolished the induction of miR-143 by NO, which was shown to mediate the cytotoxicity of NO in a p38-dependent way. These findings suggest that RBM3 protects neuroblastoma cells from NO-induced apoptosis by suppressing p38 signaling, which mediates apoptosis through miR-143 induction.

  19. Interstitial Cells: Regulators of Smooth Muscle Function

    PubMed Central

    Sanders, Kenton M.; Ward, Sean M.; Koh, Sang Don

    2014-01-01

    Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα+ cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα+ cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues. PMID:24987007

  20. Interstitial cells: regulators of smooth muscle function.

    PubMed

    Sanders, Kenton M; Ward, Sean M; Koh, Sang Don

    2014-07-01

    Smooth muscles are complex tissues containing a variety of cells in addition to muscle cells. Interstitial cells of mesenchymal origin interact with and form electrical connectivity with smooth muscle cells in many organs, and these cells provide important regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and PDGFRα(+) cells have been described, in detail, and represent distinct classes of cells with unique ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled to ICC and PDGFRα(+) cells, forming an integrated unit called the SIP syncytium. SIP cells express a variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity, propagation pathways for slow waves, transduction of inputs from motor neurons, and mechanosensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial cells are also found in a variety of other smooth muscles; however, in most cases, the physiological and pathophysiological roles for these cells have not been clearly defined. This review describes structural, functional, and molecular features of interstitial cells and discusses their contributions in determining the behaviors of smooth muscle tissues.

  1. Regulation of Gastrointestinal Smooth Muscle Function by Interstitial Cells.

    PubMed

    Sanders, Kenton M; Kito, Yoshihiko; Hwang, Sung Jin; Ward, Sean M

    2016-09-01

    Interstitial cells of mesenchymal origin form gap junctions with smooth muscle cells in visceral smooth muscles and provide important regulatory functions. In gastrointestinal (GI) muscles, there are two distinct classes of interstitial cells, c-Kit(+) interstitial cells of Cajal and PDGFRα(+) cells, that regulate motility patterns. Loss of these cells may contribute to symptoms in GI motility disorders.

  2. TGF-β1 promotes colorectal cancer immune escape by elevating B7-H3 and B7-H4 via the miR-155/miR-143 axis

    PubMed Central

    Zhu, Jianjie; Meng, Fanyi; Chen, Qi; Tao, Lihua; Li, Rui; Fu, Fengqing; Liu, Cuiping; Hu, Yuanjia; Wang, Weipeng; Zhang, Hongjian; Hua, Dong; Chen, Weichang; Zhang, Xueguang

    2016-01-01

    Transforming growth factor-beta 1 (TGF-β1) suppresses T cell function, promoting tumor immune escape. Yet, whether the depression of TGF-β1 on T cell function is mediated by co-inhibitory molecules B7-H3 and B7-H4 remains largely unclear. Here, we demonstrated that TGF-β1 elevated the expression of miR-155 in colorectal cancer cells through SMAD3 and SMAD4. The upregulated miR-155 attenuated miR-143 by inhibiting its direct target, the transcription factor CEBPB. Consequently, the direct target genes of miR-143, B7-H3 and B7-H4, were augmented in the cytoplasm and membrane of tumor cells. Over-expression of B7-H3 and B7-H4 in HCT-116 cells induced T cells to secrete TGF-β1 and the immunosuppressive cytokines IL-2, IL-6, and IL-17. Restoration of miR-143 inhibited the growth of HCT-116 xenograft tumors in mice, and also repressed the expression of B7-H3 and B7-H4 in the tumors. Thus, this study reveals the mechanism by which TGF-β1 leads to T cell-mediated tumor evasion through an increase in B7-H3 and B7-H4 expression. PMID:27626488

  3. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization*

    PubMed Central

    Hien, Tran Thi; Turczyńska, Karolina M.; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F.; Albinsson, Sebastian

    2016-01-01

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. PMID:26683376

  4. Regulation of gastrointestinal motility--insights from smooth muscle biology.

    PubMed

    Sanders, Kenton M; Koh, Sang Don; Ro, Seungil; Ward, Sean M

    2012-11-01

    Gastrointestinal motility results from coordinated contractions of the tunica muscularis, the muscular layers of the alimentary canal. Throughout most of the gastrointestinal tract, smooth muscles are organized into two layers of circularly or longitudinally oriented muscle bundles. Smooth muscle cells form electrical and mechanical junctions between cells that facilitate coordination of contractions. Excitation-contraction coupling occurs by Ca(2+) entry via ion channels in the plasma membrane, leading to a rise in intracellular Ca(2+). Ca(2+) binding to calmodulin activates myosin light chain kinase; subsequent phosphorylation of myosin initiates cross-bridge cycling. Myosin phosphatase dephosphorylates myosin to relax muscles, and a process known as Ca(2+) sensitization regulates the activity of the phosphatase. Gastrointestinal smooth muscles are 'autonomous' and generate spontaneous electrical activity (slow waves) that does not depend upon input from nerves. Intrinsic pacemaker activity comes from interstitial cells of Cajal, which are electrically coupled to smooth muscle cells. Patterns of contractile activity in gastrointestinal muscles are determined by inputs from enteric motor neurons that innervate smooth muscle cells and interstitial cells. Here we provide an overview of the cells and mechanisms that generate smooth muscle contractile behaviour and gastrointestinal motility.

  5. Regulation of gastrointestinal motility—insights from smooth muscle biology

    PubMed Central

    Sanders, Kenton M.; Koh, Sang Don; Ro, Seungil; Ward, Sean M.

    2014-01-01

    Gastrointestinal motility results from coordinated contractions of the tunica muscularis, the muscular layers of the alimentary canal. Throughout most of the gastrointestinal tract, smooth muscles are organized into two layers of circularly or longitudinally oriented muscle bundles. Smooth muscle cells form electrical and mechanical junctions between cells that facilitate coordination of contractions. Excitation–contraction coupling occurs by Ca2+ entry via ion channels in the plasma membrane, leading to a rise in intracellular Ca2+. Ca2+ binding to calmodulin activates myosin light chain kinase; subsequent phosphorylation of myosin initiates cross-bridge cycling. Myosin phosphatase dephosphorylates myosin to relax muscles, and a process known as Ca2+ sensitization regulates the activity of the phosphatase. Gastrointestinal smooth muscles are ‘autonomous’ and generate spontaneous electrical activity (slow waves) that does not depend upon input from nerves. Intrinsic pacemaker activity comes from interstitial cells of Cajal, which are electrically coupled to smooth muscle cells. Patterns of contractile activity in gastrointestinal muscles are determined by inputs from enteric motor neurons that innervate smooth muscle cells and interstitial cells. Here we provide an overview of the cells and mechanisms that generate smooth muscle contractile behaviour and gastrointestinal motility. PMID:22965426

  6. Up-regulated expression of microRNA-143 in association with obesity in adipose tissue of mice fed high-fat diet.

    PubMed

    Takanabe, Rieko; Ono, Koh; Abe, Yukiko; Takaya, Tomohide; Horie, Takahiro; Wada, Hiromichi; Kita, Toru; Satoh, Noriko; Shimatsu, Akira; Hasegawa, Koji

    2008-11-28

    MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. miR-143 has been proposed to play a role in the differentiation of adipocytes in culture. However, the mechanism regulating the expression of miR-143 in adult adipose tissue during the development of obesity in vivo is unknown. Here in, we showed that the expression of miR-143 in the mesenteric fat was up-regulated in mice fed a high-fat diet. Increased miR-143 expression was associated with an elevated body weight and mesenteric fat weight. Furthermore, miR-143 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma and aP2 as well as plasma levels of leptin, one of the important adipocytokines involved in insulin resistance. These findings provide the first evidence for the up-regulated expression of miR-143 in the mesenteric fat of high-fat diet-induced obese mice, which might contribute to the regulated expression of adipocyte genes involved in the pathophysiology of obesity.

  7. Caveolin-1 regulates contractility in differentiated vascular smooth muscle.

    PubMed

    Je, Hyun-Dong; Gallant, Cynthia; Leavis, Paul C; Morgan, Kathleen G

    2004-01-01

    Caveolin is a principal component of caveolar membranes. In the present study, we utilized a decoy peptide approach to define the degree of involvement of caveolin in PKC-dependent regulation of contractility of differentiated vascular smooth muscle. The primary isoform of caveolin in ferret aorta vascular smooth muscle is caveolin-1. Chemical loading of contractile vascular smooth muscle tissue with a synthetic caveolin-1 scaffolding domain peptide inhibited PKC-dependent increases in contractility induced by a phorbol ester or an alpha agonist. Peptide loading also resulted in a significant inhibition of phorbol ester-induced adducin Ser662 phosphorylation, an intracellular monitor of PKC kinase activity, ERK1/2 activation, and Ser789 phosphorylation of the actin binding protein caldesmon. alpha-Agonist-induced ERK1-1/2 activation was also inhibited by the caveolin-1 peptide. Scrambled peptide-loaded tissues or sham-loaded tissues were unaffected with respect to both contractility and signaling. Depolarization-induced activation of contraction was not affected by caveolin peptide loading. Similar results with respect to contractility and ERK1/2 activation during exposure to the phorbol ester or the alpha-agonist were obtained with the cholesterol-depleting agent methyl-beta-cyclodextrin. These results are consistent with a role for caveolin-1 in the coordination of signaling leading to the regulation of contractility of smooth muscle.

  8. Ionic conductances regulating the excitability of colonic smooth muscles.

    PubMed

    Koh, Sang Don; Ward, S M; Sanders, K M

    2012-08-01

    The tunica muscularis of the gastrointestinal (GI) tract contains two layers of smooth muscle cells (SMC) oriented perpendicular to each other. SMC express a variety of voltage-dependent and voltage-independent ionic conductance(s) that develop membrane potential and control excitability. Resting membrane potentials (RMP) vary through the GI tract but generally are within the range of -80 to -40 mV. RMP sets the 'gain' of smooth muscle and regulates openings of voltage-dependent Ca(2+) channels. A variety of K(+) channels contribute to setting RMP of SMC. In most regions, RMP is considerably less negative than the K(+) equilibrium potential, due to a finely tuned balance between background K(+) channels and non-selective cation channels (NSCC). Variations in expression patterns and openings of K(+) channels and NSCC account for differences of the RMP in different regions of the GI tract. Smooth muscle excitability is also regulated by interstitial cells (interstitial cells of Cajal (ICC) and PDGFRα(+) cells) that express additional conductances and are electrically coupled to SMC. Thus, 'myogenic' activity results from the integrated behavior of the SMC/ICC/PDGFRα(+) cell (SIP) syncytium. Inputs from excitatory and inhibitory motor neurons are required to produce the complex motor patterns of the gut. Motor neurons innervate three cell types in the SIP, and receptors, second messenger pathways, and ion channels in these cells mediate postjunctional responses. Studies of isolated SIP cells have begun to unravel the mechanisms responsible for neural responses. This review discusses ion channels that set and regulate RMP of SIP cells and how neurotransmitters regulate membrane potential.

  9. IP3 receptors regulate vascular smooth muscle contractility and hypertension

    PubMed Central

    Lin, Qingsong; Zhao, Guiling; Fang, Xi; Peng, Xiaohong; Tang, Huayuan; Wang, Hong; Jing, Ran; Liu, Jie; Ouyang, Kunfu

    2016-01-01

    Inositol 1, 4, 5-trisphosphate receptor–mediated (IP3R-mediated) calcium (Ca2+) release has been proposed to play an important role in regulating vascular smooth muscle cell (VSMC) contraction for decades. However, whether and how IP3R regulates blood pressure in vivo remains unclear. To address these questions, we have generated a smooth muscle–specific IP3R triple-knockout (smTKO) mouse model using a tamoxifen-inducible system. In this study, the role of IP3R-mediated Ca2+ release in adult VSMCs on aortic vascular contractility and blood pressure was assessed following tamoxifen induction. We demonstrated that deletion of IP3Rs significantly reduced aortic contractile responses to vasoconstrictors, including phenylephrine, U46619, serotonin, and endothelin 1. Deletion of IP3Rs also dramatically reduced the phosphorylation of MLC20 and MYPT1 induced by U46619. Furthermore, although the basal blood pressure of smTKO mice remained similar to that of wild-type controls, the increase in systolic blood pressure upon chronic infusion of angiotensin II was significantly attenuated in smTKO mice. Taken together, our results demonstrate an important role for IP3R-mediated Ca2+ release in VSMCs in regulating vascular contractility and hypertension. PMID:27777977

  10. Smooth muscle FGF/TGFβ cross talk regulates atherosclerosis progression.

    PubMed

    Chen, Pei-Yu; Qin, Lingfeng; Li, Guangxin; Tellides, George; Simons, Michael

    2016-07-01

    The conversion of vascular smooth muscle cells (SMCs) from contractile to proliferative phenotype is thought to play an important role in atherosclerosis. However, the contribution of this process to plaque growth has never been fully defined. In this study, we show that activation of SMC TGFβ signaling, achieved by suppression of SMC fibroblast growth factor (FGF) signaling input, induces their conversion to a contractile phenotype and dramatically reduces atherosclerotic plaque size. The FGF/TGFβ signaling cross talk was observed in vitro and in vivo In vitro, inhibition of FGF signaling increased TGFβ activity, thereby promoting smooth muscle differentiation and decreasing proliferation. In vivo, smooth muscle-specific knockout of an FGF receptor adaptor Frs2α led to a profound inhibition of atherosclerotic plaque growth when these animals were crossed on Apoe(-/-) background and subjected to a high-fat diet. In particular, there was a significant reduction in plaque cellularity, increase in fibrous cap area, and decrease in necrotic core size. In agreement with these findings, examination of human coronary arteries with various degrees of atherosclerosis revealed a strong correlation between the activation of FGF signaling, loss of TGFβ activity, and increased disease severity. These results identify SMC FGF/TGFβ signaling cross talk as an important regulator of SMC phenotype switch and document a major contribution of medial SMC proliferation to atherosclerotic plaque growth.

  11. Superparamagnetic iron oxide nanoparticles regulate smooth muscle cell phenotype

    PubMed Central

    Angelopoulos, Ioannis; Southern, Paul; Pankhurst, Quentin A.

    2016-01-01

    Abstract Superparamagnetic iron oxide nanoparticles (SPION) are used for an increasing range of biomedical applications, from imaging to mechanical actuation of cells and tissue. The aim of this study was to investigate the loading of smooth muscle cells (SMC) with SPION and to explore what effect this has on the phenotype of the cells. Adherent human SMC were loaded with ∼17 pg of unconjugated, negatively charged, 50 nm SPION. Clusters of the internalized SPION particles were held in discrete cytoplasmic vesicles. Internalized SPION did not cause any change in cell morphology, proliferation, metabolic activity, or staining pattern of actin and calponin, two of the muscle contractile proteins involved in force generation. However, internalized SPION inhibited the increased gene expression of actin and calponin normally observed when cells are incubated under differentiation conditions. The observed change in the control of gene expression of muscle contractile apparatus by SPION has not previously been described. This finding could offer novel approaches for regulating the phenotype of SMC and warrants further investigation. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2412–2419, 2016. PMID:27176658

  12. Nucleoplasmic calcium regulation in rabbit aortic vascular smooth muscle cells.

    PubMed

    Abrenica, Bernard; Pierce, Grant N; Gilchrist, James S C

    2003-03-01

    In this study, we investigated whether nucleoplasmic free Ca2+ in aortic vascular smooth muscle cells (VSMCs) might be independently regulated from cytosolic free Ca2+. Understanding mechanisms and pathways responsible for this regulation is especially relevant given the role of a numerous intranuclear Ca2+-sensitive proteins in transcriptional regulation, apoptosis and cell division. The question of an independent regulatory mechanism remains largely unsettled because the previous use of intensitometric fluorophores (e.g., Fluo-3) has been criticized on technical grounds. To circumvent the potential problem of fluorescence artifact, we utilized confocal laser scanning microscopy to image intracellular Ca2+ movements with the ratiometric fluorophore Indo-1. In cultured rabbit VSMCs, we found sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pumps and ryanodine receptor (RyR) Ca2+ channel proteins to be discretely arranged within a perinuclear locus, as determined by fluorescent staining patterns of BODIPY FL thapsigargin and BODIPY FL-X Ry. When intracellular Ca2+ stores were mobilized by addition of thapsigargin (5 microM) and activatory concentrations of ryanodine (1 microM), Indo-1 ratiometric signals were largely restricted to the nucleoplasm. Cytosolic signals, by comparison, were relatively small and even then its spatial distribution was largely perinuclear rather homogeneous. These observations indicate perinuclear RyR and SERCA proteins are intimately involved in regulating VSMC nucleoplasmic Ca2+ concentrations. We also observed a similar pattern of largely nucleoplasmic Ca2+ mobilization upon exposure of cells to the immunosuppressant drug FK506 (tacrolimus), which binds to the RyR-associated immunophillin-binding proteins FKBP12 and FKBP12.6. However, initial FK506-induced nucleoplasmic Ca2+ mobilization was followed by marked reduction of Indo-1 signal intensity close to pretreatment levels. This suggested FK506 exerts both activatory and inhibitory

  13. Elevated Glucose Levels Promote Contractile and Cytoskeletal Gene Expression in Vascular Smooth Muscle via Rho/Protein Kinase C and Actin Polymerization.

    PubMed

    Hien, Tran Thi; Turczyńska, Karolina M; Dahan, Diana; Ekman, Mari; Grossi, Mario; Sjögren, Johan; Nilsson, Johan; Braun, Thomas; Boettger, Thomas; Garcia-Vaz, Eliana; Stenkula, Karin; Swärd, Karl; Gomez, Maria F; Albinsson, Sebastian

    2016-02-12

    Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Smooth Muscle Enriched Long Noncoding RNA (SMILR) Regulates Cell Proliferation

    PubMed Central

    Ballantyne, Margaret D.; Pinel, Karine; Dakin, Rachel; Vesey, Alex T.; Diver, Louise; Mackenzie, Ruth; Garcia, Raquel; Welsh, Paul; Sattar, Naveed; Hamilton, Graham; Joshi, Nikhil; Dweck, Marc R.; Miano, Joseph M.; McBride, Martin W.; Newby, David E.; McDonald, Robert A.

    2016-01-01

    Background— Phenotypic switching of vascular smooth muscle cells from a contractile to a synthetic state is implicated in diverse vascular pathologies, including atherogenesis, plaque stabilization, and neointimal hyperplasia. However, very little is known about the role of long noncoding RNA (lncRNA) during this process. Here, we investigated a role for lncRNAs in vascular smooth muscle cell biology and pathology. Methods and Results— Using RNA sequencing, we identified >300 lncRNAs whose expression was altered in human saphenous vein vascular smooth muscle cells following stimulation with interleukin-1α and platelet-derived growth factor. We focused on a novel lncRNA (Ensembl: RP11-94A24.1), which we termed smooth muscle–induced lncRNA enhances replication (SMILR). Following stimulation, SMILR expression was increased in both the nucleus and cytoplasm, and was detected in conditioned media. Furthermore, knockdown of SMILR markedly reduced cell proliferation. Mechanistically, we noted that expression of genes proximal to SMILR was also altered by interleukin-1α/platelet-derived growth factor treatment, and HAS2 expression was reduced by SMILR knockdown. In human samples, we observed increased expression of SMILR in unstable atherosclerotic plaques and detected increased levels in plasma from patients with high plasma C-reactive protein. Conclusions— These results identify SMILR as a driver of vascular smooth muscle cell proliferation and suggest that modulation of SMILR may be a novel therapeutic strategy to reduce vascular pathologies. PMID:27052414

  15. Fibronectin matrix polymerization regulates smooth muscle cell phenotype through a Rac1 dependent mechanism.

    PubMed

    Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M; Sottile, Jane

    2014-01-01

    Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein

  16. Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction

    PubMed Central

    Eto, Masumi; Kitazawa, Toshio

    2017-01-01

    A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions. Also, the knowledge is expected to facilitate devising innovative strategies that more precisely monitor and normalize organ functions by targeting individual smooth muscles. Evidence has established a current paradigm that the myosin light chain phosphatase (MLCP) is a master regulator of smooth muscle responsiveness to stimuli. Cellular MLCP activity is negatively and positively regulated in response to G-protein activation and cAMP/cGMP production, respectively, through the MYPT1 regulatory subunit and an endogenous inhibitor protein named CPI-17. In this article we review the outcomes from two decade of research on the CPI-17 signaling and discuss emerging paradoxes in the view of signaling pathways regulating smooth muscle functions through MLCP. PMID:28260704

  17. Diversity and plasticity in signaling pathways that regulate smooth muscle responsiveness: Paradigms and paradoxes for the myosin phosphatase, the master regulator of smooth muscle contraction.

    PubMed

    Eto, Masumi; Kitazawa, Toshio

    2017-01-01

    A hallmark of smooth muscle cells is their ability to adapt their functions to meet temporal and chronic fluctuations in their demands. These functions include force development and growth. Understanding the mechanisms underlying the functional plasticity of smooth muscles, the major constituent of organ walls, is fundamental to elucidating pathophysiological rationales of failures of organ functions. Also, the knowledge is expected to facilitate devising innovative strategies that more precisely monitor and normalize organ functions by targeting individual smooth muscles. Evidence has established a current paradigm that the myosin light chain phosphatase (MLCP) is a master regulator of smooth muscle responsiveness to stimuli. Cellular MLCP activity is negatively and positively regulated in response to G-protein activation and cAMP/cGMP production, respectively, through the MYPT1 regulatory subunit and an endogenous inhibitor protein named CPI-17. In this article we review the outcomes from two decade of research on the CPI-17 signaling and discuss emerging paradoxes in the view of signaling pathways regulating smooth muscle functions through MLCP.

  18. Cystic Fibrosis Transmembrane Conductance Regulator in Sarcoplasmic Reticulum of Airway Smooth Muscle. Implications for Airway Contractility

    PubMed Central

    Cook, Daniel P.; Rector, Michael V.; Bouzek, Drake C.; Michalski, Andrew S.; Gansemer, Nicholas D.; Reznikov, Leah R.; Li, Xiaopeng; Stroik, Mallory R.; Ostedgaard, Lynda S.; Abou Alaiwa, Mahmoud H.; Thompson, Michael A.; Prakash, Y. S.; Krishnan, Ramaswamy; Meyerholz, David K.; Seow, Chun Y.

    2016-01-01

    Rationale: An asthma-like airway phenotype has been described in people with cystic fibrosis (CF). Whether these findings are directly caused by loss of CF transmembrane conductance regulator (CFTR) function or secondary to chronic airway infection and/or inflammation has been difficult to determine. Objectives: Airway contractility is primarily determined by airway smooth muscle. We tested the hypothesis that CFTR is expressed in airway smooth muscle and directly affects airway smooth muscle contractility. Methods: Newborn pigs, both wild type and with CF (before the onset of airway infection and inflammation), were used in this study. High-resolution immunofluorescence was used to identify the subcellular localization of CFTR in airway smooth muscle. Airway smooth muscle function was determined with tissue myography, intracellular calcium measurements, and regulatory myosin light chain phosphorylation status. Precision-cut lung slices were used to investigate the therapeutic potential of CFTR modulation on airway reactivity. Measurements and Main Results: We found that CFTR localizes to the sarcoplasmic reticulum compartment of airway smooth muscle and regulates airway smooth muscle tone. Loss of CFTR function led to delayed calcium reuptake following cholinergic stimulation and increased myosin light chain phosphorylation. CFTR potentiation with ivacaftor decreased airway reactivity in precision-cut lung slices following cholinergic stimulation. Conclusions: Loss of CFTR alters porcine airway smooth muscle function and may contribute to the airflow obstruction phenotype observed in human CF. Airway smooth muscle CFTR may represent a therapeutic target in CF and other diseases of airway narrowing. PMID:26488271

  19. Interferons modulate mitogen-induced protein synthesis in airway smooth muscle

    PubMed Central

    Goncharova, Elena A.; Lim, Poay N.; Chisolm, Amelia; Fogle, Homer W.; Taylor, Jerome H.; Goncharov, Dmitry A.; Eszterhas, Andrew; Panettieri, Reynold A.

    2010-01-01

    Severe asthma is characterized by increased airway smooth muscle (ASM) mass due, in part, to ASM cell growth and contractile protein expression associated with increased protein synthesis. Little is known regarding the combined effects of mitogens and interferons on ASM cytosolic protein synthesis. We demonstrate that human ASM mitogens including PDGF, EGF, and thrombin stimulate protein synthesis. Surprisingly, pleiotropic cytokines IFN-β and IFN-γ, which inhibit ASM proliferation, also increased cytosolic protein content in ASM cells. Thus IFN-β alone significantly increased protein synthesis by 1.62 ± 0.09-fold that was further enhanced by EGF to 2.52 ± 0.17-fold. IFN-γ alone also stimulated protein synthesis by 1.91 ± 0.15-fold; treatment of cells with PDGF, EGF, and thrombin in the presence of IFN-γ stimulated protein synthesis by 2.24 ± 0.3-, 1.25 ± 0.17-, and 2.67 ± 0.34-fold, respectively, compared with growth factors alone. The mammalian target of rapamycin (mTOR)/S6 kinase 1 (S6K1) inhibition with rapamycin inhibited IFN- and EGF-induced protein synthesis, suggesting that IFN-induced protein synthesis is modulated by mTOR/S6K1 activation. Furthermore, overexpression of tumor suppressor protein tuberous sclerosis complex 2 (TSC2), which is an upstream negative regulator of mTOR/S6K1 signaling, also inhibited mitogen-induced protein synthesis in ASM cells. IFN-β and IFN-γ stimulated miR143/145 microRNA expression and increased SM α-actin accumulation but had little effect on ASM cell size. In contrast, EGF increased ASM cell size but had little effect on miR143/145 expression. Our data demonstrate that both IFNs and mitogens stimulate protein synthesis but have differential effects on cell size and contractile protein expression and suggest that combined effects of IFNs and mitogens may contribute to ASM cell growth, contractile protein expression, and ASM remodeling in asthma. PMID:20382746

  20. Polo-like Kinase 1 Regulates Vimentin Phosphorylation at Ser-56 and Contraction in Smooth Muscle.

    PubMed

    Li, Jia; Wang, Ruping; Gannon, Olivia J; Rezey, Alyssa C; Jiang, Sixin; Gerlach, Brennan D; Liao, Guoning; Tang, Dale D

    2016-11-04

    Polo-like kinase 1 (Plk1) is a serine/threonine-protein kinase that has been implicated in mitosis, cytokinesis, and smooth muscle cell proliferation. The role of Plk1 in smooth muscle contraction has not been investigated. Here, stimulation with acetylcholine induced Plk1 phosphorylation at Thr-210 (an indication of Plk1 activation) in smooth muscle. Contractile stimulation also activated Plk1 in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer signal of a Plk1 sensor. Moreover, knockdown of Plk1 in smooth muscle attenuated force development. Smooth muscle conditional knock-out of Plk1 also diminished contraction of mouse tracheal rings. Plk1 knockdown inhibited acetylcholine-induced vimentin phosphorylation at Ser-56 without affecting myosin light chain phosphorylation. Expression of T210A Plk1 inhibited the agonist-induced vimentin phosphorylation at Ser-56 and contraction in smooth muscle. However, myosin light chain phosphorylation was not affected by T210A Plk1. Ste20-like kinase (SLK) is a serine/threonine-protein kinase that has been implicated in spindle orientation and microtubule organization during mitosis. In this study knockdown of SLK inhibited Plk1 phosphorylation at Thr-210 and activation. Finally, asthma is characterized by airway hyperresponsiveness, which largely stems from airway smooth muscle hyperreactivity. Here, smooth muscle conditional knock-out of Plk1 attenuated airway resistance and airway smooth muscle hyperreactivity in a murine model of asthma. Taken together, these findings suggest that Plk1 regulates smooth muscle contraction by modulating vimentin phosphorylation at Ser-56. Plk1 activation is regulated by SLK during contractile activation. Plk1 contributes to the pathogenesis of asthma. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Regulation of human airway smooth muscle cell migration and relevance to asthma.

    PubMed

    Salter, Brittany; Pray, Cara; Radford, Katherine; Martin, James G; Nair, Parameswaran

    2017-08-16

    Airway remodelling is an important feature of asthma pathogenesis. A key structural change inherent in airway remodelling is increased airway smooth muscle mass. There is emerging evidence to suggest that the migration of airway smooth muscle cells may contribute to cellular hyperplasia, and thus increased airway smooth muscle mass. The precise source of these cells remains unknown. Increased airway smooth muscle mass may be collectively due to airway infiltration of myofibroblasts, neighbouring airway smooth muscle cells in the bundle, or circulating hemopoietic progenitor cells. However, the relative contribution of each cell type is not well understood. In addition, although many studies have identified pro and anti-migratory agents of airway smooth muscle cells, whether these agents can impact airway remodelling in the context of human asthma, remains to be elucidated. As such, further research is required to determine the exact mechanism behind airway smooth muscle cell migration within the airways, how much this contributes to airway smooth muscle mass in asthma, and whether attenuating this migration may provide a therapeutic avenue for asthma. In this review article, we will discuss the current evidence with respect to the regulation of airway smooth muscle cell migration in asthma.

  2. Power fluctuations smoothing and regulations in wind turbine generator systems

    NASA Astrophysics Data System (ADS)

    Babazadehrokni, Hamed

    Wind is one of the most popular renewable energy sources and it has the potential to become the biggest energy source in future. Since the wind does not always blow constantly, the output wind power is not constant which may make some problem for the power grid. According to the grid code which is set by independent system operator, ISO, wind turbine generator systems need to follow some standards such as the predetermined acceptable power fluctuations. In order to smooth the output powers, the energy storage system and some power electronics modules are employed. The utilized power electronics modules in the wind turbine system can pursue many different goals, such as maintaining the voltage stability, frequency stability, providing the available and predetermined output active and reactive power. On the other side, the energy storage system can help achieving some of these goals but its main job is to store the extra energy when not needed and release the stored energy when needed. The energy storage system can be designed in different sizes, material and also combination of different energy storage systems (hybrid designs). Combination of power electronics devises and also energy storage system helps the wind turbine systems to smooth the output power according to the provided standards. In addition prediction of wind speed may improve the performance of wind turbine generator systems. In this research study all these three topics are studied and the obtained results are written in 10 papers which 7 of them are published and three of them are under process.

  3. Regulation of smooth muscle cell phenotype by glycosaminoglycan identity.

    PubMed

    Qu, Xin; Jimenez-Vergara, Andrea Carolina; Munoz-Pinto, Dany J; Ortiz, Diana; McMahon, Rebecca E; Cristancho, Deissy; Becerra-Bayona, Silvia; Guiza-Arguello, Viviana; Grande-Allen, K Jane; Hahn, Mariah S

    2011-03-01

    The retention of lipoproteins in the arterial intima is an initial event in early atherosclerosis and occurs, in part, through interactions between negatively charged glycosaminoglycans (GAGs) and the positively charged residues of apolipoproteins. Smooth muscle cells (SMCs) which infiltrate into the lipoprotein-enriched intima have been observed to transform into lipid-laden foam cells. This phenotypic switch is associated with SMC acquisition of a macrophage-like capacity to phagocytose lipoproteins and/or of an adipocyte-like capacity to synthesize fatty acids de novo. The aim of the present work was to explore the impact of GAG identity on SMC foam cell formation using a scaffold environment intended to be mimetic of early atherosclerosis. In these studies, we focused on chondroitin sulfate C (CSC), dermatan sulfate (DS), and an intermediate molecular weight hyaluronan (HAIMW, ∼400 kDa), the levels and/or distribution of each of which are significantly altered in atherosclerosis. DS hydrogels were associated with greater SMC phagocytosis of apolipoprotein B than HAIMW gels. Similarly, only SMCs in DS constructs maintained increased expression of the adipocyte marker A-FABP relative to HAIMW gels over 35 days of culture. The increased SMC foam cell phenotype in DS hydrogels was reflected in a corresponding decrease in SMC myosin heavy chain expression in these constructs relative to HAIMW gels at day 35. In addition, this DS-associated increase in foam cell formation was mirrored in an increased SMC synthetic phenotype, as evidenced by greater levels of collagen type I and glucose 6-phosphate dehydrogenase in DS gels than in HAIMW gels. Combined, these results support the increasing body of literature that suggests a critical role for DS-bearing proteoglycans in early atherosclerosis. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  4. Histone deacetylase 8 regulates cortactin deacetylation and contraction in smooth muscle tissues.

    PubMed

    Li, Jia; Chen, Shu; Cleary, Rachel A; Wang, Ruping; Gannon, Olivia J; Seto, Edward; Tang, Dale D

    2014-08-01

    Histone deacetylases (HDACs) are a family of enzymes that mediate nucleosomal histone deacetylation and gene expression. Some members of the HDAC family have also been implicated in nonhistone protein deacetylation, which modulates cell-cycle control, differentiation, and cell migration. However, the role of HDACs in smooth muscle contraction is largely unknown. Here, HDAC8 was localized both in the cytoplasm and the nucleus of mouse and human smooth muscle cells. Knockdown of HDAC8 by lentivirus-encoding HDAC8 shRNA inhibited force development in response to acetylcholine. Treatment of smooth muscle tissues with HDAC8 inhibitor XXIV (OSU-HDAC-44) induced relaxation of precontracted smooth muscle tissues. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3T3 cells. In this study, acetylcholine stimulation induced cortactin deacetylation in mouse and human smooth muscle tissues, as evidenced by immunoblot analysis using antibody against acetylated lysine. Knockdown of HDAC8 by RNAi or treatment with the inhibitor attenuated cortactin deacetylation and actin polymerization without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibited contraction and actin dynamics during contractile activation. These results suggest a novel mechanism for the regulation of smooth muscle contraction. In response to contractile stimulation, HDAC8 may mediate cortactin deacetylation, which subsequently promotes actin filament polymerization and smooth muscle contraction.

  5. Regulation of myosin phosphorylation and myofilament Ca2+ sensitivity in vascular smooth muscle.

    PubMed

    Hirano, Katsuya; Hirano, Mayumi; Kanaide, Hideo

    2004-12-01

    The Ca2+-dependent, reversible phosphorylation of the 20 kDa regulatory myosin light chain (MLC) plays a primary role in regulating the contraction of smooth muscle. However, it is well known that the Ca2+ signal is not the only factor which regulates such contraction, however, the alteration of the Ca2+ sensitivity in the contractile apparatus is also known to play an important role. The degree of MLC phosphorylation is determined by the balance of the activity between phosphorylation and dephosphorylation. Either the Ca2+-independent activation of MLC phosphorylation or the inhibition of MLC dephosphorylation causes a greater MLC phosphorylation for a given level of Ca2+ signal and thereby potentiates the myofilament Ca2+ sensitivity. The smooth muscle myosin light chain phosphatase (MLCP) consisting of three subunits was first isolated and cloned in the early '90s. The intensive investigation thereafter has uncovered the biochemical basis for regulating the activity of MLCP. The regulation of the MLCP activity is now considered to play a critical role in regulating the myofilament Ca2+ sensitivity. There are three major mechanisms in the regulation of MLCP; (1) the phosphorylation of a 110 kDa regulatory subunit of MLCP (2) the conformational change of the trimeric structure, and (3) the inhibition by a smooth muscle specific inhibitor protein, CPI-17. Furthermore, some kinases have been found to phosphorylate the MLC and activate the contraction of smooth muscle in a Ca2+-independent manner. Numerous protein kinases have been found to be involved in the regulation of MLC phosphorylation, and rho-kinase is one of the most frequently investigated kinases. The smooth muscle physiology is now asked to integrate the current understanding of the biochemical mechanisms and to clarify which kinases and/or proteins in the contractile apparatus play a physiological role in regulating the myofilament Ca2+ sensitivity and how such extracellular contractile stimulation

  6. Differential regulation of smooth muscle markers in human bone marrow-derived mesenchymal stem cells.

    PubMed

    Hegner, Björn; Weber, Manfred; Dragun, Duska; Schulze-Lohoff, Eckhard

    2005-06-01

    To study smooth-muscle differentiation and de-differentiation of human bone marrow-derived mesenchymal stem cells (MSCs), which have been shown to enter the circulation and to contribute to vascular repair and atherosclerosis. Human MSCs from bone marrow were cultured with 20% fetal calf serum (FCS) or with 10% FCS and various concentrations of dimethyl sulfoxide (DMSO). Expression of smooth muscle markers was determined by Western blot analysis and immunofluorescence. For signalling studies, involvement of the mammalian target of rapamycin (mTOR) pathway was tested by treatment with rapamycin. MSCs cultured with 20% FCS acquired a smooth muscle-like appearance and expressed the smooth muscle (sm) markers sm-alpha-actin, desmin, sm-calponin and myosin light chain kinase (MLCK). DMSO induced a spindle-like morphology with marked reduction of stress fibers. As judged by Western blot analysis, treatment with 2.5% DMSO strongly downregulated expression of sm-calponin (-85%), short MLCK (-98%) and sm-alpha-actin expression (-51%). Reduced calponin expression was detected by day 2 of treatment with 0.5-2.5% DMSO. After withdrawal of DMSO, MSCs regained high expression of sm-calponin. Treatment with 6 nmol/l rapamycin partly antagonized the effect of DMSO, indicating the involvement of mTOR in regulation of the smooth muscle phenotype of MSCs. DMSO strongly downregulates the smooth muscle markers sm-calponin, short MLCK and sm-alpha-actin in human MSCs, indicating a transition from a smooth muscle-like phenotype to an undifferentiated state by an mTOR-dependent mechanism. Regulating the phenotype of human MSCs may be of relevance for novel therapeutic approaches in atherosclerosis and intimal hyperplasia after vascular injury.

  7. Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling

    PubMed Central

    Miller, Eileen; Czopek, Alicja; Duthie, Karolina M.; Kirkby, Nicholas S.; van de Putte, Elisabeth E. Fransen; Christen, Sibylle; Kimmitt, Robert A.; Moorhouse, Rebecca; Castellan, Raphael F.P.; Kotelevtsev, Yuri V.; Kuc, Rhoda E.; Davenport, Anthony P.; Dhaun, Neeraj; Webb, David J.

    2017-01-01

    The role of smooth muscle endothelinB (ETB) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ETB receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ETB receptors were selectively deleted from smooth muscle by crossing floxed ETB mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ETB deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ETB was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ETB-mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ETB-mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ETB knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ETB blockade–mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ETB-mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ETB knockout mice. In the absence of other pathology, ETB receptors in vascular smooth muscle make a small but significant contribution to ETB-dependent regulation of BP. These ETB receptors have no effect on vascular contraction or neointimal remodeling. PMID:28028193

  8. Smooth Muscle Endothelin B Receptors Regulate Blood Pressure but Not Vascular Function or Neointimal Remodeling.

    PubMed

    Miller, Eileen; Czopek, Alicja; Duthie, Karolina M; Kirkby, Nicholas S; van de Putte, Elisabeth E Fransen; Christen, Sibylle; Kimmitt, Robert A; Moorhouse, Rebecca; Castellan, Raphael F P; Kotelevtsev, Yuri V; Kuc, Rhoda E; Davenport, Anthony P; Dhaun, Neeraj; Webb, David J; Hadoke, Patrick W F

    2017-02-01

    The role of smooth muscle endothelinB (ETB) receptors in regulating vascular function, blood pressure (BP), and neointimal remodeling has not been established. Selective knockout mice were generated to address the hypothesis that loss of smooth muscle ETB receptors would reduce BP, alter vascular contractility, and inhibit neointimal remodeling. ETB receptors were selectively deleted from smooth muscle by crossing floxed ETB mice with those expressing cre-recombinase controlled by the transgelin promoter. Functional consequences of ETB deletion were assessed using myography. BP was measured by telemetry, and neointimal lesion formation induced by femoral artery injury. Lesion size and composition (day 28) were analyzed using optical projection tomography, histology, and immunohistochemistry. Selective deletion of ETB was confirmed by genotyping, autoradiography, polymerase chain reaction, and immunohistochemistry. ETB-mediated contraction was reduced in trachea, but abolished from mesenteric veins, of knockout mice. Induction of ETB-mediated contraction in mesenteric arteries was also abolished in these mice. Femoral artery function was unaltered, and baseline BP modestly elevated in smooth muscle ETB knockout compared with controls (+4.2±0.2 mm Hg; P<0.0001), but salt-induced and ETB blockade-mediated hypertension were unaltered. Circulating endothelin-1 was not altered in knockout mice. ETB-mediated contraction was not induced in femoral arteries by incubation in culture medium or lesion formation, and lesion size was not altered in smooth muscle ETB knockout mice. In the absence of other pathology, ETB receptors in vascular smooth muscle make a small but significant contribution to ETB-dependent regulation of BP. These ETB receptors have no effect on vascular contraction or neointimal remodeling. © 2016 The Authors.

  9. Vascular smooth muscle cell spreading onto fibrinogen is regulated by calpains and phospholipase C.

    PubMed

    Paulhe, F; Bogyo, A; Chap, H; Perret, B; Racaud-Sultan, C

    2001-11-09

    Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of (32)P-labeled phosphatidic acid and phosphatidylinositol-3,4-bisphosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4-bisphosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains. Copyright 2001 Academic Press.

  10. Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

    PubMed Central

    Ali, Mehboob

    2015-01-01

    Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22. PMID:25617350

  11. Toll-like receptor 2 activation and serum amyloid A regulate smooth muscle cell extracellular matrix

    PubMed Central

    Bishop, Christopher A.; Best, Michael; Rich, Celeste B.; Stone, Phillip J.

    2017-01-01

    Smooth muscle cells contribute to extracellular matrix remodeling during atherogenesis. De-differentiated, synthetic smooth muscle cells are involved in processes of migration, proliferation and changes in expression of extracellular matrix components, all of which contribute to loss of homeostasis accompanying atherogenesis. Elevated levels of acute phase proteins, including serum amyloid A (SAA), are associated with an increased risk for atherosclerosis. Although infection with periodontal and respiratory pathogens via activation of inflammatory cell Toll-like receptor (TLR)2 has been linked to vascular disease, little is known about smooth muscle cell TLR2 in atherosclerosis. This study addresses the role of SAA and TLR2 activation on smooth muscle cell matrix gene expression and insoluble elastin accumulation. Cultured rat aortic smooth muscle cells were treated with SAA or TLR2 agonists and the effect on expression of matrix metallopeptidase 9 (MMP9) and tropoelastin studied. SAA up-regulated MMP9 expression. Tropoelastin is an MMP9 substrate and decreased tropoelastin levels in SAA-treated cells supported the concept of extracellular matrix remodeling. Interestingly, SAA-induced down-regulation of tropoelastin was not only evident at the protein level but at the level of gene transcription as well. Contributions of proteasomes, nuclear factor κ B and CCAAT/enhancer binding protein β on regulation of MMP9 vs. tropoleastin expression were revealed. Effects on Mmp9 and Eln mRNA expression persisted with long-term SAA treatment, resulting in decreased insoluble elastin accumulation. Interestingly, the SAA effects were TLR2-dependent and TLR2 activation by bacterial ligands also induced MMP9 expression and decreased tropoelastin expression. These data reveal a novel mechanism whereby SAA and/or infection induce changes in vascular elastin consistent with atherosclerosis. PMID:28257481

  12. Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells

    PubMed Central

    Yamazaki, Jun; Duan, Dayue; Janiak, Robert; Kuenzli, Karri; Horowitz, Burton; Hume, Joseph R

    1998-01-01

    We examined the possibility of functional and molecular expression of volume-regulated Cl− channels in vascular smooth muscle using the whole-cell patch-clamp technique and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on cells from canine pulmonary and renal arteries. Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl−-dependent outward-rectifying membrane currents with an anion permeability sequence of SCN− > I− > Br− > Cl− > aspartate−. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen. Experiments were performed to determine whether the molecular form of the volume-regulated chloride channel (ClC-3) is expressed in pulmonary and renal arteries. Quantitative RT-PCR confirmed expression of ClC-3 in both types of smooth muscle. ClC-3 expression was 76.4% of β-actin in renal artery and 48.0% of β-actin in pulmonary artery. We conclude that volume-regulated Cl− channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone. PMID:9508834

  13. Mitochondrial regulation of cytosolic Ca²⁺ signals in smooth muscle.

    PubMed

    McCarron, John G; Olson, Marnie L; Chalmers, Susan

    2012-07-01

    The cytosolic Ca²⁺ concentration ([Ca²⁺]c) controls virtually every activity of smooth muscle, including contraction, migration, transcription, division and apoptosis. These processes may be activated by large (>10 μM) amplitude [Ca²⁺]c increases, which occur in small restricted regions of the cell or by smaller (<1 μM) amplitude changes throughout the bulk cytoplasm. Mitochondria contribute to the regulation of these signals by taking up Ca²⁺. However, mitochondria's reported low affinity for Ca²⁺ is thought to require the organelle to be positioned close to ion channels and within a microdomain of high [Ca²⁺]. In cultured smooth muscle, mitochondria are highly dynamic structures but in native smooth muscle mitochondria are immobile, apparently strategically positioned organelles that regulate the upstroke and amplitude of IP₃-evoked Ca²⁺ signals and IP₃ receptor (IP₃R) cluster activity. These observations suggest mitochondria are positioned within the high [Ca²⁺] microdomain arising from an IP₃R cluster to exert significant local control of channel activity. On the other hand, neither the upstroke nor amplitude of voltage-dependent Ca²⁺ entry is modulated by mitochondria; rather, it is the declining phase of the transient that is regulated by the organelle. Control of the declining phase of the transient requires a high mitochondrial affinity for Ca²⁺ to enable uptake to occur over the normal physiological Ca²⁺ range (<1 μM). Thus, in smooth muscle, mitochondria regulate Ca²⁺ signals exerting effects over a large range of [Ca²⁺] (∼200 nM to at least tens of micromolar) to provide a wide dynamic range in the control of Ca²⁺ signals.

  14. Substance P Responsiveness of Smooth Muscle Cells is Regulated by the Integrin Ligand, Thrombospondin

    NASA Astrophysics Data System (ADS)

    Dahm, Lisa M.; Bowers, Chauncey W.

    1996-02-01

    The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties.

  15. Substance P responsiveness of smooth muscle cells is regulated by the integrin ligand, thrombospondin.

    PubMed Central

    Dahm, L M; Bowers, C W

    1996-01-01

    The extracellular factors that determine a cell's responsiveness to neurotransmitters are of particular relevance for pharmacologically diverse cell types such as neurons and smooth muscle. We previously demonstrated that matrix-associated factors are capable of dramatically and specifically suppressing the responsiveness of smooth muscle to the neuropeptide, substance P. We now demonstrate that this influence of extracellular matrix on the pharmacological phenotype of smooth muscle cells can be blocked specifically by an Arg-Gly-Asp (RGD)-containing antagonist of integrins. Of a battery of integrin ligands tested, only thrombospondin mimicked the effect of the extracellular matrix on substance P responsiveness. This effect of thrombospondin was dose dependent, RGD sensitive, and blocked by an antibody directed against the RGD-containing region of thrombospondin. Because the mRNA for thrombospondin is present in the cells of the chicken amnion, this extracellular factor may normally suppress substance P responsiveness in amniotic smooth muscle. The results suggest a role for matrix-associated integrin ligands in the regulation of cellular responses to specific neurotransmitters and hormones and in the development and maintenance of tissue-specific pharmacological properties. Images Fig. 6 PMID:8577754

  16. Capacitative Ca2+ entry and the regulation of smooth muscle tone.

    PubMed

    Gibson, A; McFadzean, I; Wallace, P; Wayman, C P

    1998-07-01

    In many non-excitable cells, activation of phospholipase C-linked receptors results in a biphasic increase in the cytosolic Ca2+ concentration; an initial transient increase, owing to the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR), is followed by a much smaller but sustained elevation, which often involves capacitative Ca2+ entry, where depletion of Ca2+ within the ER signals the opening of store-operated Ca2+ channels in the plasma membrane. However, in excitable cells such as smooth muscle, the role of capacitative Ca2+ entry is less clear and the main Ca2+ entry mechanisms responsible for sustained cellular activation have been considered to be either voltage-operated or receptor-operated Ca2+ channels. Although store-regulated Ca2+ entry was known to occur following agonist activation of smooth muscle, it was believed to be important only for the re-filling of the depleted SR and not as a source of activator Ca2+ for the contractile mechanisms. Here, Alan Gibson, Ian McFadzean, Pat Wallace and Christopher Wayman review recent evidence that capacitative Ca2+ entry might indeed be important for the regulation of smooth muscle tone, and that it might provide an important for pharmacological intervention.

  17. p21‐Activated kinase (Pak) regulates airway smooth muscle contraction by regulating paxillin complexes that mediate actin polymerization

    PubMed Central

    Zhang, Wenwu; Huang, Youliang

    2016-01-01

    Key points In airway smooth muscle, tension development caused by a contractile stimulus requires phosphorylation of the 20 kDa myosin light chain (MLC), which activates crossbridge cycling and the polymerization of a pool of submembraneous actin.The p21‐activated kinases (Paks) can regulate the contractility of smooth muscle and non‐muscle cells, and there is evidence that this occurs through the regulation of MLC phosphorylation.We show that Pak has no effect on MLC phosphorylation during the contraction of airway smooth muscle, and that it regulates contraction by mediating actin polymerization.We find that Pak phosphorylates the adhesion junction protein, paxillin, on Ser273, which promotes the formation of a signalling complex that activates the small GTPase, cdc42, and the actin polymerization catalyst, neuronal Wiskott–Aldrich syndrome protein (N‐WASP).These studies demonstrate a novel role for Pak in regulating the contractility of smooth muscle by regulating actin polymerization. Abstract The p21‐activated kinases (Pak) can regulate contractility in smooth muscle and other cell and tissue types, but the mechanisms by which Paks regulate cell contractility are unclear. In airway smooth muscle, stimulus‐induced contraction requires phosphorylation of the 20 kDa light chain of myosin, which activates crossbridge cycling, as well as the polymerization of a small pool of actin. The role of Pak in airway smooth muscle contraction was evaluated by inhibiting acetylcholine (ACh)‐induced Pak activation through the expression of a kinase inactive mutant, Pak1 K299R, or by treating tissues with the Pak inhibitor, IPA3. Pak inhibition suppressed actin polymerization and contraction in response to ACh, but it did not affect myosin light chain phosphorylation. Pak activation induced paxillin phosphorylation on Ser273; the paxillin mutant, paxillin S273A, inhibited paxillin Ser273 phosphorylation and inhibited actin polymerization and contraction

  18. Biomechanical regulation of vascular smooth muscle cell functions: from in vitro to in vivo understanding

    PubMed Central

    Qiu, Juhui; Zheng, Yiming; Hu, Jianjun; Liao, Donghua; Gregersen, Hans; Deng, Xiaoyan; Fan, Yubo; Wang, Guixue

    2014-01-01

    Vascular smooth muscle cells (VSMCs) have critical functions in vascular diseases. Haemodynamic factors are important regulators of VSMC functions in vascular pathophysiology. VSMCs are physiologically active in the three-dimensional matrix and interact with the shear stress sensor of endothelial cells (ECs). The purpose of this review is to illustrate how haemodynamic factors regulate VSMC functions under two-dimensional conditions in vitro or three-dimensional co-culture conditions in vivo. Recent advances show that high shear stress induces VSMC apoptosis through endothelial-released nitric oxide and low shear stress upregulates VSMC proliferation and migration through platelet-derived growth factor released by ECs. This differential regulation emphasizes the need to construct more actual environments for future research on vascular diseases (such as atherosclerosis and hypertension) and cardiovascular tissue engineering. PMID:24152813

  19. Endoplasmic reticulum stress in arterial smooth muscle cells: A novel regulator of vascular disease.

    PubMed

    Furmanik, Malgorzata; Shanahan, Catherine M

    2016-10-13

    Cardiovascular disease continues to be the leading cause of death in industrialised societies. The idea that the arterial smooth muscle cell (ASMC) plays a key role in regulating many vascular pathologies has been gaining importance, as has the realisation that not enough is known about the pathological cellular mechanisms regulating ASMC function in vascular remodelling. In the past decade endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been recognised as a stress response underlying many physiological and pathological processes in various vascular cell types. Here we summarize what is known about how ER stress signalling regulates phenotypic switching, trans/dedifferentiation and apoptosis of ASMCs and contributes to atherosclerosis, hypertension, aneurysms and vascular calcification.

  20. Endoplasmic Reticulum Stress in Arterial Smooth Muscle Cells: A Novel Regulator of Vascular Disease

    PubMed Central

    Furmanik, Malgorzata; Shanahan, Catherine M.

    2017-01-01

    Cardiovascular disease continues to be the leading cause of death in industrialised societies. The idea that the arterial smooth muscle cell (ASMC) plays a key role in regulating many vascular pathologies has been gaining importance, as has the realisation that not enough is known about the pathological cellular mechanisms regulating ASMC function in vascular remodelling. In the past decade endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been recognised as a stress response underlying many physiological and pathological processes in various vascular cell types. Here we summarise what is known about how ER stress signalling regulates phenotypic switching, trans/dedifferentiation and apoptosis of ASMCs and contributes to atherosclerosis, hypertension, aneurysms and vascular calcification.

  1. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells

    PubMed Central

    Fu, Shibo; Tar, Moses Tarndie; Melman, Arnold; Davies, Kelvin Paul

    2014-01-01

    Men with sickle cell disease (SCD) risk developing priapism. Recognizing that SCD is a disease of hypoxia, we investigated the effect of hypoxia on gene expression in corporal smooth muscle (CSM) cells. Rat CSM cells in vitro were treated with CoCl2 or low oxygen tension to mimic hypoxia. Hypoxic conditions increased expression of genes previously associated with priapism in animal models. Variable coding sequence a1 (Vcsa1; the rat opiorphin homologue, sialorphin), hypoxia-inducible factor 1a (Hif-1a), and A2B adenosine receptor (a2br) were increased by 10-, 4-, and 6-fold, respectively, by treatment with CoCl2, whereas low oxygen tension caused increases in expression of 3-, 4-, and 1.5-fold, respectively. Sialorphin-treated CSM cells increased expression of Hif-1a and a2br by 4-fold, and vcsa1-siRNA treatment reduced expression by ∼50%. Using a Hif-1a inhibitor, we demonstrated up-regulation of a2br by sialorphin is dependent on Hif-1a, and knockdown of vcsa1 expression with vcsa1-siRNA demonstrated that hypoxic-up-regulation of Hif-1a is dependent on vcsa1. In CSM from a SCD mouse, there was 15-fold up-regulation of opiorphin at a life stage prior to priapism. We conclude that in CSM, opiorphins are master regulators of the hypoxic response. Opiorphin up-regulation in response to SCD-associated hypoxia activates CSM “relaxant” pathways; excessive activation of these pathways results in priapism.—Fu, S., Tar, M. T., Melman, A., Davies, K. P. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells. PMID:24803544

  2. Smooth muscle–endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

    PubMed Central

    Lutter, Sophie; Xie, Sherry; Tatin, Florence

    2012-01-01

    Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels—smooth muscle and endothelial cells—and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function. PMID:22665518

  3. Molecular Regulation of Contractile Smooth Muscle Cell Phenotype: Implications for Vascular Tissue Engineering

    PubMed Central

    Beamish, Jeffrey A.; He, Ping; Kottke-Marchant, Kandice

    2010-01-01

    The molecular regulation of smooth muscle cell (SMC) behavior is reviewed, with particular emphasis on stimuli that promote the contractile phenotype. SMCs can shift reversibly along a continuum from a quiescent, contractile phenotype to a synthetic phenotype, which is characterized by proliferation and extracellular matrix (ECM) synthesis. This phenotypic plasticity can be harnessed for tissue engineering. Cultured synthetic SMCs have been used to engineer smooth muscle tissues with organized ECM and cell populations. However, returning SMCs to a contractile phenotype remains a key challenge. This review will integrate recent work on how soluble signaling factors, ECM, mechanical stimulation, and other cells contribute to the regulation of contractile SMC phenotype. The signal transduction pathways and mechanisms of gene expression induced by these stimuli are beginning to be elucidated and provide useful information for the quantitative analysis of SMC phenotype in engineered tissues. Progress in the development of tissue-engineered scaffold systems that implement biochemical, mechanical, or novel polymer fabrication approaches to promote contractile phenotype will also be reviewed. The application of an improved molecular understanding of SMC biology will facilitate the design of more potent cell-instructive scaffold systems to regulate SMC behavior. PMID:20334504

  4. PDGF-mediated autophagy regulates vascular smooth muscle cell phenotype and resistance to oxidative stress.

    PubMed

    Salabei, Joshua K; Cummins, Timothy D; Singh, Mahavir; Jones, Steven P; Bhatnagar, Aruni; Hill, Bradford G

    2013-05-01

    Vascular injury and chronic arterial diseases result in exposure of VSMCs (vascular smooth muscle cells) to increased concentrations of growth factors. The mechanisms by which growth factors trigger VSMC phenotype transitions remain unclear. Because cellular reprogramming initiated by growth factors requires not only the induction of genes involved in cell proliferation, but also the removal of contractile proteins, we hypothesized that autophagy is an essential modulator of VSMC phenotype. Treatment of VSMCs with PDGF (platelet-derived growth factor)-BB resulted in decreased expression of the contractile phenotype markers calponin and α-smooth muscle actin and up-regulation of the synthetic phenotype markers osteopontin and vimentin. Autophagy, as assessed by LC3 (microtubule-associated protein light chain 3 α; also known as MAP1LC3A)-II abundance, LC3 puncta formation and electron microscopy, was activated by PDGF exposure. Inhibition of autophagy with 3-methyladenine, spautin-1 or bafilomycin stabilized the contractile phenotype. In particular, spautin-1 stabilized α-smooth muscle cell actin and calponin in PDGF-treated cells and prevented actin filament disorganization, diminished production of extracellular matrix, and abrogated VSMC hyperproliferation and migration. Treatment of cells with PDGF prevented protein damage and cell death caused by exposure to the lipid peroxidation product 4-hydroxynonenal. The results of the present study demonstrate a distinct form of autophagy induced by PDGF that is essential for attaining the synthetic phenotype and for survival under the conditions of high oxidative stress found to occur in vascular lesions.

  5. Microtubule structures underlying the sarcoplasmic reticulum support peripheral coupling sites to regulate smooth muscle contractility.

    PubMed

    Pritchard, Harry A T; Gonzales, Albert L; Pires, Paulo W; Drumm, Bernard T; Ko, Eun A; Sanders, Kenton M; Hennig, Grant W; Earley, Scott

    2017-09-19

    Junctional membrane complexes facilitate excitation-contraction coupling in skeletal and cardiac muscle cells by forming subcellular invaginations that maintain close (≤20 nm) proximity of ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) with voltage-dependent Ca(2+) channels in the plasma membrane. In fully differentiated smooth muscle cells, junctional membrane complexes occur as distributed sites of peripheral coupling. We investigated the role of the cytoskeleton in maintaining peripheral coupling and associated Ca(2+) signaling networks within native smooth muscle cells of mouse and rat cerebral arteries. Using live-cell confocal and superresolution microscopy, we found that the tight interactions between the SR and the plasma membrane in these cells relied on arching microtubule structures present at the periphery of smooth muscle cells and were independent of the actin cytoskeleton. Loss of peripheral coupling associated with microtubule depolymerization altered the spatiotemporal properties of localized Ca(2+) sparks generated by the release of Ca(2+) through type 2 RyRs (RyR2s) on the SR and decreased the number of sites of colocalization between RyR2s and large-conductance Ca(2+)-activated K(+) (BK) channels. The reduced BK channel activity associated with the loss of SR-plasma membrane interactions was accompanied by increased pressure-induced constriction of cerebral resistance arteries. We conclude that microtubule structures maintain peripheral coupling in contractile smooth muscle cells, which is crucial for the regulation of contractility and cerebral vascular tone. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  6. Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth.

    PubMed

    Jackson, W F

    2017-01-01

    Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca(2+) channels (VGCC), Ca(2+) influx through VGCC, intracellular Ca(2+), and VSM contraction. Membrane potential also affects release of Ca(2+) from internal stores and the Ca(2+) sensitivity of the contractile machinery such that K(+) channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. VSM cells express multiple isoforms of at least five classes of K(+) channels that contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression, and function of large conductance, Ca(2+)-activated K(+) (BKCa) channels, intermediate-conductance Ca(2+)-activated K(+) (KCa3.1) channels, multiple isoforms of voltage-gated K(+) (KV) channels, ATP-sensitive K(+) (KATP) channels, and inward-rectifier K(+) (KIR) channels in both contractile and proliferating VSM cells. © 2017 Elsevier Inc. All rights reserved.

  7. Procontractile G protein–mediated signaling pathways antagonistically regulate smooth muscle differentiation in vascular remodeling

    PubMed Central

    Althoff, Till F.; Juárez, Julián Albarrán; Troidl, Kerstin; Tang, Cong; Wang, Shengpeng; Wirth, Angela; Takefuji, Mikito; Wettschureck, Nina

    2012-01-01

    Vascular smooth muscle (Sm) cells (VSMCs) are highly plastic. Their differentiation state can be regulated by serum response factor (SRF), which activates genes involved in Sm differentiation and proliferation by recruiting cofactors, such as members of the myocardin family and ternary complex factors (TCFs), respectively. However, the extracellular cues and upstream signaling mechanisms regulating SRF-dependent VSMC differentiation under in vivo conditions are poorly understood. In this study, we show that the procontractile signaling pathways mediated by the G proteins G12/G13 and Gq/G11 antagonistically regulate VSMC plasticity in different models of vascular remodeling. In mice lacking Gα12/Gα13 or their effector, the RhoGEF protein LARG, RhoA-dependent SRF-regulation was blocked and down-regulation of VSMC differentiation marker genes was enhanced. This was accompanied by an excessive vascular remodeling and exacerbation of atherosclerosis. In contrast, Sm-specific Gαq/Gα11 deficiency blocked activation of extracellular signal-regulated kinase 1/2 and the TCF Elk-1, resulting in a reduced VSMC dedifferentiation in response to flow cessation or vascular injury. These data show that the balanced activity of both G protein–mediated pathways in VSMCs is required for an appropriate vessel remodeling response in vascular diseases and suggest new approaches to modulate Sm differentiation in vascular pathologies. PMID:23129751

  8. Se Enhances MLCK Activation by Regulating Selenoprotein T (SelT) in the Gastric Smooth Muscle of Rats.

    PubMed

    Li, Jia-Ping; Zhou, Jing-Xuan; Wang, Qi; Gu, Gao-Qin; Yang, Shi-Jin; Li, Cheng-Ye; Qiu, Chang-Wei; Deng, Gan-Zhen; Guo, Meng-Yao

    2016-09-01

    Selenium (Se), a nutritionally essential trace element, is associated with health and disease. Selenoprotein T (SelT) was identified as a redoxin protein with a selenocystein, localizing in the endoplasmic reticulum. The myosin light chain kinase (MLCK) and myosin light chain (MLC) play key roles in the contraction process of smooth muscle. The present study was to detect the effect and mechanism of SelT on the contraction process of gastric smooth muscle. The WT rats were fed with different Se concentration diets, and Se and Ca(2+) concentrations were detected in the gastric smooth muscle. Western blot and qPCR were performed to determine SelT, CaM, MLCK, and MLC expressions. MLCK activity was measured by identifying the rates of [γ-32P]ATP incorporated into the MLC. The results showed Se and Ca(2+) concentrations were enhanced with Se intake in gastric smooth muscle tissues. With increasing Se, SelT, CaM, MLCK and MLC expressions increased, and MLCK and MLC activation improved in gastric smooth muscle tissue. The SelT RNA interference experiments showed that Ca(2+) release, MLCK activation, and MLC phosphorylation were regulated by SelT. Se affected the gastric smooth muscle constriction by regulating Ca(2+) release, MLCK activation, and MLC phosphorylation through SelT. Se plays a major role in regulating the contraction processes of gastric smooth muscle with the SelT.

  9. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.

    PubMed

    Bretschneider, Maria; Busch, Bianca; Mueller, Daniel; Nolze, Alexander; Schreier, Barbara; Gekle, Michael; Grossmann, Claudia

    2016-04-01

    Inappropriately activated mineralocorticoid receptor (MR) is a risk factor for vascular remodeling with unclear molecular mechanism. Recent findings suggest that post-transcriptional regulation by micro-RNAs (miRs) may be involved. Our aim was to search for MR-dependent miRs in vascular smooth muscle cells (VSMCs) and to explore the underlying molecular mechanism and the pathologic relevance. We detected that aldosteroneviathe MR reduces miR-29bin vivoin murine aorta and in human primary and cultured VSMCs (ED50= 0.07 nM) but not in endothelial cells [quantitative PCR (qPCR), luciferase assays]. This effect was mediated by an increased decay of miR-29b in the cytoplasm with unchanged miR-29 family member or primary-miR levels. Decreased miR-29b led to an increase in extracellular matrix measured by ELISA and qPCR and enhanced VSMC migration in single cell-tracking experiments. Additionally, cell proliferation and the apoptosis/necrosis ratio (caspase/lactate dehydrogenase assay) was modulated by miR-29b. Enhanced VSMC migration by aldosterone required miR-29b regulation. Control experiments were performed with scrambled RNA and empty plasmids, by comparing aldosterone-stimulated with vehicle-incubated cells. Overall, our findings provide novel insights into the molecular mechanism of aldosterone-mediated vascular pathogenesis by identifying miR-29b as a pathophysiologic relevant target of activated MR in VSMCs and by highlighting the importance of miR processing for miR regulation.-Bretschneider, M., Busch, B., Mueller, D., Nolze, A., Schreier, B., Gekle, M., Grossmann, C. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.

  10. A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction.

    PubMed

    Zhang, Wenwu; Huang, Youliang; Wu, Yidi; Gunst, Susan J

    2015-02-01

    Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.

  11. miR-125b regulates calcification of vascular smooth muscle cells.

    PubMed

    Goettsch, Claudia; Rauner, Martina; Pacyna, Nicole; Hempel, Ute; Bornstein, Stefan R; Hofbauer, Lorenz C

    2011-10-01

    Vascular calcification is a prominent feature of atherosclerosis and is closely linked to osteoporosis. Cellular differentiation is regulated by various microRNAs (miRs), including miR-125b, which is known to be involved in osteoblast differentiation. However, no specific miR has been defined that modulates vascular calcification. Herein, we assessed the impact of miR-125b in osteogenic transformation of vascular smooth muscle cells. Osteogenic transdifferentiation of human coronary artery smooth muscle cells was induced by osteogenic medium and enhanced the formation of mineralized matrix, resulting in a significantly higher mineral deposition after 21 days. Increased expression of miR-125b was time-dependent in human coronary artery smooth muscle cells and diminished during osteogenic transdifferentiation. At day 21, miR-125b was significantly reduced (-42%) compared with that in the untreated control. The expression of miR-processing enzymes, RNase III endonucleases DICER1 and DROSHA, was also decreased. Furthermore, inhibition of endogenous miR-125b promoted osteogenic transdifferentiation, as measured by increased alkaline phosphatase activity and matrix mineralization. Expression analysis revealed the osteoblast transcription factor SP7 (osterix) as a target of miR-125b. In vivo, miR-125b was decreased in calcified aortas of apolipoprotein E knockout mice. In conclusion, our results suggest that miR-125b is involved in vascular calcification in vitro and in vivo, at least partially by targeting SP7. Evaluating the role of miRs in arterial calcification in vivo may have important therapeutic implications. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  12. TSHZ3 and SOX9 Regulate the Timing of Smooth Muscle Cell Differentiation in the Ureter by Reducing Myocardin Activity

    PubMed Central

    Martin, Elise; Caubit, Xavier; Airik, Rannar; Vola, Christine; Fatmi, Ahmed; Kispert, Andreas; Fasano, Laurent

    2013-01-01

    Smooth muscle cells are of key importance for the proper functioning of different visceral organs including those of the urogenital system. In the mouse ureter, the two transcriptional regulators TSHZ3 and SOX9 are independently required for initiation of smooth muscle differentiation from uncommitted mesenchymal precursor cells. However, it has remained unclear whether TSHZ3 and SOX9 act independently or as part of a larger regulatory network. Here, we set out to characterize the molecular function of TSHZ3 in the differentiation of the ureteric mesenchyme. Using a yeast-two-hybrid screen, we identified SOX9 as an interacting protein. We show that TSHZ3 also binds to the master regulator of the smooth muscle program, MYOCD, and displaces it from the coregulator SRF, thereby disrupting the activation of smooth muscle specific genes. We found that the initiation of the expression of smooth muscle specific genes in MYOCD-positive ureteric mesenchyme coincides with the down regulation of Sox9 expression, identifying SOX9 as a possible negative regulator of smooth muscle cell differentiation. To test this hypothesis, we prolonged the expression of Sox9 in the ureteric mesenchyme in vivo. We found that Sox9 does not affect Myocd expression but significantly reduces the expression of MYOCD/SRF-dependent smooth muscle genes, suggesting that down-regulation of Sox9 is a prerequisite for MYOCD activity. We propose that the dynamic expression of Sox9 and the interaction between TSHZ3, SOX9 and MYOCD provide a mechanism that regulates the pace of progression of the myogenic program in the ureter. PMID:23671695

  13. Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle

    PubMed Central

    Fountain, S J; Cheong, A; Flemming, R; Mair, L; Sivaprasadarao, A; Beech, D J

    2004-01-01

    This study focused on the hypothesis that KCNA genes (which encode KVα1 voltage-gated K+ channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance – the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding KVα1 subunits as well as KVβ1, KVα2.1, KVγ9.3, BKCaα1 and BKCaβ1. The seven major KCNA genes were expressed and more readily detected in endothelium-denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding KVα2.1 but less or similar amounts encoding KVβ1, KVγ9.3, BKCaα1 and BKCaβ1. Patch-clamp recordings from freshly isolated smooth muscle cells revealed dominant KVα1 K+ current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K+ current than might be expected. The mechanism conferring up-regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure. PMID:14742730

  14. A role of stretch-activated potassium currents in the regulation of uterine smooth muscle contraction

    PubMed Central

    Buxton, Iain L O; Heyman, Nathanael; Wu, Yi-ying; Barnett, Scott; Ulrich, Craig

    2011-01-01

    Rates of premature birth are alarming and threaten societies and healthcare systems worldwide. Premature labor results in premature birth in over 50% of cases. Preterm birth accounts for three-quarters of infant morbidity and mortality. Children that survive birth before 34 weeks gestation often face life-long disability. Current treatments for preterm labor are wanting. No treatment has been found to be generally effective and none are systematically evaluated beyond 48 h. New approaches to the treatment of preterm labor are desperately needed. Recent studies from our laboratory suggest that the uterine muscle is a unique compartment with regulation of uterine relaxation unlike that of other smooth muscles. Here we discuss recent evidence that the mechanically activated 2-pore potassium channel, TREK-1, may contribute to contraction-relaxation signaling in uterine smooth muscle and that TREK-1 gene variants associated with human labor and preterm labor may lead to a better understanding of preterm labor and its possible prevention. PMID:21642947

  15. Selenoprotein N Was Required for the Regulation of Selenium on the Uterine Smooth Muscle Contraction in Mice.

    PubMed

    Zhou, Jingxuan; Li, Chengye; Gu, Gaoqin; Wang, Qi; Guo, Mengyao

    2017-08-23

    Selenium (Se) is an essential micronutrient affecting various aspects of health. The balance of the Se concentration has an important protective and promoter effect on physiological function in inducing muscular disorders in smooth muscle. Selenoprotein N (SelN) is closely related to Ca(2+) release. The present study aimed to determine the effects and mechanism of action of dietary Se on uterine smooth muscle contraction via SelN using a mouse model. Quantitative polymerase chain reaction (qPCR) analysis was performed to detect mRNA levels. Western blotting was performed to detect protein levels. The results of the immunohistochemical analysis showed that Se had an effect on the uterine smooth muscle. The Se-supplement increased the release of Ca(2+), Ca(2+)-calmodulin (CaM) expression, myosin light chain kinase (MLCK) expression, and myosin light chain (MLC) phosphorylation but did not affect ROCK and RhoA in uterine smooth muscle. Furthermore, the lack of Se showed an opposite impact. The effects of Se regulation were closely related to SelN. The interference of mouse SelN was performed on the uterine smooth muscle cell. Additionally, the results displayed the regulation of Se on the release of Ca(2+), CaM expression, MLCK expression, and MLC phosphorylation were significant inhibited, and there was no effect on ROCK and RhoA. In conclusion, Se played an important role in regulating the process of contraction in uterine smooth muscle with SelN.

  16. Ion channel regulation of intracellular calcium and airway smooth muscle function.

    PubMed

    Perez-Zoghbi, Jose F; Karner, Charlotta; Ito, Satoru; Shepherd, Malcolm; Alrashdan, Yazan; Sanderson, Michael J

    2009-10-01

    Airway hyper-responsiveness associated with asthma is mediated by airway smooth muscle cells (SMCs) and has a complicated etiology involving increases in cell contraction and proliferation and the secretion of inflammatory mediators. Although these pathological changes are diverse, a common feature associated with their regulation is a change in intracellular Ca(2+) concentration ([Ca(2+)](i)). Because the [Ca(2+)](i) itself is a function of the activity and expression of a variety of ion channels, in both the plasma membrane and sarcoplasmic reticulum of the SMC, the modification of this ion channel activity may predispose airway SMCs to hyper-responsiveness. Our objective is to review how ion channels determine the [Ca(2+)](i) and influence the function of airway SMCs and emphasize the potential of ion channels as sites for therapeutic approaches to asthma.

  17. Function and regulation of large conductance Ca(2+)-activated K+ channel in vascular smooth muscle cells.

    PubMed

    Hu, Xiang-Qun; Zhang, Lubo

    2012-09-01

    Large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are abundantly expressed in vascular smooth muscle cells. Activation of BK(Ca) channels leads to hyperpolarization of cell membrane, which in turn counteracts vasoconstriction. Therefore, BK(Ca) channels have an important role in regulation of vascular tone and blood pressure. The activity of BK(Ca) channels is subject to modulation by various factors. Furthermore, the function of BK(Ca) channels are altered in both physiological and pathophysiological conditions, such as pregnancy, hypertension and diabetes, which has dramatic impacts on vascular tone and hemodynamics. Consequently, compounds and genetic manipulation that alter activity and expression of the channel might be of therapeutic interest.

  18. The SWI/SNF chromatin remodeling complex regulates myocardin-induced smooth muscle-specific gene expression

    PubMed Central

    Zhou, Jiliang; Zhang, Min; Fang, Hong; El-Mounayri, Omar; Rodenberg, Jennifer M.; Imbalzano, Anthony N.; Herring, B. Paul

    2009-01-01

    Objective Transcription regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle-specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. Methods and Results We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle-specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle-specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, that lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle-specific genes. Whereas, reconstitution of wild type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle-specific genes. Brg1 and Brm directly bind to the N-terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. Conclusion These data demonstrate that the ability of myocardin to induce smooth muscle-specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes. PMID:19342595

  19. Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

    PubMed Central

    Gong, M C; Fujihara, H; Walker, L A; Somlyo, A V; Somlyo, A P

    1997-01-01

    Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector. Images PMID:9190207

  20. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells.

    PubMed

    Fu, Shibo; Tar, Moses Tarndie; Melman, Arnold; Davies, Kelvin Paul

    2014-08-01

    Men with sickle cell disease (SCD) risk developing priapism. Recognizing that SCD is a disease of hypoxia, we investigated the effect of hypoxia on gene expression in corporal smooth muscle (CSM) cells. Rat CSM cells in vitro were treated with CoCl2 or low oxygen tension to mimic hypoxia. Hypoxic conditions increased expression of genes previously associated with priapism in animal models. Variable coding sequence a1 (Vcsa1; the rat opiorphin homologue, sialorphin), hypoxia-inducible factor 1a (Hif-1a), and A2B adenosine receptor (a2br) were increased by 10-, 4-, and 6-fold, respectively, by treatment with CoCl2, whereas low oxygen tension caused increases in expression of 3-, 4-, and 1.5-fold, respectively. Sialorphin-treated CSM cells increased expression of Hif-1a and a2br by 4-fold, and vcsa1-siRNA treatment reduced expression by ∼50%. Using a Hif-1a inhibitor, we demonstrated up-regulation of a2br by sialorphin is dependent on Hif-1a, and knockdown of vcsa1 expression with vcsa1-siRNA demonstrated that hypoxic-up-regulation of Hif-1a is dependent on vcsa1. In CSM from a SCD mouse, there was 15-fold up-regulation of opiorphin at a life stage prior to priapism. We conclude that in CSM, opiorphins are master regulators of the hypoxic response. Opiorphin up-regulation in response to SCD-associated hypoxia activates CSM "relaxant" pathways; excessive activation of these pathways results in priapism.

  1. Smoothing out transitions: how pedagogy influences medical students' achievement of self-regulated learning goals.

    PubMed

    White, Casey B

    2007-08-01

    Medical school is an academic and developmental path toward a professional life demanding self-regulation and self-education. Thus, many medical schools include in their goals for medical student education their graduates' ability to self-assess and self-regulate their education upon graduation and throughout their professional lives. This study explores links between medical students' use of self-regulated learning as it relates to motivation, autonomy, and control, and how these influenced their experiences in medical school. Subjects were medical students in two distinct medical school environments, "Problem-based learning" and "Traditional." PBL students described a rough transition into medical school, but once they felt comfortable with the autonomy and control PBL gave them, they embraced the independence and responsibility. They found themselves motivated to learning for learning's sake, and able to channel their motivation into effective transitions from the classrooms into the clerkships. Traditional students had a rougher transition from the classrooms to the clerkships. In the first two years they relied on faculty to direct and control learning, and they channeled their motivation toward achieving the highest grade. In the clerkships, they found faculty expected them to be more independent and self-directed than they felt prepared to be, and they struggled to assume responsibility for their learning. Self-regulated learning can help smooth out the transitions through medical school by preparing first and second year students for expectations in the third and fourth years, which can then maximize learning in the clinical milieu, and prepare medical students for a lifetime of learning.

  2. Chemokine-Like Receptor 1 Regulates the Proliferation and Migration of Vascular Smooth Muscle Cells.

    PubMed

    Liu, Huadong; Xiong, Wei; Liu, Qiyun; Zhang, Jian; Dong, Shaohong

    2016-10-28

    BACKGROUND We aimed to explore how chemokine-like receptor 1 (CMKLR1) influences the proliferation and migration of vascular smooth muscle cells (VSMCs). MATERIAL AND METHODS Normal VSMCs, negative control VSMCs interfered by CMKLR1 gene, and VSMCs with stable knockdown of CMKLR1 gene were divided into the control group, PDGF group, negative-shRNA group, and CMKLR1-shRNA group. Both cell number counting and BrdU incorporation assays were employed to investigate the proliferation status of VSMCs. Transwell migration assay was used to measure the migration status of VSMCs. Inflammation markers, including cytokines IL-1β, IL-6, TNF-α, and chemokines MCP-1 in VSMCs, were detected by real-time quantitative RT-PCR. Western blotting assay was used to detect protein expressions of the MAPK pathway in VSMCs. RESULTS The number of VSMCs and the OD value of BrdU in PDGF group were significantly higher than those in the control group (both P<0.05). Compared with the control and negative-shRNA group, the CMKLR1-shRNA group exhibited significantly reduced VSMCs number and BrdU OD value (both P<0.05). Transwell migration assay indicated that PDGF-BB promoted whereas CMKLR1-shRNA inhibited the migration of VSMCs. The expression of IL-1β, IL-6, TNF-α, and MCP-1 were up-regulated in the PDGF group but down-regulated in the CMKLR1-shRNA group. Compared with normal VSMCs, the protein level of p-ERK1/2 was up-regulated in VSMCs treated with PDGF-BB, while it was down-regulated in the CMKLR1-shRNA group. CONCLUSIONS CMKLR1 exacerbated the proliferation and migration of VSMCs by activating ERK1/2.

  3. Rab5a-mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells

    PubMed Central

    Tan, Jin-Yun; Jia, Luo-Qi; Shi, Wei-Hao; He, Qing; Zhu, Lei; Yu, Bo

    2016-01-01

    Rab5a, a key member of the Rab family of GTPases, was determined to be a regulator of vascular smooth muscle cell (VSMC) proliferation and migration. However, the exact regulatory mechanism remains unclear. As Rab5a has been shown to be associated with autophagy, which is essential for the conversion of VSMCs from a contractile to a synthetic phenotype in order to prevent cell death due to oxidative stress. The present study hypothesized that autophagy may be responsible for the proliferation and migration of VSMCs via the Rab5a protein. The aim of the present study was to evaluate the effect of Rab5a on autophagy in VSMCs. The human aorta vascular smooth muscle cell line, T/G HA-VSMCs, was treated with small interfering (si)RNA against Rab5a and/or platelet-derived growth factor (PDGF). Following treatment, the phenotype transition of the VSMCs was evaluated by detecting the mRNA and protien expression levels of VSMC molecular markers using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In addition, autophagy in VSMCs was evaluated by western blotting for autophagy-associated proteins, flow cytometry of acidic vesicular organelles, punctate fluorescence of microtubule associated protein light chain 3 and transmission electron microscopy of typical scattered double-membrane vacuolar structures. Additionally, the proliferation, migration, cell cycle and apoptotic response of VSMCs were detected by sulforhodamine B assay, transwell assay and flow cytometry, respectively. The results revealed that transfection with siRNA against Rab5a led to a significant decrease in Rab5a protein expression, while the reduced expression trend of Rab5a was rescued by intervention with PDGF. Furthermore, cells transfected with siRNA against Rab5a inhibited the autophagy of VSMCs. Downregulated Rab5a inhibited the phenotype transition of VSMCs. Additionally, downregulated Rab5a led to slowed cell growth, decreased numbers of migrated

  4. Regulation of ERK5 by insulin and angiotensin-II in vascular smooth muscle cells

    SciTech Connect

    Sharma, Girish; Goalstone, Marc Lee; E-mail: Marc.Goalstone@uchsc.edu

    2007-03-23

    ERK5 is involved in proliferation of vascular smooth muscle cells (VSMC). The proliferative actions of insulin and angiotensin-II (A-II) in VSMC are mediated in part by ERK1/2. We hypothesized that insulin and A-II also regulate ERK5 activity in VSMC. Acute treatment (<60 min) with insulin or A-II increased phosphorylation of ERK1/2 at 15 min and ERK5 at 5 min. Chronic treatment ({<=}8 h) with insulin increased ERK1/2 phosphorylation by 4 h and ERK5 by 8 h. A-II-stimulated phosphorylation of ERK1/2 by 8 h and ERK5 by 4 h. The EC{sub 50} for insulin treatment effecting ERK1/2 and ERK5 phosphorylation was 1.5 and 0.1 nM, whereas the EC{sub 50} for A-II was 2 nM, each. Insulin plus A-II induced an additive effect only on ERK5 phosphorylation. Inhibition of insulin- and A-II-stimulated phosphorylation of ERK5 and ERK1/2 by PD98059 and Wortmannin exhibited differential and time-dependent effects. Taken together, these data indicate that insulin and A-II regulate the activity of ERK5, but different from that seen for ERK1/2.

  5. Biphasic regulation of myosin light chain phosphorylation by p21-activated kinase modulates intestinal smooth muscle contractility.

    PubMed

    Chu, Ji; Pham, Ngoc T; Olate, Nicole; Kislitsyna, Karina; Day, Mary-Clare; LeTourneau, Phillip A; Kots, Alexander; Stewart, Randolph H; Laine, Glen A; Cox, Charles S; Uray, Karen

    2013-01-11

    Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions.

  6. Phosphodiesterases Regulate BAY 41-2272-Induced VASP Phosphorylation in Vascular Smooth Muscle Cells

    PubMed Central

    Adderley, Shaquria P.; Joshi, Chintamani N.; Martin, Danielle N.; Tulis, David Anthony

    2012-01-01

    BAY 41-2272 (BAY), a stimulator of soluble guanylyl cyclase, increases cyclic nucleotides and inhibits proliferation of vascular smooth muscle cells (VSMCs). In this study, we elucidated mechanisms of action of BAY in its regulation of vasodilator-stimulated phosphoprotein (VASP) with an emphasis on VSMC phosphodiesterases (PDEs). BAY alone increased phosphorylation of VASPSer239 and VASPSer157, respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASPSer239 but inhibited phosphorylation at VASPSer157. Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASPSer239 and VASPSer157, whereas PDE5 inhibition potentiated BAY-mediated increases only at VASPSer157. In comparison, 8Br-cGMP increased phosphorylation at VASPSer239 and VASPSer157 which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASPSer239 phosphorylation and inhibition of PDE3 increased phosphorylation at VASPSer239, while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASPSer157 phosphorylation. These findings demonstrate that BAY operates via cAMP and cGMP along with regulation by PDEs to phosphorylate VASP in VSMCs and that the mechanism of action of BAY in VSMCs is different from that of direct cyclic nucleotide analogs with respect to VASP phosphorylation and the involvement of PDEs. Given a role for VASP as a critical cytoskeletal protein, these findings provide evidence for BAY as a regulator of VSMC growth and a potential therapeutic agent against vasculoproliferative disorders. PMID:22347188

  7. TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle.

    PubMed

    Vohra, Pawan K; Thompson, Michael A; Sathish, Venkatachalem; Kiel, Alexander; Jerde, Calvin; Pabelick, Christina M; Singh, Brij B; Prakash, Y S

    2013-12-01

    Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca(2+) signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca(2+) entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP-BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca(2+) (EGTA; 1mM) or intracellular Ca(2+) (BAPTA; 5μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca(2+) influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca(2+) and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.

  8. TET2 is a Master Regulator of Smooth Muscle Cell Plasticity

    PubMed Central

    Liu, Renjing; Jin, Yu; Tang, Waiho; Qin, Lingfeng; Zhang, Xinbo; Tellides, George; Hwa, John; Yu, Jun; Martin, Kathleen A.

    2014-01-01

    Background Smooth muscle cells (SMC) are remarkably plastic. Their reversible differentiation is required for growth and wound healing, but also contributes to pathologies including atherosclerosis and restenosis. While key regulators of the SMC phenotype including myocardin (MYOCD) and KLF4 have been identified, a unifying epigenetic mechanism that confers reversible SMC differentiation has not been reported. Methods and Results Using human SMC, human arterial tissue, and mouse models, we report that SMC plasticity is governed by the DNA modifying enzyme ten-eleven translocation-2 (TET2). TET2 and its 5-hydroxymethylcytosine (5-hmC) product are enriched in contractile SMC but reduced in dedifferentiated SMC. TET2 knockdown inhibits expression of key pro-contractile genes including MYOCD and SRF with concomitant transcriptional upregulation of KLF4. TET2 knockdown prevents rapamycin-induced SMC differentiation, while TET2 overexpression is sufficient to induce a contractile phenotype. TET2 overexpression also induces SMC gene expression in fibroblasts. Chromatin immunoprecipitation demonstrates that TET2 coordinately regulates phenotypic modulation through opposing effects on chromatin accessibility at the promoters of pro-contractile versus dedifferentiation-associated genes. Notably, we find that TET2 binds, and 5-hmC is enriched, in CArG-rich regions of active SMC contractile promoters (MYOCD, SRF, and MYH11). Loss of TET2 and 5-hmC positively correlates with the degree of injury in murine models of vascular injury and human atherosclerotic disease. Importantly, localized TET2 knockdown exacerbates injury response while local TET2 overexpression restores the 5-hmC epigenetic landscape, contractile gene expression, and greatly attenuates intimal hyperplasia in vivo. Conclusions We identify TET2 as a novel and necessary master epigenetic regulator of SMC differentiation. PMID:24077167

  9. A phospholipase Cγ1-activated pathway regulates transcription in human vascular smooth muscle cells.

    PubMed

    Hunter, Irene; Mascall, Keith S; Ramos, Joe W; Nixon, Graeme F

    2011-06-01

    Growth factor-induced repression of smooth muscle (SM) cell marker genes is an integral part of vascular SM (VSM) cell proliferation. This is partly regulated via translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) to the nucleus which activates the transcription factor Elk-1. The mediators involved in ERK1/2 nuclear translocation in VSM cells are unknown. The aim of this study is to examine the mechanisms which regulate growth factor-induced nuclear translocation of ERK1/2 and gene expression in VSM cells. In cultured human VSM cells, phospholipase C (PLC)γ1 expression was required for platelet-derived growth factor (PDGF)-induced ERK1/2 nuclear translocation, Elk-1 phosphorylation, and subsequent repression of SM α-actin gene expression. The mechanisms of a role for PLCγ1 in ERK1/2 nuclear localization were further examined by investigating interacting proteins. The ERK1/2-binding phosphoprotein, protein enriched in astrocytes-15 (PEA-15), was phosphorylated by PDGF and this phosphorylation required activation of PLCγ1. In cells pre-treated with PEA-15 siRNA, ERK1/2 distribution significantly increased in the nucleus and resulted in decreased SM α-actin expression and increased VSM cell proliferation. Overexpression of PEA-15 increased ERK1/2 localization in the cytoplasm. The regulatory role of PEA-15 phosphorylation was assessed. In VSM cells overexpressing a non-phosphorylatable form of PEA-15, PDGF-induced ERK1/2 nuclear localization was inhibited. These results suggest that PEA-15 phosphorylation by PLCγ1 is required for PDGF-induced ERK1/2 nuclear translocation. This represents an important level of phenotypic control by directly affecting Elk-1-dependent transcription and ultimately SM cell marker protein expression in VSM cells.

  10. Regulation of Vascular Smooth Muscle Cell Dysfunction Under Diabetic Conditions by miR-504.

    PubMed

    Reddy, Marpadga A; Das, Sadhan; Zhuo, Chen; Jin, Wen; Wang, Mei; Lanting, Linda; Natarajan, Rama

    2016-05-01

    Diabetes mellitus accelerates proatherogenic and proinflammatory phenotype of vascular smooth muscle cell (VSMC) associated with vascular complications. Evidence shows that microRNAs (miRNAs) play key roles in VSMC functions, but their role under diabetic conditions is unclear. We profiled miRNAs in VSMC from diabetic mice and examined their role in VSMC dysfunction. High throughput small RNA-sequencing identified 135 differentially expressed miRNAs in VSMC from type 2 diabetic db/db mice (db/dbVSMC) versus nondiabetic db/+ mice. Several of these miRNAs were known to regulate VSMC functions. We further focused on miR-504, because it was highly upregulated in db/dbVSMC, and its function in VSMC is unknown. miR-504 and its host gene Fgf13 were significantly increased in db/dbVSMC and in aortas from db/db mice. Bioinformatics analysis predicted that miR-504 targets including signaling adaptor Grb10 and transcription factor Egr2 could regulate growth factor signaling. We experimentally validated Grb10 and Egr2 as novel targets of miR-504. Overexpression of miR-504 in VSMC inhibited contractile genes and enhanced extracellular signal-regulated kinase 1/2 activation, proliferation, and migration. These effects were blocked by miR-504 inhibitors. Grb10 knockdown mimicked miR-504 functions and increased inflammatory genes. Egr2 knockdown-inhibited anti-inflammatory Socs1 and increased proinflammatory genes. Furthermore, high glucose and palmitic acid upregulated miR-504 and inflammatory genes, but downregulated Grb10. Diabetes mellitus misregulates several miRNAs including miR-504 that can promote VSMC dysfunction. Because changes in many of these miRNAs are sustained in diabetic VSMC even after in vitro culture, they may be involved in metabolic memory of vascular complications. Targeting such mechanisms could offer novel therapeutic strategies for diabetic complications. © 2016 American Heart Association, Inc.

  11. Vascular smooth muscle cell-derived adiponectin: a paracrine regulator of contractile phenotype

    PubMed Central

    Ding, Min; Carrao, Ana Catarina; Wagner, Robert J.; Xie, Yi; Jin, Yu; Rzucidlo, Eva M.; Yu, Jun; Li, Wei; Tellides, George; Hwa, John; Aprahamian, Tamar R.; Martin, Kathleen A.

    2011-01-01

    Adiponectin is a cardioprotective adipokine derived predominantly from visceral fat. We recently demonstrated that exogenous adiponectin induces vascular smooth muscle cell (VSMC) differentiation via repression of mTORC1 and FoxO4. Here we report for the first time that VSMC express and secrete adiponectin, which acts in an autocrine and paracrine manner to regulate VSMC contractile phenotype. Adiponectin was found to be expressed in human coronary artery and mouse aortic VSMC. Importantly, siRNA knock-down of endogenous adiponectin in VSMC significantly reduced the expression of VSMC contractile proteins. Contractile protein deficiency was also observed in primary VSMC isolated from Adiponectin-/- mice. This deficiency could be rescued by culturing Adiponectin-/- VSMC in conditioned media from wild type (WT) VSMC. Moreover, the paracrine effect of VSMC-derived adiponectin was confirmed as adiponectin neutralizing antibody blocked the rescue. Overexpressed adiponectin also exerted paracrine effects on neighboring untransfected VSMC, which was also blocked by adiponectin neutralizing antibody. Interestingly, adiponectin expression was inducible by the PPARγ agonist rosiglitazone. Our data support an important role for VSMC-derived adiponectin in maintaining VSMC contractile phenotype, contributing to critical cardioprotective functions in the vascular wall. PMID:21952104

  12. The Hemoglobin Homolog Cytoglobin in Smooth Muscle Inhibits Apoptosis and Regulates Vascular Remodeling.

    PubMed

    Jourd'heuil, Frances L; Xu, Haiyan; Reilly, Timothy; McKellar, Keneta; El Alaoui, Chaymae; Steppich, Julia; Liu, Yong Feng; Zhao, Wen; Ginnan, Roman; Conti, David; Lopez-Soler, Reynold; Asif, Arif; Keller, Rebecca K; Schwarz, John J; Thanh Thuy, Le Thi; Kawada, Norifumi; Long, Xiaochun; Singer, Harold A; Jourd'heuil, David

    2017-10-01

    The role of hemoglobin and myoglobin in the cardiovascular system is well established, yet other globins in this context are poorly characterized. Here, we examined the expression and function of cytoglobin (CYGB) during vascular injury. We characterized CYGB content in intact vessels and primary vascular smooth muscle (VSM) cells and used 2 different vascular injury models to examine the functional significance of CYGB in vivo. We found that CYGB was strongly expressed in medial arterial VSM and human veins. In vitro and in vivo studies indicated that CYGB was lost after VSM cell dedifferentiation. In the rat balloon angioplasty model, site-targeted delivery of adenovirus encoding shRNA specific for CYGB prevented its reexpression and decreased neointima formation. Similarly, 4 weeks after complete ligation of the left common carotid, Cygb knockout mice displayed little to no evidence of neointimal hyperplasia in contrast to their wild-type littermates. Mechanistic studies in the rat indicated that this was primarily associated with increased medial cell loss, terminal uridine nick-end labeling staining, and caspase-3 activation, all indicative of prolonged apoptosis. In vitro, CYGB could be reexpressed after VSM stimulation with cytokines and hypoxia and loss of CYGB sensitized human and rat aortic VSM cells to apoptosis. This was reversed after antioxidant treatment or NOS2 (nitric oxide synthase 2) inhibition. These results indicate that CYGB is expressed in vessels primarily in differentiated medial VSM cells where it regulates neointima formation and inhibits apoptosis after injury. © 2017 American Heart Association, Inc.

  13. Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

    PubMed

    Gao, Yuan Z; Saphirstein, Robert J; Yamin, Rina; Suki, Bela; Morgan, Kathleen G

    2014-10-15

    Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of N(G)-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90-200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors.

  14. Vascular smooth muscle, endothelial regulation and effects of aspirin in hypertension.

    PubMed

    Rahmani, M A

    1998-04-27

    Dysfunction of vascular smooth muscle (VSM) is at the center of occlusive disorders of the cardiovascular system such as hypertension, atherosclerosis, coronary artery disease and hypoxia. In addition to circulating biogenic amines and various neurotransmitters originating from the central nervous system and endocrine system, various autocoids of arachidonic acid metabolism in the blood as well as in the endothelium play an important regulatory role in the maintenance of the tone and the contractile function of VSM. A monolayer of endothelial cells lining the heart and large blood vessels is responsible for producing and releasing both endocrine and paracrine substances such as endothelins, nitric oxide, prostaglandins and prostacyclins. Aspirin, (acetylsalicylic acid/ASA) an ancient remedy against fever and pain, is emerging as an effective drug not only against occlusive disorders but also against various cancers and the AIDs virus. During pregnancy induced hypertension (PIH) and in occlusive disorders, aspirin provides relief through inhibition of cyclooxygenase, an enzyme required for the metabolism of arachidonic acid to produce prostaglandins and prostacyclins in platelets and in endothelial cells. Because of its unique molecular constitution, synergistic ability and solubility in the lipidic environment, various mechanisms of aspirin's actions are being currently investigated. In this review, the effect of aspirin on the regulation of VSM in the presence and absence of endothelium are discussed.

  15. Aging impairs smooth muscle-mediated regulation of aortic stiffness: a defect in shock absorption function?

    PubMed Central

    Gao, Yuan Z.; Saphirstein, Robert J.; Yamin, Rina; Suki, Bela

    2014-01-01

    Increased aortic stiffness is an early and independent biomarker of cardiovascular disease. Here we tested the hypothesis that vascular smooth muscle cells (VSMCs) contribute significantly to aortic stiffness and investigated the mechanisms involved. The relative contributions of VSMCs, focal adhesions (FAs), and matrix to stiffness in mouse aorta preparations at optimal length and with confirmed VSMC viability were separated by the use of small-molecule inhibitors and activators. Using biomechanical methods designed for minimal perturbation of cellular function, we directly quantified changes with aging in aortic material stiffness. An alpha adrenoceptor agonist, in the presence of NG-nitro-l-arginine methyl ester (l-NAME) to remove interference of endothelial nitric oxide, increases stiffness by 90–200% from baseline in both young and old mice. Interestingly, increases are robustly suppressed by the Src kinase inhibitor PP2 in young but not old mice. Phosphotyrosine screening revealed, with aging, a biochemical signature of markedly impaired agonist-induced FA remodeling previously associated with Src signaling. Protein expression measurement confirmed a decrease in Src expression with aging. Thus we report here an additive model for the in vitro biomechanical components of the mouse aortic wall in which 1) VSMCs are a surprisingly large component of aortic stiffness at physiological lengths and 2) regulation of the VSMC component through FA signaling and hence plasticity is impaired with aging, diminishing the aorta's normal shock absorption function in response to stressors. PMID:25128168

  16. MicroRNA-143-3p inhibits hyperplastic scar formation by targeting connective tissue growth factor CTGF/CCN2 via the Akt/mTOR pathway.

    PubMed

    Mu, Shengzhi; Kang, Bei; Zeng, Weihui; Sun, Yaowen; Yang, Fan

    2016-05-01

    Post-traumatic hypertrophic scar (HS) is a fibrotic disease with excessive extracellular matrix (ECM) production, which is a response to tissue injury by fibroblasts. Although emerging evidence has indicated that miRNA contributes to hypertrophic scarring, the role of miRNA in HS formation remains unclear. In this study, we found that miR-143-3p was markedly downregulated in HS tissues and fibroblasts (HSFs) using qRT-PCR. The expression of connective tissue growth factor (CTGF/CCN2) was upregulated both in HS tissues and HSFs, which is proposed to play a key role in ECM deposition in HS. The protein expression of collagen I (Col I), collagen III (Col III), and α-smooth muscle actin (α-SMA) was obviously inhibited after treatment with miR-143-3p in HSFs. The CCK-8 assay showed that miR-143-3p transfection reduced the proliferation ability of HSFs, and flow cytometry showed that either early or late apoptosis of HSFs was upregulated by miR-143-3p. In addition, the activity of caspase 3 and caspase 9 was increased after miR-143-3p transfection. On the contrary, the miR-143-3p inhibitor was demonstrated to increase cell proliferation and inhibit apoptosis of HSFs. Moreover, miR-143-3p targeted the 3'-UTR of CTGF and caused a significant decrease of CTGF. Western blot demonstrated that Akt/mTOR phosphorylation and the expression of CTGF, Col I, Col III, and α-SMA were inhibited by miR-143-3p, but increased by CTGF overexpression. In conclusion, we found that miR-143-3p inhibits hypertrophic scarring by regulating the proliferation and apoptosis of human HSFs, inhibiting ECM production-associated protein expression by targeting CTGF, and restraining the Akt/mTOR pathway.

  17. Monocyte/macrophage cytokine activity regulates vascular smooth muscle cell function within a degradable polyurethane scaffold.

    PubMed

    Battiston, K G; Ouyang, B; Labow, R S; Simmons, C A; Santerre, J P

    2014-03-01

    Tissue engineering strategies rely on the ability to promote cell proliferation and migration into porous biomaterial constructs, as well as to support specific phenotypic states of the cells in vitro. The present study investigated the use of released factors from monocytes and their derived macrophages (MDM) and the mechanism by which they regulate vascular smooth muscle cell (VSMC) response in a VSMC-monocyte co-culture system within a porous degradable polyurethane (D-PHI) scaffold. VSMCs cultured in monocyte/MDM-conditioned medium (MCM), generated from the culture of monocytes/MDM on D-PHI scaffolds for up to 28 days, similarly affected VSMC contractile marker expression, growth and three-dimensional migration when compared to direct VSMC-monocyte co-culture. Monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) were identified as two cytokines present in MCM, at concentrations that have previously been shown to influence VSMC phenotype. VSMCs cultured alone on D-PHI scaffolds and exposed to MCP-1 (5 ng ml(-1)) or IL-6 (1 ng ml(-1)) for 7 days experienced a suppression in contractile marker expression (with MCP-1 or IL-6) and increased growth (with MCP-1) compared to no cytokine medium supplementation. These effects were also observed in VSMC-monocyte co-culture on D-PHI. Neutralization of IL-6, but not MCP-1, was subsequently shown to decrease VSMC growth and enhance calponin expression for VSMC-monocyte co-cultures on D-PHI scaffolds for 7 days, implying that IL-6 mediates VSMC response in monocyte-VSMC co-cultures. This study highlights the use of monocytes and their derived macrophages in conjunction with immunomodulatory biomaterials, such as D-PHI, as agents for regulating VSMC response, and demonstrates the importance of monocyte/MDM-released factors, such as IL-6 in particular, in this process.

  18. Essential Role of TGF-β/Smad Pathway on Statin Dependent Vascular Smooth Muscle Cell Regulation

    PubMed Central

    Rodríguez-Vita, Juan; Sánchez-Galán, Eva; Santamaría, Beatriz; Sánchez-López, Elsa; Rodrigues-Díez, Raquel; Blanco-Colio, Luís Miguel; Egido, Jesús; Ortiz, Alberto; Ruiz-Ortega, Marta

    2008-01-01

    Background The 3-hydroxy-3-methylglutaryl CoA reductase inhibitors (also called statins) exert proven beneficial effects on cardiovascular diseases. Recent data suggest a protective role for Transforming Growth Factor-β (TGF-β) in atherosclerosis by regulating the balance between inflammation and extracellular matrix accumulation. However, there are no studies about the effect of statins on TGF-β/Smad pathway in atherosclerosis and vascular cells. Methodology In cultured vascular smooth muscle cells (VSMCs) statins enhanced Smad pathway activation caused by TGF-β. In addition, statins upregulated TGF-β receptor type II (TRII), and increased TGF-β synthesis and TGF-β/Smad-dependent actions. In this sense, statins, through Smad activation, render VSMCs more susceptible to TGF-β induced apoptosis and increased TGF-β-mediated ECM production. It is well documented that high doses of statins induce apoptosis in cultured VSMC in the presence of serum; however the precise mechanism of this effect remains to be elucidated. We have found that statins-induced apoptosis was mediated by TGF-β/Smad pathway. Finally, we have described that RhoA inhibition is a common intracellular mechanisms involved in statins effects. The in vivo relevance of these findings was assessed in an experimental model of atherosclerosis in apolipoprotein E deficient mice: Treatment with Atorvastatin increased Smad3 phosphorylation and TRII overexpression, associated to elevated ECM deposition in the VSMCs within atheroma plaques, while apoptosis was not detected. Conclusions Statins enhance TGF-β/Smad pathway, regulating ligand levels, receptor, main signaling pathway and cellular responses of VSMC, including apoptosis and ECM accumulation. Our findings show that TGF-β/Smad pathway is essential for statins-dependent actions in VSMCs. PMID:19088845

  19. BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells

    PubMed Central

    Wakle-Prabagaran, Monali; Lorca, Ramón A.; Ma, Xiaofeng; Stamnes, Susan J.; Amazu, Chinwendu; Hsiao, Jordy J.; Hyrc, Krzysztof L.; Wright, Michael E.; England, Sarah K.

    2016-01-01

    The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) plays an important role in regulating Ca2+ signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCa in myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCa and both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCa in an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca2+ to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca2+-free medium and inhibitors of various Ca2+ signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca2+ through store-operated Ca2+ channels. Finally, we found that the specific BKCa blocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCa channel in human myometrium and that BKCa and its immunomodulatory interacting partners regulate Ca2+ dynamics in hMSMCs during pregnancy. PMID:27044074

  20. TLR4-Activated MAPK-IL-6 Axis Regulates Vascular Smooth Muscle Cell Function.

    PubMed

    Lee, Guan-Lin; Wu, Jing-Yiing; Tsai, Chien-Sung; Lin, Chih-Yuan; Tsai, Yi-Ting; Lin, Chin-Sheng; Wang, Yi-Fu; Yet, Shaw-Fang; Hsu, Yu-Juei; Kuo, Cheng-Chin

    2016-08-24

    Migration of vascular smooth muscle cells (VSMCs) into the intima is considered to be a vital event in the pathophysiology of atherosclerosis. Despite substantial evidence supporting the pathogenic role of Toll-like receptor 4 (TLR4) in the progression of atherogenesis, its function in the regulation of VSMC migration remains unclear. The goal of the present study was to elucidate the mechanism by which TLR4 regulates VSMC migration. Inhibitor experiments revealed that TLR4-induced IL-6 secretion and VSMC migration were mediated via the concerted actions of MyD88 and TRIF on the activation of p38 MAPK and ERK1/2 signaling. Neutralizing anti-IL-6 antibodies abrogated TLR4-driven VSMC migration and F-actin polymerization. Blockade of p38 MAPK or ERK1/2 signaling cascade inhibited TLR4 agonist-mediated activation of cAMP response element binding protein (CREB). Moreover, siRNA-mediated suppression of CREB production repressed TLR4-induced IL-6 production and VSMC migration. Rac-1 inhibitor suppressed TLR4-driven VSMC migration but not IL-6 production. Importantly, the serum level of IL-6 and TLR4 endogenous ligand HMGB1 was significantly higher in patients with coronary artery diseases (CAD) than in healthy subjects. Serum HMGB1 level was positively correlated with serum IL-6 level in CAD patients. The expression of both HMGB1 and IL-6 was clearly detected in the atherosclerotic tissue of the CAD patients. Additionally, there was a positive association between p-CREB and HMGB1 in mouse atherosclerotic tissue. Based on our findings, we concluded that, upon ligand binding, TLR4 activates p38 MAPK and ERK1/2 signaling through MyD88 and TRIF in VSMCs. These signaling pathways subsequently coordinate an additive augmentation of CREB-driven IL-6 production, which in turn triggers Rac-1-mediated actin cytoskeleton to promote VSMC migration.

  1. Mechanical stretch is a highly selective regulator of gene expression in human bladder smooth muscle cells.

    PubMed

    Adam, Rosalyn M; Eaton, Samuel H; Estrada, Carlos; Nimgaonkar, Ashish; Shih, Shu-Ching; Smith, Lois E H; Kohane, Isaac S; Bägli, Darius; Freeman, Michael R

    2004-12-15

    Application of mechanical stimuli has been shown to alter gene expression in bladder smooth muscle cells (SMC). To date, only a limited number of "stretch-responsive" genes in this cell type have been reported. We employed oligonucleotide arrays to identify stretch-sensitive genes in primary culture human bladder SMC subjected to repetitive mechanical stimulation for 4 h. Differential gene expression between stretched and nonstretched cells was assessed using Significance Analysis of Microarrays (SAM). Expression of 20 out of 11,731 expressed genes ( approximately 0.17%) was altered >2-fold following stretch, with 19 genes induced and one gene (FGF-9) repressed. Using real-time RT-PCR, we tested independently the responsiveness of 15 genes to stretch and to platelet-derived growth factor-BB (PDGF-BB), another hypertrophic stimulus for bladder SMC. In response to both stimuli, expression of 13 genes increased, 1 gene (FGF-9) decreased, and 1 gene was unchanged. Six transcripts (HB-EGF, BMP-2, COX-2, LIF, PAR-2, and FGF-9) were evaluated using an ex vivo rat model of bladder distension. HB-EGF, BMP-2, COX-2, LIF, and PAR-2 increased with bladder stretch ex vivo, whereas FGF-9 decreased, consistent with expression changes observed in vitro. In silico analysis of microarray data using the FIRED algorithm identified c-jun, AP-1, ATF-2, and neurofibromin-1 (NF-1) as potential transcriptional mediators of stretch signals. Furthermore, the promoters of 9 of 13 stretch-responsive genes contained AP-1 binding sites. These observations identify stretch as a highly selective regulator of gene expression in bladder SMC. Moreover, they suggest that mechanical and growth factor signals converge on common transcriptional regulators that include members of the AP-1 family.

  2. Cyclin-dependent kinase inhibitor, p21Waf1, regulates vascular smooth muscle cell hypertrophy.

    PubMed

    Okamoto, Kenichi; Kato, Seiya; Arima, Nobuyuki; Fujii, Teruhiko; Morimatsu, Minoru; Imaizumi, Tsutomu

    2004-04-01

    In the process of vascular diseases, smooth muscle cells (SMC) undergo not only hyperplasia but also hypertrophy, resulting in vascular remodeling. A cyclin-dependent kinase inhibitor (CDKI), p21Waf1, has been shown to play an important role in SMC hyperplasia. Here we investigated a potential role of p21Waf1 in SMC hypertrophy. An exposure of cultured rat SMC to serum drove the cell cycle progression with up-regulation of various cell cycle markers and increased activities of cyclin-dependent kinases, but did not cause SMC hypertrophy. In contrast, incubation of SMC for 48 h with angiotensin II (AII, 100 nmol/l) resulted in a significant increase in the cell size measured by flowcytometric forward-angle light scatter assay, in association with an increase in the ratio of [3H]leucine/[3H]thymidine uptake, indicating SMC hypertrophy. At 48 h, p21Waf1 expression was up-regulated in SMC exposed to AII but not in those exposed to serum. These results suggest that p21Waf1 may be involved in hypertrophy. To further investigate this issue, two manipulations of the p21Waf1 gene were performed. Adenovirus-mediated over-expression of p21Waf1 not only reduced S-phasic cells but also caused hypertrophy, despite the exposure to serum. Antisense oligodeoxynucleotide for p21Waf1 inhibited the hypertrophy of SMC exposed to AII. Our data suggest that p21Waf1 may play a role in SMC hypertrophy as well.

  3. Regulation of vascular smooth muscle phenotype by cross-regulation of krüppel-like factors

    PubMed Central

    Ha, Jung Min; Yun, Sung Ji; Jin, Seo Yeon; Lee, Hye Sun; Kim, Sun Ja; Shin, Hwa Kyoung

    2017-01-01

    Regulation of vascular smooth muscle cell (VSMC) phenotype plays an essential role in many cardiovascular diseases. In the present study, we provide evidence that krüppel-like factor 8 (KLF8) is essential for tumor necrosis factor α (TNFα)-induced phenotypic conversion of VSMC obtained from thoracic aorta from 4-week-old SD rats. Stimulation of the contractile phenotype of VSMCs with TNFα significantly reduced the VSMC marker gene expression and KLF8. The gene expression of KLF8 was blocked by TNFα stimulation in an ERK-dependent manner. The promoter region of KLF8 contained putative Sp1, KLF4, and NFκB binding sites. Myocardin significantly enhanced the promoter activity of KLF4 and KLF8. The ectopic expression of KLF4 strongly enhanced the promoter activity of KLF8. Moreover, silencing of Akt1 significantly attenuated the promoter activity of KLF8; conversely, the overexpression of Akt1 significantly enhanced the promoter activity of KLF8. The promoter activity of SMA, SM22α, and KLF8 was significantly elevated in the contractile phenotype of VSMCs. The ectopic expression of KLF8 markedly enhanced the expression of SMA and SM22α concomitant with morphological changes. The overexpression of KLF8 stimulated the promoter activity of SMA. Stimulation of VSMCs with TNFα enhanced the expression of KLF5, and the promoter activity of KLF5 was markedly suppressed by KLF8 ectopic expression. Finally, the overexpression of KLF5 suppressed the promoter activity of SMA and SM22α, thereby reduced the contractility in response to the stimulation of angiotensin II. These results suggest that cross-regulation of KLF family of transcription factors plays an essential role in the VSMC phenotype. PMID:28066139

  4. Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines.

    PubMed Central

    Li, H; Freeman, M W; Libby, P

    1995-01-01

    Scavenger receptor (ScR)-mediated uptake of modified lipoproteins may contribute to the transformation of smooth muscle cells into lipid-laden foam cells during atherogenesis. This study examined the in vivo expression of ScRs in aortas, with or without balloon injury, taken from hypercholesterolemic or normocholesterolemic rabbits. Numerous intimal cells in the rabbit aortic lesions expressed ScRs as detected by immunocytochemical staining with a goat anti-rabbit ScR antibody. Single immunostaining for cell identification markers in serial sections, as well as double staining, confirmed the expression of ScRs by both intimal smooth muscle cells and macrophages. To explore potential inducers of ScR expression by smooth muscle cells in vivo, we studied the regulation of ScR expression in vitro by cytokines known to be present in atherosclerotic lesions. Tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) increased ScR mRNA levels, protein expression, and AcLDL degradative activity in cultured rabbit aortic smooth muscle cells. The induction of ScR expression in intimal smooth muscle cells in vivo could be a useful marker of smooth muscle cell activation during atherogenesis and may contribute to foam cell formation by this cell type following balloon injury and/or hypercholesterolemia. Cytokines, such as TNF-alpha or IFN-gamma, may stimulate some of the phenotypic changes that characterize the alteration in gene expression of intimal smooth muscle cells in rabbit atherosclerotic lesions. Images PMID:7814605

  5. CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide.

    PubMed

    Biedroń, Rafał; Peruń, Angelika; Józefowski, Szczepan

    2016-01-01

    Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity.

  6. CD36 Differently Regulates Macrophage Responses to Smooth and Rough Lipopolysaccharide

    PubMed Central

    Biedroń, Rafał; Peruń, Angelika; Józefowski, Szczepan

    2016-01-01

    Lipopolysaccharide (LPS) is the major pathogen-associated molecular pattern of Gram-negative bacterial infections, and includes smooth (S-LPS) and rough (R-LPS) chemotypes. Upon activation by LPS through CD14, TLR4/MD-2 heterodimers sequentially induce two waves of intracellular signaling for macrophage activation: the MyD88-dependent pathway from the plasma membrane and, following internalization, the TRIF-dependent pathway from endosomes. We sought to better define the role of scavenger receptors CD36 and CD204/SR-A as accessory LPS receptors that can contribute to pro-inflammatory and microbicidal activation of macrophages. We have found that CD36 differently regulates activation of mouse macrophages by S-LPS versus R-LPS. The ability of CD36 to substitute for CD14 in loading R-LPS, but not S-LPS onto TLR4/MD-2 allows CD14-independent macrophage responses to R-LPS. Conversely, S-LPS, but not R-LPS effectively stimulates CD14 binding to CD36, which favors S-LPS transfer from CD14 onto TLR4/MD-2 under conditions of low CD14 occupancy with S-LPS in serum-free medium. In contrast, in the presence of serum, CD36 reduces S-LPS binding to TLR4/MD-2 and the subsequent MyD88-dependent signaling, by mediating internalization of S-LPS/CD14 complexes. Additionally, CD36 positively regulates activation of TRIF-dependent signaling by both S-LPS and R-LPS, by promoting TLR4/MD-2 endocytosis. In contrast, we have found that SR-A does not function as a S-LPS receptor. Thus, by co-operating with CD14 in both R- and S-LPS loading onto TLR4/MD-2, CD36 can enhance the sensitivity of tissue-resident macrophages in detecting infections by Gram-negative bacteria. However, in later phases, following influx of serum to the infection site, the CD36-mediated negative regulation of MyD88-dependent branch of S-LPS-induced TLR4 signaling might constitute a mechanism to prevent an excessive inflammatory response, while preserving the adjuvant effect of S-LPS for adaptive immunity. PMID

  7. Sphingosine-1-Phosphate Receptor Subtypes Differentially Regulate Smooth Muscle Cell Phenotype

    PubMed Central

    Wamhoff, Brian R.; Lynch, Kevin R.; Macdonald, Timothy L.; Owens, Gary K.

    2008-01-01

    Objective The role of sphingosine-1-phosphate (S1P) receptors in acute vascular injury and smooth muscle cell (SMC) phenotypic modulation is not completely resolved. Methods and Results S1P receptor antagonists were used to test the hypothesis that specific S1P receptor subtypes differentially regulate SMC phenotypic modulation. In response to acute balloon injury of the rat carotid artery, S1P1/S1P3 receptor mRNA levels were transiently increased at 48 hours whereas S1P2 receptor expression was decreased. S1P2 expression was reinduced and increased at 7 to 10 days postinjury. Daily intraperitoneal injection of the S1P1/S1P3 antagonist VPC44116 decreased neointimal hyperplasia by ≈50%. In vitro, pharmacological inhibition of S1P1/S1P3 receptors with VPC25239 attenuated S1P-induced proliferation of rat aortic SMCs. Conversely, inhibition of S1P2 with JTE013 potentiated S1P-induced proliferation. Inhibition of S1P1/S1P3 resulted in S1P-induced activation of the SMC differentiation marker genes SMα-actin and SMMHC, whereas inhibition of S1P2 attenuated this response. S1P2-dependent activation of SMα-actin and SMMHC was shown to be mediated by L-type voltage-gated Ca2+ channels and subsequent RhoA/Rho kinase– dependent SRF enrichment of CArG box promoter regions. Conclusion Results provide evidence that S1P1/S1P3 receptors promote, whereas S1P2 receptors antagonize, SMC proliferation and phenotypic modulation in vitro in response to S1P, or in vivo after vascular injury. PMID:18535287

  8. Intracellular calcium in canine cultured tracheal smooth muscle cells is regulated by M3 muscarinic receptors.

    PubMed Central

    Yang, C. M.; Yo, Y. L.; Wang, Y. Y.

    1993-01-01

    1. The regulation of cytosolic Ca2+ concentrations ([Ca2+]i) during exposure to carbachol was measured directly in canine cultured tracheal smooth muscle cells (TSMCs) loaded with fura-2. Stimulation of muscarinic cholinoceptors (muscarinic AChRs) by carbachol produced a dose-dependent rise in [Ca2+]i which was followed by a stable plateau phase. The EC50 values of carbachol for the peak and sustained plateau responses were 0.34 and 0.33 microM, respectively. 2. Atropine (10 microM) prevented all the responses to carbachol, and when added during a response to carbachol, significantly, but not completely decreased [Ca2+]i within 5 s. Therefore, the changes in [Ca2+]i by carbachol were mediated through the muscarinic AChRs. 3. AF-DX 116 (a selective M2 antagonist) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a selective M3 antagonist) inhibited the carbachol-stimulated increase in [Ca2+]i with pKB values of 6.4 and 9.4, respectively, corresponding to low affinity for AF-DX 119 and high affinity for 4-DAMP in antagonizing this response. 4. The plateau elevation of [Ca2+]i was dependent on the presence of external Ca2+. Removal of Ca2+ by the addition of 2 mM EGTA caused the [Ca2+]i to decline rapidly to the resting level. In the absence of external Ca2+, only an initial transient peak of [Ca2+]i was seen which then declined to the resting level; the sustained elevation of [Ca2+]i could then be evoked by the addition of Ca2+ (1.8 mM) in the continued presence of carbachol.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8298822

  9. Testosterone regulates smooth muscle contractile pathways in the rat prostate: emphasis on PDE5 signaling

    PubMed Central

    Zhang, Xinhua; Zang, Ning; Wei, Yu; Yin, Jin; Teng, Ruobing; Seftel, Allen

    2012-01-01

    Testosterone (T) plays a permissive role in the development of benign prostatic hyperplasia (BPH), and phosphodiesterase 5 inhibitors (PDE5is) have been found to be effective for BPH and lower urinary tract symptoms (LUTS) in clinical trials. This study investigated the effect of T on smooth muscle (SM) contractile and regulatory signaling pathways, including PDE5 expression and functional activity in prostate in male rats (sham-operated, surgically castrated, and castrated with T supplementation). In vitro organ bath studies, real-time RT-PCR, Western blot analysis, and immunohistochemistry were performed. Castration heavily attenuated contractility, including sensitivity to phenylephrine with SM myosin immunostaining revealing a disrupted SM cell arrangement in the stroma. PDE5 was immunolocalized exclusively in the prostate stroma, and orchiectomy signficantly reduced PDE5 immunopositivity, mRNA, and protein expression, along with nNOS and ROKβ mRNA, whereas it increased eNOS plus α1a and α1b adrenoreceptor expression in castrated animals. The PDE5i zaprinast significantly increased prostate strip relaxation to the nitric oxide donor sodium nitroprusside (SNP) in control but not castrated rats. But SNP alone was more effective on castrated rats, comparable with sham treated with SNP plus zaprinast. T supplementation prevented or restored all above changes, including SNP and zaprinast in vitro responsiveness. In conclusion, our data show that T positively regulates PDE5 expression and functional activities in prostate, and T ablation not only suppresses prostate size but also reduces prostatic SM contractility, with several potential SM contraction/relaxation pathways implicated. Zaprinast findings strongly suggest a major role for PDE5/cGMP in this signaling cascade. PDE5 inhibition may represent a novel mechanism for treatment of BPH. PMID:22028410

  10. Effects of ginger and its constituents on airway smooth muscle relaxation and calcium regulation.

    PubMed

    Townsend, Elizabeth A; Siviski, Matthew E; Zhang, Yi; Xu, Carrie; Hoonjan, Bhupinder; Emala, Charles W

    2013-02-01

    The prevalence of asthma has increased in recent years, and is characterized by airway hyperresponsiveness and inflammation. Many patients report using alternative therapies to self-treat asthma symptoms as adjuncts to short-acting and long-acting β-agonists and inhaled corticosteroids (ICS). As many as 40% of patients with asthma use herbal therapies to manage asthma symptoms, often without proven efficacy or known mechanisms of action. Therefore, investigations of both the therapeutic and possible detrimental effects of isolated components of herbal treatments on the airway are important. We hypothesized that ginger and its active components induce bronchodilation by modulating intracellular calcium ([Ca(2+)](i)) in airway smooth muscle (ASM). In isolated human ASM, ginger caused significant and rapid relaxation. Four purified constituents of ginger were subsequently tested for ASM relaxant properties in both guinea pig and human tracheas: [6]-gingerol, [8]-gingerol, and [6]-shogaol induced rapid relaxation of precontracted ASM (100-300 μM), whereas [10]-gingerol failed to induce relaxation. In human ASM cells, exposure to [6]-gingerol, [8]-gingerol, and [6]-shogaol, but not [10]-gingerol (100 μM), blunted subsequent Ca(2+) responses to bradykinin (10 μM) and S-(-)-Bay K 8644 (10 μM). In A/J mice, the nebulization of [8]-gingerol (100 μM), 15 minutes before methacholine challenge, significantly attenuated airway resistance, compared with vehicle. Taken together, these novel data show that ginger and its isolated active components, [6]-gingerol, [8]-gingerol, and [6]-shogaol, relax ASM, and [8]-gingerol attenuates airway hyperresponsiveness, in part by altering [Ca(2+)](i) regulation. These purified compounds may provide a therapeutic option alone or in combination with accepted therapeutics, including β(2)-agonists, in airway diseases such as asthma.

  11. Epigenetic regulation of COL15A1 in smooth muscle cell replicative aging and atherosclerosis

    PubMed Central

    Connelly, Jessica J.; Cherepanova, Olga A.; Doss, Jennifer F.; Karaoli, Themistoclis; Lillard, Travis S.; Markunas, Christina A.; Nelson, Sarah; Wang, Tianyuan; Ellis, Peter D.; Langford, Cordelia F.; Haynes, Carol; Seo, David M.; Goldschmidt-Clermont, Pascal J.; Shah, Svati H.; Kraus, William E.; Hauser, Elizabeth R.; Gregory, Simon G.

    2013-01-01

    Smooth muscle cell (SMC) proliferation is a hallmark of vascular injury and disease. Global hypomethylation occurs during SMC proliferation in culture and in vivo during neointimal formation. Regardless of the programmed or stochastic nature of hypomethylation, identifying these changes is important in understanding vascular disease, as maintenance of a cells' epigenetic profile is essential for maintaining cellular phenotype. Global hypomethylation of proliferating aortic SMCs and concomitant decrease of DNMT1 expression were identified in culture during passage. An epigenome screen identified regions of the genome that were hypomethylated during proliferation and a region containing Collagen, type XV, alpha 1 (COL15A1) was selected by ‘genomic convergence’ for characterization. COL15A1 transcript and protein levels increased with passage-dependent decreases in DNA methylation and the transcript was sensitive to treatment with 5-Aza-2′-deoxycytidine, suggesting DNA methylation-mediated gene expression. Phenotypically, knockdown of COL15A1 increased SMC migration and decreased proliferation and Col15a1 expression was induced in an atherosclerotic lesion and localized to the atherosclerotic cap. A sequence variant in COL15A1 that is significantly associated with atherosclerosis (rs4142986, P = 0.017, OR = 1.434) was methylated and methylation of the risk allele correlated with decreased gene expression and increased atherosclerosis in human aorta. In summary, hypomethylation of COL15A1 occurs during SMC proliferation and the consequent increased gene expression may impact SMC phenotype and atherosclerosis formation. Hypomethylated genes, such as COL15A1, provide evidence for concomitant epigenetic regulation and genetic susceptibility, and define a class of causal targets that sit at the intersection of genetic and epigenetic predisposition in the etiology of complex disease. PMID:23912340

  12. Mechanism regulating proasthmatic effects of prolonged homologous beta2-adrenergic receptor desensitization in airway smooth muscle.

    PubMed

    Nino, Gustavo; Hu, Aihua; Grunstein, Judith S; Grunstein, Michael M

    2009-10-01

    Use of long-acting beta(2)-adrenergic receptor (beta2AR) agonists to treat asthma incurs an increased risk of asthma morbidity with impaired bronchodilation and heightened bronchoconstriction, reflecting the adverse effects of prolonged homologous beta2AR desensitization on airway smooth muscle (ASM) function. Since phosphodiesterase 4 (PDE4) regulates ASM relaxation and contractility, we examined whether the changes in ASM function induced by prolonged homologous beta2AR desensitization are attributed to altered expression and action of PDE4. Cultured human ASM cells and isolated rabbit ASM tissues exposed for 24 h to the long-acting beta2AR agonist salmeterol exhibited impaired acute beta2AR-mediated cAMP accumulation and relaxation, respectively, together with ASM constrictor hyperresponsiveness. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity due to enhanced expression of the PDE4D5 isoform and were prevented by pretreating the ASM preparations with the PDE4 inhibitor rolipram or with inhibitors of either PKA or ERK1/2 signaling. Extended studies using gene silencing and pharmacological approaches demonstrated that: 1) the mechanism underlying upregulated PDE4D5 expression following prolonged beta2AR agonist exposure involves PKA-dependent activation of G(i) protein signaling via its betagamma-subunits, which elicits downstream activation of ERK1/2 and its induction of PDE4D5 transcription; and 2) the induction of PDE4 activity and consequent changes in ASM responsiveness are prevented by pretreating the beta2AR agonist-exposed ASM preparations with inhibitors of G(i)-betagamma signaling. Collectively, these findings identify that the proasthmatic changes in ASM function resulting from prolonged homologous beta2AR desensitization are attributed to upregulated PDE4 expression induced by G(i)-betagamma-mediated cross-talk between the PKA and ERK1/2 signaling pathways.

  13. Effects of Ginger and Its Constituents on Airway Smooth Muscle Relaxation and Calcium Regulation

    PubMed Central

    Siviski, Matthew E.; Zhang, Yi; Xu, Carrie; Hoonjan, Bhupinder; Emala, Charles W.

    2013-01-01

    The prevalence of asthma has increased in recent years, and is characterized by airway hyperresponsiveness and inflammation. Many patients report using alternative therapies to self-treat asthma symptoms as adjuncts to short-acting and long-acting β-agonists and inhaled corticosteroids (ICS). As many as 40% of patients with asthma use herbal therapies to manage asthma symptoms, often without proven efficacy or known mechanisms of action. Therefore, investigations of both the therapeutic and possible detrimental effects of isolated components of herbal treatments on the airway are important. We hypothesized that ginger and its active components induce bronchodilation by modulating intracellular calcium ([Ca2+]i) in airway smooth muscle (ASM). In isolated human ASM, ginger caused significant and rapid relaxation. Four purified constituents of ginger were subsequently tested for ASM relaxant properties in both guinea pig and human tracheas: [6]-gingerol, [8]-gingerol, and [6]-shogaol induced rapid relaxation of precontracted ASM (100–300 μM), whereas [10]-gingerol failed to induce relaxation. In human ASM cells, exposure to [6]-gingerol, [8]-gingerol, and [6]-shogaol, but not [10]-gingerol (100 μM), blunted subsequent Ca2+ responses to bradykinin (10 μM) and S-(−)-Bay K 8644 (10 μM). In A/J mice, the nebulization of [8]-gingerol (100 μM), 15 minutes before methacholine challenge, significantly attenuated airway resistance, compared with vehicle. Taken together, these novel data show that ginger and its isolated active components, [6]-gingerol, [8]-gingerol, and [6]-shogaol, relax ASM, and [8]-gingerol attenuates airway hyperresponsiveness, in part by altering [Ca2+]i regulation. These purified compounds may provide a therapeutic option alone or in combination with accepted therapeutics, including β2-agonists, in airway diseases such as asthma. PMID:23065130

  14. Protein Kinase C as Regulator of Vascular Smooth Muscle Function and Potential Target in Vascular Disorders.

    PubMed

    Ringvold, H C; Khalil, R A

    2017-01-01

    Vascular smooth muscle (VSM) plays an important role in maintaining vascular tone. In addition to Ca(2+)-dependent myosin light chain (MLC) phosphorylation, protein kinase C (PKC) is a major regulator of VSM function. PKC is a family of conventional Ca(2+)-dependent α, β, and γ, novel Ca(2+)-independent δ, ɛ, θ, and η, and atypical ξ, and ι/λ isoforms. Inactive PKC is mainly cytosolic, and upon activation it undergoes phosphorylation, maturation, and translocation to the surface membrane, the nucleus, endoplasmic reticulum, and other cell organelles; a process facilitated by scaffold proteins such as RACKs. Activated PKC phosphorylates different substrates including ion channels, pumps, and nuclear proteins. PKC also phosphorylates CPI-17 leading to inhibition of MLC phosphatase, increased MLC phosphorylation, and enhanced VSM contraction. PKC could also initiate a cascade of protein kinases leading to phosphorylation of the actin-binding proteins calponin and caldesmon, increased actin-myosin interaction, and VSM contraction. Increased PKC activity has been associated with vascular disorders including ischemia-reperfusion injury, coronary artery disease, hypertension, and diabetic vasculopathy. PKC inhibitors could test the role of PKC in different systems and could reduce PKC hyperactivity in vascular disorders. First-generation PKC inhibitors such as staurosporine and chelerythrine are not very specific. Isoform-specific PKC inhibitors such as ruboxistaurin have been tested in clinical trials. Target delivery of PKC pseudosubstrate inhibitory peptides and PKC siRNA may be useful in localized vascular disease. Further studies of PKC and its role in VSM should help design isoform-specific PKC modulators that are experimentally potent and clinically safe to target PKC in vascular disease. © 2017 Elsevier Inc. All rights reserved.

  15. Fibroblast Growth Factor 12 Is a Novel Regulator of Vascular Smooth Muscle Cell Plasticity and Fate.

    PubMed

    Song, Sun-Hwa; Kim, Kyungjong; Jo, Eun-Kyung; Kim, Young-Wook; Kwon, Jin-Sook; Bae, Sun Sik; Sung, Jong-Hyuk; Park, Sang Gyu; Kim, Jee Taek; Suh, Wonhee

    2016-09-01

    Vascular smooth muscle cells (VSMCs) modulate their phenotype between synthetic and contractile states in response to environmental changes; this modulation plays a crucial role in the pathogenesis of restenosis and atherosclerosis. Here, we identified fibroblast growth factor 12 (FGF12) as a novel key regulator of the VSMC phenotype switch. Using murine models and human specimens, we found that FGF12 was highly expressed in contractile VSMCs of normal vessel walls but was downregulated in synthetic VSMCs from injured and atherosclerotic vessels. In human VSMCs, FGF12 expression was inhibited at the transcriptional level by platelet-derived growth factor-BB. Gain- and loss-of-function experiments showed that FGF12 was both necessary and sufficient for inducing and maintaining the quiescent and contractile phenotypes of VSMCs. FGF12 inhibited cell proliferation through the p53 pathway and upregulated the key factors involved in VSMC lineage differentiation, such as myocardin and serum response factor. Such FGF12-induced phenotypic change was mediated by the p38 MAPK (mitogen-activated protein kinase) pathway. Moreover, FGF12 promoted the differentiation of mouse embryonic stem cells and the transdifferentiation of human dermal fibroblasts into SMC-like cells. Furthermore, adenoviral infection of FGF12 substantially decreased neointima hyperplasia in a rat carotid artery injury model. In general, FGF family members induce a synthetic VSMC phenotype. Interestingly, the present study showed the unanticipated finding that FGF12 belonging to FGF family, strongly induced the quiescent and contractile VSMC phenotypes and directly promoted VSMC lineage differentiation. These novel findings suggested that FGF12 could be a new therapeutic target for treating restenosis and atherosclerosis. © 2016 American Heart Association, Inc.

  16. Mechanism regulating proasthmatic effects of prolonged homologous β2-adrenergic receptor desensitization in airway smooth muscle

    PubMed Central

    Nino, Gustavo; Hu, Aihua; Grunstein, Michael M.

    2009-01-01

    Use of long-acting β2-adrenergic receptor (β2AR) agonists to treat asthma incurs an increased risk of asthma morbidity with impaired bronchodilation and heightened bronchoconstriction, reflecting the adverse effects of prolonged homologous β2AR desensitization on airway smooth muscle (ASM) function. Since phosphodiesterase 4 (PDE4) regulates ASM relaxation and contractility, we examined whether the changes in ASM function induced by prolonged homologous β2AR desensitization are attributed to altered expression and action of PDE4. Cultured human ASM cells and isolated rabbit ASM tissues exposed for 24 h to the long-acting β2AR agonist salmeterol exhibited impaired acute β2AR-mediated cAMP accumulation and relaxation, respectively, together with ASM constrictor hyperresponsiveness. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity due to enhanced expression of the PDE4D5 isoform and were prevented by pretreating the ASM preparations with the PDE4 inhibitor rolipram or with inhibitors of either PKA or ERK1/2 signaling. Extended studies using gene silencing and pharmacological approaches demonstrated that: 1) the mechanism underlying upregulated PDE4D5 expression following prolonged β2AR agonist exposure involves PKA-dependent activation of Gi protein signaling via its βγ-subunits, which elicits downstream activation of ERK1/2 and its induction of PDE4D5 transcription; and 2) the induction of PDE4 activity and consequent changes in ASM responsiveness are prevented by pretreating the β2AR agonist-exposed ASM preparations with inhibitors of Gi-βγ signaling. Collectively, these findings identify that the proasthmatic changes in ASM function resulting from prolonged homologous β2AR desensitization are attributed to upregulated PDE4 expression induced by Gi-βγ-mediated cross-talk between the PKA and ERK1/2 signaling pathways. PMID:19666775

  17. Functional regulation of ClC-3 in the migration of vascular smooth muscle cells.

    PubMed

    Ganapathi, Sindura B; Wei, Shun-Guang; Zaremba, Angelika; Lamb, Fred S; Shears, Stephen B

    2013-01-01

    Migration of vascular smooth muscle cells (VSMCs) into neointima contributes to atherosclerosis and restenosis. This migration requires coordinated plasmalemmal fluxes of water and ions. Here, we show that aortic VSMC migration depends on the regulation of transmembrane Cl(-) flux by ClC-3, a Cl(-) channel/transporter. The contribution of ClC-3 to plasmalemmal Cl(-) current was studied in VSMCs by electrophysiological recordings. Cl(-) current was negligible in cells perfused with 0 [Ca(2+)]. Raising intracellular [Ca(2+)] to 0.5 μM activated a Cl(-) current (I(Cl.Ca)), approximately half of which was eliminated on inhibition by KN-93 of calmodulin-dependent protein kinase II. I(Cl.Ca) was also halved by inositol-3,4,5,6-tetrakisphosphate, a cellular signal with the biological function of specifically preventing calmodulin-dependent protein kinase II from activating I(Cl.Ca). Gene disruption of ClC-3 reduced I(Cl.Ca) by 50%. Moreover, I(Cl.Ca) in the ClC-3 null VSMCs was not affected by either KN-93 or inositol-3,4,5,6-tetrakisphosphate. We conclude that I(Cl.Ca) is composed of 2 components, one is ClC-3 independent whereas the other is ClC-3 dependent, activated by calmodulin-dependent protein kinase II and inhibited by inositol-3,4,5,6-tetrakisphosphate. We also assayed VSMC migration in transwell assays. Migration was halved in ClC-3 null cells versus wild-type cells. In addition, inhibition of ClC-3 by niflumic acid, KN-93, or inositol-3,4,5,6-tetrakisphosphate each reduced cell migration in wild-type cells but not in ClC-3 null cells. These cell-signaling roles of ClC-3 in VSMC migration suggest new therapeutic approaches to vascular remodeling diseases.

  18. UAP56 is a novel interacting partner of Bcr in regulating vascular smooth muscle cell DNA synthesis

    SciTech Connect

    Sahni, Abha; Wang, Nadan; Alexis, Jeffrey D.

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer UAP56 is an important regulator of DNA synthesis in vascular smooth muscle cells. Black-Right-Pointing-Pointer UAP56 binds to Bcr. Black-Right-Pointing-Pointer Interaction between Bcr and UAP56 is critical for Bcr induced DNA synthesis. -- Abstract: Bcr is a serine/threonine kinase that is a critical regulator of vascular smooth muscle cell inflammation and proliferation. We have previously demonstrated that Bcr acts in part via phosphorylation and inhibition of PPAR{gamma}. We have identified the RNA helicase UAP56 as another substrate of Bcr. In this report we demonstrate that knockdown of UAP56 blocks Bcr induced DNA synthesis in vascular smooth muscle cells (VSMC). We also found that over expression of Bcr increased the expression of cyclin E and decreased the expression of p27. Knockdown of UAP56 reversed the effect of Bcr on cyclin E and p27 expression. Furthermore, we found that Bcr binds to UAP56 and demonstrate that binding of UAP56 to Bcr is critical for Bcr induced DNA synthesis in VSMC. Our data identify UAP56 as an important binding partner of Bcr and a novel target for inhibiting vascular smooth muscle cell proliferation.

  19. Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels

    PubMed Central

    Lee, Moon Young; Park, Chanjae; Ha, Se Eun; Park, Paul J.; Berent, Robyn M.; Jorgensen, Brian G.; Corrigan, Robert D.; Grainger, Nathan; Blair, Peter J.; Slivano, Orazio J.; Miano, Joseph M.; Ward, Sean M.; Smith, Terence K.; Sanders, Kenton M.

    2017-01-01

    Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract. PMID:28152551

  20. Regulation of Contractile Responses of Vascular Smooth Muscle Cells under Conditions of Hypoxia-Reoxygenation.

    PubMed

    Gusakova, S V; Birulina, Yu G; Smagliy, L V; Kovalev, I V; Petrova, I V; Nosarev, A V; Orlov, S N

    2016-12-01

    We analyzed the effects of hypoxia and reoxygenation on changes in contractile activity in rat aortic smooth muscles. Both hypoxia and reoxygenation induced relaxation of smooth muscle cells precontracted with high-potassium Krebs solution (30 mM KCl) or α1-adrenoceptor agonist phenylephrine. Vasodilation resulted from enhancement of potassium permeability of smooth muscle cell membranes caused by activation of voltage-gated potassium channels (triggered by both precontracting agents) or by opening of ATP-sensitive potassium channels (phenylephrine). In isolated smooth muscle cells, both hypoxia and inhibition of Na(+),K(+)-ATPase with ouabain led to depletion of intracellular store of macroergic substances, reduced potassium concentration, and elevated the content of sodium ions.

  1. Substance P Regulates Environmental Tobacco Smoke-Enhanced Tracheal Smooth Muscle Responsiveness in Mice

    PubMed Central

    Xiao, Lan; Wu, Zhong-Xin

    2012-01-01

    Environmental tobacco smoke (ETS) is an environmental trigger that leads to airway inflammation and airway hyperresponsiveness (AHR) in susceptible individuals and animals, but the underlying mechanism is not fully understood. Substance P (SP) release from sensory nerve fibers has been linked to AHR. The present experiments characterize the role of SP in tracheal smooth muscle on ETS-increased airway responses. The mice were exposed to either sidestream tobacco smoke (SS), a surrogate to ETS, or filtered air (FA) for 1 day or 5 consecutive days. Contractions of tracheal smooth muscle to SP and electrical field stimulation (EFS) were not significantly altered in 1 of day SS-exposed mice. However, 5 of days SS exposure significantly increased airway smooth muscle contractions to SP and EFS. Administration of CP-99994, an antagonist of the neurokinin (NK)1 receptor, attenuates the SS exposure-enhanced tracheal smooth muscle responses to EFS. Furthermore, the immunohistochemistry showed that SP nerve fibers were increased in tracheal smooth muscle after 5 of days SS exposure. These results suggest that the increased SP production may contribute to SS-enhanced smooth muscle responsiveness in mice trachea. PMID:22927867

  2. ADP-Ribosyl cyclase in rat vascular smooth muscle cells: properties and regulation.

    PubMed

    de Toledo, F G; Cheng, J; Liang, M; Chini, E N; Dousa, T P

    2000-06-09

    We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 micromol/L) GT(1B), GD(1), and GM(3). Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by N-endoglycosidase F and to thermal inactivation at 45 degrees C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn(2+) and Cu(2+) ions; the inhibition by Zn(2+) was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn(2+) stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all-trans retinoic acid (atRA), and panagonist 9-cis-retinoic acid-upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH)(2)-Vitamin D(3) (calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED(50) congruent with 56 pmol/L). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3'-triiodothyronine (T(3)) to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+89%), but not in liver or

  3. Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension.

    PubMed

    An, Changlong; Bhetwal, Bhupal P; Sanders, Kenton M; Somlyo, Avril V; Perrino, Brian A

    2015-01-01

    Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to

  4. microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart

    PubMed Central

    Heidersbach, Amy; Saxby, Chris; Carver-Moore, Karen; Huang, Yu; Ang, Yen-Sin; de Jong, Pieter J; Ivey, Kathryn N; Srivastava, Deepak

    2013-01-01

    microRNA-1 (miR-1) is an evolutionarily conserved, striated muscle-enriched miRNA. Most mammalian genomes contain two copies of miR-1, and in mice, deletion of a single locus, miR-1-2, causes incompletely penetrant lethality and subtle cardiac defects. Here, we report that deletion of miR-1-1 resulted in a phenotype similar to that of the miR-1-2 mutant. Compound miR-1 knockout mice died uniformly before weaning due to severe cardiac dysfunction. miR-1-null cardiomyocytes had abnormal sarcomere organization and decreased phosphorylation of the regulatory myosin light chain-2 (MLC2), a critical cytoskeletal regulator. The smooth muscle-restricted inhibitor of MLC2 phosphorylation, Telokin, was ectopically expressed in the myocardium, along with other smooth muscle genes. miR-1 repressed Telokin expression through direct targeting and by repressing its transcriptional regulator, Myocardin. Our results reveal that miR-1 is required for postnatal cardiac function and reinforces the striated muscle phenotype by regulating both transcriptional and effector nodes of the smooth muscle gene expression network. DOI: http://dx.doi.org/10.7554/eLife.01323.001 PMID:24252873

  5. Atherosclerosis-prone hemodynamics differentially regulates endothelial and smooth muscle cell phenotypes and promotes pro-inflammatory priming.

    PubMed

    Hastings, Nicole E; Simmers, Michael B; McDonald, Oliver G; Wamhoff, Brian R; Blackman, Brett R

    2007-12-01

    Atherosclerosis is an inflammatory disease that preferentially forms at hemodynamically compromised regions of altered shear stress patterns. Endothelial cells (EC) and smooth muscle cells (SMC) undergo phenotypic modulation during atherosclerosis. An in vitro coculture model was developed to determine the role of hemodynamic regulation of EC and SMC phenotypes in coculture. Human ECs and SMCs were plated on a synthetic elastic lamina and human-derived atheroprone, and atheroprotective shear stresses were imposed on ECs. Atheroprone flow decreased genes associated with differentiated ECs (endothelial nitric oxide synthase, Tie2, and Kruppel-like factor 2) and SMCs (smooth muscle alpha-actin and myocardin) and induced a proinflammatory phenotype in ECs and SMCs (VCAM-1, IL-8, and monocyte chemoattractant protein-1). Atheroprone flow-induced changes in SMC differentiation markers were regulated at the chromatin level, as indicated by decreased serum response factor (SRF) binding to the smooth muscle alpha-actin-CC(a/T)(6)GG (CArG) promoter region and decreased histone H(4) acetylation. Conversely, SRF and histone H(4) acetylation were enriched at the c-fos promoter in SMCs. In the presence of atheroprotective shear stresses, ECs aligned with the direction of flow and SMCs aligned more perpendicular to flow, similar to in vivo vessel organization. These results provide a novel mechanism whereby modulation of the EC phenotype by hemodynamic shear stresses, atheroprone or atheroprotective, play a critical role in mechanical-transcriptional coupling and regulation of the SMC phenotype.

  6. Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles.

    PubMed

    Gerthoffer, William T

    2005-05-01

    Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.

  7. The role of K⁺ conductances in regulating membrane excitability in human gastric corpus smooth muscle.

    PubMed

    Lee, Ji Yeon; Ko, Eun-Ju; Ahn, Ki Duck; Kim, Sung; Rhee, Poong-Lyul

    2015-04-01

    Changes in resting membrane potential (RMP) regulate membrane excitability. K(+) conductance(s) are one of the main factors in regulating RMP. The functional role of K(+) conductances has not been studied the in human gastric corpus smooth muscles (HGCS). To examine the role of K(+) channels in regulation of RMP in HGCS we employed microelectrode recordings, patch-clamp, and molecular approaches. Tetraethylammonium and charybdotoxin did not affect the RMP, suggesting that BK channels are not involved in regulating RMP. Apamin, a selective small conductance Ca(2+)-activated K(+) channel (SK) blocker, did not show a significant effect on the membrane excitability. 4-Aminopyridine, a Kv channel blocker, caused depolarization and increased the duration of slow wave potentials. 4-Aminopyridine also inhibited a delayed rectifying K(+) current in isolated smooth muscle cells. End-product RT-PCR gel detected Kv1.2 and Kv1.5 in human gastric corpus muscles. Glibenclamide, an ATP-sensitive K(+) channel (KATP) blocker, did not induce depolarization, but nicorandil, a KATP opener, hyperpolarized HGCS, suggesting that KATP are expressed but not basally activated. Kir6.2 transcript, a pore-forming subunit of KATP was expressed in HGCS. A low concentration of Ba(2+), a Kir blocker, induced strong depolarization. Interestingly, Ba(2+)-sensitive currents were minimally expressed in isolated smooth muscle cells under whole-cell patch configuration. KCNJ2 (Kir2.1) transcript was expressed in HGCS. Unique K(+) conductances regulate the RMP in HGCS. Delayed and inwardly rectifying K(+) channels are the main candidates in regulating membrane excitability in HGCS. With the development of cell dispersion techniques of interstitial cells, the cell-specific functional significance will require further analysis.

  8. Negative-feedback regulation of excitation-contraction coupling in gastric smooth muscle.

    PubMed

    Ozaki, H; Zhang, L; Buxton, I L; Sanders, K M; Publicover, N G

    1992-12-01

    The role of phosphatidylinositol (PI) turnover in excitation-contraction coupling was investigated in canine antral smooth muscle. Acetylcholine (ACh; 0.1-1 microM) transiently increased tissue levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and increased the amplitudes of the plateau phase of slow waves and associated Ca2+ transients and phasic contractions. ACh also increased basal concentrations of cytosolic Ca2+ ([Ca2+]c), but these changes were not associated with an increase in resting tension. ATP (0.3 mM) had similar effects on Ins(1,4,5)P3 levels, basal [Ca2+]c, and resting tension. However, in contrast to the effects of ACh, ATP transiently reduced the amplitude of the plateau phase of slow waves and reduced the amplitudes of associated Ca2+ transients and phasic contractions. We investigated the possibility that two products of PI turnover, diacylglycerol (DAG) and Ins(1,4,5)P3, might provide negative feedback to regulate Ca2+ entry during slow waves. 1) DAG is known to activate protein kinase C (PKC). Activation of PKC by phorbol 12,13-dibutyrate (PDBu, 0.5 microM) reduced the amplitude of the plateau phase of slow waves and corresponding Ca2+ transients and phasic contractions. Assay of PKC showed that ACh, ATP, and PDBu stimulated enzyme activity. 2) Ins(1,4,5)P3 is known to increase [Ca2+]c by release of Ca2+ from internal stores. Basal [Ca2+]c was also increased by elevated external K+, ionomycin, thapsigargin, or caffeine. Each of these compounds reduced the amplitude and duration of slow waves. Results suggest that products of PI turnover may provide negative-feedback control of Ca2+ influx during slow waves, tending to reduce the amplitude of phasic contractile activity in gastric muscles. Differences in responses to ACh and ATP can be explained by a G protein-dependent mechanism in which ACh suppresses the voltage dependence of Ca(2+)-activated K+ channels.

  9. Kir2.1 regulates rat smooth muscle cell proliferation, migration, and post-injury carotid neointimal formation

    SciTech Connect

    Qiao, Yong; Tang, Chengchun Wang, Qingjie; Wang, Dong; Yan, Gaoliang; Zhu, Boqian

    2016-09-02

    Phenotype switching of vascular smooth muscle cells (VSMC) from the contractile type to the synthetic type is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. Inward rectifier K{sup +} channel 2.1 (Kir2.1) has been identified in VSMC. However, whether it plays a functional role in regulating cellular transformation remains obscure. In this study, we evaluated the role of Kir2.1 on VSMC proliferation, migration, phenotype switching, and post-injury carotid neointimal formation. Kir2.1 knockdown significantly suppressed platelet-derived growth factor BB-stimulated rat vascular smooth muscle cells (rat-VSMC) proliferation and migration. Deficiency in Kir2.1 contributed to the restoration of smooth muscle α-actin, smooth muscle 22α, and calponin and to a reduction in osteopontin expression in rat-VSMC. Moreover, the in vivo study showed that rat-VSMC switched to proliferative phenotypes and that knockdown of Kir2.1 significantly inhibited neointimal formation after rat carotid injury. Kir2.1 may be a potential therapeutic target in the treatment of cardiovascular diseases, such as atherosclerosis and restenosis following percutaneous coronary intervention.

  10. Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension

    PubMed Central

    An, Changlong; Bhetwal, Bhupal P.; Sanders, Kenton M.; Somlyo, Avril V.; Perrino, Brian A.

    2015-01-01

    Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/WV mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/WV gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to

  11. Smooth muscle myosin regulation by serum and cell density in cultured rat lung connective tissue cells.

    PubMed

    Babij, P; Zhao, J; White, S; Woodcock-Mitchell, J; Mitchell, J; Absher, M; Baldor, L; Periasamy, M; Low, R B

    1993-08-01

    RNA and protein analyses were used to detect expression of SM1 and SM2 smooth muscle myosin heavy chain (MHC) in cultured adult rat lung connective tissue cells (RL-90). Smooth muscle MHC mRNA expression in confluent cells grown in 10% serum was approximately 50% of the level in adult stomach. Similar results were obtained in cells cultured at low density (25% confluency) in 1% serum. However, in low-density cultures transferred to 10% serum for 24 h, the level of MHC mRNA decreased to approximately 20% of that in adult stomach. Smooth muscle alpha-actin showed a pattern of expression similar to that for smooth muscle MHC. Expression of nonmuscle MHC-A mRNA was higher in all culture conditions compared to stomach. MHC-A mRNA expression was less in low-density cultures in low serum and increased when low-density cultures were transferred to 10% serum for 24 h. MHC-B mRNA expression was less in low- vs. high-density cultures. In contrast to MHC-A, however, MHC-B mRNA expression in low-density cultures was higher in low serum. Immunofluorescence and immunoblotting with SM1-specific antibody demonstrated the presence of the SM1 protein isoform as well as reactivity to a protein band migrating slightly faster than SM2. These results demonstrate that cultured rat lung connective tissue cells express smooth muscle MHC and that expression is modulated by culture conditions.

  12. Pre- and post-junctional bradykinin B2 receptors regulate smooth muscle tension to the pig intravesical ureter.

    PubMed

    Ribeiro, Ana S F; Fernandes, Vítor S; Martínez, María Pilar; López-Oliva, María Elvira; Barahona, María Victoria; Recio, Paz; Martínez, Ana Cristina; Blaha, Igor; Orensanz, Luis M; Bustamante, Salvador; García-Sacristán, Albino; Prieto, Dolores; Hernández, Medardo

    2016-01-01

    Neuronal and non-neuronal bradykinin (BK) receptors regulate the contractility of the bladder urine outflow region. The current study investigates the role of BK receptors in the regulation of the smooth muscle contractility of the pig intravesical ureter. Western blot and immunohistochemistry were used to show the expression of BK B1 and B2 receptors and myographs for isometric force recordings. B2 receptor expression was consistently detected in the intravesical ureter urothelium and smooth muscle layer, B1 expression was not detected where a strong B2 immunoreactivity was observed within nerve fibers among smooth muscle bundles. On ureteral strips basal tone, BK induced concentration-dependent contractions, were potently reduced by extracellular Ca(2+) removal and by B2 receptor and voltage-gated Ca(2+) (VOC) channel blockade. BK contraction did not change as a consequence of urothelium mechanical removal or cyclooxygenase and Rho-associated protein kinase inhibition. On 9,11-dideoxy-9a,11a-methanoepoxy prostaglandin F2α (U46619)-precontracted samples, under non-adrenergic non-cholinergic (NANC) and nitric oxide (NO)-independent NANC conditions, electrical field stimulation-elicited frequency-dependent relaxations which were reduced by B2 receptor blockade. Kallidin, a B1 receptor agonist, failed to increase preparation basal tension or to induce relaxation on U46619-induced tone. The present results suggest that BK produces contraction of pig intravesical ureter via smooth muscle B2 receptors coupled to extracellular Ca(2+) entry mainly via VOC (L-type) channels. Facilitatory neuronal B2 receptors modulating NO-dependent or independent NANC inhibitory neurotransmission are also demonstrated. © 2014 Wiley Periodicals, Inc.

  13. E3 ubiquitin ligase isolated by differential display regulates cervical cancer growth in vitro and in vivo via microRNA-143.

    PubMed

    Li, Jibin; Wang, Xinling; Zhang, Yanshang; Zhang, Yan

    2016-08-01

    Cervical cancer is one of the most common gynecological cancers worldwide. Aberrant expression of E3 ubiquitin ligase isolated by differential display (EDD) has been detected in various types of tumor and has been demonstrated to have an important role in carcinogenesis, tumor growth and drug resistance. However, the role of EDD in cervical cancer and its underlying molecular mechanisms remains unknown. The present study aimed to investigate the role of EDD in the tumorigenicity of cervical cancer. EDD expression levels were measured using reverse transcription-quantitative polymerase chain reaction and western blotting in SiHa, HeLa, CaSki, c-41 and c-33A cervical cancer cell lines and cervical cancer tissue specimens. A functional study was performed using cell proliferation, colony formation, cell apoptosis assays in vitro and tumor growth assays in vivo with EDD either overexpressed or silenced. In the present study, EDD expression levels were significantly upregulated in cervical cancer cell lines and tissue samples. EDD knockdown significantly inhibited colony formation, cell proliferation and tumor growth and accelerated cell apoptosis in the cervical cancer cell lines and tissue samples. Furthermore, microRNA (miR)-143 expression levels were low in cervical cancer tissue samples and were negatively correlated with EDD expression. miR-143 silencing eliminated the effect of EDD on cell proliferation, colony formation and cell apoptosis in the cervical cancer cells, which suggested that miR-143 is critical for EDD-mediated regulation of cervical cancer cell growth. The results of the present study indicated that EDD may promote cervical cancer growth in vivo and in vitro by targeting miR-143. In conclusion, EDD may have an oncogenic role in cervical cancer and may serve as a potential therapeutic target for the treatment of patients with cervical cancer.

  14. Alpha-tocopherol (vitamin E) regulates vascular smooth muscle cell proliferation and protein kinase C activity.

    PubMed

    Boscoboinik, D; Szewczyk, A; Azzi, A

    1991-04-01

    Alpha-Tocopherol (vitamin E) protects against free radical damage, which has been implicated in aging, cancer initiation, and atherosclerosis. We have found that physiological concentrations of alpha-tocopherol specifically inhibited aorta smooth muscle cell (VSMC, line A7r5) proliferation and protein kinase C (PKC) activity. Other water and lipid soluble antioxidants were inactive. alpha-Tocopherol inhibition of PKC and of VSMC proliferation may represent a physiological mechanism, relevant to the onset of diseased states such as atherosclerosis.

  15. Dopamine DA1 receptors on vascular smooth muscle cells are regulated by glucocorticoid and sodium chloride.

    PubMed

    Yasunari, K; Kohno, M; Yokokawa, K; Horio, T; Takeda, T

    1994-09-01

    The modulation of dopamine DA1 receptors of cultured rat renal arterial smooth muscle cells by glucocorticoid and sodium chloride was studied. At a concentration of 10 nM, the synthetic glucocorticoid dexamethasone increased maximum receptor binding but had no effect on the dissociation constant. However, the maximum binding of [3H]Sch-23390 in cells treated with 100 mM sodium chloride did not change. However, the dissociation constant for DA1 receptor was increased by adding sodium chloride. The glucocorticoid effect on DA1 of arterial smooth muscle cells became apparent after hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml) or by the glucocorticoid receptor antagonist RU-38486, indicating that the effect required protein synthesis through glucocorticoid receptors. Treatment of cells with 1 microM dexamethasone for 24 h increased basal and DA1-stimulated adenylate cyclase activity. Basal adenylate cyclase was decreased by sodium chloride in a dose-dependent manner. These results suggest differential control of DA1 receptors on vascular smooth muscle cells by glucocorticoid or sodium chloride.

  16. Benidipine, a calcium channel blocker, regulates proliferation and phenotype of vascular smooth muscle cells.

    PubMed

    Arakawa, Emi; Hasegawa, Kazuhide

    2006-02-01

    Hyperproliferation of phenotypically modified vascular smooth muscle cells (VSMCs) is one of the major factors in the development of atherosclerosis and restenosis. Previously it was demonstrated that benidipine, a dihydropyridine-calcium channel antagonist, reduced neointimal formation in a rat balloon-injury model. In the present study, we examined the effect of benidipine on the phenotypic modulation and proliferation of VSMCs, using primary cultures of rat VSMCs. In the absence of drug treatment, protein levels of the smooth muscle specific markers, such as smooth muscle myosin heavy chain-1 (SM1), calponin 1, and alpha-actin, decreased during culture. However, treatment of VSMCs with benidipine (3 - 10 micromol/L) for 1 week reversed the effect in a concentration-related manner so that high levels of marker proteins were maintained. The expression of calponin mRNAs was reduced markedly during 1-week culture, and treatment with benidipine (3 micromol/L) significantly inhibited the reduction. Treatment with benidipine for 2 days increased the level of p21 protein and partially reduced p70 S6 kinase 1 (p70S6K1) activity. These data suggest that benidipine may arrest the growth of VSMCs, thereby preventing cell dedifferentiation. These additional properties of benidipine suggest that the drug should provide useful therapy for atherosclerosis and restenosis.

  17. Advanced glycation endproducts regulate smooth muscle cells calcification in cultured HSMCs.

    PubMed

    He, Hu-Qiang; Liu, Yong; Zeng, Hong; Sun, Xiao-Lei; Zhang, Lei; Zhang, Xue-Lin; Liao, Wen-Jun; Zhou, Xiang-Yu; He, Yan-Zheng

    2015-01-01

    To investigate the mechanism of Advanced glycation end products (AGEs) promoting the calcification of smooth muscle cells. The successfully cultured smooth muscle cells were divided into three groups: normal culture group (group A), calcified culture group (group B), calcification + AGEs group (group C); the concentration of intracellular calcium ion was detected in each group; the promotion of AGEs on the calcification of HSMCs was confirmed by VON KOSSA staining; and the expressions of β-catenin, RAGE, β-catenin, OPG and E-cadherin protein were detected by immunofluorescence and western blot. The morphology of the cells in each group showed that the amount of calcified plaques in calcification + AGES group were significantly higher than the calcification group. VON KOSSA staining showed that with increasing concentrations of AGE-BSA, the amount of its calcification gradually increased. Calcium concentration in Calcification + 20 mg/L AGEs group was significantly higher, followed by 40 mg/L AGEs group. The expression of β-catenin increased with the increasing concentrations of AGEs. AGEs can promote the calcification of human femoral artery smooth muscle cells, with a concentration gradient effect. With increasing concentrations of AGEs, the expression of RAGE increased, indicating that AGEs-induced HSMCs proliferation was correlated with RAGE expression.

  18. Tbx18 Regulates the Differentiation of Periductal Smooth Muscle Stroma and the Maintenance of Epithelial Integrity in the Prostate

    PubMed Central

    Guimarães-Camboa, Nuno; Zhang, Huimin; Troy, Joseph M.; Lu, Xiaochen; Kispert, Andreas; Evans, Sylvia M.; Stubbs, Lisa

    2016-01-01

    The T-box transcription factor TBX18 is essential to mesenchymal cell differentiation in several tissues and Tbx18 loss-of-function results in dramatic organ malformations and perinatal lethality. Here we demonstrate for the first time that Tbx18 is required for the normal development of periductal smooth muscle stromal cells in prostate, particularly in the anterior lobe, with a clear impact on prostate health in adult mice. Prostate abnormalities are only subtly apparent in Tbx18 mutants at birth; to examine postnatal prostate development we utilized a relatively long-lived hypomorphic mutant and a novel conditional Tbx18 allele. Similar to the ureter, cells that fail to express Tbx18 do not condense normally into smooth muscle cells of the periductal prostatic stroma. However, in contrast to ureter, the periductal stromal cells in mutant prostate assume a hypertrophic, myofibroblastic state and the adjacent epithelium becomes grossly disorganized. To identify molecular events preceding the onset of this pathology, we compared gene expression in the urogenital sinus (UGS), from which the prostate develops, in Tbx18-null and wild type littermates at two embryonic stages. Genes that regulate cell proliferation, smooth muscle differentiation, prostate epithelium development, and inflammatory response were significantly dysregulated in the mutant urogenital sinus around the time that Tbx18 is first expressed in the wild type UGS, suggesting a direct role in regulating those genes. Together, these results argue that Tbx18 is essential to the differentiation and maintenance of the prostate periurethral mesenchyme and that it indirectly regulates epithelial differentiation through control of stromal-epithelial signaling. PMID:27120339

  19. Reversible interactions between smooth domains of the endoplasmic reticulum and mitochondria are regulated by physiological cytosolic Ca2+ levels.

    PubMed

    Goetz, Jacky G; Genty, Hélène; St-Pierre, Pascal; Dang, Thao; Joshi, Bharat; Sauvé, Rémy; Vogl, Wayne; Nabi, Ivan R

    2007-10-15

    The 3F3A monoclonal antibody to autocrine motility factor receptor (AMFR) labels mitochondria-associated smooth endoplasmic reticulum (ER) tubules. siRNA down-regulation of AMFR expression reduces mitochondria-associated 3F3A labelling. The 3F3A-labelled ER domain does not overlap with reticulon-labelled ER tubules, the nuclear membrane or perinuclear ER markers and only partially overlaps with the translocon component Sec61alpha. Upon overexpression of FLAG-tagged AMFR, 3F3A labelling is mitochondria associated, excluded from the perinuclear ER and co-distributes with reticulon. 3F3A labelling therefore defines a distinct mitochondria-associated ER domain. Elevation of free cytosolic Ca(2+) levels with ionomycin promotes dissociation of 3F3A-labelled tubules from mitochondria and, judged by electron microscopy, disrupts close contacts (<50 nm) between smooth ER tubules and mitochondria. The ER tubule-mitochondria association is similarly disrupted upon thapsigargin-induced release of ER Ca(2+) stores or purinergic receptor stimulation by ATP. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] receptor (IP3R) colocalises to 3F3A-labelled mitochondria-associated ER tubules, and conditions that induce ER tubule-mitochondria dissociation disrupt continuity between 3F3A- and IP3R-labelled ER domains. RAS-transformed NIH-3T3 cells have increased basal cytosolic Ca(2+) levels and show dissociation of the 3F3A-labelled, but not IP3R-labelled, ER from mitochondria. Our data indicate that regulation of the ER-mitochondria association by free cytosolic Ca(2+) is a characteristic of smooth ER domains and that multiple mechanisms regulate the interaction between these organelles.

  20. YAP is up-regulated in the bronchial airway smooth muscle of the chronic asthma mouse model

    PubMed Central

    Zhou, Jing; Xu, Fei; Yu, Jing Jing; Zhang, Wei

    2015-01-01

    Asthma is characterized by leukocytic infiltration and tissue remodeling with structural changes including subepithelial fibrosis and ASM cells proliferation. The Hippo pathway is a key regulatory point involved in cell proliferation, fibroblasts, and smooth muscle cell differentiation. In order to disclose the relation between asthma and the Hippo pathway, expression of the Yes-associated protein (YAP), a key gene in the Hippo pathway, in the bronchial smooth muscle of chronic asthma model (CAM) was studied. 40 mice were randomly divided into control (wide type) and experimental group to construct CAM using chicken ovalbumin (OVA). Pathological changes of the lung tissues were observed in the CAM mice compared with the control using HE staining method. Immunohistochemistry (IHC) was used to detect if YAP protein is expressed in the lung tissues. The pathological changes of the CAM group showed that a large number of inflammatory cells infiltration including mainly lymphocytes and a small amount of eosinophilic, with the presence of certain airway smooth muscle hyperplasia, was observed in comparison with the control. IHC results showed that the YAP protein was significantly increased compared with the control groups (P < 0.01). This result was further confirmed by quantitative real-time PCR (qPCR) assay which detected the up-regulation of the YAP gene (P < 0.01) and Western blot. In conclusion, the YAP protein was significantly expressed in the bronchial airway tissues of the CAM mice, and could be used as an indicator for asthma. PMID:26617833

  1. Hyperphosphatemia induces cellular senescence in human aorta smooth muscle cells through integrin linked kinase (ILK) up-regulation.

    PubMed

    Troyano, Nuria; Nogal, María Del; Mora, Inés; Diaz-Naves, Manuel; Lopez-Carrillo, Natalia; Sosa, Patricia; Rodriguez-Puyol, Diego; Olmos, Gemma; Ruiz-Torres, María P

    2015-12-01

    Aging is conditioned by genetic and environmental factors. Hyperphosphatemia is related to some pathologies, affecting to vascular cells behavior. This work analyze whether high concentration of extracellular phosphate induces vascular smooth muscle cells senescence, exploring the intracellular mechanisms and highlighting the in vivo relevance of this phenomenon. Human aortic smooth muscle cells treated with β-Glycerophosphate (BGP, 10mM) suffered cellular senescence by increasing p53, p21 and p16 expression and the senescence associated β-galactosidase activity. In parallel, BGP induced ILK overexpression, dependent on the IGF-1 receptor activation, and oxidative stress. Down-regulating ILK expression prevented BGP-induced senescence and oxidative stress. Aortic rings from young rats treated with 10mM BGP for 48h, showed increased p53, p16 and ILK expression and SA-β-gal activity. Seven/eight nephrectomized rats feeding a hyperphosphatemic diet and fifteenth- month old mice showed hyperphosphatemia and aortic ILK, p53 and p16 expression. In conclusion, we demonstrated that high extracellular concentration of phosphate induced senescence in cultured smooth muscle through the activation of IGF-1 receptor and ILK overexpression and provided solid evidences for the in vivo relevance of these results since aged animals showed high levels of serum phosphate linked to increased expression of ILK and senescence genes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Coronary artery disease associated transcription factor TCF21 regulates smooth muscle precursor cells that contribute to the fibrous cap

    PubMed Central

    Nurnberg, S.T.; Cheng, K.; Raiesdana, A.; Kundu, R.; Miller, C.L.; Kim, J.B.; Arora, K.; Carcamo-Oribe, I.; Xiong, Y.; Tellakula, N.; Nanda, V.; Murthy, N.; Boisvert, W.A.; Hedin, U.; Perisic, L.; Aldi, S.; Maegdefessel, L.; Pjanic, M.; Owens, G.K.; Tallquist, M.D.; Quertermous, T.

    2015-01-01

    TCF21 is a basic helix–loop–helix transcription factor that has recently been implicated as contributing to susceptibility to coronary heart disease based on genome wide association studies. In order to identify transcriptionally regulated target genes in a major disease relevant cell type, we performed siRNA knockdown of TCF21 in in vitro cultured human coronary artery smooth muscle cells and compared the transcriptome of siTCF21 versus siCONTROL treated cells. The raw (FASTQ) as well as processed (BED) data from 3 technical replicates per treatment has been deposited with Gene Expression Omnibus (GSE44461). PMID:26090325

  3. Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

    1998-01-01

    Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

  4. Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

    1998-01-01

    Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

  5. Role of M1 receptor in regulation of gastric fundus smooth muscle contraction.

    PubMed

    Gajdus, Marta; Szadujkis-Szadurska, Katarzyna; Szadujkis-Szadurski, Leszek; Glaza, Izabela; Szadujkis-Szadurski, Rafał; Olkowska, Joanna

    2011-09-14

    The subject of this study is determination of the influence of drugs on gastric fundus smooth muscle contraction induced by activation of muscarinic receptors M1. Experiments tested interactions between a receptor agonist, carbachol and muscarinic receptor antagonists, atropine and pirenzepine. Testing was conducted on tissues isolated from rat's stomach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the study are average values ± SE. In order to determine statistical significance, the principles of receptor theory were used (Kenakin modification). According to tests, carbachol, in concentrations ranging between 10(-8) M to 10(-4) M, in a dosage-dependent way induces gastric fundus smooth muscle contraction. Presented results indicate that carbachol meets the conditions posed to full agonists. On the other hand, atropine, a non-selective muscarinic receptor antagonist, causes a concentration-dependent shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that atropine meets the conditions posed to competitive antagonists. The use of pirenzepine, a competitive receptor agonist M1, causes shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. From the testing conducted on the preparation of the gastric fundus we can deduce that atropine causes shift of concentration-effect curves for carbachol to the right. A similar effect is released by pirenzepine, selectively blocking muscarinic receptors of M1 type. The results indicate that in the preparation of the gastric fundus smooth muscle, M1 type receptors occur also postsynaptically.

  6. Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2.

    PubMed

    Shu, Zhouwu; Yu, Min; Zeng, Guoning; Zhang, Xin; Wu, Libiao; Tan, Xuerui

    2014-04-01

    Previous studies have shown that epigallocatechin-3-gallate (EGCG) inhibits the proliferation of vascular smooth muscle cells (VSMCs) via the extracellular-signal-regulated kinase (ERK1/2) and mitogen activated protein kinases (MAPKs) pathway. Mitofusin 2 (Mfn-2) also suppresses VSMC proliferation through Ras-Raf-ERK/MAPK, suggesting a possible link between EGCG, Mfn-2 and ERK/MAPK. However, the effect of EGCG on Mfn-2 remains unknown. In this study, we investigated the role of Mfn-2 in the regulation of VSMC proliferation by EGCG, and assessed the underlying mechanisms. The effects of EGCG on the proliferation of cultured human aortic smooth muscle cells (HASMCs) were observed by 5-ethynl-2-deoxyuridine (EdU) incorporation assay. Mfn-2 gene and protein levels, and Ras, p-c-Raf and p-ERK1/2 protein levels were determined by quantitative real-time polymerase chain reaction and western blotting, respectively. Mfn-2 gene silencing was achieved by RNA interference. EGCG 50 μmol/L profoundly inhibited the proliferation of HASMCs in culture, up-regulated Mfn-2, and down-regulated the expression of p-c-Raf and p-ERK1/2. Furthermore, RNA interference-mediated gene knockdown of Mfn-2 antagonized EGCG-induced anti-proliferation and down-regulation of Ras, p-c-Raf and p-ERK1/2. These results suggest that EGCG inhibits the proliferation of HASMCs in vitro largely via Mfn-2-mediated suppression of the Ras-Raf-ERK/MAPK signaling pathway. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. [mRNA-binding protein Human-antigen R regulates α-SMA expression in human bronchia smooth muscle cells].

    PubMed

    Yan, Di; Gu, Xianmin; Jiang, Shujuan; Wang, Yuhong

    2015-10-13

    To investigate the role of mRNA binding protein Human-antigen R (HuR) in the over-expression of α-Smooth muscle actin (α-SMA) stimulated by Platelet-derived Growth Factor (PDGF) in cultured human bronchia smooth muscle cells. Human bronchia smooth muscle cells cultured in vitro were divided into 0, 6, 12 and 24 h groups according to the time of PDGF treatment. Total HuR protein and total α-SMA protein expression were detected by Western blot. Total HuR mRNA and total α-SMA mRNA level were determined by quantitative real time-polymerase chain reaction. RNA interference technology was used to down-regulate HuR protein level to study the protective effect of HuR in PDGF-stimulated α-SMA protein expression. PDGF up-regulated the expression of HuR in a time-dependent manner. The relative expression levels of whole-cell HuR protein and mRNA in 0, 6, 12, 24 h groups were 0.23±0.09, 0.42±0.11, 0.93±0.21, 1.37±0.28; 1.00±0.00, 1.09±0.03, 1.16±0.03, 1.27±0.02 (all P<0.05). The relative expression levels of α-SMA protein and mRNA in 0, 6, 12, 24 h group also showed an increase trend marked in a time-dependent manner (1.03±0.08, 1.20±0.09, 1.39±0.11, 1.58±0.10; 1.00±0.00, 1.17±0.02, 1.23±0.02, 1.45±0.03; all P<0.05). Using RNA interference technology to down-regulate HuR protein level, there was a decrease in α-SMA protein expression. PDGF stimulation can increase the expression of HuR and α-SMA in the smooth muscle cells, and HuR protein is involved in the expression of α-SMA protein stimulated by PDGF.

  8. Differential regulation of muscarinic M2 and M3 receptor signaling in gastrointestinal smooth muscle by caveolin-1.

    PubMed

    Bhattacharya, Sayak; Mahavadi, Sunila; Al-Shboul, Othman; Rajagopal, Senthilkumar; Grider, John R; Murthy, Karnam S

    2013-08-01

    Caveolae act as scaffolding proteins for several G protein-coupled receptor signaling molecules to regulate their activity. Caveolin-1, the predominant isoform in smooth muscle, drives the formation of caveolae. The precise role of caveolin-1 and caveolae as scaffolds for G protein-coupled receptor signaling and contraction in gastrointestinal muscle is unclear. Thus the aim of this study was to examine the role of caveolin-1 in the regulation of Gq- and Gi-coupled receptor signaling. RT-PCR, Western blot, and radioligand-binding studies demonstrated the selective expression of M2 and M3 receptors in gastric smooth muscle cells. Carbachol (CCh) stimulated phosphatidylinositol (PI) hydrolysis, Rho kinase and zipper-interacting protein (ZIP) kinase activity, induced myosin phosphatase 1 (MYPT1) phosphorylation (at Thr(696)) and 20-kDa myosin light chain (MLC20) phosphorylation (at Ser(19)) and muscle contraction, and inhibited cAMP formation. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activity, phosphorylation of MYPT1 and MLC20, and muscle contraction in response to CCh were attenuated by methyl β-cyclodextrin (MβCD) or caveolin-1 small interfering RNA (siRNA). Similar inhibition of PI hydrolysis, Rho kinase, and ZIP kinase activity and muscle contraction in response to CCh and gastric emptying in vivo was obtained in caveolin-1-knockout mice compared with wild-type mice. Agonist-induced internalization of M2, but not M3, receptors was blocked by MβCD or caveolin-1 siRNA. Stimulation of PI hydrolysis, Rho kinase, and ZIP kinase activities in response to other Gq-coupled receptor agonists such as histamine and substance P was also attenuated by MβCD or caveolin-1 siRNA. Taken together, these results suggest that caveolin-1 facilitates signaling by Gq-coupled receptors and contributes to enhanced smooth muscle function.

  9. miR-128 regulates differentiation of hair follicle mesenchymal stem cells into smooth muscle cells by targeting SMAD2.

    PubMed

    Wang, Zhihao; Pang, Li; Zhao, Huiying; Song, Lei; Wang, Yuehui; Sun, Qi; Guo, Chunjie; Wang, Bin; Qin, Xiujiao; Pan, Aiqun

    2016-05-01

    Human hair follicle mesenchymal stem cells (hHFMSCs) are an important source of cardiovascular tissue engineering for their differentiation potential into smooth muscle cells (SMCs), yet the molecular pathways underlying such fate determination is unclear. MicroRNAs (miRNAs) are non-coding RNAs that play critical roles in cell differentiation. In present study, we found that miR-128 was remarkably decreased during the differentiation of hHFMSCs into SMCs induced by transforming growth factor-β1 (TGF-β1). Moreover, overexpression of miR-128 led to decreased expression of SMC cellular marker proteins, such as smooth muscle actin (SMA) and calponin, in TGF-β1-induced SMC differentiation. Further, we identified that miR-128 targeted the 3'-UTR of SMAD2 transcript for translational inhibition of SMAD2 protein, and knockdown of SMAD2 abrogated the promotional effect of antagomir-128 (miR-128 neutralizer) on SMC differentiation. These results suggest that miR-128 regulates the differentiation of hHFMSCs into SMCs via targeting SMAD2, a main transcription regulator in TGF-β signaling pathway involving SMC differentiation. The miR-128/SMAD2 axis could therefore be considered as a candidate target in tissue engineering and regenerative medicine for SMCs.

  10. DHEA attenuates PDGF-induced phenotypic proliferation of vascular smooth muscle A7r5 cells through redox regulation

    SciTech Connect

    Urata, Yoshishige; Goto, Shinji; Kawakatsu, Miho; Yodoi, Junji; Eto, Masato; Akishita, Masahiro; Kondo, Takahito

    2010-05-28

    It is known that dehydroepiandrosterone (DHEA) inhibits a phenotypic switch in vascular smooth muscle cells (VSMC) induced by platelet-derived growth factor (PDGF)-BB. However, the mechanism behind the effect of DHEA on VSMC is not clear. Previously we reported that low molecular weight-protein tyrosine phosphatase (LMW-PTP) dephosphorylates PDGF receptor (PDGFR)-{beta} via a redox-dependent mechanism involving glutathione (GSH)/glutaredoxin (GRX)1. Here we demonstrate that the redox regulation of PDGFR-{beta} is involved in the effect of DHEA on VSMC. DHEA suppressed the PDGF-BB-dependent phosphorylation of PDGFR-{beta}. As expected, DHEA increased the levels of GSH and GRX1, and the GSH/GRX1 system maintained the redox state of LMW-PTP. Down-regulation of the expression of LMW-PTP using siRNA restored the suppression of PDGFR-{beta}-phosphorylation by DHEA. A promoter analysis of GRX1 and {gamma}-glutamylcysteine synthetase ({gamma}-GCS), a rate-limiting enzyme of GSH synthesis, showed that DHEA up-regulated the transcriptional activity at the peroxisome proliferator-activated receptor (PPAR) response element, suggesting PPAR{alpha} plays a role in the induction of GRX1 and {gamma}-GCS expression by DHEA. In conclusion, the redox regulation of PDGFR-{beta} is involved in the suppressive effect of DHEA on VSMC proliferation through the up-regulation of GSH/GRX system.

  11. Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells*

    PubMed Central

    Baeten, Jeremy T.; Lilly, Brenda

    2015-01-01

    Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression. PMID:25957400

  12. Calcium regulation of vascular smooth muscle cell-derived matrix vesicles.

    PubMed

    Kapustin, Alexander N; Shanahan, Catherine M

    2012-07-01

    Vascular calcification is a pathological process common in patients with disorders of mineral metabolism and mediated by vascular smooth muscle cells (VSMCs). A key event in the initiation of VSMC calcification is the release of mineralization-competent matrix vesicles (MVs), small membrane-bound bodies with structural features enabling them to efficiently nucleate hydroxyapatite. These bodies are similar to MVs secreted by chondrocytes during bone development and their properties include the absence of calcification inhibitors, formation of nucleation sites, and accumulation of matrix metalloproteinases such as MMP-2. The mechanisms of MV biogenesis and loading remain poorly understood; however, emerging data have demonstrated that alterations in cytosolic calcium homeostasis can trigger multiple changes in MV composition that promote their mineralization.

  13. Force maintenance and myosin filament assembly regulated by Rho-kinase in airway smooth muscle.

    PubMed

    Lan, Bo; Deng, Linhong; Donovan, Graham M; Chin, Leslie Y M; Syyong, Harley T; Wang, Lu; Zhang, Jenny; Pascoe, Christopher D; Norris, Brandon A; Liu, Jeffrey C-Y; Swyngedouw, Nicholas E; Banaem, Saleha M; Paré, Peter D; Seow, Chun Y

    2015-01-01

    Smooth muscle contraction can be divided into two phases: the initial contraction determines the amount of developed force and the second phase determines how well the force is maintained. The initial phase is primarily due to activation of actomyosin interaction and is relatively well understood, whereas the second phase remains poorly understood. Force maintenance in the sustained phase can be disrupted by strains applied to the muscle; the strain causes actomyosin cross-bridges to detach and also the cytoskeletal structure to disassemble in a process known as fluidization, for which the underlying mechanism is largely unknown. In the present study we investigated the ability of airway smooth muscle to maintain force after the initial phase of contraction. Specifically, we examined the roles of Rho-kinase and protein kinase C (PKC) in force maintenance. We found that for the same degree of initial force inhibition, Rho-kinase substantially reduced the muscle's ability to sustain force under static conditions, whereas inhibition of PKC had a minimal effect on sustaining force. Under oscillatory strain, Rho-kinase inhibition caused further decline in force, but again, PKC inhibition had a minimal effect. We also found that Rho-kinase inhibition led to a decrease in the myosin filament mass in the muscle cells, suggesting that one of the functions of Rho-kinase is to stabilize myosin filaments. The results also suggest that dissolution of myosin filaments may be one of the mechanisms underlying the phenomenon of fluidization. These findings can shed light on the mechanism underlying deep inspiration induced bronchodilation.

  14. Estrogen-Induced Maldevelopment of the Penis Involves Down-Regulation of Myosin Heavy Chain 11 (MYH11) Expression, a Biomarker for Smooth Muscle Cell Differentiation1

    PubMed Central

    Okumu, L.A.; Bruinton, Sequoia; Braden, Tim D.; Simon, Liz; Goyal, Hari O.

    2012-01-01

    ABSTRACT Cavernous smooth muscle cells are essential components in penile erection. In this study, we investigated effects of estrogen exposure on biomarkers for smooth muscle cell differentiation in the penis. Neonatal rats received diethylstilbestrol (DES), with or without the estrogen receptor (ESR) antagonist ICI 182,780 (ICI) or the androgen receptor (AR) agonist dihydrotestosterone (DHT), from Postnatal Days 1 to 6. Tissues were collected at 7, 10, or 21 days of age. The smooth muscle cell biomarker MYH11 was studied in depth because microarray data showed it was significantly down-regulated, along with other biomarkers, in DES treatment. Quantitative real time-PCR and Western blot analyses showed 50%–80% reduction (P ≤ 0.05) in Myh11 expression in DES-treated rats compared to that in controls; and ICI and DHT coadministration mitigated the decrease. Temporally, from 7 to 21 days of age, Myh11 expression was onefold increased (P ≥ 0.05) in DES-treated rats versus threefold increased (P ≤ 0.001) in controls, implying the long-lasting inhibitory effect of DES on smooth muscle cell differentiation. Immunohistochemical localization of smooth muscle alpha actin, another biomarker for smooth muscle cell differentiation, showed fewer cavernous smooth muscle cells in DES-treated animals than in controls. Additionally, DES treatment significantly up-regulated Esr1 mRNA expression and suppressed the neonatal testosterone surge by 90%, which was mitigated by ICI coadministration but not by DHT coadministration. Collectively, results provided evidence that DES treatment in neonatal rats inhibited cavernous smooth muscle cell differentiation, as shown by down-regulation of MYH11 expression at the mRNA and protein levels and by reduced immunohistochemical staining of smooth muscle alpha actin. Both the ESR and the AR pathways probably mediate this effect. PMID:22976277

  15. Nitrotyrosylation of Ca2+ channels prevents c-Src kinase regulation of colonic smooth muscle contractility in experimental colitis.

    PubMed

    Ross, Gracious R; Kang, Minho; Shirwany, Najeeb; Malykhina, Anna P; Drozd, Mary; Akbarali, Hamid I

    2007-09-01

    Basal levels of c-Src kinase are known to regulate smooth muscle Ca(2+) channels. Colonic inflammation results in attenuated Ca(2+) currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase in experimental colitis. Ca(2+)-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K(+). The E(max) to CaCl(2) was significantly less in inflamed tissues (38.4 +/- 7.6%) than controls, indicative of reduced Ca(2+) influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], a selective Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD(2) from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/peroxynitrite combination, the maximal contraction to CaCl(2) was reduced by 46 +/- 7% in controls but unaffected in inflamed tissues (13 +/- 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca(2+) currents by 84.1 +/- 3.9% in normal but only 36.2 +/- 13% in inflamed tissues. Neither the Ca(2+) channel Ca(v)1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca(2+) channel protein expression but increased nitrotyrosylated-Ca(2+) channel proteins during inflammation. These data suggest that post-translational modification of Ca(2+) channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca(2+) influx.

  16. Basally activated nonselective cation currents regulate the resting membrane potential in human and monkey colonic smooth muscle

    PubMed Central

    Dwyer, Laura; Rhee, Poong-Lyul; Lowe, Vanessa; Zheng, Haifeng; Peri, Lauren; Ro, Seungil; Sanders, Kenton M.

    2011-01-01

    Resting membrane potential (RMP) plays an important role in determining the basal excitability of gastrointestinal smooth muscle. The RMP in colonic muscles is significantly less negative than the equilibrium potential of K+, suggesting that it is regulated not only by K+ conductances but by inward conductances such as Na+ and/or Ca2+. We investigated the contribution of nonselective cation channels (NSCC) to the RMP in human and monkey colonic smooth muscle cells (SMC) using voltage- and current-clamp techniques. Qualitative reverse transcriptase-polymerase chain reaction was performed to examine potential molecular candidates for these channels among the transient receptor potential (TRP) channel superfamily. Spontaneous transient inward currents and holding currents were recorded in human and monkey SMC. Replacement of extracellular Na+ with equimolar tetraethylammonium or Ca2+ with Mn2+ inhibited basally activated nonselective cation currents. Trivalent cations inhibited these channels. Under current clamp, replacement of extracellular Na+ with N-methyl-d-glucamine or addition of trivalent cations caused hyperpolarization. Three unitary conductances of NSCC were observed in human and monkey colonic SMC. Molecular candidates for basally active NSCC were TRPC1, C3, C4, C7, M2, M4, M6, M7, V1, and V2 in human and monkey SMC. Comparison of the biophysical properties of these TRP channels with basally active NSCC (bINSCC) suggests that TRPM4 and specific TRPC heteromultimer combinations may underlie the three single-channel conductances of bINSCC. In conclusion, these findings suggest that basally activated NSCC contribute to the RMP in human and monkey colonic SMC and therefore may play an important role in determining basal excitability of colonic smooth muscle. PMID:21566016

  17. Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo.

    PubMed

    Kim-Kaneyama, Joo-ri; Suzuki, Wataru; Ichikawa, Kiyoko; Ohki, Takahiro; Kohno, Yoko; Sata, Masataka; Nose, Kiyoshi; Shibanuma, Motoko

    2005-03-01

    Hic-5 is a focal adhesion protein belonging to the paxillin LIM family that shuttles in and out of the nucleus. In the present study, we examined the expression of Hic-5 among mouse tissues by immunohistochemistry and found its expression only in smooth-muscle cells in several tissues. This result is consistent with a previous report on adult human tissues and contradicts the relatively ubiquitous expression of paxillin, the protein most homologous to Hic-5. One factor characterizing smooth-muscle cells in vivo is a continuous exposure to mechanical stretching in the organs. To study the involvement of Hic-5 in cellular responses to mechanical stress, we exposed mouse embryo fibroblasts to a uni-axial cyclic stretching and found that Hic-5 was relocalized from focal adhesions to stress fibers through its C-terminal LIM domains during the stress. In sharp contrast to this, paxillin did not change its focal-adhesion-based localization. Of the factors tested, which included interacting partners of Hic-5, only CRP2 (an only-LIM protein expressed in vascular smooth-muscle cells) and GIT1 were, like Hic-5, localized to stress fibers during the cyclic stretching. Interestingly, Hic-5 showed a suppressive effect on the contractile capability of cells embedded in three-dimensional collagen gels, and the effect was further augmented when CRP2 co-localized with Hic-5 to fiber structures of those cells. These results suggested that Hic-5 was a mediator of tensional force, translocating directly from focal adhesions to actin stress fibers upon mechanical stress and regulating the contractile capability of cells in the stress fibers.

  18. The actin regulator zyxin reinforces airway smooth muscle and accumulates in airways of fatal asthmatics

    PubMed Central

    Blankman, Elizabeth; Jensen, Christopher C.; Krishnan, Ramaswamy; James, Alan L.; Elliot, John G.; Green, Francis H.; Liu, Jeffrey C.; Seow, Chun Y.; Park, Jin-Ah; Beckerle, Mary C.; Paré, Peter D.; Fredberg, Jeffrey J.; Smith, Mark A.

    2017-01-01

    Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms–and their failure in asthma–remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma. PMID:28278518

  19. Monocyte-expressed urokinase regulates human vascular smooth muscle cell migration in a coculture model.

    PubMed

    Kusch, Angelika; Tkachuk, Sergey; Lutter, Steffen; Haller, Hermann; Dietz, Rainer; Lipp, Martin; Dumler, Inna

    2002-01-01

    Interactions of vascular smooth muscle cells (VSMC) with monocytes recruited to the arterial wall at a site of injury, with resultant modulation of VSMC growth and migration, are central to the development of vascular intimal thickening. Urokinase-type plasminogen activator (uPA) expressed by monocytes is a potent chemotactic factor for VSMC and might serve for the acceleration of vascular remodeling. In this report, we demonstrate that coculture of human VSMC with freshly isolated peripheral blood-derived human monocytes results in significant VSMC migration that increases during the coculture period. Accordingly, VSMC adhesion was inhibited with similar kinetics. VSMC proliferation, however, was not affected and remained at the same basal level during the whole period of coculture. The increase of VSMC migration in coculture was equivalent to the uPA-induced migration of monocultured VSMC and was blocked by addition into coculture of soluble uPAR (suPAR). Analysis of uPA and uPAR expression in cocultured cells demonstrated that monocytes are a major source of uPA, whose expression increases in coculture five-fold, whereas VSMC display an increased expression of cell surface-associated uPAR. These findings indicate that upregulated uPA production by monocytes following vascular injury acts most likely as an endogenous activator of VSMC migration contributing to the remodeling of vessel walls.

  20. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

    PubMed

    Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C

    2015-12-01

    Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

  1. Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone

    PubMed Central

    Pearson, Helen; Britt, Rodney D.; Pabelick, Christine M.; Prakash, Y.S.; Amrani, Yassine; Pandya, Hitesh C.

    2016-01-01

    Background Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Methods Cultured fetal human ASM cells stimulated with TNF-α (0–20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. Results CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Conclusion Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases. PMID:26331770

  2. Gas6 - Axl receptor signaling is regulated by glucose in vascular smooth muscle cells

    PubMed Central

    Cavet, Megan E.; Smolock, Elaine M.; Ozturk, Oktay H.; World, Cameron; Pang, Jinjiang; Konishi, Atsushi; Berk, Bradford C.

    2009-01-01

    Objective The receptor tyrosine kinase Axl and its ligand Gas6 are involved in the development of renal diabetic disease. In vascular smooth muscle cells (VSMC) Axl is activated by reactive oxygen species and stimulates migration and cell survival, suggesting a role for Axl in the vascular complications of diabetes. Methods and Results We investigated the effect of varying glucose concentration on Axl signaling in VSMC. Glucose exerted powerful effects on Gas6-Axl signaling with greater activation of Akt and mTOR in low glucose, and greater activation of ERK1/2 in high glucose. Plasma membrane distribution and tyrosine phosphorylation of Axl were not affected by glucose. However, co-immunoprecipitation studies demonstrated that glucose changed the interaction of Axl with its binding partners. Specifically, binding of Axl to the p85 subunit of PI3-kinase was increased in low glucose, whereas binding to SHP-2 was increased in high glucose. Furthermore, Gas6-Axl induced migration was increased in high glucose, while Gas6-Axl mediated inhibition of apoptosis was greater in low glucose. Conclusion This study demonstrates a role for glucose in altering Axl signaling through coupling to binding partners, and suggests a mechanism by which Axl contributes to VSMC dysfunction in diabetes. PMID:18292389

  3. Glucagon-like peptide-1 inhibits vascular smooth muscle cell dedifferentiation through mitochondrial dynamics regulation.

    PubMed

    Torres, Gloria; Morales, Pablo E; García-Miguel, Marina; Norambuena-Soto, Ignacio; Cartes-Saavedra, Benjamín; Vidal-Peña, Gonzalo; Moncada-Ruff, David; Sanhueza-Olivares, Fernanda; San Martín, Alejandra; Chiong, Mario

    2016-03-15

    Glucagon-like peptide-1 (GLP-1) is a neuroendocrine hormone produced by gastrointestinal tract in response to food ingestion. GLP-1 plays a very important role in the glucose homeostasis by stimulating glucose-dependent insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, reducing appetite and food intake. Because of these actions, the GLP-1 peptide-mimetic exenatide is one of the most promising new medicines for the treatment of type 2 diabetes. In vivo treatments with GLP-1 or exenatide prevent neo-intima layer formation in response to endothelial damage and atherosclerotic lesion formation in aortic tissue. Whether GLP-1 modulates vascular smooth muscle cell (VSMC) migration and proliferation by controlling mitochondrial dynamics is unknown. In this report, we showed that GLP-1 increased mitochondrial fusion and activity in a PKA-dependent manner in the VSMC cell line A7r5. GLP-1 induced a Ser-637 phosphorylation in the mitochondrial fission protein Drp1, and decreased Drp1 mitochondrial localization. GLP-1 inhibited PDGF-BB-induced VSMC migration and proliferation, actions inhibited by overexpressing wild type Drp1 and mimicked by the Drp1 inhibitor Mdivi-1 and by overexpressing dominant negative Drp1. These results show that GLP-1 stimulates mitochondrial fusion, increases mitochondrial activity and decreases PDGF-BB-induced VSMC dedifferentiation by a PKA/Drp1 signaling pathway. Our data suggest that GLP-1 inhibits vascular remodeling through a mitochondrial dynamics-dependent mechanism. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Apolipoprotein serum amyloid A down-regulates smooth-muscle cell lipid biosynthesis.

    PubMed Central

    Schreiber, B M; Veverbrants, M; Fine, R E; Blusztajn, J K; Salmona, M; Patel, A; Sipe, J D

    1999-01-01

    The addition of acute-phase apolipoprotein serum amyloid A (SAA) to cultured aortic smooth-muscle cells caused a decrease in the incorporation of [(14)C]acetate into lipids. Optimal inhibition of lipid biosynthesis was achieved with 2 microM SAA, and the effect was maintained for up to 1 week when SAA was included in the culture medium. Lipid extracts were subjected to TLC and it was determined that the SAA-induced decrease in [(14)C]acetate incorporation into lipids was attributable to decreases in cholesterol, phospholipid and triglyceride levels. The accumulated mass of cholesterol and phospholipid in SAA-treated cultures was significantly less than that of controls, with no change in the accumulated protein. Moreover, SAA had no effect on either protein synthesis or DNA synthesis, suggesting that SAA specifically alters lipid synthesis. By using a peptide corresponding to the cholesterol-binding domain of acute-phase SAA (amino acids 1-18), it was shown that this region of the molecule was as effective as the full-length protein in decreasing lipid synthesis and the accumulation of cholesterol and phospholipid. The implications of these findings for atherosclerosis and Alzheimer's disease are discussed. PMID:10548527

  5. The actin regulator zyxin reinforces airway smooth muscle and accumulates in airways of fatal asthmatics.

    PubMed

    Rosner, Sonia R; Pascoe, Christopher D; Blankman, Elizabeth; Jensen, Christopher C; Krishnan, Ramaswamy; James, Alan L; Elliot, John G; Green, Francis H; Liu, Jeffrey C; Seow, Chun Y; Park, Jin-Ah; Beckerle, Mary C; Paré, Peter D; Fredberg, Jeffrey J; Smith, Mark A

    2017-01-01

    Bronchospasm induced in non-asthmatic human subjects can be easily reversed by a deep inspiration (DI) whereas bronchospasm that occurs spontaneously in asthmatic subjects cannot. This physiological effect of a DI has been attributed to the manner in which a DI causes airway smooth muscle (ASM) cells to stretch, but underlying molecular mechanisms-and their failure in asthma-remain obscure. Using cells and tissues from wild type and zyxin-/- mice we report responses to a transient stretch of physiologic magnitude and duration. At the level of the cytoskeleton, zyxin facilitated repair at sites of stress fiber fragmentation. At the level of the isolated ASM cell, zyxin facilitated recovery of contractile force. Finally, at the level of the small airway embedded with a precision cut lung slice, zyxin slowed airway dilation. Thus, at each level zyxin stabilized ASM structure and contractile properties at current muscle length. Furthermore, when we examined tissue samples from humans who died as the result of an asthma attack, we found increased accumulation of zyxin compared with non-asthmatics and asthmatics who died of other causes. Together, these data suggest a biophysical role for zyxin in fatal asthma.

  6. Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling.

    PubMed

    Wang, Yung-Chun; Cui, Xiao-Bing; Chuang, Ya-Hui; Chen, Shi-You

    2017-07-01

    Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases. © 2017 American Heart Association, Inc.

  7. Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification.

    PubMed

    Kapustin, Alexander N; Schoppet, Michael; Schurgers, Leon J; Reynolds, Joanne L; McNair, Rosamund; Heiss, Alexander; Jahnen-Dechent, Willi; Hackeng, Tilman M; Schlieper, Georg; Harrison, Paul; Shanahan, Catherine M

    2017-03-01

    The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor-dependent and PS-dependent manner. Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors. © 2017 American Heart Association, Inc.

  8. Autocrine secretion of osteopontin by vascular smooth muscle cells regulates their adhesion to collagen gels.

    PubMed Central

    Weintraub, A. S.; Giachelli, C. M.; Krauss, R. S.; Almeida, M.; Taubman, M. B.

    1996-01-01

    Osteopontin (OPN) is a secreted protein postulated to facilitate vascular smooth muscle cell (VSMC) adhesion and migration. Rat aortic VSMC lines were isolated after infection with recombinant retroviruses harboring OPN sense and antisense constructs. All lines grew normally in monolayer culture. On three-dimensional collagen gels, normal VSMCs and lines containing sense constructs (n=15) or empty vector (n=10) attached to gel and invaded the matrix. Four of five antisense clones did not adhere or invade. Antisense clones had lower OPN levels after stimulation with angiotensin II than sense clones or clones containing the empty vector (antisense, 257+/-102 ng/ml; sense, 473+/-104; vector, 434+/-66). Non-adhering antisense clones had lower mean OPN levels after angiotensin II stimulation (161+/-47 ng/ml) than sense or antisense lines with normal adhesion (486+/-63 ng/ml). The ability to adhere correlated with OPN levels >250 ng/ml. Adhesion and invasion were fully restored with addition of 100 to 200 ng/ml of exogenous OPN and were inhibited in normal VSMCs by incubation with 1 microgram/ml anti-OPN antibody. The autocrine secretion of OPN appears to play an important role in VSMC adhesion, spreading, and invasion. Images Figure 2 Figure 3 Figure 5 Figure 6 Figure 7 PMID:8686750

  9. Increasing evidence of mechanical force as a functional regulator in smooth muscle myosin light chain kinase

    PubMed Central

    Baumann, Fabian; Bauer, Magnus Sebastian; Rees, Martin; Alexandrovich, Alexander; Gautel, Mathias; Pippig, Diana Angela; Gaub, Hermann Eduard

    2017-01-01

    Mechanosensitive proteins are key players in cytoskeletal remodeling, muscle contraction, cell migration and differentiation processes. Smooth muscle myosin light chain kinase (smMLCK) is a member of a diverse group of serine/threonine kinases that feature cytoskeletal association. Its catalytic activity is triggered by a conformational change upon Ca2+/calmodulin (Ca2+/CaM) binding. Due to its significant homology with the force-activated titin kinase, smMLCK is suspected to be also regulatable by mechanical stress. In this study, a CaM-independent activation mechanism for smMLCK by mechanical release of the inhibitory elements is investigated via high throughput AFM single-molecule force spectroscopy. The characteristic pattern of transitions between different smMLCK states and their variations in the presence of different substrates and ligands are presented. Interaction between kinase domain and regulatory light chain (RLC) substrate is identified in the absence of CaM, indicating restored substrate-binding capability due to mechanically induced removal of the auto-inhibitory regulatory region. DOI: http://dx.doi.org/10.7554/eLife.26473.001 PMID:28696205

  10. Chrysin inhibits human airway smooth muscle cells proliferation through the extracellular signal-regulated kinase 1/2 signaling pathway.

    PubMed

    Yao, Jing; Zhang, Yun-Shi; Feng, Gan-Zhu; Du, Qiang

    2015-11-01

    Asthma is a chronic airway inflammatory disease characterized by an increased mass of airway smooth muscle (ASM). Chrysin (5,7-dihydroxyflavone), a natural flavonoid, has been shown to exert multiple biological activities, including anti-inflammatory, anti-proliferative and anti-oxidant effects, as well as the potency to ameliorate asthma in animal models. The objective of the present study was to identify the underlying mechanism of the therapeutic effects of chrysin. The impact of chrysin on basal and platelet-derived growth factor (PDGF)-induced proliferation and apoptosis of human airway smooth muscle cells (HASMCs) was investigated. Furthermore, the activation of the extracellular signal-regulated protein kinase (ERK) signaling pathway was evaluated in HASMCs. The results revealed that chrysin significantly inhibited basal as well as PDGF-induced HASMC proliferation, most likely through the suppression of ERK1/2 phosphorylation. However, chrysin did not significantly reduce PDGF-induced apoptosis of HASMCs. The present study indicated that chrysin may be a promising medication for controlling airway remodeling and clinical manifestations of asthma.

  11. The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators

    PubMed Central

    Llorian, Miriam; Gooding, Clare; Bellora, Nicolas; Hallegger, Martina; Buckroyd, Adrian; Wang, Xiao; Rajgor, Dipen; Kayikci, Melis; Feltham, Jack; Ule, Jernej; Eyras, Eduardo; Smith, Christopher W.J.

    2016-01-01

    Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery. PMID:27317697

  12. Transcription of G-protein coupled receptors in corporal smooth muscle is regulated by sialorphin (an endogenous neutral endopeptidase inhibitor)

    PubMed Central

    Tong, Yuehong; Tiplitsky, Scott I.; Tar, Moses; Melman, Arnold; Davies, Kelvin P.

    2009-01-01

    Purpose Several reports have suggested the rat Vcsa1 gene is down-regulated in models of erectile dysfunction (ED). Vcsa’s protein product, sialorphin, is an endogenous neutral endopeptidase (NEP), and its down-regulation could result in prolonged activation of G-protein activated signaling pathways by their peptide agonists. We investigated if down- regulation of Vcsa1 could result in adaptive change in the expression of G-protein coupled receptors (GPCR). Materials and Methods Gene expression in cultured rat corporal smooth muscle cells (CSM) following treatment with siRNA directed against Vcsa1 or the NEP gene was analyzed using microarray and quantitative RT-PCR. In rats Vcsa1 is one of the most down-regulated genes following bilateral transection of the cavernosal nerves. Using that animal model, we also investigated whether the down-regulation of Vcsa1 is accompanied by similar changes in gene expression observed in the CSM cells where Vcsa1 was knocked-down in vitro. Results Microarray analysis and quantitative RT-PCR demonstrated that CSM cells treated in vitro with siRNA against Vcsa1 resulted in up-regulation of GPCR as a functional group. In contrast, treatment of CSM cells that lowered NEP activity resulted in decreases in GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on NEP. In animals with bilaterally transected cavernous nerves the reduced expression of Vcsa1 is accompanied by increased GPCR expression in cavernosal tissue. Conclusions These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR expression. PMID:18554633

  13. Regulation of NaK-ATPase by Platelet-Derived Growth Factors in Cultured Rat Thoracic Aortic Smooth Muscle Cells.

    PubMed

    Lo, C.-S.

    1996-04-01

    Regulation of (Na(+) + K(+))-adenosine triphosphatase (NaK-ATPase) by platelet-derived growth factor (PDGF) in cultured rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances SMC proliferation and NaK-ATPase activity. Ouabain, an inhibitor of NaK-ATPase activity, prevents PDGF-BB-induced SMC proliferation. As shown by Western blot and immunochemiluminescence analysis, PDGF-BB also enhances alpha(1), truncated alpha(1), and beta(1) NaK-ATPase subunit levels. PDGF-AA and PDGF-AB show no effect on alpha(1) and truncated alpha(1) levels in slot blot analysis. Induction of NaK-ATPase subunit levels by PDGF-BB could be one of the initial events in vascular SMC proliferation. Copyright 1996 S. Karger AG, Basel

  14. Focal adhesion kinase regulates smooth muscle cell recruitment to the developing vasculature

    PubMed Central

    Cheng, Zhaokang; Sundberg-Smith, Liisa J.; Mangiante, Lee E.; Sayers, Rebecca L.; Hakim, Zeenat S.; Musunuri, Srilaxmi; Maguire, Colin T.; Majesky, Mark W.; Zhou, Zhigang; Mack, Christopher P.; Taylor, Joan M.

    2011-01-01

    Objective The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. Methods and Results We crossed fakloxp targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAKwnt and FAKnk) or coronary SMC (FAKcSMC). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with post-natal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC, but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, PDGFBB. FAK depletion resulted in un-stable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1 and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in PDGF-stimulated extracellular matrix degradation. Conclusions FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus. PMID:21757658

  15. CDKN2B Regulates TGFβ Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels

    PubMed Central

    Nanda, Vivek; Downing, Kelly P.; Ye, Jianqin; Xiao, Sophia; Kojima, Yoko; Spin, Joshua M.; DiRenzo, Daniel; Nead, Kevin T.; Connolly, Andrew J; Dandona, Sonny; Perisic, Ljubica; Hedin, Ulf; Maegdefessel, Lars; Dalman, Jessie; Guo, Liang; Zhao, XiaoQing; Kolodgie, Frank D.; Virmani, Renu; Davis, Harry R.; Leeper, Nicholas J.

    2015-01-01

    Rationale Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease (PAD), but its mechanism remains unknown. Objective To determine whether this association is secondary to an increase in atherosclerosis, or is the result of a separate angiogenesis-related mechanism. Methods and Results Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under non-atherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hind-limb ischemia and digital auto-amputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell (SMC) to support the developing neovessel. Microarray studies identified impaired TGFβ signaling in cultured CDKN2B-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-‘rheostat’ known to have antagonistic effects on the EC and SMC. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. Conclusions These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis, but may also impair TGFβ signaling and hypoxic neovessel maturation. PMID:26596284

  16. DJ-1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle-like Cells in Response to Sphingosylphosphorylcholine.

    PubMed

    Baek, Suji; Lee, Kang Pa; Jung, Seung Hyo; Cui, Long; Ko, Kinarm; Kim, Bokyung; Won, Kyung Jong

    2017-09-26

    Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox-sensitive molecules including a multifunctional protein DJ-1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)-stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α-smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC-stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ-1 in hMSCs in response to SPC. The increased abundance of oxidized DJ-1 in SPC-stimulated hMSCs was validated by immunoblot analysis. The SPC-induced increase in the expression of α-SMA was stronger in DJ-1-knockdown hMSCs than in control cells. Moreover, the expression of α-SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ-1-overexpressing hMSCs. Exogenous H2 O2 mimicked the responses induced by SPC treatment. These results indicate that the ROS-related DJ-1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  17. The nuclear receptor NOR-1/NR4A3 regulates the multifunctional glycoprotein vitronectin in human vascular smooth muscle cells.

    PubMed

    Martí-Pàmies, Ingrid; Cañes, Laia; Alonso, Judith; Rodríguez, Cristina; Martínez-González, José

    2017-10-01

    The nuclear receptor NOR-1 (NR4A3) has recently been involved in the regulation of extracellular matrix (ECM) proteins associated with neointimal thickening and the vascular control of hemostasis. We sought to find as-yet unidentified NOR-1 target genes in human vascular smooth muscle cells (VSMCs). An in silico analysis identified putative NOR-1 response elements in the proximal promoter region of several genes encoding for ECM proteins, including vitronectin (VTN). Lentiviral overexpression of NOR-1 strongly increased VTN mRNA and protein levels, whereas NOR-1 silencing significantly reduced VTN expression. Deletion and site-directed mutagenesis studies, as well as EMSA and chromatin immunoprecipitation, identified the NBRE(-202/-195) site in the VTN promoter as an essential element for NOR-1 responsiveness. Furthermore, NOR-1 and VTN colocalized in VSMCs in human atherosclerotic lesions. VTN levels were increased in cell supernatants from VSMCs that overexpress NOR-1. Cell supernatants from VSMCs overexpressing NOR-1 induced cell migration to a greater extent than supernatants from control cells, and this effect was attenuated when cell supernatants were preincubated with anti-VTN blocking antibodies or VTN was silenced in supernatant-generating cells. These results indicate that VTN is a target of NOR-1 and suggest that this multifunctional glycoprotein may participate in vascular responses mediated by this nuclear receptor.-Martí-Pàmies, I., Cañes, L., Alonso, J., Rodríguez, C., Martínez-González, J. The nuclear receptor NOR-1/NR4A3 regulates the multifunctional glycoprotein vitronectin in human vascular smooth muscle cells. © FASEB.

  18. Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.

    PubMed

    Willets, Jonathon M; Brighton, Paul J; Mistry, Rajendra; Morris, Gavin E; Konje, Justin C; Challiss, R A John

    2009-08-01

    Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

  19. β1-Subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle

    PubMed Central

    Petkov, Georgi V; Bonev, Adrian D; Heppner, Thomas J; Brenner, Robert; Aldrich, Richard W; Nelson, Mark T

    2001-01-01

    The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming α-subunit and an accessory, smooth muscle-specific, β1-subunit. Our aim was to determine the functional role of the β1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel β1-subunit ‘knock-out’ (KO) mice. The β-galactosidase reporter (lacZ gene) was targeted to the β1 locus, which provided the opportunity to examine the expression of the β1-subunit in UBSM. Based on this approach, the β1-subunit is highly expressed in UBSM. BK channels lacking β1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. The effects of the β1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from β1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from β1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the β1-subunit. The results indicate that the β1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder. PMID:11731577

  20. Role of integrin-linked kinase in vascular smooth muscle cells: Regulation by statins and angiotensin II

    SciTech Connect

    Friedrich, Erik B. . E-mail: efriedrich@med-in.uni-sb.de; Clever, Yvonne P.; Wassmann, Sven; Werner, Nikos; Boehm, Michael; Nickenig, Georg

    2006-10-27

    Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.

  1. MicroRNA-214 regulates smooth muscle cell differentiation from stem cells by targeting RNA-binding protein QKI.

    PubMed

    Wu, Yutao; Li, Zhoubin; Yang, Mei; Dai, Bing; Hu, Feng; Yang, Feng; Zhu, Jianhua; Chen, Ting; Zhang, Li

    2017-02-08

    MicroRNA-214(miR-214) has been recently reported to regulate angiogenesis and embryonic stem cells (ESCs) differentiation. However, very little is known about its functional role in vascular smooth muscle cells (VSMCs) differentiation from ESCs. In the present study, we assessed the hypothesis that miR-214 and its target genes play an important role in VSMCs differentiation. Murine ESCs were seeded on collagen-coated flasks and cultured in differentiation medium for 2 to 8 days to allow VSMCs differentiation. miR-214 was significantly upregulated during VSMCs differentiation. miR-214 overexpression and knockdown in differentiating ESCs significantly promoted and inhibited VSMCs -specific genes expression, respectively. Importantly, miR-214 overexpression in ESCs promoted VSMCs differentiation in vivo. Quaking (QKI) was predicted as one of the major targets of miR-214, which was negatively regulated by miR-214. Luciferase assay showed miR-214 substantially inhibited wild type, but not the mutant version of QKI-3-UTR-luciferase activity in differentiating ESCs, further confirming a negative regulation role of miR-214 in QKI gene expression. Mechanistically, our data showed that miR-214 regulated VSMCs gene expression during VSMCs differentiation from ESCs through suppression of QKI. We further demonstrated that QKI down-regulated the expression of SRF, MEF2C and Myocd through transcriptional repression and direct binding to promoters of the SRF, MEF2c and Myocd genes. Taken together, we have uncovered a central role of miR-214 in ESC-VSMC differentiation, and successfully identified QKI as a functional modulating target in miR-214 mediated VSMCs differentiation.

  2. c-Myb Regulates Proliferation and Differentiation of Adventitial Sca1+ Vascular Smooth Muscle Cell Progenitors by Transactivation of Myocardin.

    PubMed

    Shikatani, Eric A; Chandy, Mark; Besla, Rickvinder; Li, Cedric C; Momen, Abdul; El-Mounayri, Omar; Robbins, Clinton S; Husain, Mansoor

    2016-07-01

    Vascular smooth muscle cells (VSMCs) are believed to dedifferentiate and proliferate in response to vessel injury. Recently, adventitial progenitor cells were implicated as a source of VSMCs involved in vessel remodeling. c-Myb is a transcription factor known to regulate VSMC proliferation in vivo and differentiation of VSMCs from mouse embryonic stem cell-derived progenitors in vitro. However, the role of c-Myb in regulating specific adult vascular progenitor cell populations was not known. Our objective was to examine the role of c-Myb in the proliferation and differentiation of Sca1(+) adventitial VSMC progenitor cells. Using mice with wild-type or hypomorphic c-myb (c-myb(h/h)), BrdU (bromodeoxyuridine) uptake and flow cytometry revealed defective proliferation of Sca1(+) adventitial VSMC progenitor cells at 8, 14, and 28 days post carotid artery denudation injury in c-myb(h/h) arteries. c-myb(h/h) cKit(+)CD34(-)Flk1(-)Sca1(+)CD45(-)Lin(-) cells failed to proliferate, suggesting that c-myb regulates the activation of specific Sca1(+) progenitor cells in vivo and in vitro. Although expression levels of transforming growth factor-β1 did not vary between wild-type and c-myb(h/h) carotid arteries, in vitro differentiation of c-myb(h/h) Sca1(+) cells manifested defective transforming growth factor-β1-induced VSMC differentiation. This is mediated by reduced transcriptional activation of myocardin because chromatin immunoprecipitation revealed c-Myb binding to the myocardin promoter only during differentiation of Sca1(+) cells, myocardin promoter mutagenesis identified 2 specific c-Myb-responsive binding sites, and adenovirus-mediated expression of myocardin rescued the phenotype of c-myb(h/h) progenitors. These data support a role for c-Myb in the regulation of VSMC progenitor cells and provide novel insight into how c-myb regulates VSMC differentiation through myocardin. © 2016 American Heart Association, Inc.

  3. Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle.

    PubMed Central

    Ozaki, H; Blondfield, D P; Hori, M; Sanders, K M; Publicover, N G

    1992-01-01

    1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo-1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2-receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage-dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha-toxin-permeabilized muscles confirmed that cyclic AMP-dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3-300 microM) caused a reduction in Ca(2+)-induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing

  4. Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle.

    PubMed

    Ozaki, H; Blondfield, D P; Hori, M; Sanders, K M; Publicover, N G

    1992-02-01

    1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo-1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2-receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage-dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha-toxin-permeabilized muscles confirmed that cyclic AMP-dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3-300 microM) caused a reduction in Ca(2+)-induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing

  5. Inorganic Phosphate Accelerates the Migration of Vascular Smooth Muscle Cells: Evidence for the Involvement of miR-223

    PubMed Central

    Metzinger-Le Meuth, Valérie; Hénaut, Lucie; Djelouat, Mohamed Seif el Islam; Benchitrit, Joyce; Massy, Ziad A.; Metzinger, Laurent

    2012-01-01

    Backgound An elevated serum inorganic phosphate (Pi) level is a major risk factor for kidney disease and downstream vascular complications. We focused on the effect of Pi levels on human aortic vascular smooth muscle cells (VSMCs), with an emphasis on the role of microRNAs (miRNAs). Methodology/Principal Findings Exposure of human primary VSMCs in vitro to pathological levels of Pi increased calcification, migration rate and concomitantly reduced cell proliferation and the amount of the actin cytoskeleton. These changes were evidenced by significant downregulation of miRNA-143 (miR-143) and miR-145 and concomitant upregulation of their targets and key markers in synthetic VSMCs, such as Krüppel-like factors−4 and −5 and versican. Interestingly, we also found that miR-223 (a marker of muscle damage and a key factor in osteoclast differentiation) is expressed in VSMCs and is significantly upregulated in Pi-treated cells. Over-expressing miR-223 in VSMCs increased proliferation and markedly enhanced VSMC migration. Additionally, we found that the expression of two of the known miR-223 targets, Mef2c and RhoB, was highly reduced in Pi treated as well as miR-223 over-expressing VSMCs. To complement these in vitro findings, we also observed significant downregulation of miR-143 and miR-145 and upregulation of miR-223 in aorta samples collected from ApoE knock-out mice, which display vascular calcification. Conclusions/Significance Our results suggest that (i) high levels of Pi increase VSMC migration and calcification, (ii) altered expression levels of miR-223 could play a part in this process and (iii) miR-223 is a potential new biomarker of VSMC damage. PMID:23094093

  6. Platelet-derived growth factor regulates vascular smooth muscle phenotype via mammalian target of rapamycin complex 1

    SciTech Connect

    Ha, Jung Min; Yun, Sung Ji; Kim, Young Whan; Jin, Seo Yeon; Lee, Hye Sun; Song, Sang Heon; Shin, Hwa Kyoung; Bae, Sun Sik

    2015-08-14

    Mammalian target of rapamycin complex (mTORC) regulates various cellular processes including proliferation, growth, migration and differentiation. In this study, we showed that mTORC1 regulates platelet-derived growth factor (PDGF)-induced phenotypic conversion of vascular smooth muscle cells (VSMCs). Stimulation of contractile VSMCs with PDGF significantly reduced the expression of contractile marker proteins in a time- and dose-dependent manner. In addition, angiotensin II (AngII)-induced contraction of VSMCs was completely blocked by the stimulation of VSMCs with PDGF. PDGF-dependent suppression of VSMC marker gene expression was significantly blocked by inhibition of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinase (ERK), and mTOR whereas inhibition of p38 MAPK had no effect. In particular, inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked the PDGF-dependent phenotypic change of VSMCs whereas silencing of Rictor had no effect. In addition, loss of AngII-dependent contraction by PDGF was significantly retained by silencing of Raptor. Inhibition of mTORC1 by rapamycin or by silencing of Raptor significantly blocked PDGF-induced proliferation of VSMCs. Taken together, we suggest that mTORC1 plays an essential role in PDGF-dependent phenotypic changes of VSMCs. - Graphical abstract: Regulation of VSMC phenotype by PDGF-dependent activation of mTORC1. - Highlights: • The expression of contractile marker proteins was reduced by PDGF stimulation. • PDGF-dependent phenotypic conversion of VSMCs was blocked by inhibition of mTOR. • PDGF-induced proliferation of VSMCs was attenuated by inhibition of mTORC1. • mTORC1 plays a critical role in PDGF-dependent phenotypic conversion of VSMCs.

  7. Fibroblast growth factor (FGF) signaling regulates transforming growth factor beta (TGFβ)-dependent smooth muscle cell phenotype modulation

    PubMed Central

    Chen, Pei-Yu; Qin, Lingfeng; Li, Guangxin; Tellides, George; Simons, Michael

    2016-01-01

    Smooth muscle cells (SMCs) in normal blood vessels exist in a highly differentiate state characterized by expression of SMC-specific contractile proteins (“contractile phenotype”). Following blood vessel injury in vivo or when cultured in vitro in the presence of multiple growth factors, SMC undergo a phenotype switch characterized by the loss of contractile markers and appearance of expression of non-muscle proteins (“proliferative phenotype”). While a number of factors have been reported to modulate this process, its regulation remains uncertain. Here we show that induction of SMC FGF signaling inhibits TGFβ signaling and converts contractile SMCs to the proliferative phenotype. Conversely, inhibition of SMC FGF signaling induces TGFβ signaling converting proliferating SMCs to the contractile phenotype, even in the presence of various growth factors in vitro or vascular injury in vivo. The importance of this signaling cross-talk is supported by in vivo data that show that an SMC deletion of a pan-FGF receptor adaptor Frs2α (fibroblast growth factor receptor substrate 2 alpha) in mice profoundly reduces neointima formation and vascular remodelling following carotid artery ligation. These results demonstrate that FGF-TGFβ signaling antagonism is the primary regulator of the SMC phenotype switch. Manipulation of this cross-talk may be an effective strategy for treatment of SMC-proliferation related diseases. PMID:27634335

  8. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases.

  9. Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors.

    PubMed

    Kwon, Hyeeun; Lee, Jung-Jin; Lee, Ji-Hye; Cho, Won-Kyung; Gu, Min Jung; Lee, Kwang Jin; Ma, Jin Yeul

    2015-01-01

    Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression.

  10. mTOR regulates vascular smooth muscle cell differentiation from human bone marrow-derived mesenchymal progenitors.

    PubMed

    Hegner, Björn; Lange, Maria; Kusch, Angelika; Essin, Kirill; Sezer, Orhan; Schulze-Lohoff, Eckhard; Luft, Friedrich C; Gollasch, Maik; Dragun, Duska

    2009-02-01

    Vascular smooth muscle cells (VSMCs) and circulating mesenchymal progenitor cells (MSCs) with a VSMC phenotype contribute to neointima formation and lumen loss after angioplasty and during allograft arteriosclerosis. We hypothesized that phosphoinositol-Akt-mammalian target of rapamycin-p70S6 kinase (PI3K/Akt/mTOR/p70S6K) pathway activation regulates VSMC differentiation from MSCs. We studied effects of PI3K/Akt/mTOR signaling on phenotypic modulation of MSC and VSMC marker expression, including L-type Ca(2+) channels. Phosphorylation of Akt and p70S6K featured downregulation of VSMC markers in dedifferentiated MSCs. mTOR inhibition with rapamycin at below pharmacological concentrations blocked p70S6K phosphorylation and induced a differentiated contractile phenotype with smooth muscle (sm)-calponin, sm-alpha-actin, and SM protein 22-alpha (SM22alpha) expression. The PI3K inhibitor Ly294002 abolished Akt and p70S6K phosphorylation and reversed the dedifferentiated phenotype via induction of sm-calponin, sm-alpha-actin, SM22alpha, and myosin light chain kinase. Rapamycin acted antiproliferative without impairing MSC viability. In VSMCs, rapamycin increased a homing chemokine for MSCs, stromal cell-derived factor-1-alpha, at mRNA and protein levels. The CXCR4-mediated MSC migration toward conditioned medium of rapamycin-treated VSMCs was enhanced. We describe novel pleiotropic effects of rapamycin at very low concentrations that stabilized differentiated contractile VSMCs from MSCs in addition to exerting antiproliferative and enhanced homing effects.

  11. MicroRNAs 29b, 133b, and 211 Regulate Vascular Smooth Muscle Calcification Mediated by High Phosphorus.

    PubMed

    Panizo, Sara; Naves-Díaz, Manuel; Carrillo-López, Natalia; Martínez-Arias, Laura; Fernández-Martín, José Luis; Ruiz-Torres, María Piedad; Cannata-Andía, Jorge B; Rodríguez, Isabel

    2016-03-01

    Vascular calcification is a frequent cause of morbidity and mortality in patients with CKD and the general population. The common association between vascular calcification and osteoporosis suggests a link between bone and vascular disorders. Because microRNAs (miRs) are involved in the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells, we investigated whether miRs implicated in osteoblast differentiation and bone formation are involved in vascular calcification. Different levels of uremia, hyperphosphatemia, and aortic calcification were induced by feeding nephrectomized rats a normal or high-phosphorus diet for 12 or 20 weeks, at which times the levels of eight miRs (miR-29b, miR-125, miR-133b, miR-135, miR-141, miR-200a, miR-204, and miR-211) in the aorta were analyzed. Compared with controls and uremic rats fed a normal diet, uremic rats fed a high-phosphorous diet had lower levels of miR-133b and miR-211 and higher levels of miR-29b that correlated respectively with greater expression of osteogenic RUNX2 and with lower expression of several inhibitors of osteoblastic differentiation. Uremia per se mildly reduced miR-133b levels only. Similar results were obtained in two in vitro models of vascular calcification (uremic serum and high-calcium and -phosphorus medium), and experiments using antagomirs and mimics to modify miR-29b, miR-133b, and miR-211 expression levels in these models confirmed that these miRs regulate the calcification process. We conclude that miR-29b, miR-133b, and miR-211 have direct roles in the vascular smooth muscle calcification induced by high phosphorus and may be new therapeutic targets in the management of vascular calcification.

  12. Anti-inflammatory cytokine interleukin-19 inhibits smooth muscle cell migration and activation of cytoskeletal regulators of VSMC motility

    PubMed Central

    Gabunia, Khatuna; Jain, Surbhi; England, Ross N.

    2011-01-01

    Vascular smooth muscle cell (VSMC) migration is an important cellular event in multiple vascular diseases, including atherosclerosis, restenosis, and transplant vasculopathy. Little is known regarding the effects of anti-inflammatory interleukins on VSMC migration. This study tested the hypothesis that an anti-inflammatory Th2 interleukin, interleukin-19 (IL-19), could decrease VSMC motility. IL-19 significantly decreased platelet-derived growth factor (PDGF)-stimulated VSMC chemotaxis in Boyden chambers and migration in scratch wound assays. IL-19 significantly decreased VSMC spreading in response to PDGF. To determine the molecular mechanism(s) for these cellular effects, we examined the effect of IL-19 on activation of proteins that regulate VSMC cytoskeletal dynamics and locomotion. IL-19 decreased PDGF-driven activation of several cytoskeletal regulatory proteins that play an important role in smooth muscle cell motility, including heat shock protein-27 (HSP27), myosin light chain (MLC), and cofilin. IL-19 decreased PDGF activation of the Rac1 and RhoA GTPases, important integrators of migratory signals. IL-19 was unable to inhibit VSMC migration nor was able to inhibit activation of cytoskeletal regulatory proteins in VSMC transduced with a constitutively active Rac1 mutant (RacV14), suggesting that IL-19 inhibits events proximal to Rac1 activation. Together, these data are the first to indicate that IL-19 can have important inhibitory effects on VSMC motility and activation of cytoskeletal regulatory proteins. This has important implications for the use of anti-inflammatory cytokines in the treatment of vascular occlusive disease. PMID:21209363

  13. Externally applied cyclic strain regulates localization of focal contact components in cultured smooth muscle cells.

    PubMed

    Cunningham, James J; Linderman, Jennifer J; Mooney, David J

    2002-01-01

    Mechanical signals are critical regulators of cellular gene expression, yet little is understood of the mechanism whereby cells sense mechanical forces. In this study we have tested the hypothesis that mechanical strain applied to populations of cells via their adhesion substrate rapidly alters the cellular distribution of focal contact proteins. Focal contact-associated components (vinculin, a-actinin, paxillin) were assayed by immunofluorescence microscopy and quantitative western blotting. Application of a single step increase in strain in multiple experiments caused overall a small change in focal contact-associated vinculin. In contrast, cyclic strain induced a large and very reproducible increase in detergent-insoluble vinculin (52% relative to static) after just 1 min of strain. Insoluble paxillin was transiently enriched with a similar time course, whereas insoluble a-actinin did not change significantly in response to cyclic strain. Rhodamine-labeled chicken vinculin added to permeabilized cells preferentially localized to focal contacts in response to cyclic strain, but not a single step increase in strain. These findings establish that insoluble levels of focal contact components are altered rapidly following application of an appropriate number of mechanical perturbations, and suggest that at least one component of the mechanism does not involve soluble intermediates.

  14. Integrin α3 blockade enhances microtopographical down-regulation of α-smooth muscle actin: role of microtopography in ECM regulation.

    PubMed

    Ayala, Perla; Desai, Tejal A

    2011-07-01

    Development of functional engineered matrices for regenerative therapies can benefit from an understanding of how physical cues at the microscale affect cell behavior. In this work, we use microfabricated systems to study how stiffness and microscale topographical cues in the form of "micropegs" affect extracellular matrix synthesis. Previous work from our lab has shown that microtopographical cues in 2D and 3D systems decrease cellular proliferation and regulate matrix synthesis. In this work, the combined role of stiffness and topography on ECM synthesis is investigated in a 2D micropeg system. These studies show that fibroblasts cultured on polydimethylsiloxane (PDMS) substrates with micropegs have reduced expression of collagen type I (Col I) and collagen type VI (Col VI) compared to fibroblasts cultured on flat substrates. In addition, cells on micropegged substrates exhibit down-regulation of other important regulators of ECM synthesis such as α-smooth muscle actin (α-SMA), and integrin α3 (Int α3). Interestingly, this effect is dependent on the contractility and adhesion of the cells. When cultured in the presence of RhoA kinase (ROCK) and myosin light chain kinase (MLCK) inhibitors, no significant differences in the expression of collagen, α-SMA, Int α3, and TGFB1 are observed. Additionally, disruptions in cell adhesion prevent microtopographical regulation of ECM synthesis. When using an antibody to block the extracellular domain of Int α3, no differences in the expression of collagen are observed and blocking Int α3 results in enhanced down-regulation of α-SMA on the stiffer micropegged substrates. These findings demonstrate that regulation of extracellular matrix production by cells on a synthetic substrate can be guided via physical cues at the microscale, and add to the body of knowledge on the role of integrin-mediated mechanotransduction.

  15. Regulation of vascular smooth muscle cell turnover by endothelial cell-secreted microRNA-126: role of shear stress.

    PubMed

    Zhou, Jing; Li, Yi-Shuan; Nguyen, Phu; Wang, Kuei-Chun; Weiss, Anna; Kuo, Yi-Chun; Chiu, Jeng-Jiann; Shyy, John Y; Chien, Shu

    2013-06-21

    Endothelial microRNA-126 (miR-126) modulates vascular development and angiogenesis. However, its role in the regulation of smooth muscle cell (SMC) function is unknown. To elucidate the role of miR-126 secreted by endothelial cells (ECs) in regulating SMC turnover in vitro and in vivo, as well as the effects of shear stress on the regulation. Coculture of SMCs with ECs or treatment of SMCs with conditioned media from static EC monoculture (EC-CM) increased SMC miR-126 level and SMC turnover; these effects were abolished by inhibition of endothelial miR-126 and by the application of laminar shear stress to ECs. SMC miR-126 did not increase when treated with EC-CM from ECs subjected to inhibition of miR biogenesis, or with CM from sheared ECs. Depletion of extracellular/secreted vesicles in EC-CM did not affect the increase of SMC miR-126 by EC-CM. Biotinylated miR-126 or FLAG (DYKDDDDK epitope)-tagged Argonaute2 transfected into ECs was detected in the cocultured or EC-CM-treated SMCs, indicating a direct EC-to-SMC transmission of miR-126 and Argonaute2. Endothelial miR-126 represses forkhead box O3, B-cell lymphoma 2, and insulin receptor substrate 1 mRNAs in the cocultured SMCs, suggesting the functional roles of the transmitted miR-126. Systemic depletion of miR-126 in mice inhibited neointimal lesion formation of carotid arteries induced by cessation of blood flow. Administration of EC-CM or miR-126 mitigated the inhibitory effect. Endothelial miR-126 acts as a key intercellular mediator to increase SMC turnover, and its release is reduced by atheroprotective laminar shear stress.

  16. Calcium/calmodulin-dependent protein kinase II-delta isoform regulation of vascular smooth muscle cell proliferation.

    PubMed

    House, Suzanne J; Ginnan, Roman G; Armstrong, Shayn E; Singer, Harold A

    2007-06-01

    There is accumulating evidence that Ca(2+)-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle (VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/threonine kinase, calmodulin (CaM)-dependent protein kinase II (CaMKII), as a mediator of Ca(2+) signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKIIgamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKIIdelta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKIIdelta(2) protein and a significant decrease in CaMKIIgamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKIIdelta(2) in primary cultured cells using small-interfering RNA (siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKIIdelta(2) activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKIIdelta(2) is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum-stimulated proliferation.

  17. CaM kinase II delta2-dependent regulation of vascular smooth muscle cell polarization and migration.

    PubMed

    Mercure, Melissa Z; Ginnan, Roman; Singer, Harold A

    2008-06-01

    Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

  18. Differential regulation of hyaluronic acid synthase isoforms in human saphenous vein smooth muscle cells: possible implications for vein graft stenosis.

    PubMed

    van den Boom, M; Sarbia, M; von Wnuck Lipinski, K; Mann, P; Meyer-Kirchrath, J; Rauch, B H; Grabitz, K; Levkau, B; Schrör, K; Fischer, J W

    2006-01-06

    Autologous saphenous vein bypass grafts (SVG) are frequently compromised by neointimal thickening and subsequent atherosclerosis eventually leading to graft failure. Hyaluronic acid (HA) generated by smooth muscle cells (SMC) is thought to augment the progression of atherosclerosis. The aim of the present study was (1) to investigate HA accumulation in native and explanted arterialized SVG, (2) to identify factors that regulate HA synthase (HAS) expression and HA synthesis, and (3) to study the function of the HAS2 isoform. In native SVG, expression of all 3 HAS isoforms was detected by RT-PCR. Histochemistry revealed that native and arterialized human saphenous vein segments were characterized by marked deposition of HA in association with SMC. Interestingly, in contrast to native SVG, cyclooxygenase (COX)-2 expression by SMC and macrophages was detected only in arterialized SVG. In vitro in human venous SMC HAS isoforms were found to be differentially regulated. HAS2, HAS1, and HA synthesis were strongly induced by vasodilatory prostaglandins via Gs-coupled prostaglandin receptors. In addition, thrombin induced HAS2 via activation of PAR1 and interleukin 1beta was the only factor that induced HAS3. By small interfering RNA against HAS2, it was shown that HAS2 mediated HA synthesis is critically involved in cell cycle progression through G1/S phase and SMC proliferation. In conclusion, the present study shows that HA-rich extracellular matrix is maintained after arterialization of vein grafts and might contribute to graft failure because of its proproliferative function in venous SMC. Furthermore, COX-2-dependent prostaglandins may play a key role in the regulation of HA synthesis in arterialized vein grafts.

  19. Magnesium regulates intracellular ionized calcium concentration and cell geometry in vascular smooth muscle cells (VSMC)

    SciTech Connect

    Zhang, A.; Cheng, T.P.; Altura, B.M. )

    1991-03-11

    It has been suggested that the extracellular Mg{sup 2+} may modulate contractility of VSMC by controlling the cellular level of free Ca{sup 2+}. The present studies were designed to determine the effects of (Mg{sup 2+}) on the distribution of intracellular free Ca{sup 2+} using digital imaging fluorescence microscopy of Fura-2 fluorescence of single VSMC cultured from rat aortas. When incubated with HEPES buffer solution containing 1.2mM Mg{sup 2+}, the myocytes are spindle-shaped, and the basal level of (Ca{sup 2+}){sub i} estimated from the ratio (F340/F380) is 96.6 {plus minus} 7.9nM with a heterogeneous distribution. (Mg{sup 2+}){sub o} withdrawal from the incubation medium induces consistently a dramatic increment of (Ca{sup 2+}){sub i} up to 579.6 {plus minus} 39.3nM, about a 5.8-fold elevation compared to control experiments. Similarly, lowering (Mg{sup 2+}){sub o} to 0.3mM (the lowest physiological range) elevates (Ca{sup 2+}){sub i} to the intermediate level of 348.0 {plus minus} 31.5nM. However, the heterogeneous distribution of (Ca{sup 2+}){sub i} is still evident when (Mg{sup 2+}){sub o} is lowered. Simultaneously to the (Ca{sup 2+}){sub i} increments, cell shapes were changed. In contrast, elevation of (Mg{sup 2+}){sub o} to 4.8mM was found to decrease (Ca{sup 2+}){sub i} to 72.0 {plus minus} 4.6nM. Removal of (Ca{sup 2+}){sub o}, however, abolished the increments of (Ca{sup 2+}){sub i} induced by (Mg{sup 2+}){sub o} withdrawal. These results demonstrate that (Mg{sup 2+}){sub o} regulated (Ca{sup 2+}){sub i} and geometry of VSMC, probably through controlling plasma membrane permeability to Ca{sup 2+}.

  20. Dissociation of Crk-associated substrate from the vimentin network is regulated by p21-activated kinase upon acetylcholine activation of airway smooth muscle

    PubMed Central

    Wang, Ruping; Li, Qing-Fen; Anfinogenova, Yana; Tang, Dale D.

    2006-01-01

    The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, stimulation of tracheal smooth muscle strips with acetylcholine (ACh) resulted in the increase in ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly) in association with force development. Activation with ACh also induced vimentin phosphorylation at Ser-56 as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips upon contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The decrease in distribution of CAS in cytoskeletal vimentin elicited by ACh was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble vimentin to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser-56 and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated the dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during acetylcholine activation of tracheal smooth muscle tissues. PMID:16997882

  1. Endogenous IGF-I and alpha v beta3 integrin ligands regulate increased smooth muscle growth in TNBS-induced colitis.

    PubMed

    Hazelgrove, Krystina B; Flynn, Robert S; Qiao, Li-Ya; Grider, John R; Kuemmerle, John F

    2009-06-01

    Endogenous insulin-like growth factor-I (IGF-I) regulates intestinal smooth muscle growth by concomitantly stimulating proliferation and inhibiting apoptosis. IGF-I-stimulated growth is augmented by the alpha(v)beta(3) integrin ligands vitronectin and fibronectin. IGF-I expression in smooth muscle is increased in both TNBS-induced colitis and Crohn's disease. We hypothesized that intestinal inflammation increased vitronectin and fibronectin expression by smooth muscle and, along with IGF-I upregulation, increased intestinal muscle growth. Intestinal smooth muscle cells were examined 7 days following the induction of TNBS-induced colitis. Although alpha(v)beta(3) integrin expression was not altered by TNBS-induced colitis, vitronectin and fibronectin levels were increased by 80 +/- 10% and 90 +/- 15%, above control levels, respectively. Basal IGF-I receptor phosphorylation in inflamed muscle from TNBS-treated rats was increased by 86 +/- 8% over vehicle-treated controls. Basal ERK1/2, p70S6 kinase, and GSK-3beta phosphorylation in muscle cells of TNBS-treated rats were also increased by 140-180%. TNBS treatment increased basal muscle cell proliferation by 130 +/- 15% and decreased apoptosis by 20 +/- 2% compared with that in vehicle-treated controls. The changes in proliferation and apoptosis were reversed by an IGF-I receptor tyrosine kinase inhibitor or an alpha(v)beta(3) integrin antagonist. The results suggest that smooth muscle hyperplasia in TNBS-induced colitis partly results from the upregulation of endogenous IGF-I and ligands of alpha(v)beta(3) integrin that mediate increased smooth muscle cell proliferation and decreased apoptosis. This paper has identified one mechanism regulating smooth muscle hyperplasia, a feature of stricture formation that occurs in the chronically inflamed intestine of TNBS-induced colitis and potentially Crohn's disease.

  2. Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling

    PubMed Central

    Zhu, Pengcheng; Huang, Lei; Ge, Xiaona; Yan, Fei; Wu, Renliang; Ao, Qilin

    2006-01-01

    Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia-induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia-induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle α-actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT-PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate the hypoxia-induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle-like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia-induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle-like cells which was regulated by myocardin. PMID:17222214

  3. Signal-transduction pathways that regulate smooth muscle function I. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles.

    PubMed

    Harnett, Karen M; Cao, Weibiao; Biancani, Piero

    2005-03-01

    Contraction of esophageal (Eso) and lower esophageal sphincter (LES) circular muscle depends on distinct signal-transduction pathways. ACh-induced contraction of Eso muscle is linked to phosphatidylcholine metabolism, production of diacylglycerol and arachidonic acid (AA), and activation of the Ca(2+)-insensitive PKCepsilon. Although PKCepsilon does not require Ca(2+) for activation, either influx of extracellular Ca(2+) or release of Ca(2+) from stores is needed to activate the phospholipases responsible for hydrolysis of membrane phospholipids and production of second messengers, which activate PKCepsilon. In contrast, the LES uses two distinct intracellular pathways: 1) a PKC-dependent pathway activated by low doses of agonists or during maintenance of spontaneous tone, and 2) a Ca(2+)-calmodulin-myosin light chain kinase (MLCK)-dependent pathway activated in response to maximally effective doses of agonists during the initial phase of contraction. The Ca(2+) levels, released by agonist-induced activity of phospholipase C, determine which contractile pathway is activated in the LES. The Ca(2+)-calmodulin-MLCK-dependent contractile pathway has been well characterized in a variety of smooth muscles. The steps linking activation of PKC to myosin light chain (MLC20) phosphorylation and contraction, however, have not been clearly defined for LES, Eso, or other smooth muscles. In addition, in LES circular muscle, a low-molecular weight pancreatic-like phospholipase A2 (group I PLA2) causes production of AA, which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to heterotrimeric G proteins to induce activation of phospholipases and production of second messengers to maintain contraction of LES circular muscle. We have examined the signal-transduction pathways activated by PGF(2alpha) and by thromboxane analogs during the initial contractile phase and found that these pathways are the same as those activated by other

  4. Regulation of proliferation and gene expression in cultured human aortic smooth muscle cells by resveratrol and standardized grape extracts

    SciTech Connect

    Wang Zhirong; Chen Yan; Labinskyy, Nazar; Hsieh Tzechen; Ungvari, Zoltan; Wu, Joseph M. . E-mail: Joseph_Wu@nymc.edu

    2006-07-21

    Epidemiologic studies suggest that low to moderate consumption of red wine is inversely associated with the risk of coronary heart disease; the protection is in part attributed to grape-derived polyphenols, notably trans-resveratrol, present in red wine. It is not clear whether the cardioprotective effects of resveratrol can be reproduced by standardized grape extracts (SGE). In the present studies, we determined, using cultured human aortic smooth muscle cells (HASMC), growth and specific gene responses to resveratrol and SGE provided by the California Table Grape Commission. Suppression of HASMC proliferation by resveratrol was accompanied by a dose-dependent increase in the expression of tumor suppressor gene p53 and heat shock protein HSP27. Using resveratrol affinity chromatography and biochemical fractionation procedures, we showed by immunoblot analysis that treatment of HASMC with resveratrol increased the expression of quinone reductase I and II, and also altered their subcellular distribution. Growth of HASMC was significantly inhibited by 70% ethanolic SGE; however, gene expression patterns in various cellular compartments elicited in response to SGE were substantially different from those observed in resveratrol-treated cells. Further, SGE also differed from resveratrol in not being able to induce relaxation of rat carotid arterial rings. These results indicate that distinct mechanisms are involved in the regulation of HASMC growth and gene expression by SGE and resveratrol.

  5. Mitochondrial fission induced by platelet-derived growth factor regulates vascular smooth muscle cell bioenergetics and cell proliferation.

    PubMed

    Salabei, Joshua K; Hill, Bradford G

    2013-01-01

    Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. VSMCs were exposed to platelet-derived growth factor-BB (PDGF) and changes in mitochondrial morphology, proliferation, contractile protein expression, and mitochondrial metabolism were examined. Exposure of VSMCs to PDGF resulted in mitochondrial fragmentation and a 50% decrease in the abundance of mitofusin 2. Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation. Results of mitochondrial function assays in permeabilized cells showed few changes due to PDGF treatment in mitochondrial respiratory chain capacity and coupling. Treatment of VSMCs with Mdivi-1-an inhibitor of mitochondrial fission-inhibited PDGF-induced mitochondrial fragmentation by 50% and abolished increases in cell proliferation; however, it failed to prevent PDGF-mediated activation of autophagy and removal of contractile proteins. In addition, treatment with Mdivi-1 reversed changes in fatty acid and glucose oxidation associated with the synthetic phenotype. These results suggest that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. Mitochondrial fragmentation occurring during the transition to the synthetic phenotype could be a therapeutic target for hyperproliferative vascular disorders.

  6. Cleaved high molecular weight kininogen, a novel factor in the regulation of matrix metalloproteinases in vascular smooth muscle cells.

    PubMed

    Vosgerau, Uwe; Lauer, Diljara; Unger, Thomas; Kaschina, Elena

    2010-01-15

    We previously reported that Brown Norway Katholiek rats, which feature a deficiency of plasma kininogens, develop severe abdominal aortic aneurysm. Increased activity of matrix metalloproteinases (MMPs) in the aortic wall, leading to degradation of extracellular matrix components, is considered to play a crucial role in aneurysm formation. Using an in vitro model of vascular smooth muscle cells (VSMCs), cultured from the rat aorta, we investigated whether the cleaved form of high molecular weight kininogen, designated HKa, affects the expression of MMP-9 and MMP-2 and their tissue inhibitors (TIMPs). Treatment of VSMCs with HKa reduced in a concentration-dependent manner IL-1alpha-induced release of MMP-9 and MMP-2, associated with decreased MMP enzymatic activity levels in conditioned media, as demonstrated by gelatin zymography and fluorescein-labeled gelatin substrate assay, respectively. Real-time PCR revealed that HKa reduced corresponding MMP-9 mRNA levels. Further investigations showed that this effect did not result from a modified rate of MMP-9 mRNA degradation. TIMP-1 mRNA levels, already increased as a result of cytokine-stimulation, were significantly enhanced by HKa. Furthermore, we found elevated basal mRNA expression levels of MMP-2 and TIMP-2 in VSMCs derived from kininogen-deficient Brown Norway Katholiek rats. These results demonstrate for the first time that HKa affects the regulation of MMPs in VSMCs.

  7. Regulation of mitochondrial morphology by positive feedback interaction between PKCδ and Drp1 in vascular smooth muscle cell.

    PubMed

    Lim, Soyeon; Lee, Se-Yeon; Seo, Hyang-Hee; Ham, Onju; Lee, Changyeon; Park, Jun-Hee; Lee, Jiyun; Seung, Minji; Yun, Ina; Han, Sun M; Lee, Seahyoung; Choi, Eunhyun; Hwang, Ki-Chul

    2015-04-01

    Dynamin-related protein-1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi-1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms-related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2 O2 )-induced proliferation and migration in SMCs were attenuated by down-regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2 O2 . Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2-ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti-proliferative and -migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1-mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi-1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis.

  8. Sub-Nanomolar Sensitivity of Nitric Oxide Mediated Regulation of cGMP and Vasomotor Reactivity in Vascular Smooth Muscle

    PubMed Central

    Held, Kara F.; Dostmann, Wolfgang R.

    2012-01-01

    Nitric oxide (NO) is a potent dilator of vascular smooth muscle (VSM) by modulating intracellular cGMP ([cGMP]i) through the binding and activation of receptor guanylyl cylases (sGC). The kinetic relationship of NO and sGC, as well as the subsequent regulation of [cGMP]i and its effects on blood vessel vasodilation, is largely unknown. In isolated VSM cells exposed to both pulsed and clamped NO we observed transient and sustained increases in [cGMP]i, with sub-nanomolar sensitivity to NO (EC50 = 0.28 nM). Through the use of pharmacological inhibitors of sGC, PDE5, and PKG, a comprehensive VSM-specific modeling algorithm was constructed to elucidate the concerted activity profiles of sGC, PDE5, phosphorylated PDE5, and PDE1 in the maintenance of [cGMP]i. In small pressure-constricted arteries of the resistance vasculature we again observed both transient and sustained relaxations upon delivery of pulsed and clamped NO, while maintaining a similarly high sensitivity to NO (EC50 = 0.42 nM). Our results propose an intricate dependency of the messengers and enzymes involved in cGMP homeostasis, and vasodilation in VSM. Particularly, the high sensitivity of sGC to NO in primary tissue indicates how small changes in the concentrations of NO, irrespective of the form of NO delivery, can have significant effects on the dynamic regulation of vascular tone. PMID:22807915

  9. Intracellular Acid-extruding regulators and the effect of lipopolysaccharide in cultured human renal artery smooth muscle cells.

    PubMed

    Loh, Shih-Hurng; Lee, Chung-Yi; Tsai, Yi-Ting; Shih, Shou-Jou; Chen, Li-Wei; Cheng, Tzu-Hurng; Chang, Chung-Yi; Tsai, Chein-Sung

    2014-01-01

    Homeostasis of the intracellular pH (pHi) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na(+)-H(+) exchanger (NHE) and the Na(+)-HCO3(-) co-transporter (NBC) have been confirmed in many mammalian cells as major acid extruders. However, the role of acid-extruding regulators in human renal artery smooth muscle cells (HRASMCs) remains unclear. It has been demonstrated that lipopolysaccharide (LPS)-induced vascular occlusion is associated with the apoptosis, activating calpain and increased [Ca(2+)]i that are related to NHE1 activity in endothelia cells. This study determines the acid-extruding mechanisms and the effect of LPS on the resting pHi and active acid extruders in cultured HRASMCs. The mechanism of pHi recovery from intracellular acidosis (induced by NH4Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pHi is 7.19 ± 0.03 and 7.10 ± 0.02 for HEPES- and CO2/HCO3(-)- buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na(+)-coupled HCO3(-) transporter i.e. NBC, functionally co-exists to achieve acid-equivalent extrusion; (c) three different isoforms of NBC: NBCn1 (SLC4A7; electroneutral), NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5), are detected in protein/mRNA level; and (d) pHi and NHE protein expression/activity are significantly increased by LPS, in both a dose- and time- dependent manner, but NBCs protein expression is not. In conclusion, it is demonstrated, for the first time, that four pHi acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pHi and NHE in a dose- and time-dependent manner.

  10. Calponin Isoforms CNN1, CNN2 and CNN3: Regulators for Actin Cytoskeleton Functions in Smooth Muscle and Non-Muscle Cells

    PubMed Central

    Liu, Rong; Jin, J-P

    2016-01-01

    Calponin is an actin filament-associated regulatory protein expressed in smooth muscle and multiple types of non-muscle cells. Three homologous genes, CNN1, CNN2 and CNN3, encoding calponin isoforms 1, 2, and 3, respectively, are present in vertebrate species. All three calponin isoforms are actin-binding proteins with functions in inhibiting actin-activated myosin ATPase and stabilizing the actin cytoskeleton, while each isoform executes different physiological roles based on their cell type-specific expressions. Calponin 1 is specifically expressed in smooth muscle cells and plays a role in fine-tuning smooth muscle contractility. Calponin 2 is expressed in both smooth muscle and non-muscle cells and regulates multiple actin cytoskeleton-based functions. Calponin 3 participates in actin cytoskeleton-based activities in embryonic development and myogenesis. Phosphorylation has been extensively studied for the regulation of calponin functions. Cytoskeleton tension regulates the transcription of CNN2 gene and the degradation of calponin 2 protein. This review summarizes our knowledge learned from studies over the past three decades, focusing on the evolutionary lineage of calponin isoform genes, their tissue- and cell type-specific expressions, structure-function relationships, and mechanoregulation. PMID:26970176

  11. New endoplasmic reticulum stress regulator, Gipie, regulates the survival of vascular smooth muscle cells and the neointima formation after vascular injury.

    PubMed

    Noda, Tomonori; Maeda, Kengo; Hayano, Shinji; Asai, Naoya; Enomoto, Atsushi; Takahashi, Masahide; Murohara, Toyoaki

    2015-05-01

    The accumulation of unfolded protein in the endoplasmic reticulum (ER) initiates an adaptive stress response, termed the unfolded protein response. Previous studies suggested that ER stress might be involved in the formation of neointima after vascular injury. We recently discovered a novel regulator of ER stress, 78-kDa glucose-regulated protein-interacting protein induced by ER stress (Gipie). The objective of this study was to elucidate the role of Gipie using models of vascular disease. We investigated the functions of Gipie in cultured vascular smooth muscle cells (VSMCs) and in a vascular injury model of a rat carotid artery. The expression of Gipie was predominantly detected in synthetic VSMCs and to a much lesser extent in contractile VSMCs, which was augmented by treatment with thapsigargin. Gipie knockdown increased the phosphorylation levels of c-Jun N-terminal kinase and the number of apoptotic cells under ER stress. Moreover, Gipie knockdown decreased the mature form of collagen I in synthetic VSMCs. The expression of Gipie was rarely detected in the medial VSMCs of the intact carotid artery, whereas it was detected in most of the neointimal cells and some of the medial VSMCs after balloon injury. Depletion of Gipie in the rat carotid artery attenuated the neointimal thickening, which was accompanied by increased cell death in the neointima. Conversely, overexpression of Gipie augmented the neointimal thickening. Gipie participates in the ER stress response in VSMCs and plays an important role in neointima formation after vascular injury. © 2015 American Heart Association, Inc.

  12. Chronic exposure to fibrin and fibrinogen differentially regulates intracellular Ca2+ in human pulmonary arterial smooth muscle and endothelial cells.

    PubMed

    Firth, Amy L; Yau, Jocelyn; White, Amanda; Chiles, Peter G; Marsh, James J; Morris, Timothy A; Yuan, Jason X-J

    2009-06-01

    Acute pulmonary embolism occurs in more than half a million people a year in the United States. Chronic thromboembolic pulmonary hypertension (CTEPH) develops in approximately 4% of these patients due to unresolved thromboemboli. CTEPH is thus a relatively common, progressive, and potentially fatal disease. One currently proposed theory for the poor resolution advocates that modification of fibrinogen in CTEPH patients causes resistance of emboli to fibrinolysis. The current study investigated the regulation of cytosolic Ca(2+) ([Ca(2+)](cyt)), central to the control of cell migration, proliferation, and contraction, by chronic exposure of pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells to fibrinogen and fibrin. Basal [Ca(2+)](cyt) was substantially elevated in PAEC after culture on fibrinogen, fibrin, and thrombin and in PASMC on fibrinogen and fibrin. In PAEC, fibrinogen significantly decreased the peak [Ca(2+)](cyt) transient (P <0.001) without a change in the transient peak width (at 50% of the peak height). This response was independent of effects on the proteinase-activated receptor (PAR) 1. Furthermore, chronic exposure to thrombin, an activator of PAR, significantly reduced the peak agonist-induced Ca(2+) release in PAEC, but increased it in PASMC. The recovery rate of the agonist-induced [Ca(2+)](cyt) transients decelerated in PASMC chronically exposed to fibrin; a small increase of the peak Ca(2+) was also observed. Substantial augmentation of PASMC (but not PAEC) proliferation was observed in response to chronic fibrin exposure. In conclusion, chronic exposure to fibrinogen, fibrin, and thrombin caused differential changes in [Ca(2+)](cyt) in PAEC and PASMC. Such changes in [Ca(2+)](cyt) may contribute to vascular changes in patients who have CTEPH where the pulmonary vasculature is persistently exposed to thromboemboli.

  13. Chronic exposure to fibrin and fibrinogen differentially regulates intracellular Ca2+ in human pulmonary arterial smooth muscle and endothelial cells

    PubMed Central

    Firth, Amy L.; Yau, Jocelyn; White, Amanda; Chiles, Peter G.; Marsh, James J.; Morris, Timothy A.; Yuan, Jason X.-J.

    2009-01-01

    Acute pulmonary embolism occurs in more than half a million people a year in the United States. Chronic thromboembolic pulmonary hypertension (CTEPH) develops in ∼4% of these patients due to unresolved thromboemboli. CTEPH is thus a relatively common, progressive, and potentially fatal disease. One currently proposed theory for the poor resolution advocates that modification of fibrinogen in CTEPH patients causes resistance of emboli to fibrinolysis. The current study investigated the regulation of cytosolic Ca2+ ([Ca2+]cyt), central to the control of cell migration, proliferation, and contraction, by chronic exposure of pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells to fibrinogen and fibrin. Basal [Ca2+]cyt was substantially elevated in PAEC after culture on fibrinogen, fibrin, and thrombin and in PASMC on fibrinogen and fibrin. In PAEC, fibrinogen significantly decreased the peak [Ca2+]cyt transient (P <0.001) without a change in the transient peak width (at 50% of the peak height). This response was independent of effects on the proteinase-activated receptor (PAR) 1. Furthermore, chronic exposure to thrombin, an activator of PAR, significantly reduced the peak agonist-induced Ca2+ release in PAEC, but increased it in PASMC. The recovery rate of the agonist-induced [Ca2+]cyt transients decelerated in PASMC chronically exposed to fibrin; a small increase of the peak Ca2+ was also observed. Substantial augmentation of PASMC (but not PAEC) proliferation was observed in response to chronic fibrin exposure. In conclusion, chronic exposure to fibrinogen, fibrin, and thrombin caused differential changes in [Ca2+]cyt in PAEC and PASMC. Such changes in [Ca2+]cyt may contribute to vascular changes in patients who have CTEPH where the pulmonary vasculature is persistently exposed to thromboemboli. PMID:19363122

  14. Role of EGFR transactivation in angiotensin II signaling to extracellular regulated kinase in preglomerular smooth muscle cells.

    PubMed

    Andresen, Bradley T; Linnoila, Jenny J; Jackson, Edwin K; Romero, Guillermo G

    2003-03-01

    Angiotensin (Ang) II promotes the phosphorylation of extracellular regulated kinase (ERK); however, the mechanisms leading to Ang II-induced ERK phosphorylation are debated. The currently accepted theory involves transactivation of epidermal growth factor receptor (EGFR). We have shown that generation of phosphatidic acid (PA) is required for the recruitment of Raf to membranes and the activation of ERK by multiple agonists, including Ang II. In the present report, we confirm that phospholipase D-dependent generation of PA is required for Ang II-mediated phosphorylation of ERK in Wistar-Kyoto and spontaneously hypertensive rat preglomerular smooth muscle cells (PGSMCs). However, EGF stimulation does not activate phospholipase D or generate PA. These observations indicate that EGF recruits Raf to membranes via a mechanism that does not involve PA, and thus, Ang II-mediated phosphorylation of ERK is partially independent of EGFR-mediated signaling cascades. We hypothesized that phosphoinositide-3-kinase (PI3K) can also act to recruit Raf to membranes; therefore, inhibition of PI3K should inhibit EGF signaling to ERK. Wortmannin, a PI3K inhibitor, inhibited EGF-mediated phosphorylation of ERK (IC50, approximately 14 nmol/L). To examine the role of the EGFR in Ang II-mediated phosphorylation of ERK we utilized 100 nmol/L wortmannin to inhibit EGFR signaling to ERK and T19N RhoA to block Ang II-mediated ERK phosphorylation. Wortmannin treatment inhibited EGF-mediated but not Ang II-mediated phosphorylation of ERK. Furthermore, T19N RhoA inhibited Ang II-mediated ERK phosphorylation, whereas T19N RhoA had significantly less effect on EGF-mediated ERK phosphorylation. We conclude that transactivation of the EGFR is not primarily responsible for Ang II-mediated activation of ERK in PGSMCs.

  15. Voltage-gated K+ channels sensitive to stromatoxin-1 regulate myogenic and neurogenic contractions of rat urinary bladder smooth muscle

    PubMed Central

    Chen, Muyan; Kellett, Whitney F.

    2010-01-01

    Members of the voltage-gated K+ (KV) channel family are suggested to control the resting membrane potential and the repolarization phase of the action potential in urinary bladder smooth muscle (UBSM). Recent studies report that stromatoxin-1, a peptide isolated from tarantulas, selectively inhibits KV2.1, KV2.2, KV4.2, and KV2.1/9.3 channels. The objective of this study was to investigate whether KV channels sensitive to stromatoxin-1 participate in the regulation of rat UBSM contractility and to identify their molecular fingerprints. Stromatoxin-1 (100 nM) increased the spontaneous phasic contraction amplitude, muscle force, and tone in isolated UBSM strips. However, stromatoxin-1 (100 nM) had no effect on the UBSM contractions induced by depolarizing agents such as KCl (20 mM) or carbachol (1 μM). This indicates that, under conditions of sustained membrane depolarization, the KV channels sensitive to stromatoxin-1 have no further contribution to the membrane excitability and contractility. Stromatoxin-1 (100 nM) increased the amplitude of the electrical field stimulation-induced contractions, suggesting also a role for these channels in neurogenic contractions. RT-PCR experiments on freshly isolated UBSM cells showed mRNA expression of KV2.1, KV2.2, and KV9.3, but not KV4.2 channel subunits. Protein expression of KV2.1 and KV2.2 channels was detected using Western blot and was further confirmed by immunocytochemical detection in freshly isolated UBSM cells. These novel findings indicate that KV2.1 and KV2.2, but not KV4.2, channel subunits are expressed in rat UBSM and play a key role in opposing both myogenic and neurogenic UBSM contractions. PMID:20393158

  16. Nesprin-2-dependent ERK1/2 compartmentalisation regulates the DNA damage response in vascular smooth muscle cell ageing

    PubMed Central

    Warren, D T; Tajsic, T; Porter, L J; Minaisah, R M; Cobb, A; Jacob, A; Rajgor, D; Zhang, Q P; Shanahan, C M

    2015-01-01

    Prelamin A accumulation and persistent DNA damage response (DDR) are hallmarks of vascular smooth muscle cell (VSMC) ageing and dysfunction. Although prelamin A is proposed to interfere with DNA repair, our understanding of the crosstalk between prelamin A and the repair process remains limited. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) have emerged as key players in the DDR and are known to enhance ataxia telangiectasia-mutated protein (ATM) activity at DNA lesions, and in this study, we identified a novel relationship between prelamin A accumulation and ERK1/2 nuclear compartmentalisation during VSMC ageing. We show both prelamin A accumulation and increased DNA damage occur concomitantly, before VSMC replicative senescence, and induce the localisation of ERK1/2 to promyelocytic leukaemia protein nuclear bodies (PML NBs) at the sites of DNA damage via nesprin-2 and lamin A interactions. Importantly, VSMCs treated with DNA damaging agents also displayed prelamin A accumulation and ERK compartmentalisation at PML NBs, suggesting that prelamin A and nesprin-2 are novel components of the DDR. In support of this, disruption of ERK compartmentalisation at PML NBs, by either depletion of nesprin-2 or lamins A/C, resulted in the loss of ATM from DNA lesions. However, ATM signalling and DNA repair remained intact after lamins A/C depletion, whereas nesprin-2 disruption ablated downstream Chk2 activation and induced genomic instability. We conclude that lamins A/C and PML act as scaffolds to organise DNA-repair foci and compartmentalise nesprin-2/ERK signalling. However, nesprin-2/ERK signalling fidelity, but not their compartmentalisation at PML NBs, is essential for efficient DDR in VSMCs. PMID:25744025

  17. Hydrogen peroxide-induced extracellular signal-regulated kinase activation in cultured feline ileal smooth muscle cells.

    PubMed

    Song, Hyun Ju; Lee, Tai Sang; Jeong, Ji Hoon; Min, Young Sil; Shin, Chang Yell; Sohn, Uy Dong

    2005-01-01

    H(2)O(2) has been shown to act as a signaling molecule involved in many cellular functions such as apoptosis and proliferation. In the present study, we characterized the effects of H(2)O(2) on the activation of mitogen-activated protein (MAP) kinases and examined the factors involved in the process of extracellular signal-regulated kinase (ERK) activation by H(2)O(2) in ileal smooth muscle cells (ISMC). ISMC were cultured and exposed to H(2)O(2). Western blot analysis was performed with phosphospecific MAP kinase antibodies. Potent activation of ERK and moderate activation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase occurred within 30 min of 1 mM H(2)O(2) treatment. However, p38 MAP kinase was not activated by H(2)O(2). The activation of ERK by H(2)O(2) was reduced by the mitogen-activated/ERK-activating kinase inhibitor PD98059 [2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one], Ras inhibitor S-farnesylthiosalicylic acid, removal of extracellular Ca(2+), depletion of the intracellular Ca(2+) pool by thapsigargin, or pretreatment of ISMC with the calmodulin antagonist W-7. Also, H(2)O(2)-induced ERK activation was attenuated by a receptor tyrosine kinase inhibitor, tyrphostin 51, but not by down-regulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or by a PKC inhibitor, GF109203X [3-[1-(dimethylaminopropyl)indol-3-yl]-4-(indol-3-yl)maleimide hydrochloride]. Growth factor receptor antagonist suramin pretreatment inhibited H(2)O(2)-induced ERK activation, highlighting a role for growth factor receptors in this activation. Furthermore, the ERK activation by H(2)O(2) was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol indicating that metal-catalyzed free radical formation may mediate the initiation of signal transduction by H(2)O(2). These data suggest that short-term stimulation with H(2)O(2) activates the signaling pathways of cell mitogenic effects which are thought to be a protective

  18. Shenmai injection inhibiting the extracellular signal regulated kinase-induced human airway smooth muscle proliferation in asthma.

    PubMed

    Zhao, Li-min; Ma, Li-jun; Zhang, Luo-xian; Wu, Ji-zhen

    2010-08-01

    To investigate the relationship between the proliferation of sensitized human airway smooth muscle cells (HASMCs) and the expression of extracellular signal regulated kinase (ERK) and the effect of Shenmai Injection (SMI) on HASMCs. The HASMCs cultured in vitro were divided into three groups: (1) control group; (2) sensitized group: containing 10% asthmatic serum; (3) SMI group: further divided into three different concentration subgroups interferred with 10 microL/mL, 50 microL/mL, and 100 microL/mL SMI, respectively. The proliferation of HASMCs was detected using MTT method, the expression of proliferating cell nucleus antigen (PCNA) in HASMCs was detected using immunocytochemical staining, and the expression of phosphoration-ERK1/2 (p-ERK1/2) protein was detected using Western-blot. After passive sensitization,: the optical density value (A A(490) value) of HASMCs was significantly increased from 0.366+/-0.086 to 0.839+/- 0.168 (P<0.05). In addition, the expression of PCNA was significantly increased from 28.7%+/-5.9% in the control group to 69.8%+/-7.5% in the sensitized group (P<0.05). At the same time, the expression of p-ERK1/2 in passively sensitized HASMCs was significantly increased compared with the control group (all P<0.05). After application of 10 microL/mL, 50 microL/mL, and 100 microL/mL SMI to the cultured media of passively sensitized group, the A(570) value was significantly decreased from 0.839+/-0.168 to 0.612+/-0.100, 0.412+/-0.092, and 0.339+/-0.077, respectively (P<0.05). Moreover, the expression of PCNA was significantly decreased from 69.8%+/-7.5% to 57.8%+/-6.2%, 40.7%+/-5.4%, and 26.1%+/-5.2%, respectively. At the same time, the expression of p-ERK1/2 in each SMI group was significantly decreased compared with the sensitized group (all P<0.05). ERK signal transduction pathway may be involved in the airway remodeling in asthma. The expression of ERK can be inhibited by SMI in a dose-dependent manner, thus preventing the proliferation of

  19. Binding of the P2Y2 Nucleotide Receptor to Filamin A Regulates Migration of Vascular Smooth Muscle Cells

    PubMed Central

    Yu, Ningpu; Erb, Laurie; Shivaji, Rikka; Weisman, Gary A.; Seye, Cheikh I.

    2013-01-01

    The functional expression of the G protein– coupled P2Y2 nucleotide receptor (P2Y2R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), 2 processes involved in atherosclerosis and restenosis. Activation of the P2Y2R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y2Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y2R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y2R as bait. The FLNa binding site in the P2Y2R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y2R and abolished Tyr phosphorylation of FLNa induced by the P2Y2R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P≤0.01) and migration (3.6 fold; P≤0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y2R knockout mice. Expression of the full-length P2Y2R in SMCs isolated from P2Y2R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y2R knockout mice transfected with a mutant P2Y2R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 µmol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y2R knockout mice. Thus, this study demonstrates that P2Y2R/FLNa interaction selectively regulates spreading and migration of vascular SMCs. PMID:18202316

  20. Coronary Artery Disease Associated Transcription Factor TCF21 Regulates Smooth Muscle Precursor Cells That Contribute to the Fibrous Cap

    PubMed Central

    Raiesdana, Azad; Kundu, Ramendra; Miller, Clint L.; Kim, Juyong B.; Arora, Komal; Carcamo-Oribe, Ivan; Xiong, Yiqin; Tellakula, Nikhil; Nanda, Vivek; Murthy, Nikitha; Boisvert, William A.; Hedin, Ulf; Perisic, Ljubica; Aldi, Silvia; Maegdefessel, Lars; Pjanic, Milos; Owens, Gary K.; Tallquist, Michelle D.; Quertermous, Thomas

    2015-01-01

    Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including “vascular disease,” “disorder of artery,” and “occlusion of artery,” as well as disease-related cellular functions including “cellular movement” and “cellular growth and proliferation.” In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide

  1. Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells.

    PubMed

    Brun, Paola; Gobbo, Serena; Caputi, Valentina; Spagnol, Lisa; Schirato, Giulia; Pasqualin, Matteo; Levorato, Elia; Palù, Giorgio; Giron, Maria Cecilia; Castagliuolo, Ignazio

    2015-09-01

    Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2(-/-)) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2(-/-) mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2(-/-) mice exhibited smaller ganglia, fewer HuC/D(+ve) and nNOS(+ve) neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with

  2. MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism

    PubMed Central

    Yu, Dan; Makkar, George; Dong, Tuo; Strickland, Dudley K.; Sarkar, Rajabrata; Monahan, Thomas Stacey

    2015-01-01

    Background Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo. Methods and Results siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization. Conclusions MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular

  3. Differential regulation of phosphoinositide metabolism by alphaVbeta3 and alphaVbeta5 integrins upon smooth muscle cell migration.

    PubMed

    Paulhe, F; Racaud-Sultan, C; Ragab, A; Albiges-Rizo, C; Chap, H; Iberg, N; Morand, O; Perret, B

    2001-11-09

    Smooth muscle cell migration is a key step of atherosclerosis and angiogenesis. We demonstrate that alpha(V)beta(3) and alpha(V)beta(5) integrins synergistically regulate smooth muscle cell migration onto vitronectin. Using an original haptotactic cell migration assay, we measured a strong stimulation of phosphoinositide metabolism in migrating vascular smooth muscle cells. Phosphatidic acid production and phosphoinositide 3-kinase IA activation were triggered only upon alpha(V)beta(3) engagement. Blockade of alpha(V)beta(3) engagement or phospholipase C activity resulted in a strong inhibition of smooth muscle cell spreading on vitronectin. By contrast, blockade of alpha(V)beta(5) reinforced elongation and polarization of cell shape. Moreover, Pyk2-associated tyrosine kinase and phosphoinositide 4-kinase activities measured in Pyk2 immunoprecipitates were stimulated upon cell migration. Blockade of either alpha(V)beta(3) or alpha(V)beta(5) function, as well as inhibition of phospholipase C activity, decreased both Pyk2-associated activities. We demonstrated that the Pyk2-associated phosphoinositide 4-kinase corresponded to the beta isoform. Our data point to the metabolism of phosphoinositides as a regulatory pathway for the differential roles played by alpha(V)beta(3) and alpha(V)beta(5) upon cell migration and identify the Pyk2-associated phosphoinositide 4-kinase beta as a common target for both integrins.

  4. Hydrogen peroxide-and fetal bovine serum-induced DNA synthesis in vascular smooth muscle cells: positive and negative regulation by protein kinase C isoforms.

    PubMed

    Fiorani, M; Cantoni, O; Tasinato, A; Boscoboinik, D; Azzi, A

    1995-10-19

    Hydrogen peroxide and fetal bovine serum stimulate DNA synthesis in growth-arrested smooth muscle cells with remarkably similar kinetics and cell density dependence. However, while stimulation with fetal bovine serum results in cell proliferation, that by H2O2 is followed by cell death. Depletion of conventional and novel protein kinase C isoforms, resulting from a long treatment with phorbol-12-myristate-13-acetate, further increases H2O2-induced DNA synthesis. On the other hand, the specific protein kinase C inhibitor calphostin C abolished the increased DNA synthesis promoted by fetal bovine serum or H2O2. H2O2 increases protein kinase C activity in smooth muscle cells. This effect is markedly reduced, but not abolished, by down-regulation of the alpha, delta and epsilon protein kinase C isoforms. Thus, the zeta isoform of protein kinase C, which is not down-regulated, may be responsible for the residual H2O2 stimulation of protein kinase C. In conclusion, the results obtained show that H2O2 stimulates protein kinase C activity and DNA synthesis in growth-arrested smooth muscle cells: these events are not followed by cell proliferation but rather by cell death. This H2O2 stimulated DNA synthesis appears to be negatively controlled by alpha, delta and epsilon isoforms and positively controlled by the zeta isoform of protein kinase C.

  5. Sexual Dimorphism in the Regulation of Estrogen, Progesterone, and Androgen Receptors by Sex Steroids in the Rat Airway Smooth Muscle Cells.

    PubMed

    Zarazúa, Abraham; González-Arenas, Aliesha; Ramírez-Vélez, Gabriela; Bazán-Perkins, Blanca; Guerra-Araiza, Christian; Campos-Lara, María G

    2016-01-01

    The role of sex hormones in lung is known. The three main sex steroid receptors, estrogen, progesterone, and androgen, have not been sufficiently studied in airway smooth muscle cells (ASMC), and the sex hormone regulation on these receptors is unknown. We examined the presence and regulation of sex hormone receptors in female and male rat ASMC by Western blotting and flow cytometry. Gonadectomized rats were treated with 17β-estradiol, progesterone, 17β-estradiol + progesterone, or testosterone. ASMC were enzymatically isolated from tracheas and bronchi. The experiments were performed with double staining flow cytometry (anti-α-actin smooth muscle and antibodies to each hormone receptor). ERα, ERβ, tPR, and AR were detected in females or males. ERα was upregulated by E2 and T and downregulated by P4 in females; in males, ERα was downregulated by P4, E + P, and T. ERβ was downregulated by each treatment in females, and only by E + P and T in males. tPR was downregulated by P4, E + P, and T in females. No hormonal regulation was observed in male receptors. AR was downregulated in males treated with E + P and T. We have shown the occurrence of sex hormone receptors in ASMC and their regulation by the sex hormones in female and male rats.

  6. Sexual Dimorphism in the Regulation of Estrogen, Progesterone, and Androgen Receptors by Sex Steroids in the Rat Airway Smooth Muscle Cells

    PubMed Central

    Zarazúa, Abraham; González-Arenas, Aliesha; Ramírez-Vélez, Gabriela; Bazán-Perkins, Blanca; Guerra-Araiza, Christian; Campos-Lara, María G.

    2016-01-01

    The role of sex hormones in lung is known. The three main sex steroid receptors, estrogen, progesterone, and androgen, have not been sufficiently studied in airway smooth muscle cells (ASMC), and the sex hormone regulation on these receptors is unknown. We examined the presence and regulation of sex hormone receptors in female and male rat ASMC by Western blotting and flow cytometry. Gonadectomized rats were treated with 17β-estradiol, progesterone, 17β-estradiol + progesterone, or testosterone. ASMC were enzymatically isolated from tracheas and bronchi. The experiments were performed with double staining flow cytometry (anti-α-actin smooth muscle and antibodies to each hormone receptor). ERα, ERβ, tPR, and AR were detected in females or males. ERα was upregulated by E2 and T and downregulated by P4 in females; in males, ERα was downregulated by P4, E + P, and T. ERβ was downregulated by each treatment in females, and only by E + P and T in males. tPR was downregulated by P4, E + P, and T in females. No hormonal regulation was observed in male receptors. AR was downregulated in males treated with E + P and T. We have shown the occurrence of sex hormone receptors in ASMC and their regulation by the sex hormones in female and male rats. PMID:27110242

  7. Phosphodiesterase 3B is localized in caveolae and smooth ER in mouse hepatocytes and is important in the regulation of glucose and lipid metabolism.

    PubMed

    Berger, Karin; Lindh, Rebecka; Wierup, Nils; Zmuda-Trzebiatowska, Emilia; Lindqvist, Andreas; Manganiello, Vincent C; Degerman, Eva

    2009-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction processes mediated by cAMP and cGMP. One PDE family member, PDE3B, plays an important role in the regulation of a variety of metabolic processes such as lipolysis and insulin secretion. In this study, the cellular localization and the role of PDE3B in the regulation of triglyceride, cholesterol and glucose metabolism in hepatocytes were investigated. PDE3B was identified in caveolae, specific regions in the plasma membrane, and smooth endoplasmic reticulum. In caveolin-1 knock out mice, which lack caveolae, the amount of PDE3B protein and activity were reduced indicating a role of caveolin-1/caveolae in the stabilization of enzyme protein. Hepatocytes from PDE3B knock out mice displayed increased glucose, triglyceride and cholesterol levels, which was associated with increased expression of gluconeogenic and lipogenic genes/enzymes including, phosphoenolpyruvate carboxykinase, peroxisome proliferator-activated receptor gamma, sterol regulatory element-binding protein 1c and hydroxyl-3-methylglutaryl coenzyme A reductase. In conclusion, hepatocyte PDE3B is localized in caveolae and smooth endoplasmic reticulum and plays important roles in the regulation of glucose, triglyceride and cholesterol metabolism. Dysregulation of PDE3B could have a role in the development of fatty liver, a condition highly relevant in the context of type 2 diabetes.

  8. Regulation of intracellular pH in the smooth muscle of guinea-pig ureter: Na+ dependence.

    PubMed Central

    Aickin, C C

    1994-01-01

    1. Mechanisms involved in the regulation of intracellular pH (pHi) in smooth muscle cells of guinea-pig ureter have been investigated using double-barrelled pH-sensitive microelectrodes in isolated strips of tissue. 2. Removal of CO2-HCO3- from the superfusing solution caused a fall in the steady-state pHi except in a few cells which had been excised from the animal for many hours (usually > 24 h). The pHi value was 7.22 +/- 0.09 (n = 89; mean +/- S.D. of an observation) in solution buffered with 5% CO2-21 mM HCO3-, compared with 6.92 +/- 0.24 (n = 67) in the nominal absence of CO2-HCO3-. Recovery from experimentally induced acidosis was faster in the presence, rather than nominal absence, of CO2-HCO3- (mean half-times of 2.7 +/- 0.7 min, n = 41, and 4.6 +/- 1.3 min, n = 12, respectively). These results suggest the presence of both HCO(3-)-dependent and -independent mechanisms for the effective extrusion of acid equivalents. 3. Recovery from acidosis was dependent on external Na+ (Na+o) in both the presence and nominal absence of CO2-HCO3-, with an apparent half-maximal activation at approximately 4 and 20 mM Na+o, respectively. Removal of Na+o in the steady state caused a fall in pHi which proceeded at a faster rate in the presence rather than in the nominal absence of CO2-HCO3-. 4. Amiloride (100 microM-1 mM) reversibly inhibited the recovery from acidosis and caused a fall in the steady-state pHi when applied in the nominal absence of CO2-HCO3-, but had no measurable effect on either the recovery from acidosis or steady-state pHi in the presence of CO2-HCO3-. These results suggest that Na(+)-H+ exchange was responsible for extrusion of acid equivalents in the nominal absence of CO2 and HCO3-, but that it played little part under more physiological conditions. 5. Although Na(+)-H+ exchange appeared to be activated below a pHi of about 7.2, it was incapable of maintaining a 'normal' pHi in the nominal absence of CO2-HCO3- in freshly excised cells, where values

  9. microRNA expression profile in human coronary smooth muscle cell-derived microparticles is a source of biomarkers.

    PubMed

    de Gonzalo-Calvo, David; Cenarro, Ana; Civeira, Fernando; Llorente-Cortes, Vicenta

    2016-01-01

    microRNA (miRNA) expression profile of extracellular vesicles is a potential tool for clinical practice. Despite the key role of vascular smooth muscle cells (VSMC) in cardiovascular pathology, there is limited information about the presence of miRNAs in microparticles secreted by this cell type, including human coronary artery smooth muscle cells (HCASMC). Here, we tested whether HCASMC-derived microparticles contain miRNAs and the value of these miRNAs as biomarkers. HCASMC and explants from atherosclerotic or non-atherosclerotic areas were obtained from coronary arteries of patients undergoing heart transplant. Plasma samples were collected from: normocholesterolemic controls (N=12) and familial hypercholesterolemia (FH) patients (N=12). Both groups were strictly matched for age, sex and cardiovascular risk factors. Microparticle (0.1-1μm) isolation and characterization was performed using standard techniques. VSMC-enriched miRNAs expression (miR-21-5p, -143-3p, -145-5p, -221-3p and -222-3p) was analyzed using RT-qPCR. Total RNA isolated from HCASMC-derived microparticles contained small RNAs, including VSMC-enriched miRNAs. Exposition of HCASMC to pathophysiological conditions, such as hypercholesterolemia, induced a decrease in the expression level of miR-143-3p and miR-222-3p in microparticles, not in cells. Expression levels of miR-222-3p were lower in circulating microparticles from FH patients compared to normocholesterolemic controls. Microparticles derived from atherosclerotic plaque areas showed a decreased level of miR-143-3p and miR-222-3p compared to non-atherosclerotic areas. We demonstrated for the first time that microparticles secreted by HCASMC contain microRNAs. Hypercholesterolemia alters the microRNA profile of HCASMC-derived microparticles. The miRNA signature of HCASMC-derived microparticles is a source of cardiovascular biomarkers. Copyright © 2016 Sociedad Española de Arteriosclerosis. Publicado por Elsevier España, S.L.U. All rights

  10. 7 CFR 51.1159 - Smooth texture.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Smooth texture. 51.1159 Section 51.1159 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.1159 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and...

  11. 7 CFR 51.636 - Smooth texture.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Smooth texture. 51.636 Section 51.636 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.636 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and...

  12. 7 CFR 51.1159 - Smooth texture.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Smooth texture. 51.1159 Section 51.1159 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.1159 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and...

  13. 7 CFR 51.636 - Smooth texture.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Smooth texture. 51.636 Section 51.636 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.636 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and...

  14. 7 CFR 51.636 - Smooth texture.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Smooth texture. 51.636 Section 51.636 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.636 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and...

  15. 7 CFR 51.698 - Smooth texture.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Smooth texture. 51.698 Section 51.698 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.698 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and size...

  16. 7 CFR 51.698 - Smooth texture.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Smooth texture. 51.698 Section 51.698 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.698 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and size...

  17. 7 CFR 51.698 - Smooth texture.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Smooth texture. 51.698 Section 51.698 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.698 Smooth texture. Smooth texture means that the skin is thin and smooth for the variety and size...

  18. The smooth muscle-selective RhoGAP GRAF3 is a critical regulator of vascular tone and hypertension

    PubMed Central

    Bai, Xue; Lenhart, Kaitlin C.; Bird, Kim E.; Suen, Alisa A.; Rojas, Mauricio; Kakoki, Masao; Li, Feng; Smithies, Oliver; Mack, Christopher P.; Taylor, Joan M.

    2014-01-01

    Although hypertension is a worldwide health issue, an incomplete understanding of its etiology has hindered our ability to treat this complex disease. Here we identify arhgap42 (also known as GRAF3) as a Rho-specific GAP expressed specifically in smooth muscle cells in mice and humans. We show that GRAF3-deficient mice exhibit significant hypertension and increased pressor responses to angiotensin II and endothelin-1; these effects are prevented by treatment with the Rho-kinase inhibitor, Y-27632. RhoA activity and myosin light chain phosphorylation are elevated in GRAF3-depleted smooth muscle cells in vitro and in vivo, and isolated vessel segments from GRAF3-deficient mice show increased contractility. Taken together our data indicate that GRAF3-mediated inhibition of RhoA activity in vascular smooth muscle cells is necessary for maintaining normal blood pressure homeostasis. Moreover, these findings provide a potential mechanism for a hypertensive locus recently identified within arhgap42 and provide a foundation for the future development of innovative hypertension therapies. PMID:24335996

  19. Rare Copy Number Variants Disrupt Genes Regulating Vascular Smooth Muscle Cell Adhesion and Contractility in Sporadic Thoracic Aortic Aneurysms and Dissections

    PubMed Central

    Prakash, Siddharth K.; LeMaire, Scott A.; Guo, Dong-Chuan; Russell, Ludivine; Regalado, Ellen S.; Golabbakhsh, Hossein; Johnson, Ralph J.; Safi, Hazim J.; Estrera, Anthony L.; Coselli, Joseph S.; Bray, Molly S.; Leal, Suzanne M.; Milewicz, Dianna M.; Belmont, John W.

    2010-01-01

    Thoracic aortic aneurysms and dissections (TAAD) cause significant morbidity and mortality, but the genetic origins of TAAD remain largely unknown. In a genome-wide analysis of 418 sporadic TAAD cases, we identified 47 copy number variant (CNV) regions that were enriched in or unique to TAAD patients compared to population controls. Gene ontology, expression profiling, and network analysis showed that genes within TAAD CNVs regulate smooth muscle cell adhesion or contractility and interact with the smooth muscle-specific isoforms of α-actin and β-myosin, which are known to cause familial TAAD when altered. Enrichment of these gene functions in rare CNVs was replicated in independent cohorts with sporadic TAAD (STAAD, n = 387) and inherited TAAD (FTAAD, n = 88). The overall prevalence of rare CNVs (23%) was significantly increased in FTAAD compared with STAAD patients (Fisher's exact test, p = 0.03). Our findings suggest that rare CNVs disrupting smooth muscle adhesion or contraction contribute to both sporadic and familial disease. PMID:21092924

  20. Intravesical treatment of advanced urothelial bladder cancers with oncolytic HSV-1 co-regulated by differentially expressed microRNAs.

    PubMed

    Zhang, K-X; Matsui, Y; Lee, C; Osamu, O; Skinner, L; Wang, J; So, A; Rennie, P S; Jia, W W

    2016-05-01

    Urothelial bladder cancer is the most common malignancy of the urinary tract. Although most cases are initially diagnosed as non-muscle-invasive, more than 80% of patients will develop recurrent or metastatic tumors. No effective therapy exists currently for late-stage metastatic tumors. By intravesical application, local administration of oncolytic Herpes Simplex virus (oHSV-1) can provide a promising new therapy for this disease. However, its inherent neurotoxicity has been a perceived limitation for such application. In this study, we present a novel microRNA-regulatory approach to reduce HSV-1-induced neurotoxicity by suppressing viral replication in neurons while maintaining oncolytic selectivity toward urothelial tumors. Specifically, we designed a recombinant virus that utilizes differentially expressed endogenous microR143 (non-cancerous, ubiquitous) and microR124 (neural-specific) to regulate expression of ICP-4, a gene essential for HSV-1 replication. We found that expression of ICP-4 must be controlled by a combination of both miR143 and miR124 to achieve the most effective attenuation in HSV-1-induced toxicity while retaining maximal oncolytic capacity. These results suggest that interaction between miR143 and miR124 may be required to successfully regulate HSV-1 replication. Our resent study is the first proof-in-principle that miRNA combination can be exploited to fine-tune the replication of HSV-1 to treat human cancers.

  1. The platelet-derived growth factor receptor/STAT3 signaling pathway regulates the phenotypic transition of corpus cavernosum smooth muscle in rats

    PubMed Central

    Zhao, Jian-Feng; Fu, Hui-Ying; Zhang, Gao-Yue; Huang, Xiao-Jun; Lv, Bo-Dong

    2017-01-01

    Erectile dysfunction (ED) is a common clinical disease that is difficult to treat. We previously found that hypoxia modulates the phenotype of primary corpus cavernosum smooth muscle cells (CCSMCs) in rats, but the underlying molecular mechanism is still unknown. Platelet-derived growth factor receptor (PDGFR)-related signaling pathways are correlated with cell phenotypic transition, but research has been focused more on vascular smooth muscle and tracheal smooth muscle and less on CCSMCs. Here, we investigated the role of PDGFR-related signaling pathways in penile CCSMCs, which were successfully isolated from rats and cultured in vitro. PDGF-BB at 5, 10, or 20 ng/ml altered CCSMC morphology from the original elongated, spindle shape to a broader shape and promoted the synthetic phenotype and expression of the related proteins vimentin and collagen-I, while inhibiting the contractile phenotype and expression of the related proteins smooth muscle (SM) α-actin (α-SMA) and desmin. Inhibition of PDGFR activity via siRNA or the PDGFR inhibitor crenolanib inhibited vimentin and collagen-I expression, increased α-SMA and desmin expression, and considerably inhibited serine-threonine protein kinase (AKT) and signal transducer and activator of transcription 3 (STAT3) phosphorylation. STAT3 knockdown promoted the contractile phenotype, inhibited vimentin and collagen-I expression, and increased α-SMA and desmin expression, whereas AKT knockdown did not affect phenotype-associated proteins. STAT3 overexpression in CCSMC cells weakened the suppressive effect of PDGFR inhibition on the morphology and phenotypic transformation induced by PDGF-BB. Through activation of the PDGFR/STAT3 signaling pathway, PDGF promoted the synthetic phenotype transition; thus, regulation of this pathway might contribute to ED therapy. PMID:28245285

  2. INTERMEDIATE FILAMENTS IN SMOOTH MUSCLE

    PubMed Central

    Tang, Dale D.

    2008-01-01

    The intermediate filament (IF) network is one of the three cytoskeletal systems in smooth muscle. The type III IF proteins vimentin and desmin are major constituents of the network in smooth muscle cells and tissues. Lack of vimentin or desmin impairs contractile ability of various smooth muscle preparations, implying their important role for smooth muscle force development. The IF framework has long been viewed as a fixed cytostructure that solely provides mechanical integrity for the cell. However, recent studies suggest that the IF cytoskeleton is dynamic in mammalian cells in response to various external stimulation. In this review, the structure and biological properties of IF proteins in smooth muscle are summarized. The role of IF proteins in the modulation of smooth muscle force development and redistribution/translocation of signaling partners (such as p130 Crk-associated substrate, CAS) is depicted. This review also summarizes our latest understanding on how the IF network may be regulated in smooth muscle. PMID:18256275

  3. PKC-DEPENDENT REGULATION OF Kv7.5 CHANNELS BY THE BRONCHOCONSTRICTOR HISTAMINE IN HUMAN AIRWAY SMOOTH MUSCLE CELLS.

    PubMed

    Haick, Jennifer M; Brueggemann, Lioubov I; Cribbs, Leanne L; Denning, Mitchell F; Schwartz, Jeffrey; Byron, Kenneth L

    2017-03-10

    Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, though the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.

  4. Angiotensin II Downregulates MicroRNA-145 to Regulate Kruppel-like Factor 4 and Myocardin Expression in Human Coronary Arterial Smooth Muscle Cells under High Glucose Conditions

    PubMed Central

    Shyu, Kou-Gi; Cheng, Wen-Ping; Wang, Bao-Wei

    2015-01-01

    MicroRNA (miR)-145 is the most abundant miR in vascular smooth muscle cells (VSMCs). However, the effect of hyperglycemia on the regulation of miR-145 is unknown. We hypothesized that the hyperglycemic condition activates a proinflammatory response that mediates the expression of miR-145 in VSMCs. We investigated whether miR-145 serves as a critical regulator to regulate the downstream proliferation factors (including Kruppel-like factor 4 [Klf4] and myocardin) in VSMCs under hyperglycemic conditions. Human coronary artery smooth muscle cells (HCASMCs) were cultured under high glucose conditions. Sustained high glucose at 25 mmol/L significantly decreased the expression of miR-145 in HCASMCs. High glucose significantly increased angiotensin II (Ang II) secretion from HCASMCs and Ang II suppressed miR-145 expression in HCASMCs. Ang II repression of miR145 expression resulted in increased Klf4 and decreased myocardin expression under conditions of high glucose. Overexpression of miR-145 significantly decreased Klf4 and increased myocardin expression and inhibited HCASMC proliferation and migration induced by a high glucose state. Balloon injury of the carotid artery in diabetic rats was performed to investigate miR-145, Klf and myocardin expression. The expression of miR-145 was maximally increased at 7 d after carotid injury and gradually declined thereafter. Overexpression of miR-145 and treatment with valsartan reversed Klf4 and myocardin protein expression induced by balloon injury and improved vascular injury. In conclusion, our study reveals that Ang II downregulates miR-145 to regulate Klf4 and myocardin expression in HCASMCs under high glucose conditions. Ang II plays a critical role in the regulation of miR-145 under hyperglycemic conditions. PMID:26181633

  5. TRPA1-dependent regulation of bladder detrusor smooth muscle contractility in normal and type I diabetic rats

    PubMed Central

    Philyppov, Igor B.; Paduraru, Oksana N.; Gulak, Kseniya L.; Skryma, Roman; Prevarskaya, Natalia; Shuba, Yaroslav M.

    2016-01-01

    TRPA1 is a Ca2+-permeable cation channel that is activated by painful low temperatures (˂17 °C), irritating chemicals, reactive metabolites and mediators of inflammation. In the bladder TRPA1 is predominantly expressed in sensory afferent nerve endings, where it mediates sensory transduction. The contractile effect of its activation on detrusor smooth muscle (DSM) is explained by the release from sensory afferents of inflammatory factors – tachykinins and prostaglandins, which cause smooth muscle cell contraction. Diabetes is a systemic disease, with common complications being diabetic cystopathies and urinary incontinence. However, data on how diabetes affects bladder contractility associated with TRPA1 activation are not available. In this study, by using a rat model with streptozotocin-induced type I diabetes, contractility measurements of DSM strips in response to TRPA1-activating and modulating pharmacological agents and assessment of TRPA1 mRNA expression in bladder-innervating dorsal root ganglia, we have shown that diabetes enhances the TRPA1-dependent mechanism involved in bladder DSM contractility. This is not due to changes in TRPA1 expression, but mainly due to the general inflammatory reaction caused by diabetes. The latter leads to an increase in cyclooxygenase-2-dependent prostaglandin synthesis through the mechanisms associated with substance P activity. This results in the enhanced functional coupling between the tachykinin and prostanoid systems, and the concomitant increase of their impact on DSM contractility in response to TRPA1 activation. PMID:26935999

  6. [THE ROLE OF HYDROGEN SULFIDE IN VOLUME-DEPENDENT MECHANISMS OF REGULATION OF VASCULAR SMOOTH MUSCLE CELLS CONTRACTILE ACTIVITY].

    PubMed

    Smagliy, L V; Gusakova, S V; Birulina, Yu G; Kovalev, I V; Orlov, S N

    2015-04-01

    The hydrogen sulfide (H2S) influence on the contractile activity of vascular smooth muscle cells (SMC) was studied on endothelium-denuded aortic ring segments of male Wistar rats with method of mechanography. Contractions of SMS were induced by incubation in high potassium solution as well as in hyper-, hypo- and isosmotic solutions. 5-100 LM of H2S donor--sodium hydrosulfide (NaHS) increased mechanical tension of SMC precontracted with high potassium solution that was abolished by bumetanide--the inhibitor of Na+, K+, 2Cl(-) -cotransporter (NKCC), but 100-1000 microM of NaHS relaxed SMS. NaHS (10 microM) increased the amplitude of hyper- and isosmotic contraction, but not of hyposmotic contraction. NaHS (ImM) decreased the amplitude of hyper-, iso-, and hyposmotic contractions. The direct measurements of NKCC activity with radionuclide method showed an increase in NKCC activity under the action of 5-100 microM of NaHS. These findings suggest that low concentrations of H2S participate in the NKCC activation. This mechanism underlines constrictive action of H2S on smooth muscle cells.

  7. TRPA1-dependent regulation of bladder detrusor smooth muscle contractility in normal and type I diabetic rats.

    PubMed

    Philyppov, Igor B; Paduraru, Oksana N; Gulak, Kseniya L; Skryma, Roman; Prevarskaya, Natalia; Shuba, Yaroslav M

    2016-01-01

    TRPA1 is a Ca(2+)-permeable cation channel that is activated by painful low temperatures (<17°C), irritating chemicals, reactive metabolites and mediators of inflammation. In the bladder TRPA1 is predominantly expressed in sensory afferent nerve endings, where it mediates sensory transduction. The contractile effect of its activation on detrusor smooth muscle (DSM) is explained by the release from sensory afferents of inflammatory factors - tachykinins and prostaglandins, which cause smooth muscle cell contraction. Diabetes is a systemic disease, with common complications being diabetic cystopathies and urinary incontinence. However, data on how diabetes affects bladder contractility associated with TRPA1 activation are not available. In this study, by using a rat model with streptozotocin-induced type I diabetes, contractility measurements of DSM strips in response to TRPA1-activating and modulating pharmacological agents and assessment of TRPA1 mRNA expression in bladder-innervating dorsal root ganglia, we have shown that diabetes enhances the TRPA1-dependent mechanism involved in bladder DSM contractility. This is not due to changes in TRPA1 expression, but mainly due to the general inflammatory reaction caused by diabetes. The latter leads to an increase in cyclooxygenase-2-dependent prostaglandin synthesis through the mechanisms associated with substance P activity. This results in the enhanced functional coupling between the tachykinin and prostanoid systems, and the concomitant increase of their impact on DSM contractility in response to TRPA1 activation.

  8. A nonparametric mean-variance smoothing method to assess Arabidopsis cold stress transcriptional regulator CBF2 overexpression microarray data.

    PubMed

    Hu, Pingsha; Maiti, Tapabrata

    2011-01-01

    Microarray is a powerful tool for genome-wide gene expression analysis. In microarray expression data, often mean and variance have certain relationships. We present a non-parametric mean-variance smoothing method (NPMVS) to analyze differentially expressed genes. In this method, a nonlinear smoothing curve is fitted to estimate the relationship between mean and variance. Inference is then made upon shrinkage estimation of posterior means assuming variances are known. Different methods have been applied to simulated datasets, in which a variety of mean and variance relationships were imposed. The simulation study showed that NPMVS outperformed the other two popular shrinkage estimation methods in some mean-variance relationships; and NPMVS was competitive with the two methods in other relationships. A real biological dataset, in which a cold stress transcription factor gene, CBF2, was overexpressed, has also been analyzed with the three methods. Gene ontology and cis-element analysis showed that NPMVS identified more cold and stress responsive genes than the other two methods did. The good performance of NPMVS is mainly due to its shrinkage estimation for both means and variances. In addition, NPMVS exploits a non-parametric regression between mean and variance, instead of assuming a specific parametric relationship between mean and variance. The source code written in R is available from the authors on request.

  9. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling

    PubMed Central

    Saddouk, Fatima Z.; Sun, Li-Yan; Liu, Yong Feng; Jiang, Miao; Singer, Diane V.; Backs, Johannes; Van Riper, Dee; Ginnan, Roman; Schwarz, John J.; Singer, Harold A.

    2016-01-01

    Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions.—Saddouk, F. Z., Sun, L.-Y., Liu, Y. F., Jiang, M., Singer, D. V., Backs, J., Van Riper, D., Ginnan, R., Schwarz, J. J., Singer, H. A. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling. PMID:26567004

  10. AB283. SPR-10 Down-regulation of ryanodine receptor gene expression in murine urinary bladder smooth muscle following partial bladder outlet obstruction

    PubMed Central

    Boopathi, Ettickan; Javed, Elham; Addya, Shankar; Fortina, Paolo; Zderic, Stephen; Wein, Alan; Chacko, Samuel

    2016-01-01

    Objective Urinary bladder smooth muscle (UBSM) displays spontaneous action potentials and this potential is related to the phasic nature of spontaneous contractions in this tissue. The amplitude of a phasic contraction depends on the increase in Ca2+ entry caused by membrane depolarization. Ryanodine receptors (RyRs) in UBSM decreases the force production by decreasing the frequency of phasic contractions through interactions with large-conductance Ca2+-activated K+ (BK) and small-conductance Ca2+-activated K+ (SK) channels. Microarray and network analysis were employed to determine the changes in mRNA in 14-day obstructed murine bladders. We found that obstruction significantly down-regulated the RyRs in bladder smooth muscle (BSM). Methods Male C57Bl/6 mice were surgically obstructed and kept for 14 days. Sham-operated mice served as a control. Bladders were excised; urothelium scraped off with a scalpel, and the serosa was removed. BSM obtained from PBOO and sham control animals were used for microarray and western blotting Results Pathway-based analysis of these gene signatures showed significant number of under-expressed genes in obstructed bladder and they were mapped to proteins involved in calcium signaling. We focused our work on RyR protein expression in BSM. There was a four-fold reduction of RyR3 in BSM in 14-day obstructed groups as shown by microarray and immunoblotting compared to that of sham-operated animals. Conclusions These results confirm that the RyR gene expression is down-regulated in obstructed murine bladder smooth muscle. Funding Source(s) None

  11. The cAMP effector EPAC activates Elk1 transcription factor in prostate smooth muscle, and is a minor regulator of α1-adrenergic contraction

    PubMed Central

    2013-01-01

    Background Prostate smooth muscle tone is regulated by α1-adrenoceptor-induced contraction and cAMP-mediated relaxation. EPAC is an effector of cAMP, being involved in smooth muscle relaxation and cell cycle control outside the lower urinary tract. Here, we investigated the expression and function of EPAC in human prostate tissues from patients undergoing radical prostatectomy. Results mRNA and protein expression of EPAC was detected in all prostate tissues by RT-PCR and Western blot analysis. Immunoreactivity was observed in stromal cells, and colocalized with immunofluorescence for α-smooth muscle actin and calponin. Under normal conditions, noradrenaline- or phenylephrine-induced contraction of prostate strips in the organ bath was not affected by the EPAC activator pCPT (SP-8-pCPT-2′-O-Me-cAMPS.NA) (30 μM). However, when the cyclooxygenase inhibitor indomethacin (50 μM) was added, EPAC activators pCPT and OME (8-CPT-2′-O-Me-cAMP.Na) (30 μM) significantly reduced contractions by low concentrations of phenylephrine. These effects were not observed on noradrenaline-induced contraction. OME and pCPT caused phosphorylation of the transcription factor Elk1 in prostate tissues. Elk1 activation was confirmed by EMSA (electrophoretic mobility shift assay), where OME and pCPT incresed Elk1 binding to a specific DNA probe. Conclusions EPAC activation may reduce α1-adrenergic prostate contraction in the human prostate, although this effect is masked by cyclooxygenases and β-adrenoceptors. A main EPAC function in the human prostate may be the regulation of the transcription factor Elk1. PMID:23815815

  12. Smooth Sailing.

    ERIC Educational Resources Information Center

    Price, Beverley; Pincott, Maxine; Rebman, Ashley; Northcutt, Jen; Barsanti, Amy; Silkunas, Betty; Brighton, Susan K.; Reitz, David; Winkler, Maureen

    1999-01-01

    Presents discipline tips from several teachers to keep classrooms running smoothly all year. Some of the suggestions include the following: a bear-cave warning system, peer mediation, a motivational mystery, problem students acting as the teacher's assistant, a positive-behavior-reward chain, a hallway scavenger hunt (to ensure quiet passage…

  13. Smooth Sailing.

    ERIC Educational Resources Information Center

    Price, Beverley; Pincott, Maxine; Rebman, Ashley; Northcutt, Jen; Barsanti, Amy; Silkunas, Betty; Brighton, Susan K.; Reitz, David; Winkler, Maureen

    1999-01-01

    Presents discipline tips from several teachers to keep classrooms running smoothly all year. Some of the suggestions include the following: a bear-cave warning system, peer mediation, a motivational mystery, problem students acting as the teacher's assistant, a positive-behavior-reward chain, a hallway scavenger hunt (to ensure quiet passage…

  14. Sphingolipids regulate [Mg2+]o uptake and [Mg2+]i content in vascular smooth muscle cells: potential mechanisms and importance to membrane transport of Mg2+.

    PubMed

    Zheng, Tao; Li, Wenyan; Altura, Bella T; Shah, Nilank C; Altura, Burton M

    2011-02-01

    Sphingolipids have a variety of important signaling roles in mammalian cells. We tested the hypothesis that certain sphingolipids and neutral sphingomyelinase (N-SMase) can regulate intracellular free magnesium ions ([Mg2+]i) in vascular smooth muscle (VSM) cells. Herein, we show that several sphingolipids, including C2-ceramide, C8-ceramide, C16-ceramide, and sphingosine, as well as N-SMase, have potent and direct effects on content and mobilization of [Mg2+]i in primary cultured rat aortic smooth muscle cells. All of these sphingolipid molecules increase, rapidly, [Mg2+]i in these vascular cells in a concentration-dependent manner. The increments of [Mg2+]i, induced by these agents, are derived from influx of extracellular Mg2+ and are extracellular Ca2+ concentration-dependent. Phospholipase C and Ca2+/calmodulin/Ca2+-ATPase activity appear to be important in the sphingolipid-induced rises of [Mg2+]i. Activation of certain PKC isozymes may also be required for sphingolipid-induced rises in [Mg2+]i. These novel results suggest that sphingolipids may be homeostatic regulators of extracellular Mg2+ concentration influx (and transport) and [Mg2+]i content in vascular muscle cells.

  15. Glycosylation mediates up-regulation of a potent antiangiogenic and proatherogenic protein, thrombospondin-1, by glucose in vascular smooth muscle cells.

    PubMed

    Raman, Priya; Krukovets, Irene; Marinic, Tina E; Bornstein, Paul; Stenina, Olga I

    2007-02-23

    Accelerated development of atherosclerotic lesions remains the most frequent and dangerous complication of diabetes, accounting for 80% of deaths among diabetics. However, our understanding of the pathways mediating glucose-induced gene expression in vascular cells remains controversial and incomplete. We have identified an intracellular metabolic pathway activated by high glucose in human aortic smooth muscle cells that mediates up-regulation of thrombospondin-1 (TSP-1). TSP-1 is a potent antiangiogenic and proatherogenic protein that may represent an important link between diabetes and vascular complications. Using different glucose analogs and metabolites sharing distinct, limited metabolic steps with glucose, we demonstrated that activation of TSP-1 transcription is mediated by the hexosamine pathway of glucose catabolism, possibly resulting in modulation of the activity of nuclear proteins activity through their glycosylation. Specific inhibitors of glutamine: fructose 6-phosphate amidotransferase (GFAT), an enzyme controlling the hexosamine pathway, as well as direct inhibitors of protein glycosylation efficiently inhibited TSP-1 transcription and the activity of a TSP-1 promoter-reporter construct stimulated by high glucose. Overexpression of recombinant GFAT resulted in increased TSP-1 levels. Pharmacological inhibition of GFAT or protein glycosylation inhibited increased proliferation of human aortic smooth muscle cells caused by glucose. We have demonstrated that the hexosamine metabolic pathway mediates up-regulation of TSP-1 by high glucose. Our results suggest that the hexosamine pathway and intracellular glycosylation may control important steps in initiation and development of atherosclerotic lesions.

  16. Tape-Smoothing Tool For Adhesion Tests

    NASA Technical Reports Server (NTRS)

    Allen, Peter B.

    1992-01-01

    Small tool smoothes adhesive tape uniformly to ensure consistency and repeatability of tape-peel tests of adhesion of paint to substrate. Includes resilient pad covered with tough, smooth fabric. Internal spring regulates force applied to tape.

  17. Calcium regulation in aortic smooth muscle cells during the initial phase of tunicamycin-induced endo/sarcoplasmic reticulum stress.

    PubMed

    Ziomek, Gabriela; Cheraghi Zanjani, Parisa; Arman, Darian; van Breemen, Cornelis; Esfandiarei, Mitra

    2014-07-15

    Endo/sarcoplasmic reticulum stress and the unfolded protein response have been implicated as underlying mechanisms of cell death in many pathological conditions. We have confirmed that long-term exposure to 10µM tunicamycin induced the endo/sarcoplasmic reticulum stress in cultured vascular smooth muscle cells. Since tunicamycin is reported to induce the stress response by inhibiting protein glycosylation, we attempted to investigate a causal link between accumulation of unfolded proteins and dysregulation of cellular calcium transport. However, we found that tunicamycin caused an immediate release of calcium from the endo/sarcoplasmic reticulum, which was sensitive to thapsigargin, and an influx of calcium through the plasma membrane, resulting in a significant increase in cytoplasmic calcium and depletion of endo/sarcoplasmic reticulum calcium. Furthermore, we observed that tunicamycin also induced contraction in intact vascular smooth muscle. By applying established procedures and antagonists, we established that tunicamycin did not directly activate physiological calcium channels, such as store-operated channels, voltage gated calcium channels, ryanodine receptors or inositol trisphosphate receptors. Instead, we found that its effects on cellular calcium fluxes closely resembled those of the known calcium ionophore, ionomycin. We have concluded that tunicamycin directly permeabilizes the plasma membrane and endo/sarcoplasmic reticulum to calcium, and is, therefore, inappropriate for studying the relationship between accumulation of unfolded proteins and endo/sarcoplasmic reticulum calcium dysregulation during the endo/sarcoplasmic reticulum stress response. In contrast, we also report that two other well-known endo/sarcoplasmic reticulum stress inducers, brefeldin A and dithiothreitol, did not exhibit similar increases in calcium permeability. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Analysis of hypoxia-induced noncoding RNAs reveals metastasis-associated lung adenocarcinoma transcript 1 as an important regulator of vascular smooth muscle cell proliferation.

    PubMed

    Brock, Matthias; Schuoler, Claudio; Leuenberger, Caroline; Bühlmann, Carlo; Haider, Thomas J; Vogel, Johannes; Ulrich, Silvia; Gassmann, Max; Kohler, Malcolm; Huber, Lars C

    2017-03-01

    Vascular remodeling, a pathogenic hallmark in pulmonary hypertension, is mainly driven by a dysbalance between proliferation and apoptosis of human pulmonary artery smooth muscle cells. It has previously been shown that microRNAs are involved in the pathogenesis of pulmonary hypertension. However, the role of long noncoding RNAs has not been evaluated. long noncoding RNA expression was quantified in human pulmonary artery smooth muscle cells using PCR arrays and quantitative PCR. Knockdown of genes was performed by transfection of siRNA or GapmeR. Proliferation and migration were measured using BrdU incorporation and wound healing assays. The mouse model of hypoxia-induced PH was used to determine the physiological meaning of identified long noncoding RNAs. The expression of 84 selected long noncoding RNAs was assessed in hypoxic human pulmonary artery smooth muscle cells and the levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) were significantly increased. Depletion of hypoxia-inducible factor 1α abolished the hypoxia-induced upregulation of metastasis-associated lung adenocarcinoma transcript 1 expression. Silencing of MALAT1 significantly decreased proliferation and migration of human pulmonary artery smooth muscle cells. In vivo, MALAT1 expression was significantly increased in lungs of hypoxic mice. Of note, targeting of MALAT1 by GapmeR ameliorated heart hypertrophy in mice with pulmonary hypertension. This is the first report on functional characterization of MALAT1 in the pulmonary vasculature. Our data provide evidence that MALAT1 expression is significantly increased by hypoxia, probably by hypoxia-inducible factor 1α. Intervention experiments confirmed that MALAT1 regulates the proliferative phenotype of smooth muscle cells and silencing of MALAT1 reduced heart hypertrophy in mice with pulmonary hypertension. These data indicate a potential role of MALAT1 in the pathogenesis of pulmonary hypertension. Impact statement

  19. Cross-talk between p(38)MAPK and G iα in regulating cPLA 2 activity by ET-1 in pulmonary smooth muscle cells.

    PubMed

    Chakraborti, Sajal; Chowdhury, Animesh; Chakraborti, Tapati

    2015-02-01

    Endothelin-1 (ET-1) is known as the most potent vasoconstrictor yet described. Infusion of ET-1 into isolated rabbit lung has been shown to cause pulmonary vasoconstriction with the involvement of arachidonic acid metabolites. Given the potency of arachidonic acid metabolites, the activity of phospholipase A2 must be tightly regulated. Herein, we determined the mechanisms by which ET-1 stimulates cPLA2 activity during ET-1 stimulation of bovine pulmonary artery smooth muscle cells. We demonstrated that (i) treatment of bovine pulmonary artery smooth muscle cells with ET-1 stimulates cPLA2 activity in the cell membrane; (ii) ET-1 caused increase in O 2 (·-) production occurs via NADPH oxidase-dependent mechanism; (iii) ET-1-stimulated NADPH oxidase activity is markedly prevented upon pretreatment with PKC-ζ inhibitor, indicating that PKC-ζ plays a prominent role in this scenario; (iv) ET-1-induced NADPH oxidase-derived O 2 (·-) stimulates an aprotinin sensitive protease activity due to prominent increase in [Ca(2+)]i; (v) the aprotinin sensitive protease plays a pivotal role in activating PKC-α, which in turn phosphorylates p(38)MAPK and subsequently Giα leading to the activation of cPLA2. Taken together, we suggest that cross-talk between p(38)MAPK and Giα with the involvement of PKC-ζ, NADPH oxidase-derived O 2 (·-) , [Ca(2+)]i, aprotinin-sensitive protease and PKC-α play a pivotal role for full activation of cPLA2 during ET-1 stimulation of pulmonary artery smooth muscle cells.

  20. Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

    SciTech Connect

    Yang, Bin; Li, Wei; Zheng, Qichang; Qin, Tao; Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen; Liu, Sanguang; Song, Zifang

    2015-07-17

    Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation.

  1. Role of Myoendothelial Gap Junctions in the Regulation of Human Coronary Artery Smooth Muscle Cell Differentiation by Laminar Shear Stress.

    PubMed

    Zhang, Zongqi; Chen, Yizhu; Zhang, Tiantian; Guo, Lingyu; Yang, Wenlong; Zhang, Junfeng; Wang, Changqian

    2016-01-01

    Smooth muscle cells may dedifferentiate into the synthetic phenotype and promote atherosclerosis. Here, we explored the role of myoendothelial gap junctions in phenotypic switching of human coronary artery smooth muscle cells (HCASMCs) co-cultured with human coronary artery endothelial cells (HCAECs) exposed to shear stress. HCASMCs and HCAECs were seeded on opposite sides of Transwell inserts, and HCAECs were exposed to laminar shear stress of 12 dyn/cm2 or 5 dyn/cm2. The myoendothelial gap junctions were evaluated by using a multi-photon microscope. In co-culture with HCAECs, HCASMCs exhibited a contractile phenotype, and maintained the expression of differentiation markers MHC and H1-calponin. HCASMCs and HCAECs formed functional intercellular junctions, as evidenced by colocalization of connexin(Cx)40 and Cx43 on cellular projections inside the Transwell membrane and biocytin transfer from HCAECs to HCASMCs. Cx40 siRNA and 18-α-GA attenuated protein expression of MHC and H1-calponin in HCASMCs. Shear stress of 5 dyn/cm2 increased Cx43 and decreased Cx40 expression in HCAECs, and partly inhibited biocytin transfer from HCAECs to HCASMCs, which could be completely blocked by Cx43 siRNA or restored by Cx40 DNA transfected into HCAECs. The exposure of HCAECs to shear stress of 5 dyn/cm2 promoted HCASMC phenotypic switching, manifested by morphological changes, decrease in MHC and H1-calponin expression, and increase in platelet-derived growth factor (PDGF)-BB release, which was partly rescued by Cx43 siRNA or Cx40 DNA or PDGF receptor signaling inhibitor. The exposure of HCAECs to shear stress of 5 dyn/cm2 caused the dysfunction of Cx40/Cx43 heterotypic myoendothelial gap junctions, which may be replaced by homotypic Cx43/Cx43 channels, and induced HCASMC transition to the synthetic phenotype associated with the activation of PDGF receptor signaling, which may contribute to shear stress-associated arteriosclerosis. © 2016 The Author(s) Published by S. Karger AG

  2. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    SciTech Connect

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  3. Mechanisms controlling the smooth muscle cell death in progeria via down-regulation of poly(ADP-ribose) polymerase 1.

    PubMed

    Zhang, Haoyue; Xiong, Zheng-Mei; Cao, Kan

    2014-06-03

    Hutchinson-Gilford progeria syndrome (HGPS) is a severe human premature aging disorder caused by a lamin A mutant named progerin. Death occurs at a mean age of 13 y from cardiovascular problems. Previous studies revealed loss of vascular smooth muscle cells (SMCs) in the media of large arteries in a patient with HGPS and two mouse models, suggesting a causal connection between the SMC loss and cardiovascular malfunction. However, the mechanisms of how progerin leads to massive SMC loss are unknown. In this study, using SMCs differentiated from HGPS induced pluripotent stem cells, we show that HGPS SMCs exhibit a profound proliferative defect, which is primarily caused by caspase-independent cell death. Importantly, progerin accumulation stimulates a powerful suppression of PARP1 and consequently triggers an activation of the error-prone nonhomologous end joining response. As a result, most HGPS SMCs exhibit prolonged mitosis and die of mitotic catastrophe. This study demonstrates a critical role of PARP1 in mediating SMC loss in patients with HGPS and elucidates a molecular pathway underlying the progressive SMC loss in progeria.

  4. Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1.

    PubMed

    Osada-Oka, Mayuko; Ikeda, Takako; Akiba, Satoshi; Sato, Takashi

    2008-08-01

    The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.

  5. Ca2+/calmodulin-dependent protein kinase II-γ (CaMKIIγ) negatively regulates vascular smooth muscle cell proliferation and vascular remodeling.

    PubMed

    Saddouk, Fatima Z; Sun, Li-Yan; Liu, Yong Feng; Jiang, Miao; Singer, Diane V; Backs, Johannes; Van Riper, Dee; Ginnan, Roman; Schwarz, John J; Singer, Harold A

    2016-03-01

    Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -γ isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIγ function in vascular remodeling after injury. CaMKIIγ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIγC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIγ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIγ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIγ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIγ and -δ isoforms have nonequivalent, opposing functions. © FASEB.

  6. The long non-coding RNA GAS5 regulates transforming growth factor β (TGF-β)-induced smooth muscle cell differentiation via RNA Smad-binding elements.

    PubMed

    Tang, Rui; Zhang, Gui; Wang, Yung-Chun; Mei, Xiaohan; Chen, Shi-You

    2017-08-25

    Smooth muscle cell (SMC) differentiation is essential for vascular development, and TGF-β signaling plays a critical role in this process. Although long non-coding RNAs (lncRNAs) regulate various cellular events, their functions in SMC differentiation remain largely unknown. Here, we demonstrate that the lncRNA growth arrest-specific 5 (GAS5) suppresses TGF-β/Smad3 signaling in smooth muscle cell differentiation of mesenchymal progenitor cells. We found that forced expression of GAS5 blocked, but knockdown of GAS5 increased, the expression of SMC contractile proteins. Mechanistically, GAS5 competitively bound Smad3 protein via multiple RNA Smad-binding elements (rSBEs), which prevented Smad3 from binding to SBE DNA in TGF-β-responsive SMC gene promoters, resulting in suppression of SMC marker gene transcription and, consequently, in inhibition of TGF-β/Smad3-mediated SMC differentiation. Importantly, other lncRNAs or artificially synthesized RNA molecules that contained rSBEs also effectively inhibited TGF-β/Smad3 signaling, suggesting that lncRNA-rSBE may be a general mechanism used by cells to fine-tune Smad3 activity in both basal and TGF-β-stimulated states. Taken together, our results have uncovered an lncRNA-based mechanism that modulates TGF-β/Smad3 signaling during SMC differentiation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

    PubMed

    Mughal, W; Nguyen, L; Pustylnik, S; da Silva Rosa, S C; Piotrowski, S; Chapman, D; Du, M; Alli, N S; Grigull, J; Halayko, A J; Aliani, M; Topham, M K; Epand, R M; Hatch, G M; Pereira, T J; Kereliuk, S; McDermott, J C; Rampitsch, C; Dolinsky, V W; Gordon, J W

    2015-10-29

    Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

  8. Role of cAMP-Phosphodiesterase 1C Signaling in Regulating Growth Factor Receptor Stability, Vascular Smooth Muscle Cell Growth, Migration, and Neointimal Hyperplasia

    PubMed Central

    Cai, Yujun; Nagel, David J.; Zhou, Qian; Cygnar, Katherine D.; Zhao, Haiqing; Li, Faqian; Pi, Xinchun; Knight, Peter A.; Yan, Chen

    2015-01-01

    Objective Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, post-angioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs). Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases. Methods & Results We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. Additionally, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF-receptor-beta (PDGFRβ) known to be important in pathological vascular remodeling. PDE1C interacts with LDL-receptor-related-protein-1 (LRP1) and PDGFRβ, thus regulating PDGFRβ endocytosis and lysosome-dependent degradation in an LRP1-dependent manner. A transmembrane-adenylyl-cyclase (tmAC)-cAMP-PKA cascade modulated by PDE1C is critical in regulating PDGFRβ degradation. Conclusion These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome dependent PDGFRβ protein degradation via LRP1. PMID:25608528

  9. MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1

    SciTech Connect

    Xu, Juan; Li, Li; Yun, Hui-fang; Han, Ye-shan

    2015-08-07

    Background: Diabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. Methods: The expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. Results: We discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 3′-UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic. In addition, we examined that the up-regulation of NF-κB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Conclusion: In this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1. - Highlights: • Higher mRNA level of miR-138 was observed in SMCs from db/db mice. • The mRNA and protein level of SIRT1 in SMCs from db/db mice were greatly reduced. • miR-138 could regulate the expression of SIRT1 in SMCs. • SIRT1 overexpression reversed the up-regulation of acetylized p65 and NF-κB induced by high glucose. • MiR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose.

  10. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway

    PubMed Central

    Zhu, Dongxing; Mackenzie, Neil Charles Wallace; Shanahan, Catherine M; Shroff, Rukshana C; Farquharson, Colin; MacRae, Vicky Elizabeth

    2015-01-01

    The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention. PMID:25297851

  11. BET Bromodomains Regulate Transforming Growth Factor-β-induced Proliferation and Cytokine Release in Asthmatic Airway Smooth Muscle*

    PubMed Central

    Perry, Mark M.; Durham, Andrew L.; Austin, Philip J.; Adcock, Ian M.; Chung, Kian Fan

    2015-01-01

    Airway smooth muscle (ASM) mass is increased in asthma, and ASM cells from patients with asthma are hyperproliferative and release more IL-6 and CXCL8. The BET (bromo- and extra-terminal) family of proteins (Brd2, Brd3, and Brd4) govern the assembly of histone acetylation-dependent chromatin complexes. We have examined whether they modulate proliferation and cytokine expression in asthmatic ASM cells by studying the effect of BET bromodomain mimics JQ1/SGCBD01 and I-BET762. ASM cells from healthy individuals and nonsevere and severe asthmatics were pretreated with JQ1/SGCBD01 and I-BET762 prior to stimulation with FCS and TGF-β. Proliferation was measured by BrdU incorporation. IL-6 and CXCL8 release was measured by ELISA, and mRNA expression was measured by quantitative RT-PCR. ChIP using a specific anti-Brd4 antibody and PCR primers directed against the transcriptional start site of IL-6 and CXCL8 gene promoters was performed. Neither JQ1/SGCBD01 nor I-BET762 had any effect on ASM cell viability. JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced ASM cell proliferation and IL-6 and CXCL8 release in healthy individuals (≥ 30 nm) and in nonsevere and severe asthma patients (≥100 nm), with the latter requiring higher concentrations of these mimics. JQ1/SGCBD01 reduced Brd4 binding to IL8 and IL6 promoters induced by FCS+TGF-β. Mimics of BET bromodomains inhibit aberrant ASM cell proliferation and inflammation with lesser efficiency in those from asthmatic patients. They may be effective in reducing airway remodeling in asthma. PMID:25697361

  12. Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells*

    PubMed Central

    Nigro, Julie; Wang, Aimin; Mukhopadhyay, Durba; Lauer, Mark; Midura, Ronald J.; Sackstein, Robert; Hascall, Vincent C.

    2009-01-01

    The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (Kav shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a Kav shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (Kav of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc. PMID:19346253

  13. BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway.

    PubMed

    Zhu, Dongxing; Mackenzie, Neil Charles Wallace; Shanahan, Catherine M; Shroff, Rukshana C; Farquharson, Colin; MacRae, Vicky Elizabeth

    2015-01-01

    The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John

  14. Regulation of H1-receptor coupling and H1-receptor mRNA by histamine in bovine tracheal smooth muscle

    PubMed Central

    Pype, J L; Dupont, L J; Mak, J C W; Barnes, P J; Verleden, G M

    1998-01-01

    Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1–100 μM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72±8% (n=5) following pre-exposure to 100 μM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation.The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 μM) but not by cimetidine (an H2-receptor antagonist, 10 μM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors.Indomethacin (10 μM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R.GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization.The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 μM, 0.5, 1, 2, 4, 16 and 24 h).These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase. PMID:9535029

  15. cGMP reduces the sarcoplasmic reticulum Ca2+ loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca2+ by cGMP.

    PubMed

    Bazán-Perkins, Blanca

    2012-03-01

    Ca(2+) and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca(2+) and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca(2+) storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca(2+) has not been completely understood. To evaluate this role, intracellular Ca(2+) concentration ([Ca(2+)]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca(2+) from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca(2+)]i undershoot. Caffeine-induced Ca(2+) transient peak and [Ca(2+)]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca(2+)]i undershoot and reduced the subsequent caffeine peak (SR-Ca(2+) loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca(2+) ATPase with cyclopiazonic acid inhibited the [Ca(2+)]i undershoot as well as the SR-Ca(2+) loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca(2+) loading. Cyclic GMP enhanced [Ca(2+)]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca(2+) content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca(2+) loading and the simultaneous release of SR-Ca(2+). The reduction of SR-Ca(2+) content induced by cGMP might be a putative mechanism limiting releasable Ca(2+) in response to a particular stimulus.

  16. Adenylyl cyclase 2 selectively couples to E prostanoid type 2 receptors, whereas adenylyl cyclase 3 is not receptor-regulated in airway smooth muscle.

    PubMed

    Bogard, Amy S; Adris, Piyatilake; Ostrom, Rennolds S

    2012-08-01

    Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

  17. Mechanical Stretch Up-regulates MicroRNA-26a and Induces Human Airway Smooth Muscle Hypertrophy by Suppressing Glycogen Synthase Kinase-3β*

    PubMed Central

    Mohamed, Junaith S.; Lopez, Michael A.; Boriek, Aladin M.

    2010-01-01

    Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3′-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy. PMID:20525681

  18. Mechanical stretch up-regulates microRNA-26a and induces human airway smooth muscle hypertrophy by suppressing glycogen synthase kinase-3β.

    PubMed

    Mohamed, Junaith S; Lopez, Michael A; Boriek, Aladin M

    2010-09-17

    Airway smooth muscle hypertrophy is one of the hallmarks of airway remodeling in severe asthma. Several human diseases have been now associated with dysregulated microRNA (miRNA) expression. miRNAs are a class of small non-coding RNAs, which negatively regulate gene expression at the post-transcriptional level. Here, we identify miR-26a as a hypertrophic miRNA of human airway smooth muscle cells (HASMCs). We show that stretch selectively induces the transcription of miR-26a located in the locus 3p21.3 of human chromosome 3. The transcription factor CCAAT enhancer-binding protein α (C/EBPα) directly activates miR-26a expression through the transcriptional machinery upon stretch. Furthermore, stretch or enforced expression of miR-26a induces HASMC hypertrophy, and miR-26 knockdown reverses this effect, suggesting that miR-26a is a hypertrophic gene. We identify glycogen synthase kinase-3β (GSK-3β), an anti-hypertrophic protein, as a target gene of miR-26a. Luciferase reporter assays demonstrate that miR-26a directly interact with the 3'-untranslated repeat of the GSK-3β mRNA. Stretch or enforced expression of miR-26a attenuates the endogenous GSK-3β protein levels followed by the induction of HASMC hypertrophy. miR-26 knockdown reverses this effect, suggesting that miR-26a-induced hypertrophy occurs via its target gene GSK-3β. Overall, as a first time, our study unveils that miR-26a is a mechanosensitive gene, and it plays an important role in the regulation of HASMC hypertrophy.

  19. Positive regulation of the Egr-1/osteopontin positive feedback loop in rat vascular smooth muscle cells by TGF-{beta}, ERK, JNK, and p38 MAPK signaling

    SciTech Connect

    Yu, Hong-Wei; Liu, Qi-Feng; Liu, Gui-Nan

    2010-05-28

    Previous studies identified a positive feedback loop in rat vascular smooth muscle cells (VSMCs) in which early growth response factor-1 (Egr-1) binds to the osteopontin (OPN) promoter and upregulates OPN expression, and OPN upregulates Egr-1 expression via the extracellular signal-regulated protein kinase (ERK) signaling pathway. The current study examined whether transforming growth factor-{beta} (TGF-{beta}) activity contributes to Egr-1 binding to the OPN promoter, and whether other signaling pathways act downstream of OPN to regulate Egr-1 expression. ChIP assays using an anti-Egr-1 antibody showed that amplification of the OPN promoter sequence decreased in TGF-{beta} DNA enzyme-transfected VSMCs relative to control VSMCs. Treatment of VSMCs with PD98059 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 MAPK inhibitor) significantly inhibited OPN-induced Egr-1 expression, and PD98059 treatment was associated with the most significant decrease in Egr-1 expression. OPN-stimulated VSMC cell migration was inhibited by SP600125 or SB203580, but not by PD98059. Furthermore, MTT assays showed that OPN-mediated cell proliferation was inhibited by PD98059, but not by SP600125 or SB203580. Taken together, the results of the current study show that Egr-1 binding to the OPN promoter is positively regulated by TGF-{beta}, and that the p38 MAPK, JNK, and ERK pathways are involved in OPN-mediated Egr-1 upregulation.

  20. Endothelial Cells Can Regulate Smooth Muscle Cells in Contractile Phenotype through the miR-206/ARF6&NCX1/Exosome Axis

    PubMed Central

    Lin, Xiao; He, Yu; Hou, Xue; Zhang, Zhenming; Wang, Rui; Wu, Qiong

    2016-01-01

    Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (α-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (α-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1. PMID:27031991

  1. Inositol 1,4,5-trisphosphate (IP3) receptor up-regulation in hypertension is associated with sensitization of Ca2+ release and vascular smooth muscle contractility.

    PubMed

    Abou-Saleh, Haissam; Pathan, Asif R; Daalis, Arwa; Hubrack, Satanay; Abou-Jassoum, Hamda; Al-Naeimi, Hamda; Rusch, Nancy J; Machaca, Khaled

    2013-11-15

    Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca(2+) signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca(2+) signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca(2+) release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca(2+) influx through L-type Ca(2+) channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca(2+) release, resulting in increased VSM contraction in response to agonist stimulation.

  2. Shh mediates PDGF-induced contractile-to-synthetic phenotypic modulation in vascular smooth muscle cells through regulation of KLF4.

    PubMed

    Zeng, Qiu; Wei, Bin; Zhao, Yu; Wang, Xuehu; Fu, Qining; Liu, Hong; Li, Fenghe

    2016-07-01

    Platelet-derived growth factor (PDGF) is known to induce phenotypic switching of vascular smooth muscle cells (VSMCs) from contractile to a pathological synthetic state, which played an essential role in proliferation of VSMCs. Sonic hedgehog (Shh) contributes to the proliferation of VSMCs when induced by PDGF. Here, we investigated the probable role of Shh in PDGF-induced VSMC dedifferentiation and its underlying mechanisms. We found that PDGF stimulated Shh expression in VSMCs, which was mediated by activation of PDGFRβ/ERK1/2 cell signaling pathway. Further, we found PDGF-induced VSMC phenotypic modulation was accompanied by up-regulation of Shh/Gli family zinc finger 2 (Gli2) signaling and Krüppel-like factor 4 (KLF4). When inhibited Shh in the presence of PDGF, the expressions of KLF4 and VSMC dedifferentiation markers were down-regulated and the effect of PDGF in inducing VSMC dedifferentiation was blocked. In the absence of PDGF, Shh signaling activation increased the expression of KLF4 and promoted VSMC dedifferentiation. The results indicate Shh participated in the regulation of PDGF-induced VSMC dedifferentiation. Finally, we found that KLF4 was closely involved in this process. On inhibition of KLF4, PDGF induced VSMC dedifferentiation was abrogated, even in the presence of Shh. Taken together, the results provide critical insights into the newly discovered role of Shh in phenotypic modulation of VSMCs which depends on KLF4.

  3. Dual regulation of cofilin activity by LIM kinase and Slingshot-1L phosphatase controls platelet-derived growth factor-induced migration of human aortic smooth muscle cells.

    PubMed

    San Martín, Alejandra; Lee, Moo Yeol; Williams, Holly C; Mizuno, Kensaku; Lassègue, Bernard; Griendling, Kathy K

    2008-02-29

    Platelet-derived growth factor (PDGF) plays a central role in vascular healing, atherosclerosis, and restenosis, partly by stimulating vascular smooth muscle cell (VSMC) migration. Migration requires rapid turnover of actin filaments, which is partially controlled by cofilin. Although cofilin is negatively regulated by Ser3 phosphorylation, the upstream signaling pathways have not been defined, nor has its role in VSMC migration been studied. We hypothesized that PDGF-induced migration of VSMCs involves cofilin activation and that this is regulated by the serine kinase LIM kinase (LIMK) and the novel phosphatase Slingshot (SSH)1L. In human VSMCs, stimulation with PDGF increased G-actin incorporation into the actin cytoskeleton. PDGF transiently activated the cofilin kinase, LIMK, with a peak at 5 minutes. However, cofilin was dephosphorylated between 5 and 45 minutes, with a maximum of 43+/-5% dephosphorylation at 30 minutes, suggesting that PDGF also activates a cofilin phosphatase. We found that VSMCs express SSH1L, which is induced and activated (564+/-73 versus 1021+/-141 picomoles of PO(4); P=0.015) by PDGF. Of importance, small interfering RNA directed against SSH1L blocked cofilin dephosphorylation and decreased migration (528+/-33 versus 318+/-25 cells/field; P<0.01). Taken together, our results suggest that PDGF participates in actin dynamics by dual regulation of cofilin activity via LIMK and SSH1L.

  4. Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility*

    PubMed Central

    Abou-Saleh, Haissam; Pathan, Asif R.; Daalis, Arwa; Hubrack, Satanay; Abou-Jassoum, Hamda; Al-Naeimi, Hamda; Rusch, Nancy J.; Machaca, Khaled

    2013-01-01

    Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca2+ signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca2+ signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca2+ release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca2+ influx through L-type Ca2+ channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca2+ release, resulting in increased VSM contraction in response to agonist stimulation. PMID:24097979

  5. 7 CFR 51.1910 - Fairly smooth.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Fairly smooth. 51.1910 Section 51.1910 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... smooth. Fairly smooth means that the tomato is not conspicuously ridged or rough. ...

  6. 7 CFR 51.1910 - Fairly smooth.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Fairly smooth. 51.1910 Section 51.1910 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Consumer Standards for Fresh Tomatoes Definitions § 51.1910 Fairly smooth. Fairly smooth means that the...

  7. 7 CFR 51.1870 - Fairly smooth.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Fairly smooth. 51.1870 Section 51.1870 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Fresh Tomatoes 1 Definitions § 51.1870 Fairly smooth. Fairly smooth means that the tomato is...

  8. 7 CFR 51.1910 - Fairly smooth.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Fairly smooth. 51.1910 Section 51.1910 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Consumer Standards for Fresh Tomatoes Definitions § 51.1910 Fairly smooth. Fairly smooth means that the...

  9. 7 CFR 51.1910 - Fairly smooth.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Fairly smooth. 51.1910 Section 51.1910 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... smooth. Fairly smooth means that the tomato is not conspicuously ridged or rough. ...

  10. 7 CFR 51.1870 - Fairly smooth.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Fairly smooth. 51.1870 Section 51.1870 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Fresh Tomatoes 1 Definitions § 51.1870 Fairly smooth. Fairly smooth means that the tomato is...

  11. 7 CFR 51.1910 - Fairly smooth.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Fairly smooth. 51.1910 Section 51.1910 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Consumer Standards for Fresh Tomatoes Definitions § 51.1910 Fairly smooth. Fairly smooth means that the...

  12. [UTP regulates spontaneous transient outward currents in porcine coronary artery smooth muscle cells through PLC-IP(3) signaling pathway].

    PubMed

    Li, Peng-Yun; Zeng, Xiao-Rong; Yang, Yan; Cai, Fang; Li, Miao-Ling; Liu, Zhi-Fei; Pei, Jie; Zhou, Wen

    2008-02-25

    The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced

  13. Regulation of cell calcium and contractility in mammalian arterial smooth muscle: the role of sodium-calcium exchange.

    PubMed Central

    Ashida, T; Blaustein, M P

    1987-01-01

    1. The contraction and relaxation of rings of rat thoracic aorta and bovine tail artery were examined as a function of changes in the Na+ electrochemical gradient in order to determine the role of Na-Ca exchange in the control of contractility. 2. Inhibition of the Na+ pump in rat aorta by K+-free media or a low concentration (5 x 10(-5) M) of strophanthidin reversibly increased the contractile responses to caffeine and noradrenaline. These effects were dependent upon external Ca2+ and were observed even in the presence of a Ca2+ channel blocker (10 microM-verapamil or 10 microM-diltiazem) and an alpha-receptor blocker (10 microM-phentolamine). 3. Reduction of external Na+ concentration, [Na+]o (replaced by N-methylglucamine, tetramethylammonium or Tris), also caused an external Ca2+-dependent increase in tonic tension and, in rat aorta, an increase in the response to caffeine. These effects were also observed in the presence of verapamil and phentolamine. 4. Caffeine relaxed the bovine tail artery, but increased the sensitivity of the rat aorta to reduced [Na+]o. The latter effect was presumably due to block of Ca2+ sequestration in the sarcoplasmic reticulum, so that entering Ca2+ was more effective in raising the intracellular free Ca2+ level, [Ca2+]i. 5. Relaxation from K+-free or low-Na+ contractions, in Ca2+-free media, depended upon [Na+]o. Reduction of [Na+]o to 1.2 or 7.5 mM slowed the relaxation of rat aorta (5 mM-caffeine present) 3- to 5-fold, and the relaxation of bovine tail artery (without caffeine) 5- to 10-fold. These effects were seen in the presence of verapamil and phentolamine. 6. These observations are all consistent with an Na-Ca exchange transport system that can move Ca2+ either into or out of the arterial smooth muscle cells. Ca2+ entry is enhanced by raising [Na+]i (by Na+ pump inhibition) and/or lowering [Na+]o. Ca2+ extrusion from the contracted muscles is largely dependent upon external Na+. The latter observation implies that, when

  14. Large-conductance voltage- and Ca2+-activated K+ channel regulation by protein kinase C in guinea pig urinary bladder smooth muscle.

    PubMed

    Hristov, Kiril L; Smith, Amy C; Parajuli, Shankar P; Malysz, John; Petkov, Georgi V

    2014-03-01

    Large-conductance voltage- and Ca(2+)-activated K(+) (BK) channels are critical regulators of detrusor smooth muscle (DSM) excitability and contractility. PKC modulates the contraction of DSM and BK channel activity in non-DSM cells; however, the cellular mechanism regulating the PKC-BK channel interaction in DSM remains unknown. We provide a novel mechanistic insight into BK channel regulation by PKC in DSM. We used patch-clamp electrophysiology, live-cell Ca(2+) imaging, and functional studies of DSM contractility to elucidate BK channel regulation by PKC at cellular and tissue levels. Voltage-clamp experiments showed that pharmacological activation of PKC with PMA inhibited the spontaneous transient BK currents in native freshly isolated guinea pig DSM cells. Current-clamp recordings revealed that PMA significantly depolarized DSM membrane potential and inhibited the spontaneous transient hyperpolarizations in DSM cells. The PMA inhibitory effects on DSM membrane potential were completely abolished by the selective BK channel inhibitor paxilline. Activation of PKC with PMA did not affect the amplitude of the voltage-step-induced whole cell steady-state BK current or the single BK channel open probability (recorded in cell-attached mode) upon inhibition of all major Ca(2+) sources for BK channel activation with thapsigargin, ryanodine, and nifedipine. PKC activation with PMA elevated intracellular Ca(2+) levels in DSM cells and increased spontaneous phasic and nerve-evoked contractions of DSM isolated strips. Our results support the concept that PKC activation leads to a reduction of BK channel activity in DSM via a Ca(2+)-dependent mechanism, thus increasing DSM contractility.

  15. Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-{kappa}B in human aortic smooth muscle cells

    SciTech Connect

    Manea, Adrian; Tanase, Laurentia I.; Raicu, Monica; Simionescu, Maya

    2010-06-11

    Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-{kappa}B (NF-{kappa}B) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-{kappa}B signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-{alpha} (TNF{alpha}), a potent inducer of both Nox and NF-{kappa}B, up to 24 h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-{kappa}B pathway reduced significantly the TNF{alpha}-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-{kappa}B elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-{kappa}B significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-{kappa}B proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-{kappa}B is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-{kappa}B and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

  16. Mef2 is Regulated by CaMKIIδ2 and a HDAC4/HDAC5 Heterodimer in Vascular Smooth Muscle Cells

    PubMed Central

    Ginnan, Roman; Sun, Li Yan; Schwarz, John J.; Singer, Harold A.

    2013-01-01

    Vascular smooth muscle cells (VSM) dedifferentiate from the contractile to 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 recently demonstrated that balloon catheter injury of rat carotid arteries resulted in increased expression of CaMKIIδ2 in the medial wall and the expanding neointima. These findings led us to hypothesize that increased expression of CaMKIIδ2 is a positive mediator of synthetic VSM. HDAC4 and HDAC5 function as transcriptional corepressers and are regulated in a CaMKII-dependent manner. In this study, we report that endogenous HDAC4 and HDAC5 in VSM are activated in a Ca2+- and CaMKIIδ2-dependent manner. We further show that AngII-and PDGF-dependent phosphorylation of HDAC4 and HDAC5 is reduced when CaMKIIδ2 expression is suppressed or CaMKIIδ2 activity is attenuated. The transcriptional activator MEF2 is an important determinant of VSM phenotype and is regulated in an HDAC-dependent manner. Herein, we report that stimulation of VSM cells with ionomycin or Ang II potentiates MEF2's ability to bind DNA and increases the expression of established MEF2 target genes Nur77 and MCP1. Suppression of CaMKIIδ2 attenuates increased MEF2 DNA binding activity and upregulation of Nur77 and MCP1. Finally, we show that HDAC5 is regulated by HDAC4 in VSM. 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 VSM cells through regulation of HDAC4 and HDAC5. PMID:22360269

  17. Upregulated miR-17 Regulates Hypoxia-Mediated Human Pulmonary Artery Smooth Muscle Cell Proliferation and Apoptosis by Targeting Mitofusin 2

    PubMed Central

    Lu, Zheng; Li, Sujun; Zhao, Shunxin; Fa, Xianen

    2016-01-01

    Background Pulmonary arterial hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary artery vascular growth and remodeling. Aberrant expression of miR-17 has been shown to be involved in the pathogenesis of PAH, but its underlying molecular mechanism has not been elucidated. Material/Methods Mitofusin 2 (MFN2) expression was determined by qRT-PCR. The protein expression levels of MFN2, proliferating cell nuclear antigen (PCNA), and pro-apoptotic protein cleaved Caspase-3 were measured using Western blot analysis. Cell proliferation and apoptosis were assessed by CellTiter-Glo reagent and flow cytometry, respectively. Caspase-3/7 activity was measured using an Apo-ONE Homogeneous Caspase-3/7 assay kit. The regulation of miR-17 on MFN2 expression was assessed using luciferase reporter assay system. Results miR-17 expression was upregulated in human pulmonary artery smooth muscle cells (hPASMCs) treated with hypoxia and lung tissues of PAH patients. Inhibition of miR-17 suppressed hypoxia-induced proliferation and promoted apoptosis in hPASMCs. miR-17 inhibited MFN2 expression by binding to its 3′-UTR. Decreased cell viability and increased apoptosis and Caspase-3 activity were observed in the anti-miR-17 + siNC group compared with the anti-miR-NC + siNC group. The expression of cleaved Caspase-3 was upregulated and the expression of PCNA was downregulated in the anti-miR-17 + siNC group. Moreover, these alterations were attenuated by knockdown of MFN2. Conclusions miR-17 regulates proliferation and apoptosis in hPASMCs through MFN2 modulation. We found that miR-17 acts as a potential regulator of proliferation and apoptosis of hPASMCs, and that it might be developed as a promising new strategy for the treatment of PAH. PMID:27640178

  18. MicroRNA-31 controls phenotypic modulation of human vascular smooth muscle cells by regulating its target gene cellular repressor of E1A-stimulated genes

    SciTech Connect

    Wang, Jie; Yan, Cheng-Hui; Li, Yang; Xu, Kai; Tian, Xiao-Xiang; Peng, Cheng-Fei; Tao, Jie; Sun, Ming-Yu; Han, Ya-Ling

    2013-05-01

    Phenotypic modulation of vascular smooth muscle cells (VSMCs) plays a critical role in the pathogenesis of a variety of proliferative vascular diseases. The cellular repressor of E1A-stimulated genes (CREG) has been shown to play an important role in phenotypic modulation of VSMCs. However, the mechanism regulating CREG upstream signaling remains unclear. MicroRNAs (miRNAs) have recently been found to play a critical role in cell differentiation via target-gene regulation. This study aimed to identify a miRNA that binds directly to CREG, and may thus be involved in CREG-mediated VSMC phenotypic modulation. Computational analysis indicated that miR-31 bound to the CREG mRNA 3′ untranslated region (3′-UTR). miR-31 was upregulated in quiescent differentiated VSMCs and downregulated in proliferative cells stimulated by platelet-derived growth factor and serum starvation, demonstrating a negative relationship with the VSMC differentiation marker genes, smooth muscle α-actin, calponin and CREG. Using gain-of-function and loss-of-function approaches, CREG and VSMC differentiation marker gene expression levels were shown to be suppressed by a miR-31 mimic, but increased by a miR-31 inhibitor at both protein and mRNA levels. Notably, miR-31 overexpression or inhibition affected luciferase expression driven by the CREG 3′-UTR containing the miR-31 binding site. Furthermore, miR-31-mediated VSMC phenotypic modulation was inhibited in CREG-knockdown human VSMCs. We also determined miR-31 levels in the serum of patients with coronary artery disease (CAD), with or without in stent restenosis and in healthy controls. miR-31 levels were higher in the serum of CAD patients with restenosis compared to CAD patients without restenosis and in healthy controls. In summary, these data demonstrate that miR-31 not only directly binds to its target gene CREG and modulates the VSMC phenotype through this interaction, but also can be an important biomarker in diseases involving VSMC

  19. Hydrogen sulfide potentiates interleukin-1{beta}-induced nitric oxide production via enhancement of extracellular signal-regulated kinase activation in rat vascular smooth muscle cells

    SciTech Connect

    Jeong, Sun-Oh; Pae, Hyun-Ock; Oh, Gi-Su; Jeong, Gil-Saeng; Lee, Bok-Soo; Lee, Seoul; Kim, Du Yong; Rhew, Hyun Yul; Lee, Kang-Min; Chung, Hun-Taeg . E-mail: htchung@wonkwang.ac.kr

    2006-07-07

    Hydrogen sulfide (H{sub 2}S) and nitric oxide (NO) are endogenously synthesized from L-cysteine and L-arginine, respectively. They might constitute a cooperative network to regulate their effects. In this study, we investigated whether H{sub 2}S could affect NO production in rat vascular smooth muscle cells (VSMCs) stimulated with interleukin-1{beta} (IL-1{beta}). Although H{sub 2}S by itself showed no effect on NO production, it augmented IL-{beta}-induced NO production and this effect was associated with increased expression of inducible NO synthase (iNOS) and activation of nuclear factor (NF)-{kappa}B. IL-1{beta} activated the extracellular signal-regulated kinase 1/2 (ERK1/2), and this activation was also enhanced by H{sub 2}S. Inhibition of ERK1/2 activation by the selective inhibitor U0126 inhibited IL-1{beta}-induced NF-{kappa}B activation, iNOS expression, and NO production either in the absence or presence of H{sub 2}S. Our findings suggest that H{sub 2}S enhances NO production and iNOS expression by potentiating IL-1{beta}-induced NF-{kappa}B activation through a mechanism involving ERK1/2 signaling cascade in rat VSMCs.

  20. Oxidized low-density lipoprotein promotes osteoblast differentiation in primary cultures of vascular smooth muscle cells by up-regulating Osterix expression in an Msx2-dependent manner.

    PubMed

    Taylor, Jesse; Butcher, Martin; Zeadin, Melec; Politano, Amanda; Shaughnessy, Stephen G

    2011-02-01

    We have previously shown that oxidized low-density lipoproteins (oxLDLs) act synergistically with β-glycerophosphate to induce the osteogenic differentiation of primary bovine aortic smooth muscle cells (BASMCs). In the present study, we attempt to resolve the mechanism responsible for this effect by examining the expression of several osteoblast-specific transcription factors. Thus, by culturing BASMCs in the absence or presence of β-glycerophosphate and/or oxLDL, we demonstrate that β-glycerophosphate induces both Runx2 and Osterix (Osx) expression. In contrast, oxLDL has no effect on Runx2 expression but rather it enhances β-glycerophosphate-induced osteoblast differentiation by further up-regulating Osx expression. In an attempt to elucidate the mechanism responsible for this latter effect, we examined the ability of oxLDL to affect Msh homeobox 2 (Msx2) expression. Similar to its effect on Osx expression, oxLDL was found to synergistically enhance β-glycerophosphate-induced Msx2 expression in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2)-dependent manner. Furthermore, oxLDL's ability to enhance both β-glycerophosphate-induced Osx expression and alkaline phosphatase activity was prevented when the BASMCs were first transfected with Msx2-specific siRNA. Taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteoblast differentiation and vascular calcification in vivo.

  1. Role of PDE3A in regulation of cell cycle progression in mouse vascular smooth muscle cells and oocytes: implications in cardiovascular diseases and infertility.

    PubMed

    Begum, Najma; Shen, Weixing; Manganiello, Vincent

    2011-12-01

    Phosphodiesterase-3 (PDE3) is a major cAMP-hydrolyzing PDE in vascular smooth muscle cells (VSMCs) and oocytes. The exact role and contribution of the two PDE3 isoforms, PDE3A and PDE3B, in VSMC growth regulation and oocyte maturation was examined using PDE3A (3A) and PDE3B (3B) knockout (KO) mouse models. PDE3A-deficient VSMCs exhibit marked reduction in mitogen-induced cell growth due to cell cycle arrest at G₀-G₁ phase, which resulted from dysregulation of cAMP/protein kinase A (PKA)-activated and mitogen-activated protein kinase (MAPK)-signaling pathways, as well as from alterations in key cell cycle regulatory proteins. Similarly, PDE3A-deficient oocytes exhibit cell cycle arrest at G₂/M phase because increased cAMP/PKA signaling in KO oocytes most likely inhibits Cdc25B-catalyzed dephosphorylation/activation of Cdc2 (maturation promoting factor (MPF)), a key regulator of G₂/M transition. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Glycolaldehyde-derived advanced glycation end products (glycol-AGEs)-induced vascular smooth muscle cell dysfunction is regulated by the AGES-receptor (RAGE) axis in endothelium.

    PubMed

    Nam, Mi-Hyun; Son, Won-Rak; Lee, Young Sik; Lee, Kwang-Won

    Advanced glycation end-products (AGEs) are involved in the development of vascular smooth muscle cell (VSMC) dysfunction and the progression of atherosclerosis. However, AGEs may indirectly affect VSMCs via AGEs-induced signal transduction between monocytes and human umbilical endothelial cells (HUVECs), rather than having a direct influence. This study was designed to elucidate the signaling pathway underlying AGEs-RAGE axis influence on VSMC dysfunction using a co-culture system with monocytes, HUVECs and VSMCs. AGEs stimulated production of reactive oxygen species and pro-inflammatory mediators such as tumor necrosis factor-α and interleukin-1β via extracellular-signal-regulated kinases phosphorylation and nuclear factor-κB activation in HUVECs. It was observed that AGEs-induced pro-inflammatory cytokines increase VSMC proliferation, inflammation and vascular remodeling in the co-culture system. This result implies that RAGE plays a role in AGEs-induced VSMC dysfunction. We suggest that the regulation of signal transduction via the AGEs-RAGE axis in the endothelium can be a therapeutic target for preventing atherosclerosis.

  3. Olive Oil Polyphenols Differentially Inhibit Smooth Muscle Cell Proliferation through a G1/S Cell Cycle Block Regulated by ERK1/2

    PubMed Central

    Abe, Rei; Beckett, Joel; Abe, Ryuzo; Nixon, Alexander; Rochier, Adrienne; Yamashita, Norio; Sumpio, Bauer

    2012-01-01

    We hypothesized that polyphenols contained in olive oil play a role in reducing the risk of atherosclerosis. The aim of this study was to determine if the polyphenols in olive oil, oleuropein (Ole), hydroxytyrosol (HT), and tyrosol (Tyr) could inhibit smooth muscle cell (SMC) proliferation through its influence on cell cycle regulation. Bovine vascular SMC were cultured in the presence of Ole, HT, or Tyr at concentration of 1, 10, or 100 μmol/L. On days 1, 3, and 5, numbers of cells were counted. Cell cycle analysis was performed by flow cytometry on day 1 after SMC were stained with propidium iodide. Cell populations grown in the presence of Ole or HT at 100 μmol/L concentration were significantly inhibited after 5 days of exposure. Tyr had a similar tendency but it did not attain significance. Cell cycle analysis revealed that 66% of cells were in G1 phase in Ole group, compared with 48% in control group. To examine the cell cycle block between G1 and S phases, we performed Western blotting and found that ERK1/2 activation was inhibited by Ole or HT. We conclude that olive oil polyphenols could inhibit SMC proliferation through a cell cycle block between G1 and S phases which may be regulated by ERK1/2. These results demonstrate a mechanism by which olive oil consumption may be atheroprotective by inhibiting SMC proliferation. PMID:23730132

  4. MicroRNA-190 regulates hypoxic pulmonary vasoconstriction by targeting a voltage-gated K⁺ channel in arterial smooth muscle cells.

    PubMed

    Li, Shan-Shan; Ran, Ya-Juan; Zhang, Dan-Dan; Li, Shu-Zhen; Zhu, Daling

    2014-06-01

    Pulmonary arterial hypertension (PAH) is associated with sustained vasoconstriction, profound structural remodeling of vasculatures and alterations in Ca(2+) homeostasis in arterial smooth muscle cells (SMCs), while the underlying mechanisms are still elusive. By regulating the expression of proteins, microRNAs (miRNAs) are known to play an important role in cell fates including differentiation, apoptosis and proliferation, and may be involved in the development of PAH. Based on our previous study, hypoxia produced a significant increase of the miR-190 level in the pulmonary artery (PA), here, we used synthetic miR-190 to mimic the increase in hypoxic conditions and showed evidence for the effects of miR-190 on pulmonary arterial vasoconstriction and Ca(2+) influx in arterial SMCs. Synthetic miR-190 remarkably enhanced the vasoconstriction responses to phenylephrine (PE) and KCl. The voltage-gated K(+) channel subfamily member, Kcnq5, mRNA was shown to be a target for miR-190. Meanwhile, miR-190 antisense oligos can partially reverse the effects of miR-190 on PASMCs and PAs. Therefore, these results suggest that miR-190 appears to be a positive regulator of Ca(2+) influx, and plays an important role in hypoxic pulmonary vascular constriction. © 2014 Wiley Periodicals, Inc.

  5. cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation

    PubMed Central

    Choi, ChungSik; Sellak, Hassan; Brown, Felricia M.

    2010-01-01

    Although the regulation of smooth muscle cell (SMC) gene expression by cGMP-dependent protein kinase (PKG) is now recognized, the mechanisms underlying these effects are not fully understood. In this study, we report that PKG-I stimulates myocardin/serum response factor (SRF)-dependent gene expression in vascular SMCs. The expression of PKG in PKG-deficient cells enhanced myocardin-induced SM22 promoter activity in a concentration-dependent fashion. However, neither SRF nor myocardin expression was affected. To investigate alternative mechanisms, we examined whether PKG affects the phosphorylation of E26-like protein-1 (Elk-1), a SRF/myocardin transcription antagonist. The activation of PKG caused an increase in a higher molecular mass form of phospho-Elk-1 that was determined to be small ubiquitin-related modifier (sumo)ylated Elk-1. PKG increased Elk-1 sumoylation twofold compared with the PKG-deficient cells, and Elk-1 sumoylation was reduced using dominant-negative sumo-conjugating enzyme, DN-Ubc9, confirming PKG-dependent sumoylation of phospho-Elk-1 in vascular SMCs. In addition, PKG stimulated Elk-1 sumoylation in COS-7 cells overexpressing Elk-1, sumo-1, and PKG-I. The increased expression of PKG in vascular SMCs inhibited Elk-1 binding to SMC-specific promoters, SM22 and smooth muscle myosin heavy chain, as measured by EMSA and chromatin immunoprecipitation assay, and PKG suppressed the Elk-1 inhibition of SM22 reporter gene expression. Taken together, these data suggest that PKG-I decreases Elk-1 activity by sumo modification of Elk-1, thereby increasing myocardin-SRF activity on SMC-specific gene expression. PMID:20802137

  6. cGMP-dependent protein kinase and the regulation of vascular smooth muscle cell gene expression: possible involvement of Elk-1 sumoylation.

    PubMed

    Choi, ChungSik; Sellak, Hassan; Brown, Felricia M; Lincoln, Thomas M

    2010-11-01

    Although the regulation of smooth muscle cell (SMC) gene expression by cGMP-dependent protein kinase (PKG) is now recognized, the mechanisms underlying these effects are not fully understood. In this study, we report that PKG-I stimulates myocardin/serum response factor (SRF)-dependent gene expression in vascular SMCs. The expression of PKG in PKG-deficient cells enhanced myocardin-induced SM22 promoter activity in a concentration-dependent fashion. However, neither SRF nor myocardin expression was affected. To investigate alternative mechanisms, we examined whether PKG affects the phosphorylation of E26-like protein-1 (Elk-1), a SRF/myocardin transcription antagonist. The activation of PKG caused an increase in a higher molecular mass form of phospho-Elk-1 that was determined to be small ubiquitin-related modifier (sumo)ylated Elk-1. PKG increased Elk-1 sumoylation twofold compared with the PKG-deficient cells, and Elk-1 sumoylation was reduced using dominant-negative sumo-conjugating enzyme, DN-Ubc9, confirming PKG-dependent sumoylation of phospho-Elk-1 in vascular SMCs. In addition, PKG stimulated Elk-1 sumoylation in COS-7 cells overexpressing Elk-1, sumo-1, and PKG-I. The increased expression of PKG in vascular SMCs inhibited Elk-1 binding to SMC-specific promoters, SM22 and smooth muscle myosin heavy chain, as measured by EMSA and chromatin immunoprecipitation assay, and PKG suppressed the Elk-1 inhibition of SM22 reporter gene expression. Taken together, these data suggest that PKG-I decreases Elk-1 activity by sumo modification of Elk-1, thereby increasing myocardin-SRF activity on SMC-specific gene expression.

  7. Transcriptional profiling identifies the long noncoding RNA plasmacytoma variant translocation (PVT1) as a novel regulator of the asthmatic phenotype in human airway smooth muscle.

    PubMed

    Austin, Philip J; Tsitsiou, Eleni; Boardman, Charlotte; Jones, Simon W; Lindsay, Mark A; Adcock, Ian M; Chung, Kian Fan; Perry, Mark M

    2017-03-01

    The mechanism underlying nonsevere and severe asthma remains unclear, although it is commonly associated with increased airway smooth muscle (ASM) mass. Long noncoding RNAs (lncRNAs) are known to be important in regulating healthy primary airway smooth muscle cells (ASMCs), whereas changed expression has been observed in CD8 T cells from patients with severe asthma. Primary ASMCs were isolated from healthy subjects (n = 9) and patients classified as having nonsevere (n = 9) or severe (n = 9) asthma. ASMCs were exposed to dexamethasone and FCS. mRNA and lncRNA expression was measured by using a microarray and quantitative real-time PCR. Bioinformatic analysis was used to examine relevant biological pathways. Finally, the lncRNA plasmacytoma variant translocation 1 (PVT1) was inhibited by transfection of primary ASMCs with small interfering RNAs, and the effect on ASMC phenotype was examined. The mRNA expression profile was significantly different between patient groups after exposure to dexamethasone and FCS, and these were associated with biological pathways that might be relevant to the pathogenesis of asthma, including cellular proliferation and pathways associated with glucocorticoid activity. We also observed a significant change in lncRNA expression, yet the expression of only one lncRNA (PVT1) is decreased in patients with corticosteroid-sensitive nonsevere asthma and increased in patients with corticosteroid-insensitive severe asthma. Subsequent targeting studies demonstrated the importance of this lncRNA in controlling both proliferation and IL-6 release in ASMCs from patients with severe asthma. lncRNAs are associated with the aberrant phenotype observed in ASMCs from asthmatic patients. Targeting PVT1 might be effective in reducing airway remodeling in asthmatic patients. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. In vitro expression of the alpha-smooth muscle actin isoform by rat lung mesenchymal cells: regulation by culture condition and transforming growth factor-beta.

    PubMed

    Mitchell, J J; Woodcock-Mitchell, J L; Perry, L; Zhao, J; Low, R B; Baldor, L; Absher, P M

    1993-07-01

    alpha-Smooth muscle actin (alpha SM actin)-containing cells recently have been demonstrated in intraalveolar lesions in both rat and human tissues following lung injury. In order to develop model systems for the study of such cells, we examined cultured lung cell lines for this phenotype. The adult rat lung fibroblast-like "RL" cell lines were found to express alpha SM actin mRNA and protein and to organize this actin into stress fiber-like structures. Immunocytochemical staining of subclones of the RL87 line demonstrated the presence in the cultures of at least four cell phenotypes, one that fails to express alpha SM actin and three distinct morphologic types that do express alpha SM actin. The proportion of cellular actin that is the alpha-isoform was modulated by the culture conditions. RL cells growing at low density expressed minimal alpha SM actin. On reaching confluent densities, however, alpha SM actin increased to at least 20% of the total actin content. This effect, combined with the observation that the most immunoreactive cells were those that displayed overlapping cell processes in culture, suggests that cell-cell contact may be involved in actin isoform regulation in these cells. Similar to the response of some smooth muscle cell lines, alpha SM actin expression in RL cells also was promoted by conditions, e.g., maintenance in low serum medium, which minimize cell division. alpha SM actin expression was modulated in RL cells by the growth factor transforming growth factor-beta. Addition of this cytokine to growing cells substantially elevated the proportion of alpha SM actin protein.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. P2Y2 Receptor-mediated Lymphotoxin-α Secretion Regulates Intercellular Cell Adhesion Molecule-1 Expression in Vascular Smooth Muscle Cells*

    PubMed Central

    Seye, Cheikh I.; Agca, Yuksel; Agca, Cansu; Derbigny, Wilbert

    2012-01-01

    The proinflammatory cytokine lymphotoxin-α (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in vascular smooth muscle cells (VSMC) are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y2 nucleotide receptor (P2Y2R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y2R−/− SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Reintroduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A, which blocks the formation of vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus, suggesting that P2Y2R/filamin-mediated secretion of LTA is independent of the endoplasmic reticulum/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y2R deficient in LTA secretion. These data suggest that P2Y2R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on vascular smooth muscle cells. PMID:22298782

  10. P2Y2 receptor-mediated lymphotoxin-α secretion regulates intercellular cell adhesion molecule-1 expression in vascular smooth muscle cells.

    PubMed

    Seye, Cheikh I; Agca, Yuksel; Agca, Cansu; Derbigny, Wilbert

    2012-03-23

    The proinflammatory cytokine lymphotoxin-α (LTA) is thought to contribute to the pathogenesis of atherosclerosis. However, the mechanisms that regulate its expression in vascular smooth muscle cells (VSMC) are poorly understood. The ability of exogenous nucleotides to stimulate LTA production was evaluated in VSMC by ELISA. The P2Y(2) nucleotide receptor (P2Y(2)R) agonist UTP stimulates a strong and sustained release of LTA from WT but not P2Y(2)R(-/-) SMC. Assessment of LTA gene transcription by LTA promoter-luciferase construct indicated that LTA levels are controlled at the level of transcription. We show using RNAi techniques that knockdown of the actin-binding protein filamin-A (FLNa) severely impaired nucleotide-induced Rho activation and consequent Rho-mediated LTA secretion. Reintroduction of FLNa in FLNa RNAi SMC rescued UTP-induced LTA expression. In addition, we found that UTP-stimulated LTA secretion is not sensitive to brefeldin A, which blocks the formation of vesicles involved in protein transport from the endoplasmic reticulum to the Golgi apparatus, suggesting that P2Y(2)R/filamin-mediated secretion of LTA is independent of the endoplasmic reticulum/Golgi secretory vesicle route. Furthermore, UTP selectively induces ICAM-1 expression in WT but not SMC expressing a truncated P2Y(2)R deficient in LTA secretion. These data suggest that P2Y(2)R recruits FLNa to provide a cytoskeletal scaffold necessary for Rho signaling pathway upstream of LTA release and subsequent stimulation of ICAM-1 expression on vascular smooth muscle cells.

  11. Substrate Elastic Modulus Regulates the Morphology, Focal Adhesions, and α-Smooth Muscle Actin Expression of Retinal Müller Cells.

    PubMed

    Bu, Shao-Chong; Kuijer, Roel; van der Worp, Roelofje J; van Putten, Sander M; Wouters, Olaf; Li, Xiao-Rong; Hooymans, Johanna M M; Los, Leonoor I

    2015-09-01

    The stiffness of the extracellular matrix has been shown to regulate cell adhesion, migration, and transdifferentiation in fibrotic processes. Retinal Müller cells have been shown to be mechanosensitive; they are involved in fibrotic vitreoretinal diseases. Since fibrosis increases the rigidity of the extracellular matrix, our aim was to develop an in vitro model for studying Müller cell morphology and differentiation state in relation to matrix stiffness. A spontaneously immortalized human Müller cell line (MIO-M1) was cultured on type I collagen-coated polyacrylamide gels with Young's moduli ranging from 2 to 92 kPa. Cell surface area, focal adhesion, and the expression and morphology of α-smooth muscle actin induced by transforming growth factor β (TGF-β [10 ng/mL for 48 hours]) were analyzed by immunocytology. The images were documented by using fluorescence microscopy and confocal scanning laser microscopy. MIO-M1 cells cultured on stiff substrates exhibited a significant increase in cell surface area, stress fiber, and mature focal adhesion formation. Furthermore, Müller cells treated with TGF-β1 and TGF-β2 and cultured on stiff substrates showed an increased incorporation of α-smooth muscle actin into stress fibers when compared to those grown on soft surfaces. Compliance of the surrounding matrix seems to influence the morphology and contraction of retinal Müller cells in fibrotic conditions. Development of an in vitro model simulating both the normally compliant retinal tissue and the rigid retinal fibrotic tissue helps fill the gap between the results of petri-dish cell culture with rigid surfaces and in vivo findings.

  12. Distinct regulation of stearoyl-CoA desaturase 1 gene expression by cis and trans C18:1 fatty acids in human aortic smooth muscle cells.

    PubMed

    Minville-Walz, M; Gresti, J; Pichon, L; Bellenger, S; Bellenger, J; Narce, M; Rialland, M

    2012-04-01

    Consumption of trans fatty acids is positively correlated with cardiovascular diseases and with atherogenic risk factors. Trans fatty acids might play their atherogenic effects through lipid metabolism alteration of vascular cells. Accumulation of lipids in vascular smooth muscle cells is a feature of atherosclerosis and a consequence of lipid metabolism alteration. Stearoyl-CoA desaturase 1 (scd1) catalyses the production of monounsaturated fatty acids (e.g. oleic acid) and its expression is associated with lipogenesis induction and with atherosclerosis development. We were interested in analysing the regulation of delta-9 desaturation rate and scd1 expression in human aortic smooth muscle cells (HASMC) exposed to cis and trans C18:1 fatty acid isomers (cis-9 oleic acid, trans-11 vaccenic acid or trans-9 elaidic acid) for 48 h at 100 μM. Treatment of HASMC with these C18:1 fatty acid isomers led to differential effects on delta-9 desaturation; oleic acid repressed the desaturation rate more potently than trans-11 vaccenic acid, whereas trans-9 elaidic acid increased the delta-9 desaturation rate. We then correlated the delta-9 desaturation rate with the expression of scd1 protein and mRNA. We showed that C18:1 fatty acids controlled the expression of scd1 at the transcriptional level in HASMC, leading to an increase in scd1 mRNA content by trans-9 elaidic acid treatment, whereas a decrease in scd1 mRNA content was observed with cis-9 oleic acid and trans-11 vaccenic acid treatments. Altogether, this work highlights a differential capability of C18:1 fatty acid isomers to control scd1 gene expression, which presumes of different consequent effects on cell functions.

  13. Effect of Flow on Gene Regulation in Smooth Muscle Cells and Macromolecular Transport Across Endothelial Cell Monolayers

    NASA Technical Reports Server (NTRS)

    McIntire, Larry V.; Wagner, John E.; Papadaki, Maria; Whitson, Peggy A.; Eskin, Suzanne G.

    1996-01-01

    Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cell changes with environmental stimuli; shear stress, in particular, applied either in vivo, or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells. The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

  14. Effect of Flow on Gene Regulation in Smooth Muscle Cells and Macromolecular Transport Across Endothelial Cell Monolayers

    NASA Technical Reports Server (NTRS)

    McIntire, Larry V.; Wagner, John E.; Papadaki, Maria; Whitson, Peggy A.; Eskin, Suzanne G.

    1996-01-01

    Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cell changes with environmental stimuli; shear stress, in particular, applied either in vivo, or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells. The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

  15. Transcriptional up-regulation of antioxidant genes by PPAR{delta} inhibits angiotensin II-induced premature senescence in vascular smooth muscle cells

    SciTech Connect

    Kim, Hyo Jung; Ham, Sun Ah; Paek, Kyung Shin; Hwang, Jung Seok; Jung, Si Young; Kim, Min Young; Jin, Hanna; Kang, Eun Sil; Woo, Im Sun; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Han, Chang Woo; Seo, Han Geuk

    2011-03-25

    Research highlights: {yields} Activation of PPAR{delta} by GW501516 significantly inhibited Ang II-induced premature senescence in hVSMCs. {yields} Agonist-activated PPAR{delta} suppressed generation of Ang II-triggered ROS with a concomitant reduction in DNA damage. {yields} GW501516 up-regulated expression of antioxidant genes, such as GPx1, Trx1, Mn-SOD and HO-1. {yields} Knock-down of these antioxidant genes abolished the effects of GW501516 on ROS production and premature senescence. -- Abstract: This study evaluated peroxisome proliferator-activated receptor (PPAR) {delta} as a potential target for therapeutic intervention in Ang II-induced senescence in human vascular smooth muscle cells (hVSMCs). Activation of PPAR{delta} by GW501516, a specific agonist of PPAR{delta}, significantly inhibited the Ang II-induced premature senescence of hVSMCs. Agonist-activated PPAR{delta} suppressed the generation of Ang II-triggered reactive oxygen species (ROS) with a concomitant reduction in DNA damage. Notably, GW501516 up-regulated the expression of antioxidant genes, such as glutathione peroxidase 1, thioredoxin 1, manganese superoxide dismutase and heme oxygenase 1. siRNA-mediated down-regulation of these antioxidant genes almost completely abolished the effects of GW501516 on ROS production and premature senescence in hVSMCs treated with Ang II. Taken together, the enhanced transcription of antioxidant genes is responsible for the PPAR{delta}-mediated inhibition of premature senescence through sequestration of ROS in hVSMCs treated with Ang II.

  16. Differential regulation and recovery of intracellular Ca2+ in cerebral and small mesenteric arterial smooth muscle cells of simulated microgravity rat.

    PubMed

    Xue, Jun-Hui; Chen, Lian-Hong; Zhao, Hua-Zhou; Pu, Yong-Dong; Feng, Han-Zhong; Ma, Yu-Guang; Ma, Jin; Chang, Yao-Ming; Zhang, Zuo-Ming; Xie, Man-Jiang

    2011-01-01

    The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca(2+) determined by the alterations in the functions of plasma membrane Ca(L) channels and ryanodine-sensitive Ca(2+) releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively. Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of Ca(L) channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca(2+) releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of Ca(L) channels and ryanodine-sensitive Ca(2+) releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of Ca(L) channels and ryanodine-sensitive Ca(2+) releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of Ca(L) channels and ryanodine-sensitive Ca(2+) releases to their control levels in cerebral and small mesenteric VSMCs, respectively. The differential regulation of Ca(L) channels and ryanodine-sensitive Ca(2+) releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca(2+), which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance.

  17. Long Non-Coding RNA LnRPT is Regulated by PDGF-BB and Modulates Proliferation of Pulmonary Artery Smooth Muscle Cells.

    PubMed

    Chen, Jidong; Guo, Jiao; Cui, Xiaolei; Dai, Yan; Tang, Zhixiong; Qu, Junle; Raj, J Usha; Hu, Qinghua; Gou, Deming

    2017-09-15

    Pulmonary artery hypertension (PAH) is a rare and fatal disorder with extensive remodeling of pulmonary arteries mediated by hyperproliferation of pulmonary artery smooth muscle cell (PASMC). Aberrant platelet-derived growth factor (PDGF) activity can lead to hyperproliferation of PASMC, however, little is known about the role of long noncoding RNA (lncRNA) in this process. Using RNA sequencing (RNA-seq), we identified 725 lncRNAs in rat PASMC (RPASMC), 95 of which were expressed differentially in response to PDGF-BB treatment. Depletion of 4 lncRNAs affected proliferation of RPASMC, as measured by EdU incorporation assay. Among these, one lncRNA named LnRPT (lncRNA regulated by PDGF and TGFβ) was the most potent one to promote proliferation of PASMC when knocked down. Oppositely, proliferation of PASMC was repressed when LnRPT was overexpressed. Mechanistically, lnRPT inhibited the expression of two genes involved in the Notch signaling pathway, notch3 and jag1, as well as the cell cycle regulating genes, ccna2. In addition, downregulation of LnRPT induced by PDGF-BB was abrogated when PI3K activity was inhibited with pictilisib. Downregulation of LnRPT was also observed in the pulmonary arteries of MCT-induced PAH rats. These data provide novel insights into the effects of PDGF-BB on lncRNA expression in PASMC, and identify one lncRNA, LnRPT, which was implied in PAH development, as a regulator of PASMC proliferation through mediating the Notch signaling pathway and cell cycle.

  18. miR-125b/Ets1 axis regulates transdifferentiation and calcification of vascular smooth muscle cells in a high-phosphate environment.

    PubMed

    Wen, Ping; Cao, Hongdi; Fang, Li; Ye, Hong; Zhou, Yang; Jiang, Lei; Su, Weifang; Xu, Hongying; He, Weichun; Dai, Chunsun; Yang, Junwei

    2014-04-01

    Vascular calcification is highly prevalent in patients with chronic kidney disease (CKD) and contributes to increased risk of cardiovascular disease and mortality. Accumulated evidences suggested that vascular smooth muscle cells (VSMCs) to osteoblast-like cells transdifferentiation (VOT) plays a crucial role in promoting vascular calcification. MicroRNAs (miRNAs) are a novel class of small RNAs that negatively regulate gene expression via repression of the target mRNAs. In the present work, we sought to determine the role of miRNAs in VSMCs phenotypic transition and calcification induced by β-glycerophosphoric acid. Primary cultured rat aortic VSMCs were treated with β-glycerophosphoric acid for different periods of time. In VSMCs, after β-glycerophosphoric acid treatment, the expressions of cbf β1, osteocalcin and osteopontin were significantly increased and SM-22β expression was decreased. ALP activity was induced by β-glycerophosphoric acid in a time or dose dependent manner. Calcium deposition was detected in VSMCs incubated with calcification media; then, miR-125b expression was detected by real-time RT PCR. miR-125b expression was significantly decreased in VSMCs after incubated with β-glycerophosphoric acid. Overexpression of miR-125b could inhibit β-glycerophosphoric acid-induced osteogenic markers expression and calcification of VSMCs whereas knockdown of miR-125b promoted the phenotypic transition of VSMCs and calcification. Moreover, miR-125b targeted Ets1 and regulated its protein expression in VSMCs. Downregulating Ets1 expression by its siRNA inhibited β-glycerophosphoric acid-induced the VSMCs phenotypic transition and calcification. Our study suggests that down-regulation of miR-125b after β-glycerophosphoric acid treatment facilitates VSMCs transdifferentiation and calcification through targeting Ets1. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Ryanodine receptors in smooth muscle.

    PubMed

    Guerrero-Hernández, Agustín; Gómez-Viquez, Leticia; Guerrero-Serna, Guadalupe; Rueda, Angélica

    2002-07-01

    The sarcoplasmic reticulum (SR) of smooth muscle is endowed with two different types of Ca2+ release channels, i.e. inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs). In general, both release channels mobilize Ca2+ from the same internal store in smooth muscle. While the importance of IP3Rs in agonist-induced contraction is well established, the role of RyRs in excitation-contraction coupling of smooth muscle is not clear. The participation of smooth muscle RyRs in the amplification of Ca2+ transients induced by either opening of Ca2+-permeable channels or IP3-triggered Ca2+ release has been studied. The efficacy of both processes to activate RyRs by calcium-induced calcium release (CICR) is highly variable and not widely present in smooth muscle. Although RyRs in smooth muscle generate Ca2+ sparks that are similar to those observed in striated muscles, the contribution of these local Ca2+ events to depolarization-induced global rise in [Ca2+]i is rather limited. Recent data suggest that RyRs are involved in regulating the luminal [Ca2+] of SR and also in smooth muscle relaxation. This review summarizes studies that were carried out mainly in muscle strips or in freshly isolated myocytes, and that were aimed to determine the physiological role of RyRs in smooth muscle.

  20. Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

    PubMed Central

    Moughal, N; Stevens, P A; Kong, D; Pyne, S; Pyne, N J

    1995-01-01

    Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with

  1. Extracellular magnesium regulates nuclear and perinuclear free ionized calcium in cerebral vascular smooth muscle cells: possible relation to alcohol and central nervous system injury.

    PubMed

    Altura, B M; Zhang, A; Cheng, T P; Altura, B T

    2001-02-01

    Quantitative digital imaging microscopy, confocal laser scanning microscopy (CLSM), and multiple molecular fluorescent probes were utilized to test the hypothesis that cerebral vascular muscle cell nuclear ([Ca(2+)](n)), perinuclear ([Ca(2+)](pn)), and cytoplasmic free calcium ([Ca(2+)](i)) levels are regulated by the concentration of extracellular free magnesium ions ([Mg(2+)](o)). Primary cultured canine cerebral vascular smooth muscle cells were loaded with either fura-2/AM, indo-1/AM, or fluo-3/AM, and the subcellular Ca(2+) responses to stepwise reduction in [Mg(2+)](o) (i.e., from 1.36 to 0.17 mM) were analyzed over time. With normal 1.36 mM [Mg(2+)](o)-containing incubation media, basal mean [Ca(2+)](i) was 89.6+/-15 nM. Lowering [Mg(2+)](o) to 1.07, 0.88, 0.48, and 0.17 mM resulted in rapid (<4 min) increments in [Ca(2+)](i) going to 213+/-43, 368+/-67, 471+/-77, and 642+/-98 nM, respectively; the longer the exposure time (up to 30 min) to lowered [Mg(2+)](o), the higher the [Ca(2+)](i). Restoration of [Mg(2+)](o) to normal caused decreases in [Ca(2+)](i) to 215.9+/-42.3 nM, but only complete removal of [Ca(2+)](o) returned [Ca(2+)](i) to basal levels. Results show that basal [Ca(2+)](pn) (282+/-92 nM) exceeds basal cytoplasmic Ca(2+) (61+/-27.8 nM) and [Ca(2+)](n) (20+/-7.6 nM). However, reduction of normal [Mg(2+)](o) to 0.48 mM resulted in dramatic, rapid rises in all subcellular compartments, where [Ca(2+)](pn) (1503+/-102 nM)>cytoplasmic Ca(2+) (688+/-49 nM) approximately equal to [Ca(2+)](n) (674+/-12 nM). Nuclear Ca(2+) rose dramatically (e.g., 35-40 times basal levels). Both verapamil (1 microM) and Ni(2+) (5 mM) prevented, completely, the rises in Ca(2+) in all compartments, suggesting that Mg(2+)-dependent Ca(2+) accumulation may be dependent on nuclear, endoplasmic reticulum-Golgi, and cytoplasmic L-type voltage membrane-regulated Ca(2+) channels. The normally low [Ca(2+)](n) suggests that Ca(2+) does not transport passively across the nuclear

  2. Down-regulation of hsa-miR-148b inhibits vascular smooth muscle cells proliferation and migration by directly targeting HSP90 in atherosclerosis

    PubMed Central

    Zhang, Xinqi; Shi, Hua; Wang, Yuanlin; Hu, Jianxin; Sun, Zhaolin; Xu, Shuxiong

    2017-01-01

    The abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are crucial pathological processes that are involved in atherosclerosis. Growing evidence suggests that microRNAs (miRNAs) play critical roles in VSMCs functions. Here, we analyzed the expression of four atherosclerosis-related miRNAs and found that hsa-miR-148b was significantly down-regulated in plaques from atherosclerotic patients compared to a healthy control group. The restoration of hsa-miR-148b function in cells transfected with a hsa-miR-148b mimicmarkedly inhibited VSMCs proliferation and migration compared to a hsa-miR-148b mimic control. Furthermore, we discovered that heat shock protein 90 (HSP90) was a direct target of hsa-miR-148b in VSMCs. Hsa-miR-148b suppressed HSP90 expression by directly binding its 3’-untranslated region (UTR). In addition, the expression of hsa-miR-148b was negatively correlated with the HSP90 mRNA levels in plaques of atherosclerotic patients. Interestingly, the overexpression of HSP90 partly abrogated the hsa-miR-148b-mediated inhibition of VSMCs proliferation and migration. Our study provides the first evidence that hsa-miR-148b has anti-proliferative and migratory functions by targeting HSP90 in VSMCs and may aidin the development of new biomarkers and potential therapeutic targets for atherosclerosis. PMID:28337290

  3. Differential regulation of acidic and basic fibroblast growth factor gene expression in fibroblast growth factor-treated rat aortic smooth muscle cells.

    PubMed

    Alberts, G F; Hsu, D K; Peifley, K A; Winkles, J A

    1994-08-01

    The acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) proteins are potent vascular smooth muscle cell (SMC) mitogens that are expressed by endothelial cells and SMCs in vivo. Overexpression of these proteins in transfected cell lines can result in autocrine transformation; therefore, the precise control of fibroblast growth factor gene expression in the vessel wall may be an important mechanism regulating vascular cell growth. In the present study, we demonstrate that bFGF can induce bFGF mRNA expression, but not aFGF mRNA expression, in serum-starved rat aortic SMCs. bFGF autoinduction is maximal at 4 hours, requires de novo RNA and protein synthesis, and is mediated predominantly by a protein kinase C-dependent signaling pathway. Furthermore, aFGF treatment of rat SMCs also increases bFGF mRNA and protein expression; however, aFGF mRNA levels are only slightly modulated. These results suggest that the local release of aFGF or bFGF within the vessel wall could promote a prolonged period of elevated bFGF synthesis. This, in turn, could be of importance in the SMC hyperplasia that occurs in response to vascular injury and during atherosclerotic plaque formation.

  4. Mepivacaine-induced contraction involves phosphorylation of extracellular signal-regulated kinase through activation of the lipoxygenase pathway in isolated rat aortic smooth muscle.

    PubMed

    Lee, Hyo Min; Ok, Seong-Ho; Sung, Hui-Jin; Eun, So Young; Kim, Hye Jung; Lee, Soo Hee; Kang, Sebin; Shin, Il-Woo; Lee, Heon Keun; Chung, Young-Kyun; Choi, Mun-Jeoung; Bae, Sung Il; Sohn, Ju-Tae

    2013-04-01

    Mepivacaine is an aminoamide local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. This study investigated the arachidonic acid metabolic pathways involved in mepivacaine-induced contraction, and elucidated the associated cellular mechanism with a particular focus on extracellular signal-regulated kinase (ERK) in endothelium-denuded rat aorta. Isolated rat thoracic aortic rings were suspended for isometric tension recording. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, indomethacin, NS-398, SC-560, fluconazole, PD 98059, and verapamil. Mepivacaine-induced ERK phosphorylation, 5-lipoxygenase (5-LOX) expression, and cyclooxygenase (COX)-2 expression in rat aortic smooth muscle cells were detected by Western blot analysis in the presence or absence of inhibitors. Mepivacaine produced tonic contraction in isolated endothelium-denuded rat aorta. Quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, NS-398, PD 98059, and verapamil attenuated mepivacaine-induced contraction in a concentration-dependent manner. However, fluconazole had no effect on mepivacaine-induced contraction. PD 98059, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, and indomethacin attenuated mepivacaine-induced ERK phosphorylation. Mepivacaine upregulated 5-LOX and COX-2 expression. These results suggest that mepivacaine-induced contraction involves ERK activation, which is primarily mediated by the 5-LOX pathway and in part by the COX-2 pathway.

  5. PPARβ/δ, a Novel Regulator for Vascular Smooth Muscle Cells Phenotypic Modulation and Vascular Remodeling after Subarachnoid Hemorrhage in Rats.

    PubMed

    Zhang, Hongrong; Jiang, Li; Guo, Zongduo; Zhong, Jianjun; Wu, Jingchuan; He, Junchi; Liu, Han; He, Zhaohui; Wu, Haitao; Cheng, Chongjie; Sun, Xiaochuan

    2017-03-22

    Cerebral vascular smooth muscle cell (VSMC) phenotypic switch is involved in the pathophysiology of vascular injury after aneurysmal subarachnoid hemorrhage (aSAH), whereas the molecular mechanism underlying it remains largely speculative. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been implicated to modulate the vascular cells proliferation and vascular homeostasis. In the present study, we investigated the potential role of PPARβ/δ in VSMC phenotypic switch following SAH. Activation of PPARβ/δ by GW0742 and adenoviruses PPARβ/δ (Ad-PPARβ/δ) significantly inhibited hemoglobin-induced VSMC phenotypic switch. However, the effects of PPARβ/δ on VSMC phenotypic switch were partly obstacled in the presence of LY294002, a potent inhibitor of Phosphatidyl-Inositol-3 Kinase-AKT (PI3K/AKT). Furthermore, following study demonstrated that PPARβ/δ-induced PI3K/AKT activation can also contribute to Serum Response Factor (SRF) nucleus localization and Myocardin expression, which was highly associated with VSMC phenotypic switch. Finally, we found that Ad-PPARβ/δ positively modulated vascular remodeling in SAH rats, i.e. the diameter of basilar artery and the thickness of vessel wall. In addition, overexpression of PPARβ/δ by adenoviruses significantly improved neurological outcome. Taken together, this study identified PPARβ/δ as a useful regulator for VSMC phenotypic switch and vascular remodeling following SAH, providing novel insights into the therapeutic strategies of delayed cerebral ischemia.

  6. Comparative studies on troponin, a Ca²⁺-dependent regulator of muscle contraction, in striated and smooth muscles of protochordates.

    PubMed

    Obinata, Takashi; Sato, Naruki

    2012-01-01

    Troponin is well known as a Ca(2+)-dependent regulator of striated muscle contraction and it has been generally accepted that troponin functions as an inhibitor of muscle contraction or actin-myosin interaction at low Ca(2+) concentrations, and Ca(2+) at higher concentrations removes the inhibitory action of troponin. Recently, however, troponin became detectable in non-striated muscles of several invertebrates and in addition, unique troponin that functions as a Ca(2+)-dependent activator of muscle contraction has been detected in protochordate animals, although troponin in vertebrate striated muscle is known as an inhibitor of the contraction in the absence of a Ca(2+). Further studies on troponin in invertebrate muscle, especially in non-striated muscle, would provide new insight into the evolution of regulatory systems for muscle contraction and diverse function of troponin and related proteins. The methodology used for preparation and characterization of functional properties of protochordate striated and smooth muscles will be helpful for further studies of troponin in other invertebrate animals.

  7. PPARβ/δ, a Novel Regulator for Vascular Smooth Muscle Cells Phenotypic Modulation and Vascular Remodeling after Subarachnoid Hemorrhage in Rats

    PubMed Central

    Zhang, Hongrong; Jiang, Li; Guo, Zongduo; Zhong, Jianjun; Wu, Jingchuan; He, Junchi; Liu, Han; He, Zhaohui; Wu, Haitao; Cheng, Chongjie; Sun, Xiaochuan

    2017-01-01

    Cerebral vascular smooth muscle cell (VSMC) phenotypic switch is involved in the pathophysiology of vascular injury after aneurysmal subarachnoid hemorrhage (aSAH), whereas the molecular mechanism underlying it remains largely speculative. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) has been implicated to modulate the vascular cells proliferation and vascular homeostasis. In the present study, we investigated the potential role of PPARβ/δ in VSMC phenotypic switch following SAH. Activation of PPARβ/δ by GW0742 and adenoviruses PPARβ/δ (Ad-PPARβ/δ) significantly inhibited hemoglobin-induced VSMC phenotypic switch. However, the effects of PPARβ/δ on VSMC phenotypic switch were partly obstacled in the presence of LY294002, a potent inhibitor of Phosphatidyl-Inositol-3 Kinase-AKT (PI3K/AKT). Furthermore, following study demonstrated that PPARβ/δ-induced PI3K/AKT activation can also contribute to Serum Response Factor (SRF) nucleus localization and Myocardin expression, which was highly associated with VSMC phenotypic switch. Finally, we found that Ad-PPARβ/δ positively modulated vascular remodeling in SAH rats, i.e. the diameter of basilar artery and the thickness of vessel wall. In addition, overexpression of PPARβ/δ by adenoviruses significantly improved neurological outcome. Taken together, this study identified PPARβ/δ as a useful regulator for VSMC phenotypic switch and vascular remodeling following SAH, providing novel insights into the therapeutic strategies of delayed cerebral ischemia. PMID:28327554

  8. Protein kinase Cα stimulates hypoxia‑induced pulmonary artery smooth muscle cell proliferation in rats through activating the extracellular signal‑regulated kinase 1/2 pathway.

    PubMed

    Jiang, Rui; Shi, Yiwei; Zeng, Chao; Yu, Wenyan; Zhang, Aizhen; Du, Yongcheng

    2017-09-12

    Hypoxic pulmonary hypertension (HPH) may contribute to vascular remodeling, and pulmonary artery smooth muscle cell (PASMC) proliferation has an important role in this process. However, no relevant information concerning the role and mechanism of protein kinase C (PKC)α in hypoxia‑induced rat PASMC proliferation has been elucidated. The present study aimed to further investigate this by comparison of rat PASMC proliferation among normoxia for 72 h (21% O2), hypoxia for 72 h (3% O2), hypoxia + promoter 12‑myristate 13‑acetate control, hypoxia + safingol control, hypoxia + PD98059 control and hypoxia + U0126 control groups. The present study demonstrated that protein expression levels of PKCα in rat PASMCs were elevated. In conclusion, through activating the extracellular signal‑regulated 1/2 signaling pathway, PKCα is involved in and initiates PASMC proliferation, thus bringing about pulmonary artery hypertension. These results add to the understanding of the mechanism PKCα in PH formation and lays a theoretical basis for prevention as well as treatment of HPH.

  9. Non-raft adenylyl cyclase 2 defines a cAMP signaling compartment that selectively regulates IL-6 expression in airway smooth muscle cells: differential regulation of gene expression by AC isoforms.

    PubMed

    Bogard, Amy S; Birg, Anna V; Ostrom, Rennolds S

    2014-04-01

    Adenylyl cyclase (AC) isoforms differ in their tissue distribution, cellular localization, regulation, and protein interactions. Most cell types express multiple AC isoforms. We hypothesized that cAMP produced by different AC isoforms regulates unique cellular responses in human bronchial smooth muscle cells (BSMC). Overexpression of AC2, AC3, or AC6 had distinct effects on forskolin (Fsk)-induced expression of a number of known cAMP-responsive genes. These data show that different AC isoforms can differentially regulate gene expression. Most notable, overexpression and activation of AC2 enhanced interleukin 6 (IL-6) expression, but overexpression of AC3 or AC6 had no effect. IL-6 production by BSMC was induced by Fsk and select G protein-coupled receptor (GPCR) agonists, though IL-6 levels did not directly correlate with global cAMP levels. Treatment with PKA selective 6-Bnz-cAMP or Epac selective 8-CPT-2Me-cAMP cAMP analogs revealed a predominant role for PKA in cAMP-mediated induction of IL-6. IL-6 promoter mutations demonstrated that AP-1 and CRE transcription sites were required for Fsk to stimulate IL-6 expression. Our present study defines an AC2 cAMP signaling compartment that specifically regulates IL-6 expression in BSMC via Epac and PKA and demonstrates that other AC isoforms are excluded from this pool.

  10. 7 CFR 51.1870 - Fairly smooth.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946... Standards for Fresh Tomatoes 1 Definitions § 51.1870 Fairly smooth. Fairly smooth means that the tomato...

  11. D1-like receptors inhibit insulin-induced vascular smooth muscle cell proliferation via down-regulation of insulin receptor expression

    PubMed Central

    Zeng, Chunyu; Han, Yu; Huang, Hefei; Yu, Changqing; Ren, Hongmei; Shi, Weibin; He, Duofen; Huang, Lan; Yang, Chengming; Wang, Xukai; Zhou, Lin; Jose, Pedro A.

    2013-01-01

    Objective Vascular smooth muscle cell (VSMC) proliferation is central to the development of vascular diseases, including hypertension, which is regulated by numerous hormones and humoral factors. Our previous study showed that the stimulatory effect of norepinephrine on VSMC proliferation is inhibited by D1-like receptors and the D3 dopamine receptor, a member of the D2-like receptor family. Insulin is a proliferative hormone but it is not known if there is any interaction between insulin and D1-like receptors. We hypothesized that Dl-like receptors may have an inhibitory effect on the insulin-induced VSMC proliferation; aberrant insulin and Dl-like receptor functions could be involved in the pathogenesis of essential hypertension. Methods VSMC proliferation was determined by [3H]-thymidine incorporation; insulin receptor mRNA and protein expressions were determined by RT-PCR, immunoblotting, and immunohistochemistry. Results Insulin increased VSMC proliferation in immortalized aortic A10 cells, determined by [3H]-thymidine incorporation. Although the D1-like receptor, by itself, had no effect on VSMC proliferation, stimulation with fenoldopam, a D1-like receptor agonist, inhibited the stimulatory effect of insulin. The inhibitory effect of fenoldopam on insulin-mediated VSMC proliferation was receptor specific, because its effect could be blocked by SCH23390, a D1-like receptor antagonist. Fenoldopam also inhibited insulin receptor mRNA and protein expression, which was time dependent and concentration dependent. A PKC or MAP kinase inhibitor blocked the inhibitory effect of fenoldopam on insulin receptor expression, indicating that PKC and MAP kinase were involved in the signaling pathway. Conclusion The inhibitory effect of D1-like receptors on insulin-mediated VSMC proliferation may play an important role in the regulation of blood pressure. PMID:19293728

  12. Regulation of Guinea Pig Detrusor Smooth Muscle Excitability by 17β-Estradiol: The Role of the Large Conductance Voltage- and Ca2+-Activated K+ Channels.

    PubMed

    Provence, Aaron; Hristov, Kiril L; Parajuli, Shankar P; Petkov, Georgi V

    2015-01-01

    Estrogen replacement therapies have been suggested to be beneficial in alleviating symptoms of overactive bladder. However, the precise regulatory mechanisms of estrogen in urinary bladder smooth muscle (UBSM) at the cellular level remain unknown. Large conductance voltage- and Ca2+-activated K+ (BK) channels, which are key regulators of UBSM function, are suggested to be non-genomic targets of estrogens. This study provides an electrophysiological investigation into the role of UBSM BK channels as direct targets for 17β-estradiol, the principle estrogen in human circulation. Single BK channel recordings on inside-out excised membrane patches and perforated whole cell patch-clamp were applied in combination with the BK channel selective inhibitor paxilline to elucidate the mechanism of regulation of BK channel activity by 17β-estradiol in freshly-isolated guinea pig UBSM cells. 17β-Estradiol (100 nM) significantly increased the amplitude of depolarization-induced whole cell steady-state BK currents and the frequency of spontaneous transient BK currents in freshly-isolated UBSM cells. The increase in whole cell BK currents by 17β-estradiol was eliminated upon blocking BK channels with paxilline. 17β-Estradiol (100 nM) significantly increased (~3-fold) the single BK channel open probability, indicating direct 17β-estradiol-BK channel interactions. 17β-Estradiol (100 nM) caused a significant hyperpolarization of the membrane potential of UBSM cells, and this hyperpolarization was reversed by blocking the BK channels with paxilline. 17β-Estradiol (100 nM) had no effects on L-type voltage-gated Ca2+ channel currents recorded under perforated patch-clamp conditions. This study reveals a new regulatory mechanism in the urinary bladder whereby BK channels are directly activated by 17β-estradiol to reduce UBSM cell excitability.

  13. Glycogen synthase kinase 3{beta} regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    SciTech Connect

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-10-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 {beta} (GSK3{beta}) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3{beta} has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3{beta} (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3{beta} delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3{beta} is required for the activation of NFAT during wound repair.

  14. Targeting of a novel Ca+2/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells.

    PubMed

    Marganski, William A; Gangopadhyay, Samudra S; Je, Hyun-Dong; Gallant, Cynthia; Morgan, Kathleen G

    2005-09-16

    Subcellular targeting of kinases controls their activation and access to substrates. Although Ca2+/calmodulin-dependent protein kinase II (CaMKII) is known to regulate differentiated smooth muscle cell (dSMC) contractility, the importance of targeting in this regulation is not clear. The present study investigated the function in dSMCs of a novel variant of the gamma isoform of CaMKII that contains a potential targeting sequence in its association domain (CaMKIIgamma G-2). Antisense knockdown of CaMKIIgamma G-2 inhibited extracellular signal-related kinase (ERK) activation, myosin phosphorylation, and contractile force in dSMCs. Confocal colocalization analysis revealed that in unstimulated dSMCs CaMKIIgamma G-2 is bound to a cytoskeletal scaffold consisting of interconnected vimentin intermediate filaments and cytosolic dense bodies. On activation with a depolarizing stimulus, CaMKIIgamma G-2 is released into the cytosol and subsequently targeted to cortical dense plaques. Comparison of phosphorylation and translocation time courses indicates that, after CaMKIIgamma G-2 activation, and before CaMKIIgamma G-2 translocation, vimentin is phosphorylated at a CaMKII-specific site. Differential centrifugation demonstrated that phosphorylation of vimentin in dSMCs is not sufficient to cause its disassembly, in contrast to results in cultured cells. Loading dSMCs with a decoy peptide containing the polyproline sequence within the association domain of CaMKIIgamma G-2 inhibited targeting. Furthermore, prevention of CaMKIIgamma G-2 targeting led to significant inhibition of ERK activation as well as contractility. Thus, for the first time, this study demonstrates the importance of CaMKII targeting in dSMC signaling and identifies a novel targeting function for the association domain in addition to its known role in oligomerization.

  15. MiRNA regulation of TRAIL expression exerts selective cytotoxicity to prostate carcinoma cells.

    PubMed

    Huo, Wei; Jin, Ning; Fan, Li; Wang, Weihua

    2014-03-01

    Prostate carcinoma is the most common cancer for men and among the leading cancer-related causes. Many evidences have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) potently induces apoptosis in cancer cells, and thus, is a promising biologic agent for prostate carcinoma therapy. However, TRAIL expression mediated by the current vectors lacks tumor specificity, thereby exerting cytotoxicity to normal cells. To solve this problem, we inserted miRNA response elements (MREs), miR-143 and miR-145, expression levels of which were reduced in prostate carcinoma, as well as that of miR-122, which is specifically expressed in hepatic cells, into adenoviral vectors to control TRAIL expression (Ad-TRAIL-M3). qPCR data confirmed that miR-143, miR-145, and miR-122 levels were all decreased in prostate carcinoma cell lines and prostate cancer samples from patients. Luciferase assays showed that MREs-regulated luciferase expression was potently suppressed in normal cells, but not in prostate cancer cells. Ad-TRAIL-M3, which expresses TRAIL in a MREs-regulated manner, produced high level of TRAIL and suppressed the survival of prostate cancer cells by inducing apoptosis, while Ad-TRAIL-M3 had no TRAIL expression in normal cells and thus exerted no cytotoxicity to them. The studies on PC-3 tumor xenograft in mice further confirmed that Ad-TRAIL-M3 was able to inhibit the growth of tumors and possessed high biosafety. In conclusion, we successfully generated an adenoviral vector that expresses TRAIL in miRNA-regulated mechanism. This miRNA-based gene therapy may be promising for prostate carcinoma treatment.

  16. Advanced glycation end products promote human aortic smooth muscle cell calcification in vitro via activating NF-κB and down-regulating IGF1R expression.

    PubMed

    Wang, Yi; Zhang, Zhen-yu; Chen, Xiao-qing; Wang, Xiang; Cao, Heng; Liu, Shao-wen

    2013-04-01

    To investigate the effects of advanced glycation end products (AGEs) on calcification in human aortic smooth muscle cells (HASMCs) in vitro and the underlying mechanisms. AGEs were artificially prepared. Calcification of HASMCs was induced by adding inorganic phosphate (Pi, 2 mmol/L) in the media, and observed with Alizarin red staining. The calcium content in the supernatant was measured using QuantiChrome Calcium Assay Kit. Expression of the related mRNAs and proteins was analyzed using real-time PCR and Western blot, respectively. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NF-κB to the putative IGF1R promoter. AGEs (100 μg/mL) significantly enhanced Pi-induced calcification and the levels of osteocalcin and Cbfα1 in HASMCs. Furthermore, the treatment decreased the expression of insulin-like growth factor 1 receptor (IGF1R). Over-expression of IGF1R in HASMCs suppressed the AGEs-induced increase in calcium deposition. When IGF1R expression was knocked down in HASMCs, AGEs did not enhance the calcium deposition. Meanwhile, AGEs time-dependently decreased the amounts of IκBα and Flag-tagged p65 in the cytoplasmic extracts, and increased the amount of nuclear p65 in HASMCs. In the presence of NF-κB inhibitor PDTC (50 μmol/L), the AGEs-induced increase in calcium deposition was blocked. Over-expression of p65 significantly enhanced Pi-induced mineralization, but suppressed IGF1R mRNA level. Knockdown of p65 suppressed the AGEs-induced increase in calcium deposition, and rescued the IGF1R expression. The ChIP analysis revealed that NF-κB bound the putative IGF1R promoter at position -230 to -219 bp. The inhibition of IGF1R by NF-κB was abolished when IGF1R reporter plasmid contained mutated binding sequence for NF-κB or an NF-κB reporter vector. The results demonstrate that AGEs promote calcification of human aortic smooth muscle cells in vitro via activation of NF-κB and down-regulation of IGF1R expression.

  17. MicroRNA expression in Epstein-Barr virus-associated post-transplant smooth muscle tumours is related to leiomyomatous phenotype

    PubMed Central

    2013-01-01

    Epstein-Barr virus (EBV)-associated post-transplant smooth muscle tumours (PTSMT) are rare complications. In our previous molecular analysis, we have evaluated the expression of regulatory microRNA which are known to be EBV-related (miR-146a and miR-155) but found no deregulation in PTSMT. In this current analysis, we aimed to characterize the expression profiles of several hundred microRNA. Tissue samples from PTSMT and uterine leiomyomas were analysed by quantitative real-time PCR for the expression of 365 mature microRNA. PTSMT and leiomyomas share a highly similar microRNA profile, e.g. strong expression of miR-143/miR-145 cluster and low expression of miR-200c. Among EBV-related microRNA (miR-10b, miR-21, miR-29b, miR-34a, miR-127, miR-146a, miR-155, miR-200b, miR-203 and miR-429) only miR-10b and miR-203 were significantly deregulated. The expression pattern of microRNA in PTSMT is not associated with EBV infection but reflects the leiomyomatous differentiation of the tumour cells. PMID:23830214

  18. Urotensin II contributes to collagen synthesis and up-regulates Egr-1 expression in cultured pulmonary arterial smooth muscle cells through the ERK1/2 pathway

    SciTech Connect

    Li, Wei; Cai, Zhifeng; Liu, Mengmeng; Zhao, Cuifen; Li, Dong; Lv, Chenguang; Wang, Yuping; Xu, Tengfei

    2015-11-27

    Aim: The objective of this study was to investigate the effects of urotensin II (UII) treatment on the proliferation and collagen synthesis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) and to explore whether these effects are mediated by mitogen-activated protein kinase (MAPK) signaling pathways and early growth response 1 (Egr-1). Methods: The proliferation of cultured PASMCs stimulated with different doses of UII was detected by BrdU incorporation. The mRNA expression levels of procollagen I (procol I), procollagen III (procol III), extracellular regulated protein kinase 1/2 (ERK1/2), stress-stimulated protein kinase (Sapk), p38 MAPK (p38), and Egr-1 mRNA in cultured PASMCs after treatment with UII, the UII-specific antagonist urantide, and the ERK1/2 inhibitor PD98059 were detected by real-time polymerase chain reaction (PCR), and the protein expression levels of procol I, procol III, phosphorylated (p)-ERK1/2, p-Sapk, p-p38, and Egr-1 were detected by Western blotting. Results: Treatment with UII increased the proliferation of cultured PASMCs in a dose-dependent manner (P < 0.05). However, treatment with urantide and PD98059 inhibited the promoting effect of UII on PASMC proliferation (P < 0.05). Real-time PCR analysis showed that UII up-regulated the expression of procol I, procol III, ERK1/2, Sapk, and Egr-1 mRNA (P < 0.05), but not p38 mRNA. However, the up-regulating effect of UII was inhibited by PD98059 and urantide. Western blotting analysis showed that UII increased the synthesis of collagen I, collagen III, p-ERK1/2, p-Sapk, and Egr-1, and these effects also were inhibited by PD98059 and urantide (P < 0.05). Conclusions: Egr-1 participates in the UII-mediated proliferation and collagen synthesis of cultured rat PASMCs via activation of the ERK1/2 signaling pathway.

  19. Rosa hybrida extract suppresses vascular smooth muscle cell responses by the targeting of signaling pathways, cell cycle regulation and matrix metalloproteinase-9 expression.

    PubMed

    Lee, Se-Jung; Won, Se Yeon; Park, Sung Lyea; Song, Jun-Hui; Noh, Dae-Hwa; Kim, Hong-Man; Yin, Chang Shik; Kim, Wun-Jae; Moon, Sung-Kwon

    2016-04-01

    The pharmacological effects of Rosa hybrida are well known in the cosmetics industry. However, the role of Rosa hybrida in cardiovascular biology had not previously been investigated, to the best of our knowledge. The aim of the present study was to elucidate the effect of water extract of Rosa hybrida (WERH) on platelet‑derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs). VSMC proliferation, which was stimulated by PDGF, was inhibited in a non-toxic manner by WERH treatment, which also diminished the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and AKT. Treatment with WERH also induced G1-phase cell cycle arrest, which was due to the decreased expression of cyclins and cyclin-dependent kinases (CDKs), and induced p21WAF1 expression in PDGF-stimulated VSMCs. Moreover, WERH treatment suppressed the migration and invasion of VSMCs stimulated with PDGF. Treatment with WERH abolished the expression of matrix metalloproteinase-9 (MMP-9) and decreased the binding activity of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and specificity protein 1 (Sp1) motifs in PDGF-stimulated VSMCs. WERH treatment inhibited the proliferation of PDGF‑stimulated VSMCs through p21WAF1‑mediated G1-phase cell cycle arrest, by decreasing the kinase activity of cyclin/CDK complexes. Furthermore, WERH suppressed the PDGF-induced phosphorylation of ERK1/2 and AKT in VSMCs. Finally, treatment with WERH impeded the migration and invasion of VSMCs stimulated by PDGF by downregulating MMP-9 expression and a reduction in NF-κB, AP-1 and Sp1 activity. These results provide new insights into the effects of WERH on PDGF-stimulated VSMCs, and we suggest that WERH has the potential to act as a novel agent for the prevention and/or treatment of vascular diseases.

  20. Aspirin-induced AMP-activated protein kinase activation regulates the proliferation of vascular smooth muscle cells from spontaneously hypertensive rats

    SciTech Connect

    Sung, Jin Young; Choi, Hyoung Chul

    2011-05-06

    Highlights: {yields} Aspirin-induced AMPK phosphorylation was greater in VSMC from SHR than WKY. {yields} Aspirin-induced AMPK phosphorylation inhibited proliferation of VSMC from SHR. {yields} Low basal AMPK phosphorylation in SHR elicits increased VSMC proliferation. {yields} Inhibition of AMPK restored decreased VSMC proliferation by aspirin in SHR. {yields} Aspirin exerts anti-proliferative effect through AMPK activation in VSMC from SHR. -- Abstract: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.

  1. Sulforaphane inhibits PDGF-induced proliferation of rat aortic vascular smooth muscle cell by up-regulation of p53 leading to G1/S cell cycle arrest.

    PubMed

    Yoo, Su-Hyang; Lim, Yong; Kim, Seung-Jung; Yoo, Kyu-Dong; Yoo, Hwan-Soo; Hong, Jin-Tae; Lee, Mi-Yea; Yun, Yeo-Pyo

    2013-01-01

    Vascular diseases such as atherosclerosis and restenosis artery angioplasty are associated with vascular smooth muscle cell (VSMC) proliferation and intimal thickening arterial walls. In the present study, we investigated the inhibitory effects of sulforaphane, an isothiocyanate produced in cruciferous vegetables, on VSMC proliferation and neointimal formation in a rat carotid artery injury model. Sulforaphane at the concentrations of 0.5, 1.0, and 2.0 μM significantly inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation in a concentration-dependent manner, determined by cell count. The IC50 value of sulforaphane-inhibited VSMC proliferation was 0.8 μM. Sulforaphane increased the cyclin-dependent kinase inhibitor p21 and p53 levels, while it decreased CDK2 and cyclin E expression. The effects of sulforaphane on vascular thickening were determined 14 days after the injury to the rat carotid artery. The angiographic mean luminary diameters of the group treated with 2 and 4 μM sulforaphane were 0.25±0.1 and 0.09±0.1 mm², respectively, while the value of the control groups was 0.40±0.1 mm², indicating that sulforaphane may inhibit neointimal formation. The expression of PCNA, maker for cell cycle arrest, was decreased, while that of p53 and p21 was increased, which showed the same pattern as one in in-vitro study. These results suggest that sulforaphane-inhibited VSMC proliferation may occur through the G1/S cell cycle arrest by up-regulation of p53 signaling pathway, and then lead to the decreased neointimal hyperplasia thickening. Thus, sulforaphane may be a promising candidate for the therapy of atherosclerosis and post-angiography restenosis. © 2013.

  2. Mechanism by which nuclear factor-kappa beta (NF-kB) regulates ovine fetal pulmonary vascular smooth muscle cell proliferation.

    PubMed

    Ogbozor, Uchenna D; Opene, Michael; Renteria, Lissette S; McBride, Shaemion; Ibe, Basil O

    2015-09-01

    Platelet activating factor (PAF) modulates ovine fetal pulmonary hemodynamic. PAF acts through its receptors (PAFR) in pulmonary vascular smooth muscle cells (PVSMC) to phosphorylate and induce nuclear translocation of NF-kB p65 leading to PVSMC proliferation. However, the interaction of NF-kB p65 and PAF in the nuclear domain to effect PVSMC cell growth is not clearly defined. We used siRNA-dependent translation initiation arrest to study a mechanism by which NF-kB p65 regulates PAF stimulation of PVSMC proliferation. Our hypotheses are: (a) PAF induces NF-kB p65 DNA binding and (b) NF-kB p65 siRNA attenuates PAF stimulation of PVSMC proliferation. For DNA binding, cells were fed 10 nM PAF with and without PAFR antagonists WEB 2170, CV 3988 or BN 52021 and incubated for 12 h. DNA binding was measured by specific ELISA. For NF-kB p65 siRNA effect, starved cells transfected with the siRNA were incubated for 24 h with and without 10 nM PAF. Cell proliferation was measured by DNA synthesis while expression of NF-kB p65 and PAFR protein was measured by Western blotting. In both studies, the effect of 10% FBS alone was used as the positive control. In general, PAF stimulated DNA binding which was inhibited by PAFR antagonists. siRNAs to NF-kB p65 and PAFR significantly attenuated cell proliferation compared to 10% FBS and PAF effect. Inclusion of PAF in siRNA-treated cells did not reverse inhibitory effect of NF-kB p65 siRNA on DNA synthesis. PAFR expression was inhibited in siRNA-treated cells. These data show that PAF-stimulation of PVSMC proliferation occurs via a PAFR-NF-kB p65 linked pathway.

  3. MicroRNA-146a and -21 cooperate to regulate vascular smooth muscle cell proliferation via modulation of the Notch signaling pathway.

    PubMed

    Cao, Jian; Zhang, Kui; Zheng, Jubing; Dong, Ran

    2015-04-01

    A number of microRNAs (miRs) have been shown to participate in the regulation of vascular smooth muscle cell (VSMC) proliferation, a key step in the formation of atherosclerotic plaque, by targeting certain genes. The aim of the present study was to investigate the roles of miR‑146a and miR‑21 in VSMC growth and to study the underlying mechanisms. The expression levels of four previously reported, differentially expressed microRNAs in atherosclerotic plaque (miR‑146a/b, miR‑21, miR‑34a and miR‑210) were measured in two groups: An atherosclerotic plaque group (n=10) and a normal control group (n=10). Polymerase chain reaction (PCR) analysis revealed that the relative expression levels of miR‑146a and miR‑21 in atherosclerotic plaque samples were significantly upregulated to ~260 and 250%, respectively, compared with those in normal controls. Notch2 and Jag1 were confirmed to be target genes of miR‑146a and miR‑21 through the use of a luciferase assay, PCR and western blot analysis. Additionally, VSMCs transfected with miR‑146a expressed significantly lower levels of Notch2 protein and presented an accelerated cell proliferation, which could be attributed to a reduction in the levels of cell cycle arrest. Cotransfection of miR‑146a and miR‑21 further promoted cell cycle progression in addition to VSMC proliferation. In conclusion, the present study revealed that miR‑146a and miR‑21 were significantly upregulated in atherosclerotic plaque, and cooperated to accelerate VSMC growth and cell cycle progression by targeting Notch2 and Jag1.

  4. BK channels in rat and human pulmonary smooth muscle cells are BKα-β1 functional complexes lacking the oxygen-sensitive stress axis regulated exon insert

    PubMed Central

    Detweiler, Neil D.; Song, Li; McClenahan, Samantha J.; Versluis, Rachel J.; Kharade, Sujay V.; Kurten, Richard C.; Rhee, Sung W.

    2016-01-01

    Abstract A loss of K+ efflux in pulmonary arterial smooth muscle cells (PASMCs) contributes to abnormal vasoconstriction and PASMC proliferation during pulmonary hypertension (PH). Activation of high-conductance Ca2+-activated (BK) channels represents a therapeutic strategy to restore K+ efflux to the affected PASMCs. However, the properties of BK channels in PASMCs—including sensitivity to BK channel openers (BKCOs)—are poorly defined. The goal of this study was to compare the properties of BK channels between PASMCs of normoxic (N) and chronic hypoxic (CH) rats and then explore key findings in human PASMCs. Polymerase chain reaction results revealed that 94.3% of transcripts encoding BKα pore proteins in PASMCs from N rats represent splice variants lacking the stress axis regulated exon insert, which confers oxygen sensitivity. Subsequent patch-clamp recordings from inside-out (I-O) patches confirmed a dense population of BK channels insensitive to hypoxia. The BK channels were highly activated by intracellular Ca2+ and the BKCO lithocholate; these responses require BKα-β1 subunit coupling. PASMCs of CH rats with established PH exhibited a profound overabundance of the dominant oxygen-insensitive BKα variant. Importantly, human BK (hBK) channels in PASMCs from human donor lungs also represented the oxygen-insensitive BKα variant activated by BKCOs. The hBK channels showed significantly enhanced Ca2+ sensitivity compared with rat BK channels. We conclude that rat BK and hBK channels in PASMCs are oxygen-insensitive BKα-β1 complexes highly sensitive to Ca2+ and the BKCO lithocholate. BK channels are overexpressed in PASMCs of a rat model of PH and may provide an abundant target for BKCOs designed to restore K+ efflux. PMID:28090300

  5. Microarray analysis of ox-LDL (oxidized low-density lipoprotein)-regulated genes in human coronary artery smooth muscle cells.

    PubMed

    Minta, Joe; Jungwon Yun, James; St Bernard, Rosanne

    2010-01-01

    Recent studies suggest that circulating LDL (low-density lipoproteins) play a central role in the pathogenesis of atherosclerosis, and the oxidized form (ox-LDL) is highly atherogenic. Deposits of ox-LDL have been found in atherosclerotic plaques, and ox-LDL has been shown to promote monocyte recruitment, foam cell formation and the transition of quiescent and contractile vascular SMCs (smooth muscle cells) to the migratory and proliferative phenotype. SMC phenotype transition and hyperplasia are the pivotal events in the pathogenesis of atherosclerosis. To comprehend the complex molecular mechanisms involved in ox-LDL-mediated SMC phenotype transition, we have compared the differential gene expression profiles of cultured quiescent human coronary artery SMCs with cells induced with ox-LDL for 3 and 21 h using Affymetrix HG-133UA cDNA microarray chips. Assignment of the regulated genes into functional groups indicated that several genes involved in metabolism, membrane transport, cell-cell interactions, signal transduction, transcription, translation, cell migration, proliferation and apoptosis were differentially expressed. Our data suggests that the interaction of ox-LDL with its cognate receptors on SMCs modulates the induction of several growth factors and cytokines, which activate a variety of intracellular signalling mechanisms (including PI3K, MAPK, Jak/STAT, sphingosine, Rho kinase pathways) that contribute to SMC transition from the quiescent and contractile phenotype to the proliferative and migratory phenotype. Our study has also identified several genes (including CDC27, cyclin A1, cyclin G2, glypican 1, MINOR, p15 and apolipoprotein) not previously implicated in ox-LDL-induced SMC phenotype transition and substantially extends the list of potential candidate genes involved in atherogenesis.

  6. Opiorphin-dependent up-regulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice

    PubMed Central

    Fu, Shibo; Davies, Kelvin P.

    2015-01-01

    The precise molecular mechanisms underlying priapism associated with sickle cell disease remain to be defined. However, there is increasing evidence that up-regulated activity of the opiorphin and adenosine pathways in corporal tissue, resulting in heighted relaxation of smooth muscle, play an important role in development of priapism. A key enzyme in the adenosine pathway is CD73, an ecto-5-prime-nucleotidase (5-prime-ribonucleotide phosphohydrolase; EC 3.1.3.5) which catalyzes the conversion of adenosine mononucleotides to adenosine. In the present study we investigated how sickle cell disease and hypoxia regulate the interplay between opiorphin and CD73. In the corpora of sickle cell mice we observed significantly elevated expression of both the mouse opiorphin homologue mSmr3a (14-fold) and CD73 (2.2-fold) relative to non-sickle cell controls at a life-stage prior to the exhibition of priapism. Sickle cell disease has a pronounced hypoxic component, therefore we determined if CD73 was also modulated in in vitro corporal smooth muscle (CSM) models of hypoxia. Hypoxia significantly increased CD73 protein and mRNA expression by 1.5-fold and 2-fold, respectively. We previously demonstrated that expression of another component of the adenosine signaling pathway, the adensosine 2B receptor, can be regulated by sialorphin (the rat opiorphin homolologue), and we demonstrate that sialorphin also regulates CD73 expression in a dose and time dependent fashion. Using siRNA to knock-down sialorphin mRNA expression in CSM cells in vitro, we demonstrate that the hypoxic up-regulation of CD73 is dependent on the up-regulation of sialorphin. Overall our data provides further evidence to support a role for opiorphin in CSM in regulating the cellular response regulating response to hypoxia or sickle cell disease by activating smooth muscle relaxant pathways. PMID:25833166

  7. Zedoarondiol Inhibits Platelet-Derived Growth Factor-Induced Vascular Smooth Muscle Cells Proliferation via Regulating AMP-Activated Protein Kinase Signaling Pathway.

    PubMed

    Mao, Huimin; Tao, Tianqi; Song, Dandan; Liu, Mi; Wang, Xiaoren; Liu, Xiuhua; Shi, Dazhuo

    2016-01-01

    Vascular smooth muscle cells (VSMCs) proliferation contributes significantly to atherosclerosis and in-stent restenosis. Platelet-derived growth factor-BB (PDGF-BB) plays a vital role in VSMCs proliferation. Zedoarondiol, a sesquiterpene lactone compound, has an anti-inflammatory activity. However, the role of zedoarondiol in PDGF-BB-mediated VSMCs proliferation remains unclear. In this study, we investigated the effects of zedoarondiol on PDGF-BB-induced VSMCs proliferation and explored the possible mechanisms. The inhibitory effects of zedoarondiol on PDGF-BB-induced VSMCs proliferation were evaluated by direct cell counting and the Cell Counting Kit-8 (CCK-8) assay. DNA synthesis was examined by bromodeoxyuridine (BrdU) incorporation assay. Cell cycle was assessed by propidium iodide staining. Western blotting was performed to determine the expression of cyclin-dependent kinase 2 (CDK2), cyclin E, p53, p21, total and phosphorylated adenosine monophosphate-activated protein kinase (AMPK), acetyl CoA carboxylase (ACC), mammalian target of rapamycin (mTOR), and p70 ribosomal protein S6 kinase (p70S6K). Zedoarondiol suppressed PDGF-BB-induced VSMCs proliferation and DNA synthesis, and induced cell cycle arrest in G0/G1 phase. In addition, zedoarondiol activated AMPK and ACC, inhibited the phosphorylation of mTOR and p70S6K, increased the expression of p53 and p21, and decreased the expression of CDK2 and cyclin E. Compound C (an AMPK inhibitor) abrogated, whereas 5-aminoimidazole-4-carboxamide 1-β-ribofuranoside (AICAR, an AMPK activator) enhanced zedoarondiol-mediated inhibition of VSMCs proliferation and DNA synthesis. Zedoarondiol inhibits PDGF-BB-induced VSMCs proliferation via AMPK-mediated down-regulation of the mTOR/p70S6K pathway and up-regulation of the p53/p21 pathway. These findings suggest that zedoarondiol might be a promising compound against atherosclerosis and in-stent restenosis. © 2016 The Author(s) Published by S. Karger AG, Basel.

  8. Medpor regulates osteoblast's microRNAs.

    PubMed

    Palmieri, Annalisa; Pezzetti, Furio; Brunelli, Giorgio; Martinelli, Marcella; Scapoli, Luca; Arlotti, Marzia; Masiero, Elena; Carinci, Francesco

    2008-01-01

    Porous polyethylene (PP or Medpor) is an alloplastic material worldwide used for craniofacial reconstruction. Although several clinical studies are available, there is a lack as regard the genetic effects. Because PP is always fixed on bone and the mechanism by which PP acts on osteoblasts is unknown, we therefore attempted to address this question by using microRNA microarray techniques to investigate the translation regulation in osteoblasts exposed to PP. The miRNA oligonucleotide microarray provides a novel method to carry out genome-wide microRNA profiling in human samples. By using miRNA microarrays containing 329 probe designed from Human miRNA sequence, we identified in osteoblast-like cells line (MG-63) cultured with Medpor (Porex Corporation, Fairburn, Georgia, USA) several miRNA which expression is significantly modified. We identified 16 up-regulated miRNA (i.e. mir-337, mir-515-3p, mir-377, mir-153, mir-367, mir-152, let-7b, mir-92, mir-155, mir-424, mir-148b, mir-368, mir-18b, mir-520d, mir-20b, mir-128a) and 2 down-regulated miRNA (i.e. mir-143, mir-32). The data reported are, to our knowledge, the first study on translation regulation in osteoblasts exposed to PP. They can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects.

  9. Involvement of inositol 1,4,5-trisphosphate formation in the voltage-dependent regulation of the Ca(2+) concentration in porcine coronary arterial smooth muscle cells.

    PubMed

    Yamamura, Hisao; Ohya, Susumu; Muraki, Katsuhiko; Imaizumi, Yuji

    2012-08-01

    The involvement of inositol 1,4,5-trisphosphate (IP(3)) formation in the voltage-dependent regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined in smooth muscle cells of the porcine coronary artery. Slow ramp depolarization from -90 to 0 mV induced progressive [Ca(2+)](i) increase. The slope was reduced or increased in the presence of Cd(2+) or (±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]-phenyl)pyridine-3-carboxlic acid methyl ester (Bay K 8644), respectively. The decrease in [Ca(2+)](i) via the membrane hyperpolarization induced by K(+) channel openers (levcromakalim and Evans blue) under current clamp was identical to that under voltage clamp. The step hyperpolarization from -40 to -80 mV reduced [Ca(2+)](i) uniformly over the whole-cell area with a time constant of ∼10 s. The [Ca(2+)](i) at either potential was unaffected by heparin, an inhibitor of IP(3) receptors. Alternatively, [Ca(2+)](i) rapidly increased in the peripheral regions by depolarization from -80 to 0 mV and stayed at that level (∼400 nM) during a 60-s pulse. When the pipette solution contained IP(3) pathway blockers [heparin, 2-aminoethoxydiphenylborate, xestospongin C, or 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], the peak [Ca(2+)](i) was unchanged, but the sustained [Ca(2+)](i) was gradually reduced by ∼250 nM within 30 s. In the presence of Cd(2+), a long depolarization period slightly increased the [Ca(2+)](i), which was lower than that in the presence of heparin alone. In coronary arterial myocytes, the sustained increase in the [Ca(2+)](i) during depolarization was partly caused by the Ca(2+) release mediated by the enhanced formation of IP(3). The initial [Ca(2+)](i) elevation triggered by the Ca(2+) influx though voltage-dependent Ca(2+) channels may be predominantly responsible for the activation of phospholipase C for IP(3) formation.

  10. MicroRNA-133a regulates insulin-like growth factor-1 receptor expression and vascular smooth muscle cell proliferation in murine atherosclerosis.

    PubMed

    Gao, Song; Wassler, Michael; Zhang, Lulu; Li, Yangxin; Wang, Jun; Zhang, Yi; Shelat, Harnath; Williams, Jason; Geng, Yong-Jian

    2014-01-01

    MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis. Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE(-/-)) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE(-/-) aortas. ApoE(-/-) VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3'UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE(-/-) VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE(-/-) VSMC than in WT cells. MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1-stimulated VSMC survival and growth. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. MicroRNA-133a Regulates Insulin-like Growth Factor-1 Receptor Expression and Vascular Smooth Muscle Cell Proliferation in Murine Atherosclerosis

    PubMed Central

    Gao, Song; Wassler, Michael; Zhang, Lulu; Li, Yangxin; Wang, Jun; Zhang, Yi; Shelat, Harnath; Williams, Jason; Geng, Yong-Jian

    2014-01-01

    Objective MicroRNA-133a (miR-133a) and insulin-like growth factor-1 (IGF-1) are two different molecules known to regulate cardiovascular cell proliferation. This study tested whether miR-133a affects expression of IGF-1 receptor (IGF-1R) and proliferation of IGF-1-stimulated vascular smooth muscle cells (VSMC) in a murine model of atherosclerosis. Methods and Results Expression of IGF-1R was analyzed by immuno-fluorescence and immuno-blotting, and miR-133a by qRT-PCR in the aortas of wild-type C57BL/6J (WT) and apolipoprotein-E deficient (ApoE−/−) mice. Compared to those in WT aortas, the IGF-1R and miR-133a levels were lower in ApoE−/− aortas. ApoE−/− VSMC grew slower than WT cells in the cultures with IGF-1-containing medium. MiR-133a-specific inhibitor decreased miR-133a, IGF-1R expression, IGF-1-stimulated VSMC growth in lipoprotein-deficient media. By contrast, miR-133a precursor increased IGF-1R levels and promoted IGF-1-induced VSMC proliferation. In the luciferase-IGF-1R 3’UTR reporter system, the reporter luciferase activity was not inhibited in VSMC with miR-133a overexpression. IGF-1R mRNA half-life in ApoE−/− VSMC was shorter than that in WT VSMC. MiR-133a inhibitor reduced but precursor increased the mRNA half-life, although the effects appeared less striking in ApoE−/− VSMC than in WT cells. Conclusion MiR-133a serves as a stimulatory factor for IGF-1R expression through prolonging IGF-1R mRNA half-life. In atherosclerosis induced by ApoE deficiency, reduced miR-133a expression is associated with lower IGF-1R levels and suppressive VSMC growth. Administration of miR-133a precursor may potentiate IGF-1 stimulated VSMC survival and growth. PMID:24401233

  12. Sulfur Dioxide Inhibits Extracellular Signal-regulated Kinase Signaling to Attenuate Vascular Smooth Muscle Cell Proliferation in Angiotensin II-induced Hypertensive Mice

    PubMed Central

    Wu, Hui-Juan; Huang, Ya-Qian; Chen, Qing-Hua; Tian, Xiao-Yu; Liu, Jia; Tang, Chao-Shu; Jin, Hong-Fang; Du, Jun-Bao

    2016-01-01

    Background: Clarifying the mechanisms underlying vascular smooth muscle cell (VSMC) proliferation is important for the prevention and treatment of vascular remodeling and the reverse of hyperplastic lesions. Previous research has shown that the gaseous signaling molecule sulfur dioxide (SO2) inhibits VSMC proliferation, but the mechanism for the inhibition of the angiotensin II (AngII)-induced VSMC proliferation by SO2 has not been fully elucidated. This study was designed to investigate if SO2 inhibited VSMC proliferation in mice with hypertension induced by AngII. Methods: Thirty-six male C57 mice were randomly divided into control, AngII, and AngII + SO2 groups. Mice in AngII group and AngII + SO2 group received a capsule-type AngII pump implanted under the skin of the back at a slow-release dose of 1000 ng·kg−1·min−1. In addition, mice in AngII + SO2 received intraperitoneal injections of SO2 donor. Arterial blood pressure of tail artery was determined. The thickness of the aorta was measured by elastic fiber staining, and proliferating cell nuclear antigen (PCNA) and phosphorylated-extracellular signal-regulated kinase (P-ERK) were detected in aortic tissues. The concentration of SO2 in serum and aortic tissue homogenate supernatant was measured using high-performance liquid chromatography with fluorescence determination. In the in vitro study, VSMC of A7R5 cell lines was divided into six groups: control, AngII, AngII + SO2, PD98059 (an inhibitor of ERK phosphorylation), AngII + PD98059, and AngII + SO2 + PD98059. Expression of PCNA, ERK, and P-ERK was determined by Western blotting. Results: In animal experiment, compared with the control group, AngII markedly increased blood pressure (P < 0.01) and thickened the aortic wall in mice (P < 0.05) with an increase in the expression of PCNA (P < 0.05). SO2, however, reduced the systemic hypertension and the wall thickness induced by AngII (P < 0.05). It inhibited the increased expression of PCNA and P

  13. Developmentally regulated changes in extracellular matrix in endothelial and smooth muscle cells in the ductus arteriosus may be related to intimal proliferation

    SciTech Connect

    Boudreau, N.; Rabinovitch, M. )

    1991-02-01

    In the late gestation fetal lamb ductus arteriosus (DA), intimal proliferation is observed, characterized by smooth muscle migration and proliferation in the subendothelium. The nature of changes in the endothelial and smooth muscle extracellular matrix associated with the development of this feature are not known. We assessed the production of glycoproteins (fibronectin, laminin, and type IV collagen) and glycosaminoglycans (GAGs) (hyaluronic acid, heparan sulfate, and chondroitin sulfate) in endothelial and smooth muscle cells harvested from the DA, aorta (Ao), and pulmonary artery of fetal lambs at 100 days gestation, before the appearance of DA intimal proliferation, and at 138 days, when well-developed intimal cushions are seen. In passage 3 cells, glycoprotein synthesis was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after 48 hours incubation with (35S)methionine, and GAGs were assessed by labeling with (3H) glucosamine and separation on DEAE ion-exchange high performance liquid chromatography. Analyses were carried out on culture medium, cell layer, and solubilized matrix. Fibronectin secretion by DA smooth muscle cells from 100-day lambs was found to be twice that of Ao or pulmonary artery cells. No significant differences were seen in smooth muscle cells from 138-day lambs or when comparing endothelial cells from each of the vascular sites at both gestational ages. As well, there were no DA-specific differences in laminin or type IV collagen. No significant differences in endothelial GAG secretion were observed comparing each vascular site at both gestational ages. Analysis of endothelial-derived matrices, however, revealed increased incorporation of hyaluronic acid in the DA from 100-day lambs, 10-fold that of the pulmonary artery and Ao, and increased heparan sulfate.

  14. MicroRNA-143 regulates adipocyte differentiation.

    PubMed

    Esau, Christine; Kang, Xiaolin; Peralta, Eigen; Hanson, Elaine; Marcusson, Eric G; Ravichandran, Lingamanaidu V; Sun, Yingqing; Koo, Seongjoon; Perera, Ranjan J; Jain, Ravi; Dean, Nicholas M; Freier, Susan M; Bennett, C Frank; Lollo, Bridget; Griffey, Richard

    2004-12-10

    MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.

  15. Role of plasminogen activator inhibitor in the reciprocal regulation of bovine aortic endothelial and smooth muscle cell migration by TGF-beta 1.

    PubMed Central

    Petzelbauer, E.; Springhorn, J. P.; Tucker, A. M.; Madri, J. A.

    1996-01-01

    Vascular endothelial and smooth muscle cells exhibit reciprocal migratory responses after transforming growth factor (TGF)-beta 1 treatment. Endothelial cells exhibit a decreased migratory rate and smooth muscle cells exhibit an increased migratory rate. Previous studies have demonstrated increases in extracellular matrix and integrin synthesis and expression in response to TGF-beta 1. In this report, we illustrate the roles of plasminogen activator inhibitor in modulating the migratory rates in these two cell types. Endothelial cells appear to require a proteolytic phenotype for rapid migration, whereas vascular smooth muscle cells appear to require an anti-proteolytic phenotype. Modulation of proteinase/anti-proteinase activity ratios was accomplished via TGF-beta 1 induction, addition of exogenous plasminogen activator inhibitor, addition of anti-catalytic antibodies directed against urokinase plasminogen activator, overexpression of plasminogen activator inhibitor utilizing stable transfectants, and the use of vitronectin as a substratum. The reciprocal migratory behaviors exhibited by these two vascular cell types in response to TGF-beta 1 is discussed in the context that these two vascular cell types utilize distinct adhesive and signaling pathways in their interactions with extracellular matrix components and responsiveness to proteolytic activity. Images Figure 1 Figure 2 Figure 3 PMID:8780396

  16. GATA-6 and NF-κB Activate CPI-17 Gene Transcription and Regulate Ca2+ Sensitization of Smooth Muscle Contraction

    PubMed Central

    Boopathi, Ettickan; Hypolite, Joseph A.; Zderic, Stephen A.; Gomes, Cristiano Mendes; Malkowicz, Bruce; Liou, Hsiou-Chi; Wein, Alan J.

    2013-01-01

    Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca2+ sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH. PMID:23275439

  17. Role of protein kinase C in phospholemman mediated regulation of α₂β₁ isozyme of Na⁺/K⁺-ATPase in caveolae of pulmonary artery smooth muscle cells.

    PubMed

    Dey, Kuntal; Roy, Soumitra; Ghosh, Biswarup; Chakraborti, Sajal

    2012-04-01

    We have recently reported that α(2)β(1) and α(1)β(1) isozymes of Na(+)/K(+)-ATPase (NKA) are localized in the caveolae whereas only the α(1)β(1) isozyme of NKA is localized in the non-caveolae fraction of pulmonary artery smooth muscle cell membrane. It is well known that different isoforms of NKA are regulated differentially by PKA and PKC, but the mechanism is not known in the caveolae of pulmonary artery smooth muscle cells. Herein, we examined whether this regulation occurs through phospholemman (PLM) in the caveolae. Our results suggest that PKC mediated phosphorylation of PLM occurs only when it is associated with the α(2) isoform of NKA, whereas phosphorylation of PLM by PKA occurs when it is associated with the α(1) isoform of NKA. To investigate the mechanism of regulation of α(2) isoform of NKA by PKC-mediated phosphorylation of PLM, we have purified PLM from the caveolae and reconstituted into the liposomes. Our result revealed that (i) in the reconstituted liposomes phosphorylated PLM (PKC mediated) stimulate NKA activity, which appears to be due to an increase in the turnover number of the enzyme; (ii) phosphorylated PLM did not change the affinity of the pump for Na(+); and (iii) even after phosphorylation by PKC, PLM still remains associated with the α(2) isoform of NKA.

  18. 7 CFR 51.641 - Fairly smooth texture.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Fairly smooth texture. 51.641 Section 51.641 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... Other Than Florida, California, and Arizona) Definitions § 51.641 Fairly smooth texture. Fairly smooth...

  19. 7 CFR 51.641 - Fairly smooth texture.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Fairly smooth texture. 51.641 Section 51.641 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... Other Than Florida, California, and Arizona) Definitions § 51.641 Fairly smooth texture. Fairly smooth...

  20. 7 CFR 51.641 - Fairly smooth texture.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Fairly smooth texture. 51.641 Section 51.641 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards..., and Arizona) Definitions § 51.641 Fairly smooth texture. Fairly smooth texture means that the skin is...

  1. 7 CFR 51.701 - Fairly smooth texture.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Fairly smooth texture. 51.701 Section 51.701 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... Other Than Florida, California, and Arizona) Definitions § 51.701 Fairly smooth texture. Fairly smooth...

  2. 7 CFR 51.1008 - Fairly smooth texture.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Fairly smooth texture. 51.1008 Section 51.1008 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards....1008 Fairly smooth texture. Fairly smooth texture means that the fruit is comparatively free from...

  3. 7 CFR 51.1162 - Fairly smooth texture.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Fairly smooth texture. 51.1162 Section 51.1162 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... smooth texture. Fairly smooth texture means that the skin is fairly thin and not coarse for the variety...

  4. 7 CFR 51.701 - Fairly smooth texture.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Fairly smooth texture. 51.701 Section 51.701 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards..., and Arizona) Definitions § 51.701 Fairly smooth texture. Fairly smooth texture means that the skin is...

  5. 7 CFR 51.1162 - Fairly smooth texture.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Fairly smooth texture. 51.1162 Section 51.1162 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... Definitions § 51.1162 Fairly smooth texture. Fairly smooth texture means that the skin is fairly thin and not...

  6. 7 CFR 51.1008 - Fairly smooth texture.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Fairly smooth texture. 51.1008 Section 51.1008 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards....1008 Fairly smooth texture. Fairly smooth texture means that the fruit is comparatively free from...

  7. 7 CFR 51.641 - Fairly smooth texture.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Fairly smooth texture. 51.641 Section 51.641 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards..., and Arizona) Definitions § 51.641 Fairly smooth texture. Fairly smooth texture means that the skin is...

  8. 7 CFR 51.1162 - Fairly smooth texture.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Fairly smooth texture. 51.1162 Section 51.1162 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... Definitions § 51.1162 Fairly smooth texture. Fairly smooth texture means that the skin is fairly thin and not...

  9. 7 CFR 51.1162 - Fairly smooth texture.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Fairly smooth texture. 51.1162 Section 51.1162 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... smooth texture. Fairly smooth texture means that the skin is fairly thin and not coarse for the variety...

  10. 7 CFR 51.1162 - Fairly smooth texture.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Fairly smooth texture. 51.1162 Section 51.1162 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... smooth texture. Fairly smooth texture means that the skin is fairly thin and not coarse for the variety...

  11. 7 CFR 51.701 - Fairly smooth texture.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Fairly smooth texture. 51.701 Section 51.701 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards... Other Than Florida, California, and Arizona) Definitions § 51.701 Fairly smooth texture. Fairly smooth...

  12. 7 CFR 51.701 - Fairly smooth texture.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Fairly smooth texture. 51.701 Section 51.701 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards..., and Arizona) Definitions § 51.701 Fairly smooth texture. Fairly smooth texture means that the skin is...

  13. 7 CFR 51.641 - Fairly smooth texture.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Fairly smooth texture. 51.641 Section 51.641 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards..., and Arizona) Definitions § 51.641 Fairly smooth texture. Fairly smooth texture means that the skin is...

  14. 7 CFR 51.701 - Fairly smooth texture.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Fairly smooth texture. 51.701 Section 51.701 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards..., and Arizona) Definitions § 51.701 Fairly smooth texture. Fairly smooth texture means that the skin is...

  15. Smoothed square well potential

    NASA Astrophysics Data System (ADS)

    Salamon, P.; Vertse, T.

    2017-07-01

    The classical square well potential is smoothed with a finite range smoothing function in order to get a new simple strictly finite range form for the phenomenological nuclear potential. The smoothed square well form becomes exactly zero smoothly at a finite distance, in contrast to the Woods-Saxon form. If the smoothing range is four times the diffuseness of the Woods-Saxon shape both the central and the spin-orbit terms of the Woods-Saxon shape are reproduced reasonably well. The bound single-particle energies in a Woods-Saxon potential can be well reproduced with those in the smoothed square well potential. The same is true for the complex energies of the narrow resonances.

  16. Magnolol inhibits colonic motility through down-regulation of voltage-sensitive L-type Ca2+ channels of colonic smooth muscle cells in rats.

    PubMed

    Zhang, Man; Zang, Kai-Hong; Luo, Jia-Lie; Leung, Fung-Ping; Huang, Yu; Lin, Cheng-Yuan; Yang, Zhi-Jun; Lu, Ai-Ping; Tang, Xu-Dong; Xu, Hong-Xi; Sung, Joseph Jao-yiu; Bian, Zhao-Xiang

    2013-11-15

    This study aimed to investigate the effect of magnolol (5,5'-diallyl-2,2'-biphenyldiol) on contraction in distal colonic segments of rats and the underlying mechanisms. Colonic segments were mounted in organ baths for isometric force measurement. Whole-cell voltage-sensitive L-type Ca(2+) currents were recorded on isolated single colonic smooth muscle cells using patch-clamp technique. The spontaneous contractions and acetylcholine (ACh)- and Bay K 8644-induced contractions were inhibited by magnolol (3-100 μM). In the presence of Bay K8644 (100 nM), magnolol (10-100 μM) inhibited the contraction induced by 10 μM ACh. By contrast, tetrodotoxin (100 nM) and Nώ-nitro-L-arginine methyl ester (L-NAME 100 μM) did not change the inhibitory effect of magnolol (10 μM). In addition, magnolol (3-100 μM) inhibited the L-type Ca(2+) currents. The present results suggest that magnolol inhibits colonic smooth muscle contraction through downregulating L-type Ca(2+) channel activity.

  17. The 5-hydroxytryptamine transporter is functional in human coronary artery smooth muscle cells proliferation and is regulated by Interleukin-1 beta

    PubMed Central

    Wang, Qing-Jie; Wang, Dong; Tang, Cheng-Chun

    2015-01-01

    Abnormal human coronary artery smooth muscle cells (hCASMCs) proliferation and migration are key factors in coronary artery restenosis after percutaneous coronary intervention. Platelets release 5-hydroxytryptamine (5-HT), which is a strong mitogen for pulmonary artery smooth muscle cells proliferation and migration. Here, we investigated the effects of 5-HT and role of 5-HT transporter (5-HTT) on hCASMCs proliferation and migration. The 5-HT (10-6-10-5 mol/l) significantly increased hCASMCs proliferation and migration, and these effects were inhibited by fluoxetine (10-5 mol/l) and citalopram (10-6 mol/l), two 5-HTT blocker. Overexpression in hCASMCs enhanced 5-HT induced cells proliferation and migration. The 5-HTT and interleukin-1 beta (IL-1β) expression were increased in rat balloon injury carotid arteries. Treatment with IL-1β (10 ng/ml, 3d) upregulates 5-HTT expression in hCASMCs and increased 5-HT induced currents in Human Embryonic Kidney 293-5-HTT cells. PMID:26221231

  18. IGF-I Stimulates Cooperative Interaction between the IGF-I Receptor and CSK Homologous Kinase that Regulates SHPS-1 Phosphorylation in Vascular Smooth Muscle Cells

    PubMed Central

    Radhakrishnan, Yashwanth; Shen, Xinchun; Maile, Laura A.; Xi, Gang

    2011-01-01

    IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation. PMID:21799000

  19. Leiomodin and tropomodulin in smooth muscle

    NASA Technical Reports Server (NTRS)

    Conley, C. A.

    2001-01-01

    Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

  20. Leiomodin and tropomodulin in smooth muscle

    NASA Technical Reports Server (NTRS)

    Conley, C. A.

    2001-01-01

    Evidence is accumulating to suggest that actin filament remodeling is critical for smooth muscle contraction, which implicates actin filament ends as important sites for regulation of contraction. Tropomodulin (Tmod) and smooth muscle leiomodin (SM-Lmod) have been found in many tissues containing smooth muscle by protein immunoblot and immunofluorescence microscopy. Both proteins cofractionate with tropomyosin in the Triton-insoluble cytoskeleton of rabbit stomach smooth muscle and are solubilized by high salt. SM-Lmod binds muscle tropomyosin, a biochemical activity characteristic of Tmod proteins. SM-Lmod staining is present along the length of actin filaments in rat intestinal smooth muscle, while Tmod stains in a punctate pattern distinct from that of actin filaments or the dense body marker alpha-actinin. After smooth muscle is hypercontracted by treatment with 10 mM Ca(2+), both SM-Lmod and Tmod are found near alpha-actinin at the periphery of actin-rich contraction bands. These data suggest that SM-Lmod is a novel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth muscle may be capped by Tmod in localized clusters.

  1. Interleukin(IL)-1 Regulates Ozone-enhanced Tracheal Smooth Muscle Responsiveness by Increasing Substance P (SP) Production in Intrinsic Airway Neurons of Ferret

    PubMed Central

    Wu, Z.-X.; Barker, J. S.; Batchelor, T. P.; Dey, R.D.

    2008-01-01

    Exposure to ozone induces airway hyperresponsiveness (AHR) mediated partly by SP released from nerve terminals of intrinsic airway neurons. Our recent studies showed that IL-1, an important multifunctional proinflammatory cytokine, increases synthesis and release of SP from intrinsic airway neurons. The purpose of this study is to investigate the possible involvement of endogenous IL-1 in modulating neural responses associated with ozone-enhanced airway responsiveness. Ferrets were exposed to 2 ppm ozone or filtered air for 3 hrs. IL-1 in the bronchoalveolar lavage (BAL) fluid was significantly increased in ozone-exposed animals and responses of tracheal smooth muscle to methacholine (MCh) and electrical field stimulation (EFS) were elevated significantly. Both the SP nerve fiber density in tracheal smooth muscle and the number of SP-containing neurons in airway ganglia were significantly increased following ozone exposure. Pretreatment with IL-1 receptor antagonist (IL-1 Ra) significantly diminished ozone-enhanced airway responses to EFS as well as ozone-increased SP in the airway. To selectively investigate intrinsic airway neurons, segments of ferret trachea were maintained in culture conditions for 24 hrs to eliminate extrinsic contributions from sensory nerves. The segments were then exposed to 2 ppm ozone in vitro for 3 hrs. The changes of ozone-induced airway responses to MCh and EFS, and the SP levels in airway neurons paralleled those observed with in vivo ozone exposure. The ozone-enhanced airway responses and neuronal SP levels were inhibited by pretreatment with IL-1 Ra. These findings show that IL-1 is released during ozone exposure enhances airway responsiveness by modulating SP expression in airway neurons. PMID:18718561

  2. Smoothing error pitfalls

    NASA Astrophysics Data System (ADS)

    von Clarmann, T.

    2014-04-01

    The difference due to the content of a priori information between a constrained retrieval and the true atmospheric state is usually represented by the so-called smoothing error. In this paper it is shown that the concept of the smoothing error is questionable because it is not compliant with Gaussian error propagation. The reason for this is that the smoothing error does not represent the expected deviation of the retrieval from the true state but the expected deviation of the retrieval from the atmospheric state sampled on an arbitrary grid, which is itself a smoothed representation of the true state. The idea of a sufficiently fine sampling of this reference atmospheric state is untenable because atmospheric variability occurs on all scales, implying that there is no limit beyond which the sampling is fine enough. Even the idealization of infinitesimally fine sampling of the reference state does not help because the smoothing error is applied to quantities which are only defined in a statistical sense, which implies that a finite volume of sufficient spatial extent is needed to meaningfully talk about temperature or concentration. Smoothing differences, however, which play a role when measurements are compared, are still a useful quantity if the involved a priori covariance matrix has been evaluated on the comparison grid rather than resulting from interpolation. This is, because the undefined component of the smoothing error, which is the effect of smoothing implied by the finite grid on which the measurements are compared, cancels out when the difference is calculated.

  3. Stopping smooth pursuit.

    PubMed

    Missal, Marcus; Heinen, Stephen J

    2017-04-19

    If a visual object of interest suddenly starts to move, we will try to follow it with a smooth movement of the eyes. This smooth pursuit response aims to reduce image motion on the retina that could blur visual perception. In recent years, our knowledge of the neural control of smooth pursuit initiation has sharply increased. However, stopping smooth pursuit eye movements is less well understood and will be discussed in this paper. The most straightforward way to study smooth pursuit stopping is by interrupting image motion on the retina. This causes eye velocity to decay exponentially towards zero. However, smooth pursuit stopping is not a passive response, as shown by behavioural and electrophysiological evidence. Moreover, smooth pursuit stopping is particularly influenced by active prediction of the upcoming end of the target. Here, we suggest that a particular class of inhibitory neurons of the brainstem, the omnipause neurons, could play a central role in pursuit stopping. Furthermore, the role of supplementary eye fields of the frontal cortex in smooth pursuit stopping is also discussed.This article is part of the themed issue 'Movement suppression: brain mechanisms for stopping and stillness'. © 2017 The Author(s).

  4. Quantum State Smoothing

    NASA Astrophysics Data System (ADS)

    Guevara, Ivonne; Wiseman, Howard

    2015-10-01

    Smoothing is an estimation method whereby a classical state (probability distribution for classical variables) at a given time is conditioned on all-time (both earlier and later) observations. Here we define a smoothed quantum state for a partially monitored open quantum system, conditioned on an all-time monitoring-derived record. We calculate the smoothed distribution for a hypothetical unobserved record which, when added to the real record, would complete the monitoring, yielding a pure-state "quantum trajectory." Averaging the pure state over this smoothed distribution yields the (mixed) smoothed quantum state. We study how the choice of actual unraveling affects the purity increase over that of the conventional (filtered) state conditioned only on the past record.

  5. Quantum State Smoothing.

    PubMed

    Guevara, Ivonne; Wiseman, Howard

    2015-10-30

    Smoothing is an estimation method whereby a classical state (probability distribution for classical variables) at a given time is conditioned on all-time (both earlier and later) observations. Here we define a smoothed quantum state for a partially monitored open quantum system, conditioned on an all-time monitoring-derived record. We calculate the smoothed distribution for a hypothetical unobserved record which, when added to the real record, would complete the monitoring, yielding a pure-state "quantum trajectory." Averaging the pure state over this smoothed distribution yields the (mixed) smoothed quantum state. We study how the choice of actual unraveling affects the purity increase over that of the conventional (filtered) state conditioned only on the past record.

  6. Diamond Smoothing Tools

    NASA Technical Reports Server (NTRS)

    Voronov, Oleg

    2007-01-01

    Diamond smoothing tools have been proposed for use in conjunction with diamond cutting tools that are used in many finish-machining operations. Diamond machining (including finishing) is often used, for example, in fabrication of precise metal mirrors. A diamond smoothing tool according to the proposal would have a smooth spherical surface. For a given finish machining operation, the smoothing tool would be mounted next to the cutting tool. The smoothing tool would slide on the machined surface left behind by the cutting tool, plastically deforming the surface material and thereby reducing the roughness of the surface, closing microcracks and otherwise generally reducing or eliminating microscopic surface and subsurface defects, and increasing the microhardness of the surface layer. It has been estimated that if smoothing tools of this type were used in conjunction with cutting tools on sufficiently precise lathes, it would be possible to reduce the roughness of machined surfaces to as little as 3 nm. A tool according to the proposal would consist of a smoothing insert in a metal holder. The smoothing insert would be made from a diamond/metal functionally graded composite rod preform, which, in turn, would be made by sintering together a bulk single-crystal or polycrystalline diamond, a diamond powder, and a metallic alloy at high pressure. To form the spherical smoothing tip, the diamond end of the preform would be subjected to flat grinding, conical grinding, spherical grinding using diamond wheels, and finally spherical polishing and/or buffing using diamond powders. If the diamond were a single crystal, then it would be crystallographically oriented, relative to the machining motion, to minimize its wear and maximize its hardness. Spherically polished diamonds could also be useful for purposes other than smoothing in finish machining: They would likely also be suitable for use as heat-resistant, wear-resistant, unlubricated sliding-fit bearing inserts.

  7. Vascular smooth muscle phenotypic diversity and function

    PubMed Central

    2010-01-01

    The control of force production in vascular smooth muscle is critical to the normal regulation of blood flow and pressure, and altered regulation is common to diseases such as hypertension, heart failure, and ischemia. A great deal has been learned about imbalances in vasoconstrictor and vasodilator signals, e.g., angiotensin, endothelin, norepinephrine, and nitric oxide, that regulate vascular tone in normal and disease contexts. In contrast there has been limited study of how the phenotypic state of the vascular smooth muscle cell may influence the contractile response to these signaling pathways dependent upon the developmental, tissue-specific (vascular bed) or disease context. Smooth, skeletal, and cardiac muscle lineages are traditionally classified into fast or slow sublineages based on rates of contraction and relaxation, recognizing that this simple dichotomy vastly underrepresents muscle phenotypic diversity. A great deal has been learned about developmental specification of the striated muscle sublineages and their phenotypic interconversions in the mature animal under the control of mechanical load, neural input, and hormones. In contrast there has been relatively limited study of smooth muscle contractile phenotypic diversity. This is surprising given the number of diseases in which smooth muscle contractile dysfunction plays a key role. This review focuses on smooth muscle contractile phenotypic diversity in the vascular system, how it is generated, and how it may determine vascular function in developmental and disease contexts. PMID:20736412

  8. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.

    PubMed

    Aghamohammadzadeh, Reza; Zhang, Ying-Yi; Stephens, Thomas E; Arons, Elena; Zaman, Paula; Polach, Kevin J; Matar, Majed; Yung, Lai-Ming; Yu, Paul B; Bowman, Frederick P; Opotowsky, Alexander R; Waxman, Aaron B; Loscalzo, Joseph; Leopold, Jane A; Maron, Bradley A

    2016-07-01

    Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth

  9. miR Cluster 143/145 Directly Targets Nrl and Regulates Rod Photoreceptor Development.

    PubMed

    Sreekanth, Sreekumaran; Rasheed, Vazhanthodi A; Soundararajan, Lalitha; Antony, Jayesh; Saikia, Minakshi; Sivakumar, Krishnankutty Chandrika; Das, Ani V

    2016-11-23

    Retinal histogenesis requires coordinated and temporal functioning of factors by which different cell types are generated from multipotent progenitors. Development of rod photoreceptors is regulated by multiple transcription factors, and Nrl is one of the major factors involved in their fate specification. Presence or absence of Nrl at the postnatal stages decides the generation of cone photoreceptors or other later retinal cells. This suggests the need for regulated expression of Nrl in order to accelerate the generation of other cell types during retinal development. We found that miR cluster 143/145, comprising miR-143 and miR-145, targets and imparts a posttranscriptional inhibition of Nrl. Expression of both miRNAs was differentially regulated during retinal development and showed least expression at PN1 stage in which most of the rod photoreceptors are generated. Downregulation of rod photoreceptor regulators and markers upon miR cluster 143/145 overexpression demonstrated that this cluster indeed negatively regulates rod photoreceptors. Further, we prove that Nrl positively regulates miR cluster 143/145, thus establishing a feedback loop regulatory mechanism. This may be one possible mechanism by which Nrl is posttranscriptionally regulated to facilitate the generation of other cell types in retina.

  10. The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

    PubMed Central

    Tsapara, Anna; Luthert, Phillip; Greenwood, John; Hill, Caroline S.

    2010-01-01

    Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases. PMID:20089843

  11. The effect of alpha-tocopherol on the synthesis, phosphorylation and activity of protein kinase C in smooth muscle cells after phorbol 12-myristate 13-acetate down-regulation.

    PubMed

    Clément, S; Tasinato, A; Boscoboinik, D; Azzi, A

    1997-06-15

    Previous work had established that, in smooth muscle cells, alpha-tocopherol negatively regulates protein kinase C by preventing its activation [Tasinato, A., Boscoboinik, D., Bartoli, G. M., Maroni, P. & Azzi, A. (1995) Proc. Natl Acad. Sci. USA 92, 12190-12194]. In this study, the mechanism by which this event takes place has been analyzed. The regulation by alpha-tocopherol of protein kinase C expression, activity and phosphorylation has been followed during the synthesis of protein kinase C after its down-regulation by phorbol 12-myristate 13-acetate. The data show that protein kinase C isoenzyme alpha is synthesised significantly more (30% 72 h after down-regulation) in the presence of alpha-tocopherol. However, its activity is significantly less (45% diminution) and its phosphorylation state is also decreased (60% diminution). The effect of alpha-tocopherol appears not to be shared by the analogue beta-tocopherol, provided with similar radical-scavenging properties. The data are interpreted in terms of a diminution of protein kinase C phosphorylation, specifically caused by alpha-tocopherol, resulting in a decreased enzyme specific activity.

  12. Molecular cloning, expression of, and regulation by thyroid-hormone receptor α A in the half-smooth tongue sole Cynoglossus semilaevis during metamorphosis.

    PubMed

    Zhang, W T; Liu, K; Xiang, J S; Zhang, L Y; Liu, W J; Dong, Z D; Li, Y Z; Li, H L; Chen, S L; Wang, N

    2016-05-01

    To elucidate the effect of thyroid hormone receptor α A (thraa) on metamorphosis, the full length cDNA of half-smooth tongue sole Cynoglossus semilaevis was cloned. The relative gene transcript level of thraa at different development stages was quantified using real-time PCR. Transcription of thraa increased and declined rapidly during metamorphosis. Hyperthyroidism was induced in juveniles and larvae with exposure to T3 and T4, and hypothyroidism with thiourea (TU), 2-mercapto-1-methylimidazole (MMI). thraa mRNA was higher in fish treated for 6 days with MMI than in untreated controls, although inhibited larvae did not complete metamorphosis. The addition of exogenous T4 reversed this effect in the MMI-treated group, but not in the TU-treated group. In situ hybridization revealed progressive tail end of body growth and change during developmental stages, with corresponding changes in thraa expression. This process may be induced by thyroid hormones wi