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Sample records for rapamycin regulates vascular

  1. 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

    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 p38more » 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.« less

  2. 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.

  3. Differential Regulation of Cardiac Function and Intracardiac Cytokines by Rapamycin in Healthy and Diabetic Rats

    PubMed Central

    Luck, Christian; DeMarco, Vincent G.; Mahmood, Abuzar; Gavini, Madhavi P.

    2017-01-01

    Diabetes is comorbid with cardiovascular disease and impaired immunity. Rapamycin improves cardiac functions and extends lifespan by inhibiting the mechanistic target of rapamycin complex 1 (mTORC1). However, in diabetic murine models, Rapamycin elevates hyperglycemia and reduces longevity. Since Rapamycin is an immunosuppressant, we examined whether Rapamycin (750 μg/kg/day) modulates intracardiac cytokines, which affect the cardiac immune response, and cardiac function in male lean (ZL) and diabetic obese Zucker (ZO) rats. Rapamycin suppressed levels of fasting triglycerides, insulin, and uric acid in ZO but increased glucose. Although Rapamycin improved multiple diastolic parameters (E/E′, E′/A′, E/Vp) initially, these improvements were reversed or absent in ZO at the end of treatment, despite suppression of cardiac fibrosis and phosphoSer473Akt. Intracardiac cytokine protein profiling and Ingenuity® Pathway Analysis indicated suppression of intracardiac immune defense in ZO, in response to Rapamycin treatment in both ZO and ZL. Rapamycin increased fibrosis in ZL without increasing phosphoSer473Akt and differentially modulated anti-fibrotic IL-10, IFNγ, and GM-CSF in ZL and ZO. Therefore, fundamental difference in intracardiac host defense between diabetic ZO and healthy ZL, combined with differential regulation of intracardiac cytokines by Rapamycin in ZO and ZL hearts, underlies differential cardiac outcomes of Rapamycin treatment in health and diabetes. PMID:28408970

  4. Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer's disease.

    PubMed

    Lin, Ai-Ling; Zheng, Wei; Halloran, Jonathan J; Burbank, Raquel R; Hussong, Stacy A; Hart, Matthew J; Javors, Martin; Shih, Yen-Yu Ian; Muir, Eric; Solano Fonseca, Rene; Strong, Randy; Richardson, Arlan G; Lechleiter, James D; Fox, Peter T; Galvan, Veronica

    2013-09-01

    Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias.

  5. Chronic rapamycin restores brain vascular integrity and function through NO synthase activation and improves memory in symptomatic mice modeling Alzheimer's disease

    PubMed Central

    Lin, Ai-Ling; Zheng, Wei; Halloran, Jonathan J; Burbank, Raquel R; Hussong, Stacy A; Hart, Matthew J; Javors, Martin; Shih, Yen-Yu Ian; Muir, Eric; Solano Fonseca, Rene; Strong, Randy; Richardson, Arlan G; Lechleiter, James D; Fox, Peter T; Galvan, Veronica

    2013-01-01

    Vascular pathology is a major feature of Alzheimer's disease (AD) and other dementias. We recently showed that chronic administration of the target-of-rapamycin (TOR) inhibitor rapamycin, which extends lifespan and delays aging, halts the progression of AD-like disease in transgenic human (h)APP mice modeling AD when administered before disease onset. Here we demonstrate that chronic reduction of TOR activity by rapamycin treatment started after disease onset restored cerebral blood flow (CBF) and brain vascular density, reduced cerebral amyloid angiopathy and microhemorrhages, decreased amyloid burden, and improved cognitive function in symptomatic hAPP (AD) mice. Like acetylcholine (ACh), a potent vasodilator, acute rapamycin treatment induced the phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) and NO release in brain endothelium. Administration of the NOS inhibitor L-NG-Nitroarginine methyl ester reversed vasodilation as well as the protective effects of rapamycin on CBF and vasculature integrity, indicating that rapamycin preserves vascular density and CBF in AD mouse brains through NOS activation. Taken together, our data suggest that chronic reduction of TOR activity by rapamycin blocked the progression of AD-like cognitive and histopathological deficits by preserving brain vascular integrity and function. Drugs that inhibit the TOR pathway may have promise as a therapy for AD and possibly for vascular dementias. PMID:23801246

  6. Proteomic Identification of Novel Targets Regulated by the Mammalian Target of Rapamycin Pathway during Oligodendrocyte Differentiation

    PubMed Central

    Tyler, William A.; Jain, Mohit Raja; Cifelli, Stacey E.; Li, Qing; Ku, Li; Feng, Yue; Li, Hong; Wood, Teresa L.

    2011-01-01

    Previous work from our laboratory demonstrated that the mammalian target of rapamycin (mTOR) is active during and required for oligodendrocyte progenitor cell (OPC) differentiation. Here, we applied an iTRAQ mass spectrometry-based proteomic approach to identify novel targets of the mTOR pathway during OPC differentiation. Among the 978 proteins identified in this study, 328 (34%) exhibited a greater than 20% change (p < 0.05) in control versus rapamycin treated cultures following 4 days of differentiation in vitro. Interestingly, 197 (20%) proteins were elevated in rapamycin treated cultures, while 131 (13%) proteins were down-regulated by rapamycin. In support of our previous data, inhibiting mTOR caused a dramatic reduction pin the expression of myelin proteins. mTOR also was required for the induction of proteins involved in cholesterol and fatty acid synthesis, as well as the expression of many cytoskeletal proteins, cell signaling components, and nuclear/transcriptional regulators. Of particular interest was the identification of several critical mediators of oligodendrocyte differentiation. Specifically, mTOR activity controls the developmentally programmed up-regulation of the pro-differentiation factors Fyn and Quaking (QKI), whereas the expression of the differentiation repressor Gpr17 was elevated by mTOR inhibition. These data reveal a distinct signature of mTOR-regulated protein expression during OPC differentiation. PMID:21858874

  7. Proteomic identification of novel targets regulated by the mammalian target of rapamycin pathway during oligodendrocyte differentiation.

    PubMed

    Tyler, William A; Jain, Mohit Raja; Cifelli, Stacey E; Li, Qing; Ku, Li; Feng, Yue; Li, Hong; Wood, Teresa L

    2011-11-01

    Previous work from our laboratory demonstrated that the mammalian target of rapamycin (mTOR) is active during and required for oligodendrocyte progenitor cell (OPC) differentiation. Here, we applied an iTRAQ mass spectrometry-based proteomic approach to identify novel targets of the mTOR pathway during OPC differentiation. Among the 978 proteins identified in this study, 328 (34%) exhibited a greater than 20% change (P < 0.05) in control versus rapamycin-treated cultures following 4 days of differentiation in vitro. Interestingly, 197 (20%) proteins were elevated in rapamycin-treated cultures, while 131 (13%) proteins were downregulated by rapamycin. In support of our previous data, inhibiting mTOR caused a dramatic reduction in the expression of myelin proteins. mTOR also was required for the induction of proteins involved in cholesterol and fatty acid synthesis, as well as the expression of many cytoskeletal proteins, cell signaling components, and nuclear/transcriptional regulators. Of particular interest was the identification of several critical mediators of oligodendrocyte differentiation. Specifically, mTOR activity controls the developmentally programmed upregulation of the prodifferentiation factors Fyn and Quaking, whereas the expression of the differentiation repressor Gpr17 was elevated by mTOR inhibition. These data reveal a distinct signature of mTOR-regulated protein expression during OPC differentiation. Copyright © 2011 Wiley-Liss, Inc.

  8. Target of Rapamycin Signaling Regulates Metabolism, Growth, and Life Span in Arabidopsis[W][OA

    PubMed Central

    Ren, Maozhi; Venglat, Prakash; Qiu, Shuqing; Feng, Li; Cao, Yongguo; Wang, Edwin; Xiang, Daoquan; Wang, Jinghe; Alexander, Danny; Chalivendra, Subbaiah; Logan, David; Mattoo, Autar; Selvaraj, Gopalan; Datla, Raju

    2012-01-01

    Target of Rapamycin (TOR) is a major nutrition and energy sensor that regulates growth and life span in yeast and animals. In plants, growth and life span are intertwined not only with nutrient acquisition from the soil and nutrition generation via photosynthesis but also with their unique modes of development and differentiation. How TOR functions in these processes has not yet been determined. To gain further insights, rapamycin-sensitive transgenic Arabidopsis thaliana lines (BP12) expressing yeast FK506 Binding Protein12 were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf, and shoot growth and development leading to poor nutrient uptake and light energy utilization. Experimental limitation of nutrient availability and light energy supply in wild-type Arabidopsis produced phenotypes observed with TOR knockdown plants, indicating a link between TOR signaling and nutrition/light energy status. Genetic and physiological studies together with RNA sequencing and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and life span in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, and rRNA and protein synthesis. Gain- and loss-of-function Ribosomal Protein S6 (RPS6) mutants additionally show that TOR function involves RPS6-mediated nutrition and light-dependent growth and life span in Arabidopsis. PMID:23275579

  9. Mammalian target of rapamycin (mTOR) regulates TLR3 induced cytokines in human oral keratinocytes

    PubMed Central

    Zhao, Jiawei; Benakanakere, Manjunatha R.; Hosur, Kavita B.; Galicia, Johnah C.; Martin, Michael; Kinane, Denis F.

    2010-01-01

    Recent studies implicate the mammalian target of rapamycin (mTOR) pathway in the control of inflammatory responses following Toll-like receptor (TLR) stimulation in myeloid cells but its role in non-myeloid cells such as human keratinocytes is unknown. Here we show that TLR3 signaling can induce robust cytokine secretion including interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), IL-12p70 and interferon beta (IFN-β), and our data reveal for the first time that inhibiting mTOR with rapamycin, suppresses these TLR3 induced responses but actually enhances bioactive IL-12p70 production in human oral keratinocytes. Rapamycin inhibited the phosphorylation of the 70-kDa ribosomal protein S6 kinase (p70S6K) and the 4E binding protein 1 (4EBP-1), and suppressed the mitogen activated protein kinase (MAPK) pathway by decreasing phosphorylation of c-Jun N-terminal kinase (JNK). We also show that TLR3 induces interferon regulatory factor 3 (IRF3) activation by Akt via an mTOR-p70S6K-4EBP1 pathway. Furthermore, we provide evidence that Poly I:C induced expression of IL-1β, TNFα, IL-12p70 and IFN-β was blocked by JNK inhibitor SP600125. TLR3 preferentially phosphorylated IKKα through mTOR to activate nuclear factor kappa beta (NF-kB) in human oral keratinocytes. Taken together, these data demonstrate p70S6K, p4EBP1, JNK, NF-kB and IRF3 are involved in the regulation of inflammatory mediators by TLR3 via the mTOR pathway. mTOR is a novel pathway modulating TLR3 induced inflammatory and antiviral responses in human oral keratinocytes. PMID:20728939

  10. Profiling the role of mammalian target of rapamycin in the vascular smooth muscle metabolome in pulmonary arterial hypertension

    PubMed Central

    Kudryashova, Tatiana V.; Goncharov, Dmitry A.; Pena, Andressa; Ihida-Stansbury, Kaori; DeLisser, Horace; Kawut, Steven M.

    2015-01-01

    Abstract Increased proliferation and resistance to apoptosis of pulmonary arterial vascular smooth muscle cells (PAVSMCs), coupled with metabolic reprogramming, are key components of pulmonary vascular remodeling, a major and currently irreversible pathophysiological feature of pulmonary arterial hypertension (PAH). We recently reported that activation of mammalian target of rapamycin (mTOR) plays a key role in increased energy generation and maintenance of the proliferative, apoptosis-resistant PAVSMC phenotype in human PAH, but the downstream effects of mTOR activation on PAH PAVSMC metabolism are not clear. Using liquid and gas chromatography–based mass spectrometry, we performed pilot metabolomic profiling of human microvascular PAVSMCs from idiopathic-PAH subjects before and after treatment with the selective adenosine triphosphate–competitive mTOR inhibitor PP242 and from nondiseased lungs. We have shown that PAH PAVSMCs have a distinct metabolomic signature of altered metabolites—components of fatty acid synthesis, deficiency of sugars, amino sugars, and nucleotide sugars—intermediates of protein and lipid glycosylation, and downregulation of key biochemicals involved in glutathione and nicotinamide adenine dinucleotide (NAD) metabolism. We also report that mTOR inhibition attenuated or reversed the majority of the PAH-specific abnormalities in lipogenesis, glycosylation, glutathione, and NAD metabolism without affecting altered polyunsaturated fatty acid metabolism. Collectively, our data demonstrate a critical role of mTOR in major PAH PAVSMC metabolic abnormalities and suggest the existence of de novo lipid synthesis in PAVSMCs in human PAH that may represent a new, important component of disease pathogenesis worthy of future investigation. PMID:26697174

  11. Mammalian target of rapamycin regulates miRNA-1 and follistatin in skeletal myogenesis

    PubMed Central

    Sun, Yuting; Ge, Yejing; Drnevich, Jenny; Zhao, Yong; Band, Mark

    2010-01-01

    Mammalian target of rapamycin (mTOR) has emerged as a key regulator of skeletal muscle development by governing distinct stages of myogenesis, but the molecular pathways downstream of mTOR are not fully understood. In this study, we report that expression of the muscle-specific micro-RNA (miRNA) miR-1 is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of histone deacetylase 4 (HDAC4) results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the long-sought mTOR-regulated fusion factor. In summary, our findings unravel for the first time a link between mTOR and miRNA biogenesis and identify an mTOR–miR-1–HDAC4–follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo. PMID:20566686

  12. eIF4E-Overexpression imparts perillyl alcohol and rapamycin-mediated regulation of telomerase reverse transcriptase.

    PubMed

    Sundin, Tabetha; Peffley, Dennis; Hentosh, Patricia

    2013-08-01

    Translation is mediated partly by regulation of free eukaryotic initiation factor 4E (eIF4E) levels through PI3K-Akt-mTOR signaling. Cancer cells treated with the plant-derived perillyl alcohol (POH) or the mechanistic target of rapamycin (mTOR) inhibitor rapamycin dephosphorylate eIF4E-binding protein (4E-BP1) and attenuate cap-dependent translation. We previously showed in cancer cell lines with elevated eIF4E that POH and rapamycin regulate telomerase activity through this pathway. Here, immortalized Chinese hamster ovary (CHO) control cells and CHO cells with forced eIF4E expression (rb4E) were used to elucidate eIF4E's role in telomerase regulation by POH and rapamycin. Despite 5-fold higher eIF4E amounts in rb4E, telomerase activity, telomerase reverse transcriptase (TERT) mRNA, and TERT protein were nearly equivalent in control and rb4E cells. In control cells, telomerase activity, TERT mRNA and protein levels were unaffected by either compound. In contrast, telomerase activity and TERT protein were both attenuated by either agent in rb4E cells, but without corresponding TERT mRNA decreases indicating a translational/post-translational process. S6K, Akt, and 4E-BP1 were modulated by mTOR mediators only in the presence of increased eIF4E. Thus, eIF4E-overexpression in rb4E cells enables inhibitory effects of POH and rapamycin on telomerase and TERT protein. Importantly, eIF4E-overexpression modifies cellular protein synthetic processes and gene regulation. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Mammalian target of rapamycin (mTOR): a central regulator of male fertility?

    PubMed

    Jesus, Tito T; Oliveira, Pedro F; Sousa, Mário; Cheng, C Yan; Alves, Marco G

    2017-06-01

    Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolic phenotype and is involved in virtually all aspects of cellular function. It integrates not only nutrient and energy-sensing pathways but also actin cytoskeleton organization, in response to environmental cues including growth factors and cellular energy levels. These events are pivotal for spermatogenesis and determine the reproductive potential of males. Yet, the molecular mechanisms by which mTOR signaling acts in male reproductive system remain a matter of debate. Here, we review the current knowledge on physiological and molecular events mediated by mTOR in testis and testicular cells. In recent years, mTOR inhibition has been explored as a prime strategy to develop novel therapeutic approaches to treat cancer, cardiovascular disease, autoimmunity, and metabolic disorders. However, the physiological consequences of mTOR dysregulation and inhibition to male reproductive potential are still not fully understood. Compelling evidence suggests that mTOR is an arising regulator of male fertility and better understanding of this atypical protein kinase coordinated action in testis will provide insightful information concerning its biological significance in other tissues/organs. We also discuss why a new generation of mTOR inhibitors aiming to be used in clinical practice may also need to include an integrative view on the effects in male reproductive system.

  14. Cell cycle regulation by the nutrient-sensing mammalian target of rapamycin (mTOR) pathway.

    PubMed

    Cuyàs, Elisabet; Corominas-Faja, Bruna; Joven, Jorge; Menendez, Javier A

    2014-01-01

    Cell division involves a series of ordered and controlled events that lead to cell proliferation. Cell cycle progression implies not only demanding amounts of cell mass, protein, lipid, and nucleic acid content but also a favorable energy state. The mammalian target of rapamycin (mTOR), in response to the energy state, nutrient status, and growth factor stimulation of cells, plays a pivotal role in the coordination of cell growth and the cell cycle. Here, we review how the nutrient-sensing mTOR-signaling cascade molecularly integrates nutritional and mitogenic/anti-apoptotic cues to accurately coordinate cell growth and cell cycle. First, we briefly outline the structure, functions, and regulation of the mTOR complexes (mTORC1 and mTORC2). Second, we concisely evaluate the best known ability of mTOR to control G1-phase progression. Third, we discuss in detail the recent evidence that indicates a new genome stability caretaker function of mTOR based on the specific ability of phosphorylated forms of several mTOR-signaling components (AMPK, raptor, TSC, mTOR, and S6K1), which spatially and temporally associate with essential mitotic regulators at the mitotic spindle and at the cytokinetic cleavage furrow.

  15. Rapamycin regulates autophagy and cell adhesion in induced pluripotent stem cells.

    PubMed

    Sotthibundhu, Areechun; McDonagh, Katya; von Kriegsheim, Alexander; Garcia-Munoz, Amaya; Klawiter, Agnieszka; Thompson, Kerry; Chauhan, Kapil Dev; Krawczyk, Janusz; McInerney, Veronica; Dockery, Peter; Devine, Michael J; Kunath, Tilo; Barry, Frank; O'Brien, Timothy; Shen, Sanbing

    2016-11-15

    Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined. Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment. We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation. High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of

  16. The roles of juvenile hormone, insulin/target of rapamycin, and ecydsone signaling in regulating body size in Drosophila.

    PubMed

    Mirth, Christen Kerry; Shingleton, Alexander William

    2014-10-01

    Understanding how organisms regulate their body size has interested biologists for decades. Recent work has shown that both insulin/target of rapamycin (TOR) signaling and the steroid hormone ecdysone act to regulate rates of growth and the duration of the growth period in the fruit fly, Drosophila melanogaster. Our recent work has uncovered a third level of interaction, whereby juvenile hormone (JH) regulates levels of both ecdysone and insulin/TOR signaling to control growth rates. These studies highlight a complex network of interactions involved in regulating body and organ size.

  17. Regulation of cardiac miR-208a, an inducer of obesity, by rapamycin and nebivolol.

    PubMed

    Gul, Rukhsana; Mahmood, Abuzar; Luck, Christian; Lum-Naihe, Kelly; Alfadda, Assim A; Speth, Robert C; Pulakat, Lakshmi

    2015-11-01

    Resistance to obesity is observed in rodents and humans treated with rapamycin (Rap) or nebivolol (Neb). Because cardiac miR-208a promotes obesity, this study tested whether the modes of actions of Rap and Neb involve inhibition of miR-208a. Mouse cardiomyocyte HL-1 cells and Zucker obese (ZO) rats were used to investigate regulation of cardiac miR-208a. Angiotensin II (Ang II) increased miR-208a expression in HL-1 cells. Pretreatment with an AT1 receptor (AT1R) antagonist, losartan (1 μM), antagonized this effect, whereas a phospholipase C inhibitor, U73122 (10 μM), and an NADPH oxidase inhibitor, apocynin (0.5 mM), did not. Ang II-induced increase in miR-208a was suppressed by Rap (10 nM), an inhibitor of nutrient sensor kinase mTORC1, and Neb (1 μM), a 3rd generation β-blocker that suppressed bioavailable AT1R binding of (125) I-Ang II. Thus, suppression of AT1R expression by Neb, inhibition of AT1R activation by losartan, and inhibition of AT1R-induced activation of mTORC1 by Rap attenuated the Ang II-induced increase in miR-208a. In ZO rats, Rap treatment (750 μg kg(-1)  day(-1) ; 12 weeks) reduced obesity despite similar food intake, suppressed cardiac miR-208a, and increased cardiac MED13, a suppresser of obesity. Rap and Neb suppressed cardiac miR-208a. Suppression of miR-208a and increase in MED13 correlated with attenuated weight gain despite leptin resistance. © 2015 The Obesity Society.

  18. Targeting Rapamycin to Podocytes Using a Vascular Cell Adhesion Molecule-1 (VCAM-1)-Harnessed SAINT-Based Lipid Carrier System.

    PubMed

    Visweswaran, Ganesh Ram R; Gholizadeh, Shima; Ruiters, Marcel H J; Molema, Grietje; Kok, Robbert J; Kamps, Jan A A M

    2015-01-01

    Together with mesangial cells, glomerular endothelial cells and the basement membrane, podocytes constitute the glomerular filtration barrier (GFB) of the kidney. Podocytes play a pivotal role in the progression of various kidney-related diseases such as glomerular sclerosis and glomerulonephritis that finally lead to chronic end-stage renal disease. During podocytopathies, the slit-diaphragm connecting the adjacent podocytes are detached leading to severe loss of proteins in the urine. The pathophysiology of podocytopathies makes podocytes a potential and challenging target for nanomedicine development, though there is a lack of known molecular targets for cell selective drug delivery. To identify VCAM-1 as a cell-surface receptor that is suitable for binding and internalization of nanomedicine carrier systems by podocytes, we investigated its expression in the immortalized podocyte cell lines AB8/13 and MPC-5, and in primary podocytes. Gene and protein expression analyses revealed that VCAM-1 expression is increased by podocytes upon TNFα-activation for up to 24 h. This was paralleled by anti-VCAM-1 antibody binding to the TNFα-activated cells, which can be employed as a ligand to facilitate the uptake of nanocarriers under inflammatory conditions. Hence, we next explored the possibilities of using VCAM-1 as a cell-surface receptor to deliver the potent immunosuppressant rapamycin to TNFα-activated podocytes using the lipid-based nanocarrier system Saint-O-Somes. Anti-VCAM-1-rapamycin-SAINT-O-Somes more effectively inhibited the cell migration of AB8/13 cells than free rapamycin and non-targeted rapamycin-SAINT-O-Somes indicating the potential of VCAM-1 targeted drug delivery to podocytes.

  19. Rapamycin: one drug, many effects

    PubMed Central

    Li, Jing; Kim, Sang Gyun; Blenis, John

    2014-01-01

    The mammalian target of rapamycin (mTOR) signaling pathway is a master regulator of cell growth and metabolism. Deregulation of the mTOR pathway has been implicated in a number of human diseases such as cancer, diabetes, obesity, neurological diseases and genetic disorders. Rapamycin, a specific inhibitor of mTOR, has been shown to be useful in the treatment of certain diseases. Here we discuss its mechanism of action and highlight recent findings regarding the effects and limitations of rapamycin monotherapy and the potential utility of combination therapy with rapamycin. PMID:24508508

  20. Profiling of the fetal and adult rat liver transcriptome and translatome reveals discordant regulation by the mechanistic target of rapamycin (mTOR).

    PubMed

    Boylan, Joan M; Sanders, Jennifer A; Neretti, Nicola; Gruppuso, Philip A

    2015-07-01

    The mechanistic target of rapamycin (mTOR) integrates growth factor signaling, nutrient abundance, cell growth, and proliferation. On the basis of our interest in somatic growth in the late gestation fetus, we characterized the role of mTOR in the regulation of hepatic gene expression and translation initiation in fetal and adult rats. Our strategy was to manipulate mTOR signaling in vivo and then characterize the transcriptome and translating mRNA in liver tissue. In adult rats, we used the nonproliferative growth model of refeeding after a period of fasting and the proliferative model of liver regeneration following partial hepatectomy. We also studied livers from preterm fetal rats (embryonic day 19) in which fetal hepatocytes are asynchronously proliferating. All three models employed rapamycin to inhibit mTOR signaling. Analysis of the transcriptome in fasted-refed animals showed rapamycin-mediated induction of genes associated with oxidative phosphorylation. Genes associated with RNA processing were downregulated. In liver regeneration, rapamycin induced genes associated with lysosomal metabolism, steroid metabolism, and the acute phase response. In fetal animals, rapamycin inhibited expression of genes in several functional categories that were unrelated to effects in the adult animals. Translation control showed marked fetal-adult differences. In both adult models, rapamycin inhibited the translation of genes with complex 5' untranslated regions, including those encoding ribosomal proteins. Fetal translation was resistant to the effects of rapamycin. We conclude that the mTOR pathway in liver serves distinct physiological roles in the adult and fetus, with the latter representing a condition of rapamycin resistance. Copyright © 2015 the American Physiological Society.

  1. [Rapamycin-conditioned dendritic cells induced immune tolerance through the regulation of Treg/Th17 cells in mice].

    PubMed

    Yang, Xiaoyong; Yao, Qingchun; Hu, Xiaopeng; Wang, Wei; Yin, Hang; Ren, Liang; Liu, Hang; Zhang, Xiaodong

    2015-08-11

    To investigate the effect of tolerogenic dendritic cells (Tol-DC) generated by Rapamycin (Rapa) on the differentiation of Treg/Th17 cells and explore the possible mechanism of tolerance induction. DC progenitors from mouse bone marrow were propagated with granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin (IL)-4 stimulation for 6 days in the presence or absence of Rapa (20 ng/ml). During DC culture, morphology of cell was observed under electron microscope. Cell surface expression of CD11c, CD40 and CD80 was analyzed by flow cytometry. The antigen-presenting function of DC was determined by one-way mixed leukocyte reactions. In vivo, the recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control, Rapa, immature DC (imDC) and Tol-DC group. The survival time of the skin allograft was observed and Treg/Th17 cells were analyzed by flow cytometry in each group. The immunephenotypic analysis showed that in comparison with those in the control group and the LPS group the expression of the co-stimulatory molecules CD40 and CD80 were significantly lower in the Rapa-group and Rapa+LPS group. The ability to stimulate proliferation of T cells of the same genotype in the Rapa-group was significantly inhibited (P<0.01). In the in vivo experiment, the mice's survival time remarkably prolonged, the percentage of Treg cells was enhanced and Th17 cells was reduced in the mice's spleen in Tol-DC group. Tol-DC generated by Rapamycin can significantly induce immune tolerance through up-regulate Tregs and down-regulate Th17 cells. The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs, which can be loaded with donor antigen, and potentially used to promote organ transplant tolerance.

  2. Insulin-Like Growth Factor I Regulates G2/M Progression Through Mammalian Target of Rapamycin Signaling in Oligodendrocyte Progenitors

    PubMed Central

    Min, Jungsoo; Singh, Sukhwinder; Fitzgerald-Bocarsly, Patricia; Wood, Teresa L.

    2016-01-01

    Extrinsic factors including growth factors influence decisions of oligodendrocyte progenitor cells (OPCs) to continue cell cycle progression or exit the cell cycle and terminally differentiate into oligodendrocytes capable of producing myelin. Multiple studies have elucidated how the G1/S transition is regulated in OPCs; however, little is known about how S phase progression and the G2/M transition are regulated in these cells. Herein, we report that insulin-like growth factor (IGF)-I coordinates with FGF-2 to promote S phase progression but regulates G2/M progression independently. During S phase, IGF-I/FGF-2 enhances protein expression of cyclin A and cdk2, and further increases effective complex formation resulting in enhanced cdk2 activity. Surprisingly, however, OPCs exposed to FGF-2 in the absence of IGF-I fail to traverse through G2/M. Consistent with this observation, OPCs exposed to IGF-I, but not FGF-2, increase cell number over 48 h. IGF-I enhances cdk1 kinase activity during G2/M by promoting nuclear localization of cyclin B/cdk1 as well as of Cdc25C, an activator of cdk1. IGF-I also induces phosphorylation of histone 3 indicating traverse of cells through mitosis. Finally, we demonstrate that IGF-I-mediated G2/M regulation requires mammalian target of rapamycin activity. These data support an important function for IGF-I in G2/M progression in OPCs. PMID:22836368

  3. VESGEN Software for Mapping and Quantification of Vascular Regulators

    NASA Technical Reports Server (NTRS)

    Parsons-Wingerter, Patricia A.; Vickerman, Mary B.; Keith, Patricia A.

    2012-01-01

    VESsel GENeration (VESGEN) Analysis is an automated software that maps and quantifies effects of vascular regulators on vascular morphology by analyzing important vessel parameters. Quantification parameters include vessel diameter, length, branch points, density, and fractal dimension. For vascular trees, measurements are reported as dependent functions of vessel branching generation. VESGEN maps and quantifies vascular morphological events according to fractal-based vascular branching generation. It also relies on careful imaging of branching and networked vascular form. It was developed as a plug-in for ImageJ (National Institutes of Health, USA). VESGEN uses image-processing concepts of 8-neighbor pixel connectivity, skeleton, and distance map to analyze 2D, black-and-white (binary) images of vascular trees, networks, and tree-network composites. VESGEN maps typically 5 to 12 (or more) generations of vascular branching, starting from a single parent vessel. These generations are tracked and measured for critical vascular parameters that include vessel diameter, length, density and number, and tortuosity per branching generation. The effects of vascular therapeutics and regulators on vascular morphology and branching tested in human clinical or laboratory animal experimental studies are quantified by comparing vascular parameters with control groups. VESGEN provides a user interface to both guide and allow control over the users vascular analysis process. An option is provided to select a morphological tissue type of vascular trees, network or tree-network composites, which determines the general collections of algorithms, intermediate images, and output images and measurements that will be produced.

  4. The anti-diabetic drug metformin inhibits vascular endothelial growth factor expression via the mammalian target of rapamycin complex 1/hypoxia-inducible factor-1α signaling pathway in ELT-3 cells.

    PubMed

    Tadakawa, Mari; Takeda, Takashi; Li, Bin; Tsuiji, Kenji; Yaegashi, Nobuo

    2015-01-05

    The aim of this study was to elucidate whether metformin can regulate the expression of vascular endothelial growth factor (VEGF) in rat-derived uterine leiomyoma cells (ELT-3 cells). In vitro studies were conducted using ELT-3 cells. Under normoxic conditions, metformin suppressed VEGF protein levels in the supernatant and cells in a dose-dependent manner. In hypoxia-mimicking conditions, VEGF and hypoxia-inducible factor-1α (HIF-1α) proteins were both highly expressed and were suppressed by the metformin treatment. Metformin did not affect HIF-1α mRNA levels, which indicated that its effects occurred at the post-translational level. Metformin inhibited mammalian target of rapamycin complex 1 (mTORC1) activity by phosphorylating the mTORC1 component raptor. This study revealed the anti-angiogenic activity of metformin in ELT-3 cells by suppressing the expression of VEGF via the mTORC1/HIF-1α pathway. These results indicate that metformin may represent an effective alternative in the future treatment of uterine leiomyomas. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.

    PubMed

    Cho, Yongcheol; Di Liberto, Valentina; Carlin, Dan; Abe, Namiko; Li, Kathy H; Burlingame, Alma L; Guan, Shenheng; Michaelevski, Izhak; Cavalli, Valeria

    2014-05-30

    Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Dietary rapamycin supplementation reverses age-related vascular dysfunction and oxidative stress, while modulating nutrient-sensing, cell cycle, and senescence pathways.

    PubMed

    Lesniewski, Lisa A; Seals, Douglas R; Walker, Ashley E; Henson, Grant D; Blimline, Mark W; Trott, Daniel W; Bosshardt, Gary C; LaRocca, Thomas J; Lawson, Brooke R; Zigler, Melanie C; Donato, Anthony J

    2017-02-01

    Inhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age-related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age-related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6-8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium-dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin-treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse-wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and expression of the cell cycle-related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and

  7. Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells

    PubMed Central

    Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2013-01-01

    Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth. PMID:23165769

  8. Fluoride-Induced Autophagy via the Regulation of Phosphorylation of Mammalian Targets of Rapamycin in Mice Leydig Cells.

    PubMed

    Zhang, Jianhai; Zhu, Yuchen; Shi, Yan; Han, Yongli; Liang, Chen; Feng, Zhiyuan; Zheng, Heping; Eng, Michelle; Wang, Jundong

    2017-10-11

    Fluoride is known to impair testicular function and decrease testosterone levels, yet the underlying mechanisms remain inconclusive. The objective of this study is to investigate the roles of autophagy in fluoride-induced male reproductive toxicity using both in vivo and in vitro Leydig cell models. Using transmission electron microscopy and monodansylcadaverine staining, we observed increasing numbers of autophagosomes in testicular tissue, especially in Leydig cells of fluoride-exposed mice. Further study revealed that fluoride increased the levels of mRNA and protein expression of autophagy markers LC3, Beclin1, and Atg 5 in primary Leydig cells. Furthermore, fluoride inhibited the phosphorylation of mammalian targets of rapamycin and 4EBP1, which in turn resulted in a decrease in the levels of AKT and PI3K mRNA expression, as well as an elevation of the level of AMPK expression in both testes and primary Leydig cells. Additionally, fluoride exposure significantly changed the mRNA expression of the PDK1, TSC, and Atg13 regulator genes in primary Leydig cells but not in testicular cells. Taken together, our findings highlight the roles of autophagy in fluoride-induced testicular and Leydig cell damage and contribute to the elucidation of the underlying mechanisms of fluoride-induced male reproductive toxicity.

  9. Leucine Stimulates Insulin Secretion via Down-regulation of Surface Expression of Adrenergic α2A Receptor through the mTOR (Mammalian Target of Rapamycin) Pathway

    PubMed Central

    Yang, Jun; Dolinger, Michael; Ritaccio, Gabrielle; Mazurkiewicz, Joseph; Conti, David; Zhu, Xinjun; Huang, Yunfei

    2012-01-01

    The amino acid leucine is a potent secretagogue, capable of inducing insulin secretion. It also plays an important role in the regulation of mTOR activity, therefore, providing impetus to investigate if a leucine-sensing mechanism in the mTOR pathway is involved in insulin secretion. We found that leucine-induced insulin secretion was inhibited by both the mTOR inhibitor rapamycin as well as the adrenergic α2 receptor agonist clonidine. We also demonstrated that leucine down-regulated the surface expression of adrenergic α2A receptor via activation of the mTOR pathway. The leucine stimulatory effect on insulin secretion was attenuated in diabetic Goto-Kakizaki rats that overexpress adrenergic α2A receptors, confirming the role of leucine in insulin secretion. Thus, our data demonstrate that leucine regulates insulin secretion by modulating adrenergic α2 receptors through the mTOR pathway. The role of the mTOR pathway in metabolic homeostasis led us to a second important finding in this study; retrospective analysis of clinical data showed that co-administration of rapamycin and clonidine was associated with an increased incidence of new-onset diabetes in renal transplantation patients over those receiving rapamycin alone. We believe that inhibition of mTOR by rapamycin along with activation of adrenergic α2 receptors by clonidine represents a double-hit to pancreatic islets that synergistically disturbs glucose homeostasis. This new insight may have important implications for the clinical management of renal transplant patients. PMID:22645144

  10. Target of rapamycin signaling regulates metabolism, growth, and lifespan in Arabidopsis

    USDA-ARS?s Scientific Manuscript database

    TOR is a major nutrition and energy sensor that regulates growth and lifespan in yeast and animals. In plants growth and lifespan are intertwined with not only nutrient acquisition but also nutrition generation and unique aspects of development and differentiation. How TOR functions in these process...

  11. Mammalian target of rapamycin regulates murine and human cell differentiation through STAT3/p63/Jagged/Notch cascade

    PubMed Central

    Ma, Jianhui; Meng, Yan; Kwiatkowski, David J.; Chen, Xinxin; Peng, Haiyong; Sun, Qian; Zha, Xiaojun; Wang, Fang; Wang, Ying; Jing, Yanling; Zhang, Shu; Chen, Rongrong; Wang, Lianmei; Wu, Erxi; Cai, Guifang; Malinowska-Kolodziej, Izabela; Liao, Qi; Liu, Yuqin; Zhao, Yi; Sun, Qiang; Xu, Kaifeng; Dai, Jianwu; Han, Jiahuai; Wu, Lizi; Zhao, Robert Chunhua; Shen, Huangxuan; Zhang, Hongbing

    2009-01-01

    The receptor tyrosine kinase/PI3K/AKT/mammalian target of rapamycin (RTK/PI3K/AKT/mTOR) pathway is frequently altered in cancer, but the underlying mechanism leading to tumorigenesis by activated mTOR remains less clear. Here we show that mTOR is a positive regulator of Notch signaling in mouse and human cells, acting through induction of the STAT3/p63/Jagged signaling cascade. Furthermore, in response to differential cues from mTOR, we found that Notch served as a molecular switch to shift the balance between cell proliferation and differentiation. We determined that hyperactive mTOR signaling impaired cell differentiation of murine embryonic fibroblasts via potentiation of Notch signaling. Elevated mTOR signaling strongly correlated with enhanced Notch signaling in poorly differentiated but not in well-differentiated human breast cancers. Both human lung lymphangioleiomyomatosis (LAM) and mouse kidney tumors with hyperactive mTOR due to tumor suppressor TSC1 or TSC2 deficiency exhibited enhanced STAT3/p63/Notch signaling. Furthermore, tumorigenic potential of cells with uncontrolled mTOR signaling was suppressed by Notch inhibition. Our data therefore suggest that perturbation of cell differentiation by augmented Notch signaling might be responsible for the underdifferentiated phenotype displayed by certain tumors with an aberrantly activated RTK/PI3K/AKT/mTOR pathway. Additionally, the STAT3/p63/Notch axis may be a useful target for the treatment of cancers exhibiting hyperactive mTOR signaling. PMID:20038814

  12. The regulation of autophagy in porcine blastocysts: Regulation of PARylation-mediated autophagy via mammalian target of rapamycin complex 1 (mTORC1) signaling.

    PubMed

    Lee, Hye Ran; Kim, Duk Hyoun; Kim, Min Gyeong; Lee, Jun Sung; Hwang, Jeong Ho; Lee, Hoon Taek

    2016-05-13

    Poly(ADP-ribosyl)ation (PARylation) acts as a modulator of selective autophagic degradation of ubiquitinated aggregates for cellular quality control, functioning in pro-survival role. It was reported previously that the inhibition of PARylation resulted in autophagy defects leading accumulation of ubiquitinated aggregates SQSTM1/p62 and apoptosis in porcine blastocysts. Thus, this study aims to investigate the mechanism between PARylation and autophagy in porcine blastocysts. In vitro produced (IVP) embryos were treated with 3-aminobenzamide (3ABA, poly (ADP-ribose) polymerase inhibitor) and/or rapamycin (RAPA, an mTORC1 inhibitor) during blastocyst formation. Then, these treated blastocysts were analyzed by real-time PCR, immunocytochemistry and TUNEL Assay. We found that the 3ABA treatment increased mTORC1 downstream target, phosphorylation of thr389 p70S6K (p-p70S6K-thr389), suggesting an increase in mTORC1 activity. Co-treatment with rapamycin (RAPA), mTORC1 inhibitor, restored the 3ABA-induced autophagy defects to those of the controls by normalizing mTORC1 activity. Moreover, autophagy induction, with only RAPA treatment, increased the rate of blastocyst development (70.05 ± 0.93 vs. 50.61 ± 3.49%), total cell number (58.48 ± 2.94 vs. 49.58 ± 2.43) and blastomere survival, but decreased the accumulation of SQSTM1/p62 aggregates. In summary, mTORC1 signaling is a key mechanism of PARylation-autophagy and its inhibition improved developmental ability and embryo quality by promoting selective autophagic degradation of ubiquitinated aggregates in porcine blastocysts. Therefore, these findings have significant implications for understanding the importance of autophagy regulation for successful in vitro production of porcine embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Membrane-Mediated Regulation of Vascular Identity

    PubMed Central

    Hashimoto, Takuya; Tsuneki, Masayuki; Foster, Trenton R.; Santana, Jeans M.; Bai, Hualong; Wang, Mo; Hu, Haidi; Hanisch, Jesse J.; Dardik, Alan

    2017-01-01

    Vascular diseases span diverse pathology, but frequently arise from aberrant signaling attributed to specific membrane-associated molecules, particularly the Eph-ephrin family. Originally recognized as markers of embryonic vessel identity, Eph receptors and their membrane-associated ligands, ephrins, are now known to have a range of vital functions in vascular physiology. Interactions of Ephs with ephrins at cell-to-cell interfaces promote a variety of cellular responses such as repulsion, adhesion, attraction, and migration, and frequently occur during organ development, including vessel formation. Elaborate coordination of Eph- and ephrin-related signaling among different cell populations is required for proper formation of the embryonic vessel network. There is growing evidence supporting the idea that Eph and ephrin proteins also have postnatal interactions with a number of other membrane-associated signal transduction pathways, coordinating translation of environmental signals into cells. This article provides an overview of membrane-bound signaling mechanisms that define vascular identity in both the embryo and the adult, focusing on Eph- and ephrin-related signaling. We also discuss the role and clinical significance of this signaling system in normal organ development, neoplasms, and vascular pathologies. PMID:26992081

  14. Sphingosine 1-Phosphate Receptor Signaling Regulates Proper Embryonic Vascular Patterning*

    PubMed Central

    Mendelson, Karen; Zygmunt, Tomasz; Torres-Vázquez, Jesús; Evans, Todd; Hla, Timothy

    2013-01-01

    Sphingosine 1-phosphate (S1P) binds G-protein-coupled receptors (S1P1–5) to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here, we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development, but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development. PMID:23229546

  15. Cytoglobin regulates blood pressure and vascular tone through nitric oxide metabolism in the vascular wall

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoping; El-Mahdy, Mohamed A.; Boslett, James; Varadharaj, Saradhadevi; Hemann, Craig; Abdelghany, Tamer M.; Ismail, Raed S.; Little, Sean C.; Zhou, Danlei; Thuy, Le Thi Thanh; Kawada, Norifumi; Zweier, Jay L.

    2017-04-01

    The identity of the specific nitric oxide dioxygenase (NOD) that serves as the main in vivo regulator of O2-dependent NO degradation in smooth muscle remains elusive. Cytoglobin (Cygb) is a recently discovered globin expressed in fibroblasts and smooth muscle cells with unknown function. Cygb, coupled with a cellular reducing system, efficiently regulates the rate of NO consumption by metabolizing NO in an O2-dependent manner with decreased NO consumption in physiological hypoxia. Here we show that Cygb is a major regulator of NO degradation and cardiovascular tone. Knockout of Cygb greatly prolongs NO decay, increases vascular relaxation, and lowers blood pressure and systemic vascular resistance. We further demonstrate that downregulation of Cygb prevents angiotensin-mediated hypertension. Thus, Cygb has a critical role in the regulation of vascular tone and disease. We suggest that modulation of the expression and NOD activity of Cygb represents a strategy for the treatment of cardiovascular disease.

  16. Rapamycin preserves gut homeostasis during Drosophila aging.

    PubMed

    Fan, Xiaolan; Liang, Qing; Lian, Ting; Wu, Qi; Gaur, Uma; Li, Diyan; Yang, Deying; Mao, Xueping; Jin, Zhihua; Li, Ying; Yang, Mingyao

    2015-11-03

    Gut homeostasis plays an important role in maintaining the overall body health during aging. Rapamycin, a specific inhibitor of mTOR, exerts prolongevity effects in evolutionarily diverse species. However, its impact on the intestinal homeostasis remains poorly understood. Here, we demonstrate that rapamycin can slow down the proliferation rate of intestinal stem cells (ISCs) in the aging guts and induce autophagy in the intestinal epithelium in Drosophila. Rapamycin can also significantly affect the FOXO associated genes in intestine and up-regulate the negative regulators of IMD/Rel pathway, consequently delaying the microbial expansion in the aging guts. Collectively, these findings reveal that rapamycin can delay the intestinal aging by inhibiting mTOR and thus keeping stem cell proliferation in check. These results will further explain the mechanism of healthspan and lifespan extension by rapamycin in Drosophila.

  17. 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 Central

    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.

    2016-01-01

    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

  18. Dietary potassium regulates vascular calcification and arterial stiffness.

    PubMed

    Sun, Yong; Byon, Chang Hyun; Yang, Youfeng; Bradley, Wayne E; Dell'Italia, Louis J; Sanders, Paul W; Agarwal, Anupam; Wu, Hui; Chen, Yabing

    2017-10-05

    Vascular calcification is a risk factor that predicts adverse cardiovascular complications of several diseases including atherosclerosis. Reduced dietary potassium intake has been linked to cardiovascular diseases such as hypertension and incidental stroke, although the underlying molecular mechanisms remain largely unknown. Using the ApoE-deficient mouse model, we demonstrated for the first time to our knowledge that reduced dietary potassium (0.3%) promoted atherosclerotic vascular calcification and increased aortic stiffness, compared with normal (0.7%) potassium-fed mice. In contrast, increased dietary potassium (2.1%) attenuated vascular calcification and aortic stiffness. Mechanistically, reduction in the potassium concentration to the lower limit of the physiological range increased intracellular calcium, which activated a cAMP response element-binding protein (CREB) signal that subsequently enhanced autophagy and promoted vascular smooth muscle cell (VSMC) calcification. Inhibition of calcium signals and knockdown of either CREB or ATG7, an autophagy regulator, attenuated VSMC calcification induced by low potassium. Consistently, elevated autophagy and CREB signaling were demonstrated in the calcified arteries from low potassium diet-fed mice as well as aortic arteries exposed to low potassium ex vivo. These studies established a potentially novel causative role of dietary potassium intake in regulating atherosclerotic vascular calcification and stiffness, and uncovered mechanisms that offer opportunities to develop therapeutic strategies to control vascular disease.

  19. Mechanical regulation of epigenetics in vascular biology and pathobiology.

    PubMed

    Chen, Li-Jing; Wei, Shu-Yi; Chiu, Jeng-Jiann

    2013-04-01

    Vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) are constantly exposed to haemodynamic forces, including blood flow-induced fluid shear stress and cyclic stretch from blood pressure. These forces modulate vascular cell gene expression and function and, therefore, influence vascular physiology and pathophysiology in health and disease. Epigenetics, including DNA methylation, histone modification/chromatin remodelling and RNA-based machinery, refers to the study of heritable changes in gene expression that occur without changes in the DNA sequence. The role of haemodynamic force-induced epigenetic modifications in the regulation of vascular gene expression and function has recently been elucidated. This review provides an introduction to the epigenetic concepts that relate to vascular physiology and pathophysiology. Through the studies of gene expression, cell proliferation, angiogenesis, migration and pathophysiological states, we present a conceptual framework for understanding how mechanical force-induced epigenetic modifications work to control vascular gene expression and function and, hence, the development of vascular disorders. This research contributes to our knowledge of how the mechanical environment impacts the chromatin state of ECs and VSMCs and the consequent cellular behaviours. © 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  20. Regulation of thrombosis and vascular function by protein methionine oxidation

    PubMed Central

    Gu, Sean X.; Stevens, Jeff W.

    2015-01-01

    Redox biology is fundamental to both normal cellular homeostasis and pathological states associated with excessive oxidative stress. Reactive oxygen species function not only as signaling molecules but also as redox regulators of protein function. In the vascular system, redox reactions help regulate key physiologic responses such as cell adhesion, vasoconstriction, platelet aggregation, angiogenesis, inflammatory gene expression, and apoptosis. During pathologic states, altered redox balance can cause vascular cell dysfunction and affect the equilibrium between procoagulant and anticoagulant systems, contributing to thrombotic vascular disease. This review focuses on the emerging role of a specific reversible redox reaction, protein methionine oxidation, in vascular disease and thrombosis. A growing number of cardiovascular and hemostatic proteins are recognized to undergo reversible methionine oxidation, in which methionine residues are posttranslationally oxidized to methionine sulfoxide. Protein methionine oxidation can be reversed by the action of stereospecific enzymes known as methionine sulfoxide reductases. Calcium/calmodulin-dependent protein kinase II is a prototypical methionine redox sensor that responds to changes in the intracellular redox state via reversible oxidation of tandem methionine residues in its regulatory domain. Several other proteins with oxidation-sensitive methionine residues, including apolipoprotein A-I, thrombomodulin, and von Willebrand factor, may contribute to vascular disease and thrombosis. PMID:25900980

  1. A systematic review of economic evaluations of Tyrosine Kinase Inhibitors of Vascular Endothelial Growth Factor Receptors, mammalian target of rapamycin inhibitors and Programmed Death-1 inhibitors in metastatic renal cell cancer.

    PubMed

    Petrou, Panagiotis

    2018-02-16

    The therapeutic categories of Tyrosine Kinase Inhibitors of Vascular Endothelial Growth Factor Receptors, mammalian target of rapamycin inhibitors and Programmed Death-1 inhibitors have transformed the treatment of metastatic renal cell cancer. Nevertheless, this comes at an increased cost, in tandem with similar fiscal pressures in the broader oncology sector, which may jeopardize the sustainability of health systems. Areas covered To this direction, the economic evaluation of these agents is essential for rational and efficient resource allocation. The aim of this study is to glean, assess and present an outline of the available cost-effectiveness studies of these agents in the management of metastatic renal cell cancer. Expert Commentary We concluded that the results of economic evaluation are pertinent, apart from the product under evaluation, to the country setting as well.

  2. Autophagy regulates the apoptosis of bone marrow-derived mesenchymal stem cells under hypoxic condition via AMP-activated protein kinase/mammalian target of rapamycin pathway.

    PubMed

    Zhang, Zheng; Yang, Ming; Wang, Yabin; Wang, Le; Jin, Zhitao; Ding, Liping; Zhang, Lijuan; Zhang, Lina; Jiang, Wei; Gao, Guojie; Yang, Junke; Lu, Bingwei; Cao, Feng; Hu, Taohong

    2016-06-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been demonstrated as an ideal autologous stem cells source for cell-based therapy for myocardial infarction (MI). However, poor viability of donor stem cells after transplantation limits their therapeutic efficiency, whereas the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for maintaining homeostasis and normal function. Here, we investigated the potential role of autophagy on apoptosis in BM-MSCs induced by hypoxic injury. BM-MSCs, isolated from male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury for 6, 12, and 24 h, respectively. The autophagy state was regulated by 3-methyladenine (3MA) and rapamycin administration. Furthermore, compound C was administrated to inhibit AMPK. The apoptosis induced by H/SD was determined by TUNEL assays. Meanwhile, autophagy was measured by GFP-LC3 plasmids transfection and transmission electron microscope. Moreover, protein expressions were evaluated by Western blot assay. In the present study, we found that hypoxic stress increased autophagy and apoptosis in BM-MSCs time dependently. Meanwhile, hypoxia increased the activity of AMPK/mTOR signal pathway. Moreover, increased apoptosis in BM-MSCs under hypoxia was abolished by 3-MA, whereas was aggravated by rapamycin. Furthermore, the increased autophagy and apoptosis in BM-MSCs induced by hypoxia were abolished by AMPK inhibitor compound C. These data provide evidence that hypoxia induced AMPK/mTOR signal pathway activation which regulated the apoptosis and autophagy in BM-MSCs. Furthermore, the apoptosis of BM-MSCs under hypoxic condition was regulated by autophagy via AMPK/mTOR pathway. © 2016 International Federation for Cell Biology.

  3. Impaired sympathetic vascular regulation in humans after acute dynamic exercise

    NASA Technical Reports Server (NTRS)

    Halliwill, J. R.; Taylor, J. A.; Eckberg, D. L.

    1996-01-01

    1. The reduction in vascular resistance which accompanies acute dynamic exercise does not subside immediately during recovery, resulting in a post-exercise hypotension. This sustained vasodilatation suggests that sympathetic vascular regulation is altered after exercise. 2. Therefore, we assessed the baroreflex control of sympathetic outflow in response to arterial pressure changes, and transduction of sympathetic activity into vascular resistance during a sympatho-excitatory stimulus (isometric handgrip exercise) after either exercise (60 min cycling at 60% peak aerobic power (VO2,peak)) or sham treatment (60 min seated rest) in nine healthy subjects. 3. Both muscle sympathetic nerve activity and calf vascular resistance were reduced after exercise (-29.7 +/- 8.8 and -25.3 +/- 9.1%, both P < 0.05). The baroreflex relation between diastolic pressure and sympathetic outflow was shifted downward after exercise (post-exercise intercept, 218 +/- 38 total integrated activity (heartbeat)-1; post-sham intercept, 318 +/- 51 total integrated activity (heartbeat)-1, P < 0.05), indicating less sympathetic outflow across all diastolic pressures. Further, the relation between sympathetic activity and vascular resistance was attenuated after exercise (post-exercise slope, 0.0031 +/- 0.0007 units (total integrated activity)-1 min; post-sham slope, 0.0100 +/- 0.0033 units (total integrated activity)-1 min, P < 0.05), indicating less vasoconstriction with any increase in sympathetic activity. 4. Thus, both baroreflex control of sympathetic outflow and the transduction of sympathetic activity into vascular resistance are altered after dynamic exercise. We conclude that the vasodilation which underlies post-exercise hypotension results from both neural and vascular phenomena.

  4. Impaired sympathetic vascular regulation in humans after acute dynamic exercise.

    PubMed Central

    Halliwill, J R; Taylor, J A; Eckberg, D L

    1996-01-01

    1. The reduction in vascular resistance which accompanies acute dynamic exercise does not subside immediately during recovery, resulting in a post-exercise hypotension. This sustained vasodilatation suggests that sympathetic vascular regulation is altered after exercise. 2. Therefore, we assessed the baroreflex control of sympathetic outflow in response to arterial pressure changes, and transduction of sympathetic activity into vascular resistance during a sympatho-excitatory stimulus (isometric handgrip exercise) after either exercise (60 min cycling at 60% peak aerobic power (VO2,peak)) or sham treatment (60 min seated rest) in nine healthy subjects. 3. Both muscle sympathetic nerve activity and calf vascular resistance were reduced after exercise (-29.7 +/- 8.8 and -25.3 +/- 9.1%, both P < 0.05). The baroreflex relation between diastolic pressure and sympathetic outflow was shifted downward after exercise (post-exercise intercept, 218 +/- 38 total integrated activity (heartbeat)-1; post-sham intercept, 318 +/- 51 total integrated activity (heartbeat)-1, P < 0.05), indicating less sympathetic outflow across all diastolic pressures. Further, the relation between sympathetic activity and vascular resistance was attenuated after exercise (post-exercise slope, 0.0031 +/- 0.0007 units (total integrated activity)-1 min; post-sham slope, 0.0100 +/- 0.0033 units (total integrated activity)-1 min, P < 0.05), indicating less vasoconstriction with any increase in sympathetic activity. 4. Thus, both baroreflex control of sympathetic outflow and the transduction of sympathetic activity into vascular resistance are altered after dynamic exercise. We conclude that the vasodilation which underlies post-exercise hypotension results from both neural and vascular phenomena. Images Figure 7 PMID:8866370

  5. miR-126 regulates angiogenic signaling and vascular integrity

    PubMed Central

    Fish, Jason E.; Santoro, Massimo M.; Morton, Sarah U.; Yu, Sangho; Yeh, Ru-Fang; Wythe, Joshua D.; Bruneau, Benoit G.; Stainier, Didier Y. R.; Srivastava, Deepak

    2008-01-01

    Summary Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein, SPRED1, and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function. PMID:18694566

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

    PubMed Central

    Ringvold, Helene C.; Khalil, Raouf A.

    2016-01-01

    Vascular smooth muscle (VSM) plays an important role in maintaining vascular tone. In addition to Ca2+-dependent myosin light chain (MLC) phosphorylation, protein kinase C (PKC) is a major regulator of VSM function. PKC is a family of conventional Ca2+-dependent α, β, and γ, novel Ca2+-independent δ, ε, 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. PMID:28212798

  7. An RNA Interference Screen Identifies a Novel Regulator of Target of Rapamycin That Mediates Hypoxia Suppression of Translation in Drosophila S2 Cells

    PubMed Central

    Lee, Soo-Jung; Feldman, Renny

    2008-01-01

    In addition to its central role in energy production, oxygen has pervasive regulatory actions. Hypoxia (oxygen limitation) triggers the shutdown of major cellular processes, including gene expression. We carried out a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells for functions required to down-regulate translation during hypoxia. RNAi knockdown of specific genes allowed induction of a green fluorescent protein (GFP) reporter gene and continued protein synthesis during hypoxia. Among the identified genes, Tsc1 and Tsc2, which together form the tuberose sclerosis complex that negatively regulates target of rapamycin (TOR) kinase, gave an especially strong effect. This finding is consistent with the involvement of TOR in promoting translation. Another gene required for efficient inhibition of protein translation during hypoxia, the protein tyrosine phosphatase 61F (Ptp61F), down-regulates TOR activity under hypoxia. Lack of Ptp61F or Tsc2 improves cell survival under prolonged hypoxia in a TOR-dependent manner. Our results identify Ptp61F as a novel modulator of TOR activity and suggest that its function during hypoxia contributes to the down-regulation of protein synthesis. PMID:18653470

  8. Dipeptidyl peptidase IV inhibitors as novel regulators of vascular disease.

    PubMed

    Akoumianakis, Ioannis; Antoniades, Charalambos

    2017-09-01

    Dipeptidyl peptidase IV (DPP-IV) has been revealed as an adipokine with potential relevance in cardiovascular disease (CVD), while clinically used DPP-IV inhibitors have demonstrated beneficial cardiovascular effects in several experimental studies. Perivascular adipose tissue (PVAT) is a unique adipose tissue depot in close anatomical proximity and bidirectional functional interaction with the vascular wall, which is a source of DPP-IV and its biology may be influenced by DPP-IV inhibition. Recently, DPP-IV inhibition has been associated with decreased local inflammation and oxidative stress both in the vascular wall and the PVAT, potentially regulating atherogenesis progression in vivo. DPP-IV inhibition may thus be a promising target in cardiovascular disease. However, the exact pleiotropic mechanisms that underlie the cardiovascular effects of DPP-IV inhibition need to be clarified, while the in vivo benefit of DPP-IV inhibition in humans remains unclear. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Mechanosensitive β-catenin signaling regulates lymphatic vascular development

    PubMed Central

    Cha, Boksik; Srinivasan, R. Sathish

    2016-01-01

    The Wnt/β-catenin signaling is an evolutionarily conserved pathway that plays a pivotal role in embryonic development and adult homeostasis. However, we have limited information about the involvement of Wnt/β-catenin signaling in the lymphatic vascular system that regulates fluid homeostasis by absorbing interstitial fluid and returning it to blood circulation. In this recent publication we report that canonical Wnt/β-catenin signaling is highly active and critical for the formation of lymphovenus valves (LVVs) and lymphatic valves (LVs). β-catenin directly associates with the regulatory elements of the lymphedema-associated transcription factor, FOXC2 and activates its expression in an oscillatory shear stress (OSS)-dependent manner. The phenotype of β-catenin null embryos was rescued by FOXC2 overexpression. These results suggest that Wnt/β-catenin signaling is a mechanotransducer that links fluid force with lymphatic vascular development. [BMB Reports 2016; 49(8): 403-404] PMID:27418286

  10. Mammalian target of rapamycin pathway promotes tumor-induced angiogenesis in adenoid cystic carcinoma: its suppression by isoliquiritigenin through dual activation of c-Jun NH2-terminal kinase and inhibition of extracellular signal-regulated kinase.

    PubMed

    Sun, Zhi-Jun; Chen, Gang; Zhang, Wei; Hu, Xiang; Huang, Cong-Fa; Wang, Yu-Fan; Jia, Jun; Zhao, Yi-Fang

    2010-08-01

    Tumor-induced angiogenesis is essential for invasive growth and hematogenous metastasis of adenoid cystic carcinoma (ACC), a highly aggressive neoplasm mostly occurring in salivary glands. Previous studies have indicated that strategies directed against angiogenesis will help develop new therapeutic agents for ACC. The Chinese folk medicine licorice has been used for years as a natural remedy for angiogenesis-related diseases. In this study, we examined the effects of isoliquiritigenin (ISL), a flavonoid isolated from licorice, on the growth and viability of ACC cells and observed a concentration-dependent (0-20 microM) inhibition of cell growth without cell death at 24 h. In a further mimic coculture study, ISL effectively suppressed the ability of ACC cells to induce in vitro proliferation, migration, and tube formation of human endothelial hybridoma (EAhy926) cells as well as ex vivo and in vivo angiogenesis, whereas it exerted no effect on EAhy926 cells when added directly or in the presence of vascular endothelial growth factor (VEGF). The data also showed that the specific suppression of tumor angiogenesis by ISL was caused by down-regulation of mammalian target of rapamycin (mTOR) pathway-dependent VEGF production by ACC cells, correlating with concurrent activation of c-Jun NH(2)-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK). Most importantly, ISL also significantly decreased microvessel density within xenograft tumors, associating with the reduction of VEGF production and suppression of the mTOR pathway coregulated by JNK and ERK, as revealed by immunohistochemical studies and clustering analysis. Taken together, our results highlight the fact that ISL is a novel inhibitor of tumor angiogenesis and possesses great therapeutic potential for ACC.

  11. Rapamycin and Glucose-Target of Rapamycin (TOR) Protein Signaling in Plants*

    PubMed Central

    Xiong, Yan; Sheen, Jen

    2012-01-01

    Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs. PMID:22134914

  12. Rapamycin and glucose-target of rapamycin (TOR) protein signaling in plants.

    PubMed

    Xiong, Yan; Sheen, Jen

    2012-01-20

    Target of rapamycin (TOR) kinase is an evolutionarily conserved master regulator that integrates energy, nutrients, growth factors, and stress signals to promote survival and growth in all eukaryotes. The reported land plant resistance to rapamycin and the embryo lethality of the Arabidopsis tor mutants have hindered functional dissection of TOR signaling in plants. We developed sensitive cellular and seedling assays to monitor endogenous Arabidopsis TOR activity based on its conserved S6 kinase (S6K) phosphorylation. Surprisingly, rapamycin effectively inhibits Arabidopsis TOR-S6K1 signaling and retards glucose-mediated root and leaf growth, mimicking estradiol-inducible tor mutants. Rapamycin inhibition is relieved in transgenic plants deficient in Arabidopsis FK506-binding protein 12 (FKP12), whereas FKP12 overexpression dramatically enhances rapamycin sensitivity. The role of Arabidopsis FKP12 is highly specific as overexpression of seven closely related FKP proteins fails to increase rapamycin sensitivity. Rapamycin exerts TOR inhibition by inducing direct interaction between the TOR-FRB (FKP-rapamycin binding) domain and FKP12 in plant cells. We suggest that variable endogenous FKP12 protein levels may underlie the molecular explanation for longstanding enigmatic observations on inconsistent rapamycin resistance in plants and in various mammalian cell lines or diverse animal cell types. Integrative analyses with rapamycin and conditional tor and fkp12 mutants also reveal a central role of glucose-TOR signaling in root hair formation. Our studies demonstrate the power of chemical genetic approaches in the discovery of previously unknown and pivotal functions of glucose-TOR signaling in governing the growth of cotyledons, true leaves, petioles, and primary and secondary roots and root hairs.

  13. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions.

    PubMed

    O'Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-07-15

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K-PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K-PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K-PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K-PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K-PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K-PKB-independent mechanism that requires PLD and PA.

  14. The role of phosphoinositide 3-kinase and phosphatidic acid in the regulation of mammalian target of rapamycin following eccentric contractions

    PubMed Central

    O’Neil, T K; Duffy, L R; Frey, J W; Hornberger, T A

    2009-01-01

    Resistance exercise induces a hypertrophic response in skeletal muscle and recent studies have begun to shed light on the molecular mechanisms involved in this process. For example, several studies indicate that signalling by the mammalian target of rapamycin (mTOR) is necessary for a hypertrophic response. Furthermore, resistance exercise has been proposed to activate mTOR signalling through an upstream pathway involving the phosphoinositide 3-kinase (PI3K) and protein kinase B (PKB); however, this hypothesis has not been thoroughly tested. To test this hypothesis, we first evaluated the temporal pattern of signalling through PI3K–PKB and mTOR following a bout of resistance exercise with eccentric contractions (EC). Our results indicated that the activation of signalling through PI3K–PKB is a transient event (<15 min), while the activation of mTOR is sustained for a long duration (>12 h). Furthermore, inhibition of PI3K–PKB activity did not prevent the activation of mTOR signalling by ECs, indicating that PI3K–PKB is not part of the upstream regulatory pathway. These observations led us to investigate an alternative pathway for the activation of mTOR signalling involving the synthesis of phosphatidic acid (PA) by phospholipase D (PLD). Our results demonstrate that ECs induce a sustained elevation in [PA] and inhibiting the synthesis of PA by PLD prevented the activation of mTOR. Furthermore, we determined that similar to ECs, PA activates mTOR signalling through a PI3K–PKB-independent mechanism. Combined, the results of this study indicate that the activation of mTOR following eccentric contractions occurs through a PI3K–PKB-independent mechanism that requires PLD and PA. PMID:19470781

  15. Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function

    PubMed Central

    Yao, Yao; Wang, Junying; Yoshida, Sei; Nada, Shigeyuki; Okada, Masato

    2016-01-01

    Aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1) in glomerular podocytes leads to glomerular insufficiency and may contribute to the development of glomerular diseases, including diabetic nephropathy. Thus, an approach for preventing mTORC1 activation may allow circumvention of the onset and progression of mTORC1-dependent podocyte injury and glomerular diseases. mTORC1 activation requires inputs from both growth factors and nutrients that inactivate the tuberous sclerosis complex (TSC), a key suppressor of mTORC1, on the lysosome. Previous studies in mice revealed that the growth factor-phosphatidylinositol 3-kinase pathway and mTORC1 are essential for maintaining normal podocyte function, suggesting that direct inhibition of the phosphatidylinositol 3-kinase pathway or mTORC1 may not be an ideal approach to sustaining physiologic podocyte functions under certain disease conditions. Here, we report the role of the Ragulator complex, which recruits mTORC1 to lysosomes in response to nutrient availability in podocytes. Notably, podocytes lacking Ragulator maintain basal mTORC1 activity. Unlike podocyte-specific mTORC1-knockout mice, mice lacking functional Ragulator in podocytes did not show abnormalities in podocyte or glomerular function. However, aberrant mTORC1 activation induced by active Rheb in podocyte-specific TSC1-knockout (podo-TSC1 KO) mice did require Ragulator. Moreover, ablation of Ragulator in the podocytes of podo-TSC1 KO mice or streptozotocin-induced diabetic mice significantly blocked the development of pathologic renal phenotypes. These observations suggest that the blockade of mTORC1 recruitment to lysosomes may be a useful clinical approach to attenuate aberrant mTORC1 activation under certain disease conditions. PMID:27032892

  16. Role of Ragulator in the Regulation of Mechanistic Target of Rapamycin Signaling in Podocytes and Glomerular Function.

    PubMed

    Yao, Yao; Wang, Junying; Yoshida, Sei; Nada, Shigeyuki; Okada, Masato; Inoki, Ken

    2016-12-01

    Aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1) in glomerular podocytes leads to glomerular insufficiency and may contribute to the development of glomerular diseases, including diabetic nephropathy. Thus, an approach for preventing mTORC1 activation may allow circumvention of the onset and progression of mTORC1-dependent podocyte injury and glomerular diseases. mTORC1 activation requires inputs from both growth factors and nutrients that inactivate the tuberous sclerosis complex (TSC), a key suppressor of mTORC1, on the lysosome. Previous studies in mice revealed that the growth factor-phosphatidylinositol 3-kinase pathway and mTORC1 are essential for maintaining normal podocyte function, suggesting that direct inhibition of the phosphatidylinositol 3-kinase pathway or mTORC1 may not be an ideal approach to sustaining physiologic podocyte functions under certain disease conditions. Here, we report the role of the Ragulator complex, which recruits mTORC1 to lysosomes in response to nutrient availability in podocytes. Notably, podocytes lacking Ragulator maintain basal mTORC1 activity. Unlike podocyte-specific mTORC1-knockout mice, mice lacking functional Ragulator in podocytes did not show abnormalities in podocyte or glomerular function. However, aberrant mTORC1 activation induced by active Rheb in podocyte-specific TSC1-knockout (podo-TSC1 KO) mice did require Ragulator. Moreover, ablation of Ragulator in the podocytes of podo-TSC1 KO mice or streptozotocin-induced diabetic mice significantly blocked the development of pathologic renal phenotypes. These observations suggest that the blockade of mTORC1 recruitment to lysosomes may be a useful clinical approach to attenuate aberrant mTORC1 activation under certain disease conditions. Copyright © 2016 by the American Society of Nephrology.

  17. The effect of rapamycin on bone growth in rabbits.

    PubMed

    Phornphutkul, Chanika; Lee, Mark; Voigt, Cliff; Wu, Ke-Ying; Ehrlich, Michael G; Gruppuso, Philip A; Chen, Qian

    2009-09-01

    mTOR is a nutrient-sensing protein kinase that regulates numerous cellular processes. Our prior studies using the mTOR inhibitor, rapamycin, indicate an important role for mTOR in chondrogenesis. We extended our observations to a physiological, in vivo model of bone growth, direct infusion of rapamycin into the proximal tibial growth plates of rabbits. Rapamycin or DMSO vehicle was infused directly into growth plates by an osmotic minipump for 8 weeks. Tibial growth was followed radiographically. At the end of the experiment, growth plates were recovered for histological analysis. Six animals were studied. No untoward effects of rapamycin infusion were found. Bone growth of limbs exposed to rapamycin was slower than control limbs, particularly during the period of most rapid growth. Histological analysis revealed that growth plate height in the rapamycin-infused limbs was reduced. Both the hypertrophic and proliferative zones were significantly smaller in the rapamycin-infused limbs. Direct infusion of rapamycin into proximal tibial growth plates decreased the size of the growth plate and inhibited overall long bone growth. Rapamycin appears to affect both the proliferative and hypertrophic zones of the tibial growth plate. Our results indicate that nutrients may exert a direct effect on long bone growth via mTOR-mediated modulation of chondrogenesis at the growth plate. and suggest that the possible inhibitory effects of rapamycin on skeletal growth warrant further attention before its use in children. (c) 2009 Orthopaedic Research Society.

  18. Regulation of tyrosine phosphatases in the adventitia during vascular remodelling

    SciTech Connect

    Micke, Patrick; The Department for Genetics and Pathology, Uppsala University Hospital, 75185 Uppsala; Hackbusch, Daniel

    2009-05-15

    Protein tyrosine phosphatases (PTPs) are regulators of growth factor signalling in vascular remodelling. The aim of this study was to evaluate PTP expression in the context of PDGF-signalling in the adventitia after angioplasty. Utilising a rat carotid artery model, the adventitial layers of injured and non-injured vessels were laser microdissected. The mRNA expression of the PDGF {beta}-receptor, the ligands PDGF-A/B/C/D and the receptor-antagonising PTPs (DEP-1, TC-PTP, SHP-2, PTP1B) were determined and correlated to vascular morphometrics, proliferation markers and PDGF {beta}-receptor phosphorylation. The levels of the PDGF {beta}-receptor, PDGF-C and PDGF-D were upregulated concurrently with the antagonising PTPs DEP-1 and TC-PTPmore » at day 8, and normalised at day 14 after vessel injury. Although the proliferation parameters were time-dependently altered in the adventitial layer, the phosphorylation of the PDGF {beta}-receptor remained unchanged. The expression dynamics of specific PTPs indicate a regulatory role of PDGF-signalling also in the adventitia during vascular remodelling.« less

  19. Insulin-Like Peptides and the Target of Rapamycin Pathway Coordinately Regulate Blood Digestion and Egg Maturation in the Mosquito Aedes aegypti

    PubMed Central

    Gulia-Nuss, Monika; Robertson, Anne E.; Brown, Mark R.; Strand, Michael R.

    2011-01-01

    Background Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear. Methodology/Principal Findings Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production. Conclusions/Significance Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species. PMID:21647424

  20. Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti.

    PubMed

    Gulia-Nuss, Monika; Robertson, Anne E; Brown, Mark R; Strand, Michael R

    2011-01-01

    Mosquitoes are insects that vector many serious pathogens to humans and other vertebrates. Most mosquitoes must feed on the blood of a vertebrate host to produce eggs. In turn, multiple cycles of blood feeding promote frequent contacts with hosts and make mosquitoes ideal disease vectors. Both hormonal and nutritional factors are involved in regulating egg development in the mosquito, Aedes aegypti. However, the processes that regulate digestion of the blood meal remain unclear. Here we report that insulin peptide 3 (ILP3) directly stimulated late phase trypsin-like gene expression in blood fed females. In vivo knockdown of the mosquito insulin receptor (MIR) by RNA interference (RNAi) delayed but did not fully inhibit trypsin-like gene expression in the midgut, ecdysteroid (ECD) production by ovaries, and vitellogenin (Vg) expression by the fat body. In contrast, in vivo treatment with double-stranded MIR RNA and rapamycin completely blocked egg production. In vitro experiments showed that amino acids did not simulate late phase trypsin-like gene expression in the midgut or ECD production by the ovaries. However, amino acids did enhance ILP3-mediated stimulation of trypsin-like gene expression and ECD production. Overall, our results indicate that ILPs from the brain synchronize blood meal digestion and amino acid availability with ovarian ECD production to maximize Vg expression by the fat body. The activation of digestion by ILPs may also underlie the growth promoting effects of insulin and TOR signaling in other species.

  1. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application.

  2. Oxygen sensitivity, potassium channels, and regulation of placental vascular tone.

    PubMed

    Wareing, Mark

    2014-01-01

    The human fetoplacental vasculature is a low-resistance circulation with deoxygenated arterial relative to venous blood. The placenta lacks neuronal innervation suggesting that local physical (e.g., oxygenation; flow rate), paracrine (e.g., endothelial cell nitric oxide), and circulating (e.g., angiotensin II) factors will contribute to blood flow regulation in small fetoplacental vessels. Oxygenation (specifically hypoxia) has received particular attention. At the macro-level, hypoxic challenge increases vascular resistance, but the data's physiological relevance remains questionable. K(+) channels are a diverse family of proteins known to play important roles in the normal physiological functions of endothelial and smooth muscle cells of a variety of vascular beds. K(+) channels are categorized by their predicted transmembrane structure or gating properties. A small number of perfused placental cotyledon and isolated blood vessels studies have assessed K(+) channel activity. Specific activator/inhibitor application suggests functional voltage-gated channels, whereas toxin inhibitor studies have documented KCa channel activity. Pharmacological KATP channel activation significantly dilates preconstricted placental arteries and veins. There is a paucity of cell subtype-specific expression studies of placental K(+) channels. This review focuses on the roles of K(+) channels and oxygenation in controlling reactivity of small fetoplacental blood vessels. © 2013 John Wiley & Sons Ltd.

  3. Regulation of cyclooxygenase expression in cultured vascular cells

    SciTech Connect

    Pash, J.M.

    1988-01-01

    Arachidonic acid metabolism in vascular tissue results in synthesis of prostacylin. The key enzyme in this synthesis pathway, cyclooxygenase, is down-regulated through self-inactivation. An analogous refractory state is produced by aspirin which irreversibly acetylates the enzyme. To further understand this phenomenon, the inactivation and recovery of cyclooxygenase activity was assayed in cultured ray vascular smooth muscle cells using exogenously added arachidonic acid. Self-inactivation of cyclooxygenase was observed following treatment with micromolar amounts of arachidonic acid. The recovery of cyclooxygenase activity following self-inactivation was analogous to that observed following aspirin-inactivation in that it depended on protein synthesis and required either serummore » or EGF. Two additional factors, TGF-{beta} and uric acid, were found to enhance the stimulation of cyclooxygenase recovery by EGF. A defined medium containing 10 ng/mL EGF, 1 ng/mL TGF{beta} and 0.1 mM uric acid duplicated the cyclooxygenase recovery activity of 10% serum. Stimulation of cyclooxygenase activity by EGF and TGF-{beta} was inhibited by cycloheximide but not by actinomycin-D, indicating a link to increased translation of pre-existing mRNA. A lack of significant effect on overall protein synthesis by EGF and TGF-{beta}, measured by ({sup 35}S)-methionine incorporation under conditions where a multi-fold increase in cyclooxygenase activity was seen, indicates that the translational regulation of a small fraction of total mRNA and possibly cyclooxygenase is occurring.« less

  4. Regulator of calcineurin 1 mediates pathological vascular wall remodeling

    PubMed Central

    Esteban, Vanesa; Méndez-Barbero, Nerea; Jesús Jiménez-Borreguero, Luis; Roqué, Mercè; Novensá, Laura; Belén García-Redondo, Ana; Salaices, Mercedes; Vila, Luis; Arbonés, María L.

    2011-01-01

    Artery wall remodeling, a major feature of diseases such as hypertension, restenosis, atherosclerosis, and aneurysm, involves changes in the tunica media mass that reduce or increase the vessel lumen. The identification of molecules involved in vessel remodeling could aid the development of improved treatments for these pathologies. Angiotensin II (AngII) is a key effector of aortic wall remodeling that contributes to aneurysm formation and restenosis through incompletely defined signaling pathways. We show that AngII induces vascular smooth muscle cell (VSMC) migration and vessel remodeling in mouse models of restenosis and aneurysm. These effects were prevented by pharmacological inhibition of calcineurin (CN) or lentiviral delivery of CN-inhibitory peptides. Whole-genome analysis revealed >1,500 AngII-regulated genes in VSMCs, with just 11 of them requiring CN activation. Of these, the most sensitive to CN activation was regulator of CN 1 (Rcan1). Rcan1 was strongly activated by AngII in vitro and in vivo and was required for AngII-induced VSMC migration. Remarkably, Rcan1−/− mice were resistant to AngII-induced aneurysm and restenosis. Our results indicate that aneurysm formation and restenosis share mechanistic elements and identify Rcan1 as a potential therapeutic target for prevention of aneurysm and restenosis progression. PMID:21930771

  5. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    SciTech Connect

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  6. Nrf2/Keap1 system regulates vascular smooth muscle cell apoptosis for vascular homeostasis: role in neointimal formation after vascular injury.

    PubMed

    Ashino, Takashi; Yamamoto, Masayuki; Numazawa, Satoshi

    2016-05-20

    Abnormal increases in vascular smooth muscle cells (VSMCs) in the intimal region after a vascular injury is a key event in developing neointimal hyperplasia. To maintain vascular function, proliferation and apoptosis of VSMCs is tightly controlled during vascular remodeling. NF-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) system, a key component of the oxidative stress response that acts in maintaining homeostasis, plays an important role in neointimal hyperplasia after a vascular injury; however, the role of Nrf2/Keap1 in VSMC apoptosis has not been clarified. Here we report that 14 days after arterial injury in mice, TUNEL-positive VSMCs are detected in both the neointimal and medial layers. These layers contain cells expressing high levels of Nrf2 but low Keap1 expression. In VSMCs, Keap1 depletion induces features of apoptosis, such as positive TUNEL staining and annexin V binding. These changes are associated with an increased expression of nuclear Nrf2. Simultaneous Nrf2 depletion inhibits Keap1 depletion-induced apoptosis. At 14 days after the vascular injury, Nrf2-deficient mice demonstrated fewer TUNEL-positive cells and increased neointimal formation in the neointimal and medial areas. The results suggest that the Nrf2/Keap1 system regulates VSMC apoptosis during neointimal formation, thereby inhibiting neointimal hyperplasia after a vascular injury.

  7. Nrf2/Keap1 system regulates vascular smooth muscle cell apoptosis for vascular homeostasis: role in neointimal formation after vascular injury

    PubMed Central

    Ashino, Takashi; Yamamoto, Masayuki; Numazawa, Satoshi

    2016-01-01

    Abnormal increases in vascular smooth muscle cells (VSMCs) in the intimal region after a vascular injury is a key event in developing neointimal hyperplasia. To maintain vascular function, proliferation and apoptosis of VSMCs is tightly controlled during vascular remodeling. NF-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) system, a key component of the oxidative stress response that acts in maintaining homeostasis, plays an important role in neointimal hyperplasia after a vascular injury; however, the role of Nrf2/Keap1 in VSMC apoptosis has not been clarified. Here we report that 14 days after arterial injury in mice, TUNEL-positive VSMCs are detected in both the neointimal and medial layers. These layers contain cells expressing high levels of Nrf2 but low Keap1 expression. In VSMCs, Keap1 depletion induces features of apoptosis, such as positive TUNEL staining and annexin V binding. These changes are associated with an increased expression of nuclear Nrf2. Simultaneous Nrf2 depletion inhibits Keap1 depletion-induced apoptosis. At 14 days after the vascular injury, Nrf2-deficient mice demonstrated fewer TUNEL-positive cells and increased neointimal formation in the neointimal and medial areas. The results suggest that the Nrf2/Keap1 system regulates VSMC apoptosis during neointimal formation, thereby inhibiting neointimal hyperplasia after a vascular injury. PMID:27198574

  8. Tissue Engineered Perivascular Endothelial Cell Implants Regulate Vascular Injury

    NASA Astrophysics Data System (ADS)

    Nathan, Aruna; Nugent, Matthew A.; Edelman, Elazer R.

    1995-08-01

    Molecular biomaterial engineering permits in vivo transplantation of cells and tissues, offering the promise of restoration of physiologic control rather than pharmacologic dosing with isolated compounds. We engrafted endothelial cells on Gelfoam biopolymeric matrices with retention of viability, normal growth kinetics, immunoreactivity, and biochemical activity. The production of heparan sulfate proteoglycan and inhibition of basic fibroblast growth factor binding and activity by engrafted cells were indistinguishable from endothelial cells grown in culture. Perivascular implantation of Gelfoam-endothelial cell scaffolds around balloon-denuded rat carotid arteries reduced intimal hyperplasia 88.1%, far better than the isolated administration of heparin, the most effective endothelial mimic compound. In concert with a reduction in intimal area, cell proliferation was reduced by >90%. To our knowledge, there have been no previous reports of extravascular cell implants controlling vasculoproliferative disease. Tissue engineered cells offer the potential for potent methods of vascular growth regulation and insight into the complex autocrine-paracrine control mechanisms within the blood vessel wall.

  9. Development of a model describing regulation of casein synthesis by the mammalian target of rapamycin (mTOR) signaling pathway in response to insulin, amino acids, and acetate.

    PubMed

    Castro, J J; Arriola Apelo, S I; Appuhamy, J A D R N; Hanigan, M D

    2016-08-01

    To improve dietary protein use efficiency in lactating cows, mammary protein synthesis responses to AA, energy substrates, and hormones must be better understood. These entities exert their effects through stimulation of mRNA translation via control of initiation and elongation rates at the cellular level. A central protein kinase of this phenomenon is the mammalian target of rapamycin (mTOR), which transfers the nutritional and hormonal stimuli onto a series of proteins downstream through a cascade of phosphorylation reactions that ultimately affect protein synthesis. The objective of this work was to further develop an existing mechanistic model of mTOR phosphorylation responses to insulin and total essential AA to include the effects of specific essential AA and acetate mediated by signaling proteins including protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK), and mTOR and to add a representation of milk protein synthesis. Data from 6 experiments in MAC-T cells and mammary tissue slices previously conducted in our laboratory were assembled and used to parameterize the dynamic system of differential equations representing Akt, AMPK, and mTOR in their phosphorylated and dephosphorylated states and the resulting regulation of milk protein synthesis. The model predicted phosphorylated Akt, mTOR, AMPK, and casein synthesis rates with root mean square prediction errors of 16.8, 28.4, 33.0, and 54.9%, respectively. All other dependent variables were free of mean and slope bias, indicating an adequate representation of the data. Whereas mTOR was not very sensitive to changes in insulin or acetate levels, it was highly sensitive to leucine and isoleucine, and this signal appeared to be effectively transduced to casein synthesis. Although prior work had observed a relationship with additional essential AA, and data supporting those conclusions were present in the data set, we were unable to derive significant relationships with any essential

  10. Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity

    PubMed Central

    Xu, Bing; Zhang, Yu; Du, Xu-Fei; Li, Jia; Zi, Hua-Xing; Bu, Ji-Wen; Yan, Yong; Han, Hua; Du, Jiu-Lin

    2017-01-01

    Vascular integrity helps maintain brain microenvironment homeostasis, which is critical for the normal development and function of the central nervous system. It is known that neural cells can regulate brain vascular integrity. However, due to the high complexity of neurovascular interactions involved, understanding of the neural regulation of brain vascular integrity is still rudimentary. Using intact zebrafish larvae and cultured rodent brain cells, we find that neurons transfer miR-132, a highly conserved and neuron-enriched microRNA, via secreting exosomes to endothelial cells (ECs) to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase (eef2k). Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity. Our study indicates that miR-132 acts as an intercellular signal mediating neural regulation of the brain vascular integrity and suggests that the neuronal exosome is a novel avenue for neurovascular communication. PMID:28429770

  11. Force-specific activation of Smad1/5 regulates vascular endothelial cell cycle progression in response to disturbed flow.

    PubMed

    Zhou, Jing; Lee, Pei-Ling; Tsai, Chien-Sung; Lee, Chih-I; Yang, Tung-Lin; Chuang, Han-Sheng; Lin, Wei-Wen; Lin, Ting-Er; Lim, Seh Hong; Wei, Shu-Yi; Chen, Yuh-Lien; Chien, Shu; Chiu, Jeng-Jiann

    2012-05-15

    Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress, but the mechanism of force-specific activation of their signaling to modulate cellular function remains unclear. We have demonstrated that bone morphogenetic protein receptor (BMPR)-specific Smad1/5 can be force-specifically activated by oscillatory shear stress (OSS) in ECs to cause cell cycle progression. Smad1/5 is highly activated in ECs of atherosclerotic lesions in diseased human coronary arteries from patients with end-stage heart failure undergoing heart transplantation and from apolipoprotein E-deficient mice. Application of OSS (0.5 ± 4 dyn/cm(2)) causes the sustained activation of Smad1/5 in ECs through activations of mammalian target of rapamycin and p70S6 kinase, leading to up-regulation of cyclin A and down-regulations of p21(CIP1) and p27(KIP1) and, hence, EC cycle progression. En face examination of rat aortas reveals high levels of phospho-Smad1/5 in ECs of the inner, but not the outer, curvature of aortic arch, nor the straight segment of thoracic aorta [corrected]. Immunohistochemical and en face examinations of the experimentally stenosed abdominal aorta in rats show high levels of phospho-Smad1/5 in ECs at poststenotic sites, where OSS occurs. These OSS activations of EC Smad1/5 in vitro and in vivo are not inhibited by the BMP-specific antagonist Noggin and, hence, are independent of BMP ligand. Transfecting ECs with Smad1/5-specific small interfering RNAs inhibits the OSS-induced EC cycle progression. Our findings demonstrate the force-specificity of the activation of Smad1/5 and its contribution to cell cycle progression in ECs induced by disturbed flow.

  12. Review: novel insights into the regulation of vascular tone by sphingosine 1-phosphate.

    PubMed

    Kerage, D; Brindley, D N; Hemmings, D G

    2014-02-01

    Endothelial dysfunction leading to increased vascular tone is implicated in the pathogenesis of cardiovascular disease, hypertension and pregnancy-related complications like preeclampsia and intrauterine growth restriction. Vascular tone is regulated by a balance between vasoconstrictor and vasodilator signals. Some vascular mediators circulate in blood, whereas others are produced by the endothelium and are delivered to the underlying vascular smooth muscle cells (VSMCs). It is proposed that increased permeability of resistance arteries in preeclampsia allows access of circulating vasoactive factors to VSMCs leading to increased vascular tone. This review focuses on the role of sphingosine 1-phosphate (S1P). This sphingolipid enhances the endothelial barrier, but it can also disrupt the barrier under certain conditions. These S1P-mediated effects on the endothelial barrier have been demonstrated in cultured endothelial cells and in isolated venules. They depend on S1P concentrations, the S1P receptors expressed and the vascular bed. However, no studies have examined if vascular tone is regulated by S1P in resistance arteries through changes in endothelial permeability and the leakage of circulating vasoconstrictors. Our recent studies using the pressure myograph system show that access of infused vasoconstrictors to VSMCs is blocked under low S1P concentrations. Pathophysiological levels of infused S1P disrupt the barrier and maximally increase vascular tone by facilitating access of itself and a co-infused vasoconstrictor to the VSMCs. Interestingly, infusion of an intermediate physiological concentration of S1P showed a small increase in endothelial permeability with controlled leakage of a co-infused vasoconstrictor that led to sub-maximal vascular tone development. These and other studies delineate the important role of S1P in the regulation of vascular tone and emphasize how dysfunction of this regulation can lead to pregnancy-related disorders. Copyright

  13. Delayed Correlation of mRNA and Protein Expression in Rapamycin-treated Cells and a Role for Ggc1 in Cellular Sensitivity to Rapamycin*

    PubMed Central

    Fournier, Marjorie L.; Paulson, Ariel; Pavelka, Norman; Mosley, Amber L.; Gaudenz, Karin; Bradford, William D.; Glynn, Earl; Li, Hua; Sardiu, Mihaela E.; Fleharty, Brian; Seidel, Christopher; Florens, Laurence; Washburn, Michael P.

    2010-01-01

    To identify new molecular targets of rapamycin, an anticancer and immunosuppressive drug, we analyzed temporal changes in yeast over 6 h in response to rapamycin at the transcriptome and proteome levels and integrated the expression patterns with functional profiling. We show that the integration of transcriptomics, proteomics, and functional data sets provides novel insights into the molecular mechanisms of rapamycin action. We first observed a temporal delay in the correlation of mRNA and protein expression where mRNA expression at 1 and 2 h correlated best with protein expression changes after 6 h of rapamycin treatment. This was especially the case for the inhibition of ribosome biogenesis and induction of heat shock and autophagy essential to promote the cellular sensitivity to rapamycin. However, increased levels of vacuolar protease could enhance resistance to rapamycin. Of the 85 proteins identified as statistically significantly changing in abundance, most of the proteins that decreased in abundance were correlated with a decrease in mRNA expression. However, of the 56 proteins increasing in abundance, 26 were not correlated with an increase in mRNA expression. These protein changes were correlated with unchanged or down-regulated mRNA expression. These proteins, involved in mitochondrial genome maintenance, endocytosis, or drug export, represent new candidates effecting rapamycin action whose expression might be post-transcriptionally or post-translationally regulated. We identified GGC1, a mitochondrial GTP/GDP carrier, as a new component of the rapamycin/target of rapamycin (TOR) signaling pathway. We determined that the protein product of GGC1 was stabilized in the presence of rapamycin, and the deletion of the GGC1 enhanced growth fitness in the presence of rapamycin. A dynamic mRNA expression analysis of Δggc1 and wild-type cells treated with rapamycin revealed a key role for Ggc1p in the regulation of ribosome biogenesis and cell cycle progression

  14. Rapamycin slows aging in mice

    PubMed Central

    Wilkinson, John E.; Burmeister, Lisa; Brooks, Susan V.; Chan, Chi-Chao; Friedline, Sabrina; Harrison, David E.; Hejtmancik, James F.; Nadon, Nancy; Strong, Randy; Wood, Lauren K.; Woodward, Maria A.; Miller, Richard A.

    2012-01-01

    Summary Rapamycin increases lifespan in mice, but whether this represents merely inhibition of lethal neoplastic diseases, or an overall slowing in multiple aspects of aging is currently unclear. We report here that many forms of age-dependent change, including alterations in heart, liver, adrenal glands, endometrium, and tendon, as well as age-dependent decline in spontaneous activity, occur more slowly in rapamycin-treated mice, suggesting strongly that rapamycin retards multiple aspects of aging in mice, in addition to any beneficial effects it may have on neoplastic disease. We also note, however, that mice treated with rapamycin starting at 9 months of age have significantly higher incidence of testicular degeneration and cataracts; harmful effects of this kind will guide further studies on timing, dosage, and tissue-specific actions of rapamycin relevant to the development of clinically useful inhibitors of TOR action. PMID:22587563

  15. Enzymatic regulation of functional vascular networks using gelatin hydrogels

    PubMed Central

    Chuang, Chia-Hui; Lin, Ruei-Zeng; Tien, Han-Wen; Chu, Ya-Chun; Li, Yen-Cheng; Melero-Martin, Juan M.; Chen, Ying-Chieh

    2015-01-01

    To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl (gelatin-Ph) hydrogels that can be enzymatically crosslinked, allowing tuning of the storage modulus and the proteolytic degradation rate, for use as injectable hydrogels to support the human progenitor cell-based formation of a stable and mature vascular network. Porcine gelatin-Ph hydrogels were found to be cytocompatible with human blood-derived endothelial colony-forming cells and white adipose tissue-derived mesenchymal stem cells, resulting in >87% viability, and cell proliferation and spreading could be modulated by using hydrogels with different proteolytic degradability and stiffness. In addition, gelatin was extracted from mouse dermis and murine gelatin-Ph hydrogels were prepared. Importantly, implantation of human cell-laden porcine or murine gelatin-Ph hydrogels into immunodeficient mice resulted in the rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, the degree of enzymatic crosslinking of the gelatin-Ph hydrogels could be used to modulate cell behavior and the extent of vascular network formation in vivo. Our report details a technique for the synthesis of gelatin-Ph hydrogels from allogeneic or xenogeneic dermal skin and suggests that these hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues. PMID:25749296

  16. Enzymatic regulation of functional vascular networks using gelatin hydrogels.

    PubMed

    Chuang, Chia-Hui; Lin, Ruei-Zeng; Tien, Han-Wen; Chu, Ya-Chun; Li, Yen-Cheng; Melero-Martin, Juan M; Chen, Ying-Chieh

    2015-06-01

    To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl (gelatin-Ph) hydrogels that can be enzymatically crosslinked, allowing tuning of the storage modulus and the proteolytic degradation rate, for use as injectable hydrogels to support the human progenitor cell-based formation of a stable and mature vascular network. Porcine gelatin-Ph hydrogels were found to be cytocompatible with human blood-derived endothelial colony-forming cells and white adipose tissue-derived mesenchymal stem cells, resulting in >87% viability, and cell proliferation and spreading could be modulated by using hydrogels with different proteolytic degradability and stiffness. In addition, gelatin was extracted from mouse dermis and murine gelatin-Ph hydrogels were prepared. Importantly, implantation of human cell-laden porcine or murine gelatin-Ph hydrogels into immunodeficient mice resulted in the rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, the degree of enzymatic crosslinking of the gelatin-Ph hydrogels could be used to modulate cell behavior and the extent of vascular network formation in vivo. Our report details a technique for the synthesis of gelatin-Ph hydrogels from allogeneic or xenogeneic dermal skin and suggests that these hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights

  17. Angiopoietins regulate vascular reactivity after haemorrhagic shock in rats through the Tie2-nitric oxide pathway.

    PubMed

    Xu, Jing; Lan, Dan; Li, Tao; Yang, Guangming; Liu, Liangming

    2012-11-01

    Vascular reactivity shows biphasic changes after severe trauma or shock. Our aim was to elucidate the mechanisms of biphasic-changed vascular reactivity after haemorrhagic shock by observing the regulation of angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) on it. Haemorrhagic-shock Sprague-Dawley rats, hypoxia-treated superior mesenteric arteries (SMAs) with intact endothelia, and a cell mixture of vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) were adopted to evaluate the regulatory effects of Ang-1 and Ang-2 on vascular reactivity and their relationship to Tie2 (receptor tyrosine kinase)-Akt-endothelial nitric oxide synthase (eNOS) and Tie2-extracellular signal-regulated kinase (Erk)-inducible nitric oxide synthase (iNOS) signal pathways. Ang-1 expression, Tie2 phosphorylation, and nitric oxide (NO) release were increased at early shock. Exogenous Ang-1 maintained the vascular reactivity of SMAs after early hypoxia. Tie2-blocking antibody and the antagonists of Akt and eNOS antagonized Ang-1-induced maintenance in vascular reactivity and a slight release in NO at the early stage of shock. Ang-2 expression, Tie2 phosphorylation, and NO release were greatly increased at late shock, but exogenous Ang-2 further decreased the vascular reactivity of SMAs after late hypoxia. Tie2-blocking antibody and the antagonists of Erk and iNOS andtagonized the Ang-2-induced decrease in vascular reactivity and a large release of NO at the late stage of shock. Ang-1 and Ang-2 participated in the regulation of vascular reactivity after haemorrhagic shock. Ang-1 was mainly responsible for the hyperreactivity at early shock through the Tie2-Akt-eNOS pathway and an appropriate amount of NO release. Ang-2 was mainly responsible for the hyporeactivity at late shock through the Tie2-Erk-iNOS pathway and the release of a large amount of NO.

  18. Inflammation-regulated mRNA stability and the progression of vascular inflammatory diseases.

    PubMed

    Herman, Allison B; Autieri, Michael V

    2017-11-15

    Cardiovascular disease remains a major medical and socioeconomic burden in developed and developing societies, and will increase with an aging and increasingly sedentary society. Vascular disease and atherosclerotic vascular syndromes are essentially inflammatory disorders, and transcriptional and post-transcriptional processes play essential roles in the ability of resident vascular and inflammatory cells to adapt to environmental stimuli. The regulation of mRNA translocation, stability, and translation are key processes of post-transcriptional regulation that permit these cells to rapidly respond to inflammatory stimuli. For the most part, these processes are controlled by elements in the 3'-UTR of labile, proinflammatory transcripts. Since proinflammatory transcripts almost exclusively contain AU-rich elements (AREs), this represents a tightly regulated and specific mechanism for initiation and maintenance of the proinflammatory phenotype. RNA-binding proteins (RBPs) recognize cis elements in 3'-UTR, and regulate each of these processes, but there is little literature exploring the concept that RBPs themselves can be directly regulated by inflammatory stimuli. Conceptually, inflammation-responsive RBPs represent an attractive target of rational therapies to combat vascular inflammatory syndromes. Herein we briefly describe the cellular and molecular etiology of atherosclerosis, and summarize our current understanding of RBPs and their specific roles in regulation of inflammatory mRNA stability. We also detail RBPs as targets of current anti-inflammatory modalities and how this may translate into better treatment for vascular inflammatory diseases. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  19. Platelets regulate lymphatic vascular development through CLEC-2–SLP-76 signaling

    PubMed Central

    Bertozzi, Cara C.; Schmaier, Alec A.; Mericko, Patricia; Hess, Paul R.; Zou, Zhiying; Chen, Mei; Chen, Chiu-Yu; Xu, Bin; Lu, Min-min; Zhou, Diane; Sebzda, Eric; Santore, Matthew T.; Merianos, Demetri J.; Stadtfeld, Matthias; Flake, Alan W.; Graf, Thomas; Skoda, Radek; Maltzman, Jonathan S.; Koretzky, Gary A.

    2010-01-01

    Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK–SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre–mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76–dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development. PMID:20363774

  20. The DDAH/ADMA pathway is a critical regulator of NO signalling in vascular homeostasis

    PubMed Central

    2008-01-01

    NO is an important regulator of cardiovascular remodelling and function. ADMA, an endogenous L-arginine analogue, reduces NO production by inhibiting the activity of NOS. ADMA levels in turn, are regulated by DDAH, which metabolises ADMA. High levels of ADMA and dysregulated DDAH activity are risk factors for cardiovascular disease and morbidity. To investigate this link, the DDAH I null mouse has been recently generated and has a lethal phenotype. Studies on vascular function in the DDAH I heterozygous knockout mouse, which is viable, demonstrates a causal link between reduced DDAH I activity, increased ADMA levels and reduced NO signalling and vascular dysfunction. In another study, detailed in vitro analyses reveal that the DDAH/ADMA pathway critically regulates endothelial cell motility and angiogenesis and establishes some of the molecular mechanisms involved. These studies highlight the importance of DDAH and ADMA in regulating NO dependent vascular homeostasis. PMID:19270535

  1. The DDAH/ADMA pathway is a critical regulator of NO signalling in vascular homeostasis.

    PubMed

    Fiedler, Lorna

    2008-01-01

    NO is an important regulator of cardiovascular remodelling and function. ADMA, an endogenous L-arginine analogue, reduces NO production by inhibiting the activity of NOS. ADMA levels in turn, are regulated by DDAH, which metabolises ADMA. High levels of ADMA and dysregulated DDAH activity are risk factors for cardiovascular disease and morbidity. To investigate this link, the DDAH I null mouse has been recently generated and has a lethal phenotype. Studies on vascular function in the DDAH I heterozygous knockout mouse, which is viable, demonstrates a causal link between reduced DDAH I activity, increased ADMA levels and reduced NO signalling and vascular dysfunction. In another study, detailed in vitro analyses reveal that the DDAH/ADMA pathway critically regulates endothelial cell motility and angiogenesis and establishes some of the molecular mechanisms involved. These studies highlight the importance of DDAH and ADMA in regulating NO dependent vascular homeostasis.

  2. AIM2 regulates vascular smooth muscle cell migration in atherosclerosis.

    PubMed

    Pan, Jinyu; Lu, Lu; Wang, Xuyang; Liu, Dian; Tian, Jingjing; Liu, Hui; Zhang, Mingjun; Xu, Fengqin; An, Fengshuang

    2018-02-26

    Atherosclerosis (AS) is a common pathological basis of various cardiovascular and cerebrovascular diseases. Plaque formation is initiated and triggered by vascular smooth musclecells (VSMCs) migration in vascular wall, which gradually aggravates atherosclerosis progression. Absent in melanoma 2 (AIM2), a member of HIN-200 family, plays an important role in activating inflammasome. However, the role of AIM2 in atherosclerotic plaque progression outside of the inflammasome has not yet been reported. The potential effect and the underlying mechanism of AIM2 were investigated in apoliporotein E-deficient (ApoE-/-) mice. Murine AIM2 lentivirus, shRNA-AIM2 lentivirus and null lentivirus were constructed and injected intravenously into ApoE-/- mice, which were fed on a high fat diet. The specific mechanism of AIM2 in vascular smooth cells (VSMCs) was explored in vitro. Results showed the aortic atherosclerotic lesion area was larger with AIM2 over-expression, and the number of smooth muscle cells was enhanced in line with the increased AIM2 levels. AIM2 overexpression also induced the increasing expression of MMP2. In vitro studies revealed that different levels of ox-LDL increased AIM2 expression in a time-dependent manner. Transwell showed that AIM2 mediated migration in VSMCs. The expression of AIM2 can be inhibited when the ROS inhibitor was used. Additionally, the overexpression and inhibition of AIM2 significantly affects HG-induced migration and TGF-β/SMAD signaling pathway in VSMCs. Thus, we demonstrated that AIM2 could promote the progression of atherosclerotic plaque by increasing migration in VSMCs. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Redox-sensitive transcription factor Nrf2 regulates vascular smooth muscle cell migration and neointimal hyperplasia.

    PubMed

    Ashino, Takashi; Yamamoto, Masayuki; Yoshida, Takemi; Numazawa, Satoshi

    2013-04-01

    Reactive oxygen species are important mediators for platelet-derived growth factor (PDGF) signaling in vascular smooth muscle cells, whereas excess reactive oxygen species-induced oxidative stress contributes to the development and progression of vascular diseases, such as atherosclerosis. Activation of the redox-sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), is pivotal in cellular defense against oxidative stress by transcriptional upregulation of antioxidant proteins. This study aimed to elucidate the role of Nrf2 in PDGF-mediated vascular smooth muscle cell migration and neointimal hyperplasia. PDGF promoted nuclear translocation of Nrf2, followed by the induction of target genes, including NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, and thioredoxin-1. Nrf2 depletion by small interfering RNA enhanced PDGF-promoted Rac1 activation and reactive oxygen species production and persistently phosphorylated downstream extracellular signal-regulated kinase-1/2. Nrf2 depletion enhanced vascular smooth muscle cell migration in response to PDGF and wound scratch. In vivo, Nrf2-deficient mice showed enhanced neointimal hyperplasia in a wire injury model. These findings suggest that the Nrf2 system is important for PDGF-stimulated vascular smooth muscle cell migration by regulating reactive oxygen species elimination, which may contribute to neointimal hyperplasia after vascular injury. Our findings provide insight into the Nrf2 system as a novel therapeutic target for vascular remodeling and atherosclerosis.

  4. The vascular conducted response in cerebral blood flow regulation.

    PubMed

    Jensen, Lars Jørn; Holstein-Rathlou, Niels-Henrik

    2013-05-01

    Despite recent advances in our understanding of the molecular and cellular mechanisms behind vascular conducted responses (VCRs) in systemic arterioles, we still know very little about their potential physiological and pathophysiological role in brain penetrating arterioles controlling blood flow to the deeper areas of the brain. The scope of the present review is to present an overview of the conceptual, mechanistic, and physiological role of VCRs in resistance vessels, and to discuss in detail the recent advances in our knowledge of VCRs in brain arterioles controlling cerebral blood flow. We provide a schematic view of the ion channels and intercellular communication pathways necessary for conduction of an electrical and mechanical response in the arteriolar wall, and discuss the local signaling mechanisms and cellular pathway involved in the responses to different local stimuli and in different vascular beds. Physiological modulation of VCRs, which is a rather new finding in this field, is discussed in the light of changes in plasma membrane ion channel conductance as a function of health status or disease. Finally, we discuss the possible role of VCRs in cerebrovascular function and disease as well as suggest future directions for studying VCRs in the cerebral circulation.

  5. Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

    PubMed

    Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

    2016-01-01

    Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

  6. Combination of Rapamycin and Resveratrol for Treatment of Bladder Cancer.

    PubMed

    Alayev, Anya; Salamon, Rachel S; Schwartz, Naomi S; Berman, Adi Y; Wiener, Sara L; Holz, Marina K

    2017-02-01

    Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin-induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1-/- MEF cells, and not in wild-type MEFs. Similarly, resveratrol alone or in combination with rapamycin induced cell death in human bladder cancer cell lines. These data indicate that administration of resveratrol together with rapamycin may be a promising therapeutic option for treatment of bladder cancer. J. Cell. Physiol. 232: 436-446, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  7. Long Noncoding RNA-GAS5: A Novel Regulator of Hypertension-Induced Vascular Remodeling.

    PubMed

    Wang, Yang-Ning-Zhi; Shan, Kun; Yao, Mu-Di; Yao, Jin; Wang, Jia-Jian; Li, Xiang; Liu, Ban; Zhang, Yang-Yang; Ji, Yong; Jiang, Qin; Yan, Biao

    2016-09-01

    Vascular remodeling is an important pathological feature of hypertension, leading to increased vascular resistance and reduced compliance. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction is involved in vascular remodeling. Long noncoding RNAs are potential regulators of EC and VSMC function. Herein, we determined whether long noncoding RNA-growth arrest-specific 5 (GAS5) is involved in hypertension-related vascular remodeling. We revealed that GAS5 knockdown aggravated hypertension-induced microvascular dysfunction as shown by increased retinal neovascularization and capillary leakage. GAS5 regulated the remodeling of arteries, including caudal arteries, carotid arteries, renal arteries, and thoracic arteries. GAS5 was mainly expressed in ECs and VSMCs, and its expression was significantly downregulated in hypertension. GAS5 knockdown affected endothelial activation, endothelial proliferation, VSMC phenotypic conversion, and EC-VSMC communication in vivo and in vitro. Mechanistically, GAS5 regulated EC and VSMC function through β-catenin signaling. This study identified GAS5 as a critical regulator in hypertension and demonstrated the potential of gene therapy and drug development for treating hypertension. © 2016 American Heart Association, Inc.

  8. Nitric oxide regulates retinal vascular tone in humans.

    PubMed

    Dorner, Guido T; Garhofer, Gerhard; Kiss, Barbara; Polska, Elzbieta; Polak, Kaija; Riva, Charles E; Schmetterer, Leopold

    2003-08-01

    The purpose of the present study was to investigate the contribution of basal nitric oxide (NO) on retinal vascular tone in humans. In addition, we set out to elucidate the role of NO in flicker-induced retinal vasodilation in humans. Twelve healthy young subjects were studied in a three-way crossover design. Subjects received an intravenous infusion of either placebo or NG-monomethyl-L-arginine (L-NMMA; 3 or 6 mg/kg over 5 min), an inhibitor of NO synthase. Thereafter, diffuse luminance flicker was consecutively performed for 16, 32, and 64 s at a frequency of 8 Hz. The effect of L-NMMA on retinal arterial and venous diameter was assessed under resting conditions and during the hyperemic flicker response. Retinal vessel diameter was measured with a Zeiss retinal vessel analyzer. L-NMMA significantly reduced arterial diameter (3 mg/kg: -2%; 6 mg/kg: -4%, P < 0.001) and venous diameter (3 mg/kg: -5%; 6 mg/kg: -8%, P < 0.001). After placebo infusion, flicker induced a significant increase in retinal vessel diameter (P < 0.001). At a flicker duration of 64 s, arterial diameter increased by 4% and venous diameter increased by 3%. L-NMMA did not abolish these hyperemic responses but blunted venous vasodilation (P = 0.017) and arterial vasodilation (P = 0.02) in response to flicker stimulation. Our data indicate that NO contributes to basal retinal vascular tone in humans. In addition, NO appears to play a role in flicker-induced vasodilation of the human retinal vasculature.

  9. Nogo-A regulates vascular network architecture in the postnatal brain.

    PubMed

    Wälchli, Thomas; Ulmann-Schuler, Alexandra; Hintermüller, Christoph; Meyer, Eric; Stampanoni, Marco; Carmeliet, Peter; Emmert, Maximilian Y; Bozinov, Oliver; Regli, Luca; Schwab, Martin E; Vogel, Johannes; Hoerstrup, Simon P

    2017-02-01

    Recently, we discovered a new role for the well-known axonal growth inhibitory molecule Nogo-A as a negative regulator of angiogenesis in the developing central nervous system. However, how Nogo-A affected the three-dimensional (3D) central nervous system (CNS) vascular network architecture remained unknown. Here, using vascular corrosion casting, hierarchical, synchrotron radiation μCT-based network imaging and computer-aided network analysis, we found that genetic ablation of Nogo-A significantly increased the three-dimensional vascular volume fraction in the postnatal day 10 (P10) mouse brain. More detailed analysis of the cerebral cortex revealed that this effect was mainly due to an increased number of capillaries and capillary branchpoints. Interestingly, other vascular parameters such as vessel diameter, -length, -tortuosity, and -volume were comparable between both genotypes for non-capillary vessels and capillaries. Taken together, our three-dimensional data showing more vessel segments and branchpoints at unchanged vessel morphology suggest that stimulated angiogenesis upon Nogo-A gene deletion results in the insertion of complete capillary micro-networks and not just single vessels into existing vascular networks. These findings significantly enhance our understanding of how angiogenesis, vascular remodeling, and three-dimensional vessel network architecture are regulated during central nervous system development. Nogo-A may therefore be a potential novel target for angiogenesis-dependent central nervous system pathologies such as brain tumors or stroke.

  10. [Mathematical modeling of passive mechanisms of the human vascular regulation in orthostatic position].

    PubMed

    Bednenko, V S; Matiushev, T V; Mukhin, V A; Ryzhenkov, S P; Abashev, V Iu

    2002-01-01

    Formalized description of the vascular component of the human circulation system taking in the effects of gravity on the human organism during the standing test is presented. The structure proposed in the model by R.D. Grigorian has been used as basic to describe the vascular system. The neuroreflex and humoral regulators of circulation have been counted as constant. Organs and elements of the vascular system were represented as a network of sequential and parallel separate elastic reservoirs with distributed parameters. The pressure-volume ratio was represented by piecewise linear approximation with 3 fragments imitating main forms of the vascular cross section. Primary focus was put on investigation of circulation in the leg and the head which is of interest for evaluating body reactions to postural changes. Result of modeling have been displayed as curves of volumetric velocity, and blood volume and pressure in different fragments of cranial and crus vessels.

  11. Blood-brain barrier leakage after status epilepticus in rapamycin-treated rats II: Potential mechanisms.

    PubMed

    van Vliet, Erwin A; Otte, Willem M; Wadman, Wytse J; Aronica, Eleonora; Kooij, Gijs; de Vries, Helga E; Dijkhuizen, Rick M; Gorter, Jan A

    2016-01-01

    Blood-brain barrier (BBB) leakage may play a pro-epileptogenic role after status epilepticus. In the accompanying contrast-enhanced magnetic resonance imaging (CE-MRI) study we showed that the mammalian target of rapamycin (mTOR) inhibitor rapamycin reduced BBB leakage and seizure activity during the chronic epileptic phase. Given rapamycin's role in growth and immune response, the potential therapeutic effects of rapamycin after status epilepticus with emphasis on brain inflammation and brain vasculature were investigated. Seven weeks after kainic acid-induced status epilepticus, rats were perfusion fixed and (immuno)histochemistry was performed using several glial and vascular markers. In addition, an in vitro model for the human BBB was used to determine the effects of rapamycin on transendothelial electrical resistance as a measure for BBB integrity. (Immuno)histochemistry showed that local blood vessel density, activated microglia, and astrogliosis were reduced in rapamycin-treated rats compared to vehicle-treated rats. In vitro studies showed that rapamycin could attenuate TNFα-induced endothelial barrier breakdown. These data suggest that rapamycin improves BBB function during the chronic epileptic phase by a reduction of local brain inflammation and blood vessel density that can contribute to a milder form of epilepsy. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.

  12. Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

    SciTech Connect

    Kim, Se-Hee; Schmitt, Christopher E.; Woolls, Melissa J.

    2013-01-25

    Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A)more » signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.« less

  13. Vascular endothelial growth factor signaling regulates the segregation of artery and vein via ERK activity during vascular development

    SciTech Connect

    Kim, Se-Hee; Schmitt, Christopher E.; Woolls, Melissa J.; Holland, Melinda B.; Kim, Jun-Dae; Jin, Suk-Won

    2013-01-25

    Highlights: ► VEGF-A signaling regulates the segregation of axial vessels. ► VEGF-A signaling is mediated by PKC and ERK in this process. ► Ectopic activation of ERK is sufficient to rescue defects in vessel segregation. -- Abstract: Segregation of two axial vessels, the dorsal aorta and caudal vein, is one of the earliest patterning events occur during development of vasculature. Despite the importance of this process and recent advances in our understanding on vascular patterning during development, molecular mechanisms that coordinate the segregation of axial vessels remain largely elusive. In this report, we find that vascular endothelial growth factor-A (Vegf-A) signaling regulates the segregation of dorsal aorta and axial vein during development. Inhibition of Vegf-A pathway components including ligand Vegf-A and its cognate receptor Kdrl, caused failure in segregation of axial vessels in zebrafish embryos. Similarly, chemical inhibition of Mitogen-activated protein kinase kinase (Map2k1)/Extracellular-signal-regulated kinases (Erk) and phosphatidylinositol 3-kinases (PI3 K), which are downstream effectors of Vegf-A signaling pathway, led to the fusion of two axial vessels. Moreover, we find that restoring Erk activity by over-expression of constitutively active MEK in embryos with a reduced level of Vegf-A signaling can rescue the defects in axial vessel segregation. Taken together, our data show that segregation of axial vessels requires the function of Vegf-A signaling, and Erk may function as the major downstream effector in this process.

  14. Novel vascular cell-specific genes whose expression is regulated temporally and spatially during vascular system development.

    PubMed Central

    Demura, T; Fukuda, H

    1994-01-01

    We have isolated three cDNA clones (TED2, TED3, and TED4) for genes expressed preferentially in cells that redifferentiate into tracheary elements from mesophyll cells isolated from leaves of Zinnia elegans. Sequence analyses of TED clones revealed that TED2 encodes a hydrophobic polypeptide with a significant similarity to the guinea pig lens-specific protein (zeta-crystallin) and that the deduced polypeptide of TED3 may be a novel cell wall protein. In situ hybridization of the TED probes with young Zinnia seedlings showed that expression of the three TED genes was restricted to vascular cells and regulated in a temporal and spatial manner during vascular development. TED3 transcripts were localized specifically to a few cells that are to differentiate or are differentiating into tracheary elements in all organs examined. TED4 transcripts were present mainly in the immature primary xylem both of cotyledons and of the boundary region between the root and hypocotyl and in the procambium of roots. In contrast, TED2 transcripts accumulated not only in immature primary xylem cells but also in immature phloem cells both in roots and in the boundary region between the root and hypocotyl. In addition, TED2 transcripts were expressed in the procambium cells of roots. In cotyledons, TED2 transcripts did not accumulate in xylem or phloem cells but only in two regions that might form a new vein just outside the phloem of the main leaf vein. Taken together, our findings indicate that TED2, TED3, and TED4 can be novel and efficient markers for development of the vascular system. PMID:8069107

  15. The Fractal-based Analysis of the Regulation of Vascular Remodeling in the Quail Chorioallantoic Membrane

    NASA Technical Reports Server (NTRS)

    Smith, Genee S.

    2004-01-01

    Critical to the advancement of space exploration is the safety and well being of astronauts while in space. This study focuses on the second highest of NASA-defined risk categories for human space exploration, cardiovascular alterations. Current research of this problem is being tackled by investigating angiogenesis through vascular remodeling. Angiogenesis is the growth and formation of new blood vessels. Angiogenesis is an important part of maintaining normal development and bodily functions. The loss of control of this process, either insufficient or excessive vascular growth, is considered a common denominator in many diseases, such as cancer, diabetes, and coronary artery disease. Objectives are presently being met by observing the effects of various regulators, like thrombospondin 1 (TSP-1) and a novel vessel tortuosity factor (TF), through the use of the chorioallantoic membrane (CAM) of Japanese quail embryos, which enables the direct optical imaging of 2-dimensional vascular branching trees. Research within the CAM is being performed to deduce numerous methods of regulating vessel growth. This project centers on the ability of a novel vessel regulator to affect angiogenesis. For example, it is hypothesized that the TSP-1 will inhibit the growth of CAM vasculature. Fractal/VESGEN-based techniques and PTV analysis are the methodologies used to investigate vascular differentiation. This tactic is used to quantify results and measure the growth patterns and morphology of blood vessels. The regulatory mechanisms posed by this vessel regulator can be deduced by alterations found within the vasculature patterns of quail embryos.

  16. Autophagy regulates vascular endothelial cell eNOS and ET-1 expression induced by laminar shear stress in an ex vivo perfused system.

    PubMed

    Guo, Fengxia; Li, Xiaohong; Peng, Juan; Tang, Yaling; Yang, Qin; Liu, Lushan; Wang, Zuo; Jiang, Zhisheng; Xiao, Ming; Ni, Chuyu; Chen, Ruixing; Wei, Dangheng; Wang, Gui-xue

    2014-09-01

    Vascular endothelial cell function responds to steady laminar shear stress; however, the underlying mechanisms are not fully elucidated. In the present study, we examined the effect of steady laminar shear stress on vascular endothelial cell autophagy and endothelial cell nitric oxide synthase (eNOS) and endothelin-1 (ET-1) expression using an ex vivo perfusion system. Human vascular endothelial cells and common arteries of New Zealand rabbits were pretreated with or without rapamycin or 3-MA for 30 min. These were then placed in an ex vivo cell perfusion system or an ex vivo organ perfusion system under static conditions (0 dynes/cm2) or steady laminar shear stress (5 or 15 dynes/cm2) for 1 h. In both ex vivo perfusion vascular endothelial cells and vascular vessel segment, steady laminar shear stress promoted autophagy and eNOS expression and inhibited ET-1 expression. Compared with steady laminar shear stress treatment alone, the pretreatment of autophagy inducer rapamycin obviously strengthened the expression of eNOS and decreased the expression of ET-1 in both the 5 and 15 dynes/cm2 treatment groups. Moreover, when pretreated with the autophagy inhibitor 3-MA, the eNOS expression was obviously inhibited and the ET-1 expression was reversed. These findings demonstrate that autophagy is upregulated under steady laminar shear stress, improving endothelial cell maintenance of vascular tone function.

  17. Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells.

    PubMed

    Zhou, Fang; Jia, Xiaoling; Yang, Yang; Yang, Qingmao; Gao, Chao; Zhao, Yunhui; Fan, Yubo; Yuan, Xiaoyan

    2016-11-01

    The efficiency of biomaterials used in small vascular repair depends greatly on their ability to interact with vascular endothelial cells (VECs). Rapid endothelialization of the vascular grafts is a promising way to prevent thrombosis and intimal hyperplasia. In this work, modification of electrospun membranes of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) by three different peptides for regulation of VECs were studied in order to obtain ideal bioactive biomaterials as small diameter vascular grafts. QK (a mimetic peptide to vascular endothelial growth factor), Arg-Glu-Asp-Val (REDV, a specific adhesive peptide to VECs) and Val-Ala-Pro-Gly (VAPG, a specific adhesive peptide to vascular smooth muscle cells) were investigated. Surface properties of the modified membranes and the response of VECs were verified. It was found that protein adsorption and platelet adhesion were effectively suppressed with the introduction of QK, REDV or VAPG peptides on the PELCL electrospun membranes. Both QK- and REDV-modified electrospun membranes could accelerate the proliferation of VECs in the first 9days, and the QK-modified electrospun membrane promoted cell proliferation more significantly than the REDV-modified one. The REDV-modified PELCL membrane was the most favorable for VECs adhesion than QK- and VAPG-modified membranes. It was suggested that QK- or REDV-modified PELCL electrospun membranes may have great potential applications in cardiovascular biomaterials for rapid endothelialization in situ. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Mammalian target of rapamycin signaling in diabetic cardiovascular disease.

    PubMed

    Chong, Zhao Zhong; Maiese, Kenneth

    2012-07-16

    Diabetes mellitus currently affects more than 170 million individuals worldwide and is expected to afflict another 200 million individuals in the next 30 years. Complications of diabetes as a result of oxidant stress affect multiple systems throughout the body, but involvement of the cardiovascular system may be one of the most severe in light of the impact upon cardiac and vascular function that can result in rapid morbidity and mortality for individuals. Given these concerns, the signaling pathways of the mammalian target of rapamycin (mTOR) offer exciting prospects for the development of novel therapies for the cardiovascular complications of diabetes. In the cardiovascular and metabolic systems, mTOR and its multi-protein complexes of TORC1 and TORC2 regulate insulin release and signaling, endothelial cell survival and growth, cardiomyocyte proliferation, resistance to β-cell injury, and cell longevity. Yet, mTOR can, at times, alter insulin signaling and lead to insulin resistance in the cardiovascular system during diabetes mellitus. It is therefore vital to understand the complex relationship mTOR and its downstream pathways hold during metabolic disease in order to develop novel strategies for the complications of diabetes mellitus in the cardiovascular system.

  19. Vascular pattern of the dentate gyrus is regulated by neural progenitors.

    PubMed

    Pombero, Ana; Garcia-Lopez, Raquel; Estirado, Alicia; Martinez, Salvador

    2018-01-06

    Neurogenesis is a vital process that begins during early embryonic development and continues until adulthood, though in the latter case, it is restricted to the subventricular zone and the subgranular zone of the dentate gyrus (DG). In particular, the DG's neurogenic properties are structurally and functionally unique, which may be related to its singular vascular pattern. Neurogenesis and angiogenesis share molecular signals and act synergistically, supporting the concept of a neurogenic niche as a functional unit between neural precursors cells and their environment, in which the blood vessels play an important role. Whereas it is well known that vascular development controls neural proliferation in the embryonary and in the adult brain, by releasing neurotrophic factors; the potential influence of neural cells on vascular components during angiogenesis is largely unknown. We have demonstrated that the reduction of neural progenitors leads to a significant impairment of vascular development. Since VEGF is a potential regulator in the neurogenesis-angiogenesis crosstalk, we were interested in assessing the possible role of this molecule in the hippocampal neurovascular development. Our results showed that VEGF is the molecule involved in the regulation of vascular development by neural progenitor cells in the DG.

  20. mTORC1 inhibitors rapamycin and metformin affect cardiovascular markers differentially in ZDF rats.

    PubMed

    Nistala, Ravi; Raja, Ahmad; Pulakat, Lakshmi

    2017-03-01

    Mammalian target for rapamycin complex 1 (mTORC1) is a common target for the action of immunosuppressant macrolide rapamycin and glucose-lowering metformin. Inhibition of mTORC1 can exert both beneficial and detrimental effects in different pathologies. Here, we investigated the differential effects of rapamycin (1.2 mg/kg per day delivered subcutaneously for 6 weeks) and metformin (300 mg/kg per day delivered orally for 11 weeks) treatments on male Zucker diabetic fatty (ZDF) rats that mimic the cardiorenal pathology of type 2 diabetic patients and progress to insulin insufficiency. Rapamycin and metformin improved proteinuria, and rapamycin also reduced urinary gamma glutamyl transferase (GGT) indicating improvement of tubular health. Metformin reduced food and water intake, and urinary sodium and potassium, whereas rapamycin increased urinary sodium. Metformin reduced plasma alkaline phosphatase, but induced transaminitis as evidenced by significant increases in plasma AST and ALT. Metformin also induced hyperinsulinemia, but did not suppress fasting plasma glucose after ZDF rats reached 17 weeks of age, and worsened lipid profile. Rapamycin also induced mild transaminitis. Additionally, both rapamycin and metformin increased plasma uric acid and creatinine, biomarkers for cardiovascular and renal disease. These observations define how rapamycin and metformin differentially modulate metabolic profiles that regulate cardiorenal pathology in conditions of severe type 2 diabetes.

  1. Anti-Remodeling Effects of Rapamycin in Experimental Heart Failure: Dose Response and Interaction with Angiotensin Receptor Blockade

    PubMed Central

    Bishu, Kalkidan; Ogut, Ozgur; Kushwaha, Sudhir; Mohammed, Selma F.; Ohtani, Tomohito; Xu, Xiaolei; Brozovich, Frank V.; Redfield, Margaret M.

    2013-01-01

    While neurohumoral antagonists improve outcomes in heart failure (HF), cardiac remodeling and dysfunction progress and outcomes remain poor. Therapies superior or additive to standard HF therapy are needed. Pharmacologic mTOR inhibition by rapamycin attenuated adverse cardiac remodeling and dysfunction in experimental heart failure (HF). However, these studies used rapamycin doses that produced blood drug levels targeted for primary immunosuppression in human transplantation and therefore the immunosuppressive effects may limit clinical translation. Further, the relative or incremental effect of rapamycin combined with standard HF therapies targeting upstream regulators of cardiac remodeling (neurohumoral antagonists) has not been defined. Our objectives were to determine if anti-remodeling effects of rapamycin were preserved at lower doses and whether rapamycin effects were similar or additive to a standard HF therapy (angiotensin receptor blocker (losartan)). Experimental murine HF was produced by transverse aortic constriction (TAC). At three weeks post-TAC, male mice with established HF were treated with placebo, rapamycin at a dose producing immunosuppressive drug levels (target dose), low dose (50% target dose) rapamycin, losartan or rapamycin + losartan for six weeks. Cardiac structure and function (echocardiography, catheterization, pathology, hypertrophic and fibrotic gene expression profiles) were assessed. Downstream mTOR signaling pathways regulating protein synthesis (S6K1 and S6) and autophagy (LC3B-II) were characterized. TAC-HF mice displayed eccentric hypertrophy, systolic dysfunction and pulmonary congestion. These perturbations were attenuated to a similar degree by oral rapamycin doses achieving target (13.3±2.1 ng/dL) or low (6.7±2.5 ng/dL) blood levels. Rapamycin treatment decreased mTOR mediated regulators of protein synthesis and increased mTOR mediated regulators of autophagy. Losartan monotherapy did not attenuate remodeling, whereas

  2. Gap Junction Regulation of Vascular Tone: Implications of Modulatory Intercellular Communication During Gestation

    PubMed Central

    Ampey, Bryan C.; Morschauser, Timothy J.; Lampe, Paul D.

    2017-01-01

    In the vasculature, gap junctions (GJ) play a multifaceted role by serving as direct conduits for cell–cell intercellular communication via the facilitated diffusion of signaling molecules. GJs are essential for the control of gene expression and coordinated vascular development in addition to vascular function. The coupling of endothelial cells to each other, as well as with vascular smooth muscle cells via GJs, plays a relevant role in the control of vasomotor tone, tissue perfusion and arterial blood pressure. The regulation of cell-signaling is paramount to cardiovascular adaptations of pregnancy. Pregnancy requires highly developed cell-to-cell coupling, which is affected partly through the formation of intercellular GJs by Cx43, a gap junction protein, within adjacent cell membranes to help facilitate the increase of uterine blood flow (UBF) in order to ensure adequate perfusion for nutrient and oxygen delivery to the placenta and thus the fetus. One mode of communication that plays a critical role in regulating Cx43 is the release of endothelial-derived vasodilators such as prostacyclin (PGI2) and nitric oxide (NO) and their respective signaling mechanisms involving second messengers (cAMP and cGMP, respectively) that are likely to be important in maintaining UBF. Therefore, the assertion we present in this review is that GJs play an integral if not a central role in maintaining UBF by controlling rises in vasodilators (PGI2 and NO) via cyclic nucleotides. In this review, we discuss: (1) GJ structure and regulation; (2) second messenger regulation of GJ phosphorylation and formation; (3) pregnancy-induced changes in cell-signaling; and (4) the role of uterine arterial endothelial GJs during gestation. These topics integrate the current knowledge of this scientific field with interpretations and hypotheses regarding the vascular effects that are mediated by GJs and their relationship with vasodilatory vascular adaptations required for modulating the

  3. Morphogenesis of 3D vascular networks is regulated by tensile forces

    PubMed Central

    Rosenfeld, Dekel; Landau, Shira; Shandalov, Yulia; Raindel, Noa; Freiman, Alina; Shor, Erez; Blinder, Yaron; Vandenburgh, Herman H.; Mooney, David J.; Levenberg, Shulamit

    2016-01-01

    Understanding the forces controlling vascular network properties and morphology can enhance in vitro tissue vascularization and graft integration prospects. This work assessed the effect of uniaxial cell-induced and externally applied tensile forces on the morphology of vascular networks formed within fibroblast and endothelial cell-embedded 3D polymeric constructs. Force intensity correlated with network quality, as verified by inhibition of force and of angiogenesis-related regulators. Tensile forces during vessel formation resulted in parallel vessel orientation under static stretching and diagonal orientation under cyclic stretching, supported by angiogenic factors secreted in response to each stretch protocol. Implantation of scaffolds bearing network orientations matching those of host abdominal muscle tissue improved graft integration and the mechanical properties of the implantation site, a critical factor in repair of defects in this area. This study demonstrates the regulatory role of forces in angiogenesis and their capacities in vessel structure manipulation, which can be exploited to improve scaffolds for tissue repair. PMID:26951667

  4. Regulation of Blood–Testis Barrier (BTB) Dynamics during Spermatogenesis via the “Yin” and “Yang” Effects of Mammalian Target of Rapamycin Complex 1 (mTORC1) and mTORC2

    PubMed Central

    Mok, Ka Wai; Mruk, Dolores D.; Cheng, C. Yan

    2014-01-01

    In mammalian testes, haploid spermatozoa are formed from diploid spermatogonia during spermatogenesis, which is a complicated cellular process. While these cellular events were reported in the 1960s and 1970s, the underlying molecular mechanism(s) that regulates these events remained unexplored until the past ~10 years. For instance, adhesion proteins were shown to be integrated components at the Sertoli cell–cell interface and/or the Sertoli–spermatid interface in the late 1980s. But only until recently, studies have demonstrated that some of the adhesion proteins serve as the platform for signal transduction that regulates cell adhesion. In this chapter, a brief summary and critical discussion are provided on the latest findings regarding these cell-adhesion proteins in the testis and their relationship to spermatogenesis. Moreover, antagonistic effects of two mammalian target of rapamycin (mTOR) complexes, known as mTORC1 and mTORC2, on cell-adhesion function in the testis are discussed. Finally, a hypothetic model is presented to depict how these two mTOR-signaling complexes having the “yin” and “yang” antagonistic effects on the Sertoli cell tight junction (TJ)-permeability barrier can maintain the blood–testis barrier (BTB) integrity during the epithelial cycle while preleptotene spermatocytes are crossing the BTB. PMID:23317821

  5. Serendipity in splendid isolation: rapamycin.

    PubMed

    Rao, V Koneti

    2016-01-07

    In this issue of Blood, Bride et al report results of the first prospective multi-institutional trial of a long-term single-agent therapy for refractory cytopenias using rapamycin in 30 patients and show remarkable efficacy in children with autoimmune lymphoproliferative syndrome (ALPS).

  6. Fibronectin is an important regulator of flow-induced vascular remodeling

    PubMed Central

    Chiang, Hou-Yu; Korshunov, Vyacheslav A.; Serour, Andrew; Shi, Feng; Sottile, Jane

    2011-01-01

    Objective Fibronectin is an important regulator of cell migration, differentiation, growth, and survival. Our data show that fibronectin also plays an important role in regulating extracellular matrix (ECM) remodeling. Fibronectin circulates in the plasma, and is also deposited into the ECM by a cell dependent process. To determine whether fibronectin affects vascular remodeling in vivo, we asked whether the fibronectin polymerization inhibitor, pUR4, inhibits intima-media thickening, and prevents excess ECM deposition in arteries using a mouse model of vascular remodeling. Methods and Results To induce vascular remodeling, partial ligation of the left external and internal carotid arteries was performed in mice. pUR4 and the control peptide were applied periadventitially in pluronic gel immediately after surgery. Animals were sacrificed 7 or 14 days post surgery. Morphometric analysis demonstrated that the pUR4 fibronectin inhibitor reduced carotid intima (63%), media (27%), and adventitial thickening (40%) compared to the control peptide (III-11C). Treatment with pUR4 also resulted in a dramatic decrease in leukocyte infiltration into the vessel wall (80%), decreased ICAM-1 and VCAM-1 levels, inhibited cell proliferation (60-70%), and reduced fibronectin and collagen I accumulation in the vessel wall. In addition, the fibronectin inhibitor prevented SMC phenotypic modulation, as evidence by the maintenance of smooth muscle (SM) α-actin and SM myosin heavy chain levels in medial cells. Conclusions These data are the first to demonstrate that fibronectin plays an important role in regulating the vascular remodeling response. Collectively, these data suggest a therapeutic benefit of periadventitial pUR4 in reducing pathologic vascular remodeling. PMID:19407246

  7. Arsenite induces endothelial cytotoxicity by down-regulation of vascular endothelial nitric oxide synthase

    SciTech Connect

    Tsou, T.-C. . E-mail: tctsou@nhri.org.tw; Tsai, F.-Y.; Hsieh, Y.-W.; Li, L.-A.; Yeh, S.C; Chang, L.W.

    2005-11-01

    Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular diseases. Recent research has also linked this vascular damage to impairment of endothelial nitric oxide synthase (eNOS) function by arsenic exposure. However, the role of eNOS in regulating the arsenite-induced vascular dysfunction still remains to be clarified. In our present study, we investigated the effect of arsenite on Akt1 and eNOS and its involvement in cytotoxicity of vascular endothelial cells. Our study demonstrated that arsenite decreased the protein levels of both Akt1 and eNOS accompanied with increased levels of ubiquitination of total cell lysates. We found that inhibition of the ubiquitin-proteasome pathway by MG-132 could partially protect Akt1 and eNOS from degradation by arsenite together with a proportional protection from the arsenite-induced cytoxicity. Moreover, up-regulation of eNOS protein expression significantly attenuated the arsenite-induced cytotoxicity and eNOS activity could be significantly inhibited after incubation with arsenite for 24 h in a cell-free system. Our study indicated that endothelial eNOS activity could be attenuated by arsenite via the ubiquitin-proteasome-mediated degradation of Akt1/eNOS as well as via direct inhibition of eNOS activity. Our study also demonstrated that eNOS actually played a protective role in arsenite-induced cytoxicity. These observations supported the hypothesis that the impairment of eNOS function by arsenite is one of the mechanisms leading to vascular changes and diseases.

  8. Regulated Catalysis of Extracellular Nucleotides by Vascular CD39/ENTPD1 Is Required for Liver Regeneration

    PubMed Central

    BELDI, GUIDO; WU, YAN; SUN, XIAOFENG; IMAI, MASATO; ENJYOJI, KEIICHI; CSIZMADIA, EVA; CANDINAS, DANIEL; ERB, LAURIE; ROBSON, SIMON C.

    2010-01-01

    Background & Aims Little is known about how endothelial cells respond to injury, regulate hepatocyte turnover and reconstitute the hepatic vasculature. We aimed to determine the effects of the vascular ectonucleotidase CD39 on sinusoidal endothelial cell responses following partial hepatectomy and to dissect purinergic and growth factor interactions in this model. Methods Parameters of liver injury and regeneration, as well as the kinetics of hepatocellular and sinusoidal endothelial cell proliferation, were assessed following partial hepatectomy in mice that do not express CD39, that do not express ATP/UTP receptor P2Y2, and in controls. The effects of extracellular ATP on vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-6 responses were determined in vivo and in vitro. Phosphorylation of the endothelial VEGF receptor in response to extracellular nucleotides and growth factors was assessed in vitro. Results After partial hepatectomy, expression of the vascular ectonucleotidase CD39 increased on sinusoidal endothelial cells. Targeted disruption of CD39 impaired hepatocellular regeneration, reduced angiogenesis, and increased hepatic injury, resulting in pronounced vascular endothelial apoptosis, and decreased survival. Decreased HGF release by sinusoidal endothelial cells, despite high levels of VEGF, reduced paracrine stimulation of hepatocytes. Failure of VEGF receptor-2/KDR transactivation by extracellular nucleotides on CD39-null endothelial cells was associated with P2Y2 receptor desensitization. Conclusions Regulated phosphohydrolysis of extracellular nucleotides by CD39 coordinates both hepatocyte and endothelial cell proliferation following partial hepatectomy. Lack of CD39 activity is associated with decreased hepatic regeneration and failure of vascular reconstitution. PMID:18804472

  9. Effects of rapamycin and TOR on aging and memory: implications for Alzheimer's disease.

    PubMed

    Santos, Renato X; Correia, Sónia C; Cardoso, Susana; Carvalho, Cristina; Santos, Maria S; Moreira, Paula I

    2011-06-01

    Rapamycin is a macrolide immunosuppressant drug, originally used as an anti-fungal agent, which is widely used in transplantation medicine to prevent organ rejection. Target of rapamycin (TOR) is an evolutionarily conserved serine/threonine kinase with pleiotropic cellular functions, regulating processes such as growth and metabolism, cell survival, transcription and autophagy. TOR intervenes in two distinct enzymatic complexes with different functions, a rapamycin-sensitive complex TORC1 and a rapamycin-insensitive complex TORC2. Rapamycin has an inhibitory effect on TORC1 activity and it has been suggested to increase life span, an effect correlated with decreased protein biosynthesis and autophagy activation. In the CNS, rapamycin shows beneficial effects in neuronal survival and plasticity, thus contributing to memory improvement. In this review, evidence implying rapamycin and TOR in aging/life span extension and memory improvement will be discussed. Recent findings about the effects of rapamycin on Alzheimer's disease-associated neuropathology will be also discussed. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  10. Ryanodine receptors, calcium signaling and regulation of vascular tone in the cerebral parenchymal microcirculation

    PubMed Central

    Dabertrand, Fabrice; Nelson, Mark T.; Brayden, Joseph E.

    2012-01-01

    The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of cerebral blood flow, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of parenchymal arterioles. There are, moreover, notable functional differences between pial arteries and parenchymal arterioles. For example, in pial vascular smooth muscle cells (VSMCs), local calcium release events (“calcium sparks”) through ryanodine receptor (RyR) channels in sarcoplasmic reticulum membrane activate large conductance, calcium-sensitive potassium (BK) channels to modulate vascular diameter. In contrast, VSMCs in parenchymal arterioles express functional RyR and BK channels, but under physiological conditions these channels do not oppose pressure-induced vasoconstriction. Here we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of cerebral blood flow. PMID:23216877

  11. Neuropeptide Y regulates a vascular gateway for hematopoietic stem and progenitor cells.

    PubMed

    Singh, Pratibha; Hoggatt, Jonathan; Kamocka, Malgorzata M; Mohammad, Khalid S; Saunders, Mary R; Li, Hongge; Speth, Jennifer; Carlesso, Nadia; Guise, Theresa A; Pelus, Louis M

    2017-11-13

    Endothelial cells (ECs) are components of the hematopoietic microenvironment and regulate hematopoietic stem and progenitor cell (HSPC) homeostasis. Cytokine treatments that cause HSPC trafficking to peripheral blood are associated with an increase in dipeptidylpeptidase 4/CD26 (DPP4/CD26), an enzyme that truncates the neurotransmitter neuropeptide Y (NPY). Here, we show that enzymatically altered NPY signaling in ECs caused reduced VE-cadherin and CD31 expression along EC junctions, resulting in increased vascular permeability and HSPC egress. Moreover, selective NPY2 and NPY5 receptor antagonists restored vascular integrity and limited HSPC mobilization, demonstrating that the enzymatically controlled vascular gateway specifically opens by cleavage of NPY by CD26 signaling via NPY2 and NPY5 receptors. Mice lacking CD26 or NPY exhibited impaired HSPC trafficking that was restored by treatment with truncated NPY. Thus, our results point to ECs as gatekeepers of HSPC trafficking and identify a CD26-mediated NPY axis that has potential as a pharmacologic target to regulate hematopoietic trafficking in homeostatic and stress conditions.

  12. Evolution of plant conducting cells: perspectives from key regulators of vascular cell differentiation.

    PubMed

    Ohtani, Misato; Akiyoshi, Nobuhiro; Takenaka, Yuto; Sano, Ryosuke; Demura, Taku

    2017-01-01

    One crucial problem that plants faced during their evolution, particularly during the transition to growth on land, was how to transport water, nutrients, metabolites, and small signaling molecules within a large, multicellular body. As a solution to this problem, land plants developed specific tissues for conducting molecules, called water-conducting cells (WCCs) and food-conducting cells (FCCs). The well-developed WCCs and FCCs in extant plants are the tracheary elements and sieve elements, respectively, which are found in vascular plants. Recent molecular genetic studies revealed that transcriptional networks regulate the differentiation of tracheary and sieve elements, and that the networks governing WCC differentiation are largely conserved among land plant species. In this review, we discuss the molecular evolution of plant conducting cells. By focusing on the evolution of the key transcription factors that regulate vascular cell differentiation, the NAC transcription factor VASCULAR-RELATED NAC-DOMAIN for WCCs and the MYB-coiled-coil (CC)-type transcription factor ALTERED PHLOEM DEVELOPMENT for sieve elements, we describe how land plants evolved molecular systems to produce the specialized cells that function as WCCs and FCCs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. Vascular smooth muscle Emilin-1 is a regulator of arteriolar myogenic response and blood pressure.

    PubMed

    Litteri, Gaia; Carnevale, Daniela; D'Urso, Alessandra; Cifelli, Giuseppe; Braghetta, Paola; Damato, Antonio; Bizzotto, Dario; Landolfi, Alessandro; Ros, Francesco Da; Sabatelli, Patrizia; Facchinello, Nicola; Maffei, Angelo; Volpin, Dino; Colombatti, Alfonso; Bressan, Giorgio M; Lembo, Giuseppe

    2012-09-01

    Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-β processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1(-/-) background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1(-/-) mice. We also explored the effect of treatment with anti-TGF-β antibodies on the hypertensive phenotype of Emilin1(-/-) mice, finding that neutralization of TGF-β in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-β activity. Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-β.

  14. Rapamycin additively extends lifespan in short- and long-lived lines of the nematode Caenorhabditis remanei.

    PubMed

    Lind, Martin I; Chen, Hwei-Yen; Cortazar-Chinarro, Maria; Maklakov, Alexei A

    2017-04-01

    Despite tremendous progress in finding genes that, when manipulated, affects lifespan, little is known about the genetics underlying natural variation in lifespan. While segregating genetic variants for lifespan has been notoriously difficult to find in genome-wide association studies (GWAS), a complementary approach is to manipulate key genetic pathways in lines that differ in lifespan. If these candidate pathways are down regulated in long-lived lines, these lines can be predicted to respond less to pharmaceutical down-regulation of these pathways than short-lived lines. Experimental studies have identified the nutrient-sensing pathway TOR as a key regulator of lifespan in model organisms, and this pathway can effectively be down regulated using the drug rapamycin, which extends lifespan in all tested species. We expose short- and long-lived lines of the nematode Caenorhabditis remanei to rapamycin, and investigate if long-lived lines, which are hypothesized to already have down-regulated TOR signaling, respond less to rapamycin. We found no interaction between line and rapamycin treatment, since rapamycin extended lifespan independent of the intrinsic lifespan of the lines. This shows that rapamycin is equally effective on long and short-lived lines, and suggests that the evolution of long life may involve more factors that down-regulation of TOR. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. VEGF and endothelium-derived retinoic acid regulate lung vascular and alveolar development

    PubMed Central

    Yun, Eun Jun; Lorizio, Walter; Seedorf, Gregory; Abman, Steven H.

    2015-01-01

    Prevention or treatment of lung diseases caused by the failure to form, or destruction of, existing alveoli, as observed in infants with bronchopulmonary dysplasia and adults with emphysema, requires understanding of the molecular mechanisms of alveolar development. In addition to its critical role in gas exchange, the pulmonary circulation also contributes to alveolar morphogenesis and maintenance by the production of paracrine factors, termed “angiocrines,” that impact the development of surrounding tissue. To identify lung angiocrines that contribute to alveolar formation, we disrupted pulmonary vascular development by conditional inactivation of the Vegf-A gene during alveologenesis. This resulted in decreased pulmonary capillary and alveolar development and altered lung elastin and retinoic acid (RA) expression. We determined that RA is produced by pulmonary endothelial cells and regulates pulmonary angiogenesis and elastin synthesis by induction of VEGF-A and fibroblast growth factor (FGF)-18, respectively. Inhibition of RA synthesis in newborn mice decreased FGF-18 and elastin expression and impaired alveolarization. Treatment with RA and vitamin A partially reversed the impaired vascular and alveolar development induced by VEGF inhibition. Thus we identified RA as a lung angiocrine that regulates alveolarization through autocrine regulation of endothelial development and paracrine regulation of elastin synthesis via induction of FGF-18 in mesenchymal cells. PMID:26566904

  16. Roles of Mitogen-Activating Protein Kinase Kinase Kinase Kinase-3 (MAP4K3) in Preterm Skeletal Muscle Satellite Cell Myogenesis and Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Regulation

    PubMed Central

    Guo, Chu-yi; Yu, Mu-xue; Dai, Jie-min; Pan, Si-nian; Lu, Zhen-tong; Qiu, Xiao-shan; Zhuang, Si-qi

    2017-01-01

    Background Preterm skeletal muscle genesis is a paradigm for myogenesis. The role of mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3) in preterm skeletal muscle satellite cells myogenesis or its relationship to mammalian target of rapamycin complex 1 (mTORC1) activity have not been previously elaborated. Material/Methods Small interfering RNA (siRNA) interference technology was used to inhibit MAP4K3 expression. Leucine stimulation experiments were performed following MAP4K3-siRNA interference. The differentiation of primary preterm skeletal muscle satellite cells was observed after siRNA-MAP4K3 interference. Western blot analysis was used to determine the expression of MAP4K3, MyHC, MyoD, myogenin, p-mTOR, and p-S6K1. The immunofluorescence fusion index of MyHC and myogenin were detected. MAP4K3 effects on preterm rat satellite cells differentiation and its relationship to mTORC1 activity are reported. Results MAP4K3 siRNA knockdown inhibited myotube formation and both MyoD and myogenin expression in primary preterm rat skeletal muscle satellite cells, but MAP4K3 siRNA had no effect on the activity of mTORC1. In primary preterm rat skeletal muscle satellite cells, MAP4K3 knockdown resulted in significantly weaker, but not entirely blunted, leucine-induced mTORC1 signaling. Conclusions MAP4K3 positively regulates preterm skeletal muscle satellite cell myogenesis, but may not regulate mTORC1 activity. MAP4K3 may play a role in mTORC1 full activation in response to leucine. PMID:28731988

  17. The checkpoint kinase TOR (target of rapamycin) regulates expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) and modulates chloroplast ribosomal RNA synthesis in a unicellular red alga.

    PubMed

    Imamura, Sousuke; Nomura, Yuhta; Takemura, Tokiaki; Pancha, Imran; Taki, Keiko; Toguchi, Kazuki; Tozawa, Yuzuru; Tanaka, Kan

    2018-02-14

    Chloroplasts are plant organelles that carry out oxygenic photosynthesis. Chloroplast biogenesis depends upon chloroplast ribosomes and their translational activity. However, regulation of chloroplast ribosome biogenesis remains an important unanswered question. In this study, we found that inhibition of target of rapamycin (TOR), a general eukaryotic checkpoint kinase, results in a decline in chloroplast ribosomal RNA (rRNA) transcription in the unicellular red alga, Cyanidioschyzon merolae. Upon TOR inhibition, transcriptomics and other analyses revealed increased expression of a nuclear-encoded chloroplast RelA-SpoT homolog (RSH) gene (CmRSH4b), which encodes a homolog of the guanosine 3'-diphosphate 5'-diphosphate (ppGpp) synthetases that modulate rRNA synthesis in bacteria. Using an Escherichia coli mutant lacking ppGpp, CmRSH4b was demonstrated to have ppGpp synthetase activity. Expression analysis of a GFP-fused protein indicated that CmRSH4b localizes to the chloroplast, and overexpression of the CmRSH4b gene resulted in a decrease of chloroplast rRNA synthesis concomitant with growth inhibition and reduction of chloroplast size. Biochemical analyses using C. merolae cell lysates or purified recombinant proteins revealed that ppGpp inhibits bacteria-type RNA polymerase-dependent chloroplast rRNA synthesis as well as a chloroplast guanylate kinase. These results suggest that CmRSH4b-dependent ppGpp synthesis in chloroplasts is an important regulator of chloroplast rRNA transcription. Nuclear and mitochondrial rRNA transcription were both reduced by TOR inhibition, suggesting that the biogeneses of the three independent ribosome systems are interconnected by TOR in plant cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  18. Regulation of Vascular Growth in the Chorioallantoic Membrane of Japanese Quail Eggs

    NASA Technical Reports Server (NTRS)

    Montague, Idoreyin P.

    2004-01-01

    The Microgravity Research Program is part of NASA's Office of Biological and Physical Research (OBPR). The mission of the Microgravity Fluid Physics research program is to facilitate and conduct the best possible fluid physics research using the space environment and make this knowledge available to the scientific community and the public at large. During the summer of 2004, I worked in this division with Dr. Patricia Parsons-Wingerter. Dr. Parsons was working on several projects that used the chorioallantoic membrane (CAM) of Japanese quail eggs. The CAM develops in the eggs of birds and reptiles and is a very vascular fetal membrane composed of the fused chorion and adjacent wall of the allantois. The CAM is formed on day 4 of incubation and its primary job is to mediate gas exchanges with the extra embryonic environment. The CAM of our Japanese quail eggs is easily identifiable to us because it is transparent and it sits on top of the yolk with the embryo in the center. The CAM is of interest because of its many applications in the field of medicine as it relates to vascular remodeling and angiogenesis. Angiogenesis is simply the growth or formation of new blood vessels and anti-angiogenesis is the inhibition of said vessels. Angiogenesis occurs naturally in a healthy body for healing wounds and for restoring blood flow to tissues after injury and in females during the monthly reproductive cycle. In many serious diseases, like several types of cancer and those that affect the heart and cardiovascular system, the body loses control over angiogenesis. These diseases, which are dependent on angiogenesis, result when new blood vessels either grow excessively or insufficiently. The chorioallantoic membrane of our Japanese quail eggs gives a good model of angiogenesis. We used angiogenic regulators to inhibit or stimulate vascular growth in the CAM in a healthy manner and they induced distinct vascular patterns in vivo. Certain dominant regulators can be recognized by

  19. WNT5A-JNK regulation of vascular insulin resistance in human obesity.

    PubMed

    Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

    2016-12-01

    Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m 2 ) and five metabolically normal non-obese (BMI 26±2 kg/m 2 ) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. © The Author(s) 2016.

  20. WNT5A-JNK regulation of vascular insulin resistance in human obesity

    PubMed Central

    Farb, Melissa G; Karki, Shakun; Park, Song-Young; Saggese, Samantha M; Carmine, Brian; Hess, Donald T; Apovian, Caroline; Fetterman, Jessica L; Bretón-Romero, Rosa; Hamburg, Naomi M; Fuster, José J; Zuriaga, María A; Walsh, Kenneth; Gokce, Noyan

    2017-01-01

    Obesity is associated with the development of vascular insulin resistance; however, pathophysiological mechanisms are poorly understood. We sought to investigate the role of WNT5A-JNK in the regulation of insulin-mediated vasodilator responses in human adipose tissue arterioles prone to endothelial dysfunction. In 43 severely obese (BMI 44±11 kg/m2) and five metabolically normal non-obese (BMI 26±2 kg/m2) subjects, we isolated arterioles from subcutaneous and visceral fat during planned surgeries. Using videomicroscopy, we examined insulin-mediated, endothelium-dependent vasodilator responses and characterized adipose tissue gene and protein expression using real-time polymerase chain reaction and Western blot analyses. Immunofluorescence was used to quantify endothelial nitric oxide synthase (eNOS) phosphorylation. Insulin-mediated vasodilation was markedly impaired in visceral compared to subcutaneous vessels from obese subjects (p<0.001), but preserved in non-obese individuals. Visceral adiposity was associated with increased JNK activation and elevated expression of WNT5A and its non-canonical receptors, which correlated negatively with insulin signaling. Pharmacological JNK antagonism with SP600125 markedly improved insulin-mediated vasodilation by sixfold (p<0.001), while endothelial cells exposed to recombinant WNT5A developed insulin resistance and impaired eNOS phosphorylation (p<0.05). We observed profound vascular insulin resistance in the visceral adipose tissue arterioles of obese subjects that was associated with up-regulated WNT5A-JNK signaling and impaired endothelial eNOS activation. Pharmacological JNK antagonism markedly improved vascular endothelial function, and may represent a potential therapeutic target in obesity-related vascular disease. PMID:27688298

  1. Rapamycin Effectively Impedes Melamine-Induced Impairments of Cognition and Synaptic Plasticity in Wistar Rats.

    PubMed

    Fu, Jingxuan; Wang, Hui; Gao, Jing; Yu, Mei; Wang, Rubin; Yang, Zhuo; Zhang, Tao

    2017-03-01

    Our previous investigation demonstrated that autophagy significantly reduced melamine-induced cell death in PC12 cells via inhibiting the excessive generation of ROS. In the present study, we further examine if rapamycin, used as an autophagy activator, can play a significant role in protecting neurons and alleviating the impairment of spatial cognition and hippocampal synaptic plasticity in melamine-treated rats. Male Wistar rats were divided into three groups: control, melamine-treated, and melamine-treated + rapamycin. The animal model was established by administering melamine at a dose of 300 mg/kg/day for 4 weeks. Rapamycin was intraperitoneally given at a dose of 1 mg/kg/day for 28 consecutive days. The Morris water maze test showed that spatial learning and reversal learning in melamine-treated rats were considerably damaged, whereas rapamycin significantly impeded the cognitive function impairment. Rapamycin efficiently alleviated the melamine-induced impairments of both long-term potentiation (LTP) and depotentiation, which were damaged in melamine rats. Rapamycin further increased the expression level of autophagy markers, which were significantly enhanced in melamine rats. Moreover, rapamycin noticeably decreased the reactive oxygen species level, while the superoxide dismutase activity was remarkably increased by rapamycin in melamine rats. Malondialdehyde assay exhibited that rapamycin prominently reduced the malondialdehyde (MDA) level of hippocampal neurons in melamine-treated rats. In addition, rapamycin significantly decreased the caspase-3 activity, which was elevated by melamine. Consequently, our results suggest that regulating autophagy may become a new targeted therapy to relieve the damage induced by melamine.

  2. The E3 ubiquitin ligase protein associated with Myc (Pam) regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) signaling in vivo through N- and C-terminal domains.

    PubMed

    Han, Sangyeul; Kim, Sun; Bahl, Samira; Li, Lin; Burande, Clara F; Smith, Nicole; James, Marianne; Beauchamp, Roberta L; Bhide, Pradeep; DiAntonio, Aaron; Ramesh, Vijaya

    2012-08-31

    Pam and its homologs (the PHR protein family) are large E3 ubiquitin ligases that function to regulate synapse formation and growth in mammals, zebrafish, Drosophila, and Caenorhabditis elegans. Phr1-deficient mouse models (Phr1(Δ8,9) and Phr1(Magellan), with deletions in the N-terminal putative guanine exchange factor region and the C-terminal ubiquitin ligase region, respectively) exhibit axon guidance/outgrowth defects and striking defects of major axon tracts in the CNS. Our earlier studies identified Pam to be associated with tuberous sclerosis complex (TSC) proteins, ubiquitinating TSC2 and regulating mammalian/mechanistic target of rapamycin (mTOR) signaling. Here, we examine the potential involvement of the TSC/mTOR complex 1(mTORC1) signaling pathway in Phr1-deficient mouse models. We observed attenuation of mTORC1 signaling in the brains of both Phr1(Δ8,9) and Phr1(Magellan) mouse models. Our results establish that Pam regulates TSC/mTOR signaling in vitro and in vivo through two distinct domains. To further address whether Pam regulates mTORC1 through two functionally independent domains, we undertook heterozygous mutant crossing between Phr1(Δ8,9) and Phr1(Magellan) mice to generate a compound heterozygous model to determine whether these two domains can complement each other. mTORC1 signaling was not attenuated in the brains of double mutants (Phr1(Δ8,9/Mag)), confirming that Pam displays dual regulation of the mTORC1 pathway through two functional domains. Our results also suggest that although dysregulation of mTORC1 signaling may be responsible for the corpus callosum defects, other neurodevelopmental defects observed with Phr1 deficiency are independent of mTORC1 signaling. The ubiquitin ligase complex containing Pam-Fbxo45 likely targets additional synaptic and axonal proteins, which may explain the overlapping neurodevelopmental defects observed in Phr1 and Fbxo45 deficiency.

  3. Molecular Mechanisms Regulating the Vascular Prostacyclin Pathways and Their Adaptation during Pregnancy and in the Newborn

    PubMed Central

    Majed, Batoule H.

    2012-01-01

    Prostacyclin (PGI2) is a member of the prostanoid group of eicosanoids that regulate homeostasis, hemostasis, smooth muscle function and inflammation. Prostanoids are derived from arachidonic acid by the sequential actions of phospholipase A2, cyclooxygenase (COX), and specific prostaglandin (PG) synthases. There are two major COX enzymes, COX1 and COX2, that differ in structure, tissue distribution, subcellular localization, and function. COX1 is largely constitutively expressed, whereas COX2 is induced at sites of inflammation and vascular injury. PGI2 is produced by endothelial cells and influences many cardiovascular processes. PGI2 acts mainly on the prostacyclin (IP) receptor, but because of receptor homology, PGI2 analogs such as iloprost may act on other prostanoid receptors with variable affinities. PGI2/IP interaction stimulates G protein-coupled increase in cAMP and protein kinase A, resulting in decreased [Ca2+]i, and could also cause inhibition of Rho kinase, leading to vascular smooth muscle relaxation. In addition, PGI2 intracrine signaling may target nuclear peroxisome proliferator-activated receptors and regulate gene transcription. PGI2 counteracts the vasoconstrictor and platelet aggregation effects of thromboxane A2 (TXA2), and both prostanoids create an important balance in cardiovascular homeostasis. The PGI2/TXA2 balance is particularly critical in the regulation of maternal and fetal vascular function during pregnancy and in the newborn. A decrease in PGI2/TXA2 ratio in the maternal, fetal, and neonatal circulation may contribute to preeclampsia, intrauterine growth restriction, and persistent pulmonary hypertension of the newborn (PPHN), respectively. On the other hand, increased PGI2 activity may contribute to patent ductus arteriosus (PDA) and intraventricular hemorrhage in premature newborns. These observations have raised interest in the use of COX inhibitors and PGI2 analogs in the management of pregnancy-associated and neonatal

  4. Progeria, rapamycin and normal aging: recent breakthrough.

    PubMed

    Blagosklonny, Mikhail V

    2011-07-01

    A recent discovery that rapamycin suppresses a pro-senescent phenotype in progeric cells not only suggests a non-toxic therapy for progeria but also implies its similarity with normal aging. For one, rapamycin is also known to suppress aging of regular human cells. Here I discuss four potential scenarios, comparing progeria with both normal and accelerated aging. This reveals further indications of rapamycin both for accelerated aging in obese and for progeria.

  5. Progeria, rapamycin and normal aging: recent breakthrough

    PubMed Central

    Blagosklonny, Mikhail V.

    2011-01-01

    A recent discovery that rapamycin suppresses a pro-senescent phenotype in progeric cells not only suggests a non-toxic therapy for progeria but also implies its similarity with normal aging. For one, rapamycin is also known to suppress aging of regular human cells. Here I discuss four potential scenarios, comparing progeria with both normal and accelerated aging. This reveals further indications of rapamycin both for accelerated aging in obese and for progeria. PMID:21743107

  6. KCl cotransport regulation and protein kinase G in cultured vascular smooth muscle cells.

    PubMed

    Adragna, N C; Zhang, J; Di Fulvio, M; Lincoln, T M; Lauf, P K

    2002-05-15

    K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.

  7. Loss of the alpha7 integrin promotes extracellular signal-regulated kinase activation and altered vascular remodeling.

    PubMed

    Welser, Jennifer V; Lange, Naomi; Singer, Cherie A; Elorza, Margaret; Scowen, Paul; Keef, Kathleen D; Gerthoffer, William T; Burkin, Dean J

    2007-09-28

    Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.

  8. Biodegradable Cable-Tie Rapamycin-eluting Stents.

    PubMed

    Lee, Cheng-Hung; Hsieh, Ming-Jer; Chang, Shang-Hung; Chiang, Chang-Lin; Fan, Ching-Lung; Liu, Shih-Jung; Chen, Wei-Jan; Wang, Chao-Jan; Hsu, Ming-Yi; Hung, Kuo-Chun; Chou, Chung-Chuan; Chang, Po-Cheng

    2017-03-08

    "Cable-tie" type biodegradable stents with drug-eluting nanofiber were developed to treat rabbit denuded arteries in this study. Biodegradable stents were fabricated using poly-L-lactide film following being cut and rolled into a cable-tie type stent. Additionally, drug-eluting biodegradable nanofiber tubes were electrospun from a solution containing poly (lactic-co-glycolic acid), rapamycin, and hexafluoroisopropanol, and then mounted onto the stents. The fabricated rapamycin-eluting cable-tie stents exhibited excellent mechanical properties on evaluation of compression test and collapse pressure, and less than 8% weight loss following being immersed in phosphate-buffered saline for 16 weeks. Furthermore, the biodegradable stents delivered high rapamycin concentrations for over 4 weeks and achieved substantial reductions in intimal hyperplasia associated with elevated heme oxygenase-1 and calponin level on the denuded rabbit arteries during 6 months of follow-up. The drug-eluting cable-tie type stents developed in this study might have high potential impacts for the local drug delivery to treat various vascular diseases.

  9. The mechanistic Target of Rapamycin: The grand conducTOR of metabolism and aging

    PubMed Central

    Kennedy, Brian K.; Lamming, Dudley W.

    2016-01-01

    Summary Since the discovery that rapamycin, a small molecule inhibitor of the protein kinase mTOR (mechanistic Target Of Rapamycin), can extend the lifespan of model organisms including mice, interest in understanding the physiological role and molecular targets of this pathway has surged. While mTOR was already well known as a regulator of growth and protein translation, it is now clear that mTOR functions as a central coordinator of organismal metabolism in response to both environmental and hormonal signals. This review discusses recent developments in our understanding of how mTOR signaling is regulated by nutrients and the role of the mTOR signaling pathway in key metabolic tissues. Finally, we discuss the molecular basis for the negative metabolic side effects associated with rapamycin treatment, which may serve as barriers to the adoption of rapamycin or similar compounds for the treatment of diseases of aging and metabolism. PMID:27304501

  10. Increase of microRNA-210, Decrease of Raptor Gene Expression and Alteration of Mammalian Target of Rapamycin Regulated Proteins following Mithramycin Treatment of Human Erythroid Cells

    PubMed Central

    Bianchi, Nicoletta; Finotti, Alessia; Ferracin, Manuela; Lampronti, Ilaria; Zuccato, Cristina; Breveglieri, Giulia; Brognara, Eleonora; Fabbri, Enrica; Borgatti, Monica; Negrini, Massimo; Gambari, Roberto

    2015-01-01

    Expression and regulation of microRNAs is an emerging issue in erythroid differentiation and globin gene expression in hemoglobin disorders. In the first part of this study microarray analysis was performed both in mithramycin-induced K562 cells and erythroid precursors from healthy subjects or β-thalassemia patients producing low or high levels of fetal hemoglobin. We demonstrated that: (a) microRNA-210 expression is higher in erythroid precursors from β-thalassemia patients with high production of fetal hemoglobin; (b) microRNA-210 increases as a consequence of mithramycin treatment of K562 cells and human erythroid progenitors both from healthy and β-thalassemia subjects; (c) this increase is associated with erythroid induction and elevated expression of γ-globin genes; (d) an anti-microRNA against microRNA-210 interferes with the mithramycin-induced changes of gene expression. In the second part of the study we have obtained convergent evidences suggesting raptor mRNA as a putative target of microRNA-210. Indeed, microRNA-210 binding sites of its 3’-UTR region were involved in expression and are targets of microRNA-210-mediated modulation in a luciferase reporter assays. Furthermore, (i) raptor mRNA and protein are down-regulated upon mithramycin-induction both in K562 cells and erythroid progenitors from healthy and β-thalassemia subjects. In addition, (ii) administration of anti-microRNA-210 to K562 cells decreased endogenous microRNA-210 and increased raptor mRNA and protein expression. Finally, (iii) treatment of K562 cells with premicroRNA-210 led to a decrease of raptor mRNA and protein. In conclusion, microRNA-210 and raptor are involved in mithramycin-mediated erythroid differentiation of K562 cells and participate to the fine-tuning and control of γ-globin gene expression in erythroid precursor cells. PMID:25849663

  11. CD44 regulates vascular endothelial barrier integrity via a PECAM-1 dependent mechanism.

    PubMed

    Flynn, Kelly M; Michaud, Michael; Canosa, Sandra; Madri, Joseph A

    2013-07-01

    Vascular integrity is a critical parameter in normal growth and development. Loss of appropriate vascular barrier function is present in various immune- and injury-mediated pathological conditions. CD44 is an adhesion molecule expressed by multiple cell types, including endothelial cells (EC). The goal of the present study was to examine how loss of CD44 affected vascular permeability. Using C57BL/6 WT and CD44-KO mice, we found no significant permeability to Evan's Blue in either strain at baseline. However, there was significantly increased histamine-induced permeability in CD44-deficient mice compared to WT counterparts. Similar results were observed in vitro, where CD44-deficient endothelial monolayers were also impermeable to 40kD-FITC dextran in the absence of vasoactive challenge, but exhibited enhanced and prolonged permeability following histamine. However, CD44-KO monolayers have reduced baseline barrier strength by electrical resistance, which correlated with increased permeability, at baseline, to smaller molecular weight 4-kD FITC-dextran, suggesting weakly formed endothelial junctions. The CD44-KO EC displayed several characteristics consistent with impaired barrier function/dysfunctional EC junctions, including differential expression, phosphorylation, and localization of endothelial junction proteins, increased matrix metalloprotease expression, and altered cellular morphology. Reduced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression by CD44-KO EC in vivo and in vitro was also observed. Reconstitution of murine CD44 or PECAM-1 restored these defects to near WT status, suggesting CD44 regulates vascular permeability and integrity through a PECAM-1 dependent mechanism.

  12. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling

    PubMed Central

    Fernandez-Alonso, R; Martin-Lopez, M; Gonzalez-Cano, L; Garcia, S; Castrillo, F; Diez-Prieto, I; Fernandez-Corona, A; Lorenzo-Marcos, M E; Li, X; Claesson-Welsh, L; Marques, M M; Marin, M C

    2015-01-01

    Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity. PMID:25571973

  13. p73 is required for endothelial cell differentiation, migration and the formation of vascular networks regulating VEGF and TGFβ signaling.

    PubMed

    Fernandez-Alonso, R; Martin-Lopez, M; Gonzalez-Cano, L; Garcia, S; Castrillo, F; Diez-Prieto, I; Fernandez-Corona, A; Lorenzo-Marcos, M E; Li, X; Claesson-Welsh, L; Marques, M M; Marin, M C

    2015-08-01

    Vasculogenesis, the establishment of the vascular plexus and angiogenesis, branching of new vessels from the preexisting vasculature, involves coordinated endothelial differentiation, proliferation and migration. Disturbances in these coordinated processes may accompany diseases such as cancer. We hypothesized that the p53 family member p73, which regulates cell differentiation in several contexts, may be important in vascular development. We demonstrate that p73 deficiency perturbed vascular development in the mouse retina, decreasing vascular branching, density and stability. Furthermore, p73 deficiency could affect non endothelial cells (ECs) resulting in reduced in vivo proangiogenic milieu. Moreover, p73 functional inhibition, as well as p73 deficiency, hindered vessel sprouting, tubulogenesis and the assembly of vascular structures in mouse embryonic stem cell and induced pluripotent stem cell cultures. Therefore, p73 is necessary for EC biology and vasculogenesis and, in particular, that DNp73 regulates EC migration and tube formation capacity by regulation of expression of pro-angiogenic factors such as transforming growth factor-β and vascular endothelial growth factors. DNp73 expression is upregulated in the tumor environment, resulting in enhanced angiogenic potential of B16-F10 melanoma cells. Our results demonstrate, by the first time, that differential p73-isoform regulation is necessary for physiological vasculogenesis and angiogenesis and DNp73 overexpression becomes a positive advantage for tumor progression due to its pro-angiogenic capacity.

  14. Ryanodine receptors, calcium signaling, and regulation of vascular tone in the cerebral parenchymal microcirculation.

    PubMed

    Dabertrand, Fabrice; Nelson, Mark T; Brayden, Joseph E

    2013-05-01

    The cerebral blood supply is delivered by a surface network of pial arteries and arterioles from which arise (parenchymal) arterioles that penetrate into the cortex and terminate in a rich capillary bed. The critical regulation of CBF, locally and globally, requires precise vasomotor regulation of the intracerebral microvasculature. This vascular region is anatomically unique as illustrated by the presence of astrocytic processes that envelope almost the entire basolateral surface of PAs. There are, moreover, notable functional differences between pial arteries and PAs. For example, in pial VSMCs, local calcium release events ("calcium sparks") through ryanodine receptor (RyR) channels in SR membrane activate large conductance, calcium-sensitive potassium channels to modulate vascular diameter. In contrast, VSMCs in PAs express functional RyR and BK channels, but under physiological conditions, these channels do not oppose pressure-induced vasoconstriction. Here, we summarize the roles of ryanodine receptors in the parenchymal microvasculature under physiologic and pathologic conditions, and discuss their importance in the control of CBF. © 2012 John Wiley & Sons Ltd.

  15. Osteopontin regulates macrophage activation and osteoclast formation in hypertensive patients with vascular calcification

    PubMed Central

    Ge, Qian; Ruan, Cheng-Chao; Ma, Yu; Tang, Xiao-Feng; Wu, Qi-Hong; Wang, Ji-Guang; Zhu, Ding-Liang; Gao, Ping-Jin

    2017-01-01

    Vascular calcification (VC) is a highly regulated ectopic mineral deposition process involving immune cell infiltration in the vasculatures, which has been recognized to be promoted by hypertension. The matricellular glycoprotein osteopontin (OPN) is strongly induced in myeloid cells as a potential inflammatory mediator of vascular injury. This study aims to examine whether OPN is involved in the regulation of macrophage activation and osteoclast formation in hypertensive subjects with VC. We firstly found an increased proportion of CD11c+CD163- pro-inflammatory peripheral monocytes in hypertensive subjects with VC compared to those without VC by flow cytometric analysis. Primary cultured macrophages from hypertensive subjects with VC also showed altered expression profile of inflammatory factors and higher serum OPN level. Exogenous OPN promoted the differentiation of peripheral monocytes into an alternative, anti-inflammatory phenotype, and inhibited macrophage-to-osteoclast differentiation from these VC patients. In addition, calcified vessels showed increased osteoclasts accumulation accompanied with decreased macrophages infiltration in the of hypertensive subjects. Taken together, these demonstrated that OPN exerts an important role in the monocytes/macrophage phenotypic differentiation from hypertensive patients with VC, which includes reducing inflammatory factor expression and attenuating osteoclast formation. PMID:28091516

  16. Bim Regulates Alloimmune-Mediated Vascular Injury Through Effects on T Cell Activation and Death

    PubMed Central

    von Rossum, Anna; Enns, Winnie; Shi, Yu P.; MacEwan, Grace E.; Malekesmaeli, Mehrnoush; Brinkman, Ryan; Choy, Jonathan C.

    2014-01-01

    Objective Bim is a pro-apoptotic Bcl-2 protein known to down-regulate immune responses and to also be required for antigen-induced T cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T cell responses in a model of vascular rejection. Approach and Results Bim was required for proliferation of CD4 and CD8 T cells, and for IL-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T cell activation whereas a complete elimination of Bim was required to prevent CD4 T cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim+/−, but not Bim−/−, graft recipients. T cell proliferation in response to allograft arteries was significantly reduced in both Bim+/− and Bim−/− mice, but cell death was attenuated only in Bim−/− animals. Conclusions Bim controls both T cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries. PMID:24700126

  17. The Effects of Protein Regulators on the Vascular Remodeling of Japanese Quail Chorioallantoic Membrane

    NASA Technical Reports Server (NTRS)

    Deshpande, Arati

    2004-01-01

    Contributing to NASA s mission, the Microgravity Fluid Physics research program conducts experiments to promote space exploration and improvement of processes and products on Earth. One of the projects through this program deals with the affect of regulators on vascular remodeling and angiogenesis. This project is being led by Dr. Patricia Parsons-Wingerter. To perform the experiments, protein regulators are tested on the chorioallantoic membrane (CAM) of the Japanese quail embryos. The different types of regulators used can be broken down into two major groups of stimulators, and inhibitors. Stimulators increase the rate of blood vessel growth and inhibitors decrease of blood vessel growth. The specified regulator proteins include thrombospondin 1 (TSP-1) and a novel vessel tortuosity factor (TF), these are just the ones used in this specific experiment; other various protein regulators can also be used. The novel vessel tortuosity factor (TF) is a special kind of stimulator because it stimulates vessel tortuosity and curvature, rather than actual blood vessel growth. These regulators are being tested on Japanese quail embryos. The Japanese quail embryos naturally form a chorioallantoic membrane (CAM) from which blood flow, vascular remodeling, and angiogenesis can be observed. Chorioallantoic membranes are also easier to use because they are two dimensional when mounted onto a slide for examination. The analysis of the affect of the regulators on the CAM can be studied through PIVPROC; the program is used to analyze the altered blood flow in response to application of TF. Regulators are being thoroughly studied because cardiovascular alterations are the second highest, NASA-defined, risk categories in human space exploration. This research done on the quail is extending to even more projects that will be done on lab animals such as mice and also in human clinical studies like the diabetic retina. Not only will this research be beneficial to further space

  18. Hemodynamic activation of β-catenin and TCF signaling in vascular endothelium regulates fibronectin expression

    PubMed Central

    Gelfand, Bradley D.; Meller, Julia; Pryor, Andrew W.; Kahn, Michael; Schoppee Bortz, Pamela D.; Wamhoff, Brian R.; Blackman, Brett R.

    2011-01-01

    β-catenin/TCF signaling regulates a varied set of cellular functions including development and remodeling. Fibronectin is a TCF-regulated gene that is highly expressed in arterial endothelium during atherosclerosis development and contributes to the pathophysiology of the disease. However, the activation of endothelial β-catenin/TCF signaling and its role in fibronectin expression in atherosclerosis are not currently known. Objective To assess the activity of β-catenin/TCF signaling in atherosclerosis development and its regulation of fibronectin in vascular endothelium. Methods and Results Histological staining identified preferential nuclear localization of β-catenin in the endothelium of atheroprone aorta prior to and during lesion development. Transgenic reporter studies revealed that increased levels of TCF transcriptional activity in endothelium correlated anatomically with β-catenin nuclear localization and fibronectin deposition. Exposure of endothelial cells to human-derived atheroprone shear stress induced nuclear localization of β-catenin, transcriptional activation of TCF, and expression of fibronectin. Activation of fibronectin expression required β-catenin, TCF and the transcriptional co-activator CBP. Finally, we identified PECAM-1as a critical regulator of constitutive β-catenin and GSK-3β activities. Conclusions This data uncovers novel constitutive activation of the endothelial β-catenin/TCF signaling pathway in atherosclerosis and regulation of fibronectin through hemodynamic shear stress. PMID:21527747

  19. Regulation of Nox enzymes expression in vascular pathophysiology: Focusing on transcription factors and epigenetic mechanisms.

    PubMed

    Manea, Simona-Adriana; Constantin, Alina; Manda, Gina; Sasson, Shlomo; Manea, Adrian

    2015-08-01

    NADPH oxidases (Nox) represent a family of hetero-oligomeric enzymes whose exclusive biological function is the generation of reactive oxygen species (ROS). Nox-derived ROS are essential modulators of signal transduction pathways that control key physiological activities such as cell growth, proliferation, migration, differentiation, and apoptosis, immune responses, and biochemical pathways. Enhanced formation of Nox-derived ROS, which is generally associated with the up-regulation of different Nox subtypes, has been established in various pathologies, namely cardiovascular diseases, diabetes, obesity, cancer, and neurodegeneration. The detrimental effects of Nox-derived ROS are related to alterations in cell signalling and/or direct irreversible oxidative damage of nucleic acids, proteins, carbohydrates, and lipids. Thus, understanding of transcriptional regulation mechanisms of Nox enzymes have been extensively investigated in an attempt to find ways to counteract the excessive formation of Nox-derived ROS in various pathological states. Despite the numerous existing data, the molecular pathways responsible for Nox up-regulation are not completely understood. This review article summarizes some of the recent advances and concepts related to the regulation of Nox expression in the vascular pathophysiology. It highlights the role of transcription factors and epigenetic mechanisms in this process. Identification of the signalling molecules involved in Nox up-regulation, which is associated with the onset and development of cardiovascular dysfunction may contribute to the development of novel strategies for the treatment of cardiovascular diseases. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Function and regulation of large conductance Ca21-activated K1 channel in vascular smooth muscle cells

    PubMed Central

    Hu, Xiang-Qun; Zhang, Lubo

    2012-01-01

    Large conductance Ca21-activated K1 (BKCa) channels are abundantly expressed in vascular smooth muscle cells. Activation of BKCa channels leads to hyperpolarization of cell membrane, which in turn counteracts vasoconstriction. Therefore, BKCa channels have an important role in regulation of vascular tone and blood pressure. The activity of BKCa channels is subject to modulation by various factors. Furthermore, the function of BKCa 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. PMID:22521666

  1. The Notch Ligand Delta-Like 4 Regulates Multiple Stages of Early Hemato-Vascular Development

    PubMed Central

    Neves, Hélia; Gomes, Andreia C.; Saavedra, Pedro; Carvalho, Catarina C.; Duarte, António; Cidadão, António; Parreira, Leonor

    2012-01-01

    Background In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesoderm-derived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis. Methodology/Principal Findings Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4+/− and Dll4−/− embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for the quantitative regulation of cardiomyogenesis. Conclusions/Significance This study highlights a previously unnoticed role for Dll4 in the quantitative regulation of early hemato-vascular precursors, further indicating that it is also involved on the timely emergence of mesoderm in early embryogenesis. PMID:22514637

  2. Effect of Chronic Administration of Low Dose Rapamycin on Development and Immunity in Young Rats

    PubMed Central

    Lu, Zhenya; Liu, Furong; Chen, Linglin; Zhang, Huadan; Ding, Yuemin; Liu, Jianxiang; Wong, Michael; Zeng, Ling-Hui

    2015-01-01

    Mammalian target of rapamycin (mTOR) regulates cell growth, cell differentiation and protein synthesis. Rapamycin, an inhibitor of mTOR, has been widely used as an immunosuppressant and anti-cancer drug. Recently, mTOR inhibitors have also been reported to be a potential anti-epileptic drug, which may be effective when used in young patients with genetic epilepsy. Thus, a suitable dose of rapamycin which can maintain the normal function of mTOR and has fewer side effects ideally should be identified. In the present study, we first detected changes in marker proteins of mTOR signaling pathway during development. Then we determined the dose of rapamycin by treating rats of 2 weeks of age with different doses of rapamycin for 3 days and detected its effect on mTOR pathway. Young rats were then treated with a suitable dose of rapamycin for 4 weeks and the effect of rapamycin on mTOR, development and immunity were investigated. We found that the expression of the marker proteins of mTOR pathway was changed during development in brain hippocampus and neocortex. After 3 days of treanent, 0.03 mg/kg rapamycin had no effect on phospho-S6, whereas 0.1, 0.3, 1.0 and 3.0 mg/kg rapamycin inhibited phospho-S6 in a dose-dependent manner. However, only 1.0 mg/kg and 3.0 mg/kg rapamycin inhibited phospho-S6 after 4 weeks treatment of rapamycin. Parallel to this result, rats treated with 0.1 and 0.3 mg/kg rapamycin had no obvious adverse effects, whereas rats treated with 1.0 and 3.0 mg/kg rapamycin showed significant decreases in body, spleen and thymus weight. Additionally, rats treated with 1.0 and 3.0 mg/kg rapamycin exhibited cognitive impairment and anxiety as evident by maze and open field experiments. Furthermore, the content of IL-1β, IL-2, IFN-γ, TNF-α in serum and cerebral cortex were significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. The expression of DCX was also significantly decreased in 1.0 and 3.0 mg/kg rapamycin-treated rats. However, rats

  3. Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor

    PubMed Central

    Abe, Keisuke; Shoji, Mamoru; Chen, Jiang; Bierhaus, Angelika; Danave, Indrani; Micko, Cornelia; Casper, Katherine; Dillehay, Dirck L.; Nawroth, Peter P.; Rickles, Frederick R.

    1999-01-01

    Tissue factor (TF), a transmembrane receptor for coagulation factor VII/VIIa, is aberrantly expressed in human cancers. We demonstrated a significant correlation between TF and vascular endothelial growth factor (VEGF) production in 13 human malignant melanoma cell lines (r2 = 0.869, P < 0.0001). Two of these cell lines, RPMI-7951, a high TF and VEGF producer, and WM-115, a low TF and VEGF producer, were grown s.c. in severe combined immunodeficient mice. The high-producer cell line generated solid tumors characterized by intense vascularity, whereas the low producer generated relatively avascular tumors, as determined by immunohistologic staining of tumor vascular endothelial cells with anti-von Willebrand factor antibody. To investigate the structure-function relationship of TF and VEGF, a low-producer melanoma cell line (HT144) was transfected with a TF cDNA containing the full-length sequence, a cytoplasmic deletion mutant lacking the coding sequence for the distal three serine residues (potential substrates for protein kinase C), or an extracellular domain mutant, which has markedly diminished function for activation of factor X. Cells transfected with the full-length sequence produced increased levels of both TF and VEGF. Transfectants with the full-length sequence and the extracellular domain mutant produced approximately equal levels of VEGF mRNA. However, cells transfected with the cytoplasmic deletion mutant construct produced increased levels of TF, but little or no VEGF. Thus, the cytoplasmic tail of TF plays a role in the regulation of VEGF expression in some tumor cells. PMID:10411932

  4. Thrombospondin-1 and CD47 regulation of cardiac, pulmonary and vascular responses in health and disease.

    PubMed

    Rogers, Natasha M; Sharifi-Sanjani, Maryam; Csányi, Gábor; Pagano, Patrick J; Isenberg, Jeffrey S

    2014-07-01

    Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling is lost. Work over the last several years indicates that regulation of NO is much more complex than previously believed. It is now apparent that the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Leptin and vascular endothelial growth factor regulate angiogenesis in tooth germs.

    PubMed

    Ide, Shinji; Tokuyama, Reiko; Davaadorj, Purevsuren; Shimozuma, Masashi; Kumasaka, Shuku; Tatehara, Seiko; Satomura, Kazuhito

    2011-03-01

    Leptin, a 16 kDa non-glycolated polypeptide of 146 amino acids produced by the ob gene, has a variety of physiological roles not only in lipid metabolism, hematopoiesis, thermogenesis and ovarian function, but also in angiogenesis. This study focuses to investigate the possibility that leptin, as an angiogenic factor, may regulate the angiogenesis during tooth development. We firstly studied the expression of leptin and vascular endothelial growth factor (VEGF) during tooth development immunohistochemically. This investigation revealed that leptin is expressed in ameloblasts, odontoblasts, dental papilla cells and stratum intermedium cells. This expression pattern was similar to that of VEGF, one of the most potent angiogenic factors. Interestingly, more leptin-positive cells were observed in the upper third portion of dental papilla, which is closest to odontoblastic layer, compared to middle and lower thirds. Moreover, in the dental papilla, more CD31 and/or CD34-positive vascular endothelial cells were observed in the vicinity of ameloblasts and odontoblasts expressing leptin and VEGF. These findings strongly suggest that ameloblasts, odontoblasts and dental papilla cells induce the angiogenesis in tooth germs by secretion of leptin as well as VEGF.

  6. Thrombospondin-1 and CD47 Regulation of Cardiac, Pulmonary and Vascular Responses in Health and Disease

    PubMed Central

    Rogers, Natasha M.; Sharifi-Sanjani, Maryam; Csányi, Gábor; Pagano, Patrick J.; Isenberg, Jeffrey S.

    2014-01-01

    Cardiovascular homeostasis and health is maintained through the balanced interactions of cardiac generated blood flow and cross-talk between the cellular components that comprise blood vessels. Central to this cross-talk is endothelial generated nitric oxide (NO) that stimulates relaxation of the contractile vascular smooth muscle (VSMC) layer of blood vessels. In cardiovascular disease this balanced interaction is disrupted and NO signaling lost. Work over the last several years indicates regulation of NO is much more complex than previously believed. It is now apparent the secreted protein thrombospondin-1 (TSP1), that is upregulated in cardiovascular disease and animal models of the same, on activating cell surface receptor CD47, redundantly inhibits NO production and NO signaling. This inhibitory event has implications for baseline and disease-related responses mediated by NO. Further work has identified that TSP1-CD47 signaling stimulates enzymatic reactive oxygen species (ROS) production to further limit blood flow and promote vascular disease. Herein consideration is given to the most recent discoveries in this regard which identify the TSP1-CD47 axis as a major proximate governor of cardiovascular health. PMID:24418252

  7. Harnessing Sphingosine-1-Phosphate Signaling and Nanotopographical Cues To Regulate Skeletal Muscle Maturation and Vascularization.

    PubMed

    Tsui, Jonathan H; Janebodin, Kajohnkiart; Ieronimakis, Nicholas; Yama, David M P; Yang, Hee Seok; Chavanachat, Rakchanok; Hays, Aislinn L; Lee, Haeshin; Reyes, Morayma; Kim, Deok-Ho

    2017-12-26

    Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here, we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.

  8. Bim regulates alloimmune-mediated vascular injury through effects on T-cell activation and death.

    PubMed

    von Rossum, Anna; Enns, Winnie; Shi, Yu P; MacEwan, Grace E; Malekesmaeli, Mehrnoush; Brinkman, Ryan; Choy, Jonathan C

    2014-06-01

    Bim is a proapoptotic Bcl-2 protein known to downregulate immune responses and to also be required for antigen-induced T-cell activation. However, it is not known how the effect of Bim on these offsetting processes determines the outcome of allogeneic immune responses. We have defined the role of Bim in regulating alloantigen-driven T-cell responses in a model of vascular rejection. Bim was required for proliferation of CD4 and CD8 T cells, and for interleukin-2 production, in T cells stimulated with alloantigen in vitro. Moreover, a partial reduction in Bim expression was sufficient to attenuate T-cell activation, whereas a complete elimination of Bim was required to prevent CD4 T-cell death in response to cytokine withdrawl. When alloimmune-mediated vascular rejection was examined using an aortic interposition model, there was significantly less intimal thickening in Bim(+/-), but not Bim(-/-), graft recipients. T-cell proliferation in response to allograft arteries was significantly reduced in both Bim(+/-) and Bim(-/-) mice, but cell death was attenuated only in Bim(-/-) animals. Bim controls both T-cell activation and death in response to alloantigen stimulation. These processes act cooperatively to determine the outcome of immune responses in allograft arteries. © 2014 American Heart Association, Inc.

  9. Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion.

    PubMed

    Griner, Samantha E; Joshi, Jayashree P; Nahta, Rita

    2013-01-01

    Identification of novel molecular markers and therapeutic targets may improve survival rates for patients with ovarian cancer. In the current study, immunohistochemical (IHC) analysis of two human ovarian tumor tissue arrays showed high staining for GDF15 in a majority of tissues. Exogenous stimulation of ovarian cancer cell lines with recombinant human GDF15 (rhGDF15) or stable over-expression of a GDF15 expression plasmid promoted anchorage-independent growth, increased invasion, and up-regulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). MMP inhibition suppressed GDF15-mediated invasion. In addition, IHC analysis of human ovarian tumor tissue arrays indicated that GDF15 expression correlated significantly with high MMP2 and MMP9 expression. Exogenous and endogenous GDF15 over-expression stimulated phosphorylation of p38, Erk1/2, and Akt. Pharmacologic inhibition of p38, MEK, or PI3K suppressed GDF15-stimulated growth. Further, proliferation, growth, and invasion of GDF15 stable clones were blocked by rapamycin. IHC analysis demonstrated significant correlation between GDF15 expression and phosphorylation of mTOR. Finally, knockdown of endogenous GDF15 or neutralization of secreted GDF15 suppressed invasion and growth of a GDF15-over-expressing ovarian cancer cell line. These data indicate that GDF15 over-expression, which occurred in a majority of human ovarian cancers, promoted rapamycin-sensitive invasion and growth of ovarian cancer cells. Inhibition of mTOR may be an effective therapeutic strategy for ovarian cancers that over-express GDF15. Future studies should examine GDF15 as a novel molecular target for blocking ovarian cancer progression. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. The combination of rapamycin and MAPK inhibitors enhances the growth inhibitory effect on Nara-H cells

    PubMed Central

    NAKAMURA, OSAMU; HITORA, TOSHIAKI; YAMAGAMI, YOSHIKI; MORI, MASAKI; NISHIMURA, HIDEKI; HORIE, RYOSUKE; YAMAGUCHI, KONOSUKE; YAMAMOTO, TETSUJI

    2014-01-01

    The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway promotes the initiation of autophagy, and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) is well known to induce autophagy. Autophagy is a self-defense mechanism of cancer cells that are subjected to antitumor agents, and blocking autophagy can trigger apoptosis. In the present study, we demonstrate that an mTOR inhibitor, rapamycin, induces autophagy in the Nara-H malignant fibrous histiocytoma (MFH) cell line through the activation of ERK1/2. Rapamycin-induced apoptosis was enhanced following the inhibition of the MEK/ERK pathway. In the Nara-H cells, we examined the effects of rapamycin treatment on cell proliferation and on the phosphorylation of the mTOR pathway components and autophagy by western blot analysis. Furthermore, we examined the effects of rapamycin with or without the MEK inhibitor, U0126, on the induction of apoptosis by using fluorescence microscopy. Rapamycin inhibited Nara-H cell proliferation and decreased the phosphorylation of the mTOR pathway in the Nara-H cells. Rapamycin induced the apoptosis of Nara-H cells, and this apoptosis was enhanced by U0126. Simultaneously, phospho-ERK1/2 was activated by rapamycin. The present study demonstrates that rapamycin induces autophagy in Nara-H cells by activating the MEK/ERK signaling pathway, and the rapamycin-induced apoptosis can be enhanced by the MEK inhibitor, U0126. These results suggest that self-protective mechanisms involving mTOR inhibitors in Nara-H cells are prevented by the inhibition of the MEK/ERK pathway. The combination of an mTOR inhibitor (e.g., rapamycin) and an MEK inhibitor (e.g., U0126) may offer effective treatment for MFH, as this combination effectively activates apoptotic pathways. PMID:24676456

  11. Endothelium-derived fibronectin regulates neonatal vascular morphogenesis in an autocrine fashion.

    PubMed

    Turner, Christopher J; Badu-Nkansah, Kwabena; Hynes, Richard O

    2017-11-01

    Fibronectin containing alternatively spliced EIIIA and EIIIB domains is largely absent from mature quiescent vessels in adults, but is highly expressed around blood vessels during developmental and pathological angiogenesis. The precise functions of fibronectin and its splice variants during developmental angiogenesis however remain unclear due to the presence of cardiac, somitic, mesodermal and neural defects in existing global fibronectin KO mouse models. Using a rare family of surviving EIIIA EIIIB double KO mice, as well as inducible endothelial-specific fibronectin-deficient mutant mice, we show that vascular development in the neonatal retina is regulated in an autocrine manner by endothelium-derived fibronectin, and requires both EIIIA and EIIIB domains and the RGD-binding α5 and αv integrins for its function. Exogenous sources of fibronectin do not fully substitute for the autocrine function of endothelial fibronectin, demonstrating that fibronectins from different sources contribute differentially to specific aspects of angiogenesis.

  12. Nrf2 in ischemic neurons promotes retinal vascular regeneration through regulation of semaphorin 6A.

    PubMed

    Wei, Yanhong; Gong, Junsong; Xu, Zhenhua; Thimmulappa, Rajesh K; Mitchell, Katherine L; Welsbie, Derek S; Biswal, Shyam; Duh, Elia J

    2015-12-15

    Delayed revascularization of ischemic neural tissue is a major impediment to preservation of function in central nervous system (CNS) diseases including stroke and ischemic retinopathies. Therapeutic strategies allowing rapid revascularization are greatly needed to reduce ischemia-induced cellular damage and suppress harmful pathologic neovascularization. However, key mechanisms governing vascular recovery in ischemic CNS, including regulatory molecules governing the transition from tissue injury to tissue repair, are largely unknown. NF-E2-related factor 2 (Nrf2) is a major stress-response transcription factor well known for its cell-intrinsic cytoprotective function. However, its role in cell-cell crosstalk is less appreciated. Here we report that Nrf2 is highly activated in ischemic retina and promotes revascularization by modulating neurons in their paracrine regulation of endothelial cells. Global Nrf2 deficiency strongly suppresses retinal revascularization and increases pathologic neovascularization in a mouse model of ischemic retinopathy. Conditional knockout studies demonstrate a major role for neuronal Nrf2 in vascular regrowth into avascular retina. Deletion of neuronal Nrf2 results in semaphorin 6A (Sema6A) induction in hypoxic/ischemic retinal ganglion cells in a hypoxia-inducible factor-1 alpha (HIF-1α)-dependent fashion. Sema6A expression increases in avascular inner retina and colocalizes with Nrf2 in human fetal eyes. Extracellular Sema6A leads to dose-dependent suppression of the migratory phenotype of endothelial cells through activation of Notch signaling. Lentiviral-mediated delivery of Sema6A small hairpin RNA (shRNA) abrogates the defective retinal revascularization in Nrf2-deficient mice. Importantly, pharmacologic Nrf2 activation promotes reparative angiogenesis and suppresses pathologic neovascularization. Our findings reveal a unique function of Nrf2 in reprogramming ischemic tissue toward neurovascular repair via Sema6A regulation

  13. Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.

    PubMed

    Kapustin, Alexander N; Chatrou, Martijn L L; Drozdov, Ignat; Zheng, Ying; Davidson, Sean M; Soong, Daniel; Furmanik, Malgorzata; Sanchis, Pilar; De Rosales, Rafael Torres Martin; Alvarez-Hernandez, Daniel; Shroff, Rukshana; Yin, Xiaoke; Muller, Karin; Skepper, Jeremy N; Mayr, Manuel; Reutelingsperger, Chris P; Chester, Adrian; Bertazzo, Sergio; Schurgers, Leon J; Shanahan, Catherine M

    2015-04-10

    Matrix vesicles (MVs), secreted by vascular smooth muscle cells (VSMCs), form the first nidus for mineralization and fetuin-A, a potent circulating inhibitor of calcification, is specifically loaded into MVs. However, the processes of fetuin-A intracellular trafficking and MV biogenesis are poorly understood. The objective of this study is to investigate the regulation, and role, of MV biogenesis in VSMC calcification. Alexa488-labeled fetuin-A was internalized by human VSMCs, trafficked via the endosomal system, and exocytosed from multivesicular bodies via exosome release. VSMC-derived exosomes were enriched with the tetraspanins CD9, CD63, and CD81, and their release was regulated by sphingomyelin phosphodiesterase 3. Comparative proteomics showed that VSMC-derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins. Elevated extracellular calcium was found to induce sphingomyelin phosphodiesterase 3 expression and the secretion of calcifying exosomes from VSMCs in vitro, and chemical inhibition of sphingomyelin phosphodiesterase 3 prevented VSMC calcification. In vivo, multivesicular bodies containing exosomes were observed in vessels from chronic kidney disease patients on dialysis, and CD63 was found to colocalize with calcification. Importantly, factors such as tumor necrosis factor-α and platelet derived growth factor-BB were also found to increase exosome production, leading to increased calcification of VSMCs in response to calcifying conditions. This study identifies MVs as exosomes and shows that factors that can increase exosome release can promote vascular calcification in response to environmental calcium stress. Modulation of the exosome release pathway may be as a novel therapeutic target for prevention. © 2015 American Heart Association, Inc.

  14. Fluid shear stress as a regulator of gene expression in vascular cells: possible correlations with diabetic abnormalities

    NASA Technical Reports Server (NTRS)

    Papadaki, M.; Eskin, S. G.; Ruef, J.; Runge, M. S.; McIntire, L. V.

    1999-01-01

    Diabetes mellitus is associated with increased frequency, severity and more rapid progression of cardiovascular diseases. Metabolic perturbations from hyperglycemia result in disturbed endothelium-dependent relaxation, activation of coagulation pathways, depressed fibrinolysis, and other abnormalities in vascular homeostasis. Atherosclerosis is localized mainly at areas of geometric irregularity at which blood vessels branch, curve and change diameter, and where blood is subjected to sudden changes in velocity and/or direction of flow. Shear stress resulting from blood flow is a well known modulator of vascular cell function. This paper presents what is currently known regarding the molecular mechanisms responsible for signal transduction and gene regulation in vascular cells exposed to shear stress. Considering the importance of the hemodynamic environment of vascular cells might be vital to increasing our understanding of diabetes.

  15. Regulator of calcineurin 1 modulates vascular contractility and stiffness through the upregulation of COX-2-derived prostanoids.

    PubMed

    García-Redondo, Ana B; Esteban, Vanesa; Briones, Ana M; Díaz Del Campo, Lucía S; González-Amor, María; Méndez-Barbero, Nerea; Campanero, Miguel R; Redondo, Juan M; Salaices, Mercedes

    2018-01-05

    Cyclooxygenase-2 (COX-2) derived-prostanoids participate in the altered vascular function and mechanical properties in cardiovascular diseases. We investigated whether regulator of calcineurin 1 (Rcan1) participates in vascular contractility and stiffness through the regulation of COX-2. For this, wild type (Rcan1+/+) and Rcan1-deficient (Rcan1-/-) mice untreated or treated with the COX-2 inhibitor rofecoxib were used. Vascular function and structure were analysed by myography. COX-2 and phospo-p65 expression were studied by western blotting and immunohistochemistry and TXA2 production by ELISA. We found that Rcan1 deficiency increases COX-2 and IL-6 expression and NF-κB activation in arteries and vascular smooth muscle cells (VSMC). Adenoviral-mediated re-expression of Rcan1.4 in Rcan1-/- VSMC normalized COX-2 expression. Phenylephrine-induced vasoconstrictor responses were greater in aorta from Rcan1-/- compared to Rcan1+/+ mice. This increased response were diminished by etoricoxib, furegrelate, SQ 29548, cyclosporine A and parthenolide, inhibitors of COX-2, TXA2 synthase, TP receptors, calcineurin and NF-κB, respectively. Endothelial removal and NOS inhibition increased phenylephrine responses only in Rcan1+/+ mice. TXA2 levels were greater in Rcan1-/- mice. In small mesenteric arteries, vascular function and structure were similar in both groups of mice; however, vessels from Rcan1-/- mice displayed an increase in vascular stiffness that was diminished by rofecoxib. In conclusion, our results suggest that Rcan1 might act as endogenous negative modulator of COX-2 expression and activity by inhibiting calcineurin and NF-kB pathways to maintain normal contractility and vascular stiffness in aorta and small mesenteric arteries, respectively. Our results uncover a new role for Rcan1 in vascular contractility and mechanical properties. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Rapamycin inhibiting Jurkat T cells viability through changing mRNA expression of serine/threonine protein phosphatase 2A.

    PubMed

    Wang, Baobao; He, Qiang; Mao, Youyin; Chen, Zhimin; Jiang, Hong; Chen, Jianghua

    2012-01-01

    In this study, we analyzed the mRNA expression of serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) in the human leukemic T-cell line Jurkat cells treated with rapamycin, to determine whether rapamycin inhibiting cell viability is accompanied with the change of mRNA expression of PP2A. Jurkat cells were incubated with various concentrations of rapamycin and cultured for different hours. Cell viability was assessed by MTT assay. The mRNA expressions of PP2A subunits were measured by quantitative real-time polymerase chain reaction (PCR). We found that rapamycin had an inhibitory effect on cell viability. IC50 was 343.3 nM at 48 h.We also found rapamycin had a dose and time-dependent effect on the gene expression of PP2A. When setting the concentration of rapamycin 500 nM, the mRNA expressions of PP2A subunits (Aa, Aβ, PR55a, PR55δ, PR61γ, PR70, Ca and Cβ) were declined significantly at 48 h. When treated with various concentrations of rapamycin for 48 h, the mRNA expressions of PP2A subunits were down-regulated in the range from 10 nM to 500 nM. Rapamycin inhibiting Jurkat T cells viability may be related to the reduction of PP2A mRNA expressions. Copyright © 2011. Published by Elsevier B.V.

  17. The PP242 mammalian target of rapamycin (mTOR) inhibitor activates extracellular signal-regulated kinase (ERK) in multiple myeloma cells via a target of rapamycin complex 1 (TORC1)/eukaryotic translation initiation factor 4E (eIF-4E)/RAF pathway and activation is a mechanism of resistance.

    PubMed

    Hoang, Bao; Benavides, Angelica; Shi, Yijiang; Yang, Yonghui; Frost, Patrick; Gera, Joseph; Lichtenstein, Alan

    2012-06-22

    Activation of PI3-K-AKT and ERK pathways is a complication of mTOR inhibitor therapy. Newer mTOR inhibitors (like pp242) can overcome feedback activation of AKT in multiple myeloma (MM) cells. We, thus, studied if feedback activation of ERK is still a complication of therapy with such drugs in this tumor model. PP242 induced ERK activation in MM cell lines as well as primary cells. Surprisingly, equimolar concentrations of rapamycin were relatively ineffective at ERK activation. Activation was not correlated with P70S6kinase inhibition nor was it prevented by PI3-kinase inhibition. ERK activation was prevented by MEK inhibitors and was associated with concurrent stimulation of RAF kinase activity but not RAS activation. RAF activation correlated with decreased phosphorylation of RAF at Ser-289, Ser-296, and Ser-301 inhibitory residues. Knockdown studies confirmed TORC1 inhibition was the key proximal event that resulted in ERK activation. Furthermore, ectopic expression of eIF-4E blunted pp242-induced ERK phosphorylation. Since pp242 was more potent than rapamycin in causing sequestering of eIF-4E, a TORC1/4E-BP1/eIF-4E-mediated mechanism of ERK activation could explain the greater effectiveness of pp242. Use of MEK inhibitors confirmed ERK activation served as a mechanism of resistance to the lethal effects of pp242. Thus, although active site mTOR inhibitors overcome AKT activation often seen with rapalog therapy, feedback ERK activation is still a problem of resistance, is more severe than that seen with use of first generation rapalogs and is mediated by a TORC1- and eIF-4E-dependent mechanism ultimately signaling to RAF.

  18. Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

    PubMed Central

    Kaur, Sukhbir

    2017-01-01

    Abstract Significance: In contrast to structural elements of the extracellular matrix, matricellular proteins appear transiently during development and injury responses, but their sustained expression can contribute to chronic disease. Through interactions with other matrix components and specific cell surface receptors, matricellular proteins regulate multiple signaling pathways, including those mediated by reactive oxygen and nitrogen species and H2S. Dysregulation of matricellular proteins contributes to the pathogenesis of vascular diseases and cancer. Defining the molecular mechanisms and receptors involved is revealing new therapeutic opportunities. Recent Advances: Thrombospondin-1 (TSP1) regulates NO, H2S, and superoxide production and signaling in several cell types. The TSP1 receptor CD47 plays a central role in inhibition of NO signaling, but other TSP1 receptors also modulate redox signaling. The matricellular protein CCN1 engages some of the same receptors to regulate redox signaling, and ADAMTS1 regulates NO signaling in Marfan syndrome. In addition to mediating matricellular protein signaling, redox signaling is emerging as an important pathway that controls the expression of several matricellular proteins. Critical Issues: Redox signaling remains unexplored for many matricellular proteins. Their interactions with multiple cellular receptors remains an obstacle to defining signaling mechanisms, but improved transgenic models could overcome this barrier. Future Directions: Therapeutics targeting the TSP1 receptor CD47 may have beneficial effects for treating cardiovascular disease and cancer and have recently entered clinical trials. Biomarkers are needed to assess their effects on redox signaling in patients and to evaluate how these contribute to their therapeutic efficacy and potential side effects. Antioxid. Redox Signal. 27, 874–911. PMID:28712304

  19. Differential Effects of Rapamycin and Dexamethasone in Mouse Models of Established Allergic Asthma

    PubMed Central

    Mushaben, Elizabeth M.; Brandt, Eric B.; Hershey, Gurjit K. Khurana; Le Cras, Timothy D.

    2013-01-01

    The mammalian target of rapamycin (mTOR) plays an important role in cell growth/differentiation, integrating environmental cues, and regulating immune responses. Our lab previously demonstrated that inhibition of mTOR with rapamycin prevented house dust mite (HDM)-induced allergic asthma in mice. Here, we utilized two treatment protocols to investigate whether rapamycin, compared to the steroid, dexamethasone, could inhibit allergic responses during the later stages of the disease process, namely allergen re-exposure and/or during progression of chronic allergic disease. In protocol 1, BALB/c mice were sensitized to HDM (three i.p. injections) and administered two intranasal HDM exposures. After 6 weeks of rest/recovery, mice were re-exposed to HDM while being treated with rapamycin or dexamethasone. In protocol 2, mice were exposed to HDM for 3 or 6 weeks and treated with rapamycin or dexamethasone during weeks 4–6. Characteristic features of allergic asthma, including IgE, goblet cells, airway hyperreactivity (AHR), inflammatory cells, cytokines/chemokines, and T cell responses were assessed. In protocol 1, both rapamycin and dexamethasone suppressed goblet cells and total CD4+ T cells including activated, effector, and regulatory T cells in the lung tissue, with no effect on AHR or total inflammatory cell numbers in the bronchoalveolar lavage fluid. Rapamycin also suppressed IgE, although IL-4 and eotaxin 1 levels were augmented. In protocol 2, both drugs suppressed total CD4+ T cells, including activated, effector, and regulatory T cells and IgE levels. IL-4, eotaxin, and inflammatory cell numbers were increased after rapamycin and no effect on AHR was observed. Dexamethasone suppressed inflammatory cell numbers, especially eosinophils, but had limited effects on AHR. We conclude that while mTOR signaling is critical during the early phases of allergic asthma, its role is much more limited once disease is established. PMID:23349887

  20. p53 and rapamycin are additive.

    PubMed

    Christy, Barbara; Demaria, Marco; Campisi, Judith; Huang, Jing; Jones, Diane; Dodds, Sherry G; Williams, Charnae; Hubbard, Gene; Livi, Carolina B; Gao, Xiaoli; Weintraub, Susan; Curiel, Tyler; Sharp, Z Dave; Hasty, Paul

    2015-06-30

    Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic.

  1. p53 and rapamycin are additive

    PubMed Central

    Campisi, Judith; Huang, Jing; Jones, Diane; Dodds, Sherry G.; Williams, Charnae; Hubbard, Gene; Livi, Carolina B.; Gao, Xiaoli; Weintraub, Susan; Curiel, Tyler; Sharp, Z. Dave; Hasty, Paul

    2015-01-01

    Mechanistic target of rapamycin (mTOR) is a kinase found in a complex (mTORC1) that enables macromolecular synthesis and cell growth and is implicated in cancer etiology. The rapamycin-FK506 binding protein 12 (FKBP12) complex allosterically inhibits mTORC1. In response to stress, p53 inhibits mTORC1 through a separate pathway involving cell signaling and amino acid sensing. Thus, these different mechanisms could be additive. Here we show that p53 improved the ability of rapamycin to: 1) extend mouse life span, 2) suppress ionizing radiation (IR)-induced senescence-associated secretory phenotype (SASP) and 3) increase the levels of amino acids and citric acid in mouse embryonic stem (ES) cells. This additive effect could have implications for cancer treatment since rapamycin and p53 are anti-oncogenic. PMID:26158292

  2. Rab5a‑mediated autophagy regulates the phenotype and behavior of vascular smooth muscle cells.

    PubMed

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

    2016-11-01

    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

  3. Proper gibberellin localization in vascular tissue is required to regulate adventitious root development in tobacco

    PubMed Central

    Chen, Xiaoyang; Li, Wei

    2013-01-01

    Bioactive gibberellins (GAs) are involved in many developmental aspects of the life cycle of plants, acting either directly or through interaction with other hormones. Accumulating evidence suggests that GAs have an important effect on root growth; however, there is currently little information on the specific regulatory mechanism of GAs during adventitious root development. A study was conducted on tobacco (Nicotiana tabacum) plants for altered rates of biosynthesis, catabolism, and GA signalling constitutively or in specific tissues using a transgenic approach. In the present study, PtGA20ox, PtGA2ox1, and PtGAI were overexpressed under the control of the 35S promoter, vascular cambium-specific promoter (LMX5), or root meristem-specific promoter (TobRB7), respectively. Evidence is provided that the precise localization of bioactive GA in the stem but not in the roots is required to regulate adventitious root development in tobacco. High levels of GA negatively regulate the early initiation step of root formation through interactions with auxin, while a proper and mobile GA signal is required for the emergence and subsequent long-term elongation of established primordia. The results demonstrated that GAs have an inhibitory effect on adventitious root formation but a stimulatory effect on root elongation. PMID:23918971

  4. Regulation of myometrial circulation and uterine vascular tone by constitutive nitric oxide.

    PubMed

    Toda, Noboru; Toda, Hiroshi; Okamura, Tomio

    2013-08-15

    Pregnancy is a physiological state that involves an increase in uterine blood flow, which is mediated in part by nitric oxide (NO) liberated from the endothelium and nitrergic neurons. The main focus of this review article is to provide information about how endogenous NO regulates uterine and placental blood flow and vascular tone in experimental animals and humans in vivo or in vitro in non-pregnant and pregnant states as well as pregnancy with pre-eclampsia. Uterine arteries from non-pregnant women respond to NO liberated from the endothelium and nitrergic nerves with relaxations, and the release of endothelial NO is influenced by the phase of the estrous cycle, with its enhanced release at the follicular phase when the estrogen level is high. NO bioavailability in the uteroplacental circulatory system is gradually increased during pregnancy. Pre-eclamptic pregnancies with or without intrauterine growth restriction show impaired uteroplacental blood flow accompanied by reduced NO synthesis due to down-regulation of eNOS as well as asymmetric dimethylarginine accumulation and by augmented NO degradation by oxidative stress. Further studies are expected to provide new mechanistic insights into the fascinating process of maternal uterine adaptation in humans and novel prophylactic and therapeutic measures against pre-eclampsia. © 2013 Elsevier B.V. All rights reserved.

  5. Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells.

    PubMed

    Zhao, Jingshan; Niu, Honglin; Li, Aiying; Nie, Lei

    2016-01-01

    The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.

  6. Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

    PubMed Central

    Zhao, Jingshan; Niu, Honglin; Li, Aiying; Nie, Lei

    2016-01-01

    The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis. PMID:26863518

  7. Regulation of vascular endothelial growth factor synthesis and release by human luteal cells in vitro.

    PubMed

    Tropea, Anna; Miceli, Fiorella; Minici, Francesca; Tiberi, Federica; Orlando, Mariateresa; Gangale, Maria Francesca; Romani, Federica; Catino, Stefania; Mancuso, Salvatore; Navarra, Pierluigi; Lanzone, Antonio; Apa, Rosanna

    2006-06-01

    Vascular endothelial growth factor (VEGF) is essential for normal luteal development and function, but little is still known about the regulation of its production by human midluteal phase luteal cells. We investigated whether human chorionic gonadotropin (hCG) or local factors, including chemical hypoxia, IGF-I and IGF-II, prostaglandin (PG)E(2), and PGF(2alpha) prevail in modulating VEGF mRNA and protein production in human midluteal phase luteal cells. The effect of progesterone (P) on luteal VEGF mRNA expression and protein secretion was also evaluated. Finally, we investigated whether VEGF could directly affect luteal P secretion. In human midluteal phase luteal cells, VEGF mRNA expression was evaluated by semiquantitative RT-PCR, whereas VEGF and P release was evaluated by ELISA and RIA, respectively. hCG was unable to significantly affect luteal VEGF mRNA and protein synthesis, which in turn was significantly increased by both chemical hypoxia and IGFs. Conversely, VEGF mRNA and protein production was reduced by PGs and P. Finally, VEGF did not affect P luteal secretion. Our results suggest that local ovarian factors, rather than hCG, predominate in regulating VEGF mRNA and protein production by human midluteal phase luteal cells. For VEGF, a lack of a direct luteal steroidogenic effect was also demonstrated.

  8. Contribution of endothelium-derived hyperpolarizing factors to the regulation of vascular tone in humans.

    PubMed

    Bellien, Jeremy; Thuillez, Christian; Joannides, Robinson

    2008-08-01

    Endothelium plays a crucial role in the regulation of cardiovascular homeostasis through the release of vasoactive factors. Besides nitric oxide (NO) and prostacyclin, increasing evidences show that endothelium-derived hyperpolarizing factors (EDHF) participate in the control of vasomotor tone through the activation of calcium-activated potassium channels. In humans, the role of EDHF has been demonstrated in various vascular beds including coronary, peripheral, skin and venous vessels. The mechanisms of EDHF-type relaxations identified in humans involved the release by the endothelium of hydrogen peroxide, epoxyeicosatrienoic acids (EETs), potassium ions and electronical communication through the gap junctions. The role of EETs could be particularly important because, in addition contributing to the maintenance of the basal tone and endothelium-dependent dilation of conduit arteries, these factors share many vascular protective properties of NO. The alteration of which might be involved in the physiopathology of cardiovascular diseases. The evolution of EDHF availability in human pathology is currently under investigation with some results demonstrating an increase in EDHF release to compensate the loss of NO synthesis and to maintain the endothelial vasomotor function whereas others reported a parallel decrease in NO and EDHF-mediated relaxations. Thus, the modulation of EDHF activity emerges as a new pharmacological target and some existing therapies in particular those affecting the renin-angiotensin system have already been shown to improve endothelial function through hyperpolarizing mechanisms. In this context, the development of new specific pharmacological agents especially those increasing EETs availability may help to prevent endothelial dysfunction and therefore enhance cardiovascular protection in patients.

  9. The vascular Ca2+-sensing receptor regulates blood vessel tone and blood pressure

    PubMed Central

    Schepelmann, M.; Yarova, P. L.; Lopez-Fernandez, I.; Davies, T. S.; Brennan, S. C.; Edwards, P. J.; Aggarwal, A.; Graça, J.; Rietdorf, K.; Matchkov, V.; Fenton, R. A.; Chang, W.; Krssak, M.; Stewart, A.; Broadley, K. J.; Ward, D. T.; Price, S. A.; Edwards, D. H.; Kemp, P. J.

    2015-01-01

    The extracellular calcium-sensing receptor CaSR is expressed in blood vessels where its role is not completely understood. In this study, we tested the hypothesis that the CaSR expressed in vascular smooth muscle cells (VSMC) is directly involved in regulation of blood pressure and blood vessel tone. Mice with targeted CaSR gene ablation from vascular smooth muscle cells (VSMC) were generated by breeding exon 7 LoxP-CaSR mice with animals in which Cre recombinase is driven by a SM22α promoter (SM22α-Cre). Wire myography performed on Cre-negative [wild-type (WT)] and Cre-positive SM22αCaSRΔflox/Δflox [knockout (KO)] mice showed an endothelium-independent reduction in aorta and mesenteric artery contractility of KO compared with WT mice in response to KCl and to phenylephrine. Increasing extracellular calcium ion (Ca2+) concentrations (1–5 mM) evoked contraction in WT but only relaxation in KO aortas. Accordingly, diastolic and mean arterial blood pressures of KO animals were significantly reduced compared with WT, as measured by both tail cuff and radiotelemetry. This hypotension was mostly pronounced during the animals' active phase and was not rescued by either nitric oxide-synthase inhibition with nitro-l-arginine methyl ester or by a high-salt-supplemented diet. KO animals also exhibited cardiac remodeling, bradycardia, and reduced spontaneous activity in isolated hearts and cardiomyocyte-like cells. Our findings demonstrate a role for CaSR in the cardiovascular system and suggest that physiologically relevant changes in extracellular Ca2+ concentrations could contribute to setting blood vessel tone levels and heart rate by directly acting on the cardiovascular CaSR. PMID:26538090

  10. Towards natural mimetics of metformin and rapamycin.

    PubMed

    Aliper, Alexander; Jellen, Leslie; Cortese, Franco; Artemov, Artem; Karpinsky-Semper, Darla; Moskalev, Alexey; Swick, Andrew G; Zhavoronkov, Alex

    2017-11-15

    Aging is now at the forefront of major challenges faced globally, creating an immediate need for safe, widescale interventions to reduce the burden of chronic disease and extend human healthspan. Metformin and rapamycin are two FDA-approved mTOR inhibitors proposed for this purpose, exhibiting significant anti-cancer and anti-aging properties beyond their current clinical applications. However, each faces issues with approval for off-label, prophylactic use due to adverse effects. Here, we initiate an effort to identify nutraceuticals-safer, naturally-occurring compounds-that mimic the anti-aging effects of metformin and rapamycin without adverse effects. We applied several bioinformatic approaches and deep learning methods to the Library of Integrated Network-based Cellular Signatures (LINCS) dataset to map the gene- and pathway-level signatures of metformin and rapamycin and screen for matches among over 800 natural compounds. We then predicted the safety of each compound with an ensemble of deep neural network classifiers. The analysis revealed many novel candidate metformin and rapamycin mimetics, including allantoin and ginsenoside (metformin), epigallocatechin gallate and isoliquiritigenin (rapamycin), and withaferin A (both). Four relatively unexplored compounds also scored well with rapamycin. This work revealed promising candidates for future experimental validation while demonstrating the applications of powerful screening methods for this and similar endeavors.

  11. Towards natural mimetics of metformin and rapamycin

    PubMed Central

    Aliper, Alexander; Jellen, Leslie; Cortese, Franco; Artemov, Artem; Karpinsky-Semper, Darla; Moskalev, Alexey; Swick, Andrew G.; Zhavoronkov, Alex

    2017-01-01

    Aging is now at the forefront of major challenges faced globally, creating an immediate need for safe, widescale interventions to reduce the burden of chronic disease and extend human healthspan. Metformin and rapamycin are two FDA-approved mTOR inhibitors proposed for this purpose, exhibiting significant anti-cancer and anti-aging properties beyond their current clinical applications. However, each faces issues with approval for off-label, prophylactic use due to adverse effects. Here, we initiate an effort to identify nutraceuticals—safer, naturally-occurring compounds—that mimic the anti-aging effects of metformin and rapamycin without adverse effects. We applied several bioinformatic approaches and deep learning methods to the Library of Integrated Network-based Cellular Signatures (LINCS) dataset to map the gene- and pathway-level signatures of metformin and rapamycin and screen for matches among over 800 natural compounds. We then predicted the safety of each compound with an ensemble of deep neural network classifiers. The analysis revealed many novel candidate metformin and rapamycin mimetics, including allantoin and ginsenoside (metformin), epigallocatechin gallate and isoliquiritigenin (rapamycin), and withaferin A (both). Four relatively unexplored compounds also scored well with rapamycin. This work revealed promising candidates for future experimental validation while demonstrating the applications of powerful screening methods for this and similar endeavors. PMID:29165314

  12. Rapamycin interacts synergistically with idarubicin to induce T-leukemia cell apoptosis in vitro and in a mesenchymal stem cell simulated drug-resistant microenvironment via Akt/mammalian target of rapamycin and extracellular signal-related kinase signaling pathways.

    PubMed

    Wu, Kang-Ni; Zhao, Yan-Min; He, Ying; Wang, Bin-Sheng; Du, Kai-Li; Fu, Shan; Hu, Kai-Min; Zhang, Li-Fei; Liu, Li-Zhen; Hu, Yong-Xian; Wang, Ying-Jia; Huang, He

    2014-03-01

    T-cell acute lymphoblastic leukemias (T-ALLs) are clonal lymphoid malignancies with a poor prognosis, and still a lack of effective treatment. Here we examined the interactions between the mammalian target of rapamycin (mTOR) inhibitor rapamycin and idarubicin (IDA) in a series of human T-ALL cell lines Molt-4, Jurkat, CCRF-CEM and CEM/C1. Co-exposure of cells to rapamycin and IDA synergistically induced T-ALL cell growth inhibition and apoptosis mediated by caspase activation via the intrinsic mitochondrial pathway and extrinsic pathway. Combined treatment with rapamycin and IDA down-regulated Bcl-2 and Mcl-1, and inhibited the activation of phosphoinositide 3-kinase (PI3K)/mTOR and extracellular signal-related kinase (ERK). They also played synergistic pro-apoptotic roles in the drug-resistant microenvironment simulated by mesenchymal stem cells (MSCs) as a feeder layer. In addition, MSCs protected T-ALL cells from IDA cytotoxicity by up-regulating ERK phosphorylation, while rapamycin efficiently reversed this protective effect. Taken together, we confirm the synergistic antitumor effects of rapamycin and IDA, and provide an insight into the potential future clinical applications of combined rapamycin-IDA regimens for treating T-cell malignancies.

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

    PubMed

    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 VASP(Ser239) and VASP(Ser157), respective indicators of PKG and PKA signaling. IBMX, a non-selective inhibitor of PDEs, had no effect on BAY-induced phosphorylation at VASP(Ser239) but inhibited phosphorylation at VASP(Ser157). Selective inhibitors of PDE3 or PDE4 attenuated BAY-mediated increases at VASP(Ser239) and VASP(Ser157), whereas PDE5 inhibition potentiated BAY-mediated increases only at VASP(Ser157). In comparison, 8Br-cGMP increased phosphorylation at VASP(Ser239) and VASP(Ser157) which were not affected by selective PDE inhibitors. In the presence of 8Br-cAMP, inhibition of either PDE4 or PDE5 decreased VASP(Ser239) phosphorylation and inhibition of PDE3 increased phosphorylation at VASP(Ser239), while inhibition of PDE3 or PDE4 increased and PDE5 inhibition had no effect on VASP(Ser157) 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.

  14. Sphingosine-1-phosphate in the plasma compartment regulates basal and inflammation-induced vascular leak in mice

    PubMed Central

    Camerer, Eric; Regard, Jean B.; Cornelissen, Ivo; Srinivasan, Yoga; Duong, Daniel N.; Palmer, Daniel; Pham, Trung H.; Wong, Jinny S.; Pappu, Rajita; Coughlin, Shaun R.

    2009-01-01

    Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models. PMID:19603543

  15. 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.

  16. Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization.

    PubMed

    Datla, Srinivasa Raju; McGrail, Daniel J; Vukelic, Sasa; Huff, Lauren P; Lyle, Alicia N; Pounkova, Lily; Lee, Minyoung; Seidel-Rogol, Bonnie; Khalil, Mazen K; Hilenski, Lula L; Terada, Lance S; Dawson, Michelle R; Lassègue, Bernard; Griendling, Kathy K

    2014-10-01

    Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner. Copyright © 2014 the American Physiological Society.

  17. Regulation of vascular endothelial growth factor by melatonin in human breast cancer cells.

    PubMed

    Alvarez-García, Virginia; González, Alicia; Alonso-González, Carolina; Martínez-Campa, Carlos; Cos, Samuel

    2013-05-01

    Melatonin exerts oncostatic effects on breast cancer by interfering with the estrogen-signaling pathways. Melatonin reduces estrogen biosynthesis in human breast cancer cells, surrounding fibroblasts and peritumoral endothelial cells by regulating cytokines that influence tumor microenvironment. This hormone also exerts antiangiogenic activity in tumoral tissue. In this work, our objective was to study the role of melatonin on the regulation of the vascular endothelial growth factor (VEGF) in breast cancer cells. To accomplish this, we cocultured human breast cancer cells (MCF-7) with human umbilical vein endothelial cells (HUVECs). VEGF added to the cultures stimulated the proliferation of HUVECs and melatonin (1 mM) counteracted this effect. Melatonin reduced VEGF production and VEGF mRNA expression in MCF-7 cells. MCF-7 cells cocultured with HUVECs stimulated the endothelial cells proliferation and increased VEGF levels in the culture media. Melatonin counteracted both stimulatory effects on HUVECs proliferation and on VEGF protein levels in the coculture media. Conditioned media from MCF-7 cells increased HUVECs proliferation, and this effect was significantly counteracted by anti-VEGF and 1 mM melatonin. All these findings suggest that melatonin may play a role in the paracrine interactions between malignant epithelial cells and proximal endothelial cells through a downregulatory action on VEGF expression in human breast cancer cells, which decrease the levels of VEGF around endothelial cells. Lower levels of VEGF could be important in reducing the number of estrogen-producing cells proximal to malignant cells as well as decreasing tumoral angiogenesis. © 2012 John Wiley & Sons A/S.

  18. iNOS regulation by calcium/calmodulin-dependent protein kinase II in vascular smooth muscle.

    PubMed

    Jones, Rachel J; Jourd'heuil, David; Salerno, John C; Smith, Susan M E; Singer, Harold A

    2007-06-01

    Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKIIdelta(2) with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with gamma-tubulin. Furthermore, CaMKIIdelta(2) coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca(2+)-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKIIdelta(2) mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKIIdelta(2) using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKIIdelta(2) may be an important regulator of iNOS trafficking and activity in VSMCs.

  19. Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 regulates xylem development and growth by a conserved mechanism that modulates hormone signaling.

    PubMed

    Grienenberger, Etienne; Douglas, Carl J

    2014-04-01

    Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-β-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways.

  20. Molecular Control of Vascular Tube Morphogenesis and Stabilization: Regulation by Extracellular Matrix, Matrix Metalloproteinases, and Endothelial Cell-Pericyte Interactions

    NASA Astrophysics Data System (ADS)

    Davis, George E.; Stratman, Amber N.; Sacharidou, Anastasia

    Recent studies have revealed a critical role for both extracellular matrices and matrix metalloproteinases in the molecular control of vascular morphogenesis and stabilization in three-dimensional (3D) tissue environments. Key interactions involve endothelial cells (ECs) and pericytes, which coassemble to affect vessel formation, remodeling, and stabilization events during development and postnatal life. EC-pericyte interactions control extracellular matrix remodeling events including vascular basement membrane matrix assembly, a necessary step for endothelial tube maturation and stabilization. ECs form tube networks in 3D extracellular matrices in a manner dependent on integrins, membrane-type metalloproteinases, and the Rho GTPases, Cdc42 and Rac1. Recent work has defined an EC lumen signaling complex of proteins composed of these proteins that controls 3D matrix-specific signaling events required for these processes. The EC tube formation process results in the creation of a network of proteolytically generated vascular guidance tunnels. These tunnels are physical matrix spaces that regulate vascular tube remodeling and represent matrix conduits into which pericytes are recruited to allow dynamic cell-cell interactions with ECs. These dynamic EC-pericyte interactions induce vascular basement membrane matrix deposition, leading to vessel maturation and stabilization.

  1. SHORT-ROOT regulates vascular patterning, but not apical meristematic activity in the Arabidopsis root through cytokinin homeostasis.

    PubMed

    Hao, Yueling; Cui, Hongchang

    2012-03-01

    SHORT-ROOT (SHR) is a key regulator of radial patterning and stem-cell renewal in the Arabidopsis root. Although SHR is expressed in the stele, its function in the vascular tissue was not recognized until recently. In shr, the protoxylem is missing due to the loss of expression of microRNA165A (miR165A) and microRNA166B (miR165B). shr is also defective in lateral root formation, but the mechanism remains unclear. To dissect the SHR developmental pathway, we recently have identified its direct targets at the genome scale by chromatin immunoprecipitation followed by microarray analysis (ChIP-chip). In further studies, we have shown that SHR regulates cytokinin homeostasis through cytokinin oxidase 3 and that this role of SHR is critical to vascular patterning in the root. In this communication we report that SHR also regulates miR165A and miR166B indirectly through its effect on cytokinin homeostasis. Although cytokinin is inhibitory to root growth, the root-apical-meristem defect in shr was not alleviated by reduction of endogenous cytokinin. These results together suggest that SHR regulates vascular patterning, but not root apical meristematic activity, through cytokinin homeostasis.

  2. 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.

  3. Vascular development of the grapevine (Vitis vinifera L.) inflorescence rachis in response to flower number, plant growth regulators and defoliation.

    PubMed

    Gourieroux, Aude M; Holzapfel, Bruno P; McCully, Margaret E; Scollary, Geoffrey R; Rogiers, Suzy Y

    2017-09-01

    The grapevine inflorescence is a determinate panicle and as buds emerge, shoot, flower and rachis development occur simultaneously. The growth and architecture of the rachis is determined by genetic and environmental factors but here we examined the role of flower and leaf number as well as hormones on its elongation and vascular development. The consequences of rachis morphology and vascular area on berry size and composition were also assessed. One week prior to anthesis, Merlot and Cabernet Sauvignon field vines were exposed to manual flower removal, exogenous plant growth regulators or pre-bloom leaf removal. Manual removal of half the flowers along the vertical axis of the inflorescence resulted in a shorter rachis in both cultivars. Conversely, inflorescences treated with gibberellic acid (GA 3 ) and the synthetic cytokinin, 6-benzylaminopurine (BAP) resulted in a longer rachis while pre-bloom removal of all leaves on the inflorescence-bearing shoot did not alter rachis length relative to untreated inflorescences. Across the treatments, the cross-sectional areas of the conducting xylem and phloem in the rachis were positively correlated to rachis girth, flower number at anthesis, bunch berry number, bunch berry fresh mass and bunch sugar content at harvest. Conversely, average berry size and sugar content were not linked to rachis vascular area. These data indicate that the morphological and vascular development of the rachis was more responsive to flower number and plant growth regulators than to leaf removal.

  4. Regulation of vascular signalling by nuclear Sprouty2 in fetal lung epithelial cells: Implications for co-ordinated airway and vascular branching in lung development.

    PubMed

    Walker, David J; Land, Stephen C

    2018-02-01

    Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub-population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3' sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39 kDa form and this was accompanied by histone-3 S10K14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-1α activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Vascular disease in COPD: Systemic and pulmonary expression of PARC (Pulmonary and Activation-Regulated Chemokine)

    PubMed Central

    Muñoz-Esquerre, Mariana; Aliagas, Elisabet; López-Sánchez, Marta; Escobar, Ignacio; Huertas, Daniel; Penín, Rosa; Dorca, Jordi; Santos, Salud

    2017-01-01

    Introduction The role of Pulmonary and Activation-Regulated Chemokine (PARC) in the physiopathology of Chronic Obstructive Pulmonary Disease (COPD) is not fully understood. The aim of the present study is to analyze the expression of PARC in lung tissue and its relationship with the vascular remodeling of the systemic and pulmonary arteries of COPD subjects. Methods To achieve this objective, protein and gene expression experiments, together with ELISA assays, were performed on the lung tissue, intercostal arteries and serum samples from COPD patients, non-obstructed smokers (NOS) and never-smokers (NS). Results A total of 57 subjects were included in the analysis (23 COPD, 18 NOS and 16 NS). In the comparisons between groups, a significantly increased lung protein expression of PARC was observed in the COPD group compared to the NOS group (1.96±0.22 vs. 1.29±0.27, P-adjusted = 0.038). PARC was located predominantly in the smooth muscle cells of the remodeled pulmonary muscular arteries and the macrophage-rich area of the alveolar parenchyma. No differences were detected in PARC gene expression analyses. The protein content of PARC in the intercostal arteries were similar between groups, though little remodeling was observed in these arteries. Circulating levels of PARC were numerically higher in patients with COPD compared to NOS and NS. Conclusion The results of the present study suggest an increased lung protein expression of PARC in COPD subjects. This protein was mainly localized in the smooth muscle cells of the pulmonary muscular arteries and was associated with the severity of intimal thickening, indicating its possible role in this remodeling process. PMID:28545096

  6. Vascular Endothelial Growth Factor Receptor-3 Directly Interacts with Phosphatidylinositol 3-Kinase to Regulate Lymphangiogenesis

    PubMed Central

    Coso, Sanja; Zeng, Yiping; Opeskin, Kenneth; Williams, Elizabeth D.

    2012-01-01

    Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCγ1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis. PMID:22745786

  7. Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis.

    PubMed

    Coso, Sanja; Zeng, Yiping; Opeskin, Kenneth; Williams, Elizabeth D

    2012-01-01

    Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCγ1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

  8. Relationship between skin blood flow regulation mechanisms and vascular endothelial growth factor in patients with metabolic syndrome.

    PubMed

    Smirnova, E; Shulkina, S; Loran, E; Podtaev, S; Antonova, N

    2017-08-11

    The focus of this paper is the determination of endothelial dysfunction in patients with metabolic syndrome (MetS) and the establishment of a relationship between the traditional biomarkers of endothelial dysfunction and the vascular tone regulation indices obtained from indirect cold tests in MetS patients. Our investigation was conducted on 30 patients aged 45.5±9 years. The control group comprised 14 healthy subjects aged 48.2±2.4 years. The mechanism of vascular tone regulation was investigated using the wavelet analysis of skin temperature oscillations (WAST). The degrees of microvascular vasoconstriction and vasodilatation were determined during contralateral cold tests in the endothelial (0.02-0.0095 Hz), neurogenic (0.05-0.02 Hz) and myogenic (0.05-0.14 Hz) frequency ranges. In MetS patients, vasoconstriction indices were higher and vasodilatation indices were lower than in the subjects of the control group, which is indicative of disorders in the mechanisms of microvascular tone regulation. These indices correlate with the metabolic parameters and VEGF (vascular endothelial growth factor) levels. The correlation of vasoconstriction and vasodilatation indices with the main factors of the metabolic syndrome testifies that the biological and functional aspects of the endothelial dysfunction are closely related.

  9. The prolactin family hormones regulate vascular tone through NO and prostacyclin production in isolated rat aortic rings

    PubMed Central

    Gonzalez, Carmen; Rosas-Hernandez, Hector; Jurado-manzano, Brenda; Ramirez-Lee, Manuel Alejandro; Salazar-Garcia, Samuel; Martinez-Cuevas, Pedro Pablo; Velarde-salcedo, Aída Jimena; Morales-Loredo, Humberto; Espinosa-Tanguma, Ricardo; Ali, Syed F; Rubio, Rafael

    2015-01-01

    Aim: Prolactin family hormones include growth hormone, placental lactogen and prolactin, which are able to regulate angiogenesis via NO and prostaglandins. However, their effects on vascular tone are not fully understood. The aim of this study was to evaluate the effects of prolactin family hormones on rat vascular tone in vitro. Methods: Aortic rings were prepared from adult male rats and precontracted with phenylephrine, then treated with the hormones and drugs. The tension was measured with isometric force displacement transducer connected to a polygraph. NO production and prostacyclin release in physiological solution was determined. Cultured rat aortic endothelial cells (RAECs) were treated with the hormones and drugs, and the phosphorylation of eNOS at serine 1177 was assessed using Western bolt analysis. Results: Administration of growth hormone or placental lactogen (0.01–100 nmol/L) induced endothelium-dependent vasodilation. Both the hormones significantly increased the phosphorylation of eNOS in RAECs and NO level in physiological solution. Preincubation with L-NAME blocked growth hormone- or placental lactogen-induced vasodilation and NO production. Preincubation with an antibody against growth hormone receptors blocked growth hormone- and placental lactogen-induced vasodilation. Addition of a single dose of prolactin (0.01 nmol/L) induced sustained vessel relaxation, whereas multiple doses of prolactin induced a biphasic contraction-relaxation effect. The vascular effects of prolactin depended on endothelium. Prolactin significantly increased the level of prostacyclin I2 in physiological solution. Preincubation with indomethacin or an antibody against prolactin receptors blocked prolactin-induced vasodilation. Conclusion: The prolactin family hormones regulate rat vascular tone, selectively promoting either relaxation or contraction of vascular smooth muscle via activation of either growth hormone receptors or prolactin receptors within the

  10. Angiotensin-(1-7) regulates Angiotensin II-induced VCAM-1 expression on vascular endothelial cells

    SciTech Connect

    Zhang, Feng; Ren, Jingyi; Chan, Kenneth; Chen, Hong

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We for the first time found that Ang-(1-7) inhibits Ang II-induced VCAM-1 expression. Black-Right-Pointing-Pointer The inhibitory effect of Ang-(1-7) on VCAM-1 is mediated by MAS receptor. Black-Right-Pointing-Pointer The effect of Ang-(1-7) is due to the suppression of NF-kappaB translocation. -- Abstract: Angiotensin II (Ang II) and Angiotensin-(1-7) (Ang-(1-7)) are key effector peptides in the renin-angiotensin system. Increased circulatory Ang II level is associated with the development of hypertension and atherosclerosis, whereas Ang-(1-7) is a counter-regulatory mediator of Ang II which appears to be protective against cardiovascular disease. However, whether Ang-(1-7) regulates the action of Ang II on vascular endothelial cells (EC) remains unclear. We investigated the effects of Ang II and Ang-(1-7) in the context of atherogenesis, specifically endothelial cell VCAM-1 expression that is implicated in early plaque formation. The results show that Ang II increased VCAM-1 mRNA expression and protein displayed on EC surface, while Ang-(1-7) alone exerted no effects. However, Ang-(1-7) significantly suppressed Ang II-induced VCAM-1 expression. Ang-(1-7) also inhibited the Ang II-induced VCAM-1 promoter activity driven by transcription factor NF-KappaB. Furthermore, immunofluorescence assay and ELISA showed that Ang II facilitated the nuclear translocation of NF-kappaB in ECs, and this was attenuated by the presence of Ang-(1-7). The inhibitory effects of Ang-(1-7) on Ang II-induced VCAM-1 promoter activity and NF-kappaB nuclear translocation were all reversed by the competitive antagonist of Ang-(1-7) at the Mas receptor. Our results suggest that Ang-(1-7) mediates its affects on ECs through the Mas receptor, and negatively regulates Ang II-induced VCAM-1 expression by attenuating nuclear translocation of NF-kappaB.

  11. Extracellular signal-regulated kinase-5: Novel mediator of insulin and tumor necrosis factor α-stimulated vascular cell adhesion molecule-1 expression in vascular cells.

    PubMed

    Mackesy, Daniel Z; Goalstone, Marc L

    2014-11-01

    Atherosclerosis may be stimulated by the increased presence of insulin and tumor necrosis-factor-α (TNFα) with subsequent expression of vascular cell adhesion molecule-1 (VCAM-1). We hypothesized that extracellular signal-regulated kinase-5 (ERK5) plays an important role in insulin and TNFα-stimulated total and cell surface VCAM-1 expression. Rat aorta vascular endothelial cells were first transfected with either no inhibitory RNA, inactive (scrambled) inhibitory ERK5 RNA (scERK5) or active inhibitory ERK5 RNA (siERK5) and then treated with either (i) no analog; (ii) insulin (1 nM), or TNFα (1 ng/mL) alone, or (iii) insulin plus TNFα for 6 h. Thereafter either total VCAM-1 protein or surface VCAM-1 protein was determined. Genetic inhibition of ERK5 decreased TNFα-stimulated total VCAM-1 expression by 57% and surface expression by 27%. In contrast, genetic inhibition of ERK5 did not significantly decrease insulin-stimulated total or surface VCAM-1 expression. Interestingly, genetic inhibition of ERK5 did not significantly decrease insulin plus TNFα-stimulated total VCAM-1 expression, but significantly (P < 0.05) decreased insulin plus TNFα-stimulated surface VCAM-1 expression 41%. We report here that ERK5 plays a minor role in insulin-stimulation of VCAM-1, but plays a significant role in TNFα-stimulation of both total and cell surface VCAM-1 protein expression. Taken together, these results demonstrate that not only does ERK5 have differential mediation of insulin and TNFα-stimulated VCAM-1 expression, but also has differential regulation of insulin plus TNFα-stimulated total and surface VCAM-1 expression, suggesting that other intermediates of the insulin and TNFα intracellular pathways are contributing to atherogenesis. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

  12. Multi-Scale Optical Imaging of the Delayed Type Hypersensitivity Reaction Attenuated by Rapamycin

    PubMed Central

    Luo, Meijie; Zhang, Zhihong; Li, Hui; Qiao, Sha; Liu, Zheng; Fu, Ling; Shen, Guanxin; Luo, Qingming

    2014-01-01

    Neutrophils and monocytes/macrophages (MMs) play important roles in the development of cell-mediated delayed type hypersensitivity (DTH). However, the dynamics of neutrophils and MMs during the DTH reaction and how the immunosuppressant rapamycin modulates their behavior in vivo are rarely reported. Here, we take advantage of multi-scale optical imaging techniques and a footpad DTH reaction model to non-invasively investigate the dynamic behavior and properties of immune cells from the whole field of the footpad to the cellular level. During the classic elicitation phase of the DTH reaction, both neutrophils and MMs obviously accumulated at inflammatory foci at 24 h post-challenge. Rapamycin treatment resulted in advanced neutrophil recruitment and vascular hyperpermeability at an early stage (4 h), the reduced accumulation of neutrophils (> 50% inhibition ratio) at 48 h, and the delayed involvement of MMs in inflammatory foci. The motility parameters of immune cells in the rapamycin-treated reaction at 4 h post-challenge displayed similar mean velocities, arrest durations, mean displacements, and confinements as the classic DTH reaction at 24 h. These results indicate that rapamycin treatment shortened the initial preparation stage of the DTH reaction and attenuated its intensity, which may be due to the involvement of T helper type 2 cells or regulatory T cells. PMID:24465276

  13. How longevity research can lead to therapies for Alzheimer's disease: The rapamycin story.

    PubMed

    Richardson, Arlan; Galvan, Veronica; Lin, Ai-Ling; Oddo, Salvatore

    2015-08-01

    The discovery that rapamycin increases lifespan in mice and restores/delays many aging phenotypes has led to the speculation that rapamycin has 'anti-aging' properties. The major question discussed in this review is whether a manipulation that has anti-aging properties can alter the onset and/or progression of Alzheimer's disease, a disease in which age is the major risk factor. Rapamycin has been shown to prevent (and possibly restore in some cases) the deficit in memory observed in the mouse model of Alzheimer's disease (AD-Tg) as well as reduce Aβ and tau aggregation, restore cerebral blood flow and vascularization, and reduce microglia activation. All of these parameters are widely recognized as symptoms central to the development of AD. Furthermore, rapamycin has also been shown to improve memory and reduce anxiety and depression in several other mouse models that show cognitive deficits as well as in 'normal' mice. The current research shows the feasibility of using pharmacological agents that increase lifespan, such as those identified by the National Institute on Aging Intervention Testing Program, to treat Alzheimer's disease. Published by Elsevier Inc.

  14. Inhibition of mTOR kinase via rapamycin blocks persistent predator stress-induced hyperarousal.

    PubMed

    Fifield, Kathleen; Hebert, Mark; Angel, Rebecca; Adamec, Robert; Blundell, Jacqueline

    2013-11-01

    Traumatic, stressful life events are thought to trigger acquired anxiety disorders such as post-traumatic stress disorder (PTSD). Recent data suggests that the mammalian target of rapamycin (mTOR) plays a key role in the formation of traumatic memories. The predator stress paradigm allows us to determine whether mTOR mediates the formation of both context-dependent (associative) and context-independent (non-associative) fear memories. Predator stress involves an acute, unprotected exposure of a rat to a cat which causes long-lasting non-associative fear memories manifested as generalized hyperarousal and increased anxiety-like behavior. Here, we show that rapamycin, an mTOR inhibitor, attenuates predator stress-induced hyperarousal, lasting at least three weeks. In addition, rapamycin blocks a subset of anxiety-like behaviors as measured in the elevated plus maze and hole board. Furthermore, when re-exposed to the predator stress context, rapamycin-treated stressed rats showed increased activity compared to vehicle controls suggesting that rapamycin blocks predator stress-induced associative fear memory. Taken together with past research, our results indicate that mTOR regulation of protein translation is required for the formation of both associative and non-associative fear memories. Overall, these data suggest that mTOR activation may contribute to the development of acquired anxiety disorders such as PTSD. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Role of chemokine RANTES in the regulation of perivascular inflammation, T-cell accumulation, and vascular dysfunction in hypertension

    PubMed Central

    Mikolajczyk, Tomasz P.; Nosalski, Ryszard; Szczepaniak, Piotr; Budzyn, Klaudia; Osmenda, Grzegorz; Skiba, Dominik; Sagan, Agnieszka; Wu, Jing; Vinh, Antony; Marvar, Paul J.; Guzik, Bartlomiej; Podolec, Jakub; Drummond, Grant; Lob, Heinrich E.; Harrison, David G.; Guzik, Tomasz J.

    2016-01-01

    Recent studies have emphasized the role of perivascular inflammation in cardiovascular disease. We studied mechanisms of perivascular leukocyte infiltration in angiotensin II (Ang II)-induced hypertension and their links to vascular dysfunction. Chronic Ang II infusion in mice increased immune cell content of T cells (255 ± 130 to 1664 ± 349 cells/mg; P < 0.01), M1 and M2 macrophages, and dendritic cells in perivascular adipose tissue. In particular, the content of T lymphocytes bearing CC chemokine receptor (CCR) 1, CCR3, and CCR5 receptors for RANTES chemokine was increased by Ang II (CCR1, 15.6 ± 1.5% vs. 31 ± 5%; P < 0.01). Hypertension was associated with an increase in perivascular adipose tissue expression of the chemokine RANTES (relative quantification, 1.2 ± 0.2 vs. 3.5 ± 1.1; P < 0.05), which induced T-cell chemotaxis and vascular accumulation of T cells expressing the chemokine receptors CCR1, CCR3, and CCR5. Mechanistically, RANTES−/− knockout protected against vascular leukocyte, and in particular T lymphocyte infiltration (26 ± 5% in wild type Ang II vs. 15 ± 4% in RANTES−/−), which was associated with protection from endothelial dysfunction induced by Ang II. This effect was linked with diminished infiltration of IFN-γ-producing CD8+ and double-negative CD3+CD4−CD8− T cells in perivascular space and reduced vascular oxidative stress while FoxP3+ T-regulatory cells were unaltered. IFN-γ ex vivo caused significant endothelial dysfunction, which was reduced by superoxide anion scavenging. In a human cohort, a significant inverse correlation was observed between circulating RANTES levels as a biomarker and vascular function measured as flow-mediated dilatation (R = −0.3, P < 0.01) or endothelial injury marker von Willebrand factor (R = +0.3; P < 0.01). Thus, chemokine RANTES is important in the regulation of vascular dysfunction through modulation of perivascular inflammation.—Mikolajczyk, T. P., Nosalski, R., Szczepaniak, P

  16. 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. Copyright © 2016 by the American Society of Nephrology.

  17. MicroRNA regulates vascular endothelial growth factor expression in chondrosarcoma cells.

    PubMed

    Sun, Xiaojuan; Wei, Lei; Chen, Qian; Terek, Richard M

    2015-03-01

    Systemic treatments to prevent or treat chondrosarcoma metastasis are lacking and targeted therapy has yet to be developed. Hypoxia develops in tumors as they grow and hypoxia-related alterations in gene expression underlie some of the traits of cancer. One critical trait is the ability to induce sustained angiogenesis, which is usually related to expression of vascular endothelial growth factor (VEGF). A potential hypoxia-related mechanism resulting in altered gene expression involves microRNA. Little is known about microRNA expression in chondrosarcoma and its potential role in regulation of VEGF expression. Our purposes were (1) to determine if there is hypoxia-regulated microRNA overexpressed in chondrosarcoma; (2) if that contributes to increased VEGF expression; and (3) can VEGF expression be inhibited with a specific antagomir? MicroRNA expression was analyzed in two primary human chondrosarcomas and articular cartilage using array analysis and a cutoff of a fourfold difference in expression between tumor and normal tissue. The effects of hypoxia and hypoxia-inducible factor-1α (HIF-1α) transfection and silencing with siRNA on expression of candidate microRNAs were analyzed in chondrosarcoma cell line JJ. VEGF expression was measured with quantitative polymerase chain reaction and enzyme-linked immunosorbent assay after specific microRNA transfection and knockdown. miR-181a was identified by array analysis and confirmed with quantitative reverse transcription-polymerase chain reaction, which showed that miR-181a was overexpressed in both human chondrosarcomas (33- and 55-fold) and the JJ cell line (sixfold) compared with cartilage and chondrocytes, respectively. In vitro, hypoxia and HIF-1α transfection each further increased miR-181a expression twofold in JJ cells. miR-181a transfection of JJ cells doubled expression of VEGF mRNA and increased secreted VEGF protein by 46% in normoxia, an effect that could be either direct or indirect. Similar enhancement

  18. Interferon regulatory factor-1 as a positive regulator for high glucose-induced proliferation of vascular smooth muscle cells.

    PubMed

    Yu, Sun; Xi, Zhang; Hai-Yan, Chen; Ya-Li, Chi; Shao-Hu, Xiong; Chuan-Sen, Zhang; Xiang-Qun, Yang; Jin-Ping, Guo; Hai-Yan, Lin; Lei, Dong

    2012-08-01

    High glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. However, molecular mediators responding for the proliferation of VSMCs remain to be determined. In this study, VSMCs were isolated from the rat thoracic aorta, and two cell models with Irf-1 knockdown and overexpression were established by transfecting cells with pGCsi-FU-Irf-1 and pGC-FU-Irf-1, respectively. Subsequently, high glucose was added to cells to induce proliferation. Proliferation assays were performed to see whether Irf-1 was involved in high glucose-induced proliferation of VSMCs. In addition, the expression of Irf-1 was detected in VSMCs stimulated with high glucose and the thoracic aorta of diabetic rats to confirm the relationship between Irf-1 expression and the proliferation of hyperglycemia-dependent VSMCs. The results showed that Irf-1 expression was significantly higher in the thoracic aorta of diabetic rats and VSMCs stimulated with high glucose than that in nondiabetic rats and untreated cells. Overexpression of Irf-1 accelerated the proliferation of VSMCs, and down-regulation of Irf-1 expression significantly depressed the proliferative ability of VSMCs under high-glucose conditions, indicating that Irf-1 was a positive regulator for high glucose-induced proliferation of VSMCs. It could be presumed that Irf-1 is associated with the accelerated proliferation of VSMCs in diabetic vascular diseases and may prove to be a potential target gene for disease treatment. Copyright © 2012 Wiley Periodicals, Inc.

  19. Coplanar polychlorinated biphenyl-induced CYP1A1 is regulated through caveolae signaling in vascular endothelial cells

    PubMed Central

    Lim, Eun Jin; Májková, Zuzana; Xu, Shifen; Bachas, Leonidas; Arzuaga, Xabier; Smart, Eric; Tseng, Michael T.; Toborek, Michal; Hennig, Bernhard

    2008-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that can induce inflammatory processes in the vascular endothelium. We hypothesize that the plasma membrane microdomains called caveolae are critical in endothelial activation and toxicity induced by PCBs. Caveolae are particularly abundant in endothelial cells and play a major role in endothelial trafficking and the regulation of signaling pathways associated with the pathology of vascular diseases. We focused on the role of caveolae and their major protein component, caveolin-1 (Cav-1), on aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 1A1 (CYP1A1) by coplanar PCBs. Endothelial cell exposure to PCB77 increased both caveolin-1 and CYP1A1 levels in a time-dependent manner in total cell lysates, with a maximum increase at 6 h. Furthermore, PCB77 accumulated mainly in the caveolae-rich fraction, as determined by gas chromatograph-mass spectrometry. Immunoprecipitation analysis revealed that PCB77 increased AhR binding to caveolin-1. Silencing of caveolin-1 significantly attenuated PCB77-mediated induction of CYP1A1 and oxidative stress. Similar effects were observed in caveolin-1 null mice treated with PCB77. These data suggest that caveolae may play a role in regulating vascular toxicity induced by persistent environmental pollutants such as coplanar PCBs. This may have implications in understanding mechanisms of inflammatory diseases induced by environmental pollutants. PMID:18786521

  20. Slingshot isoform-specific regulation of cofilin-mediated vascular smooth muscle cell migration and neointima formation.

    PubMed

    Torres, Rebecca A; Drake, Douglas A; Solodushko, Viktoriya; Jadhav, Rashmi; Smith, Erika; Rocic, Petra; Weber, David S

    2011-11-01

    We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the "wound," and platelet-derived growth factor (PDGF)-induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

  1. Podocalyxin Regulates Murine Lung Vascular Permeability by Altering Endothelial Cell Adhesion

    PubMed Central

    Debruin, Erin J.; Hughes, Michael R.; Sina, Christina; Liu, Alex; Cait, Jessica; Jian, Zhiqi; Lopez, Martin; Lo, Bernard; Abraham, Thomas; McNagny, Kelly M.

    2014-01-01

    Despite the widespread use of CD34-family sialomucins (CD34, podocalyxin and endoglycan) as vascular endothelial cell markers, there is remarkably little known of their vascular function. Podocalyxin (gene name Podxl), in particular, has been difficult to study in adult vasculature as germ-line deletion of podocalyxin in mice leads to kidney malformations and perinatal death. We generated mice that conditionally delete podocalyxin in vascular endothelial cells (PodxlΔEC mice) to study the homeostatic role of podocalyxin in adult mouse vessels. Although PodxlΔEC adult mice are viable, their lungs display increased lung volume and changes to the matrix composition. Intriguingly, this was associated with increased basal and inflammation-induced pulmonary vascular permeability. To further investigate the etiology of these defects, we isolated mouse pulmonary endothelial cells. PodxlΔEC endothelial cells display mildly enhanced static adhesion to fibronectin but spread normally when plated on fibronectin-coated transwells. In contrast, PodxlΔEC endothelial cells exhibit a severely impaired ability to spread on laminin and, to a lesser extent, collagen I coated transwells. The data suggest that, in endothelial cells, podocalyxin plays a previously unrecognized role in maintaining vascular integrity, likely through orchestrating interactions with extracellular matrix components and basement membranes, and that this influences downstream epithelial architecture. PMID:25303643

  2. Protective effects of hydrogen-rich medium on lipopolysaccharide-induced monocytic adhesion and vascular endothelial permeability through regulation of vascular endothelial cadherin.

    PubMed

    Yu, Y; Wang, W N; Han, H Z; Xie, K L; Wang, G L; Yu, Y H

    2015-06-11

    We observed the effect of hydrogen-rich medium on lipopolysaccharide (LPS)-induced human umbilical vein endothelial cells (HUVECs), hyaline leukocyte conglutination, and permeability of the endothelium. Endotheliocytes were inoculated on 6-well plates and randomly divided into 4 groups: control, H2, LPS, LPS+H2, H2, and LPS+H2 in saturated hydrogen-rich medium. We applied Wright's stain-ing to observe conglutination of hyaline leukocytes and HUVECs, flow cytometry to determine the content of vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), enzyme-linked immunosorbent assay to measure the E-selectin concentration in the cell liquor, the transendothelial electrical resistance (TEER) to test the permeability of endothelial cells, and Western blot and immunofluorescence to test the expression and distribution of vascular endothelial (VE)-cadherin. Compared with control cells, there was an increase in endothelium-hyaline leukocyte conglutination, a reduction in VCAM-1, ICAM-1, and E-selectin, and the TEER value increased obviously. Compared with LPS, there was an obvious reduction in the conglutination of LPS+H2 cells, a reduction in VCAM-1, ICAM-1, and E-selectin levels, and a reduction in the TEER-resistance value, while the expression of VE-cadherin increased. Fluorescence results showed that, compared with control cells, the VE-cadherin in LPS cells was in-complete at the cell joints. Compared with LPS cells, the VE-cadherin in LPS+H2 cells was even and complete at the cell joints. Liquid rich in hydrogen could reduce LPS-induced production of adhesion molecules and endothelium-hyaline leukocyte conglutination, and influence the expression and distribution of VE-cadherin to regulate the permeability of the endothelium.

  3. Regulatory T Cell-Enriching Microparticles for Promoting Vascularized Composite Allotransplant Survival

    DTIC Science & Technology

    2016-10-01

    hindlimb transplants. Particles containing IL-2,TGFb, and rapamycin were fabricated and this triple cocktail, along with all pairwise iterations of...suppressive regulatory T cells. These particles , referred to as Expansion MP (IL2, TGF, and Rapamycin microparticles) and Recruitment MP (CCL22 loaded...observed effects these particles have on allograft survival. Key Words CTA Composite Tissue Allotransplantation VCA Vascularized Composite

  4. Glutathione regulation of redox-sensitive signals in tumor necrosis factor-{alpha}-induced vascular endothelial dysfunction

    SciTech Connect

    Tsou, T.-C. . E-mail: tctsou@nhri.org.tw; Yeh, S.C.; Tsai, F.-Y.; Chen, J.-W.; Chiang, H.-C.

    2007-06-01

    We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-{alpha})-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte-endothelial monolayer binding. Since TNF-{alpha} induces various biological effects on vascular cells, TNF-{alpha} dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-{alpha} concentrations, we adopted the lower TNF-{alpha} (0.2 ng/ml) to rule out the possible involvement of other TNF-{alpha}-induced biological effects. Inhibition of glutathione synthesis by L-buthionine-(S,R)-sulfoximine (BSO) resulted in down-regulations of the TNF-{alpha}-induced adhesion molecule expression and monocyte-endothelial monolayer binding. BSO attenuated the TNF-{alpha}-induced nuclear factor-kappaB (NF-{kappa}B) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-{alpha}. Inhibition of ERK, JNK, or NF-{kappa}B attenuates TNF-{alpha}-induced ICAM-1 promoter activation and monocyte-endothelial monolayer binding. Our study indicates that TNF-{alpha} induces adhesion molecule expression and monocyte-endothelial monolayer binding mainly via activation of NF-{kappa}B in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-{alpha}. Although AP-1 activation by the lower TNF-{alpha} was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-{alpha}-induced adhesion molecule expression.

  5. 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.

  6. Glutathione regulation of redox-sensitive signals in tumor necrosis factor-{alpha}-induced vascular endothelial dysfunction

    SciTech Connect

    Tsou, T.-C.; Yeh, S.C.; Tsai, F.-Y.

    2007-06-01

    We investigated the regulatory role of glutathione in tumor necrosis factor-alpha (TNF-{alpha})-induced vascular endothelial dysfunction as evaluated by using vascular endothelial adhesion molecule expression and monocyte-endothelial monolayer binding. Since TNF-{alpha} induces various biological effects on vascular cells, TNF-{alpha} dosage could be a determinant factor directing vascular cells into different biological fates. Based on the adhesion molecule expression patterns responding to different TNF-{alpha} concentrations, we adopted the lower TNF-{alpha} (0.2 ng/ml) to rule out the possible involvement of other TNF-{alpha}-induced biological effects. Inhibition of glutathione synthesis by L-buthionine-(S,R)-sulfoximine (BSO) resulted in down-regulations of the TNF-{alpha}-induced adhesion molecule expression and monocyte-endothelial monolayermore » binding. BSO attenuated the TNF-{alpha}-induced nuclear factor-kappaB (NF-{kappa}B) activation, however, with no detectable effect on AP-1 and its related mitogen-activated protein kinases (MAPKs). Deletion of an AP-1 binding site in intercellular adhesion molecule-1 (ICAM-1) promoter totally abolished its constitutive promoter activity and its responsiveness to TNF-{alpha}. Inhibition of ERK, JNK, or NF-{kappa}B attenuates TNF-{alpha}-induced ICAM-1 promoter activation and monocyte-endothelial monolayer binding. Our study indicates that TNF-{alpha} induces adhesion molecule expression and monocyte-endothelial monolayer binding mainly via activation of NF-{kappa}B in a glutathione-sensitive manner. We also demonstrated that intracellular glutathione does not modulate the activation of MAPKs and/or their downstream AP-1 induced by lower TNF-{alpha}. Although AP-1 activation by the lower TNF-{alpha} was not detected in our systems, we could not rule out the possible involvement of transiently activated MAPKs/AP-1 in the regulation of TNF-{alpha}-induced adhesion molecule expression.« less

  7. Rapamycin selectively alters serum chemistry in diabetic mice.

    PubMed

    Tabatabai-Mir, Hooman; Sataranatarajan, Kavithalakshmi; Lee, Hak Joo; Bokov, Alex F; Fernandez, Elizabeth; Diaz, Vivian; Choudhury, Goutam Ghosh; Richardson, Arlan; Kasinath, Balakuntalam S

    2012-01-01

    The study was undertaken to explore the effect of rapamycin, an anti-inflammatory agent, on the metabolic profile of type 2 diabetic mice. Seven-month-old diabetic db/db mice and their lean littermate non-diabetic controls (db/m) were randomized to receive control chow or chow mixed with rapamycin (2.24 mg/kg/day) (each group n =20, males and females) for 4 months and sacrificed. Serum samples were analyzed for the measurement of glucose, creatinine, blood urea nitrogen (BUN), alkaline phosphatase (ALP), alanine aminotransferase (ALT), total cholesterol, total triglyceride, and total protein, using the automated dry chemistry analysis. Rapamycin elevated serum glucose in female diabetic mice. Serum creatinine tended to be higher in diabetic mice but was not affected by rapamycin; there was no difference in BUN levels among the groups. Serum ALP was elevated in diabetic mice and rapamycin lowered it only in female diabetic mice; serum ALT levels were increased in female diabetic mice, unaffected by rapamycin. Serum total protein was elevated in diabetic mice of both genders but was not affected by rapamycin. Diabetic mice from both genders had elevated serum cholesterol and triglycerides; rapamycin did not affect serum cholesterol but decreased serum total triglycerides in male diabetic mice. We conclude that rapamycin elicits complex metabolic responses in aging diabetic mice, worsening hyperglycemia in females but improving ALP in female diabetic and total triglycerides in male diabetic mice, respectively. The metabolic effects of rapamycin should be considered while performing studies with rapamycin in mice.

  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. Up-regulation of vascular endothelial growth factor/vascular permeability factor in mouse skin carcinogenesis correlates with malignant progression state and activated H-ras expression levels.

    PubMed

    Larcher, F; Robles, A I; Duran, H; Murillas, R; Quintanilla, M; Cano, A; Conti, C J; Jorcano, J L

    1996-12-01

    Angiogenesis is a crucial process for tumor growth and metastasis regulated by the balance of positive and negative factors. Vascular endothelial growth factor (VEGF/VPF) is a specific mitogen for endothelial cells that has been shown to be overexpressed in a variety of tumors and other inflammatory diseases. To analyze the implication of VEGF/VPF during tumorigenesis, we have studied its expression at different stages of tumor development using the mouse skin carcinogenesis model. VEGF/VPF mRNA was induced in skin in vivo after 12-O-tetradecanoylphorbol-13-acetate treatment. Constitutive up-regulation of VEGF/VPF at the mRNA and protein levels was also observed in premalignant papillomas and, at a higher level, in squamous carcinomas, suggesting a correlation between VEGF/VPF expression and tumor progression. A direct positive correlation between VEGF/VPF mRNA expression and the level of activated H-ras gene was found in a series of cell lines representing different stages of epidermal tumor development. Consequently, a clone of one of these cell lines, HaCa4, which has lost most of its v-ras expression, down-regulated VEGF mRNA expression concomitantly with its metastatic potential. Direct evidence of H-ras involvement in VEGF induction was obtained when an immortalized mouse keratinocyte cell line transduced with a retrovirus carrying v-H-ras showed highly increased VEGF/VPF mRNA levels. These data show that in mouse skin carcinogenesis, the VEGF/VPF angiogenic stimulus occurs early during premalignant papilloma development and further increases at later stages. Moreover, we demonstrate that increasing the activated H-ras dose, a phenomenon that takes place sequentially throughout mouse skin tumor development, may play an additional role by facilitating malignant in vivo progression through the modulation of VEGF/VPF-mediated angiogenesis.

  10. Activation of AKT by O-linked N-acetylglucosamine induces vascular calcification in diabetes mellitus.

    PubMed

    Heath, Jack M; Sun, Yong; Yuan, Kaiyu; Bradley, Wayne E; Litovsky, Silvio; Dell'Italia, Louis J; Chatham, John C; Wu, Hui; Chen, Yabing

    2014-03-28

    Vascular calcification is a serious cardiovascular complication that contributes to the increased morbidity and mortality of patients with diabetes mellitus. Hyperglycemia, a hallmark of diabetes mellitus, is associated with increased vascular calcification and increased modification of proteins by O-linked N-acetylglucosamine (O-GlcNAcylation). We sought to determine the role of protein O-GlcNAcylation in regulating vascular calcification and the underlying mechanisms. Low-dose streptozotocin-induced diabetic mice exhibited increased aortic O-GlcNAcylation and vascular calcification, which was also associated with impaired aortic compliance in mice. Elevation of O-GlcNAcylation by administration of Thiamet-G, a potent inhibitor for O-GlcNAcase that removes O-GlcNAcylation, further accelerated vascular calcification and worsened aortic compliance of diabetic mice in vivo. Increased O-GlcNAcylation, either by Thiamet-G or O-GlcNAcase knockdown, promoted calcification of primary mouse vascular smooth muscle cells. Increased O-GlcNAcylation in diabetic arteries or in the O-GlcNAcase knockdown vascular smooth muscle cell upregulated expression of the osteogenic transcription factor Runx2 and enhanced activation of AKT. O-GlcNAcylation of AKT at two new sites, T430 and T479, promoted AKT phosphorylation, which in turn enhanced vascular smooth muscle cell calcification. Site-directed mutation of AKT at T430 and T479 decreased O-GlcNAcylation, inhibited phosphorylation of AKT at S473 and binding of mammalian target of rapamycin complex 2 to AKT, and subsequently blocked Runx2 transactivity and vascular smooth muscle cell calcification. O-GlcNAcylation of AKT at 2 new sites enhanced AKT phosphorylation and activation, thus promoting vascular calcification. Our studies have identified a novel causative effect of O-GlcNAcylation in regulating vascular calcification in diabetes mellitus and uncovered a key molecular mechanism underlying O-GlcNAcylation-mediated activation of

  11. Light-triggered in vivo activation of adhesive peptides regulates cell adhesion, inflammation and vascularization of biomaterials

    NASA Astrophysics Data System (ADS)

    Lee, Ted T.; García, José R.; Paez, Julieta I.; Singh, Ankur; Phelps, Edward A.; Weis, Simone; Shafiq, Zahid; Shekaran, Asha; Del Campo, Aránzazu; García, Andrés J.

    2015-03-01

    Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have recently been realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell-adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials.

  12. Light-triggered in vivo Activation of Adhesive Peptides Regulates Cell Adhesion, Inflammation and Vascularization of Biomaterials

    PubMed Central

    Lee, Ted T.; García, José R.; Paez, Julieta; Singh, Ankur; Phelps, Edward A.; Weis, Simone; Shafiq, Zahid; Shekaran, Asha; del Campo, Aránzazu; García, Andrés J.

    2014-01-01

    Materials engineered to elicit targeted cellular responses in regenerative medicine must display bioligands with precise spatial and temporal control. Although materials with temporally regulated presentation of bioadhesive ligands using external triggers, such as light and electric fields, have been recently realized for cells in culture, the impact of in vivo temporal ligand presentation on cell-material responses is unknown. Here, we present a general strategy to temporally and spatially control the in vivo presentation of bioligands using cell adhesive peptides with a protecting group that can be easily removed via transdermal light exposure to render the peptide fully active. We demonstrate that non-invasive, transdermal time-regulated activation of cell-adhesive RGD peptide on implanted biomaterials regulates in vivo cell adhesion, inflammation, fibrous encapsulation, and vascularization of the material. This work shows that triggered in vivo presentation of bioligands can be harnessed to direct tissue reparative responses associated with implanted biomaterials. PMID:25502097

  13. Stromal vascular cells and adipogenesis: Cells within adipose depots regulate adipogenesis

    USDA-ARS?s Scientific Manuscript database

    A collection of investigations indicate the importance of adipose tissue stromal/stem cells to vasculogenesis and angiogenesis during adipogenesis. Early in development the stromal-vascular (S-V) elements control and dictate the extent of adipogenesis in a depot dependent manner. For instance, the...

  14. The Inhibitory Effect of Shikonin on the Agonist-Induced Regulation of Vascular Contractility

    PubMed Central

    Je, Hyun Dong; Kim, Hyeong-Dong; La, Hyen-Oh

    2015-01-01

    Shikonin, a natural flavonoid found in the roots of Lithospermum erythrorhizon, has been shown to possess many biological functions. The present study was undertaken to investigate the influence of shikonin on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Shikonin significantly relaxed fluoride-, thromboxane A2- or phorbol ester-induced vascular contraction suggesting as a possible anti-hypertensive on the agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, shikonin significantly inhibited fluoride-induced increases in pMYPT1 levels and phorbol ester-induced increases in pERK1/2 levels suggesting the mechanism involving the inhibition of Rho-kinase activity and the subsequent phosphorylation of MYPT1 and the inhibition of MEK activity and the subsequent phosphorylation of ERK1/2. This study provides evidence regarding the mechanism underlying the relaxation effect of shikonin on agonist-induced vascular contraction regardless of endothelial function. PMID:25995821

  15. Endothelium-Independent Effect of Fisetin on the Agonist-Induced Regulation of Vascular Contractility

    PubMed Central

    Je, Hyun Dong; Sohn, Uy Dong; La, Hyen-Oh

    2016-01-01

    Fisetin, a natural flavonoid found in a variety of vegetables and fruits, has been shown to possess many biological functions. The present study was undertaken to investigate the influence of fisetin on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Fisetin significantly relaxed fluoride-, thromboxane A2- or phorbol ester-induced vascular contraction suggesting as a possible anti-hypertensive on the agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, fisetin significantly inhibited fluoride-induced increases in pMYPT1 levels and phorbol ester-induced increases in pERK1/2 levels suggesting the mechanism involving the inhibition of Rho-kinase activity and the subsequent phosphorylation of MYPT1 and MEK activity and the subsequent phosphorylation of ERK1/2. This study provides evidence regarding the mechanism underlying the relaxation effect of fisetin on agonist-induced vascular contraction regardless of endothelial function. PMID:26759702

  16. Prenatal Mechanistic Target of Rapamycin Complex 1 (m TORC1) Inhibition by Rapamycin Treatment of Pregnant Mice Causes Intrauterine Growth Restriction and Alters Postnatal Cardiac Growth, Morphology, and Function.

    PubMed

    Hennig, Maria; Fiedler, Saskia; Jux, Christian; Thierfelder, Ludwig; Drenckhahn, Jörg-Detlef

    2017-08-04

    Fetal growth impacts cardiovascular health throughout postnatal life in humans. Various animal models of intrauterine growth restriction exhibit reduced heart size at birth, which negatively influences cardiac function in adulthood. The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and growth factor availability with cell growth, thereby regulating organ size. This study aimed at elucidating a possible involvement of mTORC1 in intrauterine growth restriction and prenatal heart growth. We inhibited mTORC1 in fetal mice by rapamycin treatment of pregnant dams in late gestation. Prenatal rapamycin treatment reduces mTORC1 activity in various organs at birth, which is fully restored by postnatal day 3. Rapamycin-treated neonates exhibit a 16% reduction in body weight compared with vehicle-treated controls. Heart weight decreases by 35%, resulting in a significantly reduced heart weight/body weight ratio, smaller left ventricular dimensions, and reduced cardiac output in rapamycin- versus vehicle-treated mice at birth. Although proliferation rates in neonatal rapamycin-treated hearts are unaffected, cardiomyocyte size is reduced, and apoptosis increased compared with vehicle-treated neonates. Rapamycin-treated mice exhibit postnatal catch-up growth, but body weight and left ventricular mass remain reduced in adulthood. Prenatal mTORC1 inhibition causes a reduction in cardiomyocyte number in adult hearts compared with controls, which is partially compensated for by an increased cardiomyocyte volume, resulting in normal cardiac function without maladaptive left ventricular remodeling. Prenatal rapamycin treatment of pregnant dams represents a new mouse model of intrauterine growth restriction and identifies an important role of mTORC1 in perinatal cardiac growth. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  17. Rapamycin Modulates Markers of Mitochondrial Biogenesis and Fatty Acid Oxidation in the Adipose Tissue of db/db Mice.

    PubMed

    Deepa, Sathyaseelan S; Walsh, Michael E; Hamilton, Ryan T; Pulliam, Daniel; Shi, Yun; Hill, Shauna; Li, Yan; Van Remmen, Holly

    2013-06-01

    Excess nutrient uptake leads to obesity, insulin resistance, and type 2 diabetes. Mammalian target of the rapamycin (mTOR), a major component of the nutrient-sensing pathway also regulates mitochondrial oxidative function. Rapamycin, a pharmacological inhibitor of mTOR, causes glucose intolerance and inhibits mitochondrial oxidative function. While a number of studies have focused on the effect of rapamycin on control wild-type mice, ours is the first to study the effect of rapamycin on mitochondrial gene expression and insulin sensitivity in the db/db mouse, a model of diabetic dyslipidemia. Female db/+ and db/db mice were fed ad libitum a rapamycin-containing diet or a control diet for 6 months, starting at two months of age. Body weight, fat mass, lean mass and food intake were measured monthly. Effect of rapamycin or control diet on markers of adipogenesis, fatty acid oxidation and mitochondrial biogenesis in the gonadal white adipose tissue (WAT) as well as different serum parameters were assessed. Whole body insulin sensitivity was measured by insulin tolerance test. Rapamycin feeding to db/db mice decreased body weight (58%) and fat mass (33%), elevated markers of fatty acid oxidation and mitochondrial biogenesis in WAT, reduced circulating non-esterified free fatty acids (NEFA), elevated circulating adiponectin and improved insulin sensitivity, compared to control diet fed db/db mice. These data demonstrate that rapamycin exhibits an anti-obesity effect and improves whole body insulin sensitivity in db/db mice and suggest an unexpected effect of simultaneous inhibition mTOR and leptin signaling in mice.

  18. Rapamycin transiently induces mitochondrial remodeling to reprogram energy metabolism in old hearts

    PubMed Central

    Chiao, Ying Ann; Kolwicz, Stephen C.; Basisty, Nathan; Gagnidze, Arni; Zhang, Julia; Gu, Haiwei; Djukovic, Danijel; Beyer, Richard P.; Raftery, Daniel; MacCoss, Michael; Tian, Rong; Rabinovitch, Peter S.

    2016-01-01

    Rapamycin, an inhibitor of mTOR signaling, has been shown to reverse diastolic dysfunction in old mice in 10 weeks, highlighting its therapeutic potential for a poorly treatable condition. However, the mechanisms and temporal regulation of its cardiac benefits remain unclear. We show that improved diastolic function in old mice begins at 2-4 weeks, progressing over the course of 10-week treatment. While TORC1-mediated S6 phosphorylation and TORC2 mediated AKT and PKCα phosphorylation are inhibited throughout the course of treatment, rapamycin inhibits ULK phosphorylation and induces autophagy during just the first week of treatment, returning to baseline at two weeks and after. Concordantly, markers of mitochondrial biogenesis increase over the first two weeks of treatment and return to control levels thereafter. This transient induction of autophagy and mitochondrial biogenesis suggests that damaged mitochondria are replaced by newly synthesized ones to rejuvenate mitochondrial homeostasis. This remodeling is shown to rapidly reverse the age-related reduction in fatty acid oxidation to restore a more youthful substrate utilization and energetic profile in old isolated perfused hearts, and modulates the myocardial metabolome in vivo. This study demonstrates the differential and dynamic mechanisms following rapamycin treatment and highlights the importance of understanding the temporal regulation of rapamycin effects. PMID:26872208

  19. TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation via the WOX4 Homeobox Gene in Arabidopsis[W

    PubMed Central

    Hirakawa, Yuki; Kondo, Yuki; Fukuda, Hiroo

    2010-01-01

    The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell–cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth. PMID:20729381

  20. Reduced contribution of endothelin to the regulation of systemic and pulmonary vascular tone in severe familial hypercholesterolaemia

    PubMed Central

    Bender, Shawn B; de Beer, Vincent J; Tharp, Darla L; van Deel, Elza D; Bowles, Douglas K; Duncker, Dirk J; Laughlin, M Harold; Merkus, Daphne

    2014-01-01

    Vascular dysfunction has been associated with familial hypercholesterolaemia (FH), a severe form of hyperlipidaemia. We recently demonstrated that swine with FH exhibit reduced exercise-induced systemic, but not pulmonary, vasodilatation involving reduced nitric oxide (NO) bioavailability. Since NO normally limits endothelin (ET) action, we examined the hypothesis that reduced systemic vasodilatation during exercise in FH swine results from increased ET-mediated vasoconstriction. Systemic and pulmonary vascular responses to exercise were examined in chronically instrumented normal and FH swine in the absence and presence of the ETA/B receptor antagonist tezosentan. Intrinsic reactivity to ET was further assessed in skeletal muscle arterioles. FH swine exhibited ∼9-fold elevation in total plasma cholesterol versus normal swine. Similar to our recent findings, systemic, not pulmonary, vasodilatation during exercise was reduced in FH swine. Blockade of ET receptors caused marked systemic vasodilatation at rest and during exercise in normal swine that was significantly reduced in FH swine. The reduced role of ET in FH swine in vivo was not the result of decreased arteriolar ET responsiveness, as responsiveness was increased in isolated arterioles. Smooth muscle ET receptor protein content was unaltered by FH. However, circulating plasma ET levels were reduced in FH swine. ET receptor antagonism caused pulmonary vasodilatation at rest and during exercise in normal, but not FH, swine. Therefore, contrary to our hypothesis, FH swine exhibit a generalised reduction in the role of ET in regulating vascular tone in vivo probably resulting from reduced ET production. This may represent a unique vascular consequence of severe familial hypercholesterolaemia. PMID:24421352

  1. Importance of the splanchnic vascular bed in human blood pressure regulation.

    NASA Technical Reports Server (NTRS)

    Rowell, L. B.; Detry, J.-M. R.; Blackmon, J. R.; Wyss, C.

    1972-01-01

    Three-part experiment in which five subjects were exposed to lower body negative pressure (LBNP) at -50 mm Hg below the iliac crests. Duration of LBNP to earliest vagal symptoms was 7 to 21 min; all data are expressed as changes from control period to the last measurements before these symptoms. In part I, forearm blood flow (by Whitney gauge) fell 45% during LBNP. In part II, splanchnic blood flow (from arterial clearance hepatic extraction of indocyanine green) fell 32% and splanchnic vascular resistance rose 30%. In part III, cardiac output fell 28%, stroke volume 51%, and central blood volume 21%. Total peripheral resistance and heart rate rose 19% and 52%. Of the reduction in total vascular conductance, decreased splanchnic conductance accounted for approximately 33%; skin plus muscle conductance decreased similarly.

  2. miR-21 regulates chronic hypoxia-induced pulmonary vascular remodeling

    PubMed Central

    Yang, Shanzhong; Banerjee, Sami; de Freitas, Andressa; Cui, Huachun; Xie, Na; Abraham, Edward

    2012-01-01

    Chronic hypoxia causes pulmonary vascular remodeling leading to pulmonary hypertension (PH) and right ventricle (RV) hypertrophy. Aberrant expression of microRNA (miRNA) is closely associated with a number of pathophysiologic processes. However, the role of miRNAs in chronic hypoxia-induced pulmonary vascular remodeling and PH has not been well characterized. In this study, we found increased expression of miR-21 in distal small arteries in the lungs of hypoxia-exposed mice. Putative miR-21 targets, including bone morphogenetic protein receptor (BMPR2), WWP1, SATB1, and YOD1, were downregulated in the lungs of hypoxia-exposed mice and in human pulmonary artery smooth muscle cells (PASMCs) overexpressing miR-21. We found that sequestration of miR-21, either before or after hypoxia exposure, diminished chronic hypoxia-induced PH and attenuated hypoxia-induced pulmonary vascular remodeling, likely through relieving the suppressed expression of miR-21 targets in the lungs of hypoxia-exposed mice. Overexpression of miR-21 enhanced, whereas downregulation of miR-21 diminished, the proliferation of human PASMCs in vitro and the expression of cell proliferation associated proteins, such as proliferating cell nuclear antigen, cyclin D1, and Bcl-xL. Our data suggest that miR-21 plays an important role in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and also suggest that miR-21 is a potential target for novel therapeutics to treat chronic hypoxia associated pulmonary diseases. PMID:22227207

  3. The molecular actions of oestrogen in the regulation of vascular health.

    PubMed

    Usselman, Charlotte W; Stachenfeld, Nina S; Bender, Jeffrey R

    2016-03-01

    What is the topic of this review? This review summarizes the beneficial actions of oestrogen on the vasculature, highlighting both molecular mechanisms and functional outcomes. What advances does it highlight? The net effect of oestrogen on the vascular health of women continues to be debated. Recent advances have provided strong evidence for the role of membrane-bound oestrogen receptors in the maintenance of normal endothelial function. On a broader scale, functional outcomes of oestrogen actions on the vasculature may mediate the reduced risk of cardiovascular disease in premenopausal women. The conflicting implications of the large-scale clinical menopausal hormone therapy trials in humans versus the findings of studies on experimental animals underscore the limitations within our understanding of the molecular actions of oestrogen. However, recent research has provided improved insight into the actions of oestrogen on the endothelium and vascular smooth muscle. This review outlines the actions of oestrogen as it contributes to vascular structure, function and health. © 2016 The Authors. Experimental Physiology © 2016 The Physiological Society.

  4. Estrogen-regulated uterine vascularization modulates the guinea pig intrauterine environment.

    PubMed

    Garris, D R

    1997-01-01

    The effects of experimentally-induced, uterine vascular restriction on uterine blood flow (UBF) and uterine blood volume (UBV) capacity, as well as the dependent intrauterine oxygen tension (IUpO2) measurements used as an indication of luminal nutrient availability, were examined using ovariectomized, estrogen (E)-treated guinea pigs. Following 3 days of E treatment, both UBF and UBV measurements were found to be elevated and associated with a causally-related increase in intraluminal uterine oxygen availability levels. Following the acute clamping of the uterine arteries, both UBF and UBV levels decreased dramatically and induced a rapid fall in associated intrauterine luminal oxygen tension measurements. As a result of chronic (i.e., 6 h) restriction of segmental blood flow to the uterus by vascular cauterization, both UBV and IUPO2 levels were suppressed as compared with sham-operated control levels, whereas UBF rates were not significantly altered. The results of the present studies are the first quantitative demonstration that either acute or chronic reductions in uterine vascular capacity or competency can induce rapid and dramatic changes in the intrauterine nutritional environment recognized to be essential for the initiation, support and maintenance of nidation and subsequent fetal-placental development.

  5. A βPix–Pak2a signaling pathway regulates cerebral vascular stability in zebrafish

    PubMed Central

    Liu, Jing; Fraser, Sherri D.; Faloon, Patrick W.; Rollins, Evvi Lynn; Vom Berg, Johannes; Starovic-Subota, Olivera; Laliberte, Angie L.; Chen, Jau-Nian; Serluca, Fabrizio C.; Childs, Sarah J.

    2007-01-01

    The vasculature tailors to the needs of different tissues and organs. Molecular, structural, and functional specializations are observed in different vascular beds, but few genetic models give insight into how these differences arise. We identify a unique cerebrovascular mutation in the zebrafish affecting the integrity of blood vessels supplying the brain. The zebrafish bubblehead (bbh) mutant exhibits hydrocephalus and severe cranial hemorrhage during early embryogenesis, whereas blood vessels in other regions of the embryo appear intact. Here we show that hemorrhages are associated with poor cerebral endothelial–mesenchymal contacts and an immature vascular pattern in the head. Positional cloning of bbh reveals a hypomorphic mutation in βPix, a binding partner for the p21-activated kinase (Pak) and a guanine nucleotide exchange factor for Rac and Cdc42. βPix is broadly expressed during embryonic development and is enriched in the brain and in large blood vessels. By knockdown of specific βPix splice variants, we show that they play unique roles in embryonic vascular stabilization or hydrocephalus. Finally, we show that Pak2a signaling is downstream of βPix. These data identify an essential in vivo role for βPix and Pak2a during embryonic development and illuminate a previously unrecognized pathway specifically involved in cerebrovascular stabilization. PMID:17573532

  6. A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression.

    PubMed

    Gupta, Rajat M; Hadaya, Joseph; Trehan, Aditi; Zekavat, Seyedeh M; Roselli, Carolina; Klarin, Derek; Emdin, Connor A; Hilvering, Catharina R E; Bianchi, Valerio; Mueller, Christian; Khera, Amit V; Ryan, Russell J H; Engreitz, Jesse M; Issner, Robbyn; Shoresh, Noam; Epstein, Charles B; de Laat, Wouter; Brown, Jonathan D; Schnabel, Renate B; Bernstein, Bradley E; Kathiresan, Sekar

    2017-07-27

    Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Slingshot isoform-specific regulation of cofilin-mediated vascular smooth muscle cell migration and neointima formation

    PubMed Central

    Torres, Rebecca A.; Drake, Douglas A.; Solodushko, Viktoriya; Jadhav, Rashmi; Smith, Erika; Rocic, Petra; Weber, David S.

    2011-01-01

    Objective We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating VSMC migration and neoinitima formation following vascular injury. Methods and Results Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each slingshot phosphatase isoforms (SSH1,-2,-3) by siRNA, respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the “wound” and PDGF-induced migration was virtually eliminated versus a 3.5-fold increase in non-treated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, while SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 (4.7 fold) and cofilin (3.9 fold) expression were increased in balloon injured versus non-injured carotid arteries and expression was prevalent in the neointima. Conclusions These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent, and that this mechanism potentially contributes to neointima formation following vascular injury in vivo. PMID:21868701

  8. 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.

  9. MicroRNA857 Is Involved in the Regulation of Secondary Growth of Vascular Tissues in Arabidopsis1

    PubMed Central

    Zhao, Yuanyuan; Lin, Sen; Qiu, Zongbo; Cao, Dechang; Wen, Jialong; Deng, Xin; Wang, Xiaohua; Lin, Jinxing; Li, Xiaojuan

    2015-01-01

    MicroRNAs (miRNAs) are endogenous small RNAs that repress target gene expression posttranscriptionally, and are critically involved in various developmental processes and responses to environmental stresses in eukaryotes. MiRNA857 is not widely distributed in plants and is encoded by a single gene, AtMIR857, in Arabidopsis (Arabidopsis thaliana). The functions of miR857 and its mechanisms in regulating plant growth and development are still unclear. Here, by means of genetic analysis coupled with cytological studies, we investigated the expression pattern and regulation mechanism of miR857 and its biological functions in Arabidopsis development. We found that miR857 regulates its target gene, Arabidopsis LACCASE7, at the transcriptional level, thereby reducing laccase activity. Using stimulated Raman scattering and x-ray microtomography three-dimensional analyses, we showed that miR857 was involved in the regulation of lignin content and consequently morphogenesis of the secondary xylem. In addition, miR857 was activated by SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 in response to low copper conditions. Collectively, these findings demonstrate the role of miR857 in the regulation of secondary growth of vascular tissues in Arabidopsis and reveal a unique control mechanism for secondary growth based on the miR857 expression in response to copper deficiency. PMID:26511915

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

    SciTech Connect

    Urata, Yoshishige; Goto, Shinji; Kawakatsu, Miho

    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 statemore » 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.« less

  11. Central role of endogenous Toll-like receptor-2 activation in regulating inflammation, reactive oxygen species production, and subsequent neointimal formation after vascular injury

    SciTech Connect

    Shishido, Tetsuro . E-mail: Tetsuro_Shishido@URMC.Rochester.edu; Nozaki, Naoki; Takahashi, Hiroki; Arimoto, Takanori; Niizeki, Takeshi; Koyama, Yo; Abe, Jun-ichi; Takeishi, Yasuchika; Kubota, Isao

    2006-07-14

    Background: It is now evident that inflammation after vascular injury has significant impact on the restenosis after revascularization procedures such as angioplasty, stenting, and bypass grafting. However, the mechanisms that regulate inflammation and repair after vascular injury are incompletely understood. Here, we report that vascular injury-mediated cytokine expression, reactive oxygen species (ROS) production, as well as subsequent neointimal formation requires Toll-like receptor-2 (TLR-2) mediated signaling pathway in vivo. Methods and results: Vascular injury was induced by cuff-placement around the femoral artery in non-transgenic littermates (NLC) and TLR-2 knockout (TLR-2KO) mice. After cuff-placement in NLC mice, expression of TLR-2 was significantly increased in both smooth muscle medial layer and adventitia. Interestingly, we found that inflammatory genes expression such as tumor necrosis factor-{alpha}, interleukin-1{beta} (IL-1{beta}), IL-6, and monocyte chemoattractant protein-1 were markedly decreased in TLR-2KO mice compared with NLC mice. In addition, ROS production after vascular injury was attenuated in TLR-2KO mice compared with NLC mice. Since we observed the significant role of endogenous TLR-2 activation in regulating inflammatory responses and ROS production after vascular injury, we determined whether inhibition of endogenous TLR-2 activation can inhibit neointimal proliferation after vascular injury. Neointimal hyperplasia was markedly suppressed in TLR-2KO mice compared with WT mice at both 2 and 4 weeks after vascular injury. Conclusions: These findings suggested that endogenous TLR-2 activation might play a central role in the regulation of vascular inflammation as well as subsequent neointimal formation in injured vessels.

  12. Inhibition of Mammalian Target of Rapamycin Signaling with Rapamycin Prevents Trauma-Induced Heterotopic Ossification.

    PubMed

    Qureshi, Ammar T; Dey, Devaveena; Sanders, Erin M; Seavey, Jonathan G; Tomasino, Allison M; Moss, Kaitlyn; Wheatley, Benjamin; Cholok, David; Loder, Shawn; Li, John; Levi, Benjamin; Davis, Thomas A

    2017-11-01

    A pressing clinical need exists for 63% to 65% of combat-wounded service members and 11% to 20% of civilians who develop heterotopic ossification (HO) after blast-related extremity injury and traumatic injuries, respectively. The mammalian target of rapamycin pathway is a central cellular sensor of injury. We evaluated the prophylactic effects of rapamycin, a selective inhibitor of mammalian target of rapamycin signaling, on HO formation in a rat model of blast-related, polytraumatic extremity injury. Rapamycin was administered intraperitoneally daily for 14 days at 0.5 mg/kg or 2.5 mg/kg. Ectopic bone formation was monitored by micro-computed tomography and confirmed by histologic examination. Connective tissue progenitor cells, platelet-derived growth factor receptor-α-positive cells, and α-smooth muscle actin-positive blood vessels were assayed at postoperative day 7 by colony formation and immunofluorescence. Early gene expression changes were determined by low-density microarray. There was significant attenuation of 1) total new bone and soft tissue ectopic bone with 0.5 mg/kg (38.5% and 14.7%) and 2.5 mg/kg rapamycin (90.3% and 82.9%), respectively, 2) connective tissue progenitor cells, 3) platelet-derived growth factor receptor-α-positive cells, 4) α-smooth muscle actin-positive blood vessels, and 5) of key extracellular matrix remodeling (CD44, Col1a1, integrins), osteogenesis (Sp7, Runx2, Bmp2), inflammation (Cxcl5, 10, IL6, Ccl2), and angiogenesis (Angpt2) genes. No wound healing complications were noted. Our data demonstrate the efficacy of rapamycin in inhibiting blast trauma-induced HO by a multipronged mechanism. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  13. miR-181b regulates vascular stiffness age dependently in part by regulating TGF-β signaling

    PubMed Central

    Hori, Daijiro; Dunkerly-Eyring, Brittany; Nomura, Yohei; Biswas, Debjit; Steppan, Jochen; Henao-Mejia, Jorge; Adachi, Hideo; Santhanam, Lakshmi; Berkowitz, Dan E.; Steenbergen, Charles; Flavell, Richard A.

    2017-01-01

    Background Endothelial dysfunction and arterial stiffening play major roles in cardiovascular diseases. The critical role for the miR-181 family in vascular inflammation has been documented. Here we tested whether the miR-181 family can influence the pathogenesis of hypertension and vascular stiffening. Methods and results qPCR data showed a significant decrease in miR-181b expression in the aorta of the older mice. Eight miR-181a1/b1-/- mice and wild types (C57BL6J:WT) were followed weekly for pulse wave velocity (PWV) and blood pressure measurements. After 20 weeks, the mice were tested for endothelial function and aortic modulus. There was a progressive increase in PWV and higher systolic blood pressure in miR-181a1/b1-/- mice compared with WTs. At 21 weeks, aortic modulus was significantly greater in the miR-181a1/b1-/- group, and serum TGF-β was found to be elevated at this time. A luciferase reporter assay confirmed miR-181b targets TGF-βi (TGF-β induced) in the aortic VSMCs. In contrast, wire myography revealed unaltered endothelial function along with higher nitric oxide production in the miR-181a1/b1-/- group. Cultured VECs and VSMCs from the mouse aorta showed more secreted TGF-β in VSMCs of the miR-181a1/b1-/- group; whereas, no change was observed from VECs. Circulating levels of angiotensin II were similar in both groups. Treatment with losartan (0.6 g/L) prevented the increase in PWV, blood pressure, and vascular stiffness in miR-181a1/b1-/- mice. Immunohistochemistry and western blot for p-SMAD2/3 validated the inhibitory effect of losartan on TGF-β signaling in miR-181a1/b1-/- mice. Conclusions Decreased miR-181b with aging plays a critical role in ECM remodeling by removing the brake on the TGF-β, pSMAD2/3 pathway. PMID:28323879

  14. Catalytic mammalian target of rapamycin inhibitors as antineoplastic agents.

    PubMed

    Mohindra, Nisha A; Platanias, Leonidas C

    2015-01-01

    The mammalian target of rapamycin (mTOR) pathway is a major therapeutic target in the treatment of hematological malignancies, as it controls cellular events of high importance for regulation of mRNA translation and protein production. Rapalogs, or first-generation mTOR inhibitors, have produced only modest clinical benefits so far. Limitations to rapalogs likely result from the partial inhibition of mTORC1 substrates and lack of effects on mTORC2. Efforts toward the development of agents with more potent and complete inhibitory effects on the mTOR pathway have resulted in the development of catalytic mTOR inhibitors. Key preclinical and early clinical investigations of several catalytic mTOR inhibitors and potential resistance mechanisms to their activities are summarized here.

  15. Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures

    PubMed Central

    Ramakrishnan, Venkat M.; Tien, Kevin T.; McKinley, Thomas R.; Bocard, Braden R.; McCurry, Terry M.; Williams, Stuart K.; Hoying, James B.; Boyd, Nolan L.

    2016-01-01

    Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0–150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt

  16. Hydrogen Sulfide Stimulates Ischemic Vascular Remodeling Through Nitric Oxide Synthase and Nitrite Reduction Activity Regulating Hypoxia‐Inducible Factor‐1α and Vascular Endothelial Growth Factor–Dependent Angiogenesis

    PubMed Central

    Bir, Shyamal C.; Kolluru, Gopi K.; McCarthy, Paul; Shen, Xinggui; Pardue, Sibile; Pattillo, Christopher B.; Kevil, Christopher G.

    2012-01-01

    Background Hydrogen sulfide (H2S) therapy is recognized as a modulator of vascular function during tissue ischemia with the notion of potential interactions of nitric oxide (NO) metabolism. However, little is known about specific biochemical mechanisms or the importance of H2S activation of NO metabolism during ischemic tissue vascular remodeling. The goal of this study was to determine the effect of H2S on NO metabolism during chronic tissue ischemia and subsequent effects on ischemic vascular remodeling responses. Methods and Results The unilateral, permanent femoral artery ligation model of hind‐limb ischemia was performed in C57BL/6J wild‐type and endothelial NO synthase–knockout mice to evaluate exogenous H2S effects on NO bioavailability and ischemic revascularization. We found that H2S selectively restored chronic ischemic tissue function and viability by enhancing NO production involving both endothelial NO synthase and nitrite reduction mechanisms. Importantly, H2S increased ischemic tissue xanthine oxidase activity, hind‐limb blood flow, and angiogenesis, which were blunted by the xanthine oxidase inhibitor febuxostat. H2S treatment increased ischemic tissue and endothelial cell hypoxia‐inducible factor‐1α expression and activity and vascular endothelial growth factor protein expression and function in a NO‐dependent manner that was required for ischemic vascular remodeling. Conclusions These data demonstrate that H2S differentially regulates NO metabolism during chronic tissue ischemia, highlighting novel biochemical pathways to increase NO bioavailability for ischemic vascular remodeling. PMID:23316304

  17. YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development

    PubMed Central

    Neto, Filipa; Klaus-Bergmann, Alexandra; Ong, Yu Ting; Alt, Silvanus; Vion, Anne-Clémence; Szymborska, Anna; Carvalho, Joana R; Hollfinger, Irene; Bartels-Klein, Eireen; Franco, Claudio A

    2018-01-01

    Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels. PMID:29400648

  18. YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development.

    PubMed

    Neto, Filipa; Klaus-Bergmann, Alexandra; Ong, Yu Ting; Alt, Silvanus; Vion, Anne-Clémence; Szymborska, Anna; Carvalho, Joana R; Hollfinger, Irene; Bartels-Klein, Eireen; Franco, Claudio A; Potente, Michael; Gerhardt, Holger

    2018-02-05

    Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels. © 2018, Neto et al.

  19. Thrombospondin-1 is a Central Regulator of Nitric Oxide Signaling in Vascular Physiology

    PubMed Central

    Isenberg, Jeff S.; Frazier, William A.; Roberts, David D.

    2008-01-01

    Thrombospondin-1 is secreted protein that modulates vascular cell behavior via several cell surface receptors. In vitro, nanomolar concentrations of thrombospondin-1 are required to alter endothelial and vascular smooth muscle cell adhesion, proliferation, motility, and survival. Yet, much lower levels of thrombospondin-1 are clearly functional in vivo. This discrepancy was explained with the discovery that the potency of thrombospondin-1 increases more than 100-fold in the presence of physiological levels of NO. Thrombospondin-1 binding to CD47 inhibits NO signaling by preventing cGMP synthesis and activation of its target cGMP-dependent protein kinase. This potent antagonism of NO signaling allows thrombospondin-1 to acutely constrict blood vessels, accelerate platelet aggregation and, if sustained, inhibit angiogenic responses. Acute antagonism of NO signaling by thrombospondin-1 is important for hemostasis but becomes detrimental for tissue survival of ischemic injuries. New therapeutic approaches targeting thrombospondin-1 or CD47 can improve recovery from ischemic injuries and overcome a deficit in NO-responsiveness in aging. PMID:18193160

  20. Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis

    PubMed Central

    DeFalco, Tony; Bhattacharya, Indrashis; Williams, Alyna V.; Sams, Dustin M.; Capel, Blanche

    2014-01-01

    Organogenesis of the testis is initiated when expression of Sry in pre-Sertoli cells directs the gonad toward a male-specific fate. The cells in the early bipotential gonad undergo de novo organization to form testis cords that enclose germ cells inside tubules lined by epithelial Sertoli cells. Although Sertoli cells are a driving force in the de novo formation of testis cords, recent studies in mouse showed that reorganization of the vasculature and of interstitial cells also play critical roles in testis cord morphogenesis. However, the mechanism driving reorganization of the vasculature during fetal organogenesis remained unclear. Here we demonstrate that fetal macrophages are associated with nascent gonadal and mesonephric vasculature during the initial phases of testis morphogenesis. Macrophages mediate vascular reorganization and prune errant germ cells and somatic cells after testis architecture is established. We show that gonadal macrophages are derived from primitive yolk-sac hematopoietic progenitors and exhibit hallmarks of M2 activation status, suggestive of angiogenic and tissue remodeling functions. Depletion of macrophages resulted in impaired vascular reorganization and abnormal cord formation. These findings reveal a previously unappreciated role for macrophages in testis morphogenesis and suggest that macrophages are an intermediary between neovascularization and organ architecture during fetal organogenesis. PMID:24912173

  1. Regulation of angiotensin II actions by enhancers and super-enhancers in vascular smooth muscle cells.

    PubMed

    Das, Sadhan; Senapati, Parijat; Chen, Zhuo; Reddy, Marpadga A; Ganguly, Rituparna; Lanting, Linda; Mandi, Varun; Bansal, Anita; Leung, Amy; Zhang, Selena; Jia, Ye; Wu, Xiwei; Schones, Dustin E; Natarajan, Rama

    2017-11-13

    Angiotensin II (AngII) promotes hypertension and atherosclerosis by activating growth-promoting and pro-inflammatory gene expression in vascular smooth muscle cells (VSMCs). Enhancers and super-enhancers (SEs) play critical roles in driving disease-associated gene expression. However, enhancers/SEs mediating VSMC dysfunction remain uncharacterized. Here, we show that AngII alters vascular enhancer and SE repertoires in cultured VSMCs in vitro, ex vivo, and in AngII-infused mice aortas in vivo. AngII-induced enhancers/SEs are enriched in binding sites for signal-dependent transcription factors and dependent on key signaling kinases. Moreover, CRISPR-Cas9-mediated deletion of candidate enhancers/SEs, targeting SEs with the bromodomain and extra-terminal domain inhibitor JQ1, or knockdown of overlapping long noncoding RNAs (lncRNAs) blocks AngII-induced genes associated with growth-factor signaling and atherosclerosis. Furthermore, JQ1 ameliorates AngII-induced hypertension, medial hypertrophy and inflammation in vivo in mice. These results demonstrate AngII-induced signals integrate enhancers/SEs and lncRNAs to increase expression of genes involved in VSMC dysfunction, and could uncover novel therapies.

  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. PINK1 and its familial Parkinson's disease-associated mutation regulate brain vascular endothelial inflammation.

    PubMed

    Yunfu, Wang; Guangjian, Liu; Ping, Zhong; Yanpeng, Sun; Xiaoxia, Fang; Wei, Hu; Jiang, Yuan; Jingquan, Hu; Songlin, Wang; Hongyan, Zhang; Yong, Liu; Shi, Chen

    2014-05-01

    Parkinson's disease (PD) is a debilitating disorder that affects movement. Inflammation-mediated endothelial dysfunction has been found to be involved in neurodegenerative diseases, including PD. More than 40 PTEN-induced putative kinase 1 (PINK1) mutations have been found in PD patients. The effects of PINK1 in vascular inflammation are as yet unknown. In this study, our findings revealed that PINK1 can be increased by the inflammatory cytokine tumor necrosis factor-α in primary human brain microvascular endothelial cells (HBMECs). We found that wild-type PINK1 prevents expression of the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1), thus inhibiting the attachment of monocytes to brain endothelial cells. However, PINK1G309D, the loss-of-function mutation associated with early-onset familial PD, promotes expression of VCAM-1 and exacerbates attachment of monocytes to brain endothelial cells. Mechanism studies revealed that overexpression of wild-type PINK1 inhibits the VCAM-1 promoter by inhibiting the transcriptional activity of interferon regulatory factor 1 (IRF-1). However, PINK1G309D promotes the VCAM-1 promoter by increasing the transcriptional activity of IRF-1.

  4. 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.

  5. Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture.

    PubMed

    Gidfar, Sanaz; Milani, Farnoud Y; Milani, Behrad Y; Shen, Xiang; Eslani, Medi; Putra, Ilham; Huvard, Michael J; Sagha, Hossein; Djalilian, Ali R

    2017-01-05

    Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated β-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-β-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

  6. miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat

    PubMed Central

    Merlet, Elise; Atassi, Fabrice; Motiani, Rajender K.; Mougenot, Nathalie; Jacquet, Adeline; Nadaud, Sophie; Capiod, Thierry; Trebak, Mohamed; Lompré, Anne-Marie; Marchand, Alexandre

    2013-01-01

    Aims Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. Methods and results MicroRNAs microarray and qRT–PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca2+-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca2+ entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. Conclusion Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis. PMID:23447642

  7. Genetic variants of Adam17 differentially regulate TGFβ signaling to modify vascular pathology in mice and humans.

    PubMed

    Kawasaki, Kyoko; Freimuth, Julia; Meyer, Dominique S; Lee, Marie M; Tochimoto-Okamoto, Akiko; Benzinou, Michael; Clermont, Frederic F; Wu, Gloria; Roy, Ritu; Letteboer, Tom G W; Ploos van Amstel, Johannes Kristian; Giraud, Sophie; Dupuis-Girod, Sophie; Lesca, Gaeten; Westermann, Cornelius J J; Coffey, Robert J; Akhurst, Rosemary J

    2014-05-27

    Outcome of TGFβ1 signaling is context dependent and differs between individuals due to germ-line genetic variation. To explore innate genetic variants that determine differential outcome of reduced TGFβ1 signaling, we dissected the modifier locus Tgfbm3, on mouse chromosome 12. On a NIH/OlaHsd genetic background, the Tgfbm3b(C57) haplotype suppresses prenatal lethality of Tgfb1(-/-) embryos and enhances nuclear accumulation of mothers against decapentaplegic homolog 2 (Smad2) in embryonic cells. Amino acid polymorphisms within a disintegrin and metalloprotease 17 (Adam17) can account, at least in part, for this Tgfbm3b effect. ADAM17 is known to down-regulate Smad2 signaling by shedding the extracellular domain of TGFβRI, and we show that the C57 variant is hypomorphic for down-regulation of Smad2/3-driven transcription. Genetic variation at Tgfbm3 or pharmacological inhibition of ADAM17, modulates postnatal circulating endothelial progenitor cell (CEPC) numbers via effects on TGFβRI activity. Because CEPC numbers correlate with angiogenic potential, this suggests that variant Adam17 is an innate modifier of adult angiogenesis, acting through TGFβR1. To determine whether human ADAM17 is also polymorphic and interacts with TGFβ signaling in human vascular disease, we investigated hereditary hemorrhagic telangiectasia (HHT), which is caused by mutations in TGFβ/bone morphogenetic protein receptor genes, ENG, encoding endoglin (HHT1), or ACVRL1 encoding ALK1 (HHT2), and considered a disease of excessive abnormal angiogenesis. HHT manifests highly variable incidence and severity of clinical features, ranging from small mucocutaneous telangiectases to life-threatening visceral and cerebral arteriovenous malformations (AVMs). We show that ADAM17 SNPs associate with the presence of pulmonary AVM in HHT1 but not HHT2, indicating genetic variation in ADAM17 can potentiate a TGFβ-regulated vascular disease.

  8. Retinoic acid-loaded polymeric nanoparticles enhance vascular regulation of neural stem cell survival and differentiation after ischaemia

    NASA Astrophysics Data System (ADS)

    Ferreira, R.; Fonseca, M. C.; Santos, T.; Sargento-Freitas, J.; Tjeng, R.; Paiva, F.; Castelo-Branco, M.; Ferreira, L. S.; Bernardino, L.

    2016-04-01

    Stroke is one of the leading causes of death and disability worldwide. However, current therapies only reach a small percentage of patients and may cause serious side effects. We propose the therapeutic use of retinoic acid-loaded nanoparticles (RA-NP) to safely and efficiently repair the ischaemic brain by creating a favourable pro-angiogenic environment that enhances neurogenesis and neuronal restitution. Our data showed that RA-NP enhanced endothelial cell proliferation and tubule network formation and protected against ischaemia-induced death. To evaluate the effect of RA-NP on vascular regulation of neural stem cell (NSC) survival and differentiation, endothelial cell-conditioned media (EC-CM) were collected. EC-CM from healthy RA-NP-treated cells reduced NSC death and promoted proliferation while EC-CM from ischaemic RA-NP-treated cells decreased cell death, increased proliferation and neuronal differentiation. In parallel, human endothelial progenitor cells (hEPC), which are part of the endogenous repair response to vascular injury, were collected from ischaemic stroke patients. hEPC treated with RA-NP had significantly higher proliferation, which further highlights the therapeutic potential of this formulation. To conclude, RA-NP protected endothelial cells from ischaemic death and stimulated the release of pro-survival, proliferation-stimulating factors and differentiation cues for NSC. RA-NP were shown to be up to 83-fold more efficient than free RA and to enhance hEPC proliferation. These data serve as a stepping stone to use RA-NP as vasculotrophic and neurogenic agents for vascular disorders and neurodegenerative diseases with compromised vasculature.

  9. Vascular Endothelial Growth Factor (VEGF) Bioavailability Regulates Angiogenesis and Intestinal Stem and Progenitor Cell Proliferation during Postnatal Small Intestinal Development.

    PubMed

    Schlieve, Christopher R; Mojica, Salvador Garcia; Holoyda, Kathleen A; Hou, Xiaogang; Fowler, Kathryn L; Grikscheit, Tracy C

    2016-01-01

    Vascular endothelial growth factor (VEGF) is a highly conserved, master regulatory molecule required for endothelial cell proliferation, organization, migration and branching morphogenesis. Podocoryne carnea and drosophila, which lack endothelial cells and a vascular system, express VEGF homologs, indicating potential roles beyond angiogenesis and vasculogenesis. The role of VEGF in the development and homeostasis of the postnatal small intestine is unknown. We hypothesized regulating VEGF bioavailability in the postnatal small intestine would exhibit effects beyond the vasculature and influence epithelial cell stem/progenitor populations. VEGF mutant mice were created that overexpressed VEGF in the brush border of epithelium via the villin promotor following doxycycline treatment. To decrease VEGF bioavailability, sFlt-1 mutant mice were generated that overexpressed the soluble VEGF receptor sFlt-1 upon doxycycline administration in the intestinal epithelium. Mice were analyzed after 21 days of doxycycline administration. Increased VEGF expression was confirmed by RT-qPCR and ELISA in the intestine of the VEGF mutants compared to littermates. The VEGF mutant duodenum demonstrated increased angiogenesis and vascular leak as compared to littermate controls. The VEGF mutant duodenum revealed taller villi and increased Ki-67-positive cells in the transit-amplifying zone with reduced Lgr5 expression. The duodenum of sFlt-1 mutants revealed shorter villi and longer crypts with reduced proliferation in the transit-amplifying zone, reduced expression of Dll1, Bmp4 and VE-cadherin, and increased expression of Sox9 and EphB2. Manipulating VEGF bioavailability leads to profound effects on not only the intestinal vasculature, but epithelial stem and progenitor cells in the intestinal crypt. Elucidation of the crosstalk between VEGF signaling in the vasculature, mesenchyme and epithelial stem/progenitor cell populations may direct future cell therapies for intestinal

  10. Lysyl Oxidase Is Predictive of Unfavorable Outcomes and Essential for Regulation of Vascular Endothelial Growth Factor in Hepatocellular Carcinoma.

    PubMed

    Zhu, Jiye; Huang, Shan; Wu, Guobin; Huang, Chaoyuan; Li, Xianjian; Chen, Zhigang; Zhao, Lei; Zhao, Yinnong

    2015-10-01

    Lysyl oxidase (LOX) is frequently overexpressed in a variety of malignancies and involved in tumor invasion and metastasis. Furthermore, it has been shown that LOX is closely related to vascular endothelial growth factor (VEGF). In this study, we aimed to investigate the exact role of LOX and the correlation between LOX and VEGF in hepatocellular carcinoma (HCC). The expression levels of LOX in HCC tissue and adjacent noncancerous tissue were evaluated by quantitative reverse transcription polymerase chain reaction and immunohistochemical analysis. The effect of LOX knockdown on cell proliferation, migration, and invasion was investigated in vitro. The role of LOX in the regulation of VEGF was further characterized in HCC cells that had been treated with transforming growth factor beta (TGF-β). Our study showed that LOX was up-regulated in HCC cell lines and tissue. HCC patients with elevated expression of LOX had relatively shorter disease-free survival and overall survival. Knockdown of LOX reduced the proliferation, migration, and invasion of HCC cells. Additionally, the expression level of LOX positively correlated with that of VEGF. After treatment with TGF-β, the levels of LOX and VEGF were both up-regulated in a dose-dependent manner. In the cells treated with siRNA of LOX, levels of VEGF and phosphorylated p38 were significantly decreased and could not be up-regulated by TGF-β. Inhibition of p38 MAPK signaling abrogated TGF-β-mediated up-regulation of VGEF but did not affect LOX expression. LOX appears to be a predictor of less favorable outcomes and may regulate the expression of VEGF via p38 MAPK signaling.

  11. Intermittent hypoxia augments pulmonary vascular smooth muscle reactivity to NO: regulation by reactive oxygen species.

    PubMed

    Norton, Charles E; Jernigan, Nikki L; Kanagy, Nancy L; Walker, Benjimen R; Resta, Thomas C

    2011-10-01

    Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension. IH causes oxidative stress that may limit bioavailability of the endothelium-derived vasodilator nitric oxide (NO) and thus contribute to this hypertensive response. We therefore hypothesized that increased vascular superoxide anion (O(2)(-)) generation reduces NO-dependent pulmonary vasodilation following IH. To test this hypothesis, we examined effects of the O(2)(-) scavenger tiron on vasodilatory responses to the endothelium-dependent vasodilator ionomycin and the NO donor S-nitroso-N-acetylpenicillamine in isolated lungs from hypocapnic-IH (H-IH; 3 min cycles of 5% O(2)/air flush, 7 h/day, 4 wk), eucapnic-IH (E-IH; cycles of 5% O(2), 5% CO(2)/air flush), and sham-treated (air/air cycled) rats. Next, we assessed effects of endogenous O(2)(-) on NO- and cGMP-dependent vasoreactivity and measured O(2)(-) levels using the fluorescent indicator dihydroethidium (DHE) in isolated, endothelium-disrupted small pulmonary arteries from each group. Both E-IH and H-IH augmented NO-dependent vasodilation; however, enhanced vascular smooth muscle (VSM) reactivity to NO following H-IH was masked by an effect of endogenous O(2)(-). Furthermore, H-IH and E-IH similarly increased VSM sensitivity to cGMP, but this response was independent of either O(2)(-) generation or altered arterial protein kinase G expression. Finally, both H-IH and E-IH increased arterial O(2)(-) levels, although this response was more pronounced following H-IH, and H-IH exposure resulted in greater protein tyrosine nitration indicative of increased NO scavenging by O(2)(-). We conclude that IH increases pulmonary VSM sensitivity to NO and cGMP. Furthermore, endogenous O(2)(-) limits NO-dependent vasodilation following H-IH through an apparent reduction in bioavailable NO.

  12. Statins as Regulators of Redox State in the Vascular Endothelium: Beyond Lipid Lowering

    PubMed Central

    Margaritis, Marios; Channon, Keith M.

    2014-01-01

    Abstract Significance: Endothelial dysfunction and the imbalance between nitric oxide (NO) and reactive oxygen species production in the vascular endothelium are important early steps in atherogenesis, a major socioeconomic health problem. Statins have well-established roles in primary and secondary prevention of cardiovascular disease (CVD), due to both their lipid-lowering capacity and their pleiotropic properties. It is therefore important to understand the mechanisms by which statins can modify endothelial function and affect atherogenesis. Recent Advances: In the last decade, the concept of statin pleiotropy has been reinforced by a large number of cell culture, animal, and translational studies. Statins have been shown to suppress the activity of pro-oxidant enzymes (such as NADPH oxidase) and pro-inflammatory transcriptional pathways in the endothelium. At the same time, they enhance endothelial NO synthase expression and activity while they also improve its enzymatic coupling. This leads to increased NO bioavailability and improved endothelial function. Critical Issues: Despite significant recent advances, the exact mechanisms of statin pleitropy are still only partially understood. The vast majority of the published literature relies on animal studies, while the actual mechanistic studies in humans are limited. Future Directions: The success of statins as endothelium redox-modifying agents with a direct impact on clinical outcome highlights the importance of the endothelium as a therapeutic target in CVD. Better understanding of the mechanisms that underlie endothelial dysfunction could lead to the design of novel therapeutic strategies that target the vascular endothelium for the prevention and treatment of CVD. Antioxid. Redox Signal. 20, 1198–1215. PMID:24111702

  13. Vitamin D regulates the production of vascular endothelial growth factor: A triggering cause in the pathogenesis of rheumatic heart disease?

    PubMed

    Sarkar, Subendu; Chopra, Seema; Rohit, Manoj Kumar; Banerjee, Dibyajyoti; Chakraborti, Anuradha

    2016-10-01

    Rheumatic heart disease (RHD) remains a major cause of cardiac related mortality and morbidity in the developing countries due to poor diagnosis and lack of proper therapeutics. The definite reason of heart valve injury during RHD is poorly understood. Valvular endothelial cells play an important role in pathogenesis of different cardiovascular diseases. Besides, the regulation of vitamin D (calciferol) and VEGF (vascular endothelial growth factor) results in the functional changes in endothelial cells. However, the crosstalk between vitamin D and VEGF in the pathogenesis of RHD is not yet unfurled. Evidences in the concerned fields are documented by searching through Google Scholar and Pubmed. Literature based survey has revealed that vascular endothelium, especially endothelial cells play important roles in valvular remodelling during cardiovascular diseases. Endothelial cell dysfunction leads to heart valve remodelling, which furthermore initiates the pathogenesis of valvular heart disease. Vitamin D has the potential to maintain the concentration of VEGF in the circulation and induce the function of endothelial cells. Hence, we hypothesize that vitamin D and VEGF homeostasis can alter the function of endothelial cells, which may subsequently trigger the valvular remodelling or even damage of heart valves during the progression of RHD pathogenesis. Our hypothesis shed light on the evidence based knowledge translation of plausible cellular phenomena due to vitamin D/VEGF homeostasis during valvular vandalism in RHD. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Vascular Cell Adhesion Molecule-1 Expression and Signaling During Disease: Regulation by Reactive Oxygen Species and Antioxidants

    PubMed Central

    Marchese, Michelle E.; Abdala-Valencia, Hiam

    2011-01-01

    Abstract The endothelium is immunoregulatory in that inhibiting the function of vascular adhesion molecules blocks leukocyte recruitment and thus tissue inflammation. The function of endothelial cells during leukocyte recruitment is regulated by reactive oxygen species (ROS) and antioxidants. In inflammatory sites and lymph nodes, the endothelium is stimulated to express adhesion molecules that mediate leukocyte binding. Upon leukocyte binding, these adhesion molecules activate endothelial cell signal transduction that then alters endothelial cell shape for the opening of passageways through which leukocytes can migrate. If the stimulation of this opening is blocked, inflammation is blocked. In this review, we focus on the endothelial cell adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1). Expression of VCAM-1 is induced on endothelial cells during inflammatory diseases by several mediators, including ROS. Then, VCAM-1 on the endothelium functions as both a scaffold for leukocyte migration and a trigger of endothelial signaling through NADPH oxidase-generated ROS. These ROS induce signals for the opening of intercellular passageways through which leukocytes migrate. In several inflammatory diseases, inflammation is blocked by inhibition of leukocyte binding to VCAM-1 or by inhibition of VCAM-1 signal transduction. VCAM-1 signal transduction and VCAM-1-dependent inflammation are blocked by antioxidants. Thus, VCAM-1 signaling is a target for intervention by pharmacological agents and by antioxidants during inflammatory diseases. This review discusses ROS and antioxidant functions during activation of VCAM-1 expression and VCAM-1 signaling in inflammatory diseases. Antioxid. Redox Signal. 15, 1607–1638. PMID:21050132

  15. Short-term rapamycin treatment in mice has few effects on the transcriptome of white adipose tissue compared to dietary restriction.

    PubMed

    Fok, Wilson C; Livi, Carolina; Bokov, Alex; Yu, Zhen; Chen, Yidong; Richardson, Arlan; Pérez, Viviana I

    2014-09-01

    Rapamycin, a drug that has been shown to increase lifespan in mice, inhibits the target of rapamycin (TOR) pathway, a major pathway that regulates cell growth and energy status. It has been hypothesized that rapamycin and dietary restriction (DR) extend lifespan through similar mechanisms/pathways. Using microarray analysis, we compared the transcriptome of white adipose tissue from mice fed rapamycin or DR-diet for 6 months. Multidimensional scaling and heatmap analyses showed that rapamycin had essentially no effect on the transcriptome as compared to DR. For example, only six transcripts were significantly altered by rapamycin while mice fed DR showed a significant change in over 1000 transcripts. Using ingenuity pathway analysis, we found that stearate biosynthesis and circadian rhythm signaling were significantly changed by DR. Our findings showing that DR, but not rapamycin, has an effect on the transcriptome of the adipose tissue, suggesting that these two manipulations increase lifespan through different mechanisms/pathways. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Adaptive Regulation of Endothelin Receptor Type A and Type B in Vascular Smooth Muscle Cells during Pregnancy in Rats

    PubMed Central

    Ou, Minghui; Dang, Yiping; Mazzuca, Marc Q.; Basile, Rebecca; Khalil, Raouf A.

    2013-01-01

    Normal pregnancy is associated with systemic vasodilation and decreased vascular contraction, partly due to increased release of endothelium-derived vasodilator substances. Endothelin-1 (ET-1) is an endothelium-derived vasoconstrictor acting via endothelin receptor type A (ETAR) and possibly type B (ETBR) in vascular smooth muscle cells (VSMCs), with additional vasodilator effects via endothelial ETBR. However, the role of ET-1 receptor subtypes in the regulation of vascular function during pregnancy is unclear. We investigated whether the decreased vascular contraction during pregnancy reflects changes in the expression/activity of ETAR and ETBR. Contraction was measured in single aortic VSMCs isolated from virgin, mid-pregnant (mid-Preg, day 12) and late-Preg (day 19) Sprague-Dawley rats, and the mRNA expression, protein amount, tissue and cellular distribution of ETAR and ETBR were examined using RT-PCR, Western blots, immunohistochemistry and immunofluorescence. Phenylephrine (Phe, 10−5 M), KCl (51 mM) and ET-1 (10−6 M) caused VSMC contraction that was in late-Preg < mid-Preg and virgin rats. In VSMCs treated with ETBR antagonist BQ788, ET-1 caused significant contraction that was still in late-Preg < mid-Preg and virgin rats. In VSMCs treated with the ETAR antagonist BQ123, ET-1 caused a small contraction; and the ETBR agonists IRL-1620 and sarafotoxin 6c (S6c) caused similar contraction that was in late-Preg < mid-Preg and virgin rats. RT-PCR revealed similar ETAR, but greater ETBR mRNA expression in pregnant vs. virgin rats. Western blots revealed similar ETAR, and greater protein amount of ETBR in endothelium-intact vessels, but reduced ETBR in endothelium-denuded vessels of pregnant vs. virgin rats. Immunohistochemistry revealed prominent ETBR staining in the intima, but reduced ETAR and ETBR in the aortic media of pregnant rats. Immunofluorescence signal for ETAR and ETBR was less in VSMCs of pregnant vs. virgin rats. The pregnancy-associated decrease

  17. MicroRNA-196b Regulates the Homeobox B7-Vascular Endothelial Growth Factor Axis in Cervical Cancer

    PubMed Central

    How, Christine; Hui, Angela B. Y.; Alajez, Nehad M.; Shi, Wei; Boutros, Paul C.; Clarke, Blaise A.; Yan, Rui; Pintilie, Melania; Fyles, Anthony; Hedley, David W.; Hill, Richard P.; Milosevic, Michael; Liu, Fei-Fei

    2013-01-01

    The down-regulation of microRNA-196b (miR-196b) has been reported, but its contribution to cervical cancer progression remains to be investigated. In this study, we first demonstrated that miR-196b down-regulation was significantly associated with worse disease-free survival (DFS) for cervical cancer patients treated with combined chemo-radiation. Secondly, using a tri-modal approach for target identification, we determined that homeobox-B7 (HOXB7) was a bona fide target for miR-196b, and in turn, vascular endothelial growth factor (VEGF) was a downstream transcript regulated by HOXB7. Reconstitution of miR-196b expression by transient transfection resulted in reduced cell growth, clonogenicity, migration and invasion in vitro, as well as reduced tumor angiogenesis and tumor cell proliferation in vivo. Concordantly, siRNA knockdown of HOXB7 or VEGF phenocopied the biological effects of miR-196b over-expression. Our findings have demonstrated that the miR-196b/HOXB7/VEGF pathway plays an important role in cervical cancer progression; hence targeting this pathway could be a promising therapeutic strategy for the future management of this disease. PMID:23861821

  18. Angiopoietin-1 and Vascular Endothelial Growth Factor Regulation of Leukocyte Adhesion to Endothelial Cells: Role of Nuclear Receptor-77

    PubMed Central

    Ismail, Hodan; Mofarrahi, Mahroo; Echavarria, Raquel; Harel, Sharon; Verdin, Eric; Lim, Hyung W.; Jin, Zheng-Gen; Sun, Jianxin; Zeng, Huiyan; Hussain, Sabah N.A.

    2012-01-01

    Objective Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion. Methods and Results Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs. Conclusion Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation. PMID:22628435

  19. KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine

    PubMed Central

    Goodwill, Adam G.; Fu, Lijuan; Noblet, Jillian N.; Casalini, Eli D.; Berwick, Zachary C.; Kassab, Ghassan S.; Tune, Johnathan D.

    2016-01-01

    Hydrogen peroxide (H2O2) and voltage-dependent K+ (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. PMID:26825518

  20. Inhibiting the Mammalian target of rapamycin blocks the development of experimental cerebral malaria.

    PubMed

    Gordon, Emile B; Hart, Geoffrey T; Tran, Tuan M; Waisberg, Michael; Akkaya, Munir; Skinner, Jeff; Zinöcker, Severin; Pena, Mirna; Yazew, Takele; Qi, Chen-Feng; Miller, Louis H; Pierce, Susan K

    2015-06-02

    Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host's immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4(+) and CD8(+) T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism. Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection

  1. Mammalian Target of Rapamycin (mTOR) Inhibition with Rapamycin Improves Cardiac Function in Type 2 Diabetic Mice

    PubMed Central

    Das, Anindita; Durrant, David; Koka, Saisudha; Salloum, Fadi N.; Xi, Lei; Kukreja, Rakesh C.

    2014-01-01

    Elevated mammalian target of rapamycin (mTOR) signaling contributes to the pathogenesis of diabetes, with increased morbidity and mortality, mainly because of cardiovascular complications. Because mTOR inhibition with rapamycin protects against ischemia/reperfusion injury, we hypothesized that rapamycin would prevent cardiac dysfunction associated with type 2 diabetes (T2D). We also investigated the possible mechanisms and novel protein targets involved in rapamycin-induced preservation of cardiac function in T2D mice. Adult male leptin receptor null, homozygous db/db, or wild type mice were treated daily for 28 days with vehicle (5% DMSO) or rapamycin (0.25 mg/kg, intraperitoneally). Cardiac function was monitored by echocardiography, and protein targets were identified by proteomics analysis. Rapamycin treatment significantly reduced body weight, heart weight, plasma glucose, triglyceride, and insulin levels in db/db mice. Fractional shortening was improved by rapamycin treatment in db/db mice. Oxidative stress as measured by glutathione levels and lipid peroxidation was significantly reduced in rapamycin-treated db/db hearts. Rapamycin blocked the enhanced phosphorylation of mTOR and S6, but not AKT in db/db hearts. Proteomic (by two-dimensional gel and mass spectrometry) and Western blot analyses identified significant changes in several cytoskeletal/contractile proteins (myosin light chain MLY2, myosin heavy chain 6, myosin-binding protein C), glucose metabolism proteins (pyruvate dehydrogenase E1, PYGB, Pgm2), and antioxidant proteins (peroxiredoxin 5, ferritin heavy chain 1) following rapamycin treatment in db/db heart. These results show that chronic rapamycin treatment prevents cardiac dysfunction in T2D mice, possibly through attenuation of oxidative stress and alteration of antioxidants and contractile as well as glucose metabolic protein expression. PMID:24371138

  2. Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

    SciTech Connect

    Chung, Eun Joo; Sowers, Anastasia; Thetford, Angela

    2016-11-15

    Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials: C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-timemore » polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results: Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P=.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P<.001). Conclusions: Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.« less

  3. Mammalian Target of Rapamycin Inhibition With Rapamycin Mitigates Radiation-Induced Pulmonary Fibrosis in a Murine Model

    SciTech Connect

    Chung, Eun Joo; Sowers, Anastasia; Thetford, Angela; McKay-Corkum, Grace; Chung, Su I.; Mitchell, James B.; Citrin, Deborah E.

    2016-11-15

    Purpose: Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials: C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results: Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days (P=.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P=.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P<.001). Conclusions: Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

  4. Rapamycin extends murine lifespan but has limited effects on aging.

    PubMed

    Neff, Frauke; Flores-Dominguez, Diana; Ryan, Devon P; Horsch, Marion; Schröder, Susanne; Adler, Thure; Afonso, Luciana Caminha; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Garrett, Lillian; Hans, Wolfgang; Hettich, Moritz M; Holtmeier, Richard; Hölter, Sabine M; Moreth, Kristin; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Rozman, Jan; Naton, Beatrix; Ordemann, Rainer; Adamski, Jerzy; Beckers, Johannes; Bekeredjian, Raffi; Busch, Dirk H; Ehninger, Gerhard; Graw, Jochen; Höfler, Heinz; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Stypmann, Jörg; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabe de Angelis, Martin; Ehninger, Dan

    2013-08-01

    Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.

  5. Osteoprotegerin Inhibits Calcification of Vascular Smooth Muscle Cell via Down Regulation of the Notch1-RBP-Jκ/Msx2 Signaling Pathway

    PubMed Central

    Zhou, Shaoqiong; Fang, Xing; Xin, Huaping; Li, Wei

    2013-01-01

    Objective Vascular calcification is a common pathobiological process which occurs among the elder population and in patients with diabetes and chronic kidney disease. Osteoprotegerin, a secreted glycoprotein that regulates bone mass, has recently emerged as an important regulator of the development of vascular calcification. However, the mechanism is not fully understood. The purpose of this study is to explore novel signaling mechanisms of osteoprotegerin in the osteoblastic differentiation in rat aortic vascular smooth muscle cells (VSMCs). Methods and Results VSMCs were isolated from thoracic aorta of Sprague Dawley rats. Osteoblastic differentiation of VSMCs was induced by an osteogenic medium. We confirmed by Von Kossa staining and direct cellular calcium measurement that mineralization was significantly increased in VSMCs cultured in osteogenic medium; consistent with an enhanced alkaline phosphatase activity. This osteoblastic differentiation in VSMCs was significantly reduced by the addition of osteoprotegerin in a dose responsive manner. Moreover, we identified, by real-time qPCR and western blotting, that expression of Notch1 and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both the mRNA and protein levels, these effects were dose-dependently abolished by the treatment of osteoprotegerin. Furthermore, we identified that Msx2, a downstream target of the Notch1/RBP-Jκ signaling, was markedly down-regulated by the treatment of osteoprotegerin. Conclusion Osteoprotegerin inhibits vascular calcification through the down regulation of the Notch1-RBP-Jκ signaling pathway. PMID:23874840

  6. Osteoprotegerin inhibits calcification of vascular smooth muscle cell via down regulation of the Notch1-RBP-Jκ/Msx2 signaling pathway.

    PubMed

    Zhou, Shaoqiong; Fang, Xin; Fang, Xing; Xin, Huaping; Li, Wei; Qiu, Hongyu; Guan, Siming

    2013-01-01

    Vascular calcification is a common pathobiological process which occurs among the elder population and in patients with diabetes and chronic kidney disease. Osteoprotegerin, a secreted glycoprotein that regulates bone mass, has recently emerged as an important regulator of the development of vascular calcification. However, the mechanism is not fully understood. The purpose of this study is to explore novel signaling mechanisms of osteoprotegerin in the osteoblastic differentiation in rat aortic vascular smooth muscle cells (VSMCs). VSMCs were isolated from thoracic aorta of Sprague Dawley rats. Osteoblastic differentiation of VSMCs was induced by an osteogenic medium. We confirmed by Von Kossa staining and direct cellular calcium measurement that mineralization was significantly increased in VSMCs cultured in osteogenic medium; consistent with an enhanced alkaline phosphatase activity. This osteoblastic differentiation in VSMCs was significantly reduced by the addition of osteoprotegerin in a dose responsive manner. Moreover, we identified, by real-time qPCR and western blotting, that expression of Notch1 and RBP-Jκ were significantly up-regulated in VSMCs cultured in osteogenic medium at both the mRNA and protein levels, these effects were dose-dependently abolished by the treatment of osteoprotegerin. Furthermore, we identified that Msx2, a downstream target of the Notch1/RBP-Jκ signaling, was markedly down-regulated by the treatment of osteoprotegerin. Osteoprotegerin inhibits vascular calcification through the down regulation of the Notch1-RBP-Jκ signaling pathway.

  7. Calcium channel regulation in vascular smooth muscle cells: Synergistic effects of statins and calcium channel blockers

    PubMed Central

    Clunn, Gerard F.; Sever, Peter S.; Hughes, Alun D.

    2009-01-01

    In the Anglo-Scandinavian Cardiac Outcomes Trial–Lipid Lowering Arm (ASCOT-LLA) we have reported a positive interaction between atorvastatin and amlodipine-based antihypertensive strategy in terms of the prevention of coronary events. In cellular and molecular studies on human vascular smooth muscle cells (VSMC) we have reported that transformation from a differentiated to a synthetic or dedifferentiated phenotype is associated with loss of function of L-type calcium channels and hence loss of potential responsiveness to calcium channel blockers (CCB). Statins directly inhibit cell cycle progression and dedifferentiation of VSMC due to their ability to inhibit the synthesis of isoprenoid cholesterol intermediates. We hypothesize that statins promote a more differentiated VSMC phenotype that results in upregulation of L-type calcium channels and restoration of a CCB-sensitive calcium influx pathway in VSMC, favourably affecting the balance that exists between VSMC proliferation, apoptosis and matrix metalloproteinase production with an associated increase in stability of atheromatous plaques. PMID:19523699

  8. Calcium channel regulation in vascular smooth muscle cells: synergistic effects of statins and calcium channel blockers.

    PubMed

    Clunn, Gerard F; Sever, Peter S; Hughes, Alun D

    2010-02-18

    In the Anglo-Scandinavian Cardiac Outcomes Trial-Lipid Lowering Arm (ASCOT-LLA) we have reported a positive interaction between atorvastatin and amlodipine-based antihypertensive strategy in terms of the prevention of coronary events. In cellular and molecular studies on human vascular smooth muscle cells (VSMC) we have reported that transformation from a differentiated to a synthetic or dedifferentiated phenotype is associated with loss of function of L-type calcium channels and hence loss of potential responsiveness to calcium channel blockers (CCB). Statins directly inhibit cell cycle progression and dedifferentiation of VSMC due to their ability to inhibit the synthesis of isoprenoid cholesterol intermediates. We hypothesize that statins promote a more differentiated VSMC phenotype that results in upregulation of L-type calcium channels and restoration of a CCB-sensitive calcium influx pathway in VSMC, favourably affecting the balance that exists between VSMC proliferation, apoptosis and matrix metalloproteinase production with an associated increase in stability of atheromatous plaques. Copyright 2009 Elsevier Ireland Ltd. All rights reserved.

  9. Flowers regulate the growth and vascular development of the inflorescence rachis in Vitis vinifera L.

    PubMed

    Gourieroux, Aude M; McCully, Margaret E; Holzapfel, Bruno P; Scollary, Geoffrey R; Rogiers, Suzy Y

    2016-11-01

    The rachis, the structural framework of the grapevine (Vitis vinifera L.) inflorescence (and subsequent bunch), consists of a main axis and one or more orders of lateral branches with the flower-bearing pedicels at their fine tips. The rachis is crucial both for support, and transport from the shoot. Earlier suggestions that the flowers per se affect normal rachis development are investigated further in this study. Different percentages (0, 25, 50, 75 or 100) of flowers were removed manually one week before anthesis on field-grown vines. Treatment effects on subsequent rachis development (curvature, vitality, anatomy, starch deposit) were assessed. Sections, both fixed and embedded, and fresh hand-cut were observed by fluorescence and bright-field optics after appropriate staining. Emphasis was on measurement of changes in cross-sectional area of secondary xylem and phloem, and on maturation of fibres and periderm. Specific defects in rachis development were dependent on the percent and location of flower removal one week prior to anthesis. The rachises curved inwards where most of the flowers were removed. When fully de-flowered, they became progressively necrotic from the laterals back to the primary axes and from the distal to the proximal end of those axes, with a concurrent disorganisation of their anatomy. A few remaining groups of flowers prevented desiccation and abscission of the rachis axes proximal to the group, but not distally. Flower removal (50%) reduced rachis elongation, while 75% removal reduced xylem and phloem area and delayed phloem fibre and periderm development. 75% flower removal did not affect starch present in the rachis during berry development. Developing flowers affect the growth and vitality of the rachis and the development of its vascular and support structures. The extent of these effects depends on the cultivar and the number and position of flowers remaining after some are removed one week before anthesis. Copyright © 2016

  10. Native vascular related NAC transcription factors are efficient regulator of multiple classes of secondary wall associated genes in banana.

    PubMed

    Negi, Sanjana; Tak, Himanshu; Ganapathi, T R

    2017-12-01

    Secondary-wall deposition in xylem vessel elements is regulated by vascular-related NAC transcription factors (VNDs). We show that three banana VNDs (MusaVND1, MusaVND2 and MusaVND3) directly regulate multiple secondary-wall associated genes by binding to their 5'-upstream regulatory region. Transgenic banana harboring either P MusaVND1 :GUS, P MusaVND2 :GUS or P MusaVND3 :GUS showed specific GUS staining in lignified tissues. MusaVND1, MusaVND2 and MusaVND3 encodes transcriptional-activators as its C-terminal region drive expression of reporter genes in vivo in yeast. Purified MusaVND1, MusaVND2 and MusaVND3 proteins in gel shift assay bind to 19-bp secondary-wall NAC binding element (SNBE) while it fails to bind mutated SNBE. Putative SNBE sites in the 5'-upstream regulatory region of important secondary-wall associated genes related to programmed cell death (XCP1), cell-wall modification (IRX1/CesA8, IRX3/CesA7,IRX5/CesA4, IRX8, IRX10 and IRX12) and transcriptional regulation (MYB52, MYB48/59, MYB85, MYB58/72, MYB46, and MYB83) in banana was identified and mobility of these regulatory regions got retarded by MusaVND1, MusaVND2 and MusaVND3. Transcript level of these important secondary wall associated genes were elevated in transgenic banana overexpressing either MusaVND1, MusaVND2 or MusaVND3. Present study suggested promoters with prospective utilization in wall modification in banana (a potential biofuel crop) and suggest a complex transcriptional regulation of secondary wall deposition in plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Metabolic and Epigenetic Interactions Regulate Vascular Phenotypic Change and Maintenance in Pulmonary Hypertension

    DTIC Science & Technology

    2016-10-01

    regulated genes in PH-Fibs compared to CO-Fibs. Glycolysis/gluconeogenesis and pyruvate metabolism were identified as two of the most significantly...metabolites of the pentose phosphate pathway were observed, consistent with the necessity to generate reducing equivalents (NADPH) to recycle oxidized...in PH- fibroblasts. Increased fluxes through the non-oxidative phase of the pentose phosphate pathway and accumulation of products of lipid anabolism

  12. MicroRNAs as critical regulators of the endothelial to mesenchymal transition in vascular biology.

    PubMed

    Kim, Jongmin

    2018-02-01

    The endothelial to mesenchymal transition (EndMT) is a newly recognized, fundamental biological process involved in development and tissue regeneration, as well as pathological processes such as the complications of diabetes, fibrosis and pulmonary arterial hypertension. The EndMT process is tightly controlled by diverse signaling networks, similar to the epithelial to mesenchymal transition. Accumulating evidence suggests that microRNAs (miRNAs) are key regulators of this network, with the capacity to target multiple messenger RNAs involved in the EndMT process as well as in the regulation of disease progression. Thus, it is highly important to understand the molecular basis of miRNA control of EndMT. This review highlights the current fund of knowledge regarding the known links between miRNAs and the EndMT process, with a focus on the mechanism that regulates associated signaling pathways and discusses the potential for the EndMT as a therapeutic target to treat many diseases. [BMB Reports 2018; 51(2): 65-72].

  13. Estrogen and testosterone in concert with EFNB3 regulate vascular smooth muscle cell contractility and blood pressure

    PubMed Central

    Wang, Yujia; Thorin, Eric; Tremblay, Johanne; Lavoie, Julie L.; Luo, Hongyu; Peng, Junzheng; Qi, Shijie; Wu, Tao; Chen, Fei; Shen, Jianzhong; Hu, Shenjiang; Wu, Jiangping

    2016-01-01

    EPH kinases and their ligands, ephrins (EFNs), have vital and diverse biological functions, although their function in blood pressure (BP) control has not been studied in detail. In the present study, we report that Efnb3 gene knockout (KO) led to increased BP in female but not male mice. Vascular smooth muscle cells (VSMCs) were target cells for EFNB3 function in BP regulation. The deletion of EFNB3 augmented contractility of VSMCs from female but not male KO mice, compared with their wild-type (WT) counterparts. Estrogen augmented VSMC contractility while testosterone reduced it in the absence of EFNB3, although these sex hormones had no effect on the contractility of VSMCs from WT mice. The effect of estrogen on KO VSMC contractility was via a nongenomic pathway involving GPER, while that of testosterone was likely via a genomic pathway, according to VSMC contractility assays and GPER knockdown assays. The sex hormone-dependent contraction phenotypes in KO VSMCs were reflected in BP in vivo. Ovariectomy rendered female KO mice normotensive. At the molecular level, EFNB3 KO in VSMCs resulted in reduced myosin light chain kinase phosphorylation, an event enhancing sensitivity to Ca2+ flux in VSMCs. Our investigation has revealed previously unknown EFNB3 functions in BP regulation and show that EFNB3 might be a hypertension risk gene in certain individuals. PMID:26851246

  14. Rapamycin targeting mTOR and hedgehog signaling pathways blocks human rhabdomyosarcoma growth in xenograft murine model

    SciTech Connect

    Kaylani, Samer Z.; Xu, Jianmin; Srivastava, Ritesh K.; Kopelovich, Levy; Pressey, Joseph G.; Athar, Mohammad

    2013-06-14

    Graphical abstract: Intervention of poorly differentiated RMS by rapamycin: In poorly differentiated RMS, rapamycin blocks mTOR and Hh signaling pathways concomitantly. This leads to dampening in cell cycle regulation and induction of apoptosis. This study provides a rationale for the therapeutic intervention of poorly differentiated RMS by treating patients with rapamycin alone or in combination with other chemotherapeutic agents. -- Highlights: •Rapamycin abrogates RMS tumor growth by modulating proliferation and apoptosis. •Co-targeting mTOR/Hh pathways underlie the molecular basis of effectiveness. •Reduction in mTOR/Hh pathways diminish EMT leading to reduced invasiveness. -- Abstract: Rhabdomyosarcomas (RMS) represent the most common childhood soft-tissue sarcoma. Over the past few decades outcomes for low and intermediate risk RMS patients have slowly improved while patients with metastatic or relapsed RMS still face a grim prognosis. New chemotherapeutic agents or combinations of chemotherapies have largely failed to improve the outcome. Based on the identification of novel molecular targets, potential therapeutic approaches in RMS may offer a decreased reliance on conventional chemotherapy. Thus, identification of effective therapeutic agents that specifically target relevant pathways may be particularly beneficial for patients with metastatic and refractory RMS. The PI3K/AKT/mTOR pathway has been found to be a potentially attractive target in RMS therapy. In this study, we provide evidence that rapamycin (sirolimus) abrogates growth of RMS development in a RMS xenograft mouse model. As compared to a vehicle-treated control group, more than 95% inhibition in tumor growth was observed in mice receiving parenteral administration of rapamycin. The residual tumors in rapamycin-treated group showed significant reduction in the expression of biomarkers indicative of proliferation and tumor invasiveness. These tumors also showed enhanced apoptosis

  15. Kallistatin reduces vascular senescence and aging by regulating microRNA-34a-SIRT1 pathway.

    PubMed

    Guo, Youming; Li, Pengfei; Gao, Lin; Zhang, Jingmei; Yang, Zhirong; Bledsoe, Grant; Chang, Eugene; Chao, Lee; Chao, Julie

    2017-08-01

    Kallistatin, an endogenous protein, protects against vascular injury by inhibiting oxidative stress and inflammation in hypertensive rats and enhancing the mobility and function of endothelial progenitor cells (EPCs). We aimed to determine the role and mechanism of kallistatin in vascular senescence and aging using cultured EPCs, streptozotocin (STZ)-induced diabetic mice, and Caenorhabditis elegans (C. elegans). Human kallistatin significantly decreased TNF-α-induced cellular senescence in EPCs, as indicated by reduced senescence-associated β-galactosidase activity and plasminogen activator inhibitor-1 expression, and elevated telomerase activity. Kallistatin blocked TNF-α-induced superoxide levels, NADPH oxidase activity, and microRNA-21 (miR-21) and p16 INK 4a synthesis. Kallistatin prevented TNF-α-mediated inhibition of SIRT1, eNOS, and catalase, and directly stimulated the expression of these antioxidant enzymes. Moreover, kallistatin inhibited miR-34a synthesis, whereas miR-34a overexpression abolished kallistatin-induced antioxidant gene expression and antisenescence activity. Kallistatin via its active site inhibited miR-34a, and stimulated SIRT1 and eNOS synthesis in EPCs, which was abolished by genistein, indicating an event mediated by tyrosine kinase. Moreover, kallistatin administration attenuated STZ-induced aortic senescence, oxidative stress, and miR-34a and miR-21 synthesis, and increased SIRT1, eNOS, and catalase levels in diabetic mice. Furthermore, kallistatin treatment reduced superoxide formation and prolonged wild-type C. elegans lifespan under oxidative or heat stress, although kallistatin's protective effect was abolished in miR-34 or sir-2.1 (SIRT1 homolog) mutant C. elegans. Kallistatin inhibited miR-34, but stimulated sir-2.1 and sod-3 synthesis in C. elegans. These in vitro and in vivo studies provide significant insights into the role and mechanism of kallistatin in vascular senescence and aging by regulating miR-34a-SIRT1

  16. 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.

  17. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide?

    PubMed Central

    Ali, M Y; Ping, C Y; Mok, Y-YP; Ling, L; Whiteman, M; Bhatia, M; Moore, P K

    2006-01-01

    Background and Purpose: The aim of these experiments was to evaluate the significance of the chemical reaction between hydrogen sulphide (H2S) and nitric oxide (NO) for the control of vascular tone. Experimental Approach: The effect of sodium hydrosulphide (NaHS; H2S donor) and a range of NO donors, such as sodium nitroprusside (SNP), either alone or together, was determined using phenylephrine (PE)-precontracted rat aortic rings and on the blood pressure of anaesthetised rats. Key Results: Mixing NaHS with NO donors inhibited the vasorelaxant effect of NO both in vitro and in vivo. Low concentrations of NaHS or H2S gas in solution reversed the relaxant effect of acetylcholine (ACh, 400 nM) and histamine (100 μM) but not isoprenaline (400 nM). The effect of NaHS on the ACh response was antagonized by CuSO4 (200 nM) but was unaffected by glibenclamide (10 μM). In contrast, high concentrations of NaHS (200–1600 μM) relaxed aortic rings directly, an effect reduced by glibenclamide but unaffected by CuSO4. Intravenous infusion of a low concentration of NaHS (10 μmol kg-1 min-1) into the anaesthetized rat significantly increased mean arterial blood pressure. L-NAME (25 mg kg-1, i.v.) pretreatment reduced this effect. Conclusions and Implications: These results suggest that H2S and NO react together to form a molecule (possibly a nitrosothiol) which exhibits little or no vasorelaxant activity either in vitro or in vivo. We propose that a crucial, and hitherto unappreciated, role of H2S in the vascular system is the regulation of the availability of NO. PMID:17016507

  18. Rapamycin Inhibition of mTOR Reduces Levels of the Na+/H+ Exchanger 3 in Intestines of Mice and Humans, Leading to Diarrhea.

    PubMed

    Yang, Jun; Zhao, Xiaofeng; Patel, Archana; Potru, Rachana; Azizi-Ghannad, Sadra; Dolinger, Michael; Cao, James; Bartholomew, Catherine; Mazurkiewicz, Joseph; Conti, David; Jones, David; Huang, Yunfei; Zhu, Xinjun Cindy

    2015-07-01

    . This process appears to require the autophagic activity mediated by ATG7. Loss of mTOR regulation of NHE3 could mediate the development of diarrhea in patients undergoing rapamycin therapy. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

  19. Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling

    PubMed Central

    Wilson, Christopher W.; Parker, Leon H.; Hall, Christopher J.; Smyczek, Tanya; Mak, Judy; Crow, Ailey; Posthuma, George; De Mazière, Ann; Sagolla, Meredith; Chalouni, Cecile; Vitorino, Philip; Roose-Girma, Merone; Warming, Søren; Klumperman, Judith; Crosier, Philip S.

    2013-01-01

    Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1–dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling. PMID:23886837

  20. MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane

    PubMed Central

    Kurz, Angela R.M.; Pruenster, Monika; Rohwedder, Ina; Ramadass, Mahalakshmi; Schäfer, Kerstin; Harrison, Ute; Nussbaum, Claudia; Immler, Roland; Wiessner, Johannes R.; Lim, Dae-Sik; Walzog, Barbara; Dietzel, Steffen; Moser, Markus; Klein, Christoph; Vestweber, Dietmar; Catz, Sergio D.

    2016-01-01

    Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20–like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1–/–) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1–/– neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1–/– neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency. PMID:27701149

  1. Transient receptor potential vanilloid 4 channel regulates vascular endothelial permeability during colonic inflammation in dextran sulphate sodium-induced murine colitis.

    PubMed

    Matsumoto, Kenjiro; Yamaba, Riho; Inoue, Ken; Utsumi, Daichi; Tsukahara, Takuya; Amagase, Kikuko; Tominaga, Makoto; Kato, Shinichi

    2018-01-01

    The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel involved in physical sensing in various tissue types. The present study aimed to elucidate the function and expression of TRPV4 channels in colonic vascular endothelial cells during dextran sulphate sodium (DSS)-induced colitis. The role of TRPV4 channels in the progression of colonic inflammation was examined in a murine DSS-induced colitis model using immunohistochemical analysis, Western blotting and Evans blue dye extrusion assay. DSS-induced colitis was significantly attenuated in TRPV4-deficient (TRPV4 KO) as compared to wild-type mice. Repeated intrarectal administration of GSK1016790A, a TRPV4 agonist, exacerbated the severity of DSS-induced colitis. Bone marrow transfer experiments demonstrated the important role of TRPV4 in non-haematopoietic cells for DSS-induced colitis. DSS treatment up-regulated TRPV4 expression in the vascular endothelia of colonic mucosa and submucosa. DSS treatment increased vascular permeability, which was abolished in TRPV4 KO mice. This DSS-induced increase in vascular permeability was further enhanced by i.v. administration of GSK1016790A, and this effect was abolished by the TRPV4 antagonist RN1734. TRPV4 was co-localized with vascular endothelial (VE)-cadherin, and VE-cadherin expression was decreased by repeated i.v. administration of GSK1016790A during colitis. Furthermore, GSK106790A decreased VE-cadherin expression in mouse aortic endothelial cells exposed to TNF-α. These findings indicate that an up-regulation of TRPV4 channels in vascular endothelial cells contributes to the progression of colonic inflammation by increasing vascular permeability. Thus, TRPV4 is an attractive target for the treatment of inflammatory bowel diseases. © 2017 The British Pharmacological Society.

  2. Microparticulate Caspase-1 Regulates Gasdermin-D and Pulmonary Vascular Endothelial Cell Injury.

    PubMed

    Mitra, Srabani; Exline, Matthew; Habyarimana, Fabien; Gavrilin, Mikhail; Baker, Paul; Masters, Seth L; Wewers, Mark D; Sarkar, Anasuya

    2018-01-24

    Lung endothelial cell apoptosis and injury occurs throughout all stages of acute lung injury (ALI/ARDS) and impacts disease progression. Caspases 1, 4 and 5 are essential for completion of the apoptotic program known as pyroptosis that also involves pro-inflammatory cytokines. Because GSDM-D mediates pyroptotic death and is essential for pore formation, we hypothesized that it may direct caspase-1 encapsulated microparticle (MP) release and mediate endothelial cell death. Our current work provides evidence that GSDM-D is released by LPS stimulated THP1 monocytic cells where it is packaged into microparticles along with active caspase-1. Furthermore, only MP released from stimulated monocytic cells that contain both cleaved GSDM-D and active caspase-1 induce endothelial cell apoptosis. MPs pretreated with caspase-1 inhibitor, YVAD, or pan-caspase inhibitor, ZVAD, do not contain cleaved GSDM-D. MPs from caspase-1KO cells are also deficient in p30 active GSDM-D, further confirming that caspase-1 regulates GSDM-D function. Although control MPs contained cleaved GSDM-D without caspase-1, these fractions were unable to induce cell death, suggesting that encapsulation of both caspase-1 and GSDM-D is essential for cell death induction. Release of microparticulate active caspase-1 was abrogated in GSDM-KO cells, although cytosolic caspase-1 activation was not impaired. Lastly, higher levels of microparticulate GSDM-D was detected in septic ARDS patient plasma when compared to healthy donors. Taken together, these findings suggest that GSDM-D regulates the release of microparticulate active caspase-1 from monocytes essential for induction of cell death and thereby may play a critical role in sepsis-induced endothelial cell injury.

  3. 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.

  4. 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

    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 negativemore » 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.« less

  5. Rapamycin suppresses brain aging in senescence-accelerated OXYS rats.

    PubMed

    Kolosova, Nataliya G; Vitovtov, Anton O; Muraleva, Natalia A; Akulov, Andrey E; Stefanova, Natalia A; Blagosklonny, Mikhail V

    2013-06-01

    Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span inC elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wister rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging.

  6. Rapamycin suppresses brain aging in senescence-accelerated OXYS rats

    PubMed Central

    Kolosova, Nataliya G.; Vitovtov, Anton O.; Muraleva, Natalia A; Akulov, Andrey E.; Stefanova, Natalia A.; Blagosklonny, Mikhail V.

    2013-01-01

    Cellular and organismal aging are driven in part by the MTOR (mechanistic target of rapamycin) pathway and rapamycin extends life span in C elegans, Drosophila and mice. Herein, we investigated effects of rapamycin on brain aging in OXYS rats. Previously we found, in OXYS rats, an early development of age-associated pathological phenotypes similar to several geriatric disorders in humans, including cerebral dysfunctions. Behavioral alterations as well as learning and memory deficits develop by 3 months. Here we show that rapamycin treatment (0.1 or 0.5 mg/kg as a food mixture daily from the age of 1.5 to 3.5 months) decreased anxiety and improved locomotor and exploratory behavior in OXYS rats. In untreated OXYS rats, MRI revealed an increase of the area of hippocampus, substantial hydrocephalus and 2-fold increased area of the lateral ventricles. Rapamycin treatment prevented these abnormalities, erasing the difference between OXYS and Wistar rats (used as control). All untreated OXYS rats showed signs of neurodegeneration, manifested by loci of demyelination. Rapamycin decreased the percentage of animals with demyelination and the number of loci. Levels of Tau and phospho-Tau (T181) were increased in OXYS rats (compared with Wistar). Rapamycin significantly decreased Tau and inhibited its phosphorylation in the hippocampus of OXYS and Wistar rats. Importantly, rapamycin treatment caused a compensatory increase in levels of S6 and correspondingly levels of phospo-S6 in the frontal cortex, indicating that some downstream events were compensatory preserved, explaining the lack of toxicity. We conclude that rapamycin in low chronic doses can suppress brain aging. PMID:23817674

  7. A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

    SciTech Connect

    Latham, Antony M.; Odell, Adam F.; Mughal, Nadeem A.; Issitt, Theo; Ulyatt, Clare; Walker, John H.; Homer-Vanniasinkam, Shervanthi; Ponnambalam, Sreenivasan

    2012-11-01

    Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states. -- Highlights: Black-Right-Pointing-Pointer Endothelial cells mount a stress response under conditions of low serum. Black

  8. Rapamycin extends life- and health span because it slows aging

    PubMed Central

    Blagosklonny, Mikhail V.

    2013-01-01

    Making headlines, a thought-provocative paper by Neff, Ehninger and coworkers claims that rapamycin extends life span but has limited effects on aging. How is that possibly possible? And what is aging if not an increase of the probability of death with age. I discuss that the JCI paper actually shows that rapamycin slows aging and also extends lifespan regardless of its direct anti-cancer activities. Aging is, in part, MTOR-driven: a purposeless continuation of developmental growth. Rapamycin affects the same processes in young and old animals: young animals' traits and phenotypes, which continuations become hyperfunctional, harmful and lethal later in life. PMID:23934728

  9. Rapamycin extends life- and health span because it slows aging.

    PubMed

    Blagosklonny, Mikhail V

    2013-08-01

    Making headlines, a thought-provocative paper by Neff, Ehninger and coworkers claims that rapamycin extends life span but has limited effects on aging. How is that possibly possible? And what is aging if not an increase of the probability of death with age. I discuss that the JCI paper actually shows that rapamycin slows aging and also extends lifespan regardless of its direct anti-cancer activities. Aging is, in part, MTOR-driven: a purposeless continuation of developmental growth. Rapamycin affects the same processes in young and old animals: young animals' traits and phenotypes, which continuations become hyperfunctional, harmful and lethal later in life.

  10. Rapamycin, anti-aging, and avoiding the fate of Tithonus

    PubMed Central

    Richardson, Arlan

    2013-01-01

    The discovery that rapamycin increased the lifespan of mice was recognized by Science as one of the top 10 scientific breakthroughs of 2009. In addition to increasing lifespan, Neff and colleagues show that while rapamycin improves several functions/pathologies that change with age, it has little effect on the majority of the physiological and structural parameters they evaluated. What do these data tell us about the ability of rapamycin to delay aging and improve quality of life, i.e., prevent the fate of Tithonus? PMID:24063054

  11. Rapamycin, anti-aging, and avoiding the fate of Tithonus.

    PubMed

    Richardson, Arlan

    2013-08-01

    The discovery that rapamycin increased the lifespan of mice was recognized by Science as one of the top 10 scientific breakthroughs of 2009. In addition to increasing lifespan, Neff and colleagues show that while rapamycin improves several functions/pathologies that change with age, it has little effect on the majority of the physiological and structural parameters they evaluated. What do these data tell us about the ability of rapamycin to delay aging and improve quality of life, i.e., prevent the fate of Tithonus?

  12. Regulated Expression of Vascular Cell Adhesion Molecule-1 in Human Malignant Melanoma

    PubMed Central

    Jonjic, Nives; Martìn-Padura, Inés; Pollicino, Teresa; Bernasconi, Sergio; Jílek, Petr; Bigotti, Aldo; Mortarini, Roberta; Anichini, Andrea; Parmiani, Giorgio; Colotta, Francesco; Dejana, Elisabetta; Mantovani, Alberto; Natali, Pier Giorgio

    1992-01-01

    Expression of the endothelial adhesion molecule VCAM-1 was studied in human malignant melanoma lines by flow cytometry. Clones 2/4 and 2/14(derived from the same lesion) had appreciable levels of VCAM-1 expression, whereas clone 2/21 and thelines A2058, Mel24, and A375 were negative. Clone 2/14 was selected for further analysis. Exposure to tumor necrosis factor (TNF) markedly augmented VCAM-1 on melanoma cells. Surface VCAM-1 was associated with expression of specific transcripts that were augmented by TNF. Analysis by reverse transcriptase and polymerase chain reaction using appropriate primers revealed that TNF-stimulated melanoma cells expressed both 7 and 6 immunoglobulin domain transcripts with predominance of the longer species. Tumor necrosis factor-stimulated melanoma cells bound more VLA-4-expressing cells (melanoma and monocytes) than resting tumor cells and anti-VCAM-1 monoclonal antibodies significantly inhibited binding, thus suggesting that surface VCAM-1 on melanoma is functional. Analysis of melanoma tissue sections demonstrated that VCAM-1 is not a marker of transformation of melanocytes because it can be detected in benign nevi. Although, unlike ICAM-1, VCAM-1 is not correlated with tumor progression, its expression in a fraction of primary melanomas indicates that it may play a role in regulating host immune response and homotypic interactions in some malignant melanomas ImagesFigure 2Figure 3Figure 5 PMID:1281617

  13. NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress

    PubMed Central

    Alonso, Judith; Galán, María; Martí-Pàmies, Ingrid; Romero, José María; Camacho, Mercedes; Rodríguez, Cristina; Martínez-González, José

    2016-01-01

    Vascular cell survival is compromised under pathological conditions such as abdominal aortic aneurysm (AAA). We have previously shown that the nuclear receptor NOR-1 is involved in the survival response of vascular cells to hypoxia. Here, we identify the anti-apoptotic protein cIAP2 as a downstream effector of NOR-1. NOR-1 and cIAP2 were up-regulated in human AAA samples, colocalizing in vascular smooth muscle cells (VSMC). While NOR-1 silencing reduced cIAP2 expression in vascular cells, lentiviral over-expression of this receptor increased cIAP2 mRNA and protein levels. The transcriptional regulation of the human cIAP2 promoter was analyzed in cells over-expressing NOR-1 by luciferase reporter assays, electrophoretic mobility shift analysis and chromatin immunoprecipitation, identifying a NGFI-B site (NBRE-358/-351) essential for NOR-1 responsiveness. NOR-1 and cIAP2 were up-regulated by hypoxia and by a hypoxia mimetic showing a similar time-dependent pattern. Deletion and site-directed mutagenesis studies show that NOR-1 mediates the hypoxia-induced cIAP2 expression. While NOR-1 over-expression up-regulated cIAP2 and limited VSMC apoptosis induced by hypoxic stress, cIAP2 silencing partially prevented this NOR-1 pro-survival effect. These results indicate that cIAP2 is a target of NOR-1, and suggest that this anti-apoptotic protein is involved in the survival response to hypoxic stress mediated by NOR-1 in vascular cells. PMID:27654514

  14. NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress.

    PubMed

    Alonso, Judith; Galán, María; Martí-Pàmies, Ingrid; Romero, José María; Camacho, Mercedes; Rodríguez, Cristina; Martínez-González, José

    2016-09-22

    Vascular cell survival is compromised under pathological conditions such as abdominal aortic aneurysm (AAA). We have previously shown that the nuclear receptor NOR-1 is involved in the survival response of vascular cells to hypoxia. Here, we identify the anti-apoptotic protein cIAP2 as a downstream effector of NOR-1. NOR-1 and cIAP2 were up-regulated in human AAA samples, colocalizing in vascular smooth muscle cells (VSMC). While NOR-1 silencing reduced cIAP2 expression in vascular cells, lentiviral over-expression of this receptor increased cIAP2 mRNA and protein levels. The transcriptional regulation of the human cIAP2 promoter was analyzed in cells over-expressing NOR-1 by luciferase reporter assays, electrophoretic mobility shift analysis and chromatin immunoprecipitation, identifying a NGFI-B site (NBRE-358/-351) essential for NOR-1 responsiveness. NOR-1 and cIAP2 were up-regulated by hypoxia and by a hypoxia mimetic showing a similar time-dependent pattern. Deletion and site-directed mutagenesis studies show that NOR-1 mediates the hypoxia-induced cIAP2 expression. While NOR-1 over-expression up-regulated cIAP2 and limited VSMC apoptosis induced by hypoxic stress, cIAP2 silencing partially prevented this NOR-1 pro-survival effect. These results indicate that cIAP2 is a target of NOR-1, and suggest that this anti-apoptotic protein is involved in the survival response to hypoxic stress mediated by NOR-1 in vascular cells.

  15. Cyclic strain-mediated regulation of vascular endothelial cell migration and tube formation.

    PubMed

    Von Offenberg Sweeney, Nicholas; Cummins, Philip M; Cotter, Eoin J; Fitzpatrick, Paul A; Birney, Yvonne A; Redmond, Eileen M; Cahill, Paul A

    2005-04-08

    Hemodynamic forces exerted by blood flow (cyclic strain, shear stress) affect the initiation and progression of angiogenesis; however, the precise signaling mechanism(s) involved are unknown. In this study, we examine the role of cyclic strain in regulating bovine aortic endothelial cell (BAEC) migration and tube formation, indices of angiogenesis. Considering their well-documented mechanosensitivity, functional inter-dependence, and involvement in angiogenesis, we hypothesized roles for matrix metalloproteinases (MMP-2/9), RGD-dependent integrins, and urokinase plasminogen activator (uPA) in this process. BAECs were exposed to equibiaxial cyclic strain (5% strain, 1Hz for 24h) before their migration and tube formation was assessed by transwell migration and collagen gel tube formation assays, respectively. In response to strain, both migration and tube formation were increased by 1.83+/-0.1- and 1.84+/-0.1-fold, respectively. Pertussis toxin, a Gi-protein inhibitor, decreased strain-induced migration by 45.7+/-32% and tube formation by 69.8+/-13%, whilst protein tyrosine kinase (PTK) inhibition with genistein had no effect. siRNA-directed attenuation of endothelial MMP-9 (but not MMP-2) expression/activity decreased strain-induced migration and tube formation by 98.6+/-41% and 40.7+/-31%, respectively. Finally, integrin blockade with cRGD peptide and siRNA-directed attenuation of uPA expression reduced strain-induced tube formation by 85.7+/-15% and 84.7+/-31%, respectively, whilst having no effect on migration. Cyclic strain promotes BAEC migration and tube formation in a Gi-protein-dependent PTK-independent manner. Moreover, we demonstrate for the first time a putative role for MMP-9 in both strain-induced events, whilst RGD-dependent integrins and uPA appear only to be involved in strain-induced tube formation.

  16. FOXO3a reactivation mediates the synergistic cytotoxic effects of rapamycin and cisplatin in oral squamous cell carcinoma cells

    SciTech Connect

    Fang Liang; Wang Huiming; Zhou Lin; Yu Da

    2011-02-15

    FOXO3a, a well-known transcriptional regulator, controls a wide spectrum of biological processes. The Phosphoinositide-3-kinase (PI3K)/Akt signaling pathway inactivates FOXO3a via phosphorylation-induced nuclear exclusion and degradation. A loss or gain of FOXO3a activity has been correlated with efficiency of chemotherapies in various cancers including oral squamous cell carcinoma (OSCC). Therefore, in the current study, we have investigated the FOXO3a activity modulating and antitumor effects of rapamycin and cisplatin in OSCC cells. Cisplatin inhibited proliferation and induced apoptosis in a dose-dependent way in OSCC Tca8113 cells. Rapamycin alone had no effect on cell proliferation and apoptosis. Rapamycin downregulated the expression of S-phase kinase associated protein-2 (Skp2) and increased the FOXO3a protein stability but induced the upregulation of feedback Akt activation-mediated FOXO3a phosphorylation. Cisplatin decreased the phosphorylation of FOXO3a via Akt inhibition. Rapamycin combined with cisplatin as its feedback Akt activation inhibitor revealed the most dramatic FOXO3a nuclear localization and reactivation with the prevention of its feedback loop and exposed significant synergistic effects of decreased cell proliferation and increased apoptosis in vitro and decreased tumor size in vivo. Furthermore, the downstream effects of FOXO3a reactivation were found to be accumulation of p27 and Bim. In conclusion, rapamycin/cisplatin combination therapy boosts synergistic antitumor effects through the significant FOXO3a reactivation in OSCC cells. These results may represent a novel mechanism by which rapamycin/cisplatin combination therapy proves to be a potent molecular-targeted strategy for OSCC.

  17. FOXO3a reactivation mediates the synergistic cytotoxic effects of rapamycin and cisplatin in oral squamous cell carcinoma cells

    SciTech Connect

    Fang Liang; Wang Huiming, E-mail: hmwang_1960@yahoo.com.cn; Zhou Lin

    2011-02-15

    FOXO3a, a well-known transcriptional regulator, controls a wide spectrum of biological processes. The Phosphoinositide-3-kinase (PI3K)/Akt signaling pathway inactivates FOXO3a via phosphorylation-induced nuclear exclusion and degradation. A loss or gain of FOXO3a activity has been correlated with efficiency of chemotherapies in various cancers including oral squamous cell carcinoma (OSCC). Therefore, in the current study, we have investigated the FOXO3a activity modulating and antitumor effects of rapamycin and cisplatin in OSCC cells. Cisplatin inhibited proliferation and induced apoptosis in a dose-dependent way in OSCC Tca8113 cells. Rapamycin alone had no effect on cell proliferation and apoptosis. Rapamycin downregulated the expression of S-phasemore » kinase associated protein-2 (Skp2) and increased the FOXO3a protein stability but induced the upregulation of feedback Akt activation-mediated FOXO3a phosphorylation. Cisplatin decreased the phosphorylation of FOXO3a via Akt inhibition. Rapamycin combined with cisplatin as its feedback Akt activation inhibitor revealed the most dramatic FOXO3a nuclear localization and reactivation with the prevention of its feedback loop and exposed significant synergistic effects of decreased cell proliferation and increased apoptosis in vitro and decreased tumor size in vivo. Furthermore, the downstream effects of FOXO3a reactivation were found to be accumulation of p27 and Bim. In conclusion, rapamycin/cisplatin combination therapy boosts synergistic antitumor effects through the significant FOXO3a reactivation in OSCC cells. These results may represent a novel mechanism by which rapamycin/cisplatin combination therapy proves to be a potent molecular-targeted strategy for OSCC.« less

  18. Up-Regulation of Pressure-activated Ca2+-permeable Cation Channel in Intact Vascular Endothelium of Hypertensive Rats

    NASA Astrophysics Data System (ADS)

    Hoyer, J.; Kohler, R.; Haase, W.; Distler, A.

    1996-10-01

    In endothelial cells, stretch-activated cation channels have been proposed to act as mechanosensors for changes in hemodynamic forces. We have identified a novel mechanosensitive pressure-activated channel in intact endothelium from rat aorta and mesenteric artery. The 18-pS cation channel responded with a multifold increase in channel activity when positive pressure was applied to the luminal cell surface with the patch pipette and inactivated at negative pipette pressure. Channel permeability ratio for K+, Na+, and Ca2+ ions was 1:0.98:0.23. Ca2+ influx through the channel was sufficient to activate a neighboring Ca2+-dependent K+ channel. Hemodynamic forces are chronically disturbed in arterial hypertension. Endothelial cell dysfunction has been implicated in the pathogenesis of arterial hypertension. In two comparative studies, density of the pressure-activated channel was found to be significantly higher in spontaneously hypertensive rats and renovascular hypertensive rats compared with their respective normotensive controls. Channel activity presumably leads to mechanosensitive Ca2+ influx and induces cell hyperpolarization by K+ channel activity. Both Ca2+ influx and hyperpolarization are known to induce a vasodilatory endothelial response by stimulating endothelial nitric oxide (NO) production. Up-regulation of channel density in hypertension could, therefore, represent a counterregulatory mechanism of vascular endothelium.

  19. Oxidant-redox regulation of pulmonary vascular responses to hypoxia and nitric oxide-cGMP signaling.

    PubMed

    Wolin, Michael S; Gupte, Sachin A; Neo, Boon Hwa; Gao, Qun; Ahmad, Mansoor

    2010-01-01

    Most current theories for the mechanism of hypoxic pulmonary vasoconstriction (HPV) include a role for reactive oxygen species and/or changes in redox regulation, but extreme controversy exists regarding which systems and redox changes mediate the HPV response. Nitric oxide (NO) appears to help to maintain low pulmonary arterial pressure, suppress HPV, and prevent the development of pulmonary hypertension. Our studies have found a key role for glucose-6-phosphate dehydrogenase in bovine pulmonary arterial smooth muscle functioning to maintain elevated levels of cytosolic NADPH which fuels the generation of vasodilator levels of hydrogen peroxide. HPV results from hypoxia removing vasodilation by peroxide. Decreased superoxide generation by Nox4 oxidase and its conversion to peroxide by Cu,Zn-SOD appear to be potential factors in sensing hypoxia, and decreased cGMP-associated vasodilation and removal of redox controlled vasodilator mechanisms by increased cytosolic NADPH may be key coordinators of the HPV response. Oxidant generation associated with vascular disease processes, including the removal of NO by superoxide, and attenuation of its ability to stimulate cGMP production by oxidation of the heme and thiols of soluble guanylate cyclase attenuate potential beneficial actions of NO on pulmonary arterial function. While pulmonary hypertension appears to have multiple poorly understood effects on redox-associated processes, potentially influencing responses to hypoxia and NO-cGMP signaling, much remains to be elucidated regarding how these processes may be important factors in the progression, expression and therapeutic treatment of pulmonary hypertension.

  20. Hypoxic Regulation of Vascular Endothelial Growth Factor mRNA Stability Requires the Cooperation of Multiple RNA Elements

    PubMed Central

    Dibbens, J. A.; Miller, D. L.; Damert, A.; Risau, W.; Vadas, M. A.; Goodall, G. J.

    1999-01-01

    Vascular endothelial growth factor (VEGF) is a key regulator of developmental, physiological, and tumor angiogenesis. Upregulation of VEGF expression by hypoxia appears to be a critical step in the neovascularization of solid cancers. The VEGF mRNA is intrinsically labile, but in response to hypoxia the mRNA is stabilized. We have systematically analyzed the regions in the VEGF mRNA that are responsible for its lability under normoxic conditions and for stabilization in response to hypoxia. We find that the VEGF mRNA not only contains destabilizing elements in its 3′ untranslated region (3′UTR), but also contains destabilizing elements in the 5′UTR and coding region. Each region can independently promote mRNA degradation, and together they act additively to effect rapid degradation under normoxic conditions. Stabilization of the mRNA in response to hypoxia is completely dependent on the cooperation of elements in each of the 5′UTR, coding region, and 3′UTR. Combinations of any of two of these three regions were completely ineffective in responding to hypoxia, whereas combining all three regions allowed recapitulation of the hypoxic stabilization seen with the endogenous VEGF mRNA. We conclude that multiple regions in the VEGF mRNA cooperate both to ensure the rapid degradation of the mRNA under normoxic conditions and to allow stabilization of the mRNA in response to hypoxia. Our findings highlight the complexity of VEGF gene expression and also reveal a mechanism of gene regulation that could become the target for strategies of therapeutic intervention. PMID:10198046

  1. Rapamycin prevents strong phosphorylation of p53 on serine 46 and attenuates activation of the p53 pathway in A549 lung cancer cells exposed to actinomycin D.

    PubMed

    Krześniak, Małgorzata; Zajkowicz, Artur; Matuszczyk, Iwona; Rusin, Marek

    2014-07-01

    The activation of the p53 pathway by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a molecule that mimics metabolic stress, is attenuated by rapamycin, an inhibitor of mTOR kinase, immunosuppressant, and cancer drug. Rapamycin also extends lifespan in experimental animals. Because AICAR is a relatively weak activator of p53, we investigated whether stimulation of p53 by the strong activator actinomycin D is also sensitive to the inhibitory effect of rapamycin. In A549 lung cancer cells, activation of p53 by actinomycin D was associated with phosphorylation of p53 on Ser46. Rapamycin inhibited the accumulation of phospho-Ser46 p53, attenuated upregulation of some p53 target genes, and altered cell-cycle progression. Moreover, in cells exposed to actinomycin D, rapamycin attenuated the accumulation of PML, a protein that in some conditions stimulates Ser46 phosphorylation. However, Ser46 phosphorylation was not diminished in PML-knockdown cells, suggesting that in our system PML does not play a major role in stimulating p53 phosphorylation on Ser46. Knockdown of p53 diminished the upregulation of PML by stress-inducing agents, consistent with the idea that PML is a p53-regulated gene. Our data suggest that the attenuation of p53 phosphorylation on Ser46 may play a significant role in the biological activity of anti-aging rapamycin. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive.

    PubMed

    Laurent, Sébastien; Richard, Adrien; Mulner-Lorillon, Odile; Morales, Julia; Flament, Didier; Glippa, Virginie; Bourdon, Jérémie; Gosselin, Pauline; Siegel, Anne; Cormier, Patrick; Bellé, Robert

    2014-01-01

    Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR.

  3. Rapamycin and quasi-programmed aging: four years later.

    PubMed

    Blagosklonny, Mikhail V

    2010-05-15

    In 2006, Cell Cycle featured the concept that aging is not caused by molecular damage (nor by free radicals) but instead is a purposeless quasi-program (program-like, but not a program) driven in part by TOR (Target of Rapamycin). Taken together with the analysis of clinical data, this pointed to Sirolimus (rapamycin) as a genuine anti-aging drug which will prolong life in humans and prevent age-related diseases by slowing down aging. Since that time many predictions of this concept have been confirmed. Rapamycin was shown to suppress aging in mammalian cells, prolong life span in mice and flies, improve immunity and stem cell function in old animals, thus confirming twelve predictions as discussed herein. One prediction remains to be confirmed: rapamycin will become the cornerstone of anti-aging therapy in our life time.

  4. Topical rapamycin for facial angiofibromas in tuberous sclerosis complex

    PubMed Central

    2017-01-01

    Abstract Facial angiofibromas are a common cutaneous manifestation of tuberous sclerosis complex. Although angiofibromas are usually asymptomatic, they can be highly disfiguring and can have a significant impact on patient quality of life. Treatment for facial angiofibromas is challenging. Recently, topical rapamycin has been proposed as an effective option to treat angiofibromas. Herein is reported a case of a 27-year-old woman whose facial angiofibromas were successfully treated with topical rapamycin without relevant side effects. PMID:28690858

  5. Rapamycin Inhibition of mTOR Reduces Levels of the Na+/H+ Exchanger 3 in Intestines of Mice and Humans, Leading to Diarrhea

    PubMed Central

    Yang, Jun; Zhao, Xiaofeng; Patel, Archana; Potru, Rachana; Azizi-Ghannad, Sadra; Dolinger, Michael; Mazurkiewicz, Joseph; Conti, David; Jones, David; Huang, Yunfei; Zhu, Xinjun

    2016-01-01

    process appears to require the autophagic activity mediated by ATG7. Loss of mTOR regulation of NHE3 could mediate the development of diarrhea in patients undergoing rapamycin therapy. PMID:25836987

  6. Metabolic Effects of Acute Thiamine Depletion Are Reversed by Rapamycin in Breast and Leukemia Cells

    PubMed Central

    Liu, Shuqian; Miriyala, Sumitra; Keaton, Mignon A.; Jordan, Craig T.; Wiedl, Christina; Clair, Daret K. St.; Moscow, Jeffrey A.

    2014-01-01

    Thiamine-dependent enzymes (TDEs) control metabolic pathways that are frequently altered in cancer and therefore present cancer-relevant targets. We have previously shown that the recombinant enzyme thiaminase cleaves and depletes intracellular thiamine, has growth inhibitory activity against leukemia and breast cancer cell lines, and that its growth inhibitory effects were reversed in leukemia cell lines by rapamycin. Now, we first show further evidence of thiaminase therapeutic potential by demonstrating its activity against breast and leukemia xenografts, and against a primary leukemia xenograft. We therefore further explored the metabolic effects of thiaminase in combination with rapamycin in leukemia and breast cell lines. Thiaminase decreased oxygen consumption rate and increased extracellular acidification rate, consistent with the inhibitory effect of acute thiamine depletion on the activity of the TDEs pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes; these effects were reversed by rapamycin. Metabolomic studies demonstrated intracellular thiamine depletion and the presence of the thiazole cleavage product in thiaminase-treated cells, providing validation of the experimental procedures. Accumulation of ribose and ribulose in both cell lines support the thiaminase-mediated suppression of the TDE transketolase. Interestingly, thiaminase suppression of another TDE, branched chain amino ketoacid dehydrogenase (BCKDH), showed very different patterns in the two cell lines: in RS4 leukemia cells it led to an increase in BCKDH substrates, and in MCF-7 breast cancer cells it led to a decrease in BCKDH products. Immunoblot analyses showed corresponding differences in expression of BCKDH pathway enzymes, and partial protection of thiaminase growth inhibition by gabapentin indicated that BCKDH inhibition may be a mechanism of thiaminase-mediated toxicity. Surprisingly, most of thiaminase-mediated metabolomic effects were also reversed by rapamycin

  7. Defects in skin γδ T cell function contribute to delayed wound repair in rapamycin-treated mice1

    PubMed Central

    Mills, Robyn E.; Taylor, Kristen R.; Podshivalova, Katie; McKay, Dianne B.; Jameson, Julie M.

    2008-01-01

    Disruptions in the normal program of tissue repair can result in poor wound healing, which perturbs the integrity of barrier tissues such as the skin. Such defects in wound repair occur in transplant recipients treated with the immunosuppressant drug rapamycin (sirolimus). Intraepithelial lymphocytes, such as γδT cells in the skin, mediate tissue repair through the production of cytokines and growth factors. The capacity of skin-resident T cells to function during rapamycin treatment was analyzed in a mouse model of wound repair. Rapamycin treatment renders skin γδ T cells unable to proliferate, migrate and produce normal levels of growth factors. The observed impairment of skin γδ T cell function is directly related to the inhibitory action of rapamycin on mammalian target of rapamycin (mTOR). Skin γδ T cells treated with rapamycin are refractory to IL-2 stimulation and attempt to survive in the absence of cytokine and growth factor signaling by undergoing autophagy. Normal wound closure can be restored in rapamycin-treated mice by addition of the skin γδ T cell-produced factor, insulin-like growth factor-1. These studies not only reveal that mTOR is a master regulator of γδ T cell function but also provide a novel mechanism for the increased susceptibility to nonhealing wounds that occurs during rapamycin administration. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final

  8. Rapamycin inhibits anal carcinogenesis in two preclinical animal models.

    PubMed

    Stelzer, Marie K; Pitot, Henry C; Liem, Amy; Lee, Denis; Kennedy, Gregory D; Lambert, Paul F

    2010-12-01

    The incidence of anal cancer is increasing especially among HIV-infected persons in the HAART era. Treatment of this cancer is based upon traditional chemoradiotherapeutic approaches, which are associated with high morbidity and of limited effectiveness for patients with high-grade disease. The mammalian target of rapamycin (mTOR) pathway has been implicated in several human cancers, and is being investigated as a potential therapeutic target. In archival human anal cancers, we observed mTOR pathway activation. To assess response of anal cancer to mTOR inhibition, we utilized two newly developed mouse models, one in which anal cancers are induced to arise in HPV16 transgenic mice and the second a human anal cancer xenograft model. Using the transgenic mouse model, we assessed the preventative effect of rapamycin on neoplastic disease. We saw significant changes in the overall incidence of tumors, and tumor growth rate was also reduced. Using both the transgenic mouse and human anal xenograft mouse models, we studied the therapeutic effect of rapamycin on preexisting anal cancer. Rapamycin was found to significantly slow, if not stop, the growth of both mouse and human anal cancers. As has been seen in other cancers, rapamycin treatment led to an activation of the MAPK pathway. These results provide us cause to pursue further the evaluation of rapamycin as a therapeutic agent in the control of anal cancer. ©2010 AACR.

  9. Rapamycin extends murine lifespan but has limited effects on aging

    PubMed Central

    Neff, Frauke; Flores-Dominguez, Diana; Ryan, Devon P.; Horsch, Marion; Schröder, Susanne; Adler, Thure; Afonso, Luciana Caminha; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Garrett, Lillian; Hans, Wolfgang; Hettich, Moritz M.; Holtmeier, Richard; Hölter, Sabine M.; Moreth, Kristin; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Rozman, Jan; Naton, Beatrix; Ordemann, Rainer; Adamski, Jerzy; Beckers, Johannes; Bekeredjian, Raffi; Busch, Dirk H.; Ehninger, Gerhard; Graw, Jochen; Höfler, Heinz; Klingenspor, Martin; Klopstock, Thomas; Ollert, Markus; Stypmann, Jörg; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabe de Angelis, Martin; Ehninger, Dan

    2013-01-01

    Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin’s effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin’s longevity effects from effects on aging itself. PMID:23863708

  10. [Moxibustion Improves Learning Ability by Regulating Hippocampal Neurotrophic Factor Expression and Notch Signaling in Vascular Dementia Rats].

    PubMed

    Wang, Ying; Cai, Sheng-Chao; Song, Xiao-Ge; Zhu, Wan-Li; Yang, Kun; Qian, Jian-Jian; Ding, Nuo-Nuo

    2017-10-25

    To observe the effect of moxibustion on the learning ability and expression of neurotrophic factors and Notch signaling in vascular dementia (VD) rats, so as to explore its neurogenesis mechanism underlying improvement of VD. Sixty SD rats were equally and randomly divided into sham operation (sham), model, medication and moxibustion groups ( n =15 rats/group). The VD model was established by occlusion of the bilateral cervical common arteries and reperfusion. Moxibustion was applied to "Dazhui"(GV 14), "Guanyuan"(CV 4) and "Mingmen"(GV 4)for 15 minutes, once daily, 6 times a week for 4 weeks. Rats of the medication group were treated by gavage of nimodipine (2 mg·kg -1 ·d -1 ),3 times a day, 6 days a week for 4 weeks. Morris water maze tests were performed to detect the rat's learning-memory ability. The infarcted size of the brain was detected by using 2,3,5-triphenyltetrazolium chloride (TTC) staining, and H.E. staining was used to detect the histopathological changes. The expression level of glia fibrillary acidic protein (GFAP), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), Notch 1 (a receptor), Hes 3 (a downstream effector) mRNAs and proteins in the hippocampal tissues were detected by quantitative real-time PCR and Western blot, respectively. After 4 weeks' intervention, modeling-induced increase of escape latency was significantly shortened in both moxibustion and medication groups relevant to the model group ( P <0.05), and the infarct size was reduced and the damage degree of nerve cells in the brain tissue alleviated. The expression levels of BDNF, NGF, GFAP, Hes 3, Notch 1 genes and proteins were significantly up-regulated in the model group relevant to the sham operation group ( P <0.05, P <0.01). After the intervention, the expression levels of hippocampal BDNF, NGF, GFAP, Hes 3 and Notch 1 mRNAs and proteins in the moxibustion group, and NGF and GFAP mRNAs, and BDNF, NGF, GFAP, Hes 3 and Notch 1 proteins in the medication

  11. Expression of miRNA-155 in carotid atherosclerotic plaques of apolipoprotein E knockout (ApoE-/-) mice and the interventional effect of rapamycin.

    PubMed

    Ma, Juanjuan; Yang, Shaonan; Ma, Aijun; Pan, Xudong; Wang, Hongliang; Li, Na; Liu, Shihai; Wu, Mei

    2017-05-01

    Carotid atherosclerosis (AS) is an inflammatory process and is the primary pathogenesis of cerebrovascular disease. Many factors are responsible for development of atherosclerosis such as inflammation and autophagy. It is reported that microRNAs (miRNAs) could regulate the development of atherosclerosis through targeting autophagy-related genes. Many studies have demonstrated that miRNA-155 could regulate autophagy in macrophages or tumor cells. However, the role of miRNA-155 on autophagy in carotid plaques is not yet known. In this study, we explore the expression of miRNA-155 and autophagy-related proteins in carotid plaques of ApoE-/- mice and the interventional effect of rapamycin. We compared the expression of miRNA-155 and autophagy-related proteins between the control, model and rapamycin groups using qRT-PCR and western blot. Compared to the control group, we found the miRNA-155 and LC3-II expression was up-regulated (P<0.05), expression ratio of phosphorylated mammalian target of rapamycin to total mammalian target of rapamycin (p-mTOR/mTOR) was down-regulated in model group (P<0.05), but atherosclerotic lesions were still aggravated. These results following rapamycin group indicated that miRNA-155 and LC3-II expression was significantly up-regulated (P<0.05), the expression ratio of p-mTOR/mTOR was significantly down-regulated (P<0.05), and atherosclerotic lesions were reduced. Our results showed in the early stages of atherosclerotic plaques development, effective autophagy could attenuate atherosclerosis in ApoE-/- mice. Furthermore, our results also demonstrated that rapamycin might promote the activation of the autophagy by enhancing the expression of miRNA-155, which delays the development of atherosclerotic plaques. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Phosphorylation of VE-cadherin is modulated by haemodynamic forces and contributes to the regulation of vascular permeability in vivo

    PubMed Central

    Orsenigo, Fabrizio; Giampietro, Costanza; Ferrari, Aldo; Corada, Monica; Galaup, Ariane; Sigismund, Sara; Ristagno, Giuseppe; Maddaluno, Luigi; Young Koh, Gou; Franco, Davide; Kurtcuoglu, Vartan; Poulikakos, Dimos; Baluk, Peter; McDonald, Donald; Grazia Lampugnani, Maria; Dejana, Elisabetta

    2012-01-01

    Endothelial adherens junctions maintain vascular integrity. Arteries and veins differ in their permeability but whether organization and strength of their adherens junctions vary has not been demonstrated in vivo. Here we report that vascular endothelial cadherin, an endothelial specific adhesion protein located at adherens junctions, is phosphorylated in Y658 and Y685 in vivo in veins but not in arteries under resting conditions. This difference is due to shear stress-induced junctional Src activation in veins. Phosphorylated vascular endothelial-cadherin is internalized and ubiquitinated in response to permeability-increasing agents such as bradykinin and histamine. Inhibition of Src blocks vascular endothelial cadherin phosphorylation and bradykinin-induced permeability. Point mutation of Y658F and Y685F prevents vascular endothelial cadherin internalization, ubiquitination and an increase in permeability by bradykinin in vitro. Thus, phosphorylation of vascular endothelial cadherin contributes to a dynamic state of adherens junctions, but is not sufficient to increase vascular permeability in the absence of inflammatory agents. PMID:23169049

  13. Ca2+/calmodulin-stimulated PDE1 regulates the beta-catenin/TCF signaling through PP2A B56 gamma subunit in proliferating vascular smooth muscle cells.

    PubMed

    Jeon, Kye-Im; Jono, Hirofumi; Miller, Clint L; Cai, Yujun; Lim, Soyeon; Liu, Xuan; Gao, Pingjin; Abe, Jun-Ichi; Li, Jian-Dong; Yan, Chen

    2010-12-01

    The phenotypic change of vascular smooth muscle cells (VSMCs), from a 'contractile' phenotype to a 'synthetic' phenotype, is crucial for pathogenic vascular remodeling in vascular diseases such as atherosclerosis and restenosis. Ca(2+)/calmodulin-stimulated phosphodiesterase 1 (PDE1) isozymes, including PDE1A and PDE1C, play integral roles in regulating the proliferation of synthetic VSMCs. However, the underlying molecular mechanism(s) remain unknown. In this study, we explore the role and mechanism of PDE1 isoforms in regulating β-catenin/T-cell factor (TCF) signaling in VSMCs, a pathway important for vascular remodeling through promoting VSMC growth and survival. We found that inhibition of PDE1 activity markedly attenuated β-catenin/TCF signaling by downregulating β-catenin protein. The effect of PDE1 inhibition on β-catenin protein reduction is exerted via promoting glycogen synthase kinase 3 (GSK3)β activation, β-catenin phosphorylation and subsequent β-catenin protein degradation. Moreover, PDE1 inhibition specifically upregulated phosphatase protein phosphatase 2A (PP2A) B56γ subunit gene expression, which is responsible for the effects of PDE1 inhibition on GSK3β and β-catenin/TCF signaling. Furthermore, the effect of PDE1 inhibition on β-catenin was specifically mediated by PDE1A but not PDE1C isozyme. Interestingly, in synthetic VSMCs, PP2A B56γ, phospho-GSK3β and phospho-β-catenin were all found in the nucleus, suggesting that PDE1A regulates nuclear β-catenin protein stability through the nuclear PP2A-GSK3β-β-catenin signaling axis. Taken together, these findings provide direct evidence for the first time that PP2A B56γ is a critical mediator for PDE1A in the regulation of β-catenin signaling in proliferating VSMCs. © 2010 The Authors Journal compilation © 2010 FEBS.

  14. Prostaglandin E2 regulation of amnion cell vascular endothelial growth factor expression: relationship with intramembranous absorption rate in fetal sheep.

    PubMed

    Cheung, Cecilia Y; Beardall, Michael K; Anderson, Debra F; Brace, Robert A

    2014-08-01

    We hypothesized that prostaglandin E2 (PGE2) stimulates amniotic fluid transport across the amnion by upregulating vascular endothelial growth factor (VEGF) expression in amnion cells and that amniotic PGE2 concentration correlates positively with intramembranous (IM) absorption rate in fetal sheep. The effects of PGE2 at a range of concentrations on VEGF 164 and caveolin-1 gene expressions were analyzed in cultured ovine amnion cells. IM absorption rate, amniotic fluid (AF) volume, and PGE2 concentration in AF were determined in late-gestation fetal sheep during control conditions, isovolumic fetal urine replacement (low IM absorption rate), or intra-amniotic fluid infusion (high IM absorption rate). In ovine amnion cells, PGE2 induced dose- and time-dependent increases in VEGF 164 mRNA levels and reduced caveolin-1 mRNA and protein levels. VEGF receptor blockade abolished the caveolin-1 response, while minimally affecting the VEGF response to PGE2. In sheep fetuses, urine replacement reduced amniotic PGE2 concentration by 58%, decreased IM absorption rate by half, and doubled AF volume (P < 0.01). Intra-amniotic fluid infusion increased IM absorption rate and AF volume (P < 0.01), while amniotic PGE2 concentration was unchanged. Neither IM absorption rate nor AF volume correlated with amniotic PGE2 concentration under each experimental condition. Although PGE2 at micromolar concentrations induced dose-dependent responses in VEGF and caveolin-1 gene expression in cultured amnion cells consistent with a role of PGE2 in activating VEGF to mediate AF transport across the amnion, amniotic PGE2 at physiological nanomolar concentrations does not appear to regulate IM absorption rate or AF volume. Copyright © 2014 the American Physiological Society.

  15. Oxidative stress regulates IGF1R expression in vascular smooth-muscle cells via p53 and HDAC recruitment.

    PubMed

    Kavurma, Mary M; Figg, Nichola; Bennett, Martin R; Mercer, John; Khachigian, Levon M; Littlewood, Trevor D

    2007-10-01

    Apoptosis of VSMCs (vascular smooth-muscle cells) leads to features of atherosclerotic plaque instability. We have demonstrated previously that plaque-derived VSMCs have reduced IGF1 (insulin-like growth factor 1) signalling, resulting from a decrease in the expression of IGF1R (IGF1 receptor) compared with normal aortic VSMCs [Patel, Zhang, Siddle, Soos, Goddard, Weissberg and Bennett (2001) Circ. Res. 88, 895-902]. In the present study, we show that apoptosis induced by oxidative stress is inhibited by ectopic expression of IGF1R. Oxidative stress repressed IGF1R expression at multiple levels, and this was also blocked by mutant p53. Oxidative stress also induced p53 phosphorylation and apoptosis in VSMCs. p53 negatively regulated IGF1R promoter activity and expression and, consistent with this, p53-/- VSMCs demonstrated increased IGF1R expression, both in vitro and in advanced atherosclerotic plaques in vivo. Oxidative-stress-induced interaction of endogenous p53 with TBP (TATA-box-binding protein) was dependent on p53 phosphorylation. Oxidative stress also increased the association of p53 with HDAC1 (histone deacetylase 1). Trichostatin A, a specific HDAC inhibitor, or p300 overexpression relieved the repression of IGF1R following oxidative stress. Furthermore, acetylated histone-4 association with the IGF1R promoter was reduced in cells subjected to oxidative stress. These results suggest that oxidative-stress-induced repression of IGF1R is mediated by the association of phosphorylated p53 with the IGF1R promoter via TBP, and by the subsequent recruitment of chromatin-modifying proteins, such as HDAC1, to the IGF1R promoter-TBP-p53 complex.

  16. Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

    PubMed Central

    LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

    2014-01-01

    Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

  17. Rapamycin-Sensitive Late-LTP is Enhanced in the Hippocampus of IL-6 Transgenic Mice.

    PubMed

    Olde Engberink, Anneke; Hernandez, Ruben; de Graan, Pierre; Gruol, Donna L

    2017-12-26

    The neuroimmune factor IL-6 has been shown to regulate hippocampal long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission that plays a central role in memory and learning. This IL-6 action was demonstrated with relatively short IL-6 exposure, and may reflect physiological actions of IL-6. IL-6 is also expressed chronically at elevated levels in the central nervous system (CNS) under pathological conditions such as neurological disorders. Little is known about the effects IL-6 on LTP under such conditions, an issue that we are addressing by electrophysiological recordings from CA1 pyramidal neurons of hippocampal slices from transgenic mice that persistently express elevated levels of IL-6 in the CNS (IL-6 tg). The current studies examined the long-lasting phase of LTP (late LTP; L-LTP) and the potential involvement mammalian target of rapamycin (mTOR), a known regulator of L-LTP and a downstream partner of IL-6 signal transduction pathways. Results show that basal synaptic transmission and L-LTP were increased in hippocampal slices from IL-6 tg mice compared to slices from non-transgenic (non-tg) control mice. An inhibitor of mTOR, rapamycin, reduced L-LTP in slices from both genotypes, and eliminated the difference in magnitude of L-LTP between IL-6 and non-tg hippocampus. There were no genotypic effect of rapamycin on basal synaptic transmission, but synaptic responses during the LTP induction protocol were reduced in IL-6 tg slices, an effect that could contribute to the reduction of L-LTP in the IL-6 tg slices. These results indicate that persistently increased levels of IL-6 can lead to alterations in mTOR regulation of L-LTP, possibly affecting learning and memory. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. Mycophenolate Mofetil and Rapamycin Induce Apoptosis in the Human Monocytic U937 Cell Line Through Two Different Pathways.

    PubMed

    Nowak, Maxime; Tardivel, Sylviane; Nguyen-Khoa, Thao; Abreu, Sonia; Allaoui, Fatima; Fournier, Natalie; Chaminade, Pierre; Paul, Jean-Louis; Lacour, Bernard

    2017-10-01

    Transplant vasculopathy may be considered as an accelerated form of atherosclerosis resulting in chronic rejection of vascularized allografts. After organ transplantation, a diffuse intimal thickening is observed, leading to the development of an atherosclerosis plaque due to a significant monocyte infiltration. This results from a chronic inflammatory process induced by the immune response. In this study, we investigated the impact of two immunosuppressive drugs used in therapy initiated after organ transplantation, mycophenolate mofetil, and rapamycin, on the apoptotic response of monocytes induced or not by oxidized LDL. Here we show the pro-apoptotic effect of these two drugs through two distinct signaling pathways and we highlight a synergistic effect of rapamycin on apoptosis induced by oxidized LDL. In conclusion, since immunosuppressive therapy using mycophenolate mofetil or rapamycin can increase the cell death in a monocyte cell line, this treatment could exert similar effects on human monocytes in transplant patients, and thus, prevent transplant vasculopathy, atherosclerosis development, and chronic allograft rejection. J. Cell. Biochem. 118: 3480-3487, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Rapamycin and aging: When, for how long, and how much?

    PubMed Central

    Kaeberlein, Matt

    2015-01-01

    The drug rapamycin is the only pharmacological agent thus far shown to reproducibly extend lifespan and delay a subset of age-associated pathologies in multiple strains of mice. Unfortunately, the vast majority of aging-related studies on rapamycin in mice have been performed at a single dose of the drug delivered in encapsulated form through the diet. Recently, the National Institute on Aging Interventions Testing Program reported that a three-fold higher dose of dietary rapamycin results in a significantly greater increase in lifespan. This observation demonstrates that current studies of the effects of rapamycin on lifespan and healthspan in mice are being performed under conditions that are sub-optimal. Here I argue that the failure to properly determine the dose and timing response profile for rapamycin with respect to healthy aging represents a major barrier for the field. This barrier continues to hamper our ability to gain mechanistic insights and may threaten efforts to translate these findings into interventions that promote healthy aging in people. PMID:25269671

  20. Thrombospondin-1 (TSP1) Contributes to the Development of Vascular Inflammation by Regulating Monocytic Cell Motility in Mouse Models of Abdominal Aortic Aneurysm

    PubMed Central

    Liu, Zhenjie; Morgan, Stephanie; Ren, Jun; Wang, Qiwei; Annis, Douglas S.; Mosher, Deane F.; Zhang, Jing; Sorenson, Christine M; Sheibani, Nader; Liu, Bo

    2015-01-01

    Rationale Histological examination of abdominal aortic aneurysm (AAA) tissues demonstrates extracellular matrix (ECM) destruction and infiltration of inflammatory cells. Previous work with mouse models of AAA has shown that anti-inflammatory strategies can effectively attenuate aneurysm formation. Thrombospondin-1 (TSP1) is a matricellular protein involved in the maintenance of vascular structure and homeostasis through the regulation of biological functions such as cell proliferation, apoptosis, and adhesion. Expression levels of TSP1 correlate with vascular disease conditions. Objective To use TSP1 deficient (Thbs1−/−) mice to test the hypothesis that TSP1 contributes to pathogenesis of AAAs. Methods and Results Mouse experimental AAA was induced either through perivascular treatment with calcium phosphate, intraluminal perfusion with porcine elastase, or systemic administration of Angiotensin II. Induction of AAA increased TSP1 expression in aortas of C57BL/6 or apoE−/− mice. Compared to Thbs1+/+ mice, Thbs1−/− mice developed significantly smaller aortic expansion when subjected to AAA inductions, which was associated with diminished infiltration of macrophages. Thbs1−/− monocytic cells had reduced adhesion and migratory capacity in vitro compared to wildtype counterparts. Adoptive transfer of Thbs1+/+ monocytic cells or bone marrow reconstitution rescued aneurysm development in Thbs1−/− mice. Conclusions TSP1 expression plays a significant role in regulation of migration and adhesion of mononuclear cells, contributing to vascular inflammation during AAA development. PMID:25940549

  1. odd skipped related1 reveals a novel role for endoderm in regulating kidney vs. vascular cell fate

    PubMed Central

    Mudumana, Sudha P.; Hentschel, Dirk; Liu, Yan; Vasilyev, Aleksandr; Drummond, Iain A.

    2009-01-01

    Summary The kidney and vasculature are intimately linked functionally and during development, where nephric and blood/vascular progenitor cells occupy adjacent bands of mesoderm in zebrafish and frog embryos. Developmental mechanisms underlying the differentiation of kidney vs. blood/vascular lineages remain unknown. The odd skipped related1 (osr1) gene encodes a zinc finger transcription factor that is expressed in the germ ring mesendoderm and subsequently in the endoderm and intermediate mesoderm, prior to the expression of definitive kidney or blood/vascular markers. Knockdown of osr1 in zebrafish embryos resulted in a complete, segment-specific loss of anterior kidney progenitors and a compensatory increase in the number of angioblast cells in the same trunk region. Histology revealed a subsequent absence of kidney tubules, enlarged cardinal vein, and expansion of the posterior venous plexus. Altered kidney vs. vascular development correlated with expanded endoderm development in osr1 knockdowns. Combined osr1 loss of function and blockade of endoderm development by knockdown of sox32/casanova rescued anterior kidney development. The results indicate that osr1 activity is required to limit endoderm differentiation from mesendoderm and, in the absence of osr1, excess endoderm alters mesoderm differentiation, shifting the balance from kidney toward vascular development. PMID:18787069

  2. DNA Damage: A Main Determinant of Vascular Aging.

    PubMed

    Bautista-Niño, Paula K; Portilla-Fernandez, Eliana; Vaughan, Douglas E; Danser, A H Jan; Roks, Anton J M

    2016-05-18

    Vascular aging plays a central role in health problems and mortality in older people. Apart from the impact of several classical cardiovascular risk factors on the vasculature, chronological aging remains the single most important determinant of cardiovascular problems. The causative mechanisms by which chronological aging mediates its impact, independently from classical risk factors, remain to be elucidated. In recent years evidence has accumulated that unrepaired DNA damage may play an important role. Observations in animal models and in humans indicate that under conditions during which DNA damage accumulates in an accelerated rate, functional decline of the vasculature takes place in a similar but more rapid or more exaggerated way than occurs in the absence of such conditions. Also epidemiological studies suggest a relationship between DNA maintenance and age-related cardiovascular disease. Accordingly, mouse models of defective DNA repair are means to study the mechanisms involved in biological aging of the vasculature. We here review the evidence of the role of DNA damage in vascular aging, and present mechanisms by which genomic instability interferes with regulation of the vascular tone. In addition, we present potential remedies against vascular aging induced by genomic instability. Central to this review is the role of diverse types of DNA damage (telomeric, non-telomeric and mitochondrial), of cellular changes (apoptosis, senescence, autophagy), mediators of senescence and cell growth (plasminogen activator inhibitor-1 (PAI-1), cyclin-dependent kinase inhibitors, senescence-associated secretory phenotype (SASP)/senescence-messaging secretome (SMS), insulin and insulin-like growth factor 1 (IGF-1) signaling), the adenosine monophosphate-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-nuclear factor kappa B (NFκB) axis, reactive oxygen species (ROS) vs. endothelial nitric oxide synthase (eNOS)-cyclic guanosine monophosphate (c

  3. The Role of Estrogen Receptor α and β in Regulating Vascular Smooth Muscle Cell Proliferation is Based on Sex

    PubMed Central

    Hogg, Melissa E.; Vavra, Ashley K.; Banerjee, Monisha N.; Martinez, Janet; Jiang, Qun; Keefer, Larry K.; Chambon, Pierre; Kibbe, Melina R.

    2012-01-01

    Background We previously demonstrated that vascular smooth muscle cells (VSMC) proliferation and development of neointimal hyperplasia as well as the ability of nitric oxide (NO) to inhibit these processes is dependent on sex and hormone status. The aim of this study was to evaluate the role of estrogen receptor (ER) in mediating proliferation in male and female VSMC. Materials and Methods Proliferation was assessed in primary rat aortic male and female VSMC using 3H-thymidine incorporation in the presence or absence of ER alpha (α) inhibitor methyl-piperidino-pyrazole, the ER beta (β) inhibitor (R,R)-5,11-Diethyl-5,6,11,12-tetrahydro-2,8-chrysenediol, the combined ERαβ inhibitor ICI 182,780, and/or the NO donor DETA/NO. Proliferation was also assessed in primary aortic mouse VSMC harvested from wildtype (WT), ERα knockout (ERα KO), and ERβ knockout (ERβ KO) mice in the presence or absence of DETA/NO and the ERα, ERβ, and ERαβ inhibitors. Protein levels were assessed using Western blot analysis. Results Protein expression of ERα and ERβ was present and equal in male and female VSMC, and did not change after exposure to NO. Inhibition of either ERα or ERβ had no effect on VSMC proliferation in the presence or absence of NO in either sex. However, inhibition of ERαβ in rat VSMC mitigated NO-mediated inhibition in female but not male VSMC (p<0.05). Evaluation of proliferation in the knockout mice revealed distinct patterns. Male ERαKO and ERβKO VSMC proliferated faster than male WT VSMC (p<0.05). Female ERβKO proliferated faster than female WT VSMC (p<0.05), but female ERαKO VSMC proliferated slower than female WT VSMC (p<0.05). Last, we evaluated the effect of combined inhibition of ERα and ERβ in these knockout strains. Combined ERαβ inhibition abrogated NO-mediated inhibition of VSMC proliferation in female WT and knockout VSMC (p<0.05), but not in male VSMC. Conclusions These data clearly demonstrate a role for the ER in mediating VSMC

  4. Puerarin up-regulates methyl-CpG binding protein 2 phosphorylation in hippocampus of vascular dementia rats.

    PubMed

    Wang, Hu-Qing; Zhang, Meng; Zhao, Jia-Xin; Wu, Hai-Qin; Gao, Zhen; Zhang, Gui-Lian; Zhang, Ru

    2018-01-09

    To observe the effect of puerarin on methyl-CpG binding protein 2 (MeCP2) phosphorylation (pMeCP2) in the hippocampus of a rat model of vascular dementia (VD). Thirty-six healthy Sprague-Dawley rats were randomly assigned to the sham-operated group, dementia group and puerarintreated group using a random number table (n=12 per group). The modifified permanent bilateral common carotid artery occlusion method was used to establish the VD model. The sham-operated and dementia groups were given 2 mL/d of saline, while the puerarin-treated group was given 100 mg/(kg•d) of puerarin for 17 days. The learning and memory abilities were evaluated by the Morris water maze test. Hematoxylin-eosin staining, immunohistochemical (IHC) staining and Western blot analysis were carried out to observe changes in neuron morphology and in level of pMeCP2 in the hippocampus, respectively. The morphologies of rat hippocampal neurons in the puerarintreated group were markedly improved compared with the dementia group. The escape latency of the dementia group was significantly longer than the sham-operated group (P<0.05), while the puerarin-treated group was obviously shorter than the dementia group (P<0.05). Cross-platform times of the dementia group were signifificantly decreased compared with the sham-operated group (P<0.05), while the puerarin-treated group was obviously increased compared with the dementia group (P<0.05). IHC staining showed no significant difference in the number of MeCP2 positive cells among 3 groups (P<0.05). The number of pMeCP2 positive cells in the CA1 region of hippocampus in the dementia group was signifificantly increased compared with the sham-operated group, and the puerarin-treated group was signifificantly increased compared with the dementia group (both P>0.05). Western blot analysis showed no signifificant difference of MeCP2 expression among 3 groups (P>0.05). The expression of pMeCP2 in the dementia group was signifificantly increased compared with

  5. SIRT1 attenuates PAF-induced MMP-2 production via down-regulation of PAF receptor expression in vascular smooth muscle cells.

    PubMed

    Kim, Yun H; Bae, Jin U; Lee, Seung J; Park, So Y; Kim, Chi D

    2015-09-01

    Silent mating type information regulation 2 homolog 1 (SIRT1) is known as a key regulator in the protection of various vascular disorders, however, no direct evidences have been reported in the progression of atherosclerosis. Considering the pivotal role of matrix metalloproteinase-2 (MMP-2) in plaque destabilization, this study investigated the role of SIRT1 on MMP-2 production in vascular smooth muscle cells (VSMCs) induced by platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In VSMCs stimulated with resveratrol, SIRT1 activator, PAF receptor (PAFR) was internalized and then its protein levels were diminished. It was attenuated in cells pretreated with proteasome or lysosome inhibitor. Also, the degradation of PAFR in SIRT1-stimulated cells was significantly attenuated by β-arrestin2 depletion. In cells treated with nicotinamide, SIRT1 deacetylase inhibitor, PAFR internalization by resveratrol or reSIRT1 was inhibited, demonstrating that deacetylation of SIRT1 is an important step in SIRT1-induced PAFR down-regulation. Moreover, PAF-induced MMP-2 production in VSMCs and aorta was attenuated by resveratrol. In the aorta of SIRT1 transgenic mice, the PAF-induced MMP-2 expression was prominently attenuated compared to that in wild type mice. Taken together, it was suggested that SIRT1 down-regulated PAFR in VSMCs via β-arrestin2-mediated internalization and degradation, leading to an inhibition of PAF-induced MMP-2 production. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 Regulates Xylem Development and Growth by a Conserved Mechanism That Modulates Hormone Signaling1[W][OPEN

    PubMed Central

    Grienenberger, Etienne; Douglas, Carl J.

    2014-01-01

    Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-β-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways. PMID:24567189

  7. Rapamycin improves sociability in the BTBR T+Itpr3tf/J mouse model of autism spectrum disorders

    PubMed Central

    Burket, Jessica A.; Benson, Andrew D.; Tang, Amy H.; Deutsch, Stephen I.

    2017-01-01

    Overactivation of the mammalian target of rapamycin (mTOR) has been implicated in the pathogenesis of syndromic forms of autism spectrum disorders (ASDs), such as tuberous sclerosis complex, neurofibromatosis 1, and fragile X syndrome. Administration of mTORC1 (mTOR complex 1) inhibitors (e.g. rapamycin) in syndromic mouse models of ASDs improved behavior, cognition, and neuropathology. However, since only a minority of ASDs are due to the effects of single genes (~10%), there is a need to explore inhibition of mTOR activity in mouse models that may be more relevant to the majority of nonsyndromic presentations, such as the genetically inbred BTBR T+Itpr3tf/J (BTBR) mouse model of ASDs. BTBR mice have social impairment and exhibit increased stereotypic behavior. In prior work, d-cycloserine, a partial glycineB site agonist that targets the N-methyl-d-aspartate (NMDA) receptor, was shown to improve sociability in both Balb/c and BTBR mouse models of ASDs. Importantly, NMDA receptor activation regulates mTOR signaling activity. The current study investigated the ability of rapamycin (10 mg/kg, i.p. × four days), an mTORC1 inhibitor, to improve sociability and stereotypic behavior in BTBR mice. Using a standard paradigm to assess mouse social behavior, rapamycin improved several measures of sociability in the BTBR mouse, suggesting that mTOR overactivation represents a therapeutic target that mediates or contributes to impaired sociability in the BTBR mouse model of ASDs. Interestingly, there was no effect of rapamycin on stereotypic behaviors in this mouse model. PMID:24295733

  8. Resveratrol and rapamycin: are they anti-aging drugs?

    PubMed

    Kaeberlein, Matt

    2010-02-01

    Studies of the basic biology of aging have advanced to the point where anti-aging interventions, identified from experiments in model organisms, are beginning to be tested in people. Resveratrol and rapamycin, two compounds that target conserved longevity pathways and may mimic some aspects of dietary restriction, represent the first such interventions. Both compounds have been reported to slow aging in yeast and invertebrate species, and rapamycin has also recently been found to increase life span in rodents. In addition, both compounds also show impressive effects in rodent models of age-associated diseases. Clinical trials are underway to assess whether resveratrol is useful as an anti-cancer treatment, and rapamycin is already approved for use in human patients. Compounds such as these, identified from longevity studies in model organisms, hold great promise as therapies to target multiple age-related diseases by modulating the molecular causes of aging.

  9. α(1D)-Adrenoceptor regulates the vasopressor action of α(1A)-adrenoceptor in mesenteric vascular bed of α(1D)-adrenoceptor knockout mice.

    PubMed

    Martínez-Salas, S G; Campos-Peralta, J M; Pardo, J P; Hernández-Muñoz, R; Ibarra, M; Tanoue, A; Tsujimoto, G; Villalobos-Molina, R

    2011-01-01

    1 The pressor action of the α(1A)-adrenoceptor (α(1A)-AR) agonist A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) and the α(1)-ARs agonist phenylephrine and their blockade by selective α(1)-ARs antagonists in the isolated mesenteric vascular bed of wild-type (WT) mice and α(1D)-AR knockout (KO α(1D)-AR) mice were evaluated. 2 The apparent potency of A61603 to increase the perfusion pressure in the mesenteric vascular bed of WT and KO α(1D)-AR mice is 86 and 138 times the affinity of phenylephrine, respectively. 3 A61603 also enhanced the perfusion pressure by ≈1.7 fold in the mesenteric vascular bed of WT mice compared with KO α(1D)-AR mice. 4 Because of its high affinity, low concentrations of the α(1A)-AR selective antagonist RS100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl] propyl]-2,4-(1H)-pyrimidinedione) shifted the agonist concentration-response curves to the right in the mesenteric vascular bed of WT and KO α(1D)-AR mice. 5 The α(1D)-AR selective antagonist BMY7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione) did not modify the A61603 or the phenylephrine-induced pressor effect. 6 The α(1B/D)-ARs alkylating antagonist chloroethylclonidine (CEC) shifted the agonist concentration-response curves to the right and decreased the maximum phenylephrine-induced vascular contraction in KO α(1D)-AR mice when compared to WT mice; however, CEC only slightly modified the contraction induced by A61603. 7 The results indicate that the isolated mesenteric vascular bed of WT and KO α(1D)-AR mice expresses α(1A)-AR, that the pressor action of α(1A)-AR is up-regulated for α(1D)-AR in WT mice and suggest an important role of α(1B)-AR in the vascular pressure evoked by phenylephrine in KO α(1D)-AR mice. © 2011 Blackwell Publishing Ltd.

  10. Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise

    PubMed Central

    Philp, Andrew; Schenk, Simon; Perez-Schindler, Joaquin; Hamilton, D Lee; Breen, Leigh; Laverone, Erin; Jeromson, Stewart; Phillips, Stuart M; Baar, Keith

    2015-01-01

    Abstract The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1 h of treadmill running (18 m min−1; 5° grade), 1 h after i.p. administration of rapamycin (1.5 mg · kg−1) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50 mg · kg−1) of l-[ring-13C6]phenylalanine was administered via i.p. injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6 h after completing exercise (n = 4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6 h, whereas this response was transiently blunted (30 min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P < 0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6 h recovery period. Peroxisome proliferator activated receptor-γ coactivator-1α, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P < 0.05) and this response was augmented by rapamycin (P < 0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation. Key points Previous studies have shown that endurance exercise increases myofibrillar (MyoPS) and

  11. Target of Rapamycin (TOR) in Nutrient Signaling and Growth Control

    PubMed Central

    Loewith, Robbie; Hall, Michael N.

    2011-01-01

    TOR (Target Of Rapamycin) is a highly conserved protein kinase that is important in both fundamental and clinical biology. In fundamental biology, TOR is a nutrient-sensitive, central controller of cell growth and aging. In clinical biology, TOR is implicated in many diseases and is the target of the drug rapamycin used in three different therapeutic areas. The yeast Saccharomyces cerevisiae has played a prominent role in both the discovery of TOR and the elucidation of its function. Here we review the TOR signaling network in S. cerevisiae. PMID:22174183

  12. The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

    PubMed

    Mukaida, Shiho; Ogawa, Takumi; Ohishi, Kazuko; Tanizawa, Yasuhiro; Ohta, Daisaku; Arita, Masanori

    2016-06-01

    Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species.

  13. Rapamycin increases lifespan and inhibits spontaneous tumorigenesis in inbred female mice.

    PubMed

    Anisimov, Vladimir N; Zabezhinski, Mark A; Popovich, Irina G; Piskunova, Tatiana S; Semenchenko, Anna V; Tyndyk, Margarita L; Yurova, Maria N; Rosenfeld, Svetlana V; Blagosklonny, Mikhail V

    2011-12-15

    The nutrient-sensing TOR (target of rapamycin) pathway is involved in cellular and organismal aging. Rapamycin, an inhibitor of TOR, extends lifespan in yeast, fruit flies and genetically heterogeneous mice. Here, we demonstrate that lifelong administration of rapamycin extends lifespan in female 129/Sv mice characterized by normal mean lifespan of 2 y. Importantly, rapamycin was administrated intermittently (2 weeks per month) starting from the age of 2 mo. Rapamycin inhibited age-related weight gain, decreased aging rate, increased lifespan (especially in the last survivors) and delayed spontaneous cancer. 22.9% of rapamycin-treated mice survived the age of death of the last mouse in control group. Thus we demonstrated for the first time in normal inbred mice that lifespan can be extended by rapamycin. This opens an avenue to develop optimal doses and schedules of rapamycin as an anti-aging modality.

  14. Down-regulation of vascular PPAR-γ contributes to endothelial dysfunction in high-fat diet-induced obese mice exposed to chronic intermittent hypoxia.

    PubMed

    Zhang, Yanan; Zhang, Chunlian; Li, Haiou; Hou, Jingdong

    2017-10-14

    Obstructive sleep apnea (OSA), characterized by chronic intermittent hypoxia (CIH), is associated with endothelial dysfunction. The prevalence of OSA is linked to an epidemic of obesity. CIH has recently been reported to cause endothelial dysfunction in diet-induced obese animals by exaggerating oxidative stress and inflammation, but the underlying mechanism remains unclear. PPAR-γ, a ligand-inducible transcription factor that exerts anti-oxidant and anti-inflammatory effects, is down-regulated in the peripheral tissues in diet-induce obesity. We tested the hypothesis that down-regulation of vascular PPAR-γ in diet-induced obesity enhances inflammation and oxidative stress in response to CIH, resulting in endothelial dysfunction. Male C57BL/6 mice were fed either a high-fat diet (HFD) or a low-fat diet (LFD) and simultaneously exposed to CIH or intermittent air for 6 weeks. An additional HFD group received a combination of CIH and PPAR-γ agonist pioglitazone for 6 weeks. Endothelial-dependent vasodilation was impaired only in HFD group exposed to CIH, compared with other groups, but was restored by concomitant pioglitazone treatment. Molecular studies revealed that vascular PPAR-γ expression and activity were reduced in HFD groups, compared with LFD groups, but were reversed by pioglitazone treatment. In addition, CIH elevated vascular expression of NADPH oxidase 4 and dihydroethidium fluorescence, and increased expression of proinflammatory cytokines TNF-α and IL-1β in both LFD and HFD groups, but these increases was significantly greater in HFD group, along with decreased vascular eNOS activity. Pioglitazone treatment of HFD group prevented CIH-induced changes in above molecular markers. The results suggest that HFD-induced obesity down-regulates vascular PPAR-γ, which results in exaggerated oxidative stress and inflammation in response to CIH, contributing to endothelial dysfunction. This finding may provide new insights into the mechanisms by which OSA

  15. Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis.

    PubMed

    Huber, Alexandre; Bodenmiller, Bernd; Uotila, Aino; Stahl, Michael; Wanka, Stefanie; Gerrits, Bertran; Aebersold, Ruedi; Loewith, Robbie

    2009-08-15

    The target of rapamycin complex 1 (TORC1) is an essential multiprotein complex conserved from yeast to humans. Under favorable growth conditions, and in the absence of the macrolide rapamycin, TORC1 is active, and influences virtually all aspects of cell growth. Although two direct effectors of yeast TORC1 have been reported (Tap42, a regulator of PP2A phosphatases and Sch9, an AGC family kinase), the signaling pathways that couple TORC1 to its distal effectors were not well understood. To elucidate these pathways we developed and employed a quantitative, label-free mass spectrometry approach. Analyses of the rapamycin-sensitive phosphoproteomes in various genetic backgrounds revealed both documented and novel TORC1 effectors and allowed us to partition phosphorylation events between Tap42 and Sch9. Follow-up detailed characterization shows that Sch9 regulates RNA polymerases I and III, the latter via Maf1, in addition to translation initiation and the expression of ribosomal protein and ribosome biogenesis genes. This demonstrates that Sch9 is a master regulator of protein synthesis.

  16. Differential regulation of blood flow‐induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling

    PubMed Central

    Stone, Oliver A.; Carter, James G.; Lin, P. Charles; Paleolog, Ewa; Machado, Maria J. C.

    2017-01-01

    Key points Combining nitric oxide (NO)‐mediated increased blood flow with angiopoietin‐1–Tie2 receptor signalling induces arteriolargenesis – the formation of arterioles from capillaries – in a model of physiological angiogenesis.This NO–Tie‐mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling.Inhibition of VEGF signalling increases pericyte coverage in microvessels.Together these findings indicate that generation of functional neovasculature requires close titration of NO–Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. Abstract Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains‐2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin‐1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)‐mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over‐expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms‐like tyrosine kinase receptor‐1 (sFlt1). We found that NO‐mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin‐1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte

  17. Lipid-soluble Cigarette Smoke Particles Induced Vascular Endothelin Type A Receptor Up-Regulation through Activation of ERK1/2 Signal Pathways.

    PubMed

    Zhang, Yaping; Zhang, Wei; Edvinsson, Lars; Xu, Cang-Bao

    2017-04-01

    Abnormal contraction of vessels termed 'vasospasm' is associated with various cardiovascular diseases. Smoking is a well-known risk factor that increases vasospasm. However, the molecular mechanisms by which smoking leads to vasospasm and cardiovascular disease are not fully understood. This study was designed to examine whether DMSO-extracted cigarette smoke particles (DSP) could induce up-regulation of vascular endothelin type A (ETA ) receptors, and whether ETA receptor is up-regulated through activation of extracellular regulated protein kinases 1 and 2 (ERK1/2) signal pathways. Mesenteric arterial segments from rats were cultured in the presence of DSP, water-extracted cigarette smoke particles (WSP) or equivalent concentration of nicotine for up to 24 hr. The results showed that DSP, but not WSP or nicotine, induced ETA receptor up-regulation with increased ETA receptor-mediated contraction (myograph, p < 0.001). Simultaneously, the expression of ETA receptor mRNA (real-time PCR, p < 0.001) and protein (immunohistochemistry) were enhanced in the smooth muscle cells, suggesting that the lipid-soluble substances contained in cigarette smoke were responsible for the effects of DSP. Actinomycin D (a general transcriptional inhibitor) decreased ETA receptor mRNA expression and attenuated receptor-mediated contraction (p < 0.001), while DSP accelerated ETA receptor mRNA degradation (p < 0.01) and promoted the translation of ETA receptor mRNA into protein. Furthermore, the up-regulation of ETA receptors was significantly attenuated by inhibition of ERK1/2 signal pathways (p < 0.001). In conclusion, DSP most likely activate ERK1/2 signal pathway-mediated transcriptional and post-transcriptional (translational) mechanisms that lead to vascular ETA receptor up-regulation, which might contribute to vasospasm and the development of smoking-associated cardiovascular diseases. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  18. Growth of Streptomyces Hygroscopicus in Rotating-Wall Bioreactor Under Simulated Microgravity Inhibits Rapamycin Production

    NASA Technical Reports Server (NTRS)

    Fang, A.; Pierson, D. L.; Mishra, S. K.; Demain, A. L.

    2000-01-01

    Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions.

  19. HCdc14A is involved in cell cycle regulation of human brain vascular endothelial cells following injury induced by high glucose, free fatty acids and hypoxia.

    PubMed

    Su, Jingjing; Zhou, Houguang; Tao, Yinghong; Guo, Zhuangli; Zhang, Shuo; Zhang, Yu; Huang, Yanyan; Tang, Yuping; Hu, Renming; Dong, Qiang

    2015-01-01

    Cell cycle processes play a vital role in vascular endothelial proliferation and dysfunction. Cell division cycle protein 14 (Cdc14) is an important cell cycle regulatory phosphatase. Previous studies in budding yeast demonstrated that Cdc14 could trigger the inactivation of mitotic cyclin-dependent kinases (Cdks), which are required for mitotic exit and cytokinesis. However, the exact function of human Cdc14 (hCdc14) in cell cycle regulation during vascular diseases is yet to be elucidated. There are two HCdc14 homologs: hCdc14A and hCdc14B. In the current study, we investigated the potential role of hCdc14A in high glucose-, free fatty acids (FFAs)-, and hypoxia-induced injury in cultured human brain vascular endothelial cells (HBVECs). Data revealed that high glucose, FFA, and hypoxia down-regulated hCdc14A expression remarkably, and also affected the expression of other cell cycle-related proteins such as cyclin B, cyclin D, cyclin E, and p53. Furthermore, the combined addition of the three stimuli largely blocked cell cycle progression, decreased cell proliferation, and increased apoptosis. We also determined that hCdc14A was localized mainly to centrosomes during interphase and spindles during mitosis using confocal microscopy, and that it could affect the expression of other cycle-related proteins. More importantly, the overexpression of hCdc14A accelerated cell cycle progression, enhanced cell proliferation, and promoted neoplastic transformation, whereas the knockdown of hCdc14A using small interfering RNA produced the opposite effects. Therefore, these findings provide novel evidence that hCdc14A might be involved in cell cycle regulation in cultured HBVECs during high glucose-, FFA-, and hypoxia-induced injury. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

    SciTech Connect

    Moriyama, Yuki; Ohata, Yoshihisa; Mori, Shoko; Matsukawa, Shinya; Michiue, Tatsuo; Asashima, Makoto; Kuroda, Hiroki

    2011-01-28

    Research highlights: {yields} Does famous anti-aging drug rapamycin work from the beginning of life? The answer is yes. {yields} This study shows that developmental speed of frog embryo was dose-dependently decreased by rapamycin treatment. {yields} In additions, morphogenetic effects such as less pigmentations and gut malformation are occurred by rapamycin. -- Abstract: Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood. Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.

  1. Differing Effects of Systemically Administered Rapamycin on Consolidation and Reconsolidation of Context vs. Cued Fear Memories

    ERIC Educational Resources Information Center

    Glover, Ebony M.; Ressler, Kerry J.; Davis, Michael

    2010-01-01

    Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) kinase, has attracted interest as a possible prophylactic for post-traumatic stress disorder (PTSD)-associated fear memories. We report here that although rapamycin (40 mg/kg, i.p.) disrupted the consolidation and reconsolidation of fear-potentiated startle paradigm to a…

  2. Rapamycin treatment causes developmental delay, pigmentation defects, and gastrointestinal malformation on Xenopus embryogenesis

    SciTech Connect

    Moriyama, Yuki; Ohata, Yoshihisa; Mori, Shoko

    2011-01-28

    Research highlights: {yields} Does famous anti-aging drug rapamycin work from the beginning of life? The answer is yes. {yields} This study shows that developmental speed of frog embryo was dose-dependently decreased by rapamycin treatment. {yields} In additions, morphogenetic effects such as less pigmentations and gut malformation are occurred by rapamycin. -- Abstract: Rapamycin is a drug working as an inhibitor of the TOR (target of rapamycin) signaling pathway and influences various life phenomena such as cell growth, proliferation, and life span extension in eukaryote. However, the extent to which rapamycin controls early developmental events of amphibians remains to be understood.more » Here we report an examination of rapamycin effects during Xenopus early development, followed by a confirmation of suppression of TOR downstream kinase S6K by rapamycin treatment. First, we found that developmental speed was declined in dose-dependent manner of rapamycin. Second, black pigment spots located at dorsal and lateral skin in tadpoles were reduced by rapamycin treatment. Moreover, in tadpole stages severe gastrointestinal malformations were observed in rapamycin-treated embryos. Taken together with these results, we conclude that treatment of the drug rapamycin causes enormous influences on early developmental period.« less

  3. ULK1, mammalian target of rapamycin, and mitochondria: linking nutrient availability and autophagy.

    PubMed

    Kundu, Mondira

    2011-05-15

    A fundamental function of autophagy conserved from yeast to mammals is mobilization of macromolecules during times of limited nutrient availability, permitting organisms to survive under starvation conditions. In yeast, autophagy is initiated following nitrogen or carbon deprivation, and autophagy mutants die rapidly under these conditions. Similarly, in mammals, autophagy is upregulated in most organs following initiation of starvation, and is critical for survival in the perinatal period following abrupt termination of the placental nutrient supply. The nutrient-sensing kinase, mammalian target of rapamycin, coordinates cellular proliferation and growth with nutrient availability, at least in part by regulating protein synthesis and autophagy-mediated degradation. This review focusses on the regulation of autophagy by Tor, a mammalian target of rapamycin, and Ulk1, a mammalian homolog of Atg1, in response to changes in nutrient availability. Given the importance of mitochondria in maintaining bioenergetic homestasis, and potentially as a source of membrane for autophagosomes during starvation, possible roles for mitochondria in this process are also discussed.

  4. Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling.

    PubMed

    Jin, Yi; Muhl, Lars; Burmakin, Mikhail; Wang, Yixin; Duchez, Anne-Claire; Betsholtz, Christer; Arthur, Helen M; Jakobsson, Lars

    2017-06-01

    Loss-of-function (LOF) mutations in the endothelial cell (EC)-enriched gene endoglin (ENG) cause the human disease hereditary haemorrhagic telangiectasia-1, characterized by vascular malformations promoted by vascular endothelial growth factor A (VEGFA). How ENG deficiency alters EC behaviour to trigger these anomalies is not understood. Mosaic ENG deletion in the postnatal mouse rendered Eng LOF ECs insensitive to flow-mediated venous to arterial migration. Eng LOF ECs retained within arterioles acquired venous characteristics and secondary ENG-independent proliferation resulting in arteriovenous malformation (AVM). Analysis following simultaneous Eng LOF and overexpression (OE) revealed that ENG OE ECs dominate tip-cell positions and home preferentially to arteries. ENG knockdown altered VEGFA-mediated VEGFR2 kinetics and promoted AKT signalling. Blockage of PI(3)K/AKT partly normalized flow-directed migration of ENG LOF ECs in vitro and reduced the severity of AVM in vivo. This demonstrates the requirement of ENG in flow-mediated migration and modulation of VEGFR2 signalling in vascular patterning.

  5. Possible role of mechanical force in regulating regeneration of the vascularized fat flap inside a tissue engineering chamber.

    PubMed

    Ye, Yuan; Yuan, Yi; Lu, Feng; Gao, Jianhua

    2015-12-01

    In plastic and reconstructive surgery, adipose tissue is widely used as effective filler for tissue defects. Strategies for treating soft tissue deficiency, which include free adipose tissue grafts, use of hyaluronic acid, collagen injections, and implantation of synthetic materials, have several clinical limitations. With the aim of overcoming these limitations, researchers have recently utilized tissue engineering chambers to produce large volumes of engineered vascularized fat tissue. However, the process of growing fat tissue in a chamber is still relatively limited, and can result in unpredictable or dissatisfactory final tissue volumes. Therefore, detailed understanding of the process is both necessary and urgent. Many studies have shown that mechanical force can change the function of cells via mechanotransduction. Here, we hypothesized that, besides the inflammatory response, one of the key factors to control the regeneration of vascularized fat flap inside a tissue engineering chamber might be the balance of mechanical forces. To test our hypothesis, we intend to change the balance of forces by means of measures in order to make the equilibrium point in favor of the direction of regeneration. If those measures proved to be feasible, they could be applied in clinical practice to engineer vascularized adipose tissue of predictable size and shape, which would in turn help in the advancement of tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling

    PubMed Central

    Jin, Yi; Muhl, Lars; Burmakin, Mikhail; Wang, Yixin; Duchez, Anne-Claire; Betsholtz, Christer; Arthur, Helen M.; Jakobsson, Lars

    2017-01-01

    Loss-of-function (LOF) mutations in the endothelial cell (EC) enriched gene endoglin (ENG) causes the human disease hereditary haemorrhagic telangiectasia-1, characterized by vascular malformations promoted by vascular endothelial growth factor A (VEGFA). How ENG deficiency alters EC behaviour to trigger these anomalies is not understood. Mosaic ENG deletion in the postnatal mouse rendered Eng LOF ECs insensitive to flow-mediated venous to arterial migration. Eng LOF ECs retained within arterioles acquired venous characteristics and secondary ENG-independent proliferation resulting in arterio-venous malformation (AVM). Analysis following simultaneous Eng LOF and overexpression (OE) revealed that ENG OE ECs dominate tip cell positions and home preferentially to arteries. ENG knock-down altered VEGFA-mediated VEGFR2 kinetics and promoted AKT signalling. Blockage of PI3K/AKT partly normalised flow-directed migration of ENG LOF ECs in vitro and reduced the severity of AVM in vivo. This demonstrates the requirement of ENG in flow-mediated migration and modulation of VEGFR2 signalling in vascular patterning. PMID:28530660

  7. Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc kinase-dependent connexin43 phosphorylation.

    PubMed

    Hangaard, Lise; Bouzinova, Elena V; Staehr, Christian; Dam, Vibeke S; Kim, Sukhan; Xie, Zijian; Aalkjaer, Christian; Matchkov, Vladimir V

    2017-04-01

    Communication between vascular smooth muscle cells (VSMCs) is dependent on gap junctions and is regulated by the Na-K-ATPase. The Na-K-ATPase is therefore important for synchronized VSMC oscillatory activity, i.e., vasomotion. The signaling between the Na-K-ATPase and gap junctions is unknown. We tested here the hypothesis that this signaling involves cSrc kinase. Intercellular communication was assessed by membrane capacitance measurements of electrically coupled VSMCs. Vasomotion in isometric myograph, input resistance, and synchronized [Ca2+]i transients were used as readout for intercellular coupling in rat mesenteric small arteries in vitro. Phosphorylation of cSrc kinase and connexin43 (Cx43) were semiquantified by Western blotting. Micromole concentration of ouabain reduced the amplitude of norepinephrine-induced vasomotion and desynchronized Ca2+ transients in VSMC in the arterial wall. Ouabain also increased input resistance in the arterial wall. These effects of ouabain were antagonized by inhibition of tyrosine phosphorylation with genistein, PP2, and by an inhibitor of the Na-K-ATPase-dependent cSrc activation, pNaKtide. Moreover, inhibition of cSrc phosphorylation increased vasomotion amplitude and decreased the resistance between cells in the vascular wall. Ouabain inhibited the electrical coupling between A7r5 cells, but pNaKtide restored the electrical coupling. Ouabain increased cSrc autophosphorylation of tyrosine 418 (Y418) required for full catalytic activity whereas pNaKtide antagonized it. This cSrc activation was associated with Cx43 phosphorylation of tyrosine 265 (Y265). Our findings demonstrate that Na-K-ATPase regulates intercellular communication in the vascular wall via cSrc-dependent Cx43 tyrosine phosphorylation. Copyright © 2017 the American Physiological Society.

  8. Modulation of CaV1.2 calcium channel by neuropeptide W regulates vascular myogenic tone via G protein-coupled receptor 7.

    PubMed

    Ji, Li; Zhu, Huayuan; Chen, Hong; Fan, Wenyong; Chen, Junjie; Chen, Jing; Zhu, Guoqing; Wang, Juejin

    2015-12-01

    Neuropeptide W (NPW), an endogenous ligand for the G protein-coupled receptor 7 (GPR7), was first found to make important roles in central nerve system. In periphery, NPW was also present and regulated intracellular calcium homeostasis by L-type calcium channels. This study was designed to discover the effects of NPW-GPR7 on the function of CaV1.2 calcium channels in the vascular smooth muscle cells (VSMCs) and vasotone of arterial vessels. By whole-cell patch clamp, we studied the effects of NPW-23, the active form of NPW, on the CaV1.2 channels in the heterologously transfected human embryonic kidney 293 cells and VSMCs isolated from rat. Living system was used to explore the physiological function of NPW-23 in arterial myogenic tone. To investigate the pathological relevance, NPW mRNA level of mesenteric arteries was measured in the hypertensive and normotensive rats. NPW's receptor GPR7 was coexpressed with CaV1.2 channels in arterial smooth muscle. NPW-23 increased the ICa,L in transfected human embryonic kidney 293 cells and VSMCs via GPR7, which could be abrogated by phospholipase C (PLC)/protein kinase C (PKC) inhibitors, not protein kinase A or protein kinase G inhibitor. After NPW-23 application, the expression of pan phospho-PKC was increased; moreover, intracellular diacylglycerol level, the second messenger catalyzed by PLC, was increased 1.5-2-fold. Application with NPW-23 increased pressure-induced vasotone of the rat mesenteric arteries. Importantly, the expression of NPW was decreased in the hypertensive rats. NPW-23 regulates ICa,L via GPR7, which is mediated by PLC/PKC signaling, and such a mechanism plays a role in modulating vascular myogenic tone, which may involve in the development of vascular hypertension.

  9. BEST: A Randomized Phase II Study of Vascular Endothelial Growth Factor, RAF Kinase, and Mammalian Target of Rapamycin Combination Targeted Therapy With Bevacizumab, Sorafenib, and Temsirolimus in Advanced Renal Cell Carcinoma--A Trial of the ECOG-ACRIN Cancer Research Group (E2804).

    PubMed

    Flaherty, Keith T; Manola, Judith B; Pins, Michael; McDermott, David F; Atkins, Michael B; Dutcher, Janice J; George, Daniel J; Margolin, Kim A; DiPaola, Robert S

    2015-07-20

    On the basis of evidence that resistance to vascular endothelial growth factor (VEGF) receptor inhibition is caused by hypoxia-driven residual VEGF and other proangiogenic factors, combinations of agents from these classes were hypothesized to improve treatment outcomes relative to single-agent VEGF pathway blockade. A total of 361 patients with metastatic clear cell renal cell carcinoma were randomly assigned equally to arm A (bevacizumab monotherapy 10 mg/kg intravenously [IV] every 2 weeks), B (bevacizumab 10 mg/kg IV every 2 weeks and temsirolimus 25 mg IV every week), C (bevacizumab 5 mg/kg IV every 2 weeks and sorafenib 200 mg orally twice daily on days 1 to 5, 8 to 12, 15 to 19, and 22 to 26), or D (sorafenib 200 mg twice daily and temsirolimus 25 mg IV weekly). Progression-free survival was the primary end point. Among 331 eligible treated patients, median PFS was 7.5 months for bevacizumab alone (90% CI, 5.8 to 10.8 months), 7.6 months for bevacizumab plus temsirolimus (90% CI, 6.7 to 9.2 months), 9.2 months for bevacizumab plus sorafenib (90% CI, 7.5 to 11.4 months), and 7.4 months for sorafenib plus temsirolimus (90% CI, 5.6 to 7.9 months). Hazard ratios from stratified Cox proportional hazards models were 1.01, 0.89, and 1.07 (with respective P values of .95, .49, and .68) for the three combinations, respectively, compared with bevacizumab alone. Adverse events did not differ significantly among treatment arms. The activity of sorafenib, temsirolimus, and bevacizumab administered in doublet combinations did not significantly improve median progression-free survival in comparison with bevacizumab monotherapy. © 2015 by American Society of Clinical Oncology.

  10. BEST: A Randomized Phase II Study of Vascular Endothelial Growth Factor, RAF Kinase, and Mammalian Target of Rapamycin Combination Targeted Therapy With Bevacizumab, Sorafenib, and Temsirolimus in Advanced Renal Cell Carcinoma—A Trial of the ECOG–ACRIN Cancer Research Group (E2804)

    PubMed Central

    Flaherty, Keith T.; Manola, Judith B.; Pins, Michael; McDermott, David F.; Atkins, Michael B.; Dutcher, Janice J.; George, Daniel J.; Margolin, Kim A.; DiPaola, Robert S.

    2015-01-01

    Purpose On the basis of evidence that resistance to vascular endothelial growth factor (VEGF) receptor inhibition is caused by hypoxia-driven residual VEGF and other proangiogenic factors, combinations of agents from these classes were hypothesized to improve treatment outcomes relative to single-agent VEGF pathway blockade. Patients and Methods A total of 361 patients with metastatic clear cell renal cell carcinoma were randomly assigned equally to arm A (bevacizumab monotherapy 10 mg/kg intravenously [IV] every 2 weeks), B (bevacizumab 10 mg/kg IV every 2 weeks and temsirolimus 25 mg IV every week), C (bevacizumab 5 mg/kg IV every 2 weeks and sorafenib 200 mg orally twice daily on days 1 to 5, 8 to 12, 15 to 19, and 22 to 26), or D (sorafenib 200 mg twice daily and temsirolimus 25 mg IV weekly). Progression-free survival was the primary end point. Results Among 331 eligible treated patients, median PFS was 7.5 months for bevacizumab alone (90% CI, 5.8 to 10.8 months), 7.6 months for bevacizumab plus temsirolimus (90% CI, 6.7 to 9.2 months), 9.2 months for bevacizumab plus sorafenib (90% CI, 7.5 to 11.4 months), and 7.4 months for sorafenib plus temsirolimus (90% CI, 5.6 to 7.9 months). Hazard ratios from stratified Cox proportional hazards models were 1.01, 0.89, and 1.07 (with respective P values of .95, .49, and .68) for the three combinations, respectively, compared with bevacizumab alone. Adverse events did not differ significantly among treatment arms. Conclusion The activity of sorafenib, temsirolimus, and bevacizumab administered in doublet combinations did not significantly improve median progression-free survival in comparison with bevacizumab monotherapy. PMID:26077237

  11. The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

    PubMed

    Chou, Chih-Wei; Zhuo, You-Lin; Jiang, Zhe-Yu; Liu, Yi-Wen

    2014-01-01

    While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated β-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms. These results demonstrate

  12. Rapamycin-induced metabolic defects are reversible in both lean and obese mice

    PubMed Central

    Liu, Yuhong; Diaz, Vivian; Fernandez, Elizabeth; Strong, Randy; Ye, Lan; Baur, Joseph A.; Lamming, Dudley W.; Richardson, Arlan; Salmon, Adam B.

    2014-01-01

    The inhibition of mTOR (mechanistic target of rapamycin) by the macrolide rapamycin has many beneficial effects in mice, including extension of lifespan and reduction or prevention of several age-related diseases. At the same time, chronic rapamycin treatment causes impairments in glucose metabolism including hyperglycemia, glucose intolerance and insulin resistance. It is unknown whether these metabolic effects of rapamycin are permanent or whether they can be alleviated. Here, we confirmed that rapamycin causes glucose intolerance and insulin resistance in both inbred and genetically heterogeneous mice fed either low fat or high fat diets, suggesting that these effects of rapamycin are independent of genetic background. Importantly, we also found that these effects were almost completely lost within a few weeks of cessation of treatment, showing that chronic rapamycin treatment does not induce permanent impairment of glucose metabolism. Somewhat surprisingly, chronic rapamycin also promoted increased accumulation of adipose tissue in high fat fed mice. However, this effect too was lost when rapamycin treatment was ended suggesting that this effect of rapamycin is also not permanent. The reversible nature of rapamycin's alterations of metabolic function suggests that these potentially detrimental side-effects might be managed through alternative dosing strategies or concurrent treatment options. PMID:25324470

  13. Rapamycin extends life and health in C57BL/6 mice.

    PubMed

    Zhang, Yiqiang; Bokov, Alex; Gelfond, John; Soto, Vanessa; Ikeno, Yuji; Hubbard, Gene; Diaz, Vivian; Sloane, Lauren; Maslin, Keith; Treaster, Stephen; Réndon, Samantha; van Remmen, Holly; Ward, Walter; Javors, Martin; Richardson, Arlan; Austad, Steven N; Fischer, Kathleen

    2014-02-01

    Target of rapamycin inhibition by rapamycin feeding has previously been shown to extend life in genetically heterogeneous mice. To examine whether it similarly affected mouse health, we fed encapsulated rapamycin or a control diet to C57BL/6Nia mice of both sexes starting at 19 months of age. We performed a range of health assessments 6 and 12 months later. Rapamycin feeding significantly reduced mTOR activity in most but not all tissues. It also reduced total and resting metabolic rate during the light (inactive) phase of the light:dark cycle in females only but had no effect on spontaneous activity or metabolism during the dark (active) phase of either sex. Males only had less fragmented sleep when fed rapamycin, whereas stride length and rotarod performance were improved in both sexes. Survival was also improved by this late-life rapamycin feeding, and some pathological lesions were delayed. We found no adverse health consequences associated with rapamycin treatment.

  14. Vascular ring

    MedlinePlus

    ... with aberrant subclavian and left ligamentum ateriosus; Congenital heart defect - vascular ring; Birth defect heart - vascular ring ... accounts for less than 1% of all congenital heart problems. The condition occurs as often in males ...

  15. Rapamycin nanoparticles localize in diseased lung vasculature and prevent pulmonary arterial hypertension.

    PubMed

    Segura-Ibarra, Victor; Amione-Guerra, Javier; Cruz-Solbes, Ana S; Cara, Francisca E; Iruegas-Nunez, David A; Wu, Suhong; Youker, Keith A; Bhimaraj, Arvind; Torre-Amione, Guillermo; Ferrari, Mauro; Karmouty-Quintana, Harry; Guha, Ashrith; Blanco, Elvin

    2017-05-30

    Vascular remodeling resulting from pulmonary arterial hypertension (PAH) leads to endothelial fenestrations. This feature can be exploited by nanoparticles (NP), allowing them to extravasate from circulation and accumulate in remodeled pulmonary vessels. Hyperactivation of the mTOR pathway in PAH drives pulmonary arterial smooth muscle cell proliferation. We hypothesized that rapamycin (RAP)-loaded NPs, an mTOR inhibitor, would accumulate in diseased lungs, selectively targeting vascular mTOR and preventing PAH progression. RAP poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL) NPs were fabricated. NP accumulation and efficacy were examined in a rat monocrotaline model of PAH. Following intravenous (IV) administration, NP accumulation in diseased lungs was verified via LC/MS analysis and confocal imaging. Pulmonary arteriole thickness, right ventricular systolic pressures, and ventricular remodeling were determined to assess the therapeutic potential of RAP NPs. Monocrotaline-exposed rats showed increased NP accumulation within lungs compared to healthy controls, with NPs present to a high extent within pulmonary perivascular regions. RAP, in both free and NP form, attenuated PAH development, with histological analysis revealing minimal changes in pulmonary arteriole thickness and no ventricular remodeling. Importantly, NP-treated rats showed reduced systemic side effects compared to free RAP. This study demonstrates the potential for nanoparticles to significantly impact PAH through site-specific delivery of therapeutics. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Endothelial cell mineralocorticoid receptors regulate deoxycorticosterone/salt-mediated cardiac remodeling and vascular reactivity but not blood pressure.

    PubMed

    Rickard, Amanda J; Morgan, James; Chrissobolis, Sophocles; Miller, Alyson A; Sobey, Christopher G; Young, Morag J

    2014-05-01

    Recent studies have identified novel pathological roles for mineralocorticoid receptors (MR) in specific cell types in cardiovascular disease. The mechanisms by which MR promotes inflammation and fibrosis involve multiple cell-specific events. To identify the role of MR in endothelial cells (EC-MR), the current study explored the vascular responses to aldosterone in wild-type (WT) and EC-null mice (EC-MRKO). Nitric oxide function was impaired in the thoracic aorta and mesenteric arteries of aldosterone-treated WT mice. Although endothelial nitric oxide function was equivalently impaired in the mesenteric arteries of aldosterone-treated EC-MRKO mice, endothelial function was unaffected in the aorta, suggesting a differential role for EC-MR depending on the vascular bed. Second, the contribution of EC-MR to cardiovascular inflammation, fibrosis, and hypertension was determined in WT and EC-MRKO treated with deoxycorticosterone/salt for 8 days or 8 weeks. At 8 days, loss of EC-MR prevented macrophage infiltration and the expression of proinflammatory genes in the myocardium. Increased cardiac fibrosis was not detected in either genotype at this time, mRNA levels of profibrotic genes were significantly lower in EC-MRKO mice versus WT. At 8 weeks, deoxycorticosterone/salt treatment increased macrophage recruitment and proinflammatory gene expression in WT but not in EC-MRKO. Collagen deposition and connective tissue growth factor expression were significantly reduced in EC-MRKO versus WT. Interestingly, systolic blood pressure was equivalently elevated in deoxycorticosterone/salt treated WT and EC-MRKO. Our data demonstrate that (1) EC-MR signaling contributes to vascular nitric oxide function in large conduit arteries but not in resistance vessels and (2) an independent role for EC-MR in the inflammatory and profibrotic response to deoxycorticosterone/salt.

  17. The Role of Target of Rapamycin Signaling Networks in Plant Growth and Metabolism1

    PubMed Central

    Sheen, Jen

    2014-01-01

    The target of rapamycin (TOR) kinase, a master regulator that is evolutionarily conserved among yeasts (Saccharomyces cerevisiae), plants, animals, and humans, integrates nutrient and energy signaling to promote cell proliferation and growth. Recent breakthroughs made possible by integrating chemical, genetic, and genomic analyses have greatly increased our understanding of the molecular functions and dynamic regulation of the TOR kinase in photosynthetic plants. TOR signaling plays fundamental roles in embryogenesis, meristem activation, root and leaf growth, flowering, senescence, and life span determination. The molecular mechanisms underlying TOR-mediated ribosomal biogenesis, translation promotion, readjustment of metabolism, and autophagy inhibition are now being uncovered. Moreover, monitoring photosynthesis-derived Glc and bioenergetics relays has revealed that TOR orchestrates unprecedented transcriptional networks that wire central metabolism and biosynthesis for energy and biomass production. In addition, these networks integrate localized stem/progenitor cell proliferation through interorgan nutrient coordination to control developmental transitions and growth. PMID:24385567

  18. Target of rapamycin signaling orchestrates growth-defense trade-offs in plants.

    PubMed

    De Vleesschauwer, David; Filipe, Osvaldo; Hoffman, Gena; Seifi, Hamed Soren; Haeck, Ashley; Canlas, Patrick; Van Bockhaven, Jonas; De Waele, Evelien; Demeestere, Kristof; Ronald, Pamela; Hofte, Monica

    2018-01-01

    Plant defense to microbial pathogens is often accompanied by significant growth inhibition. How plants merge immune system function with normal growth and development is still poorly understood. Here, we investigated the role of target of rapamycin (TOR), an evolutionary conserved serine/threonine kinase, in the plant defense response. We used rice as a model system and applied a combination of chemical, genetic, genomic and cell-based analyses. We demonstrate that ectopic expression of TOR and Raptor (regulatory-associated protein of mTOR), a protein previously demonstrated to interact with TOR in Arabidopsis, positively regulates growth and development in rice. Transcriptome analysis of rice cells treated with the TOR-specific inhibitor rapamycin revealed that TOR not only dictates transcriptional reprogramming of extensive gene sets involved in central and secondary metabolism, cell cycle and transcription, but also suppresses many defense-related genes. TOR overexpression lines displayed increased susceptibility to both bacterial and fungal pathogens, whereas plants with reduced TOR signaling displayed enhanced resistance. Finally, we found that TOR antagonizes the action of the classic defense hormones salicylic acid and jasmonic acid. Together, these results indicate that TOR acts as a molecular switch for the activation of cell proliferation and plant growth at the expense of cellular immunity. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  19. Rapid estrogen receptor-α signaling mediated by ERK activation regulates vascular tone in male and ovary-intact female mice.

    PubMed

    Kim, Seong Chul; Boese, Austin C; Moore, Matthew H; Cleland, Rea M; Chang, Lin; Delafontaine, Patrice; Yin, Ke-Jie; Lee, Jean-Pyo; Hamblin, Milton H

    2018-02-01

    Estrogen has been shown to affect vascular reactivity. Here, we assessed the estrogen receptor-α (ERα) dependency of estrogenic effects on vasorelaxation via a rapid nongenomic pathway in both male and ovary-intact female mice. We compared the effect of a primary estrogen, 17β-estradiol (E 2 ) or 4,4',4″-(4-propyl-[1H]pyrazole-1,3,5-triyl)tris-phenol (PPT; selective ERα agonist). We found that E 2 and PPT induced greater aortic relaxation in female mice than in male mice, indicating ERα mediation, which was further validated by using ERα antagonism. Treatment with 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP dihydrochloride; ERα antagonist) attenuated PPT-mediated vessel relaxation in both sexes. ERα-mediated vessel relaxation was further validated by the absence of significant PPT-mediated relaxation in aortas isolated from ERα knockout mice. Treatment with a specific ERK inhibitor, PD-98059, reduced E 2 -induced vessel relaxation in both sexes but to a lesser extent in female mice. Furthermore, PD-98059 prevented PPT-induced vessel relaxation in both sexes. Both E 2 and PPT treatment activated ERK as early as 5-10 min, which was attenuated by PD-98059 in aortic tissue, cultured primary vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). Aortic rings denuded of endothelium showed no differences in vessel relaxation after E 2 or PPT treatment, implicating a role of ECs in the observed sex differences. Here, our results are unique to show estrogen-stimulated rapid ERα signaling mediated by ERK activation in aortic tissue, as well as VSMCs and ECs in vitro, in regulating vascular function by using side-by-side comparisons in male and ovary-intact female mice in response to E 2 or PPT. NEW & NOTEWORTHY Here, we assessed the estrogen receptor-α dependency of estrogenic effects in vasorelaxation of both male and ovary-intact female mice by performing side-by-side comparisons. Also, we

  20. Regulation of the collagen phenotype expression of gamma-irradiated vascular smooth muscle cells by heparan mimetics (RGTA).

    PubMed

    Alexakis, C; Strup, C; Mestries, P; Mathé, D; Caruelle, J P; Barritault, D; Kern, P

    2004-09-15

    Restenosis is characterized by vascular smooth muscle cell (VSMC) proliferation and accumulation of collagen III in a hypertrophic and disorganized extracellular matrix. Restenosis is prevented by antimitotic agents or irradiation but no significant progress has been made to control collagen expression deregulation. Previously, we have shown that a new family of biopolymers named RGTA (heparan mimetics elaborated by grafting on dextran of carboxylate, sulfate, and benzylamide units) stimulate in vivo tissue repair and reduce fibrosis in various models. Using VSMC in vitro (pig aortic VSMC irradiated with a 60Co source and labeled with [3H]Proline), we now show that gamma-irradiation reduced cell survival by 50% and collagen synthesis 6-fold with a major increase in the ratio of collagen III to collagen I biosynthesis taken as a fibrotic index. RGTA added to the cells enhanced their survival up to 80% and reduced collagen III/I ratio back to values found in normal vascular tissues. These results suggest that RGTA combined with gamma-radiation could be an efficient strategy against restenosis.

  1. Vascular Endothelial Growth Factor Ligands and Receptors That Regulate Human Cytotrophoblast Survival Are Dysregulated in Severe Preeclampsia and Hemolysis, Elevated Liver Enzymes, and Low Platelets Syndrome

    PubMed Central

    Zhou, Yan; McMaster, Michael; Woo, Kirstin; Janatpour, Mary; Perry, Jean; Karpanen, Terhi; Alitalo, Kari; Damsky, Caroline; Fisher, Susan J.

    2002-01-01

    Human placental development combines elements of tumorigenesis and vasculogenesis. The organ’s specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell’s expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin α1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia. PMID:11943725

  2. 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.

  3. Vascular Tone Regulation Induced by C-Type Natriuretic Peptide: Differences in Endothelium-Dependent and -Independent Mechanisms Involved in Normotensive and Spontaneously Hypertensive Rats

    PubMed Central

    Caniffi, Carolina; Cerniello, Flavia M.; Gobetto, María N.; Sueiro, María L.; Arranz, Cristina

    2016-01-01

    Given that the role of C-type natriuretic peptide (CNP) in the regulation of vascular tone in hypertensive states is unclear, we hypothesized that impaired response of the nitric oxide system to CNP in spontaneously hypertensive rats (SHR) could affect vascular relaxation induced by the peptide in this model of hypertension, and that other endothelial systems or potassium channels opening could also be involved. We examined the effect of CNP on isolated SHR aortas, and the hindlimb vascular resistance (HVR) in response to CNP administration compared to normotensive rats. Aortas were mounted in an isometric organ bath and contracted with phenylephrine. CNP relaxed arteries in a concentration-dependent manner but was less potent in inducing relaxation in SHR. The action of CNP was diminished by removal of the endothelium, inhibition of nitric oxide synthase by Nω-nitro-L-arginine methyl ester, and inhibition of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one in both groups. In contrast, blockade of cyclooxygenase or subtype 2 bradykinin receptor increased CNP potency only in SHR. In both Wistar and SHR, CNP relaxation was blunted by tetraethylammonium and partially inhibited by BaCl2 and iberiotoxin, indicating that it was due to opening of the Kir and BKCa channels. However, SHR seem to be more sensitive to Kir channel blockade and less sensitive to BKCa channel blockade than normotensive rats. In addition, CNP decreases HVR in Wistar and SHR, but the effect of CNP increasing blood flow was more marked in SHR. We conclude that CNP induces aorta relaxation by activation of the nitric oxide system and opening of potassium channels, but the response to the peptide is impaired in conductance vessel of hypertensive rats. PMID:27936197

  4. 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

  5. Cargo and Carrier Effects of Rapamycin-Loaded Perfluorocarbon Nanoparticles

    NASA Astrophysics Data System (ADS)

    Bibee, Kristin Page

    Nanoparticle-based drug delivery has been championed as a means to increase local delivery of therapeutics while decreasing systemic drug exposure. By targeting the particles, and therefore the drugs, to diseased cells of interest, healthy cells will be spared and side effects avoided. This delivery mechanism would be particularly useful for drugs that interfere with cell growth and proliferation pathways, as blocking proliferation in normal cells leads to significant patient morbidity. Rapamycin is a macrolide and a known inhibitor of mTORC1, a protein complex that plays a crucial role in protein translation and cell growth. This work demonstrates the effects of rapamycin complexed with a nanoparticle carrier on two distinct pathologies: a new triple negative breast cancer cell line and a conventional mouse model of muscular dystrophy (mdx). Rapamycin is able to alter mitochondrial function and thus metabolism in both free and nanoparticle-delivered form without killing the cells. Although nanoparticles are considered to be a benign carrier, this work shows that perfluorocarbon nanoparticles are able to induce autophagy in vitro. The benefits of autophagy induction in cancer cells is cell and stage specific, but has been reported to be useful for radiosensitization of triple negative breast cancers. Additionally, the particles are shown to induce autophagy in the mdx model of Duchenne Muscular Dystrophy and, when loaded with rapamycin, dramatically improve strength even in older animals with muscular dystrophy. Overall, this work enhances our understanding of the cellular effects of perfluorocarbon nanoparticles in two different disease models and enhances prospects for clinical translation of nanoparticle-based drug delivery.

  6. Haploinsufficiency of Target of Rapamycin Attenuates Cardiomyopathies in Adult Zebrafish

    PubMed Central

    Ding, Yonghe; Sun, Xiaojing; Huang, Wei; Hoage, Tiffany; Redfield, Margaret; Kushwaha, Sudhir; Sivasubbu, Sridhar; Lin, Xueying; Ekker, Stephen; Xu, Xiaolei

    2011-01-01

    Rationale Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. Here, we explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. Objective We generate the second adult zebrafish cardiomyopathy model induced by doxorubicin (DOX). By genetically analyzing both the DOX and our previous established anemia-induced cardiomyopathy models, we aim to decipher the functions of TOR signaling in cardiomyopathies of different etiology. Methods and Results Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling via short-term rapamycin treatment. To assess the long term effects of TOR reduction, we utilized a zebrafish target of rapamycin (ztor) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the anti-hypertrophy, anti-apoptosis, and proautophagy function of TOR signaling inhibition. Conclusions Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least two cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis. PMID:21757652

  7. Identification of novel rapamycin derivatives as low-level impurities in active pharmaceutical ingredients.

    PubMed

    Zech, Stephan G; Carr, Michael; Mohemmad, Qurish K; Narasimhan, Narayana I; Murray, Christopher; Rozamus, Leonard W; Dalgarno, David C

    2011-09-01

    We describe the identification of novel rapamycin derivatives present as low-level impurities in active pharmaceutical ingredients using an integrated, multidisciplinary approach. Rapamycin, a fermentation-derived natural product is itself used clinically and provides the starting material for several rapamycin analog drugs, typically used in oncology. LC-MS proved a sensitive means to analyze impurity profiles in batches of rapamycin. MS fragmentation was used to gain structural insight into these impurities, usually fermentation by-products, structurally very similar to rapamycin. In cases where MS fragmentation was unable to provide unambiguous structural identification, the impurities were isolated and purified using orthogonal HPLC methods. Using the higher mass sensitivity of small-volume NMR microprobes, submilligram amounts of isolated impurities were sufficient for further characterization by multidimensional NMR spectroscopy. Full assignment of the (1)H and (13)C NMR signals revealed the structure of these impurities at an atomic level. This systematic workflow enabled the identification of several novel rapamycin congeners from active pharmaceutical ingredient without the need for large-scale isolation of impurities. For illustration, two novel rapamycin derivatives are described in this study: 12-ethyl-rapamycin and 33-ethyl-rapamycin, which exemplify previously unreported modifications on the carbon skeleton of the rapamycin macrocycle. The methodologies described here can be of wide use for identification of closely related structures found; for example as fermentation by-products, metabolites or degradants of natural product-based drugs.

  8. Sulforaphane protected the injury of human vascular endothelial cell induced by LPC through up-regulating endogenous antioxidants and phase II enzymes.

    PubMed

    Li, Baolong; Tian, Sicong; Liu, Xu; He, Canxia; Ding, Zhongqing; Shan, Yujuan

    2015-06-01

    Sulforaphane (SFN), which is an isothiocyanate (ITC) that is found in cruciferous vegetables, has received considerable attention because of its beneficial effects. In this study, the protection by SFN in the lysophosphatidylcholine (LPC)-induced injury of human vascular endothelial EA.hy.926 cells was investigated. ROS intensity was obtained by fluorescence microscopic imaging. Levels of MDA, GSH and the activity of SOD were determined spectrophotometrically. Expressions of GST, GSH-Px, TrxR and Nrf-2 proteins were measured by western blotting analysis. SFN largely decreased ROS production, similar to vitamin E. The MDA level was decreased by SFN to a level that was comparable to the negative group. Incubation with 0.5, 1.25, 2.5 μmol L(-1) SFN for 24 h restored the activity of SOD by 58%, 64%, and 123%, respectively. SOD activities were individually increased by 53%, 97%, 103% after treatment with 2.5 μmol L(-1) SFN for 12 h, 24 h, and 48 h, respectively. SFN restored and up-regulated the expressions of GST, GSH-Px and TrxR both in dose- and time-dependent ways. Although VE presents comparable induction of phase 2 enzymes as 1.25 μmol L(-1) SFN, it cannot induce the translocation of Nrf-2 to the nucleus. SFN protected the injury of vascular endothelial cell by LPC by enhancing anti-oxidative capabilities mediated by Nrf-2 translocation.

  9. Platelet-derived growth factor (PDGF) regulates Slingshot phosphatase activity via Nox1-dependent auto-dephosphorylation of serine 834 in vascular smooth muscle cells.

    PubMed

    Maheswaranathan, Mithunan; Gole, Hope K A; Fernandez, Isabel; Lassègue, Bernard; Griendling, Kathy K; San Martín, Alejandra

    2011-10-14

    Migration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown that PDGF-induced migration in VSMCs requires Slingshot-1L (SSH1L) phosphatase activity. In the present work, the mechanism of SSH1L activation by PDGF is further investigated. We identified a 14-3-3 consensus binding motif encompassing Ser-834 in SSH1L that is constitutively phosphorylated. PDGF induces SSH1L auto-dephosphorylation at Ser-834 in wild type (wt), but not in Nox1(-/y) cells. A SSH1L-S834A phospho-deficient mutant has significantly lower binding capacity for 14-3-3 when compared with the phospho-mimetic SSH1L-S834D mutant, and acts as a constitutively active phosphatase, lacking of PDGF-mediated regulation. Given that Nox1 produces reactive oxygen species, we evaluated their participation in this SSH1L activation mechanism. We found that H(2)O(2) activates SSH1L and this is accompanied by SSH1L/14-3-3 complex disruption and 14-3-3 oxidation in wt, but not in Nox1(-/y) cells. Together, these data demonstrate that PDGF activates SSH1L in VSMC by a mechanism that involves Nox1-mediated oxidation of 14-3-3 and Ser-834 SSH1L auto-dephosphorylation.

  10. Platelet-derived Growth Factor (PDGF) Regulates Slingshot Phosphatase Activity via Nox1-dependent Auto-dephosphorylation of Serine 834 in Vascular Smooth Muscle Cells*

    PubMed Central

    Maheswaranathan, Mithunan; Gole, Hope K. A.; Fernandez, Isabel; Lassègue, Bernard; Griendling, Kathy K.; San Martín, Alejandra

    2011-01-01

    Migration of vascular smooth muscle cells (VSMCs) contributes to vascular pathology. PDGF induces VSMC migration by a Nox1-based NADPH oxidase mediated mechanism. We have previously shown that PDGF-induced migration in VSMCs requires Slingshot-1L (SSH1L) phosphatase activity. In the present work, the mechanism of SSH1L activation by PDGF is further investigated. We identified a 14-3-3 consensus binding motif encompassing Ser-834 in SSH1L that is constitutively phosphorylated. PDGF induces SSH1L auto-dephosphorylation at Ser-834 in wild type (wt), but not in Nox1−/y cells. A SSH1L-S834A phospho-deficient mutant has significantly lower binding capacity for 14-3-3 when compared with the phospho-mimetic SSH1L-S834D mutant, and acts as a constitutively active phosphatase, lacking of PDGF-mediated regulation. Given that Nox1 produces reactive oxygen species, we evaluated their participation in this SSH1L activation mechanism. We found that H2O2 activates SSH1L and this is accompanied by SSH1L/14-3-3 complex disruption and 14-3-3 oxidation in wt, but not in Nox1−/y cells. Together, these data demonstrate that PDGF activates SSH1L in VSMC by a mechanism that involves Nox1-mediated oxidation of 14-3-3 and Ser-834 SSH1L auto-dephosphorylation. PMID:21857021

  11. Rapamycin causes regression of astrocytomas in tuberous sclerosis complex.

    PubMed

    Franz, David Neal; Leonard, Jennifer; Tudor, Cynthia; Chuck, Gail; Care, Marguerite; Sethuraman, Gopalan; Dinopoulos, Argirios; Thomas, George; Crone, Kerry R

    2006-03-01

    Tuberous sclerosis complex (TSC) is a genetic disorder characterized by the formation of hamartomas in multiple organs. Five to 15% of affected individuals display subependymal giant cell astrocytomas, which can lead to substantial neurological and postoperative morbidity due to the production of hydrocephalus, mass effect, and their typical location adjacent to the foramen of Monro. We sought to see whether therapy with oral rapamycin could affect growth or induce regression in astrocytomas associated with TSC. Five subjects with clinically definite TSC and either subependymal giant cell astrocytomas (n = 4) or a pilocytic astrocytoma (n = 1) were treated with oral rapamycin at standard immunosuppressive doses (serum levels 5-15 ng/ml) from 2.5 to 20 months. All lesions demonstrated growth on serial neuroimaging studies. Magnetic resonance imaging scans were performed before and at regular intervals following initiation of therapy. All lesions exhibited regression and, in one case, necrosis. Interruption of therapy resulted in regrowth of subependymal giant cell astrocytomas in one patient. Resumption of therapy resulted in further regression. Treatment was well tolerated. Oral rapamycin therapy can induce regression of astrocytomas associated with TSC and may offer an alternative to operative therapy of these lesions.

  12. Improvement of microbial strain and fermentation process of rapamycin biosynthesis.

    PubMed

    Baby Rani, Polavarapu; Battula, Suneel Kumar; Rao, A K S Bhujanga; Gunja, Madhavi; Narasu, Mangamoori Lakshmi

    2013-01-01

    The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N'-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.

  13. Myocardin inhibited the gap protein connexin 43 via promoted miR-206 to regulate vascular smooth muscle cell phenotypic switch.

    PubMed

    Li, Hui; Xiang, Yuan; Fan, Li-Juan; Zhang, Xiao-Yu; Li, Jia-Peng; Yu, Cheng-Xi; Bao, Le-Yuan; Cao, Dong-Sun; Xing, Wei-Bing; Liao, Xing-Hua; Zhang, Tong-Cun

    2017-06-15

    Myocardin is regarded as a key mediator for the change of smooth muscle phenotype. The gap junction protein connexin 43 (Cx43) has been shown to be involved in vascular smooth muscle cells (VSMCs) proliferation and the development of atherosclerosis. However, the role of myocardin on gap junction of cell communication and the relation between myocardin and Cx43 in VSMC phenotypic switch has not been investigated. The goal of the present study is to investigate the molecular mechanism by which myocardin affects Cx43-regulated VSMC proliferation. Data presented in this study demonstrated that inhibition of the Cx43 activation process impaired VSMC proliferation. On the other hand, overexpression miR-206 inhibited VSMC proliferation. In additon, miR-206 silences the expression of Cx43 via targeting Cx43 3' Untranslated Regions. Importantly, myocardin can significantly promote the expression of miR-206. Cx43 regulates VSMCs' proliferation and metastasis through miR-206, which could be promoted by myocardin and used as a marker for diagnosis and a target for therapeutic intervention. Thus myocardin affected the gap junction by inhibited Cx43 and myocardin-miR-206-Cx43 formed a loop to regulate VSMC phenotypic switch. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Functional microRNA library screening identifies the hypoxamir miR-24 as a potent regulator of smooth muscle cell proliferation and vascularization.

    PubMed

    Fiedler, Jan; Stöhr, Andrea; Gupta, Shashi Kumar; Hartmann, Dorothee; Holzmann, Angelika; Just, Annette; Hansen, Arne; Hilfiker-Kleiner, Denise; Eschenhagen, Thomas; Thum, Thomas

    2014-09-10

    Smooth muscle cells (SMCs) are key components within the vasculature. Dependent on the stimulus, SMC can either be in a proliferative (synthetic) or differentiated state. Alterations of SMC phenotype also appear in several disease settings, further contributing to disease progression. Here, we asked whether microRNAs (miRNAs, miRs), which are strong posttranscriptional regulators of gene expression, could alter SMC proliferation. Results and Innovation: Employing a robotic-assisted high-throughput screening method using miRNA libraries, we identified hypoxia-regulated miR-24 as a master regulator of SMC proliferation. Proteome profiling showed a strong miR-24-dependent impact on cellular stress-associated factors, overall resulting in reduced stress resistance. In vitro, synthetic miR-24 overexpression had detrimental effects on SMC functional capacity inducing apoptosis, migration defects, enhanced autophagy, and loss of contractile marker genes. Impaired SMC function was mediated in part by the herein identified direct target gene heme oxygenase 1. Ex vivo, miR-24 was shown to inhibit the development of vasculature in a model of engineered heart tissue. Collectively, we report the identification of the hypoxamir-24 as an inhibitor of SMC proliferation, contributing to loss of vascularization.

  15. EPHB4 Protein Expression in Vascular Smooth Muscle Cells Regulates Their Contractility, and EPHB4 Deletion Leads to Hypotension in Mice*

    PubMed Central

    Wang, Yujia; Thorin, Eric; Luo, Hongyu; Tremblay, Johanne; Lavoie, Julie L.; Wu, Zenghui; Peng, Junzheng; Qi, Shijie; Wu, Jiangping

    2015-01-01

    EPH kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. This work presents evidence that EPHB4 on vascular smooth muscle cells (VSMCs) is involved in blood pressure regulation. We generated gene KO mice with smooth muscle cell-specific deletion of EPHB4. Male KO mice, but not female KO mice, were hypotensive. VSMCs from male KO mice showed reduced contractility when compared with their WT counterparts. Signaling both from EFNBs to EPHB4 (forward signaling) and from EPHB4 to EFNB2 (reverse signaling) modulated VSMC contractility. At the molecular level, the absence of EPHB4 in VSMCs resulted in compromised signaling from Ca2+/calmodulin-dependent protein kinase II (CaMKII) to myosin light chain kinase (MLCK) to myosin light chain, the last of which controls the contraction force of motor molecule myosin. Near the cell membrane, an adaptor protein GRIP1, which can associate with EFNB2, was found to be essential in mediating EPHB4-to-EFNB reverse signaling, which regulated VSMC contractility, based on siRNA gene knockdown studies. Our research indicates that EPHB4 plays an essential role in regulating small artery contractility and blood pressure. PMID:25903126

  16. EPHB4 Protein Expression in Vascular Smooth Muscle Cells Regulates Their Contractility, and EPHB4 Deletion Leads to Hypotension in Mice.

    PubMed

    Wang, Yujia; Thorin, Eric; Luo, Hongyu; Tremblay, Johanne; Lavoie, Julie L; Wu, Zenghui; Peng, Junzheng; Qi, Shijie; Wu, Jiangping

    2015-05-29

    EPH kinases are the largest family of receptor tyrosine kinases, and their ligands, ephrins (EFNs), are also cell surface molecules. This work presents evidence that EPHB4 on vascular smooth muscle cells (VSMCs) is involved in blood pressure regulation. We generated gene KO mice with smooth muscle cell-specific deletion of EPHB4. Male KO mice, but not female KO mice, were hypotensive. VSMCs from male KO mice showed reduced contractility when compared with their WT counterparts. Signaling both from EFNBs to EPHB4 (forward signaling) and from EPHB4 to EFNB2 (reverse signaling) modulated VSMC contractility. At the molecular level, the absence of EPHB4 in VSMCs resulted in compromised signaling from Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to myosin light chain kinase (MLCK) to myosin light chain, the last of which controls the contraction force of motor molecule myosin. Near the cell membrane, an adaptor protein GRIP1, which can associate with EFNB2, was found to be essential in mediating EPHB4-to-EFNB reverse signaling, which regulated VSMC contractility, based on siRNA gene knockdown studies. Our research indicates that EPHB4 plays an essential role in regulating small artery contractility and blood pressure. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. CD147 induces up-regulation of vascular endothelial growth factor in U937-derived foam cells through PI3K/AKT pathway.

    PubMed

    Zong, JiaXin; Li, YunTian; Du, DaYong; Liu, Yang; Yin, YongJun

    2016-11-01

    Intraplaque angiogenesis has been recognized as an important risk factor for the rupture of advanced atherosclerotic plaques in recent years. CD147, also called Extracellular Matrix Metalloproteinase Inducer, has been found the ability to promote angiogenesis in many pathological conditions such as cancer diseases and rheumatoid arthritis via the up-regulation of vascular endothelial growth factor (VEGF), a critical mediator of angiogenesis. We investigated whether CD147 would also induce the up-regulation of VEGF in the foam cells formation process and explored the probable signaling pathway. The results showed the expression of CD147 and VEGF was significantly higher in U937-derived foam cells. After CD147 stealth siRNA transfection treatment, the production of VEGF was reduced depended on the inhibition efficiency of CD147 siRNAs.The special signaling pathway inhibitors LY294002, SP600125, SB203580 and U0126 were added to cultures respectively and the results showed LY294002 dose-dependently inhibited the expression of VEGF. The reduction of phospho-Akt was observed in both LY294002 and siRNA groups, suggested that the phosphatidylinositol 3-kinase/Akt pathway may be the probable signaling pathway underlying CD147 induced up-regulation of VEGF in U937-derived foam cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Down-regulation of vascular endothelial growth factor expression by anti-Her2/neu single chain antibodies.

    PubMed

    Nejatollahi, Foroogh; Asgharpour, Mahdi; Jaberipour, Mansooreh

    2012-03-01

    HER-2/neu is overexpressed in 25-30% of breast tumors. Signaling through HER-2/neu leads to an increase in the production of vascular endothelial growth factor (VEGF) and enhances angiogenesis. We evaluated the effects of three specific anti-HER2/neu single chain-Fv (scFv) antibodies on the expression level of VEGF in HER2/neu-expressing breast cancer cell lines. A nonimmunized human scFv library was panned against three epitopes of HER2/neu. BT-474 human breast cancer cell line was treated with three specific anti-HER2/neu scFv antibodies and the amount of VEGF gene transcript was determined by quantitative real-time PCR. The expression of VEGF protein was analyzed by western blot. All three scFv antibodies along with their combination inhibited VEGF expression at both the gene and protein levels. Our results show that anti-HER2/neu recombinant antibodies can be considered as anti-angiogenic agents in HER2/neu-positive breast cancers.

  19. Activated CD47 regulates multiple vascular and stress responses: implications for acute kidney injury and its management

    PubMed Central

    Rogers, Natasha M.; Yao, Mingyi; Novelli, Enrico M.; Thomson, Angus W.; Roberts, David D.

    2012-01-01

    Ischemia-reperfusion injury (IRI) remains a significant source of early and delayed renal transplant failure. Therapeutic interventions have yet to resolve this ongoing clinical challenge although the reasons for this remain unclear. The cell surface receptor CD47 is widely expressed on vascular cells and in tissues. It has one known soluble ligand, the stress-released matricellular protein thrombospondin-1 (TSP1). The TSP1-CD47 ligand receptor axis controls a number of important cellular processes, inhibiting survival factors such as nitric oxide, cGMP, cAMP, and VEGF, while activating injurious pathways such as production of reactive oxygen species. A role of CD47 in renal IRI was recently revealed by the finding that the TSP1-CD47 axis is induced in renal tubular epithelial cells (RTEC) under hypoxia and following IRI. The absence of CD47 in knockout mice increases survival, mitigates RTEC damage, and prevents subsequent kidney failure. Conversely, therapeutic blockade of TSP1-CD47 signaling provides these same advantages to wild-type animals. Together, these findings suggest an important role for CD47 in renal IRI as a proximate promoter of injury and as a novel therapeutic target. PMID:22874763

  20. Macrophages regulate salt-dependent volume and blood pressure by a vascular endothelial growth factor-C-dependent buffering mechanism.

    PubMed

    Machnik, Agnes; Neuhofer, Wolfgang; Jantsch, Jonathan; Dahlmann, Anke; Tammela, Tuomas; Machura, Katharina; Park, Joon-Keun; Beck, Franz-Xaver; Müller, Dominik N; Derer, Wolfgang; Goss, Jennifer; Ziomber, Agata; Dietsch, Peter; Wagner, Hubertus; van Rooijen, Nico; Kurtz, Armin; Hilgers, Karl F; Alitalo, Kari; Eckardt, Kai-Uwe; Luft, Friedrich C; Kerjaschki, Dontscho; Titze, Jens

    2009-05-01

    In salt-sensitive hypertension, the accumulation of Na(+) in tissue has been presumed to be accompanied by a commensurate retention of water to maintain the isotonicity of body fluids. We show here that a high-salt diet (HSD) in rats leads to interstitial hypertonic Na(+) accumulation in skin, resulting in increased density and hyperplasia of the lymphcapillary network. The mechanisms underlying these effects on lymphatics involve activation of tonicity-responsive enhancer binding protein (TonEBP) in mononuclear phagocyte system (MPS) cells infiltrating the interstitium of the skin. TonEBP binds the promoter of the gene encoding vascular endothelial growth factor-C (VEGF-C, encoded by Vegfc) and causes VEGF-C secretion by macrophages. MPS cell depletion or VEGF-C trapping by soluble VEGF receptor-3 blocks VEGF-C signaling, augments interstitial hypertonic volume retention, decreases endothelial nitric oxide synthase expression and elevates blood pressure in response to HSD. Our data show that TonEBP-VEGF-C signaling in MPS cells is a major determinant of extracellular volume and blood pressure homeostasis and identify VEGFC as an osmosensitive, hypertonicity-driven gene intimately involved in salt-induced hypertension.

  1. Cytosolic Ca2+ concentration and rate of increase of the cytosolic Ca2+ concentration in the regulation of vascular permeability in Rana in vivo.

    PubMed

    Glass, C A; Pocock, T M; Curry, F E; Bates, D O

    2005-05-01

    Vascular permeability is assumed to be regulated by the cytosolic Ca(2+) concentration ([Ca(2+)](c)) of the endothelial cells. When permeability is increased, however, the maximum [Ca(2+)](c) appears to occur after the maximum permeability increase, suggesting that Ca(2+)-dependent mechanisms other than the absolute Ca(2+) concentration may regulate permeability. Here we investigate whether the rate of increase of the [Ca(2+)](c) (d[Ca(2+)](c)/dt) may more closely approximate the time course of the permeability increase. Hydraulic conductivity (L(p)) and endothelial [Ca(2+)](c) were measured in single perfused frog mesenteric microvessels in vivo. The relationships between the time courses of the increased L(p), [Ca(2+)](c) and d[Ca(2+)](c)/dt were examined. L(p) peaked significantly earlier than [Ca(2+)](c) in all drug treatments examined (Ca(2+) store release, store-mediated Ca(2+) influx, and store-independent Ca(2+) influx). When L(p) was increased in a store-dependent manner the time taken for L(p) to peak (3.6 +/- 0.9 min during store release, 1.2 +/- 0.3 min during store-mediated Ca(2+) influx) was significantly less than the time taken for [Ca(2+)](c) to peak (9.2 +/- 2.8 min during store release, 2.1 +/- 0.7 min during store-mediated influx), but very similar to that for the peak d[Ca(2+)](c)/dt to occur (4.3 +/- 2.0 min during store release, 1.1 +/- 0.5 min during Ca(2+) influx). Additionally, when the increase was independent of intracellular Ca(2+) stores, L(p) (0.38 +/- 0.03 min) and d[Ca(2+)](c)/dt (0.30 +/- 0.1 min) both peaked significantly before the [Ca(2+)](c) (1.05 +/- 0.31 min). These data suggest that the regulation of vascular permeability by endothelial cell Ca(2+) may be regulated by the rate of change of the [Ca(2+)](c) rather than the global [Ca(2+)].

  2. Dicer1 activity in the stromal compartment regulates nephron differentiation and vascular patterning during mammalian kidney organogenesis

    PubMed Central

    Nakagawa, Naoki; Xin, Cuiyan; Roach, Allie M.; Naiman, Natalie; Shankland, Stuart J.; Ligresti, Giovanni; Ren, Shuyu; Szak, Suzanne; Gomez, Ivan G.; Duffield, Jeremy S.

    2015-01-01

    MicroRNAs, activated by the enzyme Dicer1, control post-transcriptional gene expression. Dicer1 has important roles in the epithelium during nephrogenesis, but its function in stromal cells during kidney development is unknown. To study this we inactivated Dicer1 in renal stromal cells. This resulted in hypoplastic kidneys, abnormal differentiation of the nephron tubule and vasculature, and perinatal mortality. In mutant kidneys, genes involved in stromal cell migration and activation were suppressed as were those involved in epithelial and endothelial differentiation and maturation. Consistently, polarity of the proximal tubule was incorrect, distal tubule differentiation was diminished, and elongation of Henle’s loop attenuated resulting in lack of inner medulla and papilla in stroma-specific Dicer1 mutants. Glomerular maturation and capillary loop formation were abnormal while peritubular capillaries, with enhanced branching and increased diameter, formed later. In Dicer1-null renal stromal cells, expression of factors associated with migration, proliferation and morphogenic functions including α-smooth muscle actin, integrin-α8, -β1, and the WNT pathway transcriptional regulator LEF1 were reduced. Dicer1 mutation in stroma led to loss of expression of distinct microRNAs. Of these, miR-214, -199a-5p and -199a-3p regulate stromal cell functions ex vivo, including WNT pathway activation, migration and proliferation. Thus, Dicer1 activity in the renal stromal compartment regulates critical stromal cell functions that, in turn, regulate differentiation of the nephron and vasculature during nephrogenesis. PMID:25651362

  3. Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway.

    PubMed

    Altamirano, Francisco; Oyarce, César; Silva, Patricio; Toyos, Marcela; Wilson, Carlos; Lavandero, Sergio; Uhlén, Per; Estrada, Manuel

    2009-08-01

    Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of beta-myosin heavy chain, alpha-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP(3)/Ca(2+) and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.

  4. [Rapamycin improves learning and memory ability in ICR mice with pilocarpine-induced temporal lobe epilepsy].

    PubMed

    Zhang, Huadan; Xie, Yacong; Weng, Ling; Zhang, Yuchen; Shi, Qiongyao; Chen, Tao; Zeng, Linghui

    2013-11-01

    To investigate the effect of rapamycin, an mTOR inhibitor, on learning and memory ability of mice with pilocarpine (PILO)-induced seizure. One hundred and sixty male adult ICR mice were randomly grouped as vehicle control (n=20), rapamycin control (n=20), PILO model (n=40), rapamycin pre-treatment (n=40) and rapamycin post-treatment (n=40). PILO model and rapamycin treatment groups were injected with PILO to induce temporal lobe seizure. Rapamycin was administrated for 3 days before or after seizure. Morris water maze, Y maze and open field were used for the assessment of learning and memory, and FJB and Timm staining were conducted to detect the neuronal cell death and mossy fiber sprouting, respectively. No significant cell death was observed in the mice with PILO-induced seizure. The learning and memory were impaired in mice 7 to 10 days after PILO-induced seizure, which was evident by prolongation of avoiding latency (P<0.05), decrease in number of correct reaction (P<0.01) and number of crossing (P<0.05). Treatment with rapamycin both pre-and post- PILO injection reversed seizure-induced cognitive impairment. In addition, rapamycin inhibited the mossy fiber sprouting after seizure (P<0.001). Rapamycin improves learning and memory ability in ICR mice after PILO-induced seizure, and its mechanism needs to be further studied.

  5. Curcumin inhibits cellular cholesterol accumulation by regulating SREBP-1/caveolin-1 signaling pathway in vascular smooth muscle cells.

    PubMed

    Yuan, Hao-Yu; Kuang, Shuang-Yu; Zheng, Xing; Ling, Hong-Yan; Yang, Yun-Bo; Yan, Peng-Ke; Li, Kai; Liao, Duan-Fang

    2008-05-01

    To investigate the protective effect and the possible mechanism of curcumin on anti-atherosclerosis. Morphological changes of atherosclerotic lesions taken from apoE knockout (apoE-/-) mice were determined by hematoxylin- eosin staining. Intracellular lipid droplets and lipid levels were assayed by oil red O staining and HPLC. The protein expression of caveolin-1 was quantified by Western blotting. Translocation and the expression of sterol response element-binding protein-1 (SREBP-1) were indirectly detected by an immunofluorescence analysis. The administration of 20 mg. kg(-1 ). d(-1 )curcumin to apoE-/- mice for 4 months induced a 50% reduction of atherosclerotic lesions and yielded a 5- fold increase in the caveolin-1 expression level as compared to the model group. Rat vascular smooth muscle cells (VSMC) pretreated with 50 mg/L ox-lipid density lipoprotein(ox-LDL) for 48 h increased cellular lipid contents, and stimulated SREBP-1 translocation, but decreased the caveolin-1 expression level. Lipid-loaded cells exposed to curcumin at various concentrations (12.5, 25, and 50 micromol/L) for different durations (0, 6, 12, 24, and 48 h) significantly diminished the number and area of cellular lipid droplets, total cholesterol, cholesterol ester, and free cholesterol accompanying the elevation of the caveolin-1 expression level (approximately 3-fold); the translocation of SREBP-1 from the cytoplasm to the nucleus was inhibited compared with the models. Lipid-loaded VSMC exposed to N-acetyl- Leu-Leu-norleucinal, a SREBP-1 protease inhibitor, showed increased nuclear translocation of SREBP-1, reduced caveolin-1 expression level, and upregulated cellular lipid levels. Curcumin inhibits ox-LDL-induced cholesterol accumulation in cultured VSMC through increasing the caveolin-1 expression via the inhibition of nuclear translocation of SREBP-1.

  6. Shh mediates PDGF-induced contractile-to-synthetic phenotypic modulation in vascular smooth muscle cells through regulation of KLF4

    SciTech Connect

    Zeng, Qiu; Wei, Bin; Zhao, Yu

    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 andmore » 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. - Highlights: • Shh as a downstream effector of PDGF participates in PDGF-induced VSMC phenotypic modulation. • Shh can promote VSMC phenotypic switching from contractile to synthetic state. • Shh mediates VSMC phenotypic modulation through regulation of KLF4.« less

  7. Shh mediates PDGF-induced contractile-to-synthetic phenotypic modulation in vascular smooth muscle cells through regulation of KLF4

    SciTech Connect

    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. - Highlights: • Shh as a downstream effector of PDGF participates in PDGF-induced VSMC phenotypic modulation. • Shh can promote VSMC phenotypic switching from contractile to synthetic state. • Shh mediates VSMC phenotypic modulation through regulation of KLF4.

  8. The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

    PubMed

    Celec, Peter; Gardlík, Roman; Pálffy, Roland; Hodosy, Július; Stuchlík, Stanislav; Drahovská, Hana; Stuchlíková, Martina; Minárik, Gabriel; Lukács, Ján; Jurkovicová, Ingrid; Hulín, Ivan; Turna, Ján; Jakubovský, Ján; Kopáni, Martin; Danisovic, Lubos; Jandzík, Dávid; Kúdela, Matús; Yonemitsu, Yoshikazu

    2005-01-01

    Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous

  9. Vascular Cures

    MedlinePlus

    ... on October 20, 2017 $100 Million for Cardiovascular Artificial Intelligence Posted on October 20, 2017 Sean English Named 2017 Wylie Scholar Posted on June 27, 2017 THERE ARE MANY WAYS TO GIVE TO VASCULAR CURES Support Our Work Vascular Cures is the only national non-profit ...

  10. Triglyceride-Rich Lipoprotein Modulates Endothelial Vascular Cell Adhesion Molecule (VCAM)-1 Expression via Differential Regulation of Endoplasmic Reticulum Stress

    PubMed Central

    Wang, Ying I.; Bettaieb, Ahmed; Sun, Chongxiu; DeVerse, J. Sherrod; Radecke, Christopher E.; Mathew, Steven; Edwards, Christina M.; Haj, Fawaz G.; Passerini, Anthony G.; Simon, Scott I.

    2013-01-01

    Circulating triglyceride-rich lipoproteins (TGRL) from hypertriglyceridemic subjects exacerbate endothelial inflammation and promote monocyte infiltration into the arterial wall. We have recently reported that TGRL isolated from human blood after a high-fat meal can elicit a pro- or anti-atherogenic state in human aortic endothelial cells (HAEC), defined as up- or down-regulation of VCAM-1 expression in response to tumor necrosis factor alpha (TNFα) stimulation, respectively. A direct correlation was found between subjects categorized at higher risk for cardiovascular disease based upon serum triglycerides and postprandial production of TGRL particles that increased VCAM-1-dependent monocyte adhesion to inflamed endothelium. To establish how TGRL metabolism is linked to VCAM-1 regulation, we examined endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) pathways. Regardless of its atherogenicity, the rate and extent of TGRL internalization and lipid droplet formation by HAEC were uniform. However, pro-atherogenic TGRL exacerbated ER membrane expansion and stress following TNFα stimulation, whereas anti-atherogenic TGRL ameliorated such effects. Inhibition of ER stress with a chemical chaperone 4-phenylbutyric acid decreased TNFα-induced VCAM-1 expression and abrogated TGRL’s atherogenic effect. Activation of ER stress sensors PKR-like ER-regulated kinase (PERK) and inositol requiring protein 1α (IRE1α), and downstream effectors including eukaryotic initiation factor-2α (eIF2α), spliced X-box-binding protein 1 (sXBP1) and C/EBP homologous protein (CHOP), directly correlated with the atherogenic activity of an individual’s TGRL. Modulation of ER stress sensors also correlated with changes in expression of interferon regulatory factor 1 (IRF-1), a transcription factor of Vcam-1 responsible for regulation of its expression. Moreover, knockdown studies using siRNA defined a causal relationship between the PERK/eIF2α/CHOP pathway and IRF-1

  11. Nuclear Localization of Vascular Endothelial Growth Factor-D and Regulation of c-Myc–Dependent Transcripts in Human Lung Fibroblasts

    PubMed Central

    Pacheco-Rodriguez, Gustavo; Malide, Daniela; Meza-Carmen, Victor; Kato, Jiro; Cui, Ye; Padilla, Philip I.; Samidurai, Arun; Gochuico, Bernadette R.

    2014-01-01

    Lymphangiogenesis and angiogenesis are processes that are, in part, regulated by vascular endothelial growth factor (VEGF)-D. The formation of lymphatic structures has been implicated in multiple lung diseases, including pulmonary fibrosis. VEGF-D is a secreted protein produced by fibroblasts and macrophages, which induces lymphangiogenesis by signaling via VEGF receptor-3, and angiogenesis through VEGF receptor-2. VEGF-D contains a central VEGF homology domain, which is the biologically active domain, with flanking N- and C-terminal propeptides. Full-length VEGF-D (∼ 50 kD) is proteolytically processed in the extracellular space, to generate VEGF homology domain that contains the VEGF-D receptor–binding sites. Here, we report that, independent of its cell surface receptors, full-length VEGF-D accumulated in nuclei of fibroblasts, and that this process appears to increase with cell density. In nuclei, full-length VEGF-D associated with RNA polymerase II and c-Myc. In cells depleted of VEGF-D, the transcriptionally regulated genes appear to be modulated by c-Myc. These findings have potential clinical implications, as VEGF-D was found in fibroblast nuclei in idiopathic pulmonary fibrosis, a disease characterized by fibroblast proliferation. These findings are consistent with actions of full-length VEGF-D in cellular homeostasis in health and disease, independent of its receptors. PMID:24450584

  12. A systematic expression analysis implicates Plexin-B2 and its ligand Sema4C in the regulation of the vascular and endocrine system.

    PubMed

    Zielonka, Matthias; Xia, Jingjing; Friedel, Roland H; Offermanns, Stefan; Worzfeld, Thomas

    2010-09-10

    Plexins serve as receptors for semaphorins and play important roles in the developing nervous system. Plexin-B2 controls decisive developmental programs in the neural tube and cerebellum. However, whether Plexin-B2 also regulates biological functions in adult nonneuronal tissues is unknown. Here we show by two methodologically independent approaches that Plexin-B2 is expressed in discrete cell types of several nonneuronal tissues in the adult mouse. In the vasculature, Plexin-B2 is selectively expressed in functionally specialized endothelial cells. In endocrine organs, Plexin-B2 localizes to the pancreatic islets of Langerhans and to both cortex and medulla of the adrenal gland. Plexin-B2 expression is also detected in certain types of immune and epithelial cells. In addition, we report on a systematic comparison of the expression patterns of Plexin-B2 and its ligand Sema4C, which show complementarity or overlap in some but not all tissues. Furthermore, we demonstrate that Plexin-B2 and its family member Plexin-B1 display largely nonredundant expression patterns. This work establishes Plexin-B2 and Sema4C as potential regulators of the vascular and endocrine system and provides an anatomical basis to understand the biological functions of this ligand-receptor pair. Copyright 2010 Elsevier Inc. All rights reserved.

  13. Ginsenoside Rg3 up-regulates the expression of vascular endothelial growth factor in human dermal papilla cells and mouse hair follicles.

    PubMed

    Shin, Dae Hyun; Cha, Youn Jeong; Yang, Kyeong Eun; Jang, Ik-Soon; Son, Chang-Gue; Kim, Bo Hyeon; Kim, Jung Min

    2014-07-01

    Crude Panax ginseng has been documented to possess hair growth activity and is widely used to treat alopecia, but the effects of ginsenoside Rg3 on hair growth have not to our knowledge been determined. The aim of the current study was to identify the molecules through which Rg3 stimulates hair growth. The thymidine incorporation for measuring cell proliferation was determined. We used DNA microarray analysis to measure gene expression levels in dermal papilla (DP) cells upon treatment with Rg3. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF) in human DP cells were measured by real-time polymerase chain reaction and immunohistochemistry, respectively. We also used immunohistochemistry assays to detect in vivo changes in VEGF and 3-stemness marker expressions in mouse hair follicles. Reverse transcription polymerase chain reaction showed dose-dependent increases in VEGF mRNA levels on treatment with Rg3. Immunohistochemical analysis showed that expression of VEGF was significantly up-regulated by Rg3 in a dose-dependent manner in human DP cells and in mouse hair follicles. In addition, the CD8 and CD34 were also up-regulated by Rg3 in the mouse hair follicles. It may be concluded that Rg3 might increase hair growth through stimulation of hair follicle stem cells and it has the potential to be used in hair growth products. Copyright © 2013 John Wiley & Sons, Ltd.

  14. Acetylsalicylic acid regulates overexpressed small GTPase RhoA in vascular smooth muscle cells through prevention of new synthesis and enhancement of protein degradation.

    PubMed

    Li, Dong-Bo; Fu, Zhi-Xuan; Ruan, Shu-Qin; Hu, Shen-Jiang; Li, Xia

    2012-04-01

    RhoA has been shown to play a major role in vascular processes and acetylsalicylic acid (aspirin) is known to exert a cytoprotective effect via multiple mechanisms. In the present study, we aimed at investigating the effect of aspirin on RhoA expression under a stress state in rat VSMCs (vascular smooth muscle cells) and the underlying mechanisms. The expression of iNOS (inducible nitric oxide synthase) and iNOS activity as well as NO concentration was significantly promoted by LPS (lipopolysaccharide) accompanying the elevation of RhoA expression, which was blocked by the addition of the iNOS inhibitor L-NIL [L-N6-(1-iminoethyl)lysine dihydrochloride]. Aspirin (30 μM) significantly attenuated the elevation of RhoA, while indomethacin and salicylate had no similar effect. The sGC (soluble guanylate cyclase) inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) showed the same effect as aspirin in down-regulating RhoA but was reversed by the addition of the cGMP analogue 8-Br-PET-cGMP (β-phenyl-1,N2-ethano-8-bromoguanosine 3',5'-cyclic monophosphorothioate). 8-Br-PET-cGMP solely enhanced the RhoA expression that was abrogated by preincubation with aspirin. Degradation analysis indicated that aspirin enhanced the protein degradation rate of RhoA and GDP-bound RhoA seemed to be more susceptible to aspirin-enhanced degradation compared with the GTP-bound form. Our results indicate that aspirin attenuates the LPS-induced overexpression of RhoA both by inhibiting new synthesis and accelerating protein degradation, which may help elucidate the multiple beneficial effects of aspirin.

  15. Apocynin attenuates angiotensin II-induced vascular smooth muscle cells osteogenic switching via suppressing extracellular signal-regulated kinase 1/2.

    PubMed

    Feng, Weijing; Zhang, Kun; Liu, Yu; Chen, Jie; Cai, Qingqing; Zhang, Yinyin; Wang, Mongheng; Wang, Jingfeng; Huang, Hui

    2016-12-13

    Vascular calcification (VC) is a significant risk factor for cardiovascular morbidity and mortality. We recently reported that apocynin had benefits for preventing cardiovascular diseases. However, whether apocynin could attenuate VC is unknown. Here, we investigated the role of apocynin in VC and its underlying mechanisms. 163 participants with high or normal ankle-brachial index (ABI) were enrolled in this study for analyzing the demographic and biochemical data. In vitro, vascular smooth muscle cells (VSMCs) were exposed to calcification medium containing b-glycerophosphate and angiotensin II (Ang II) for 24 hours. The results showed that serum level of Ang II was significantly increased in patients with high ABI (P<0.05). In cultured VSMCs, Ang II significantly exacerbated osteogenic switching. The expression of osteogenic phenotype markers, including bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2) and osteopontin (OPN), were significantly upregulated, whereas contractile markers expression, including alpha smooth muscle actin (a-SMA) and smooth muscle 22 alpha (SM22a) were simultaneously downregulated. However, these effects were greatly attenuated by apocynin. Apocynin enhanced expression of a-SMA by 5.3%, and reduced expression of BMP2, Runx2, OPN by 3.37%, 0.61% and 3.07%, respectively. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was upregulated by Ang II, and this effect was also reversed by apocynin. Intriguingly, pretreatment with U0126, an inhibitor of ERK1/2, had similar effects with apocynin. Apocynin may act as a novel molecular candidate to protect against VSMCs osteogenic switching through suppressing ERK1/2 pathway.

  16. Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

    PubMed

    Denancé, Nicolas; Ranocha, Philippe; Oria, Nicolas; Barlet, Xavier; Rivière, Marie-Pierre; Yadeta, Koste A; Hoffmann, Laurent; Perreau, François; Clément, Gilles; Maia-Grondard, Alessandra; van den Berg, Grardy C M; Savelli, Bruno; Fournier, Sylvie; Aubert, Yann; Pelletier, Sandra; Thomma, Bart P H J; Molina, Antonio; Jouanin, Lise; Marco, Yves; Goffner, Deborah

    2013-01-01

    Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  17. Role of Nitric Oxide Synthase on Blood Pressure Regulation and Vascular Function in Pregnant Rats on a High-Fat Diet.

    PubMed

    Palei, Ana C; Spradley, Frank T; Granger, Joey P

    2017-03-01

    While obesity is a leading risk factor for preeclampsia, the mechanisms whereby obese women are more susceptible to pregnancy-induced hypertension are unclear. As high-fat diet (HFD) is an important contributor to the development of obesity, we tested the hypothesis that pregnant rats on HFD have hypertension and endothelial dysfunction due to reduced nitric oxide synthase (NOS). Twelve-week-old Sprague-Dawley female rats were fed normal diet (ND, 13% fat kcal) or HFD (40% fat kcal) for 9 weeks. Timed-pregnant rats were then generated and the effect of HFD on mean arterial blood pressure (MAP) and vascular function was assessed on gestational day (GD) 19. MAP was not different between HFD and ND pregnant rats. Intriguingly, sensitivity to acetylcholine-induced endothelium-dependent vasorelaxation was enhanced in small mesenteric arteries of HFD dams compared to ND controls (logEC50 -7.9 ± 0.3 vs. -6.7 ± 0.3 M; P < 0.05). Additionally, HFD dams exhibited higher mesenteric artery expression of NOS3 and plasma levels of NO metabolites than ND controls (1738.0 ± 316.4 vs. 1094.0 ± 82.5 pg/mg and 72.5 ± 8.7 vs. 39.7 ± 4.5 µM, respectively; both P < 0.05). Further, to determine the role of NOS in modulating blood pressure in HFD pregnant rats, animals were treated with the nonselective inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride (100 mg/l, drinking water) from GD 14 to 19. It was found that NOS inhibition increased MAP equally in HFD and ND groups. Contrary to our initial hypothesis, HFD dams were normotensive and presented increased endothelial function and NO/NOS3 levels. This enhanced NOS-mediated vascular function does not appear to have a major impact on blood pressure regulation of HFD-fed pregnant rats.

  18. Reperfusion Therapy with Rapamycin Attenuates Myocardial Infarction through Activation of AKT and ERK

    PubMed Central

    Filippone, Scott M.; Samidurai, Arun; Roh, Sean K.; Cain, Chad K.; He, Jun; Salloum, Fadi N.; Kukreja, Rakesh C.

    2017-01-01

    Prompt coronary reperfusion is the gold standard for minimizing injury following acute myocardial infarction. Rapamycin, mammalian target of Rapamycin (mTOR) inhibitor, exerts preconditioning-like cardioprotective effects against ischemia/reperfusion (I/R) injury. We hypothesized that Rapamycin, given at the onset of reperfusion, reduces myocardial infarct size through modulation of mTOR complexes. Adult C57 male mice were subjected to 30 min of myocardial ischemia followed by reperfusion for 1 hour/24 hours. Rapamycin (0.25 mg/kg) or DMSO (7.5%) was injected intracardially at the onset of reperfusion. Post-I/R survival (87%) and cardiac function (fractional shortening, FS: 28.63 ± 3.01%) were improved in Rapamycin-treated mice compared to DMSO (survival: 63%, FS: 17.4 ± 2.6%). Rapamycin caused significant reduction in myocardial infarct size (IS: 26.2 ± 2.2%) and apoptosis (2.87 ± 0.64%) as compared to DMSO-treated mice (IS: 47.0 ± 2.3%; apoptosis: 7.39 ± 0.81%). Rapamycin induced phosphorylation of AKT S473 (target of mTORC2) but abolished ribosomal protein S6 phosphorylation (target of mTORC1) after I/R. Rapamycin induced phosphorylation of ERK1/2 but inhibited p38 phosphorylation. Infarct-limiting effect of Rapamycin was abolished with ERK inhibitor, PD98059. Rapamycin also attenuated Bax and increased Bcl-2/Bax ratio. These results suggest that reperfusion therapy with Rapamycin protects the heart against I/R injury by selective activation of mTORC2 and ERK with concurrent inhibition of mTORC1 and p38. PMID:28373901

  19. Time-dependent effects of rapamycin on consolidation of predator stress-induced hyperarousal.

    PubMed

    Fifield, Kathleen; Hebert, Mark; Williams, Kimberly; Linehan, Victoria; Whiteman, Jesse D; Mac Callum, Phillip; Blundell, Jacqueline

    2015-06-01

    Previous studies have indicated that rapamycin, a potent inhibitor of the mammalian target of rapamycin (mTOR) pathway, blocks consolidation of shock-induced associative fear memories. Moreover, rapamycin's block of associative fear memories is time-dependent. It is unknown, however, if rapamycin blocks consolidation of predator stress-induced non-associative fear memories. Furthermore, the temporal pattern of mTOR activation following predator stress is unknown. Thus, the goal of the current studies was to determine if rapamycin blocks consolidation of predator stress-induced fear memories and if so, whether rapamycin's effect is time-dependent. Male rats were injected systemically with rapamycin at various time points following predator stress. Predator stress involves an acute, unprotected exposure of a rat to a cat, which causes long-lasting non-associative fear memories manifested as generalized hyperarousal and increased anxiety-like behaviour. We show that rapamycin injected immediately after predator stress blocked consolidation of stress-induced startle. However, rapamycin injected 9, 24 or 48h post predator stress potentiated stress-induced startle. Consistent with shock-induced associative fear memories, we show that mTOR signalling is essential for consolidation of predator stress-induced hyperarousal. However, unlike shock-induced fear memories, a second, persistent, late phase mTOR-dependent process following predator stress actually dampens startle. Consistent with previous findings, our data support the potential role for rapamycin in treatment of stress related disorders such as posttraumatic stress disorder. However, our data suggest timing of rapamycin administration is critical. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Mammalian target of rapamycin inhibitors, temsirolimus and torin 1, attenuate stemness-associated properties and expression of mesenchymal markers promoted by phorbol-myristate-acetate and oncostatin-M in glioblastoma cells.

    PubMed

    Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

    2017-03-01

    The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors-rapamycin, temsirolimus, torin 1, and PP242-suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as "stemness-inhibiting drugs" and a promising therapeutic approach to target glioma stem cells.

  1. LincRNA-p21 Regulates Neointima Formation, Vascular Smooth Muscle Cell Proliferation, Apoptosis and Atherosclerosis by Enhancing p53 Activity

    PubMed Central

    Han, Yu; Chen, Jinghai; Huang, Zhan-Peng; Chen, Caiyu; Cai, Yue; Huang, Hefei; Yang, Yujia; Liu, Yukai; Xu, Zaicheng; He, Duofen; Zhang, Xiaoqun; Hu, Xiaoyun; Pinello, Luca; Zhong, Dan; He, Fengtian; Yuan, Guo-Cheng; Wang, Da-Zhi; Zeng, Chunyu

    2014-01-01

    Background Long non-coding RNAs (lncRNAs) recently have been implicated in many biological processes and diseases. Atherosclerosis is a major risk factor for cardiovascular disease. However, the functional role of lncRNAs in atherosclerosis is largely unknown. Methods and Results We identified lincRNA-p21 as a key regulator of cell proliferation and apoptosis during atherosclerosis. The expression of lincRNA-p21 was dramatically down-regulated in atherosclerotic plaques of ApoE−/− mice, an animal model for atherosclerosis. Through loss- and gain-of function approaches, we showed that lincRNA-p21 represses cell proliferation and induces apoptosis in vascular smooth muscle cells (VSMCs) and mouse mononuclear macrophage cells in vitro. Moreover, we found that inhibition of lincRNA-p21 results in neointimal hyperplasia in vivo in a carotid artery injury model. Genome-wide analysis revealed that lincRNA-p21 inhibition dysregulated many p53 targets. Furthermore, lincRNA-p21, a transcriptional target of p53, feeds back to enhance p53 transcriptional activity, at least in part, via binding to mouse double minute 2 (MDM2), an E3 ubiquitin-protein ligase. The association of lincRNA-p21 and MDM2 releases MDM2 repression of p53, enabling p53 to interact with p300 and bind to the promoters/enhancers of its target genes. Finally, we show that lincRNA-p21 expression is decreased in coronary artery disease patients. Conclusions Our studies identify lincRNA-p21 as a novel regulator of cell proliferation and apoptosis and suggest that this lncRNA could serve as a therapeutic target to treat atherosclerosis and related cardiovascular disorders. PMID:25156994

  2. 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

    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 ofmore » 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.« less

  3. 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.

  4. Mammalian target of rapamycin complex 1 signalling is essential for germinal centre reaction.

    PubMed

    Li, Bingshou; Li, Zhirong; Wang, Pengcheng; Huang, Qizhao; Xu, Lifan; He, Ran; Ye, Lilin; Bai, Qiang

    2017-10-01

    The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that has been shown to be essential for the differentiation and function of various immune cells. Earlier in vitro studies showed that mTOR signalling regulates B-cell biology by supporting their activation and proliferation. However, how mTOR signalling temporally regulates in vivo germinal centre B (GCB) cell development and differentiation into short-lived plasma cells, long-lived plasma cells and memory cells is still not well understood. In this study, we used a combined conditional/inducible knock-out system to investigate the temporal regulation of mTOR complex 1 (mTORC1) in the GCB cell response to acute lymphocytic choriomeningitis virus infection by deleting Raptor, a main component of mTORC1, specifically in B cells in pre- and late GC phase. Early Raptor deficiency strongly inhibited GCB cell proliferation and differentiation and plasma cell differentiation. Nevertheless, late GC Raptor deficiency caused only decreases in the size of memory B cells and long-lived plasma cells through poor maintenance of GCB cells, but it did not change their differentiation. Collectively, our data revealed that mTORC1 signalling supports GCB cell responses at both early and late GC phases during viral infection but does not regulate GCB cell differentiation into memory B cells and plasma cells at the late GC stage. © 2017 John Wiley & Sons Ltd.

  5. Mechanistic Target of Rapamycin-Independent Antidepressant Effects of (R)-Ketamine in a Social Defeat Stress Model.

    PubMed

    Yang, Chun; Ren, Qian; Qu, Youge; Zhang, Ji-Chun; Ma, Min; Dong, Chao; Hashimoto, Kenji

    2018-01-01

    The role of the mechanistic target of rapamycin (mTOR) signaling in the antidepressant effects of ketamine is controversial. In addition to mTOR, extracellular signal-regulated kinase (ERK) is a key signaling molecule in prominent pathways that regulate protein synthesis. (R)-Ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here we investigated whether mTOR signaling and ERK signaling play a role in the antidepressant effects of two enantiomers. The effects of mTOR inhibitors (rapamycin and AZD8055) and an ERK inhibitor (SL327) on the antidepressant effects of ketamine enantiomers in the chronic social defeat stress (CSDS) model (n = 7 or 8) and on those of ketamine enantiomers in these signaling pathways in mouse brain regions were examined. The intracerebroventricular infusion of rapamycin or AZD8055 blocked the antidepressant effects of (S)-ketamine, but not (R)-ketamine, in the CSDS model. Furthermore, (S)-ketamine, but not (R)-ketamine, significantly attenuated the decreased phosphorylation of mTOR and its downstream effector, ribosomal protein S6 kinase, in the prefrontal cortex of susceptible mice after CSDS. Pretreatment with SL327 blocked the antidepressant effects of (R)-ketamine but not (S)-ketamine. Moreover, (R)-ketamine, but not (S)-ketamine, significantly attenuated the decreased phosphorylation of ERK and its upstream effector, mitogen-activated protein kinase/ERK kinase, in the prefrontal cortex and hippocampal dentate gyrus of susceptible mice after CSDS. This study suggests that mTOR plays a role in the antidepressant effects of (S)-ketamine, but not (R)-ketamine, and that ERK plays a role in (R)-ketamine's antidepressant effects. Thus, it is unlikely that the activation of mTOR signaling is necessary for antidepressant actions of (R)-ketamine. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  6. Loss of cardiac carnitine palmitoyltransferase 2 results in rapamycin-resistant, acetylation-independent hypertrophy.

    PubMed

    Pereyra, Andrea S; Hasek, Like Y; Harris, Kate L; Berman, Alycia G; Damen, Frederick W; Goergen, Craig J; Ellis, Jessica M

    2017-11-10

    Cardiac hypertrophy is closely linked to impaired fatty acid oxidation, but the molecular basis of this link is unclear. Here, we investigated the loss of an obligate enzyme in mitochondrial long-chain fatty acid oxidation, carnitine palmitoyltransferase 2 (CPT2), on muscle and heart structure, function, and molecular signatures in a muscle- and heart-specific CPT2-deficient mouse (Cpt2 M-/- ) model. CPT2 loss in heart and muscle reduced complete oxidation of long-chain fatty acids by 87 and 69%, respectively, without altering body weight, energy expenditure, respiratory quotient, or adiposity. Cpt2M -/- mice developed cardiac hypertrophy and systolic dysfunction, evidenced by a 5-fold greater heart mass, 60-90% reduction in blood ejection fraction relative to control mice, and eventual lethality in the absence of cardiac fibrosis. The hypertrophy-inducing mammalian target of rapamycin complex 1 (mTORC1) pathway was activated in Cpt2M -/- hearts; however, daily rapamycin exposure failed to attenuate hypertrophy in Cpt2M -/- mice. Lysine acetylation was reduced by ∼50% in Cpt2M -/- hearts, but trichostatin A, a histone deacetylase inhibitor that improves cardiac remodeling, failed to attenuate Cpt2M -/- hypertrophy. Strikingly, a ketogenic diet increased lysine acetylation in Cpt2M -/- hearts 2.3-fold compared with littermate control mice fed a ketogenic diet, yet it did not improve cardiac hypertrophy. Together, these results suggest that a shift away from mitochondrial fatty acid oxidation initiates deleterious hypertrophic cardiac remodeling independent of fibrosis. The data also indicate that CPT2-deficient hearts are impervious to hypertrophy attenuators, that mitochondrial metabolism regulates cardiac acetylation, and that signals derived from alterations in mitochondrial metabolism are the key mediators of cardiac hypertrophic growth. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Mammalian Target of Rapamycin (mTor) Mediates Tau Protein Dyshomeostasis

    PubMed Central

    Tang, Zhi; Bereczki, Erika; Zhang, Haiyan; Wang, Shan; Li, Chunxia; Ji, Xinying; Branca, Rui M.; Lehtiö, Janne; Guan, Zhizhong; Filipcik, Peter; Xu, Shaohua; Winblad, Bengt; Pei, Jin-Jing

    2013-01-01

    Previous evidence from post-mortem Alzheimer disease (AD) brains and drug (especially rapamycin)-oriented in vitro and in vivo models implicated an aberrant accumulation of the mammalian target of rapamycin (mTor) in tangle-bearing neurons in AD brains and its role in the formation of abnormally hyperphosphorylated tau. Compelling evidence indicated that the sequential molecular events such as the synthesis and phosphorylation of tau can be regulated through p70 S6 kinase, the well characterized immediate downstream target of mTor. In the present study, we further identified that the active form of mTor per se accumulates in tangle-bearing neurons, particularly those at early stages in AD brains. By using mass spectrometry and Western blotting, we identified three phosphoepitopes of tau directly phosphorylated by mTor. We have developed a variety of stable cell lines with genetic modification of mTor activity using SH-SY5Y neuroblastoma cells as background. In these cellular systems, we not only confirmed the tau phosphorylation sites found in vitro but also found that mTor mediates the synthesis and aggregation of tau, resulting in compromised microtubule stability. Changes of mTor activity cause fluctuation of the level of a battery of tau kinases such as protein kinase A, v-Akt murine thymoma viral oncogene homolog-1, glycogen synthase kinase 3β, cyclin-dependent kinase 5, and tau protein phosphatase 2A. These results implicate mTor in promoting an imbalance of tau homeostasis, a condition required for neurons to maintain physiological function. PMID:23585566

  8. Subacute calorie restriction and rapamycin discordantly alter mouse liver proteome homeostasis and reverse aging effects.

    PubMed

    Karunadharma, Pabalu P; Basisty, Nathan; Dai, Dao-Fu; Chiao, Ying A; Quarles, Ellen K; Hsieh, Edward J; Crispin, David; Bielas, Jason H; Ericson, Nolan G; Beyer, Richard P; MacKay, Vivian L; MacCoss, Michael J; Rabinovitch, Peter S

    2015-08-01

    Calorie restriction (CR) and rapamycin (RP) extend lifespan and improve health across model organisms. Both treatments inhibit mammalian target of rapamycin (mTOR) signaling, a conserved longevity pathway and a key regulator of protein homeostasis, yet their effects on proteome homeostasis are relatively unknown. To comprehensively study the effects of aging, CR, and RP on protein homeostasis, we performed the first simultaneous measurement of mRNA translation, protein turnover, and abundance in livers of young (3 month) and old (25 month) mice subjected to 10-week RP or 40% CR. Protein abundance and turnover were measured in vivo using (2) H3 -leucine heavy isotope labeling followed by LC-MS/MS, and translation was assessed by polysome profiling. We observed 35-60% increased protein half-lives after CR and 15% increased half-lives after RP compared to age-matched controls. Surprisingly, the effects of RP and CR on protein turnover and abundance differed greatly between canonical pathways, with opposite effects in mitochondrial (mt) dysfunction and eIF2 signaling pathways. CR most closely recapitulated the young phenotype in the top pathways. Polysome profiles indicated that CR reduced polysome loading while RP increased polysome loading in young and old mice, suggesting distinct mechanisms of reduced protein synthesis. CR and RP both attenuated protein oxidative damage. Our findings collectively suggest that CR and RP extend lifespan in part through the reduction of protein synthetic burden and damage and a concomitant increase in protein quality. However, these results challenge the notion that RP is a faithful CR mimetic and highlight mechanistic differences between the two interventions. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  9. A central role for the mammalian target of rapamycin in LPS-induced anorexia in mice.

    PubMed

    Yue, Yunshuang; Wang, Yi; Li, Dan; Song, Zhigang; Jiao, Hongchao; Lin, Hai

    2015-01-01

    Bacterial lipopolysaccharide (LPS), also known as endotoxin, induces profound anorexia. However, the LPS-provoked pro-inflammatory signaling cascades and the neural mechanisms underlying the development of anorexia are not clear. Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. This study aimed to determine whether the mTOR pathway is involved in LPS-induced anorexia. Effects of LPS on hypothalamic gene/protein expression in mice were measured by RT-PCR or western blotting analysis. To determine whether inhibition of mTOR signaling could attenuate LPS-induced anorexia, we administered an i.c.v. injection of rapamycin, an mTOR inhibitor, on LPS-treated male mice. In this study, we showed that LPS stimulates the mTOR signaling pathway through the enhanced phosphorylation of mTOR(Ser2448) and p70S6K(Thr389). We also showed that LPS administration increased the phosphorylation of FOXO1(Ser256), the p65 subunit of nuclear factor kappa B (P<0.05), and FOXO1/3a(Thr) (24) (/) (32) (P<0.01). Blocking the mTOR pathway significantly attenuated the LPS-induced anorexia by decreasing the phosphorylation of p70S6K(Thr389), FOXO1(Ser256), and FOXO1/3a(Thr) (24) (/) (32). These results suggest promising approaches for the prevention and treatment of LPS-induced anorexia. © 2015 Society for Endocrinology.

  10. Regulating effect of tea polyphenols on endothelin, intracellular calcium concentration, and mitochondrial membrane potential in vascular endothelial cells injured by Angiotensin II.

    PubMed

    Li, Minggao; Ma, Guixi; Han, Lei; Li, Jing

    2014-05-01

    Polyphenols are the main active component of tea and are considered an antiatherosclerosis agent, protecting vascular endothelial cells (VECs) from injury and preventing cardiovascular diseases. Endothelin level, intracellular calcium concentration, and mitochondrial membrane potential in VECs directly reflect the function and injury status of cells. The objective of this study was to study the regulating effects of tea polyphenols on these factors in VECs injured by angiotensin II (Ang-II) and explore the protective effect of tea polyphenols on human VECs. In this study, human aortic vascular endothelial cells were divided into 4 groups: (1) a control group; (2) Ang-II group: Ang-II at a concentration of 10(-7) mol/L was added to the cells; (3) low-concentration tea polyphenols + Ang-II group: tea polyphenols at a concentration of 5 mg/L was added in addition to Ang-II; (4) high-concentration tea polyphenols + Ang-II group: tea polyphenols at a concentration of 25 mg/L was added in addition to Ang-II. One hundred microliters of supernatant was extracted before treatment and at 0.5, 6, and 24 hours after treatment in each group to establish the content of endothelin. The results showed the following: (1) tea polyphenols decreased the expression of endothelin-1 messenger RNA, which was increased by Ang-II (P < 0.01). (2) Tea polyphenols inhibited endothelin secretion induced by Ang-II (P < 0.01), and the inhibition of low-concentration tea polyphenols was superior to that of high concentration tea polyphenols (P < 0.01). (3) Tea polyphenols ameliorated the changes in intracellular calcium concentration and mitochondrial membrane potential induced by Ang-II (P < 0.01). This study suggests that tea polyphenols may effectively protect VECs against injury by regulating endothelin, intracellular calcium concentration, and mitochondrial membrane potential in VECs injured by Ang-II. Additionally, lower dose would be used clinically rather than the higher dose for obtaining

  11. Regulated in Development and DNA Damage 1 Is Necessary for Hyperglycemia-induced Vascular Endothelial Growth Factor Expression in the Retina of Diabetic Rodents*

    PubMed Central

    Dennis, Michael D.; Kimball, Scot R.; Fort, Patrice E.; Jefferson, Leonard S.

    2015-01-01

    Vascular endothelial growth factor (VEGF) is considered a major role player in the pathogenesis of diabetic retinopathy, yet the mechanisms regulating its expression are not fully understood. Our laboratory previously demonstrated that diabetes-induced VEGF expression in the retina was dependent on the repressor of mRNA translation 4E-BP1. Interaction of 4E-BP1 with the cap-binding protein eIF4E regulates protein expression by controlling the selection of mRNAs for translation. The process is regulated by the master kinase mTOR in complex 1 (mTORC1), which phosphorylates 4E-BP1, thus promoting its disassociation from eIF4E. In the present study, we investigated the role of the Akt/mTORC1 repressor REDD1 (regulated in development and DNA damage) in diabetes-induced VEGF expression. REDD1 expression was induced by hyperglycemia in the retina of diabetic rodents and by hyperglycemic conditions in Müller cells concomitant with increased VEGF expression. In Müller cells, hyperglycemic conditions attenuated global rates of protein synthesis and cap-dependent mRNA translation concomitant with up-regulated cap-independent VEGF mRNA translation, as assessed by a bicistronic luciferase reporter assay. Hyperglycemic conditions also attenuated mTORC1 signaling and enhanced 4E-BP1 binding to eIF4E. Furthermore, ectopic expression of REDD1 in Müller cells was sufficient to promote both increased 4E-BP1 binding to eIF4E and VEGF expression. Whereas the retina of wild-type mice exhibited increased expression of VEGF and tumor necrosis factor alpha (TNF-α) 4 weeks after streptozotocin administration, the retina of REDD1 knock-out mice failed to do so. Overall, the results demonstrate that REDD1 contributes to the pathogenesis of diabetes in the retina by mediating the pathogenic effects of hyperglycemia. PMID:25548280

  12. Cooperative Role of Mineralocorticoid Receptor and Caveolin-1 in Regulating the Vascular Response to Low Nitric Oxide–High Angiotensin II–Induced Cardiovascular Injury

    PubMed Central

    Pojoga, Luminita H.; Yao, Tham M.; Opsasnick, Lauren A.; Siddiqui, Waleed T.; Reslan, Ossama M.; Adler, Gail K.; Williams, Gordon H.

    2015-01-01

    not affected by EPL. Vascular relaxation to the NO donor sodium nitroprusside was increased with L-NAME + AngII in WT mice but not in cav-1−/− mice. Plasma aldosterone levels increased and cardiac MR expression decreased in L-NAME + AngII treated WT and cav-1−/− mice and did not change with EPL. Thus, during L-NAME + AngII induced hypertension, MR blockade increases contraction and alters vascular relaxation via NO-cGMP, and these changes are absent in cav-1 deficiency states. The data suggest a cooperative role of MR and cav-1 in regulating vascular contraction and NO-cGMP–mediated relaxation during low NO–high AngII–dependent cardiovascular injury. PMID:26183312

  13. LY303511 (2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one) acts via phosphatidylinositol 3-kinase-independent pathways to inhibit cell proliferation via mammalian target of rapamycin (mTOR)- and non-mTOR-dependent mechanisms.

    PubMed

    Kristof, Arnold S; Pacheco-Rodriguez, Gustavo; Schremmer, Bruno; Moss, Joel

    2005-09-01

    Mammalian target of rapamycin (mTOR), a serine/threonine kinase, regulates cell growth and proliferation in part via the activation of p70 S6 kinase (S6K). Rapamycin is an antineo-plastic agent that, in complex with FKBP12, is a specific inhibitor of mTOR through interaction with its FKBP12-rapamycin binding domain, thereby causing G(1) cell cycle arrest. However, cancer cells often develop resistance to rapamycin, and alternative inhibitors of mTOR are desired. 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) blocks mTOR kinase activity, but it also inhibits phosphatidylinositol 3-kinase (PI3K), an enzyme that regulates cellular functions other than proliferation. We hypothesized that a close structural analog, 2-piperazinyl-8-phenyl-4H-1-benzopyran-4-one (LY303511) might inhibit mTOR-dependent cell proliferation without unwanted effects on PI3K. In human lung epithelial adenocarcinoma (A549) cells, LY303511, like rapamycin, inhibited mTOR-dependent phosphorylation of S6K, but not PI3K-dependent phosphorylation of Akt. LY303511 blocked proliferation in A549 as well as in primary pulmonary artery smooth muscle cells, without causing apoptosis. In contrast to rapamycin, LY303511 reduced G(2)/M progression as well as G(2)/M-specific cyclins in A549 cells. Consistent with an additional mTOR-independent kinase target, LY303511 inhibited casein kinase 2 activity, a known regulator of G(1) and G(2)/M progression. In addition to its antiproliferative effect in vitro, LY303511 inhibited the growth of human prostate adenocarcinoma tumor implants in athymic mice. Given its inhibition of cell proliferation via mTOR-dependent and independent mechanisms, LY303511 has therapeutic potential with antineoplastic actions that are independent of PI3K inhibition.

  14. Rapamycin and bafilomycin A1 alter autophagy and megakaryopoiesis.

    PubMed

    Wang, Qi; You, Tao; Fan, Hongqiong; Wang, Yinyan; Chu, Tinatian; Poncz, Mortimer; Zhu, Li

    2017-01-01

    Autophagy is an effective strategy for cell development by recycling cytoplasmic constituents. Genetic deletion of autophagy mediator Atg7 in hematopoietic stem cells (HSCs) can lead to failure of megakaryopoiesis and enhanced autophagy has been implicated in various hematological disorders such as immune thrombocytopenia and myelodysplastic syndrome. Here, we examined the hypothesis that optimal autophagy is essential for megakaryopoiesis and thrombopoiesis by altering autophagy using pharmacological approaches. When autophagy was induced by rapamycin or inhibited by bafilomycin A1 in fetal liver cells, we observed a significant decrease in high ploidy megakaryocytes, a reduction of CD41 and CD61 co-expressing cells, and less proplatelet or platelet formation. Additionally, reduced cell size was shown in megakaryocytes derived from rapamycin, but not bafilomycin A1-treated mouse fetal liver cells. However, when autophagy was altered in mature megakaryocytes, we observed no significant change in proplatelet formation, which was consistent with normal platelet counts, megakaryocyte numbers, and ploidy in Atg7 flox/flox PF4-Cre mice with megakaryocyte- and platelet-specific deletion of autophagy-related gene Atg7. Therefore, our findings suggest that either induction or inhibition of autophagy in the early stage of megakaryopoiesis suppresses megakaryopoiesis and thrombopoiesis.

  15. Caloric Restriction and Rapamycin Differentially Alter Energy Metabolism in Yeast.

    PubMed

    Choi, Kyung-Mi; Hong, Seok-Jin; van Deursen, Jan M; Kim, Sooah; Kim, Kyoung Heon; Lee, Cheol-Koo

    2017-12-12

    Rapamycin (RM), a drug that inhibits the mechanistic target of rapamycin (mTOR) pathway and responds to nutrient availability, seemingly mimics the effects of caloric restriction (CR) on healthy life span. However, the extent of the mechanistic overlap between RM and CR remains incompletely understood. Here, we compared the impact of CR and RM on cellular metabolic status. Both regimens maintained intracellular ATP through the chronological aging process and showed enhanced mitochondrial capacity. Comparative transcriptome analysis showed that CR had a stronger impact on global gene expression than RM. We observed a like impact on the metabolome and identified distinct metabolites affected by CR and RM. CR severely reduced the level of energy storage molecules including glycogen and lipid droplets, whereas RM did not. RM boosted the production of enzymes responsible for the breakdown of glycogen and lipid droplets. Collectively, these results provide insights into the distinct energy metabolism mechanisms induced by CR and RM, suggesting that these two anti-aging regimens might extend life span through distinctive pathways. © The Author(s) 2017. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Rapamycin protects neurons from brain contusion-induced inflammatory reaction via modulation of microglial activation

    PubMed Central

    SONG, QI; XIE, DUJIANG; PAN, SHIYONG; XU, WEIJUN

    2015-01-01

    The inflammatory reaction is important in secondary injury following traumatic brain injury (TBI). Rapamycin has been demonstrated as a neuroprotective agent in a mouse model of TBI, however, there is a lack of data regarding the effects of rapamycin on the inflammatory reaction following TBI. Therefore, the present study was designed to assess the effects of treatment with rapamycin on inflammatory reactions and examine the possible involvement of microglial activation following TBI. Male imprinting control region mice were randomly divided into four groups: Sham group (n=23), TBI group (n=23), TBI + dimethyl sulfoxide (DMSO) group (n=31) and TBI + rapamycin group (n=31). Rapamycin was dissolved in DMSO (50 mg/ml) and injected 30 min after TBI (2 mg/Kg; intraperitoneally). A weight-drop model of TBI was induced, and the brain tissues were harvested 24 h after TBI. The findings indicated that the administration of