Many attempts to modulate leukocyte-endothelial interaction to prevent or reduce excessive inflammatory reactions were made in the past. However, the basic regulatory principles of the endothelial inflammatory process remain unclear. It seems that the inhibition of individual components of the inflammatory cascade, for example, by a single antibody against an adhesion molecule, may not be enough to achieve a sustained effect on vascular inflammation.In the past years, microRNAs have been identified as important regulators of gene expression in a wide range of Molecular Medicine© 2017 American Heart Association, Inc. Rationale:The interaction of circulating cells within the vascular wall is a critical event in chronic inflammatory processes, such as atherosclerosis, but the control of the vascular inflammatory state is still largely unclear.Objective: This study was undertaken to characterize the function of the endothelial-enriched microRNA miR-100 during vascular inflammation and atherogenesis. Methods and Results:Based on a transcriptome analysis of endothelial cells after miR-100 overexpression, we identified miR-100 as a potent suppressor of endothelial adhesion molecule expression, resulting in attenuated leukocyte-endothelial interaction in vitro and in vivo as shown by flow cytometry and intravital imaging. Mechanistically, miR-100 directly repressed several components of mammalian target of rapamycin complex 1-signaling, including mammalian target of rapamycin and raptor, which resulted in a stimulation of endothelial autophagy and attenuated nuclear factor κB signaling in vitro and in vivo. In a low-density lipoprotein receptordeficient atherosclerotic mouse model, pharmacological inhibition of miR-100 resulted in enhanced plaque lesion formation and a higher macrophage content of the plaque, whereas a systemic miR-100 replacement therapy had protective effects and attenuated atherogenesis, resulting in a decrease of plaque area by 45%. Finally, analysis of miR-100 expression in >70 samples obtained during carotid endarterectomy revealed that local miR-100 expression was inversely correlated with inflammatory cell content in patients. Conclusions: In summary, we describe an anti-inflammatory function of miR-100 in the vascular response to injury and inflammation and identify an important novel modulator of mammalian target of rapamycin signaling and autophagy
Background Diet-induced obesity can result in the development of a diverse spectrum of cardiovascular and metabolic diseases, including type 2 diabetes, dyslipidemia, non-alcoholic liver steatosis and atherosclerotic disease. MicroRNAs have been described to be important regulators of metabolism and disease development. Methods In the current study, we investigated the effects of ubiquitous miR-100 overexpression on weight gain and the metabolic phenotype in a newly generated transgenic mouse strain under normal chow and high fat diet and used microarray expression analysis to identify new potential target genes of miR-100. Results While transgenic overexpression of miR-100 did not significantly affect weight and metabolism under a normal diet, miR-100 overexpressing mice showed a reduced weight gain under a high fat diet compared to wildtype mice, despite an equal calorie intake. This was accompanied by less visceral and subcutaneous fat development and lover serum LDL cholesterol. In addition, transgenic miR-100 mice were more glucose tolerant and insulin sensitive and demonstrated increased energy expenditure under high fat diet feeding. A comprehensive gene expression profiling revealed the differential expression of several genes involved in lipid storage- and metabolism, among them CD36 and Cyp4A14. Our data showed a direct regulation of CD36 by miR-100, leading to a reduced fatty acid uptake in primary hepatocytes overexpressing miR-100 and the downregulation of several downstream mediators of lipid metabolism such as ACC1, FABP4, FAS and PPARγ in the liver. Conclusions Our findings demonstrate a protective role of miR-100 in high fat diet induced metabolic syndrome and liver steatosis, partially mediated by the direct repression of CD36 and attenuation of hepatic lipid storage, implicating miR-100 as a possible therapeutic target in liver steatosis.
1032 The PI3K/AKT/mTOR pathway is a major downstream signaling pathway of the bcr-abl oncogene that is the hallmark of Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (Ph+ ALL) and of CML. Ph+ ALL is a subtype of ALL with a particularly poor prognosis despite the availability of tyrosine kinase inhibitors (TKI) that effectively suppress BCR-ABL kinase activity. Resistance of Ph+ ALL to TKI has been suggested to involve activation of the PI3K signaling pathway, which has also been shown in several other hematologic malignancies to contribute to leukemogenesis and disease progression. Its role in ALL subtypes other than Ph+ ALL has not been clearly established. Moreover, the relative contributions of the individual components of the PI3K/AKT/mTOR signaling pathway to leukemogenesis remain to be resolved. mTOR is a serine/threonine kinase and catalytic subunit of the two biochemically distinct complexes mTORC1 and mTORC2. mTORC1 controls cell growth in response to nutrients and growth factors, whereas mTORC2 is thought to mediate cell proliferation and cell survival. AKT activates mTORC1, which promotes cell growth in part by directly phosphorylating the translational regulators S6K1 and 4E-BP1. Linking mTORC1 regulation to oncogenic PI3K activity provided strong rationale for targeting mTORC1 in cancer, but the effectiveness of targeting mTORC1 is mitigated by strong, mTORC1-dependent negative feedback loops that become inactive on mTORC1 inhibition. mTORC2 directly phosphorylates AKT on a critical regulatory site required for maximal AKT kinase activity. This prompted efforts to develop mTOR inhibitors that target both complexes. We compared the effects of selective inhibitors of PI3K (NVP-BKM120) and mTORC1 (RAD001) with those of dual PI3K/mTORC1/C2 inhibitors (NVP-BEZ235 & NVP-BGT226) on Ph+ and Ph neg. B-precursor ALL. Long-term serum-free cultures of primary human Ph+ B-ALL (n=6) and Ph- B-ALL (n=6) cells were exposed to increasing concentrations of these inhibitors (NVP-BKM120 (50nM-10uM), RAD001 (5nM-20uM), NVP-BGT226 (1nM-500nM), NVP-BEZ235 (10nM-1uM). All inhibitors were kindly provided by Novartis, Basel, Switzerland. Some of the Ph+ ALL cells are partially resistant to 1st and 2nd generation TKI. Cell proliferation and apoptosis were monitored by XTT-assays and FACS analysis using annexin V/propidium iodide. Phosphorylation of the proteins 4E-BP1 (Thr37/46) & S6 Ribosomal Protein (Ser235/236) downstream of mTOR was assessed by Western Blotting in a time and concentration dependent manner. In both Ph+ ALL and Ph- ALL, inhibition of PI3K activity by BKM120 suppressed proliferation and induced apoptosis at high nanomolar (IC50≤1μM) and low micromolar (IC50≤5μM) concentrations, respectively. Inhibition of only mTORC1 by RAD001 slightly inhibited proliferation, but failed to induce apoptosis. Combined inhibition of PI3K and both mTOR complexes mTORC1/C2 by NVP-BEZ235 or NVP-BGT226 resulted in a significantly more pronounced suppression of cell growth (BEZ235 (IC50≤200nM), BGT226 (IC50≤20nM) and induction of apoptosis at nanomolar concentrations (BEZ235 (IC50≤250nM), BGT226 (IC50≤25nM)) as compared to both selective inhibitors (NVP-BKM120 and RAD001). The anti-proliferative and pro-apoptotic effects of these inhibitors was independent of bcr-abl status. Comparison of the effect of selective PI3K and mTOR inhibitors on mTOR signaling revealed differential regulation of S6 and 4E-BP1. Whereas selective inhibition of PI3K and mTORC1 by BKM 120 and RAD001, respectively, resulted in dephosphorylation only of the S6 protein, combined inhibition of PI3K and mTORC1 was associated primarily with a decrease of S6 phosphorylation and only minor dephosphorylation of 4E-BP1. On the other hand, exposure to the dual PI3K/mTORC1/C2 inhibitors resulted in nearly complete dephosphorylation of both S6 and 4E-BP1. These data indicate that in ALL, mTORC2 contributes substantially to regulation of the downstream target 4E-BP1 by mTORC1. Our observation that compounds inhibiting PI3K and both mTOR complexes (mTORC1/mTORC2) have significantly greater growth inhibitory and pro-apoptotic effects than selective inhibition of PI3K and mTORC1 support a functional role of mTORC2 in survival and growth of B-precursor ALL cells. Combined targeting of these complexes may provide a novel therapeutic approach for both Ph+ ALL resistant to ABL TKI and Ph- ALL. Disclosures: Ottmann: Novartis: Consultancy, Honoraria, Research Funding; BMS: Honoraria, Research Funding.
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