Aim: Paeoniflorin has shown to attenuate bleomycin-induced pulmonary fibrosis (PF) in mice. Because the epithelial-mesenchymal transition (EMT) in type 2 lung endothelial cells contributes to excessive fibroblasts and myofibroblasts during multiple fibrosis of tissues, we investigated the effects of paeoniflorin on TGF-β mediated pulmonary EMT in bleomycin-induced PF mice. Methods: PF was induced in mice by intratracheal instillation of bleomycin (5 mg/kg). The mice were orally treated with paeoniflorin or prednisone for 21 d. After the mice were sacrificed, lung tissues were collected for analysis. An in vitro EMT model was established in alveolar epithelial cells (A549 cells) incubated with TGF-β1 (2 ng/mL). EMT identification and the expression of related proteins were performed using immunohistochemistry, transwell assay, ELISA, Western blot and RT-qPCR. Results: In PF mice, paeoniflorin (50, 100 mg·kg -1 ·d -1 ) or prednisone (6 mg·kg -1 ·d -1 ) significantly decreased the expression of FSP-1 and α-SMA, and increased the expression of E-cadherin in lung tissues. In A549 cells, TGF-β1 stimulation induced EMT, as shown by the changes in cell morphology, the increased cell migration, and the increased vimentin and α-SMA expression as well as type I and type III collagen levels, and by the decreased E-cadherin expression. In contrast, effects of paeoniflorin on EMT disappeared when the A549 cells were pretreated with TGF-β1 for 24 h. TGF-β1 stimulation markedly increased the expression of Snail and activated Smad2/3, Akt, ERK, JNK and p38 MAPK in A549 cells. Co-incubation with paeoniflorin (1-30 µmol/L) dose-dependently attenuated TGF-β1-induced expression of Snail and activation of Smad2/3, but slightly affected TGF-β1-induced activation of Akt, ERK, JNK and p38 MAPK. Moreover, paeoniflorin markedly increased Smad7 level, and decreased ALK5 level in A549 cells. Conclusion: Paeoniflorin suppresses the early stages of TGF-β mediated EMT in alveolar epithelial cells, likely by decreasing the expression of the transcription factors Snail via a Smad-dependent pathway involving the up-regulation of Smad7.
BackgroundCirculating tumor cells (CTCs), an advantageous target of liquid biopsy, is an important biomarker for the prognosis and monitoring of cancer. Currently, detection techniques for CTCs are mainly based on the physical and/or epithelial characteristics of tumor cells. However, biofunctional activity markers that can indicate the high metastatic capacity of CTCs are lacking.MethodsFunctional microarray, quantitative real-time polymerase chain reaction, and Western blot were used on five prostate cancer cell lines with different metastatic capacities to identify the metastasis-related metabolic genes. The identified genes were detected in the CTCs of 64 clinical samples using the RNA in situ hybridization. A multi-criteria weighted model was used to determine the optimal metabolic markers for the CTCs test. Based on five fluorescent signals targeting DAPI, CD45, metabolic, epithelial (EpCAM/CKs), and mesenchymal (Vimentin/Twist) markers, the filtration-enriched CTCs were classified as GM+CTCs/GM−CTCs (metabolic types) or E-CTCs/H-CTCs/M-CTCs (EMT types). Correlation analysis and ROC curve were conducted on 54 prostate cancer samples to evaluate the clinical significance of CTCs subtypes.ResultsEight metastasis-related metabolic genes were identified, including HK2, PDP2, G6PD, PGK1, PHKA1, PYGL, PDK1, and PKM2. Among them, PGK1 and G6PD were determined as optimal glucose metabolic (GM) markers for CTCs. GM+CTCs (marked by PGK1/G6PD) were detectable in 64.8% (35/54) of prostate cancer patients, accounting for 46.5% (134/288) of total CTCs. An increased GM+CTCs level was associated with advanced tumor stage and metastasis (P < 0.05). In the discrimination of cancer metastasis from non-metastasis, GM+CTCs presented a higher AUC of the ROC curve (0.780) compared with the EMT CTCs subtypes (E-CTCs 0.729, H-CTCs 0.741, and M-CTCs 0.648). A triple tPSA–Gleason–GM+CTCs marker increased the AUC to 0.904, which was better than that of the tPSA–Gleason–H-CTCs marker (0.874).ConclusionsThe metabolic marker (PGK1/G6PD) is determined as the indicator for the biofunctional activity analysis of CTCs, compared with the existing morphological (EMT) classification on CTCs. The metabolic characterization of CTCs demonstrates that hypermetabolic GM+CTCs are promising biomarkers for prostate cancer metastasis.Electronic supplementary materialThe online version of this article (10.1186/s13046-018-0789-0) contains supplementary material, which is available to authorized users.
DNA nanostructures as scaffolds for drug delivery, biosensing, and bioimaging are hindered by its vulnerability in physiological settings, less favorable of incorporating arbitrary guest molecules and other desirable functionalities. Noncanonical self‐assembly of DNA nanostructures with small molecules in an alternative system is an attractive strategy to expand their applications in multidisciplinary fields and is rarely explored. This work reports a nitrogen‐enriched carbon dots (NCDs)‐mediated DNA nanostructure self‐assembly strategy. Given the excellent photoluminescence and photodynamic properties of NCDs, the obtained DNA/NCDs nanocomplex holds great potential for bioimaging and anticancer therapy. NCDs can mediate DNA nanoprism (NPNCD) self‐assembly isothermally at a large temperature and pH range in a magnesium‐free manner. To explore the suitability of NPNCD in potential biomedical applications, the cytotoxicity and cellular uptake efficiency of NPNCD are evaluated. NPNCD with KRAS siRNA (NPNCDK) is further conjugated for KRAS‐mutated nonsmall cell lung cancer therapy. The NPNCDK shows excellent gene knockdown efficiency and anticancer effect in vitro. The current study suggests that conjugating NCDs with programmable DNA nanostructures is a powerful strategy to endow DNA nanostructures with new functionalities, and NPNCD may be a potential theranostic platform with further fine‐tuned properties of CDs such as near‐red fluorescence or photothermal activities.
Aims Macrophage-mediated inflammatory response represents a key pathophysiological process in a host of cardiovascular diseases including heart failure. Regardless of etiology, heart failure is invariably preceded by cardiac hypertrophy. In the present study we investigated the effect of macrophage-specific deletion of myocardin-related transcription factor A (MRTF-A) on cardiac hypertrophy and the underlying mechanism. Methods and Results We report that when subjected to transverse aortic constriction (TAC), macrophage MRTF-A conditional knockout (CKO) mice developed a less severe phenotype of cardiac hypertrophy compared to wild type (WT) littermates and were partially protected from the loss of heart function. In addition, there was less extensive cardiac fibrosis in the CKO mice than WT mice following the TAC procedure. Further analysis revealed that cardiac inflammation, as assessed by levels of pro-inflammatory cytokines and chemokines, was dampened in CKO mice paralleling reduced infiltration of macrophages in the heart. Mechanistically, MRTF-A deficiency attenuated the expression of integrin beta 2 (ITGB2/CD18) in macrophage thereby disrupting adhesion of macrophages to vascular endothelial cells. MRTF-A was recruited by Sp1 to the ITGB2 promoter and cooperated with Sp1 to activate ITGB2 transcription in macrophages. Administration of a CD18 blocking antibody attenuated TAC induced cardiac hypertrophy in mice. Interaction between MRTF-A and the histone demethylase KDM3A likely contributed to IGTB2 transcription and consequently adhesion of macrophages to endothelial cells. Conclusions Our data suggest that MRTF-A may regulate macrophage trafficking and contribute to the pathogenesis of cardiac hypertrophy by activating ITGB2 transcription.
Executive function (EF), its importance for scholastic achievement and the question of whether or not EF is malleable, have become a topic of intense interest. Education or schooling is often seen as effective approaches to enhance EF due to the specific school-related requirements as compared to kindergarten or pre-school. However, no study to date has investigated whether targeted training focusing on those domains might be comparable with regular schooling in improving EF and fluid intelligence (Gf). The aim of the present study was to replicate and extend the previously demonstrated schooling effects on EF by using a school-cutoff design, and to further investigate whether a theoretically motivated intervention targeting specific EF, i.e., working memory (WM) or inhibitory control (IC), could achieve comparable effects with schooling in both, WM and IC, as well as Gf. 91 6-year-old kindergarteners and first-graders with similar chronological age participated the study. We compared the performance of a first-grade schooling group with that of two kindergarten training groups as well as a business-as-usual kindergarten control group. Participants were assessed in WM, IC and Gf at baseline, immediately after the intervention (posttest), as well as 3 months after training completion (follow-up). The results showed that the schooling group indeed outperformed the kindergarten groups at baseline in several cognitive tasks. Furthermore, both the WM and IC training showed pronounced gains in the trained tasks, as well as varying degrees of improvement in non-trained outcome measures. Most importantly, both training groups achieved comparable performance with the schooling group, which was especially apparent in Gf at follow-up. Our findings provide further evidence for the malleability of EF demonstrating that both, long-term and short-term interventions can facilitate the acquisition of those important skills, and as such, our work has important implications for educational practice.
Vascular endothelial cells contribute to the pathogenesis of cardiovascular diseases by producing and disseminating angiocrine factors. Nitric oxide (NO), catalyzed by endothelial NO synthase (eNOS), is one of the prototypical angiocrine factors. eNOS activity is modulated by site-specific phosphorylation. We have previously shown that endothelial-specific knockdown of BRG1 in Apoe –/– mice attenuates the development of atherosclerosis, in which eNOS-dependent NO catalysis plays an antagonizing role. Here we report that attenuation of atherogenesis in mice by BRG1 knockdown was accompanied by partial restoration of NO biosynthesis by 44% in the arteries and a simultaneous up-regulation of eNOS serine 1177 phosphorylation by 59%. Indeed, BRG1 depletion or inhibition ameliorated oxLDL-induced loss of NO bioavailability and eNOS phosphorylation in cultured endothelial cells. Further analysis revealed that BRG1 regulated eNOS phosphorylation and NO synthesis by activating the transcription of protein phosphatase 2A (PP2A) structural subunit a (encoded by PR65A ). BRG1 interacted with ETS1, was recruited by ETS1 to the PR65A promoter, and cooperated with ETS1 to activate PR65A transcription. Finally, depletion of ETS1, similar to BRG1, repressed PR65A induction, normalized eNOS phosphorylation, and rescued NO biosynthesis in endothelial cells treated with oxLDL. In conclusion, our data characterize a novel transcriptional cascade that regulates NO bioavailability in vascular endothelial cells.
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