A new promising trend in personalized medicine is the use of autologous cells (macrophages or stem cells) for cell-based therapy and also as a “Trojan horse” for targeted delivery of a drug carrier. ...The natural ability of macrophages for chemotaxis allows them to deliver cargo to the damaged area, significantly reducing side effects on healthy organ tissues. Therefore, it is important to develop tools to track their behavior in the organism. While labeled containers can serve as anchored tags for imaging macrophages in vivo, they can affect the properties and functions of macrophages. This work demonstrates that 3 μm sized capsules based on biocompatible polyelectrolytes and fluorescently labeled with both Cy7 and RITC dyes do not affect cell functionalization in vitro, such as viability, proliferation, and movement of transformed monocyte/macrophage-like cells (RAW 264.7) and primary bone marrow derived macrophages (BMDM) at maximal loading of five capsules per cell. In addition, capsules allowed fluorescent detection of ex vivo loaded cells 24 h after the tail vein injection in vivo and visualization of microcapsule-laden macrophages ex vivo using confocal microscopy. We have delivered about 62.5% of injected BMDM containing 12.5 million capsules with 3.75 μg of high-molecular-weight cargo (0.3 pg/capsule) to the liver. Our results demonstrate that 3 μm polyelectrolyte fluorescently labeled microcapsules can be used for safe macrophage loading, allowing cell tracking and drug delivery, which will facilitate development of macrophage-based cell therapy protocols.
4-methylumbelliferone (4MU) is an inhibitor of hyaluronan deposition and an active substance of hymecromone, a choleretic and antispasmodic drug. 4MU reported to be anti-fibrotic in mouse models; ...however, precise mechanism of action still requires further investigation. Here we describe the cellular and molecular mechanisms of 4MU action on CCl
-induced liver fibrosis in mice using NGS transcriptome, Q-PCR and immunohistochemical analysis. Collagen and hyaluronan deposition were prevented by 4MU. The CCl
stimulated expression of Col1a and αSMA were reduced, while the expression of the ECM catabolic gene Hyal1 was increased in the presence of 4MU. Bioinformatic analysis identified an activation of TGF-beta and Wnt/beta-catenin signaling pathways, and inhibition of the genes associated with lipid metabolism by CCL
treatment, while 4MU restored key markers of these pathways to the control level. Immunohistochemical analysis reveals the suppression of hepatic stellate cells (HSCs) transdifferentiation to myofibroblasts by 4MU treatment. The drug affected the localization of HSCs and macrophages in the sites of fibrogenesis. CCl
treatment induced the expression of FSTL1, which was downregulated by 4MU. Our results support the hypothesis that 4MU alleviates CCl
-induced liver fibrosis by reducing hyaluronan deposition and downregulating FSTL1 expression, accompanied by the suppression of HSC trans-differentiation and altered macrophage localization.
Reactive oxygen species (ROS) are highly reactive products of the cell metabolism derived from oxygen molecules, and their abundant level is observed in many diseases, particularly tumors, such as ...hepatocellular carcinoma (HCC). In vivo imaging of ROS is a necessary tool in preclinical research to evaluate the efficacy of drugs with antioxidant activity and for diagnosis and monitoring of diseases. However, most known sensors cannot be used for in vivo experiments due to low stability in the blood and rapid elimination from the body. In this work, we focused on the development of an effective delivery system of fluorescent probes for intravital ROS visualization using the HCC model. We have synthesized various lipid nanoparticles (LNPs) loaded with ROS-inducible hydrocyanine pro-fluorescent dye or plasmid DNA (pDNA) with genetically encoded protein sensors of hydrogen peroxide (HyPer7). LNP with an average diameter of 110 ± 12 nm, characterized by increased stability and pDNA loading efficiency (64 ± 7%), demonstrated preferable accumulation in the liver compared to 170 nm LNPs. We evaluated cytotoxicity and demonstrated the efficacy of hydrocyanine-5 and HyPer7 formulated in LNP for ROS visualization in mouse hepatocytes (AML12 cells) and in the mouse xenograft model of HCC. Our results demonstrate that obtained LNP could be a valuable tool in preclinical research for visualization ROS in liver diseases.
Autologous macrophage transfer is an emerging platform for cell therapy. It is anticipated that conventional macrophage reprogramming based on ex vivo polarization using cytokines and ligands of TLRs ...may enhance the therapeutic effect. We describe an alternative approach based on small interfering RNA (siRNA) knockdown of selected molecular cues of macrophage polarization, namely EGR2, IRF3, IRF5, and TLR4 in Raw264.7 monocyte/macrophage cell line and mouse-bone-marrow-derived macrophages (BMDMs). The impact of IRF5 knockdown was most pronounced, curtailing the expression of other inflammatory mediators such as IL-6 and NOS2, especially in M1-polarized macrophages. Contrary to IRF5, EGR2 knockdown potentiated M1-associated markers while altogether abolishing M2 marker expression, which is indicative of the principal role of EGR2 in the maintenance of alternative phenotypes. IRF3 knockdown suppressed M1 polarization but upregulated Arg 1, a canonical marker of alternative polarization in M1 macrophages. As anticipated, the knockdown of TLR4 also attenuated the M1 phenotype but, akin to IRF3, significantly induced Arginase 1 in M0 and M1, driving the phenotype towards M2. This study validates RNAi as a viable option for the alteration and maintenance of macrophage phenotypes.
The variability in the prognosis of individuals with hepatocellular carcinoma (HCC) suggests that HCC may comprise several distinct biological phenotypes. These phenotypes may result from activation ...of different oncogenic pathways during tumorigenesis and/or from a different cell of origin. Here we address whether the transcriptional characteristics of HCC can provide insight into the cellular origin of the tumor. We integrated gene expression data from rat fetal hepatoblasts and adult hepatocytes with HCC from human and mouse models. Individuals with HCC who shared a gene expression pattern with fetal hepatoblasts had a poor prognosis. The gene expression program that distinguished this subtype from other types of HCC included markers of hepatic oval cells, suggesting that HCC of this subtype may arise from hepatic progenitor cells. Analyses of gene networks showed that activation of AP-1 transcription factors in this newly identified HCC subtype might have key roles in tumor development.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Genetically modified mice have been extensively used for analyzing the molecular events that occur during tumor development. In many, if not all, cases, however, it is uncertain to what extent the ...mouse models reproduce features observed in the corresponding human conditions. This is due largely to lack of precise methods for direct and comprehensive comparison at the molecular level of the mouse and human tumors. Here we use global gene expression patterns of 68 hepatocellular carcinomas (HCCs) from seven different mouse models and 91 human HCCs from predefined subclasses to obtain direct comparison of the molecular features of mouse and human HCCs. Gene expression patterns in HCCs from Myc, E2f1 and Myc E2f1 transgenic mice were most similar to those of the better survival group of human HCCs, whereas the expression patterns in HCCs from Myc Tgfa transgenic mice and in diethylnitrosamine-induced mouse HCCs were most similar to those of the poorer survival group of human HCCs. Gene expression patterns in HCCs from Acox1−/−
mice and in ciprofibrate-induced HCCs were least similar to those observed in human HCCs. We conclude that our approach can effectively identify appropriate mouse models to study human cancers.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
4-methylumbelliferone (4MU) is a well-known hyaluronic acid synthesis inhibitor and an approved drug for the treatment of cholestasis. In animal models, 4MU decreases inflammation, reduces fibrosis, ...and lowers body weight, serum cholesterol, and insulin resistance. It also inhibits tumor progression and metastasis. The broad spectrum of effects suggests multiple and yet unknown targets of 4MU. Aiming at 4MU target deconvolution, we have analyzed publicly available data bases, including: 1. Small molecule library Bio Assay screening (PubChemBioAssay); 2. GO pathway databases screening; 3. Protein Atlas Database. We also performed comparative liver transcriptome analysis of mice on normal diet and mice fed with 4MU for two weeks. Potential targets of 4MU public data base analysis fall into two big groups, enzymes and transcription factors (TFs), including 13 members of the nuclear receptor superfamily regulating lipid and carbohydrate metabolism. Transcriptome analysis revealed changes in the expression of genes involved in bile acid metabolism, gluconeogenesis, and immune response. It was found that 4MU feeding decreased the accumulation of the glycogen granules in the liver. Thus, 4MU has multiple targets and can regulate cell metabolism by modulating signaling via nuclear receptors.
Abstract
Reactive oxygen/nitrogen species (ROS/RNS) are formed during normal cellular metabolism and contribute to its regulation, while many pathological processes are associated with ROS/RNS ...imbalances. Modern methods for measuring ROS/RNS are mainly based on the use of inducible fluorescent dyes and protein-based sensors, which have several disadvantages for in vivo use. Intravital electrochemical nanosensors can be used to quantify ROS/RNS with high sensitivity without exogenous tracers and allow dynamic ROS/RNS measurements in vivo. Here, we developed a method for quantifying total ROS/RNS levels in the liver and evaluated our setup in live mice using three common models of liver disease associated with ROS activation: acute liver injury with CCl
4
, partial hepatectomy (HE), and induced hepatocellular carcinoma (HCC). We have demonstrated using intravital electrochemical detection that any exposure to the peritoneum in vivo leads to an increase in total ROS/RNS levels, from a slight increase to an explosion, depending on the procedure. Analysis of the total ROS/RNS level in a partial hepatectomy model revealed oxidative stress, both in mice 24 h after HE and in sham-operated mice. We quantified dose-dependent ROS/RNS production in CCl
4
-induced injury with underlying neutrophil infiltration and cell death. We expect that in vivo electrochemical measurements of reactive oxygen/nitrogen species in the liver may become a routine approach that provides valuable data in research and preclinical studies.
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