The Regulation of Exosome-Derived miRNA on Heterogeneity of Macrophages in Atherosclerotic Plaques.

Exosomes are nanosized vesicles secreted by most cells, which can deliver a variety of functional lipids, proteins, and RNAs into the target cells to participate in complex intercellular communications. Cells respond to certain physical, chemical, and biological stimuli by releasing exosomes. Exosomes are rich in small molecules of RNA, including miRNAs and mRNAs, which have been demonstrated to have certain functions in recipient cells. Recent studies on single-cell RNA sequences have revealed the transcription and the heterogeneity of macrophages in Ldlr-/-mice fed with a high-fat diet. Five macrophage populations were found in the atherosclerotic plaques. It is worth noting that these subset populations of macrophages seem to be endowed with different functions in lipid metabolism and catabolism. A total of 100 differentially expressed mRNAs were selected for these subset populations. Importantly, these macrophage populations were also present in human advanced atherosclerosis. To clarify the specific functions and the regulatory mechanism of these macrophage populations, we extracted exosome RNAs from the plasma of patients with chronic coronary artery disease (CAD) and performed RNA sequencing analysis. Compared with the healthy control, a total of 14 miRNAs were significantly expressed in these patients. A total of 5,248 potential mRNAs were predicted by the bioinformatics platform. Next, we determined the outcome of the intersection of these predicted mRNAs with 100 mRNAs expressed in the above-mentioned five macrophage populations. Based on the screening of miRNA-mRNA pairs, a co-expression network was drawn to find out the key RNAs. Three down-regulated miRNAs and five up-regulated mRNAs were selected for validation by real-time RT-PCR. The results showed that the expression of miR-4498 in plasma exosomes was lower than that in the healthy control, and the expressions of Ctss, Ccr2 and Trem2 mRNA in peripheral blood mononuclear cells isolated from CAD patients were higher. In order to clarify the regulatory mechanism, we established a co-culture system in vitro. Studies have shown that the uptake of exosomes from CAD patients can up-regulate the expression of Ctss, Trem2, and Ccr2 mRNA in THP-1 cells induced by lipopolysaccharide. Our findings revealed a unique relationship between the transcriptional signature and the phenotypic heterogeneity of macrophage in the atherosclerotic microenvironment.

Tanshinone IIA protects mice against atherosclerotic injury by activating the TGF-ß/PI3K/Akt/eNOS pathway.

OBJECTIVE: Explored the mechanism of action of tanshinone IIA (TIIA) against atherosclerosis. METHODS: ApoE mice were divided into two groups of 10: model and TIIA. A control group of 10 wild-type mice was created. ApoE mice were fed a high-fat diet for 12 weeks. The TIIA group received TIIA once daily. Mice were anesthetized, blood collected by cardiac puncture, and the aortic sinus/arch collected for histology and molecular studies, respectively. RESULTS: Mice intima in the model group had large areas of plaque formation, serum levels of total cholesterol (TC), triglycerides, and low-density lipoprotein-cholesterol (LDL-C) increased significantly, and high-density lipoprotein-cholesterol (HDL-C) levels decreased significantly in the model group after 12 weeks. Staining [hematoxylin and eosin (H&E), Oil-Red-O] showed that the aorta had lesions, a higher degree of plaque formation, and considerable lipid deposition in model-group mice. After TIIA treatment, expression of HDL-C was increased significantly and that of TC, triglycerides and LDL-C decreased significantly, and plaque size and lipid deposition improved obviously. Analyses of protein phosphorylation in aortic tissue suggested that the transforming growth factor (TGF)-ß/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway was activated in TIIA-treated mice. CONCLUSION: TIIA can lower levels of serum lipids, stabilize atherosclerotic plaques, reduce endothelial injury, and inflammatory damage by activation of the TGF-ß/PI3K/Akt/eNOS pathway.

HMGN5 knockdown sensitizes prostate cancer cells to ionizing radiation.

BACKGROUND: High Mobility Group N (HMGN) proteins are a family of chromatin structural proteins that specifically bind to nucleosome core particles. HMGN5 is a novel and characteristic member of the HMGN protein family. We have previously found that HMGN5 is upregulated in prostate cancer and its downregulation had been demonstrated to induce apoptosis and G2-M cell cycle arrest. METHODS: The radiosensitization effect of HMGN5 knockdown on PC3 and DU145 cells was assessed using clonogenic assay, flow cytometry, and comet assay. The DNA double-strand break (DSB) repair kinetics of HMGN5 knockdown and control cells after radiation exposure was evaluated using immunocytofluorescence. The mitochondrial reactive oxygen species (ROS) levels were estimated using Dihydrorhodamine 123 (DHR 123) probes. Expression of mitochondrial antioxidant MnSOD was measured by real-time PCR and Western blot. The expression of antiapoptotic proteins Bcl-2 and Bcl-xL as well as cleavage of caspase-3, caspase-9, and PARP were also measured using Western blot. RESULTS: HMGN5 knockdown cells exhibit decreased clonogenic survival and increased apoptosis rate in response to 2-8 Gy ionizing radiation (IR). Loss of HMGN5 does not affect the DSB repair kinetics after radiation exposure. HMGN5 knockdown cells demonstrated increased mitochondrial ROS level and suppressed induction of MnSOD upon radiation compared with control cells upon radiation. Further, MnSOD knockdown resulted in inhibited cell viability as well as increased mitochondrial ROS level and apoptosis upon radiation in PC3 and DU145 cells. Finally, HMGN5 knockdown cells showed significantly decreased levels of antiapoptotic proteins Bcl-2 and Bcl-xL as well as increased cleavage of caspase-3, caspase-9, and PARP compared with control cells after radiation. CONCLUSIONS: HMGN5 knockdown sensitizes prostate cancer cells to ionizing radiation, and the radiosensitization effect may be partially mediated through suppressed induction of MnSOD and enhanced activation of apoptosis pathway in response to IR.

A preclinical in vivo investigation of high-intensity focused ultrasound combined with radiotherapy.

This study aims to perform an in vivo investigation evaluating the injury to the pancreas and adjacent tissue of swine resulting with high-intensity focused ultrasound (HIFU) combined with radiotherapy (RT). The protocol was approved by the animal ethics committee at the Peking University First Hospital. A total of 12 domestic swine were divided into four groups: control, HIFU only, RT only and HIFU + RT. The injury to the pancreas, adjacent tissue and tissue within the acoustic path of the HIFU beam was assessed based on gross and histologic findings. For the targeted region of the pancreas, the score of the combined group was higher than that of the HIFU group and there was significant difference. For the acoustic path tissue, there was no significant difference except between the control group and the other groups. HIFU combined with RT increased the injury to the targeted pancreas, without increased injury to tissue outside of the targeted region.