Oxidized low-density lipoprotein stimulates dendritic cells maturation via LOX-1-mediated MAPK/NF-κB pathway.

Dendritic cells (DCs) play a crucial role as central orchestrators of immune system response in atherosclerosis initiation and progression. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the immune maturation of DCs, but the underlying mechanisms remain unclear. We isolated mouse bone marrow progenitors and stimulated them with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce immature DCs. We then treated DCs with oxidized low-density lipoprotein (oxLDL) to induce maturation. LOX-1 siRNA was used to investigate the modulation of LOX-1 on the development of DCs and the underlying signal pathways. CD11c-positive DCs were successfully derived from mouse bone marrow progenitors. OxLDL promoted the expressions of DCs maturation markers and pro-inflammatory cytokines. OxLDL also upregulated LOX-1 expression and activated MAPK/NF-κB pathways. LOX-1 siRNA could attenuate the expression of MAPK/NF-κB pathways and inflammatory cytokines. In conclusion, oxLDL induced the maturation of DCs via LOX-1-mediated MAPK/NF-κB pathway, which contributed to the initiation and progression of atherosclerosis.

Oxidized low-density lipoprotein stimulates dendritic cells maturation via LOX-1-mediated MAPK/NF-κB pathway

Dendritic cells (DCs) play a crucial role as central orchestrators of immune system response in atherosclerosis initiation and progression. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is involved in the immune maturation of DCs, but the underlying mechanisms remain unclear. We isolated mouse bone marrow progenitors and stimulated them with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 to induce immature DCs. We then treated DCs with oxidized low-density lipoprotein (oxLDL) to induce maturation. LOX-1 siRNA was used to investigate the modulation of LOX-1 on the development of DCs and the underlying signal pathways. CD11c-positive DCs were successfully derived from mouse bone marrow progenitors. OxLDL promoted the expressions of DCs maturation markers and pro-inflammatory cytokines. OxLDL also upregulated LOX-1 expression and activated MAPK/NF-κB pathways. LOX-1 siRNA could attenuate the expression of MAPK/NF-κB pathways and inflammatory cytokines. In conclusion, oxLDL induced the maturation of DCs via LOX-1-mediated MAPK/NF-κB pathway, which contributed to the initiation and progression of atherosclerosis.

MiR-335 promotes cell proliferation by inhibiting MEF2D and sensitizes cells to 5-Fu treatment in gallbladder carcinoma.

OBJECTIVE: Gallbladder carcinoma is a malignant tumor in the bile duct with poor prognosis. Although aberrant expression of miR-335 has been reported in the tumor tissues of gallbladder carcinoma, the biological role of miR-335 was still largely unknown. This study was intended to explore the role of miR-335 in the progression of gallbladder carcinoma. PATIENTS AND METHODS: The gallbladder carcinoma cell lines GBC-SD and SGC-996 were used in our study. MiR-335 mimic, miR-335 inhibitor, and si-myocyte enhancer factor 2D (MEF2D) were transfected into gallbladder carcinoma cells, respectively. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay analysis was used to determine cell viability. The colony formation was also analyzed. Cell cycle progression was determined using flow cytometer. To verify the target gene of miR-335, the luciferase assay was used. RESULTS: MiR-335 overexpression inhibited cell viability and colony formation of GBC-SD and SGC-996 cells. The percentage of cells in first gap phase (G1)/resting phase (G0) was significantly increased, and the expression of cell division cycle 2 (cdc2) and cell division cycle 25 (cdc25) was decreased after miR-335 was overexpressed, indicating its role in inducing the cell cycle arrest of GBC-SD and SGC-996 cells. MEF2D was up-regulated in gallbladder cancer and associated with tumor size and clinical stage. Down-regulation of MEF2D inhibited cell viability and colony formation, induced cell cycle arrest in G1/G0 phase, and decreased the expression of cdc2 and cdc25 in GBC-SD and SGC-996 cells. Bioinformatics analysis by TargetScan and luciferase assay verified that MEF2D could be targeted by miR-335. Importantly, the effects of miR-335 inhibitor on cell growth were rescued by small interfering RNA of MEF2D (siMEF2D) in GBC-SD and SGC-996 cells. Besides, miR-335 overexpression increased cell sensitivity to 5-Fluoracil (Fu) treatment and decreased the expression levels of ATP-binding cassette transporter B1 (ABCB1) and ATP-binding cassette G2 (ABCG2) in GBC-SD and SGC-996 cells. CONCLUSIONS: MiR-335 participates in the progression of gallbladder carcinoma by targeting MEF2D. MiR-335 may be a potential therapeutic target for gallbladder carcinoma.

Development of a rinsing separation method for exosome isolation and comparison to conventional methods.

OBJECTIVE: Exosomes contain valuable biomarkers for many diseases. Tragically, standardized isolation methods and subsequent characterization criteria for exosomes remain limited. Therefore, we developed a new exosome isolation method, termed rinsing separation, and compared its advantages and weaknesses relative to the existing ultracentrifugation and ExoQuick precipitation methods. MATERIALS AND METHODS: Rinsing separation utilizes heparin and glutaraldehyde as a fixative to isolate exosomes, and was developed using the culture supernatant from mesenchymal stem cells (MSCs). The isolated exosomes were characterized and compared by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blot. RESULTS: Consistent with known exosome parameters, exosomes isolated using each method ranged in size from 30 to 150 nm and demonstrated the characteristic cup-shaped morphology. Moreover, the exosome markers CD63 and TSG101 were observed in the lysate of all exosome samples that were isolated using each method. Several advantages and drawbacks were noted for each exosome isolation method. Most notably, ultracentrifugation resulted in fewer, but highly pure, exosomes, and samples generated using the ExoQuick precipitation method contained the most contaminating debris. Samples obtained using pour rinsing separation method represented an amalgam of these two fractions, but were isolated in significantly less time. CONCLUSIONS: In this study, we propose rinsing separation as a new method of isolating exosomes. This method is convenient, and the resulting exosomes are highly pure. Moreover, rinsing separation offers time- and cost-efficiency advantages, making it a promising approach for exosome isolation for clinical applications.

[Safety and efficacy of rotational atherectomy in the interventional treatment of coronary chronic total occlusion lesions].

Objective: To investigate the safety and efficacy of rotational atherectomy in the interventional treatment of coronary chronic total occlusion lesions. Methods: In this retrospective study,a total of 31 consecutive patients with coronary chronic total occlusion(CTO) lesions underwent rotational atherectomy in our hospital from February 2004 to December 2016 were enrolled,and the clinical features were analyzed. Coronary atherectomy was performed if balloon failed to cross the CTO lesions or balloon could not be fully dilated in the CTO lesions after wire crossing. The definition of procedure success was defined as residual stenosis less than 20% after implantation of drug eluting stent and rotational atherectomy. After the procedure, the patients were followed up to observe major adverse cardiac and cerebral vascular events which including cardiogenic death, myocardial infarction, cerebrovascular accident, and target lesion revascularization. Results: The 1.25 mm diameter burr was firstly selected in 80.6% (25/31) patients,and 96.8%(30/31) patients used only 1 burr to complete the rotational atherectomy procedure. The complication rate was 9.8% (3/31) including 1 patient with coronary dissection and 3 patients with slow flow or no flow. There was 1 patent with both coronary dissection and slow flow. The procedure success rate was 96.8%(30/31). Interventional treatment related myocardial infarction occurred in 3 patients during hospitalization.The 30 patients with procedure success were followed up 36(11, 96) months. The incidence rate of major adverse cardiac and cerebral vascular events was 13.3% (4/30), of which the cardiogenic death rate was 3.3% (1/30), the myocardial infarction rate was 6.7% (2/30), cerebrovascular accident rate was 3.3%(1/30),and the target lesion revascularization rate was 6.7% (2/30). Conclusion: Rotational atherectomy is safe and effective in the interventional treatment of coronary CTO lesions.

Molecular authentication of Gynostemma pentaphyllum through development and application of random amplification polymorphic DNA sequence-characterized amplified region marker.

Due to the morphological similarities of aerial parts, it is difficult to distinguish Gynostemma pentaphyllum from Cayratia japonica, which is usually an adulterant of the former. To develop a reliable method for the identification and authentication of G. pentaphyllum, a combination of random amplification polymorphic DNA (RAPD) technique with sequence-characterized amplified region (SCAR) markers was studied. Twenty-five samples of G. pentaphyllum and two samples of C. japonica were collected from different regions in Guangxi or bought from different provinces in China. Through the RAPD analysis, significant genetic polymorphism was observed among the intraspecies samples of G. pentaphyllum. Furthermore, a specific marker, J-750, was obtained for authentication. Therefore, the SCAR marker for G. pentaphyllum (359 bp) was developed from the RAPD amplicon. With PCR amplification using the SCAR primers, a specific band of 359 bp was distinctly visible for all tested samples of G. pentaphyllum, but was absent in the samples of C. japonica. Furthermore, the results revealed that the SCAR marker was useful for the identification and authentication of G. pentaphyllum irrespective of whether samples were fresh, dry, or of commercial origin. The SCAR marker obtained in this study successfully authenticated G. pentaphyllum through an integrated PCR system containing SCAR and control primer combinations of two pairs. In addition, it was also used for simultaneous discrimination of G. pentaphyllum from C. japonica.

Impact of age on epicardial and pericoronary adipose tissue volume.

OBJECTIVE: The aim of the present study was to investigate the impact of age on epicardial and pericoronary adipose tissue volume. PATIENTS AND METHODS: Eighty healthy individuals with normal body mass index underwent multi-slice computed tomography (MSCT) with coronary computed tomography angiography, and their scanning images were stored and analysed. Among them, 62 subjects were male, and 18 subjects were female. The patients were grouped by age: 10 subjects were < 35 yrs, 20 were 35-44 yrs, 20 were 45-54 yrs, 20 were 55-64 yrs, and 10 were > 65 yrs. Pericoronary adipose tissue (PCAT), and the volume and the thickness of epicardial adipose tissue (EAT) were measured. RESULTS: The correlation analysis showed that age was positively correlated to EAT volume. Moreover, spearman correlation analysis showed that the volumes of PCAT in the left main-left anterior descending artery (LM-LAD) and right coronary artery (RCA) gradually increased with increasing age, but not with the left circumflex artery (LCX) and EAT thickness (p > 0.05). CONCLUSIONS: These findings suggest that the volumes of EAT and the adipose tissue surrounding the LM-LAD and RCA increase with increasing age.

Assessment of urinary N-acetyl-ß-glucosaminidase as an early marker of contrast-induced nephropathy.

Early detection and timely intervention are important for improving contrast-induced nephropathy (CIN) prognosis. Whether urinary N-acetyl-ß-glucosaminidase (NAG) is a useful marker for early detection of CIN was investigated in 590 patients undergoing diagnostic coronary angiography (CA) and/or therapeutic percutaneous coronary intervention (PCI) for acute coronary syndromes or stable angina, and who received low-osmolality nonionic contrast agent. Urinary NAG, osmolality and serum creatinine were measured before and 1, 2 and 6 days after contrast agent exposure. CIN occurred in 33 patients; its incidence in high-risk patients (pre-existing renal dysfunction with/without diabetes mellitus) was significantly higher than in others. In patients with CIN, urinary NAG and serum creatinine levels on days 1 and 2 were significantly higher than at baseline and compared with patients without CIN; mean levels were gradually returning to baseline by day 6. Compared with serum creatinine, urinary NAG levels peaked earlier in CIN patients and increased much more. The results suggest that, following CA and/or PCI, CIN occurs to a certain degree and that NAG may be a useful early CIN marker as it is noninvasive, simple, inexpensive and sensitive.