High Mobility Group B-1 (HMGB-1) Promotes Apoptosis of Macrophage-Derived Foam Cells by Inducing Endoplasmic Reticulum Stress.

BACKGROUND/AIMS: High mobility group B-1 (HMGB-1)-induced endoplasmic reticulum stress (ERS) has been implicated in inflammation and dendritic cell maturation. C/EBP-homologous protein (CHOP) is a vital component of ERS and apoptosis and plays a critical role in atherosclerosis. However, only a little information is available about the role of HMGB-1 in foam cell formation. Thus, the role of HMGB-1-induced ERS/CHOP pathway in apoptosis and formation of macrophage-derived foam cells is investigated. METHODS: RAW264.7 cells were treated with oxidized low-density lipoprotein (oxLDL) in the absence and/or presence of HMGB-1, N-acetylcysteine (NAC, an antioxidant), glycyrrhizin (Gly, an HMGB-1 inhibitor), tunicamycin (TM, an ERS inducer), and 4-phenylbutyrate (4-PBA, an ERS inhibitor). Reactive oxygen species (ROS) production was examined by dihydroethidium (DHE) staining. Oil Red O staining, intracellular total cholesterol assay, and Dil-oxLDL uptake assay evaluated the accumulation of lipids in macrophages. Cell apoptosis was measured by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Western blot detected the expression of HMGB-1/ERS/CHOP pathway. RESULTS: oxLDL induced HMGB-1 translocation and secretion in a dose- and time-dependent manner, which was inhibited by NAC. oxLDL-induced lipid accumulation in macrophages was promoted synergistically by HMGB-1 that was attenuated by Gly. Moreover, TM synergized with oxLDL induced lipid accumulation and apoptosis of macrophages; however, 4-PBA alleviated the oxLDL-induced apoptotic foam cells. Additionally, the inhibition of ERS with 4-PBA suppressed the expression of HMGB-1-induced CHOP. CONCLUSIONS: OxLDL triggered HMGB-1 secretion in macrophages via oxidative stress. Furthermore, HMGB-1 promoted the formation and apoptosis of macrophage-derived foam cells via activation of ERS/CHOP pathway.

Diabetes-Induced Oxidative Stress in Endothelial Progenitor Cells May Be Sustained by a Positive Feedback Loop Involving High Mobility Group Box-1.

Oxidative stress is considered to be a critical factor in diabetes-induced endothelial progenitor cell (EPC) dysfunction, although the underlying mechanisms are not fully understood. In this study, we investigated the role of high mobility group box-1 (HMGB-1) in diabetes-induced oxidative stress. HMGB-1 was upregulated in both serum and bone marrow-derived monocytes from diabetic mice compared with control mice. In vitro, advanced glycation end productions (AGEs) induced, expression of HMGB-1 in EPCs and in cell culture supernatants in a dose-dependent manner. However, inhibition of oxidative stress with N-acetylcysteine (NAC) partially inhibited the induction of HMGB-1 induced by AGEs. Furthermore, p66shc expression in EPCs induced by AGEs was abrogated by incubation with glycyrrhizin (Gly), while increased superoxide dismutase (SOD) activity in cell culture supernatants was observed in the Gly treated group. Thus, HMGB-1 may play an important role in diabetes-induced oxidative stress in EPCs via a positive feedback loop involving the AGE/reactive oxygen species/HMGB-1 pathway.

[Construction of recombinant adenovirus vector of calcitonin gene-related peptide gene and transfection to neonatal rat cardiomyocytes].

OBJECTIVE: To construct a recombinant adenovirus vector of calcitonin gene-related peptide (CGRP) by AdEasy system and to validate its expression in myocardial cells. METHODS: The full-length of CGRP gene cDNA was acquired by RT-PCR and cloned into pShuttle-CMV. After linearization with Pme I, the recombinant plasmid (pShuttle-CMV-CGRP) was transformed into E.coli BJ5183 by electroporation to construct the recombinant adenovirus plasmid AdEasy-pShuttle-CGRP. The recombinant adenovirus plasmids were transformed into E.coli XL10-Gold cells to be amplified. Then the recombinant plasmid was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. PCR technique was used to detect target gene. The recombinant adenovirus particles were purified by CsC1 density gradient. The purified recombinant adenovirus was transfected to neonatal rat cardiomyocytes,and the recombinant adenovirus production was observed by fluorescent microscope. Expression of CGRP in hearts 7 days after intravenous delivery of adenoviral vectors AV-CGRP was determined by radioimmunoassay. RESULT: The RT-PCR products confirmed a full-length cDNA of CGRP gene in PUC(57) by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pShuttle-CMV. The recombinant adenovirus plasmid AdEasy-pShuttle-CGRP was digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3 kb and 30 kb. PCR analysis and fluorescent microscope observation confirmed that the CGRP gene was inserted into the adenovirus vector with very strong power of transfection. The recombinant adenovirus particles infected neonatal rat cardiomyocytes successfully. Radioimmunoassay showed that delivery of AV-CGRP significantly increased the expression of CGRP in mice hearts. CONCLUSION: The recombinant adenovirus vector of CGRP gene has been constructed,and it can infect neonatal rat cardiomyocytes successfully. Somatic delivery of CGRP gene can significantly increase the expression of CGRP in mice hearts. The results may provide a sound foundation for further study on the value of CGRP as the target for gene therapy in both laboratory and clinical trials.