Smoking is a major risk factor for atherosclerosis. In this study, we evaluated the effects of benzo[a]pyrene (BaP, a prominent component of tobacco smoke) on the function and pro-inflammatory response of human endothelial progenitor cells (EPCs). EPCs were isolated from umbilical cord blood and treated with different concentrations (10, 20 and 50 µmol/l) of BaP. The proliferation, migration, adhesion and angiogenesis of BaP-treated EPCs were evaluated using the cell counting kit-8 (CCK-8), Transwell assay, adhesion assay and in vitro tube formation assay, respectively. The activation of nuclear factor-κB (NF-κB) was evaluated by measuring the mRNA expression of NF-κB p65 and p50 by real-time RT-PCR and NF-κB translocation assay. Reactive oxygen species (ROS) production was determined by the reduction of fluorescent 2',7'-dichlorofluorescein diacetate (DCFH-DA). The results demonstrated that BaP treatment significantly inhibited the proliferation, migration, adhesion and angiogenesis of EPCs in vitro. In addition, BaP induced the release of interleukin (IL)-1β and tumor necrosis factor-α from these cells. Moreover, the exposure of EPCs to BaP induced ROS generation and the activation of NF-κB. Experiments with EPCs pre-treated with pyrrolidine dithiocarbamate, an inhibitor of NF-κB, revealed that the BaP-mediated inhibition of proliferation, migration, adhesion and angiogenesis of EPCs is mainly regulated by NF-κB. Thus, tobacco smoke may induce oxidant-mediated stress responses in EPCs and impair their function via the activation of the NF-κB pathway.

Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro.In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by ␤-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.

BaP promotes Ang II-induced AAA formation in mice via elevating infiltration of macrophages, activating nuclear factor-κB, upregulating the expression of MMP-2, MMP-9, and MMP-12, and increasing the apoptosis of vascular muscle cells in its synergistic effect with Ang II in aortic wall.

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Background. Abdominal aortic aneurysm (AAA) is a fatal disease characterized by high morbidity and mortality in old population. Globally, effective drugs for AAA are still limited. Si-Miao-Yong-An decoction (SMYAD), a traditional Chinese medicine (TCM) formula with a high medical value, was reported to be successfully used in an old AAA patient. Thus, we reason that SMYAD may serve as a potential anti-AAA regime. Objective. The exact effects and detailed mechanisms of SMYAD on AAA were explored by using the experimental study and bioinformatics analysis. Methods. Firstly, C57BL/6N mice induced by Bap and Ang II were utilized to reproduce the AAA model, and the effects of SMYAD were systematically assessed according to histology, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). Then, network pharmacology was applied to identify the biological processes, pathways, and hub targets of SMYAD against AAA; moreover, molecular docking was utilized to identify the binding ability and action targets. Results. In an animal experiment, SMYAD was found to effectively alleviate the degree of pathological expansion of abdominal aorta and reduce the incidence of Bap/Ang II-induced AAA, along with reducing the damage to elastic lamella, attenuating infiltration of macrophage, and lowering the circulating IL-6 level corresponding to the animal study, and network pharmacology revealed the detailed mechanisms of SMYAD on AAA that were related to pathways of inflammatory response, defense response, apoptotic, cell migration and adhesion, and reactive oxygen species metabolic process. Then, seven targets, IL-6, TNF, HSP90AA1, RELA, PTGS2, ESR1, and MMP9, were identified as hub targets of SMYAD against AAA. Furthermore, molecular docking verification revealed that the active compounds of SMYAD had good binding ability and clear binding site with core targets related to AAA formation. Conclusion. SMYAD can suppress AAA development through multicompound, multitarget, and multipathway, which provides a research direction for further study.