Kallistatin (KS) has been recognized as a plasma protein with anti-inflammatory functions. Macrophages are the primary inflammatory cells in atherosclerotic plaques. However, it is unknown whether KS plays a role in macrophage development and the pathogenesis of atherosclerosis. This study investigated the role of KS in macrophage development, a key pathological process in atherosclerosis. An atherosclerosis model was established in ApoE mice via partial left carotid artery (PLCA) ligation. An adenovirus vector (Ad. HKS) containing the human KS gene was delivered via the tail vein before PLCA ligation. The mice were divided into two groups: the PLCA + Ad. HKS and PLCA + adenovirus vector (Ad. Null) groups and followed for 2 and 4 weeks. Human KS was expressed in the mice after KS gene delivery. In addition, KS significantly inhibited plaque formation and reduced inflammation in the plaques and liver 4 weeks after gene delivery. Moreover, KS gene delivery significantly increased the expression of interleukin-10 and Arginase 1, which are M2 macrophage markers, and reduced the expression of inducible nitric oxide synthase and monocyte chemotactic protein 1, which are M1 macrophage markers. Furthermore, in cultured RAW 264.7 macrophages, KS significantly stimulated M2 marker expression and differentiation and decreased M1 marker expression, as determined by flow cytometry and real-time polymerase chain reaction. These effects were blocked by Krüppel-like factor 4 small-interfering RNA oligonucleotides. These findings demonstrate that KS inhibits atherosclerotic plaque formation and regulates M1/M2 macrophage polarization via Krüppel-like factor 4 activation.

Background. Circulating endothelial progenitor cells (EPCs) play important roles in vascular repair. However, the mechanisms of high-glucose- (HG-) induced cord blood EPC senescence and the role of B2 receptor (B2R) remain unknown. Methods. Cord blood samples from 26 patients with gestational diabetes mellitus (GDM) and samples from 26 healthy controls were collected. B2R expression on circulating CD34+ cells of cord blood mononuclear cells (CBMCs) was detected using flow cytometry. The plasma concentrations of 8-isoprostaglandin F2α (8-iso-PGF2α) and nitric oxide (NO) were measured. EPCs were treated with HG (40 mM) alone or with bradykinin (BK) (1 nM). The B2R and eNOS small interfering RNAs (siRNAs) and the PI3K antagonist LY294002 were added to block B2R, eNOS, and PI3K separately. To determine the number of senescent cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed. The level of mitochondrial reactive oxygen species (ROS) in EPCs was assessed by Mito-Sox staining. Cell viability was evaluated by Cell Counting Kit-8 (CCK-8) assays. Mitochondrial DNA (mtDNA) copy number and the relative length of telomeres were detected by real time-PCR. The distribution of human telomerase reverse transcriptase (hTERT) in the nucleus, cytosol, and mitochondria of EPCs was detected by immunofluorescence. The expression of B2R, p16, p21, p53, P-Ser473AKT, T-AKT, eNOS, and hTERT was demonstrated by Western blot. Results. B2R expression on circulating CD34+ cells of CBMCs was significantly reduced in patients with GDM compared to healthy controls. Furthermore, B2R expression on circulating CD34+ cells of CBMCs was inversely correlated with plasma 8-iso-PGF2α concentrations and positively correlated with plasma NO levels. BK treatment decreased EPC senescence and ROS generation. Furthermore, BK treatment of HG-exposed cells led to elevated P-Ser473AKT and eNOS protein expression compared with HG treatment alone. BK reduced hTERT translocation in HG-induced senescent EPCs. B2R siRNA, eNOS siRNA, and antagonist of the PI3K signalling pathway blocked the protective effects of BK. Conclusion. BK, acting through PI3K-AKT-eNOS signalling pathways, reduced hTERT translocation, increased the relative length of telomeres while reducing mtDNA copy number, and finally protected against EPC senescence induced by HG.