Cyclophilin A Impairs Efferocytosis and Accelerates Atherosclerosis by Overexpressing CD 47 and Down-Regulating Calreticulin

Impairment of efferocytosis in apoptotic macrophages is a known determinant of the severity of atherosclerosis and the vulnerability of plaques to rupture. The precise mechanisms involved in impaired efferocytosis are unclear. Given the well-recognized role of the inflammatory cytokine cyclophilin A (Cyp A) in modulating several atherogenic mechanisms in high-glucose primed monocytes, we investigated the role of Cyp A in macrophage efferocytosis. The efficiency of efferocytosis in RAW 264.7 macrophages grown in vitro and primed with cyclophilin A was assessed using flow cytometry and confocal assays. Cholesterol content in cells was measured using cell-based cholesterol efflux assay. Proteomic analysis and bioinformatics tools were employed to decipher the link between cyclophilin A and the known ligand receptors involved in efferocytosis. Cyclophilin A was found to impair efferocytosis in apoptotic macrophages by reducing ABCA1-mediated cholesterol efflux in foam cells derived from macrophages. Cyclophilin A-primed macrophages showed an increase in expression of the don’t-eat-me signal CD 47 and a decrease in the expression of the eat-me signal, calreticulin. Phagocytosis was restored upon silencing of cyclophilin A. New Zealand white rabbits were fed a high-fat diet, and lesions in their aortae were analyzed histologically for evidence of atherosclerosis and the expression of Cyp A, CD 47 and calreticulin, the ligand receptor involved in efferocytosis. Gene and protein expressions in aortae and macrophages were analyzed by real-time PCR and Western blotting. Cyclophilin A, via its effects on the expression of CD 47 and calreticulin, impairs efferocytosis in apoptotic macrophages. Together with its impact on cholesterol efflux from macrophages, these effects can amplify other mechanisms of Cyp A in accelerating the progression of atherosclerosis.

Cyclophilin A regulates A549 cells apoptosis via stabilizing Twist1 protein

Cyclophilin A (CypA) is an essential member of the immunophilin family. As an intracellular target of immunosuppressive drug cyclosporin A (CsA) or a peptidyl-prolyl cis/trans isomerase (PPIase), it catalyzes the cis-trans isomerization of proline amidic peptide bonds, through which, it regulates a variety of biological processes, such as intracellular signaling, transcription, and apoptosis. In this study, we found that intracellular CypA enhanced Twist1 phosphorylation at Ser68 and inhibited apoptosis in A549 cells. Mechanistically, CypA could mediate the phosphorylation of Twist1 at Ser68 via p38 MAPK, which inhibited its ubiquitination-mediated degradation. In addition, CypA increased Twist–p65 interaction and nuclear accumulation, which regulated Twist1-dependent expression of CDH1 and CDH2. Our findings collectively indicated the role of CypA in Twist1-mediated A549 cells apoptosis through stabilizing Twist1 protein.

Tanshinone IIA Shows Higher Antiproliferative Activities than Sinapic Acid in 4 Cancer Cell Lines and Simultaneously Induces Apoptosis and Necroptosis in Human Lung Cancer A549 Cells

Tanshinone IIA (Tan IIA) and sinapic acid (SA) are 2 components separately isolated from 2 Asian medicinal plants, Hydnophytum formicarum Jack and Salvia miltiorrhiza Bunge. The antitumor activities of them were worth exploring, therefore, we examined their antitumor activities in A549, HCT116, HeLa, and Colo320 cancer cell lines by means of WST-1 assay. The results show that Tan IIA exerted far higher (IC50 from 1.0 ± 0.0 to 166.3 ± 24.0 µg/mL) antiproliferative activities than SA (IC50 from 2236.3 ± 484.1 to >10 000.0 µg/mL). Of the 4 cell lines, A549 cells were the most sensitive to Tan IIA; thus, we used Western blotting to explore the cytotoxic mechanisms of Tan IIA in A549 cells and found that they rely on simultaneous induction of apoptosis and necroptosis in the cells. Apoptosis was hallmarked by the induction of cleaved caspase-3 by Tan IIA and necroptosis by the necroptotic marker proteins cyclophilin A and high mobility group box 1 (HMGB1), as well as increased lactate dehydrogenase (LDH) activities. The necroptotic effect was confirmed by the necroptosis inhibitor necrostatin-1 (Nec-1), which eliminated these effects and restored cell survival rates. The levels of cyclophilin A decreased in response to the pan-caspase inhibitor z-VAD-fmk, and those of cleaved caspase-3 decreased in response to Nec-1. Conclusively, Tan IIA has the potential to prevent lung cancer and the mechanism seems to be apoptosis and necroptosis, of which the relationship is mutually interdependent. This is the first report of Tan IIA eliciting necroptosis in A549 cells. Tan IIA may be used for necroptosis-based cancer therapy, especially to overcome apoptosis resistance.

Intravenous injection of cyclophilin A realizes the transient and reversible opening of barrier of neural vasculature through basigin in endothelial cells

Neural vasculature forms the blood–brain barrier against the delivery of systemically administered therapeutic drugs into parenchyma of neural tissues. Therefore, procedures to open the blood–brain barrier with minimal damage to tissues would lead to the great progress in therapeutic strategy for intractable neural diseases. In this study, through analyses with mouse in vitro brain microvascular endothelial cells and in vivo neural vasculature, we demonstrate that the administration of cyclophilin A (CypA), a ligand of basigin which is expressed in barrier-forming endothelial cells, realizes the artificial opening of blood–brain barrier. Monolayers of endothelial cells lost their barrier properties through the disappearance of claudin-5, an integral tight junction molecule, from cell membranes in a transient and reversible manner. Furthermore, the intravenous injection of a single dose of CypA into mice resulted in the opening of blood–brain barrier for a certain period which enabled the enhanced delivery of systemically administered doxorubicin into the parenchyma of neural tissues. These findings that the pre-injection of a single dose of CypA realizes an artificial, transient as well as reversible opening of blood–brain barrier are considered to be a great step toward the establishment of therapeutic protocols to overcome the intractability of neural diseases.