Midkine Promote Atherosclerosis by Regulating the Expression of ATP-Binding Cassette Transporter A1 via Activator Protein-1

Purpose: Midkine (MK) has been shown to facilitate atherosclerotic plaque formation by downregulating the expression of ATP-binding cassette transporter A1 (ABCA1). However, the mechanism by which MK regulates ABCA1 to promote atherosclerosis remains incompletely understood. In this study, we sought to investigate the molecular mechanism by which MK's regulation of ABCA1 influences the pathogenesis of atherosclerosis.

Methods: Male apoE^-/- mice were subjected to a high-fat diet to establish an atherosclerosis model. The model mice received intraperitoneal injections of MK and activator protein-1 (AP-l) inhibitor SR11302. The ATP-binding cassette transporter A1 (ABCA1) and AP1 expression were detected using immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR), and western blotting (WB). RAW264.7 macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) to generate foam cells. These foam cells were treated with MK, SR11302, JNK Inhibitor SP600125, and PI3K Inhibitor wortmannin. The expression of ABCA1, AP-1, JNK, and PI3K were detected using qPCR and WB. The Cholesterol efflux and lipid accumulation of cells were analyzed using scintillation counting and oil red O staining, respectively.

Results: MK-treated mice exhibited an accelerated development of atherosclerotic lesion (30% in the MK group vs. 20% in the control group), along with hepatic steatosis and lipid disorder. The expression of c-Fos and AP-1 were up-regulated by MK in macrophages. Compared with the MK-treated group, inhibition of AP-1 using SR11302 or transfection with c-Fos siRNA markedly enhanced the Cholesterol efflux (12.73% in the MK + SR11302 group vs. 9.98% in the MK group, 12.73% in the MK + si-c-fos group vs. 10.02 % in the MK group), reduced lipid accumulation, and increased the protein levels of ABCA1 in macrophages. Compared to the MK-treated group, mice treated with both MK and SR11302 showed downregulated ABCA1 expression in aortic sinus lesions, a larger lesion area (22.59% vs. 18.54%), and significantly elevated levels of plasma total Cholesterol (TC), low-density lipoprotein (LDL), and triglycerides (TG). These results suggest that MK-induced pharmacological inhibition of AP-1 augmented ABCA1 expression in plaques, ameliorated lipid disorders, and abrogated atherosclerosis progression in apoE^-/- mice. In addition, in vitro experiments revealed that the MK-induced up-regulation of c-Fos expression was effectively suppressed by inhibitors of JNK and PI3K.

Conclusions: Our findings unveil a novel mechanistic pathway in atherosclerosis, whereby MK promotes the development of atherosclerosis by up-regulating AP-1 in macrophages via the PI3K/Akt/JNK signaling cascade.

ATP-binding cassette transporter A1; Activator protein-1; Atherosclerosis; Midkine.