SMTP-44D Inhibits Atherosclerotic Plaque Formation in Apolipoprotein-E Null Mice Partly by Suppressing the AGEs-RAGE Axis

Abstract

SMTP-44D has been reported to have anti-oxidative and anti-inflammatory reactions, including reduced expression of receptor for advanced glycation end products (RAGE) in experimental diabetic neuropathy. Although activation of RAGE with its ligands, and advanced glycation end products (AGEs), play a crucial role in atherosclerotic cardiovascular disease, a leading cause of death in diabetic patients, it remains unclear whether SMTP-44D could inhibit experimental atherosclerosis by suppressing the AGEs-RAGE axis. In this study, we investigated the effects of SMTP-44D on atherosclerotic plaque formation and expression of AGEs in apolipoprotein-E null (Apoe^-/-) mice. We further studied here whether and how SMTP-44D inhibited foam cell formation of macrophages isolated from Apoe^-/- mice ex vivo. Although administration of SMTP-44D to Apoe^-/- mice did not affect clinical or biochemical parameters, it significantly decreased the surface area of atherosclerotic lesions and reduced the atheromatous plaque size, macrophage infiltration, and AGEs accumulation in the aortic roots. SMTP-44D bound to immobilized RAGE and subsequently attenuated the interaction of AGEs with RAGE in vitro. Furthermore, foam cell formation evaluated by Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and gene expression of RAGE, cyclin-dependent kinase 5 (Cdk5) and CD36 in macrophages isolated from SMTP-44D-treated Apoe^-/- mice were significantly decreased compared with those from saline-treated mice. Gene expression levels of RAGE and Cdk5 were highly correlated with each other, the latter of which was also positively associated with that of CD36. The present study suggests that SMTP-44D may inhibit atherosclerotic plaque formation in Apoe^-/- mice partly by blocking the AGEs-RAGE-induced ox-LDL uptake into macrophages via the suppression of Cdk5-CD36 pathway.

Keywords: AGEs; CD36; Cdk5; RAGE; SMTP-44D; atherosclerosis; macrophages.

Figure 1

Effects of SMTP-44D on atherosclerotic lesions, macrophage infiltration, and accumulation of AGEs in the entire aorta and aortic roots in Apoe^−/− mice administered with SMTP-44D or saline. (A–H) Representative images are shown. The aortic surface was stained with Oil red O. White scale bars correspond to a length of 5 mm. (A,E), while the aortic roots were stained with Oil red O (B,F), MOMA-2 (C,G), and AGEs (D,H). Magnification x40. Black scale bars correspond to a length of 200 μm. Surface area of the atherosclerotic lesions (I), and the cross-sectional area of atheromatous plaque size (J), macrophage infiltration (K), and AGEs accumulation (L) in the aortic roots were quantified and shown as mean ± standard deviation. Number = 6 for each group. ^★★★ p < 0.005, ^★★ p < 0.01, and ^★ p < 0.05 vs. saline-treated mice.

Figure 2

Interaction of SMTP-44D at 56 μmol/L and 5.6 μmol/L to immobilized RAGE was analyzed by bio-layer interferometry using BIAcore system (A). Effects of 56 μmol/L SMTP-44D on the interaction of 250 μg/mL AGEs with immobilized RAGE were evaluated (B). The binding protein is expressed as RU.

Figure 3

Effects of SMTP-44D on Dil-ox-LDL uptake into, and RAGE, Cdk5, and CD36 gene expression in, Apoe^−/− mice. Peritoneal macrophages were extracted from Apoe^−/− mice injected with SMTP-44D at 30 mg/kg/day or saline every other day for 4 weeks. (A–F) Representative immunofluorescent staining images in peritoneal macrophages. Dil-ox-LDL positive cells were stained in red (A,D), while F4/80 were in purple (B,E). (C,F) Merge images. Scale bars, 50 μm. Quantification of fluorescence intensity in red. Dil-ox-LDL uptake was shown as a relative value compared to control mice (G). Gene expression levels of RAGE (H), Cdk5 (I), and CD36 (J) derived from Apoe^−/− mice, and their correlation (K,L). Total RNAs were reverse-transcribed, and the resulting cDNAs were amplified by real-time PCR. Data were normalized by the intensity of GAPDH mRNA-derived signals and expressed as a relative to the control values. Number = 6 for each group. Error bars are standard deviation. ^★ p < 0.05 and ^★★ p < 0.01 vs. control.

Figure 4

Possible anti-atherosclerotic actions of SMTP-44D. SMTP-44D could inhibit the AGEs-RAGE-induced macrophage foam cell formation through the suppression of the CD36-Cdk5 pathway. AGEs, advanced glycation end products; RAGE, receptor for AGEs; Cdk5, cyclin-dependent kinase 5; ox-LDL, oxidized low-density lipoprotein.