Effect Of Resveatrol On Advanced Glycation End Product-induced Foam Cell Formation And Vascular Smooth Muscle Cell Calcification

Diabetes is associated with high frequency and high severity of atherosclerosis and vascular calcification which causes most morbidity and mortality in diabetic patients. This has highlighted the importance and urgency of studying the mechanism of diabetic atherosclerosis and vascular calcification for developing therapeutic options.Diabetic patients have increased production and reduced clearance of advanced glycation end products (AGEs). It has been well documented that AGEs and their receptor (RAGE) axis is involved in the pathogenesis of cardiovascular disease due to oxidative stress and inflammatory responses. Furthermore, impaired macrophage lipid metabolism directly participates in the foam cell formation in diabetes. In another hand, previous studies found that AGEs increased the osteoblastic trans-differentiation of vascular smooth muscle cell (VSMC). However, these studies were inconsistent and whether the macrophage lipid accumulation and VSMC calcification was actually affected by RAGE activation was still unclear.Resveratrol (trans-3,5,4'-trihydroxystilbene, RESV), a natural polyphenol phytoalexin, possesses various bioactivities. A recent study indicated that resveratrol inhibits foam cell formation via suppression of reactive oxygen species (ROS) generation and macrophage activation. However, it's still unknown whether resveratrol could prevent the impairment of AGE on the lipid homeostasis in macrophages, which might partially contribute to the prevention of diabetic atherosclerosis. Furthermore, resveratrol could regulate the differentiation of osteoblast in skeletal. Considering that osteoblast differentiation and VSMC calcification share a lot of similarites, it is possible that resveratol may regulate the VSMC calcification in diabetic patients.To test the above hypothesis, we first find that resveratrol down-regulates RAGE expression and prevents the impairment of AGEs on macrophage lipid homeostasis through PPARγactivation. Then we document that resveratrol inhibits the AGEs-induced calcification of VSMC by suppression of AGEs-RAGE-ERK1/2 pathway. Finally, the cardiovascular protective effects of resveratol were validated in diabetic apoE knockout (apoE-/-) mice in vivo.Materials and Methods:The present study includes in vivo and in vitro experiments. In vivo models include apoE-/- mouse and STZ-induced-diabetic apoE-/- mouse fed with or without resveratrol. In vitro experiments were carried out in THP-1 derived macrophages and in VSMC primarily cultured from the aortas of C57BL/6J mice.1. The effects of different concentrations of AGEs on RAGE expression and cholesterol accumulation in macrophages were detected by western blot and fluorometric assay respectively.2. The effect of resveratrol on AGEs-induced protein expression of RAGE, SR-A, ABCA1 and ABCG1 was detected by immunoblotting.3. The effect of resveratrol on AGEs-induced acceleration of cholesterol accumulation and foam cell formation were detected by fluorometric assay and Oil-Red O staining.4. The effects of different concentrations of resveratrol on cbfa1 and sm22a expression in VSMC were detected by Real-time PCR.5. The ALP expression and calcium accumulation in VSMC were detected with specific assay kits.6. The effect of resveratrol on AGEs-induced RAGE expression and ERK1/2 phosphorylation in VSMC were detected by immunoblotting.7. The effects of resveratrol, anti-RAGE antibody and ERK1/2 inhibitor on the mRNA expression of cbfa1 and sm22a were detected by real-time PCR.8. Diabetic atherosclerotic mice models were established by treating apoE-/- mice with STZ and high-fat diet.9. Blood levels of glucose and lipids were detected by specific assay kits.10. The calcificaton of VSMC and aortas were shown by alizarin red staining.Results:1. AGE promotes cholesterol accumulation through induction of RAGE in macrophages.2. Resveratrol reduces AGEs-induced RAGE expression via activation of PPARγbut not PPARαnor AMPK. 3. Resveratrol reverses the effect of AGEs on the expression of proteins involved in lipid homeostasis via PPARγactivation.4. Resveratrol prevents the AGEs-induced acceleration of cholesterol accumulation and inhibits foam cell formation via PPARγ.5. Resveratrol inhibits AGEs-induced expression of cbfa1 and ALP, upregulates sm22a expression, and attenuates calcium accumulation in VSMC.5. Resveratrol reduces AGEs-induced RAGE expression and ERK1/2 phosphorylation in VSMC.6. Blocking RAGE and ERK1/2 pathway mimics the protective effect of resveratrol on AGEs-induced calcification in VSMC.7. Dietary resveratrol lowers the levels of plasma lipids in diabetic apoE-/- mice.8. Long-term intervention with resveratrol reduces atherosclerotic lesions and artery tunica media calcification in diabetic apoE-/- mice.Conclusions:1. Resveratrol inhibits AGEs-induced foam cell formation through regulating lipid transport in macrophages by suppressing RAGE expression and reduces atherosclerotic lesions in diabetic apoE-/- mice.2. Resveratrol suppresses AGEs-induced calcification of VSMC through the inhibition of RAGE-ERK1/2 pathway and ameliorates artery tunica media calcification in diabetic apoE-/- mice.