Silent Information Regulator 1 Negatively Regulates Atherosclerotic Angiogenesis via Mammalian Target of Rapamycin Complex 1 Signaling Pathway

Abstract

BackgroundThis study aimed to investigate the interactions between silent information regulator 1 (SIRT1) and mammalian target of rapamycin (mTOR) in intraplaque angiogenesis and their potential mechanisms through in vivo and in vitro studies. MethodsAn atherosclerosis model was established in 12 rabbits on a high-cholesterol diet. The rabbits were equally divided into 3 groups: a control group (high-lipid diet), RAP group (high-lipid diet supplemented with rapamycin) and RAP + NAM group (high-lipid diet supplemented with rapamycin and nicotinamide). At the end of 4 weeks, the area of plaques in the aorta was determined and the protein expression of CD31 and vascular endothelial growth factor (VEGF) was detected through hematoxylin and eosin staining and immunohistochemical staining, respectively. For in vitro study, a hypoxia model was established in human umbilical vein endothelial cells (HUVECs) by using the chemical method (CoCl2). The MTT assay, scratch assay and tube formation assay were performed to evaluate the proliferation and angiogenesis abilities of HUVECs. Reverse transcription polymerase chain reaction was used to examine the mRNA levels of SIRT1, hypoxia-inducible factor-1α (HIF-1α), mTOR and p70 ribosomal S6 kinase (p70S6K). Western blotting was used to examine the protein levels of SIRT1, HIF-1α, mTOR, p-mTOR, p-raptor and p-p70S6K. ResultsThe results of the in vivo study indicated a significant inhibitory effect of rapamycin on plaque size and intraplaque angiogenesis (0.05 ± 0.02mm2 versus 5.44 ± 0.50mm2, P < 0.05). This effect was attenuated by nicotinamide (0.76 ± 0.15mm2 versus 0.05 ± 0.02mm2, P < 0.05). Compared with the RAP group, CD31- and VEGF-positive vessels were abundant in the RAP + NAM group. The RAP group showed lower expression of p-mTOR, p-p70S6K and HIF-1α than did the control group (P < 0.05), whereas the RAP + NAM group showed slightly higher expression of these factors than did the RAP group (P < 0.05). Furthermore, in vitro studies revealed that the inhibitory effect of rapamycin on the angiogenic ability of HUVECs and its significant inhibitory effects on the protein level of HIF-1α and the phosphorylation of proteins involved in the mTORC1 pathway, including mTOR, raptor and p70S6K (P < 0.05), were enhanced by cotreatment with SRT1720 and rapamycin (P < 0.05). In contrast to mTOR and SIRT1, the mRNA levels of p70S6K and HIF-1α were reduced by rapamycin (P < 0.05) and further reduced by cotreatment with SRT1720 and rapamycin. ConclusionsThe study results indicate that SIRT1 might negatively regulate atherosclerotic angiogenesis via mTORC1 and HIF-1α signaling pathway and cointervention of SIRT1 and mTOR may serve as a crucial therapeutic strategy in cardiovascular medicine.