SIRT3 mediates the effects of PCSK9 inhibitors on inflammation, autophagy, and oxidative stress in endothelial cells

Abstract

Background: Proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors (i) are a class of lipid-lowering drugs suggested to hold a plethora of beneficial effects independent of their LDL cholesterol-lowering properties. However, the mechanism underlying such observations is debated. Methods: Human aortic endothelial cells (TeloHAEC) were pre-treated with 100 µg/mL of the PCSK9i evolocumab and then exposed to 20 ng/mL of IL-6, a major driver of cardiovascular diseases (CVD), in both naïve state and after siRNA-mediated suppression of the NAD-dependent deacetylase sirtuin-3 (SIRT3). Inflammation, autophagy, and oxidative stress were assessed through Western Blots, ELISAs, and/or immunofluorescence coupled by flow cytometry. To explore the human relevance of the findings, we also evaluated the expression of IL-6, SIRT3, IL-1β, the ratio LC3B II/I, and PCSK9 within the plaques of patients undergoing carotid endarterectomy (n=277), testing possible correlations between these proteins. Results: PCSK9i improved a range of phenotypes including the activation of inflammatory pathways, oxidative stress, and autophagy. Indeed, treatment with PCSK9i was able to counteract the IL-6 induced increase in inflammasome activation, the accrual of autophagic cells, and mitochondrial ROS accumulation. Of note, silencing of SIRT3 reverted the beneficial effects observed with PCSK9i treatment on all these phenomena. In atheroma specimens, the expression of PCSK9 was inversely related to that of SIRT3 while positively correlating with IL-6, IL-1β, and the ratio LC3B II/I. Conclusions:Overall, these data suggest that PCSK9i bear intrinsic anti-inflammatory, anti-autophagic, and antioxidant properties in endothelial cells, and that these pleiotropic effects might be mediated, at least in part, by SIRT3. These results provide an additional mechanism supporting the emerging knowledge relative to the benefit of PCSK9i on CVD beyond LDL-lowering and uncover SIRT3 as a putative mediator of such pleiotropy.

PCSK9 inhibitors ameliorate arterial stiffness in ACS patients: evidences from mendelian randomization, cohort studies and basic experiments

“…According to published methods, 10 mmol/L β-gp, 20ug/m dexamethasone and 50ug/mL Lascorbic acid were added to the routine medium to induce osteogenesis of VSMCs for 7 days, the medium were changed every 2/3 days [23][24][25][26][27][28] . To mimic the effects of PCSK9i in vivo, 100 µg/mL of reagent-grade puri ed evolocumab (Selleck, America) was used to co-incubate with VSMCs for 7 days during osteogenesis induction [29][30][31] . To mimic the stimulation of VSMCs by PCSK9 protein in vivo, refer to previous research [32] , 0 µg/ml, 0.55 µg/ml, 1.1 µg/ml, 2.2 µg/ml, and 4.4 µg/ml of recombinant human PCSK9 (MedChemExpress, USA) were co-incubated with VSMCs for 7 days, and the concentration exhibiting the highest intracellular calcium content was chosen for subsequent experimental procedures.…”

Sirtuins and autophagy in lipid metabolism

“…PCSK9i has intrinsic anti-inflammatory, antiautophagy, and antioxidant properties in endothelial cells, and these effects have been shown to be at least partially mediated by SIRT3. 38 Assisted by mitochondrial HSP60, SIRT3 can improve the development of nonalcoholic fatty liver disease by promoting fatty acid oxidation while suppressing mitochondrial stress and inflammation. 39 Knockdown of SIRT6 can increase liver oxidative stress and inflammation in mice fed a high-fat diet, while overexpression of SIRT6 can relieve liver oxidative stress and inflammation.…”

“…Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors (i) are a class of lipid‐lowering drugs that are believed to have numerous beneficial effects independent of their LDL cholesterol‐lowering properties. PCSK9i has intrinsic anti‐inflammatory, antiautophagy, and antioxidant properties in endothelial cells, and these effects have been shown to be at least partially mediated by SIRT3 38 . Assisted by mitochondrial HSP60, SIRT3 can improve the development of nonalcoholic fatty liver disease by promoting fatty acid oxidation while suppressing mitochondrial stress and inflammation 39 …”

PCSK9 promotes vascular neointimal hyperplasia through non-lipid regulation of vascular smooth muscle cell proliferation, migration, and autophagy

“…Evolocumab reduces infarct size via suppressing autophagy in cardiac tissue [28]. PCSK9i possess intrinsic properties for antiin ammatory, anti-autophagic, and antioxidant in endothelial cells [29]. However, it is unknown whether PCSK9i are able to counteract with the autophagy and proliferation in VSMC.…”