Background and Aims

Sirtuin 6 (SIRT6) is a histone deacetylase with key functions in genome maintenance, metabolism and aging. We investigated how SIRT6 expression is regulated, and its role in vascular smooth muscle cells (VSMCs) in atherosclerosis.

Methods

Human VSMCs (hVSMCs) were derived from atherosclerotic plaques or healthy aortas. Lentiviruses were used to stably knockdown SIRT6 using shRNA, or overexpress SIRT6 or its deacetylase-inactive mutant (SIRT6^H133Y). ApoE^-/- mice were generated that overexpress SIRT6 or SIRT6^H133Y in VSMCs only.

Results

Human plaque VSMCs showed reduced SIRT6 protein, but not mRNA, expression compared to aortic VSMCs, which was mimicked by treating healthy VSMCs with palmitate. SIRT6 was degraded via the proteasome, but not poly-ubiquitinated upon palmitate, while the expression of ubiquitin ligase CHIP was reduced. Silencing of CHIP further enhanced SIRT6 degradation, while CHIP overexpression promoted SIRT6 stability. Loss of SIRT6 resulted in cellular senescence, shown by reduced lifespan and increased expression of p16^ink4a and SAβG, while SIRT6 (but not SIRT6^H133Y) overexpression halted senescence, without affecting apoptosis or DNA damage. SIRT6 knockdown also altered cell metabolism, characterised by impaired fatty acid oxidation and increased glycolysis, which was prevented by overexpressing SIRT6 but not SIRT6^H133Y. Finally, SM22α-hSIRT6/ApoE^-/- mice showed reduced aortic plaque burden after 16 weeks of high fat diet compared to controls. Further analysis of plaque composition will be available at the conference.

Conclusions

Sirtuin 6 protein expression is decreased in human plaque VSMCs, and is positively regulated by CHIP ligase in a non-canonical fashion. SIRT6 prevents VSMC senescence and protects against atherogenesis, identifying a potential therapeutic target.

Background and Aims

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology and several evidence indicates that apoptosis of VSMCs is associated with features of high-risk/vulnerable atherosclerotic lesions. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques.

Methods

We generated an apolipoprotein E deficient (ApoE^-/-) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7.

Results

We observed that impaired VSMC autophagy promotes an unstable plaque phenotype as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate the apoptosis of VSMCs and plaque vulnerability.

Conclusions

Overall, our results indicate that mitochondrial quality control could be a promising therapeutic target to stabilize atherosclerotic plaques.

Background and Aims

Besides providing a structural framework for the artery wall, the extracellular matrix (ECM) is also essential in preserving atherosclerotic plaque integrity. ECM regulates smooth muscle cell (SMC) migration and proliferation and serves as an extracellular pool of cytokines and growth factors such as TGF-β. Response Gene to Complement (RGC)-32 has been shown to stimulate the proliferation as well as migration of aortic SMC. Our goal was to explore the effect of RGC-32 on TGF-β induced differentiation of SMC and ECM production.

Methods

The effect of RGC-32 on ECM production and SMC differentiation was studied by silencing RGC-32 expression in human aortic SMCs (ASMC), using transfection of RGC-32 siRNA in the presence of Lipofectamine 3000. The expression of collagens I, fibronectin, myocardin, SM22 (or transgelin) and α-smooth muscle actin (α-SMA) were determined by real-time PCR and western blot analysis.

Results

We investigated the role of RGC-32 in TGF-β-induced SMC differentiation markers and ECM production in ASMC. In cultured ASMC α-SMA, collagen I, fibronectin, myocardin and SM22 were significantly induced at 18 h of stimulation with TGF-β. Silencing of RGC-32 in ASMC was followed by a significant reduction in TGF-β induced expression of myocardin (p<0.001), SM22 (p<0.002), α-SMA (p<0.02), fibronectin (p<0.05) and collagen I (p<0.03).

Conclusions

RGC-32 modulates myocardin and other differentiation markers expression in aortic SMC indicating that RGC-32 is an important player not only in TGF-β-mediated ECM production, but also in SMC differentiation. Thus, RGC-32 represents a potential target for therapeutic intervention in atherosclerosis.

Background and Aims

During atherosclerosis, intimal smooth muscle cells (SMCs) acquire a synthetic phenotype. We previously isolated spindle-shaped (S) and rhomboid (R) SMCs from porcine coronary artery. R-SMCs display the features of synthetic/dedifferentiated SMCs. S100A4 was identified as being a marker of R-SMCs in vitro and of intimal SMCs, in pig and man. Our aim was to decipher the role of extracellular S100A4 in phenotypic transition of SMCs and its causal relation to pathogenesis of atherosclerosis.

Methods

We performed treatments of porcine S-SMCs with oligomeric and dimeric recombinant S100A4 to clarify the mechanisms underlying S100A4 dependent SMC phenotypic changes.

To investigate whether altered S100A4 expression is causally related to the formation of atherosclerotic lesions, ApoE^-/- mice were fed with high-cholesterol diet for 9 weeks, and during the 3 last weeks, they were injected with neutralizing monoclonal S100A4 antibody or with control IgG1.

Results

Treatment of S-SMCs with oligomeric S100A4 and platelet-derived growth factor-BB (PDGF-BB) together induced a complete SMC transition toward a R-phenotype associated with NFκB activation and strong upregulation of pro-inflammatory genes compared with oligomeric S100A4 or PDGF-BB alone. In vivo, neutralization of extracellular S100A4 induced: decreased area of atherosclerotic lesions and necrotic core, decreased expression of CD68 and increased expression of α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chains (SMMHCs) in atherosclerostic plaques, when compared to control groups.

Conclusions

Our results indicate that extracellular S100A4 is causally related to the atherosclerotic plaque progression and that it could be a new target to influence the evolution of atherosclerotic plaque, leading to plaque stabilization.

Background and Aims

Genome-wide association studies have discovered a link between genetic variants on chromosome 15q26.1 and increased risk of coronary artery disease (CAD). This genetic locus contains the FES gene, which encodes a cytoplasmic protein-tyrosine kinase involved in the regulation of cell behavior. It is unclear whether the 15q26.1 variants modulate FES expression, and it is also unknown whether FES plays a role in atherosclerosis.

Methods

Included in the Results section.

Results

Single-cell RNA-sequencing analysis showed that in human atherosclerotic plaques, cells that expressed FES were predominately monocytes/macrophages. Analyses of isogenic monocyte cell lines generated by CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing showed that monocytes with an engineered 15q26.1 CAD risk genotype had reduced FES expression and increased migratory activity. Small-interfering-RNA-mediated knockdown of FES in monocytes also promoted monocyte migration. Fes-/-Apoe-/-mice fed a high fat diet developed larger atherosclerotic plaques with a greater abundance of monocytes/macrophages and more matrix metalloproteinase-9, than Apoe-/- mice. Immunohistochemical analysis of a collection of ex vivo human coronary atherosclerotic plaques showed that plaques from patients of the 15q26.1 CAD risk genotype contained greater numbers of monocytes/macrophages.

Conclusions

We provide compelling evidence that the CAD risk variants at the 15q26.1 locus reduced FES expression in monocytes and that FES depletion results in larger atherosclerotic plaques with more monocytes/macrophages. This study is the first demonstration that FES plays a protective role against atherosclerosis, and suggests that enhancing FES activity could be a potentially novel therapeutic approach for CAD intervention.