Vascular smooth muscle cell (VSMC) senescence promotes atherosclerosis and features of plaque instability, in part, through lipid-mediated oxidative DNA damage and telomere dysfunction. SIRT6 ...(Sirtuin 6) is a nuclear deacetylase involved in DNA damage response signaling, inflammation, and metabolism; however, its role in regulating VSMC senescence and atherosclerosis is unclear.
We examined SIRT6 expression in human VSMCs, the role, regulation, and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis.
SIRT6 protein, but not mRNA, expression was markedly reduced in VSMCs in human and mouse atherosclerotic plaques, and in human VSMCs derived from plaques or undergoing replicative or palmitate-induced senescence versus healthy aortic VSMCs. The ubiquitin ligase CHIP (C terminus of HSC70-interacting protein) promoted SIRT6 stability, but CHIP expression was reduced in human and mouse plaque VSMCs and by palmitate in a p38- and c-Jun N-terminal kinase-dependent manner. SIRT6 bound to telomeres, while SIRT6 inhibition using shRNA or a deacetylase-inactive mutant (SIRT6
) shortened human VSMC lifespan and induced senescence, associated with telomeric H3K9 (histone H3 lysine 9) hyperacetylation and 53BP1 (p53 binding protein 1) binding, indicative of telomere damage. In contrast, SIRT6 overexpression preserved telomere integrity, delayed cellular senescence, and reduced inflammatory cytokine expression and changes in VSMC metabolism associated with senescence. SIRT6, but not SIRT6
, promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE
(apolipoprotein E) mice were generated that overexpress SIRT6 or SIRT6
in VSMCs only. SM22α-hSIRT6/ApoE
mice had reduced atherosclerosis, markers of senescence and inflammation compared with littermate controls, while plaques of SM22α-hSIRT6
/ApoE
mice showed increased features of plaque instability.
SIRT6 protein expression is reduced in human and mouse plaque VSMCs and is positively regulated by CHIP. SIRT6 regulates telomere maintenance and VSMC lifespan and inhibits atherogenesis, all dependent on its deacetylase activity. Our data show that endogenous SIRT6 deacetylase is an important and unrecognized inhibitor of VSMC senescence and atherosclerosis.
Summary
Aging is the largest risk factor for cardiovascular disease, yet the molecular mechanisms underlying vascular aging remain unclear. Mitochondrial DNA (mtDNA) damage is linked to aging, but ...whether mtDNA damage or mitochondrial dysfunction is present and directly promotes vascular aging is unknown. Furthermore, mechanistic studies in mice are severely hampered by long study times and lack of sensitive, repeatable and reproducible parameters of arterial aging at standardized early time points. We examined the time course of multiple invasive and noninvasive arterial physiological parameters and structural changes of arterial aging in mice, how aging affects vessel mitochondrial function, and the effects of gain or loss of mitochondrial function on vascular aging. Vascular aging was first detected by 44 weeks (wk) of age, with reduced carotid compliance and distensibility, increased β‐stiffness index and increased aortic pulse wave velocity (PWV). Aortic collagen content and elastin breaks also increased at 44 wk. Arterial mtDNA copy number (mtCN) and the mtCN‐regulatory proteins TFAM, PGC1α and Twinkle were reduced by 44 wk, associated with reduced mitochondrial respiration. Overexpression of the mitochondrial helicase Twinkle (Tw+) increased mtCN and improved mitochondrial respiration in arteries, and delayed physiological and structural aging in all parameters studied. Conversely, mice with defective mitochondrial polymerase‐gamma (PolG) and reduced mtDNA integrity demonstrated accelerated vascular aging. Our study identifies multiple early and reproducible parameters for assessing vascular aging in mice. Arterial mitochondrial respiration reduces markedly with age, and reduced mtDNA integrity and mitochondrial function directly promote vascular aging.
Abstract
Aims
Quiescent, differentiated adult vascular smooth muscle cells (VSMCs) can be induced to proliferate and switch phenotype. Such plasticity underlies blood vessel homeostasis and ...contributes to vascular disease development. Oligoclonal VSMC contribution is a hallmark of end-stage vascular disease. Here, we aim to understand cellular mechanisms underpinning generation of this VSMC oligoclonality.
Methods and results
We investigate the dynamics of VSMC clone formation using confocal microscopy and single-cell transcriptomics in VSMC-lineage-traced animal models. We find that activation of medial VSMC proliferation occurs at low frequency after vascular injury and that only a subset of expanding clones migrate, which together drives formation of oligoclonal neointimal lesions. VSMC contribution in small atherosclerotic lesions is typically from one or two clones, similar to observations in mature lesions. Low frequency (<0.1%) of clonal VSMC proliferation is also observed in vitro. Single-cell RNA-sequencing revealed progressive cell state changes across a contiguous VSMC population at onset of injury-induced proliferation. Proliferating VSMCs mapped selectively to one of two distinct trajectories and were associated with cells showing extensive phenotypic switching. A proliferation-associated transitory state shared pronounced similarities with atypical SCA1+ VSMCs from uninjured mouse arteries and VSMCs in healthy human aorta. We show functionally that clonal expansion of SCA1+ VSMCs from healthy arteries occurs at higher rate and frequency compared with SCA1− cells.
Conclusion
Our data suggest that activation of proliferation at low frequency is a general, cell-intrinsic feature of VSMCs. We show that rare VSMCs in healthy arteries display VSMC phenotypic switching akin to that observed in pathological vessel remodelling and that this is a conserved feature of mouse and human healthy arteries. The increased proliferation of modulated VSMCs from healthy arteries suggests that these cells respond more readily to disease-inducing cues and could drive oligoclonal VSMC expansion.
Graphical Abstract
Graphical Abstract
BACKGROUND—Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis ...or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species.
METHODS AND RESULTS—mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE mice deficient for mitochondrial polymerase-γ proofreading activity (polG/ApoE). polG/ApoE mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG/ApoE mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG/ApoE bone marrow increased the features of plaque vulnerability, and polG/ApoE monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden.
CONCLUSIONS—We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.
Stiffening of the aorta is an important independent risk factor for myocardial infarction and stroke. Yet its genetics is complex and little is known about its molecular drivers. We have identified ...for the first time, tagSNPs in the genes for extracellular matrix proteins, aggrecan and fibulin-1, that modulate stiffness in young healthy adults. We confirmed SNP associations with ex vivo stiffness measurements and expression studies in human donor aortic tissues. Both aggrecan and fibulin-1 were found in the aortic wall, but with marked differences in the distribution and glycosylation of aggrecan reflecting loss of chondroitin-sulphate binding domains. These differences were age-dependent but the striking finding was the acceleration of this process in stiff versus elastic young aortas. These findings suggest that aggrecan and fibulin-1 have critical roles in determining the biomechanics of the aorta and their modification with age could underpin age-related aortic stiffening.
RATIONALE:Atherosclerosis is characterized by lipid accumulation in the vessel wall, inflammation, and both macrophage and vascular smooth muscle cell (VSMC) apoptosis. However, whereas VSMC ...apoptosis in mice with established atherosclerotic plaques or hyperlipidemia increases serum levels of the proatherogenic cytokines monocyte chemotactic protein (MCP)-1, tumor necrosis factor α, and interleukin (IL)-6, the link between hyperlipidemia, apoptosis and inflammation, and the mechanisms by which apoptotic cells promote inflammation in atherosclerosis are unknown.
OBJECTIVE:To determine whether hyperlipidemia affects apoptotic cell clearance, and identify the molecular pathways downstream of VSMC apoptosis that may promote inflammation.
METHODS AND RESULTS:We find that human VSMCs are potent and efficient phagocytes of apoptotic human VSMCs, but phagocytosis is significantly reduced by oxidized low-density lipoprotein in vitro or hyperlipidemia in vivo. Necrotic human aortic VSMCs release IL-1α, which induces IL-6 and MCP-1 production from viable human VSMCs in vitro. In contrast, secondary necrotic VSMCs release both IL-1α and caspase-activated IL-1β, augmenting IL-6 and MCP-1 production. Conditionally inducing VSMC apoptosis in situ in hyperlipidemic SM22α-hDTR/ApoE mice to levels seen in human plaques increases serum MCP-1, tumor necrosis factor α, and IL-6, which is prevented by blocking IL-1.
CONCLUSIONS:We conclude that VSMC necrosis releases IL-1α, whereas secondary necrosis of apoptotic VSMCs releases both IL-1α and β. IL-1 from necrotic VSMCs induces the surrounding viable VSMCs to produce proinflammatory cytokines. Thus, failed clearance of apoptotic VSMCs caused by hyperlipidemia in vivo may promote the increased serum cytokines and chronic inflammation associated with atherosclerosis.
Fiber structures and pathological features, e.g., inflammation and glycosaminoglycan (GAG) deposition, are the primary determinants of aortic mechanical properties which are associated with the ...development of an aneurysm. This study is designed to quantify the association of tissue ultimate strength and extensibility with the structural percentage of different components, in particular, GAG, and local fiber orientation. Thoracic aortic aneurysm (TAA) tissues from eight patients were collected. Ninety-six tissue strips of thickened intima, media, and adventitia were prepared for uni-extension tests and histopathological examination. Area ratios of collagen, elastin, macrophage and GAG, and collagen fiber dispersion were quantified. Collagen, elastin, and GAG were layer-dependent and the inflammatory burden in all layers was low. The local GAG ratio was negatively associated with the collagen ratio (r2 = 0.173, p < 0.05), but positively with elastin (r2 = 0.037, p < 0.05). Higher GAG deposition resulted in larger local collagen fiber dispersion in the media and adventitia, but not in the intima. The ultimate stretch in both axial and circumferential directions was exclusively associated with elastin ratio (axial: r2 = 0.186, p = 0.04; circumferential: r2 = 0.175, p = 0.04). Multivariate analysis showed that collagen and GAG contents were both associated with ultimate strength in the circumferential direction, but not with the axial direction (collagen: slope = 27.3, GAG: slope = -18.4, r2 = 0.438, p = 0.002). GAG may play important roles in TAA material strength. Their deposition was found to be associated positively with the local collagen fiber dispersion and negatively with ultimate strength in the circumferential direction.
Deletion of exon 9 from Cullin‐3 (CUL3, residues 403–459: CUL3Δ403–459) causes pseudohypoaldosteronism type IIE (PHA2E), a severe form of familial hyperkalaemia and hypertension (FHHt). CUL3 binds ...the RING protein RBX1 and various substrate adaptors to form Cullin‐RING‐ubiquitin‐ligase complexes. Bound to KLHL3, CUL3‐RBX1 ubiquitylates WNK kinases, promoting their ubiquitin‐mediated proteasomal degradation. Since WNK kinases activate Na/Cl co‐transporters to promote salt retention, CUL3 regulates blood pressure. Mutations in both KLHL3 and WNK kinases cause PHA2 by disrupting Cullin‐RING‐ligase formation. We report here that the PHA2E mutant, CUL3Δ403–459, is severely compromised in its ability to ubiquitylate WNKs, possibly due to altered structural flexibility. Instead, CUL3Δ403–459 auto‐ubiquitylates and loses interaction with two important Cullin regulators: the COP9‐signalosome and CAND1. A novel knock‐in mouse model of CUL3WT/Δ403–459 closely recapitulates the human PHA2E phenotype. These mice also show changes in the arterial pulse waveform, suggesting a vascular contribution to their hypertension not reported in previous FHHt models. These findings may explain the severity of the FHHt phenotype caused by CUL3 mutations compared to those reported in KLHL3 or WNK kinases.
Synopsis
Molecular defects observed in a mutant form of the ubiquitin E3 protein CUL3 causing a severe form of familial hypertension (PHA2E) result in E3 ligase loss of function. A novel mouse model with the same CUL3 disease mutation was generated, and it closely recapitulates the human PHA2E phenotype.
Mutations in CUL3 that cause hereditary hypertension prevent the ubiquitylation and degradation of WNK kinases probably due to increased flexibility of the cullin backbone.
The mutant CUL3Δ403–459 protein shows marked auto‐ubiquitylation and loss of binding to the critical cullin regulators, CSN and CAND1.
The phenotype of knock‐in mice carrying the CUL3Δ403–459 mutation closely recapitulates PHA2E; kidney DCTs contain striking accumulation of WNK and SPAK proteins.
The mice expressing CUL3Δ403–459 have a vascular phenotype suggesting increased vascular tone that may contribute to the severity of the hypertension seen in PHA2E compared to other forms (PHA2A‐D).
Molecular defects observed in a mutant form of the ubiquitin E3 protein CUL3 causing a severe form of familial hypertension (PHA2E) result in E3 ligase loss of function. A novel mouse model with the same CUL3 disease mutation was generated, and it closely recapitulates the human PHA2E phenotype.
Apoptosis is a key process occurring in atherosclerosis, both in humans and in animal models. Apoptosis occurs in all cell types studied thus far, and thus lineage marking is often necessary. ...Apoptosis should be ascertained using a combination of morphological features and activation of specific pathways (e.g., terminal UTP nick end labeling-TUNEL). Both TUNEL and cryptic epitope antibodies (e.g., cleaved caspase 3) can be used, although they will often give different frequencies. Apoptotic frequency but not rate can be estimated from these methods, as we do not know the timing of apoptosis or how much of the process is marked by each method. We describe the morphological and immunohistochemical methods used in our laboratory to detect apoptotic cells in animal and human atherosclerotic plaques.
Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography (
FFDG PET),
FFDG lacks cell ...specificity, and coronary imaging is unreliable because of myocardial spillover.
This study tested the efficacy of gallium-68-labeled DOTATATE (
Ga-DOTATATE), a somatostatin receptor subtype-2 (SST
)-binding PET tracer, for imaging atherosclerotic inflammation.
We confirmed
Ga-DOTATATE binding in macrophages and excised carotid plaques.
Ga-DOTATATE PET imaging was compared to
FFDG PET imaging in 42 patients with atherosclerosis.
Target SSTR2 gene expression occurred exclusively in "proinflammatory" M1 macrophages, specific
Ga-DOTATATE ligand binding to SST
receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo
Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval CI: 0.28 to 0.99; p = 0.02).
Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBR
) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range IQR: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003).
Ga-DOTATATE mTBR
predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve ROC AUC: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p <0.0001) and
FFDG uptake (r = 0.73; 95% CI: 0.64 to 0.81; p < 0.0001).
FFDG mTBR
differentiated culprit from nonculprit carotid lesions (median difference: 0.12; IQR: 0.0 to 0.23; p = 0.008) and high-risk from lower-risk coronary arteries (ROC AUC: 0.76; 95% CI: 0.62 to 0.91; p = 0.002); however, myocardial
FFDG spillover rendered coronary
FFDG scans uninterpretable in 27 patients (64%). Coronary
Ga-DOTATATE PET scans were readable in all patients.
We validated
Ga-DOTATATE PET as a novel marker of atherosclerotic inflammation and confirmed that
Ga-DOTATATE offers superior coronary imaging, excellent macrophage specificity, and better power to discriminate high-risk versus low-risk coronary lesions than
FFDG. (Vascular Inflammation Imaging Using Somatostatin Receptor Positron Emission Tomography VISION; NCT02021188).
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