Restoring mitochondrial DNA copy number preserves mitochondrial function and delays vascular aging in mice.

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

Poor maternal nutrition programmes a pro-atherosclerotic phenotype in ApoE-/- mice.

Numerous animal studies have consistently shown that early life exposure to LP (low-protein) diet programmes risk factors for CVD (cardiovascular disease) such as dyslipidaemia, high BP (blood pressure) and cardiac dysfunction in the offspring. However, studies on the effect of maternal under-nutrition on offspring development of atherosclerosis are scarce. Applying our LP model to the ApoE(-/-) atherosclerosis-prone mouse model, we investigated the development of atherosclerotic lesions in the aortic root of 6-month-old offspring. In addition, markers of plaque progression including SMA (smooth muscle actin) and Mac3 (macrophage marker 3) were studied. Pregnant dams were fed on a control (20% protein) or on an isocaloric LP diet (8% protein) throughout pregnancy and lactation. After weaning, male offspring were maintained on 20% normal laboratory chow. At 6 months of age, LP offspring showed a significantly greater plaque area (P<0.05) with increased cholesterol clefts and significantly higher indices of DNA damage compared with controls (P<0.05). The expression of HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) (P<0.05) and LDL (low-density lipoprotein) receptor in the liver of LP offspring were increased. Furthermore, LP offspring had higher LDL-cholesterol levels (P<0.05) and a trend towards elevated insulin. There were no differences in other lipid measurements and fasting glucose between groups. These observations suggest that early exposure to an LP diet accelerates the development and increases the progression of atherosclerotic lesions in young adult offspring. Future studies are needed to elucidate the specific mechanisms linking in utero exposure to a diet low in protein to the development of atherosclerosis.This work was supported by the British Heart Foundation [grant numbers FS/09/029/27902, FS/09/050], the Biotechnology and Biological Sciences Research Council and the Cambridge Commonwealth Trust

Characterisation of the Cullin-3 mutation that causes a severe form of familial hypertension and hyperkalaemia

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

Hematopoietic IKBKE limits the chronicity of inflammasome priming and metaflammation

Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation

SIRT6 Protects Smooth Muscle Cells From Senescence and Reduces Atherosclerosis.

RATIONALE: 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. OBJECTIVE: We examined SIRT6 expression in human VSMCs, the role, regulation, and downstream pathways activated by SIRT6, and how VSMC SIRT6 regulates atherogenesis. METHODS AND RESULTS: 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 (SIRT6H133Y) 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 SIRT6H133Y, promoted proliferation and lifespan of mouse VSMCs, and prevented senescence-associated metabolic changes. ApoE-/- (apolipoprotein E) mice were generated that overexpress SIRT6 or SIRT6H133Y in VSMCs only. SM22α-hSIRT6/ApoE-/- mice had reduced atherosclerosis, markers of senescence and inflammation compared with littermate controls, while plaques of SM22α-hSIRT6H133Y/ApoE-/- mice showed increased features of plaque instability. CONCLUSIONS: 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

Poor maternal nutrition programmes a pro-atherosclerotic phenotype in ApoE

A B S T R A C T Numerous animal studies have consistently shown that early life exposure to LP (low-protein) diet programmes risk factors for CVD (cardiovascular disease) such as dyslipidaemia, high BP (blood pressure) and cardiac dysfunction in the offspring. However, studies on the effect of maternal under-nutrition on offspring development of atherosclerosis are scarce. Applying our LP model to the ApoE − / − atherosclerosis-prone mouse model, we investigated the development of atherosclerotic lesions in the aortic root of 6-month-old offspring. In addition, markers of plaque progression including SMA (smooth muscle actin) and Mac3 (macrophage marker 3) were studied. Pregnant dams were fed on a control (20 % protein) or on an isocaloric LP diet (8 % protein) throughout pregnancy and lactation. After weaning, male offspring were maintained on 20 % normal laboratory chow. At 6 months of age, LP offspring showed a significantly greater plaque area (P &lt; 0.05) with increased cholesterol clefts and significantly higher indices of DNA damage compared with controls (P &lt; 0.05). The expression of HMG-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase) (P &lt; 0.05) and LDL (low-density lipoprotein) receptor in the liver of LP offspring were increased. Furthermore, LP offspring had higher LDL-cholesterol levels (P &lt; 0.05) and a trend towards elevated insulin. There were no differences in other lipid measurements and fasting glucose between groups. These observations suggest that early exposure to an LP diet accelerates the development and increases the progression of atherosclerotic lesions in young adult offspring. Future studies are needed to elucidate the specific mechanisms linking in utero exposure to a diet low in protein to the development of atherosclerosis

Association of Collagen, Elastin, Glycosaminoglycans, and Macrophages With Tissue Ultimate Material Strength and Stretch in Human Thoracic Aortic Aneurysms: A Uniaxial Tension Study.

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.EPSRC (EP/P021654/1), NSERC (6799-427538-2012), NIHR Cambridge Biomedical Research Centre (BRC-1215-20014), EPSRC Doctoral Training Award at the University of Cambridge, China Scholarship Council