Endothelial SIRT6 deficiency promotes arterial thrombosis in mice.

OBJECTIVE: Arterial thrombosis may be initiated by endothelial inflammation or denudation, activation of blood-borne elements or the coagulation system. Tissue factor (TF), a central trigger of the coagulation cascade, is regulated by the pro-inflammatory NF-κB-dependent pathways. Sirtuin 6 (SIRT6) is a nuclear member of the sirtuin family of NAD+-dependent deacetylases and is known to inhibit NF-κB signaling. Its constitutive deletion in mice shows early lethality with hypoglycemia and accelerated aging. Of note, the role of SIRT6 in arterial thrombosis remains unknown. Thus, we hypothesized that endothelial SIRT6 protects from arterial thrombosis by modulating inhibition of NF-κB-associated pathways. APPROACH AND RESULTS: Using a laser-induced carotid thrombosis model, in vivo arterial occlusion occurred 45% faster in 12-week-old male endothelial-specific Sirt6-/- mice as compared to Sirt6fl/fl controls (n ≥ 9 per group; p = 0.0012). Levels of procoagulant TF were increased in animals lacking endothelial SIRT6 as compared to control littermates. Similarly, in cultured human aortic endothelial cells, SIRT6 knockdown increased TF mRNA, protein and activity. Moreover, SIRT6 knockdown increased mRNA levels of NF-κB-associated genes tumor necrosis factor alpha (TNF-α), poly [ADP-ribose] polymerase 1 (PARP-1), vascular cell adhesion molecule 1 (VCAM-1), and cyclooxygenase-2 (COX-2); at the protein level, COX-2, VCAM-1, TNF-α, and cleaved PARP-1 remained increased after Sirt6 knockdown. CONCLUSIONS: Endothelium-specific Sirt6 deletion promotes arterial thrombosis in mice. In cultured human aortic endothelial cells, SIRT6 silencing enhances TF expression and activates pro-inflammatory pathways including TNF-α, cleaved PARP-1, VCAM-1 and COX-2. Hence, endogenous endothelial SIRT6 exerts a protective role in experimental arterial thrombosis.

Endothelial SIRT6 blunts stroke size and neurological deficit by preserving blood-brain barrier integrity: a translational study.

AIMS: Aging is an established risk factor for stroke; genes regulating longevity are implicated in the pathogenesis of ischaemic stroke where to date, therapeutic options remain limited. The blood-brain barrier (BBB) is crucially involved in ischaemia/reperfusion (I/R) brain injury thus representing an attractive target for developing novel therapeutic agents. Given the role of endothelial cells in the BBB, we hypothesized that the endothelial-specific expression of the recently described longevity gene SIRT6 may exhibit protective properties in stroke. METHODS AND RESULTS: SIRT6 endothelial expression was reduced following stroke. Endothelial-specific Sirt6 knockout (eSirt6-/-) mice, as well as animals in which Sirt6 overexpression was post-ischaemically induced, underwent transient middle cerebral artery occlusion (tMCAO). eSirt6-/- animals displayed increased infarct volumes, mortality, and neurological deficit after tMCAO, as compared to control littermates. Conversely, post-ischaemic Sirt6 overexpression decreased infarct size and neurological deficit. Analysis of ischaemic brain sections revealed increased BBB damage and endothelial expression of cleaved caspase-3 in eSIRT6-/- mice as compared to controls. In primary human brain microvascular endothelial cells (HBMVECs), hypoxia/reoxygenation (H/R) reduced SIRT6 expression and SIRT6 silencing impaired the barrier function (transendothelial resistance) similar to what was observed in mice exposed to I/R. Further, SIRT6-silenced HBMVECs exposed to H/R showed reduced viability, increased cleaved caspase-3 expression and reduced activation of the survival pathway Akt. In ischaemic stroke patients, SIRT6 expression was higher in those with short-term neurological improvement as assessed by NIHSS scale and correlated with stroke outcome. CONCLUSION: Endothelial SIRT6 exerts a protective role in ischaemic stroke by blunting I/R-mediated BBB damage and thus, it may represent an interesting novel therapeutic target to be explored in future clinical investigation.

Sirt6 deletion in bone marrow-derived cells increases atherosclerosis - Central role of macrophage scavenger receptor 1.

AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.

The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts.

Sirtuin 3 is a nicotinamide adenine dinucleotide dependent mitochondrial deacetylase that governs mitochondrial metabolism and oxidative defense. The demise in myocardial function following myocardial ischemia has been associated with mitochondrial dysfunction. Sirt3 maintains myocardial contractile function and protects from cardiac hypertrophy. The role of Sirt3 in ischemia is controversial. Our objective was to understand, under what circumstances Sirt3 is protective in different facets of ischemia, using an in vitro proof-of-concept approach based on simulated ischemia in cultured cardiomyoblasts. Cultured H9c2 cardiomyoblasts were subjected to hypoxia and/or serum deprivation, the combination of which we refer to as simulated ischemia. Apoptosis, as assessed by Annexin V staining in life-cell imaging and propidium-iodide inclusion in flow cytometry, was enhanced following simulated ischemia. Interestingly, serum deprivation was a stronger trigger of apoptosis than hypoxia. Knockdown of Sirt3 further increased apoptosis upon serum deprivation, whereas no such effect occurred upon additional hypoxia. Similarly, only upon serum deprivation but not upon simulated ischemia, silencing of Sirt3 led to a deterioration of mitochondrial function in extracellular flux analysis. In the absence of oxygen these Sirt3-dependent effects were abolished. These data indicate, that Sirt3-mediated myocardial protection is oxygen-dependent. Thus, mitochondrial respiration takes center-stage in Sirt3-dependent prevention of stress-induced myocardial damage.

Mild endothelial dysfunction in Sirt3 knockout mice fed a high-cholesterol diet: protective role of a novel C/EBP-ß-dependent feedback regulation of SOD2.

Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3 (-/-) mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3 (-/-) mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3 (-/-) mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-ß)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-ß and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-ß-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.

Deletion of Sirt3 does not affect atherosclerosis but accelerates weight gain and impairs rapid metabolic adaptation in LDL receptor knockout mice: implications for cardiovascular risk factor development.

Sirt3 is a mitochondrial NAD(+)-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species homeostasis. Sirt3 deficiency has been reported to accelerate the development of the metabolic syndrome. However, the role of Sirt3 in atherosclerosis remains enigmatic. We aimed to investigate whether Sirt3 deficiency affects atherosclerosis, plaque vulnerability, and metabolic homeostasis. Low-density lipoprotein receptor knockout (LDLR(-/-)) and LDLR/Sirt3 double-knockout (Sirt3(-/-)LDLR(-/-)) mice were fed a high-cholesterol diet (1.25 % w/w) for 12 weeks. Atherosclerosis was assessed en face in thoraco-abdominal aortae and in cross sections of aortic roots. Sirt3 deletion led to hepatic mitochondrial protein hyperacetylation. Unexpectedly, though plasma malondialdehyde levels were elevated in Sirt3-deficient mice, Sirt3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter). Likewise, plaque macrophage and T cell infiltration as well as endothelial activation remained unaltered. Electron microscopy of aortic walls revealed no difference in mitochondrial microarchitecture between both groups. Interestingly, loss of Sirt3 was associated with accelerated weight gain and an impaired capacity to cope with rapid changes in nutrient supply as assessed by indirect calorimetry. Serum lipid levels and glucose tolerance were unaffected by Sirt3 deletion in LDLR(-/-) mice. Sirt3 deficiency does not affect atherosclerosis in LDLR(-/-) mice. However, Sirt3 controls systemic levels of oxidative stress, limits expedited weight gain, and allows rapid metabolic adaptation. Thus, Sirt3 may contribute to postponing cardiovascular risk factor development.

Deletion of fibroblast activation protein provides atheroprotection.

AIMS: Fibroblast activation protein (FAP) is upregulated at sites of tissue remodelling including chronic arthritis, solid tumours, and fibrotic hearts. It has also been associated with human coronary atherosclerotic plaques. Yet, the causal role of FAP in atherosclerosis remains unknown. To investigate the cause-effect relationship of endogenous FAP in atherogenesis, we assessed the effects of constitutive Fap deletion on plaque formation in atherosclerosis-prone apolipoprotein E (Apoe) or low-density lipoprotein receptor (Ldlr) knockout mice. METHODS AND RESULTS: Using en face analyses of thoraco-abdominal aortae and aortic sinus cross-sections, we demonstrate that Fap deficiency decreased plaque formation in two atherosclerotic mouse models (-46% in Apoe and -34% in Ldlr knockout mice). As a surrogate of plaque vulnerability fibrous cap thickness was used; it was increased in Fap-deficient mice, whereas Sirius red staining demonstrated that total collagen content remained unchanged. Using polarized light, atherosclerotic lesions from Fap-deficient mice displayed increased FAP targets in terms of enhanced collagen birefringence in plaques and increased pre-COL3A1 expression in aortic lysates. Analyses of the Stockholm Atherosclerosis Gene Expression data revealed that FAP expression was increased in human atherosclerotic compared to non-atherosclerotic arteries. CONCLUSIONS: Our data provide causal evidence that constitutive Fap deletion decreases progression of experimental atherosclerosis and increases features of plaque stability with decreased collagen breakdown. Thus, inhibition of FAP expression or activity may not only represent a promising therapeutic target in atherosclerosis but appears safe at the experimental level for FAP-targeted cancer therapies.

Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac JunD messenger ribonucleic acid expression was reduced while JunB increased. Cardiac-specific JunD overexpressing mice (JunDTg/0 ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.

Deleterious role of endothelial lectin-like oxidized low-density lipoprotein receptor-1 in ischaemia/reperfusion cerebral injury.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is implicated in cardiovascular disease by modulating apoptosis and oxidative stress. We hypothesized that LOX-1 may be involved in pathophysiology of stroke by mediating ischaemia/reperfusion (I/R)-dependent cell death. Transient middle cerebral artery occlusion (tMCAO) was performed in wild-type (WT) mice, endothelial-specific LOX-1 transgenic mice (eLOX-1TG) and WT animals treated with LOX-1 silencing RNA (siRNA). In WT mice exposed to tMCAO, LOX-1 expression and function were increased in the MCA. Compared to WT animals, eLOX-1TG mice displayed increased stroke volumes and worsened outcome after I/R. Conversely, LOX-1-silencing decreased both stroke volume and neurological impairment. Similarly, in HBMVECs, hypoxia/reoxygenation increased LOX-1 expression, while LOX-1 overexpressing cells showed increased death following hypoxia reoxygenation. Increased caspase-3 activation was observed following LOX-1 overexpression both in vivo and in vitro, thus representing a likely mediator. Finally, monocytes from ischaemic stroke patients exhibited increased LOX-1 expression which also correlated with disease severity. Our data unequivocally demonstrate a key role for LOX-1 in determining outcome following I/R brain damage. Our findings could be corroborated in human brain endothelial cells and monocytes from patients, underscoring their translational relevance and suggesting siRNA-mediated LOX-1 knockdown as a novel therapeutic strategy for stroke patients.

Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity.

Aims: Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Methods and results: Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3-/- mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3-/- compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3-/- mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3-/- bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3-/- mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3-/- neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Conclusions: Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

Ticagrelor, but not clopidogrel, reduces arterial thrombosis via endothelial tissue factor suppression.

AIMS: The P2Y12 antagonist ticagrelor reduces mortality in patients with acute coronary syndrome (ACS), compared with clopidogrel, and the mechanisms underlying this effect are not clearly understood. Arterial thrombosis is the key event in ACS; however, direct vascular effects of either ticagrelor or clopidogrel with focus on arterial thrombosis and its key trigger tissue factor have not been previously investigated. METHODS AND RESULTS: Human aortic endothelial cells were treated with ticagrelor or clopidogrel active metabolite (CAM) and stimulated with tumour necrosis factor-alpha (TNF-α); effects on procoagulant tissue factor (TF) expression and activity, its counter-player TF pathway inhibitor (TFPI) and the underlying mechanisms were determined. Further, arterial thrombosis by photochemical injury of the common carotid artery, and TF expression in the murine endothelium were examined in C57BL/6 mice treated with ticagrelor or clopidogrel. Ticagrelor, but not CAM, reduced TNF-α-induced TF expression via proteasomal degradation and TF activity, independently of the P2Y12 receptor and the equilibrative nucleoside transporter 1 (ENT1), an additional target of ticagrelor. In C57BL/6 mice, ticagrelor prolonged time to arterial occlusion, compared with clopidogrel, despite comparable antiplatelet effects. In line with our in vitro results, ticagrelor, but not clopidogrel, reduced TF expression in the endothelium of murine arteries. CONCLUSION: Ticagrelor, unlike clopidogrel, exhibits endothelial-specific antithrombotic properties and blunts arterial thrombus formation. The additional antithrombotic properties displayed by ticagrelor may explain its greater efficacy in reducing thrombotic events in clinical trials. These findings may provide the basis for new indications for ticagrelor.