Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells

Aims The mammalian silent information regulator-two 1 (Sirt1) blunts the noxious effects of cardiovascular risk factors such as type 2 diabetes mellitus and obesity. Nevertheless, the role of Sirt1 in regulating the expression of tissue factor (TF), the key trigger of coagulation, and arterial thrombus formation remains unknown. Methods and results Human as well as mouse cell lines were used for in vitro experiments, and C57Bl/6 mice for in vivo procedures. Sirt1 inhibition by splitomicin or sirtinol enhanced cytokine-induced endothelial TF protein expression as well as surface activity, while TF pathway inhibitor protein expression did not change. Sirt1 inhibition further enhanced TF mRNA expression, TF promoter activity, and nuclear translocation as well as DNA binding of the p65 subunit of nuclear factor-kappa B (NFκB/p65). Sirt1 siRNA enhanced TF protein and mRNA expression, and this effect was reduced in NFκB/p65−/− mouse embryonic fibroblasts reconstituted with non-acetylatable Lys310-mutant NFκB/p65. Activation of the mitogen-activated protein kinases p38, c-Jun NH2-terminal kinase, and p44/42 (ERK) remained unaffected. In vivo, mice treated with the Sirt1 inhibitor splitomicin exhibited enhanced TF activity in the arterial vessel wall and accelerated carotid artery thrombus formation in a photochemical injury model. Conclusion We provide pharmacological and genetic evidence that Sirt1 inhibition enhances TF expression and activity by increasing NFκB/p65 activation in human endothelial cells. Furthermore, Sirt1 inhibition induces arterial thrombus formation in vivo. Hence, modulation of Sirt1 may offer novel therapeutic options for targeting thrombosi

Deletion of the ageing gene p66Shc reduces early stroke size following ischaemia/reperfusion brain injury

Aims Stroke is a leading cause of morbidity and mortality, and its incidence increases with age. Both in animals and in humans, oxidative stress appears to play an important role in ischaemic stroke, with or without reperfusion. The adaptor protein p66Shc is a key regulator of reactive oxygen species (ROS) production and a mediator of ischaemia/reperfusion damage in ex vivo hearts. Hence, we hypothesized that p66Shc may be involved in ischaemia/reperfusion brain damage. To this end, we investigated whether genetic deletion of p66Shc protects from ischaemia/reperfusion brain injury. Methods and results Transient middle cerebral artery occlusion (MCAO) was performed to induce ischaemia/reperfusion brain injury in wild-type (Wt) and p66Shc knockout mice (p66Shc−/−), followed by 24 h of reperfusion. Cerebral blood flow and blood pressure measurements revealed comparable haemodynamics in both experimental groups. Neuronal nuclear antigen immunohistochemical staining showed a significantly reduced stroke size in p66Shc−/− when compared with Wt mice (P < 0.05, n = 7-8). In line with this, p66Shc−/− mice exhibited a less impaired neurological function and a decreased production of free radicals locally and systemically (P < 0.05, n = 4-5). Following MCAO, protein levels of gp91phox nicotinamide adenine dinucleotide phosphate oxidase subunit were increased in brain homogenates of Wt (P < 0.05, n = 4), but not of p66Shc−/− mice. Further, reperfusion injury in Wt mice induced p66Shc protein in the basilar and middle cerebral artery, but not in brain tissue, suggesting a predominant involvement of vascular p66Shc. Conclusion In the present study, we show that the deletion of the ageing gene p66Shc protects mice from ischaemia/reperfusion brain injury through a blunted production of free radicals. The ROS mediator p66Shc may represent a novel therapeutical target for the treatment of ischaemic strok

Reduced nitric oxide bioavailability mediates cerebroarterial dysfunction independent of cerebral amyloid angiopathy in a mouse model of Alzheimer's disease

In Alzheimer's disease (AD), cerebral arteries, in contrast to cerebral microvessels, show both cerebral amyloid angiopathy- (CAA) dependent and -independent vessel wall pathology. However, it remains unclear whether CAA-independent vessel wall pathology affects arterial function thereby chronically reducing cerebral perfusion, and if so which mechanisms mediate this effect. To this end, we assessed the ex vivo vascular function of the basilar artery and a similar-sized peripheral artery (femoral artery) in the Swedish-Arctic (SweArc) transgenic AD mouse model at different disease stages. Further, we used quantitative immunohistochemistry to analyze CAA, endothelial morphology, and molecular pathways pertinent to vascular relaxation. We found that endothelium-dependent, but not smooth muscle-dependent vasorelaxation was significantly impaired in basilar and femoral arteries of 15-month-old SweArc mice compared to that of age-matched wildtype (WT) and 6-month-old SweArc mice. This impairment was accompanied by significantly reduced levels of cyclic GMP (cGMP), indicating a reduced nitric oxide (NO) bioavailability. However, no age- and genotype-related differences in oxidative stress as measured by lipid peroxidation were observed. Although parenchymal capillaries, arterioles, and arteries showed abundant CAA in the 15-month-old SweArc mice, no CAA or changes in endothelial morphology were detected histologically in the basilar and femoral artery. Thus, our results suggest that in this AD mouse model dysfunction of large intracranial, extracerebral arteries important for brain perfusion is mediated by reduced NO bioavailability rather than by CAA. This finding supports the growing body of evidence highlighting the therapeutic importance of targeting the cerebrovasculature in AD

Adaptor protein p66(Shc) mediates hypertension-associated, cyclic stretch-dependent, endothelial damage

Increased cyclic stretch to the vessel wall, as observed in hypertension, leads to endothelial dysfunction through increased free radical production and reduced nitric oxide bioavailability. Genetic deletion of the adaptor protein p66(Shc) protects mice against age-related and hyperglycemia-induced endothelial dysfunction, as well as atherosclerosis and stroke. Furthermore, p66(Shc) mediates vascular dysfunction in hypertensive mice. However, the direct role of p66(Shc) in mediating mechanical force-induced free radical production is unknown; thus, we studied the effect of cyclic stretch on p66(Shc) activation in primary human aortic endothelial cells and aortic endothelial cells isolated from normotensive and hypertensive rats. Exposure of human aortic endothelial cells to cyclic stretch led to a stretch- and time-dependent p66(Shc) phosphorylation at Ser36 downstream of integrin α5β1 and c-Jun N-terminal kinase. In parallel, nicotinamide adenine dinucleotide phosphate oxidase activation, as well as production of reactive oxygen species, increased, whereas nitric oxide bioavailability decreased. Silencing of p66(Shc) blunted stretch-increased superoxide anion production and nicotinamide adenine dinucleotide phosphate oxidase activation and restored nitric oxide bioavailability. In line with the above, activation of p66(Shc) increased in isolated aortic endothelial cells of spontaneously hypertensive rats compared with normotensive ones. Pathological stretch by activating integrin α5β1 and c-Jun N-terminal kinase phosphorylates p66(Shc) at Ser36, augments reactive oxygen species production via nicotinamide adenine dinucleotide phosphate oxidase, and in turn reduces nitric oxide bioavailability. This novel molecular pathway may be relevant for endothelial dysfunction and vascular disease in hypertension