SIRT5 is upregulated in PBMCs of AIS patients and in the MCA of WT mice exposed to tMCAO; SIRT5 mediates I/R-induced brain damage by increasing BBB permeability through degradation of occludin. This effect was reproduced in HBMVECs exposed to H/R, mediated by the PI3K/Akt pathway. Our findings shed new light on the mechanisms of I/R-dependent brain damage and suggest SIRT5 as a novel therapeutic target.

Background and Purpose— Inflammation is a major pathogenic component of ischemia/reperfusion brain injury, and as such, interventions aimed at inhibiting inflammatory mediators promise to be effective strategies in stroke therapy. JunD—a member of the AP-1 (activated protein-1) family of transcription factors—was recently shown to regulate inflammation by targeting IL (interleukin)-1β synthesis and macrophage activation. The purpose of the present study was to assess the role of JunD in ischemia/reperfusion-induced brain injury. Methods— WT (wild type) mice randomly treated with either JunD or scramble (control) siRNA were subjected to 45 minutes of transient middle cerebral artery occlusion followed by 24 hours of reperfusion. Stroke size, neurological deficit, plasma/brain cytokines, and oxidative stress determined by 4-hydroxynonenal immunofluorescence staining were evaluated 24 hours after reperfusion. Additionally, the role of IL-1β was investigated by treating JunD siRNA mice with an anti–IL-1β monoclonal antibody on reperfusion. Finally, JunD expression was assessed in peripheral blood monocytes isolated from patients with acute ischemic stroke. Results— In vivo JunD knockdown resulted in increased stroke size, reduced neurological function, and increased systemic inflammation, as confirmed by higher neutrophil count and lymphopenia. Brain tissue IL-1β levels were augmented in JunD siRNA mice as compared with scramble siRNA, whereas no difference was detected in IL-6, TNF-α (tumor necrosis factor-α), and 4-hydroxynonenal levels. The deleterious effects of silencing of JunD were rescued by treating mice with an anti–IL-1β antibody. In addition, JunD expression was decreased in peripheral blood monocytes of patients with acute ischemic stroke at 6 and 24 hours after onset of stroke symptoms compared with sex- and age-matched healthy controls. Conclusions— JunD blunts ischemia/reperfusion-induced brain injury via suppression of IL-1β.

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Aims Arterial stiffness is a hallmark of vascular aging that precedes and strongly predicts the development of cardiovascular diseases. Age-dependent stiffening of large elastic arteries is primarily attributed to increased levels of matrix metalloproteinase-2 (MMP-2). However, the mechanistic link between age-dependent arterial stiffness and MMP-2 remains unclear. Thus, we aimed to investigate the efficacy of MMP-2 knockdown using small interfering RNA (siRNA) on age-dependent arterial stiffness. Methods and Results Pulse wave velocity (PWV) was assessed in right carotid artery of wild type (WT) mice from different age groups. MMP-2 levels in the carotid artery and plasma of young (3 months) and old (20-25 months) WT mice were determined. Carotid PWV as well as vascular and circulating MMP-2 were elevated with increasing age in mice. Old WT mice (18-21-month-old) were treated for 4 weeks with either MMP-2 or scrambled (Scr) siRNA via tail vein injection. Carotid PWV was assessed at baseline, 2 and 4 weeks after start of the treatment. MMP-2 knockdown reduced vascular MMP-2 levels and attenuated age-dependent carotid stiffness. siMMP-2 treated mice showed increased elastin to collagen ratio, lower plasma desmosine (DES), enhanced phosphorylation of endothelial nitric oxide synthase (eNOS) and higher levels of vascular cyclic guanosine monophosphate (cGMP). An age-dependent increase in direct protein-protein interaction between MMP-2 and eNOS was also observed. Lastly, DES, an elastin breakdown product, was measured in a patient cohort (n = 64, 23-86 years old), where carotid-femoral PWV was also assessed; here, plasma levels of DES directly correlated with age and arterial stiffness. Conclusion MMP-2 knockdown attenuates age-dependent carotid stiffness by blunting elastin degradation and augmenting eNOS bioavailability. Given the increasing clinical use of siRNA technology, MMP2 knockdown should be investigated further as a possible strategy to mitigate age-dependent arterial stiffness and related CV diseases. Translational Perspective Arterial stiffness is a hallmark of vascular aging that precedes and strongly predicts the development of cardiovascular diseases. This study provides translational evidence to support a key role for MMP-2 on the development of age-associated arterial stiffness. Silencing of MMP-2 using siRNA technology shows an effect on aged mice where it attenuates age-dependent carotid stiffness by reducing elastin degradation and increasing eNOS bioavailability. Additionally, in humans we show that elastin breakdown increases with age and increased PWV. These findings indicate MMP-2 knockdown as a promising novel strategy to attenuate age-dependent arterial stiffness and cardiovascular diseases.

Background: Endothelial cells regulate the formation of blood clots; thus, genes selectively expressed in these cells could primarily determine thrombus formation.Apold1 (apolipoprotein L domain containing 1) is a gene expressed by endothelial cells; whether Apold1 directly contributes to arterial thrombosis has not yet been investigated. Here, we assessed the effect of Apold1 deletion on arterial thrombus formation using an in vivo model of carotid thrombosis induced by photochemical injury. Material and methods: Apold1 knockout (Apold1 −/− ) mice and wild-type (WT) littermates underwent carotid thrombosis induced by photochemical injury, and time to occlusion was recorded. Tissue factor (TF) activity and activation of mitogenactivated protein kinases (MAPKs) and phosphatidyl-inositol-3 kinase (PI3K)/ Akt pathways were analysed by colorimetric assay and Western blotting in both Apold1 −/− and WT mice. Finally, platelet reactivity was assessed using light transmission aggregometry. Results: After photochemical injury, Apold1 −/− mice exhibited shorter time to occlusion as compared to WT mice. Moreover, TF activity was increased in carotid arteries of Apold1 −/− when compared to WT mice. Underlying mechanistic markers such as TF mRNA and MAPKs activation were unaffected in Apold1 −/− mice. In contrast, phosphorylation of Akt was reduced in Apold1 −/− as compared to WT mice.