Publications by authors named "Vanasa Nageswaran"

4 Publications

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Sirtuin 5 promotes arterial thrombosis by blunting the fibrinolytic system.

Cardiovasc Res 2020 Sep 15. Epub 2020 Sep 15.

Center for Molecular Cardiology, Schlieren Campus, University of Zurich, Wagistrasse 12, Schlieren, Switzerland.

Aims: Arterial thrombosis as a result of plaque rupture or erosion is a key event in acute cardiovascular events. Sirtuin 5 (SIRT5) belongs to the lifespan-regulating sirtuin superfamily and has been implicated in acute ischemic stroke and cardiac hypertrophy. This project aims at investigating the role of SIRT5 in arterial thrombus formation.

Methods And Results: Sirt5 transgenic (Sirt5Tg/0) as well as knock-out (Sirt5-/-) mice underwent photochemically-induced carotid endothelial injury to trigger arterial thrombosis. Primary human aortic endothelial cells (HAECs) treated with SIRT5 silencing-RNA (si-SIRT5) as well as peripheral blood mononuclear cells (PBMCs) from acute coronary syndrome (ACS) patients and non-ACS controls (case-control study, total n = 171) were used to increase the translational relevance of our data. Compared to WT controls, Sirt5Tg/0 mice displayed accelerated arterial thrombus formation following endothelial-specific damage. Conversely, in Sirt5-/-mice arterial thrombosis was blunted. Platelet function was unaltered, as assessed by ex vivo collagen-induced aggregometry. Similarly, activation of the coagulation cascade as assessed by vascular and plasma tissue factor (TF) and TF pathway inhibitor (TFPI) expression was unaltered. Increased thrombus embolization episodes and circulating D-dimer levels suggested augmented activation of the fibrinolytic system in Sirt5-/- mice. Accordingly, Sirt5-/- mice showed reduced plasma and vascular expression of the fibrinolysis inhibitor plasminogen activator inhibitor (PAI)-1. In HAECs, SIRT5-silencing inhibited PAI-1 gene and protein expression in response to TNF-α. This effect was mediated by increased AMPK activation and reduced phosphorylation of the MAP kinase ERK 1/2, but not JNK and p38 as shown both in vivo and in vitro. Lastly, both PAI-1 and SIRT5 gene expression are increased in ACS patients compared to non-ACS controls after adjustment for cardiovascular risk factors, while PAI-1 expression increased across tertiles of SIRT5.

Conclusions: SIRT5 promotes arterial thrombosis by modulating fibrinolysis through endothelial PAI-1 expression. Hence, SIRT5 may be an interesting therapeutic target in the context of atherothrombotic events.

Translational Perspectives: This study illustrates a novel role for Sirtuin 5 in arterial thrombosis by regulating fibrinolysis through plasminogen activator inhibitor 1 (PAI-1). These results shed new light onto the pathophysiology of arterial thrombus formation which underlies most of the acute atherosclerotic complications. Also, they further affirm the intrinsic relationship between lifespan regulating genes, vascular dysfunction and age-related cardiovascular disease, thus indicating these genes as potential targets for cardiovascular prevention and therapy. Further studies will be needed to assess the predictive ability of SIRT5 in patients with acute cardiovascular or cerebrovascular events. Also, the design of specific SIRT5 inhibitors will allow trials aiming at investigating the efficacy of SIRT5 blockage in the clinical setting.
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September 2020

Apold1 deficiency associates with increased arterial thrombosis in vivo.

Eur J Clin Invest 2020 Feb;50(2):e13191

Center for Molecular Cardiology, University of Zurich, Schlieren, Switzerland.

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 mitogen-activated 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. Additionally, Apold1 mice displayed increased platelet reactivity to stimulation with collagen compared with WT animals.

Conclusions: Deficiency of Apold1 results in a prothrombotic phenotype, accompanied by increased vascular TF activity, decreased PI3K/Akt activation and increased platelet reactivity. These findings suggest Apold1 as an interesting new therapeutic target in the context of arterial thrombosis.
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February 2020

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

Eur Heart J 2020 04;41(16):1575-1587

Center for Molecular Cardiology, Schlieren Campus, University of Zurich, Wagistrasse 12, Schlieren 8952, Switzerland.

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.
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April 2020

Rapid and sensitive detection of calreticulin type 1 and 2 mutations by real-time quantitative PCR.

Mol Diagn Ther 2015 Oct;19(5):329-34

Charité, Med. Klinik für Hämatologie, Onkologie, Tumorimmunologie, Hindenburgdamm 30, 12200, Berlin, Germany.

Background: The majority of patients with JAK2 V617F-negative essential thrombocythemia or primary myelofibrosis harbor mutations involving the calreticulin (CALR) gene. These mutations are located in CALR exon 9 and lead to a frameshift with subsequent alteration of the CALR protein C-terminus. They have emerged as valuable molecular markers for the diagnosis of clonal myeloproliferative diseases. Although a variety of CALR mutations have been described, two mutations, denoted type 1 and type 2, account for around 85 % of cases. The type 1 mutation encompasses a 52 bp deletion and the type 2 mutation a 5 bp TTGTC insertion.

Methods: This work describes the development and testing of quantitative real-time PCRs (qPCRs) for detecting these two mutations.

Results: The final type 1 CALR qPCR displayed a sensitivity of <0.1 % mutant alleles and the type 2 CALR qPCR had a sensitivity of <0.01 % mutant alleles. Additionally, two new CALR mutations are reported.

Conclusion: These sensitive and specific qPCRs should be helpful in establishing the diagnosis and in monitoring minimal residual disease in patients during or after therapy.
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October 2015