Abstract
Background: Besides its well-known functions in hemostasis, thrombin plays a role in various non-hemostatic biological and pathophysiologic processes. We examined the potential of thrombin to promote premature atrial endothelial cells (ECs) senescence. Methods and Results: Primary ECs were isolated from porcine atrial tissue. Endothelial senescence was assessed by measuring beta-galactosidase (SA-β-gal) activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, protein level by Western blot, and matrix metalloproteinases (MMPs) activity using zymography. Atrial endothelial senescence was induced by thrombin at clinically relevant concentrations. Thrombin induced the up-regulation of p53, a key regulator in cellular senescence and of p21 and p16, two cyclin-dependent kinase inhibitors. Nicotinamide adenine dinucleotide phosphate NADPH oxidase, cyclooxygenases and the mitochondrial respiration complex contributed to oxidative stress and senescence. Enhanced expression levels of vascular cell adhesion molecule (VCAM)-1, tissue factor, transforming growth factor (TGF)-β and MMP-2 and 9 characterized the senescence-associated secretory phenotype of atrial ECs. In addition, the pro-senescence endothelial response to thrombin was associated with an overexpression of both angiotensin converting enzyme and AT1 receptors and was inhibited by perindoprilat and losartan. Conclusions: Thrombin promotes premature ageing and senescence of atrial ECs and may pave the way to deleterious remodeling of atrial tissue by a local up-regulation of the angiotensin system and by promoting pro-inflammatory, pro-thrombotic, pro-fibrotic and pro-remodeling responses. Hence, targeting thrombin and/or angiotensin systems may efficiently prevent atrial endothelial senescence.
Highlights
Atrial fibrillation (AF) is the most common sustained arrhythmia, especially during ageing, and portends an increased risk of thromboembolism, ischemic stroke and mortality [1]
The major characteristic of senescence is cell-cycle arrest, recent studies revealed that cells undergoing either replicative or premature senescence acquire a senescence-associated secretory phenotype (SASP) which leads to the release of three categories of factors: inflammatory mediators, growth factors, and modifiers of the extracellular matrix (ECM) [13]
We have recently demonstrated that endothelial senescence favors thrombogenicity through several mechanisms including tissue factor (TF) up-regulation, pro-coagulant MPs release, endothelial nitric oxide synthase down-regulation and reduced endothelial cells (ECs)-mediated nitric oxide (NO)-dependent inhibition of platelet aggregation [15]
Summary
Atrial fibrillation (AF) is the most common sustained arrhythmia, especially during ageing, and portends an increased risk of thromboembolism, ischemic stroke and mortality [1]. Besides its role in thrombus formation, recent findings have highlighted that thrombin conveys “non-hematological” functions through cell protease activated receptor (PAR). At the interface between blood and atria tissue, ECs play a determinant role in the tuning of hemostatic functions even though the underlying mechanisms remain poorly investigated. Endothelial senescent cells secrete matrix metalloproteinases MMPs, plasminogen activator inhibitor 1, growth factors, proteases, and cytokines such as IL-1, IL-6 and IL-8, all with potential strong autocrine and paracrine actions [14]. We have recently demonstrated that endothelial senescence favors thrombogenicity through several mechanisms including tissue factor (TF) up-regulation, pro-coagulant MPs release, endothelial nitric oxide synthase (eNOS) down-regulation and reduced ECs-mediated nitric oxide (NO)-dependent inhibition of platelet aggregation [15]. The possibility that atrial endothelial senescence shifts the cell phenotype towards pro-thrombotic, pro-fibrotic, pro-inflammatory and pro-remodeling patterns was examined. Since angiotensin II (AngII) via Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived oxidative stress is a potent inducer of premature endothelial senescence and ECs express both angiotensin-converting enzyme (ACE) and angiotensin type 1 receptors (AT1R) [15], the potential role of the local AngII/AT1R in the noxious impact of thrombin on premature endothelial senescence and dysfunction was determined
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