Dual regulatory effects of nitric oxide on plasminogen activator inhibitor type 1 expression in endothelial cells

Abstract

In this report we compared the mechanism by which nitric oxide (NO), generated exogenously and endogenously, affects the plasminogen activator inhibitor type 1 (PAI-1) expression in endothelial cells. For this purpose, we stimulated the endothelial cell line EA.hy 926 with tumour necrosis factor a (TNFa) in the presence of the exogenously NO-releasing donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine, or regulators of nitric oxide synthase (NOS) inhibitor N-nitro-l-arginine-methyl ester hydrochloride and substrate l-Arg. Expression of PAI-1 in EA.hy 926 cells was determined by measuring the level of mRNA, using relative quantitative reverse transcriptase PCR, and protein, using ELISA. In addition, we estimated the level of activation of two mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK1/2), in the cells before and after treatment with TNFa, in the presence or absence of NO donors and inhibitors. In contrast to exogenously released NO that significantly reduced mostly basal PAI-1 expression, endogenously generated NO by NOS potentiated TNFa-induced upregulation of PAI-1 expression. Exogenously and endogenously generated NO causes different effects on activation of the MAPKs ERK1/2 and JNK1/2. Specifically, the SNP-released NO activates only ERK1/2, while endogenously generated NO in a pathway induced by TNFa activates both MAPKs. Thus our data indicate that due to different cellular locations and mechanisms of generation, NO may participate in various signalling pathways leading to opposite effects on PAI-1 expression in endothelial cells.Keywords: plasminogen activator inhibitor type 1 expression; nitric oxide donors; tumor necrosis factor a activation; mitogen-activated protein kinases.Plasminogen-activator inhibitor type 1 (PAI-1) is an essential component of the plasmin/plasminogen activator cascade that regulates both vascular fibrinolysis and extravascular generation of plasmin. Plasmin/plasminogen activator-mediated proteolysis not only solubilizes fibrin, and digests various basement membrane components [1], but also is implicated in the activation of collagenase [2] and transforming growth factor b [3] and in the release of basic fibroblast growth factor complexed with heparan sulfate from endothelial cells [4]. It also regulates the proteolytic activity generated extravascularly after activation of plasminogen accompanying tissue repair. In addition, PAI-1 appears to be involved in controlling migration of endothelial cells [5] and vascular smooth muscle cells [6], a critical process in vascular remodelling and atherogenesis. PAI-1 expression has been demonstrated in various cell types, and multiple factors have been described that play a role in the regulation of PAI-1 synthesis and secretion [7,8]. Increased levels of PAI-1 have been identified in atherosclerotic human arteries [9], in neontima formed after vein graft failure [10], and balloon-injured blood vessels [11]. Elevated PAI-1 synthesis and secre...

Downregulation of Endocardial Nitric Oxide Synthase Expression and Nitric Oxide Production in Atrial Fibrillation

“…An increase in PAI-1 attributable to a loss of NO · at these sites may also contribute to lesion formation. 10,11 Some of the PAI-1 expressed and released by endothelial cells remains associated with the cell surface. It can reduce endothelial fibrinolytic activity, thus promoting local thrombus formation.…”

“…9 The antithrombotic effects of NO · on arterial endothelium are also attributable to inhibition of the expression of the prothrombotic protein plasminogen activator inhibitor-1 (PAI-1). 10,11 Nitric oxide is synthesized by nitric oxide synthase (NOS), which is present in large concentrations in the endothelium. In the endothelium, it has been shown that NOS expression is regulated by flowmediated shear stress and is downregulated at sites with low flow.…”

“…11 To determine if this reciprocal relationship was also present in the atrial endocardium, PAI-1 protein levels were determined by Figure 2. Basal NO · production in atria, atrial appendage, and aortic tissues isolated from control and atrial fibrillation animals.…”

Cerivastatin, a HMG-CoA Reductase Inhibitor, Reduces Plasminogen Activator Inhibitor-1 (PAI-1) Expression in Endothelial Cells by Down-Regulation of Cellular Signaling and the Inhibition of PAI-1 Promoter Activity

“…Prior to the treatment with either LOV or CRV, the cell medium was changed and the cells starved for 12 h in DMEM supplemented with 0.1% FCS to ensure the appropriate cell synchronisation. Such conditions neither affected cell viability nor caused any detectable increase cell activation (not shown) (23). Then LOV (10 -80 mM) or CRV (0.01 -20 mM) were added and the cells were further incubated for 24 h in 48-well microplates.…”

“…Kinase assays were performed as described before (23). The treatment of cells with TNF-a (50 ng /ml) for 20 min was used to reach a maximum activation of ERK 1 /2.…”