Elsevier

Atherosclerosis

Volume 185, Issue 2, April 2006, Pages 246-253
Atherosclerosis

Human monoclonal IgG anticardiolipin antibodies induce nitric oxide synthase expression

https://doi.org/10.1016/j.atherosclerosis.2005.06.044Get rights and content

Abstract

Objective

Antiphospholipid antibodies are associated with increased risk of thrombosis, particularly as in antiphospholipid syndrome. This study aims to determine the acute effects of anticardiolipin antibodies on nitric oxide production and vascular function.

Methods

Ex vivo aortic rings from male Sprague Dawley rats were incubated with IgG monoclonal anticardiolipin antibody (IS4) or a non-specific IgG control. In organ baths, response to phenylephrine and acetlycholine was determined alone and with nitro-l-arginine methyl ester (l-NAME), 1400 W, d-arginine, l-arginine, sodium nitroprusside and cardiolipin. In vivo antibodies were injected into anaesthetised, spontaneously breathing male Sprague Dawley rats. Haemodynamic variables and serum nitric oxide were measured. Immunohistochemistry for iNOS and eNOS was performed in kidney vessels.

Results

Phenylepherine contraction was decreased in the IS4 group compared to controls (p < 0.001). l-NAME, 1400 W and cardiolipin, abolished this effect. l-Arginine caused significant relaxation in the IS4 group (p = 0.005). Mean arterial pressure in rats injected with IS4 was reduced compared to IgG and saline controls (p < 0.001). NO in plasma increased significantly after IS4 administration (p < 0.001). Immunohistochemistry showed increased iNOS expression in kidney arteries in the IS4 group, with no change in eNOS.

Conclusion

Anticardiolipin antibodies induce NO production acutely via increased expression of iNOS in both ex vivo and in vivo models.

Introduction

Antiphospholipid antibodies are the hallmark of the antiphospholipid antibody syndrome (APS), an autoimmune disease characterised clinically by venous and arterial thromboses and recurrent foetal losses. These antibodies have varying affinities for anionic (negatively charged) phospholipids (e.g. cardiolipin, phosphatidylglycerol, phosphatidylinositol and phosphatidylserine), plasma phospholipid-binding proteins (e.g. β2-glycoprotein 1[B2-GP1], prothrombin and protein C) or complexes of these proteins and phospholipids [1]. Antiphospholipid (aPL) antibodies may have pro-coagulant effects through the inhibition of the anticoagulant activity of β2-GP1 [2], decreased activation of protein C and antithrombin III, reduction in levels of free protein S and inhibition of factor XII/prekallikrein-mediated fibrinolytic activity [3]. Other reported actions of aPL include complement activation [4], platelet activation and aggregation [5], increased tissue factor expression [6] and decreased function of annexin V [7]. An interaction between aPL antibodies and the vascular endothelium has also been reported, and enhanced expression of adhesion molecules by endothelial cells after incubation with anticardiolipin (aCL) antibodies, suggests a direct activation of the endothelium by aCL [8].

Oxidative stress is now recognised as one of the major factors contributing to vascular disease in general and atherosclerosis in particular, and also seems to play an important role in the pathophysiology of APS [9]. F2-isoprostanes are elevated in patients with systemic lupus erythematosus (SLE) and with aCL antibodies [10], and the lipid peroxidation levels correlate with aCL antibody titres in patients with APS [11]. Furthermore, the activity of paraoxonase (an antioxidant enzyme present in high-density lipoprotein particles), is reduced in patients with APS, as is the total antioxidant capacity (TAC) of plasma [12].

One additional mechanism by which reactive oxygen species might contribute to vascular thrombosis is by rapid chemical inactivation of the vasodilator and antithrombotic mediator NO derived from the endothelium. However, a direct relationship between aCL antibodies and NO activity has never been established, although NO metabolites in urine have been reported to be decreased in patients with APS and their levels to inversely correlate with the aCL antibody titres [9].

We, therefore, examined the effects of aCL antibodies on NO pathways and vascular function in vivo and in vitro. We find that aCL antibodies are proinflammatory and can induce iNOS expression and function and increase oxidant stress in blood vessels, which may contribute to the increased risk of thrombosis in patients with APS.

Section snippets

Hybridoma cells and antibodies

IS4, a human IgG monoclonal antibody which binds to cardiolipin and β2-GP1, was derived from patients with antiphospholipid syndrome (a kind gift from Pojen Chen, UCLA, CA, USA) [13]. Cells were cultured in RPMI 1640 medium containing 1% l-glutamine, 1% sodium pyruvate, 2% MEM non-essential amino-acids, 1% penicillin/streptomycin, 0.2% gentamycin (all from Gibco, UK) and 10% FCS (Sigma, UK). Antibodies were purified in an LPS free environment (Cymbus Biotechnology limited). Human non-specific

Organ chamber experiments

The PE contraction of rat aortic rings was markedly decreased in vessels pre-treated with IS4 for 4 h, when compared to control IgG or vehicle (n = 8, p < 0.001; Fig. 1A). The impairment in PE contraction induced by IS4 was equal in magnitude to that produced by LPS (n = 4).

The impaired response to PE in IS4 and LPS treated vessels was abolished by the addition of l-NAME or 1400 W indicating that the PE hyporeactivity was secondary to an increased activation of iNOS (Fig. 1A and B).

PE hyporeactivity to

Discussion

In this study the ex vivo model demonstrated that short-term incubation with IS4 leads to phenylephrine and acetylcholine hyporeactivity in rat aortic tissue, with no change in response to the endothelium-independent vasodilator sodium nitroprusside. These changes are reversible following incubation with l-NAME or 1400 W and were prevented when the antibody was pre-incubated with its antigen cardiolipin. Addition of l-arginine pre-constricted tissues led to significant relaxation in the IS4

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