Aspirin, but not clopidogrel, reduces monocyte plaque content through a modulatory action on endothelial-derived netrin-1 via a COX-dependent epigenetic modification

Gabriella Passacquale, Alkystis Phinikaridou, Christina Warboys, Alessio Alfieri, Marcelo Andia, Rene Botnar, Albert Ferro. King's College London, London, UK

Introduction: The neuroimmune guidance cue netrin-1 is an important regulator of atheroma development by virtue of its ability to inhibit monocyte motility. Endothelial-derived netrin-1, which is reduced under pro-inflammatory conditions, repels monocyte vascular infiltration from the bloodstream; on the other hand, monocyte netrin-1, which is enhanced by pro-atherogenic stimuli, compromises their egress from plaques. Hence, an important and unresolved question is whether systemic therapeutic manipulation of netrin-1 might produce detrimental rather than beneficial anti-atherogenic effects due to an indiscriminate action on different cell types with opposite effects on monocyte arterial accumulation.

Aims: We aimed to identify whether i) aspirin, which is used as anti-platelet agent in cardiovascular prevention but also exerts anti-inflammatory properties on the endothelium, modulates the endothelial expression of netrin-1 in vitro and ii) what is the net effect of aspirin on netrin-1 production and atherosclerosis progression in vivo.

Methods: Human umbilical vein endothelial cells (HUVECs) were incubated overnight with TNFα (10 ng/ml) either alone or in combination with aspirin (0.5 mM), the cyclooxygenase (COX)-1 inhibitor SC-560 (30 nM), the COX-2 inhibitor NS-398 (10 μM) or indomethacin (100 μM). Netrin-1 was measured in cells and tissues by RT-PCR and immunofluorescence staining and by ELISA in cell supernatant. Monocyte migration toward treated endothelial cells was evaluated using a Boyden chamber system. Given its crucial role in regulating TNFα-dependent gene expression, histone acetylation was studied by western blotting and immunofluorescence staining. Histone deacetylase (HDAC) and histone acetyltransferase (HAT) activities were measured by enzyme immunoassay. The HDAC inhibitor trichostatin A (TSA, 400 nM) was used as a positive control. ApoE-/- mice on 8-weeks high fat diet were treated with aspirin (5 mg kg-1day-1) or clopidogrel (25 mg kg-1day-1) and compared to untreated animals. Netrin-1 was measured in the plasma by ELISA and within the arterial wall by immunofluorescence staining. Circulating and plaque monocytes were characterized by flow cytometry.

Results: TNFα down-regulated the synthesis of secreted netrin-1 by HUVECs (86.25±57.2 ng/ml vs 295.5±64.4 ng/ml in control; p<0.01), thus increasing monocyte chemotaxis toward the endothelium (122.4±20.6 vs 66±30 cells/field in control; p<0.01). Aspirin restored netrin-1 secretion by TNFα-stimulated cells (199.6±30 ng/ml; p<0.01) and reduced monocyte chemotaxis (83±35.7 cells/field; p<0.01). Aspirin, in co-treatment with TNFα , increased histone acetylation (ratio acetylated/total histone: 0.2±0.01 with aspirin/TNFα co-treatment vs 0.02±0.02 in untreated cells; p<0.05 and vs 0.05±0.02 in TNFα-treated cells; p<0.05). Among the other COX-inhibitors, only indomethacin reproduced the effects of aspirin. TSA stimulation either alone (618.3±77 ng/ml; p<0.01 in control) or in combination with TNFα (993.6±61 ng/ml; p<0.01 vs control), enhanced endothelial netrin-1 secretion. In ApoE-/- mice, aspirin, but not clopidogrel, increased the plasma level (170.7±56.13 ng/ml vs 53.73 ng/ml in control mice; p<0.01) and endothelial expression of netrin-1. Unlike clopidogrel, aspirin reduced plaque monocyte content (26.6±3.7% vs 44.2±7.1% in untreated animals; p<0.01), despite both drugs suppressing the blood monocytosis that accompanies plaque development in ApoE-/- mice (monocytes: 29±3% and 28.4±4.5% in aspirin and clopidogrel groups respectively vs 42.8±6% in control mice; p<0.01 and p<0.05 respectively).

Conclusions: Aspirin restores endothelial-derived netrin-1 production under pro-inflammatory conditions through a platelet-independent action on the endothelium sustained by COX-inhibition and subsequent histone hyper-acetylation. Aspirin-dependent increase in netrin-1 beneficially affects plaque composition by reducing monocyte accumulation.