Histone deacetylase 3 antagonizes aspirin-stimulated endothelial nitric oxide production by reversing aspirin-induced lysine acetylation of endothelial nitric oxide synthase.

Rationale: Low-dose acetylsalicylic acid (aspirin) is widely used in the treatment and prevention of vascular atherothrombosis. Cardiovascular doses of aspirin also reduce systemic blood pressure and improve endothelium-dependent vasorelaxation in patients with atherosclerosis or risk factors for atherosclerosis. Aspirin can acetylate proteins, other than its pharmacological target cyclooxygenase, at lysine residues. The role of lysine acetylation in mediating the effects of low-dose aspirin on the endothelium is not known.

Objective: To determine the role of lysine acetylation of endothelial nitric oxide synthase (eNOS) in the regulation of endothelial NO production by low-dose aspirin and to examine whether the lysine deacetylase histone deacetylase (HDAC)3 antagonizes the effect of low-dose aspirin on endothelial NO production by reversing acetylation of functionally critical eNOS lysine residues.

Methods And Results: Low concentrations of aspirin induce lysine acetylation of eNOS, stimulating eNOS enzymatic activity and endothelial NO production in a cyclooxygenase-1-independent fashion. Low-dose aspirin in vivo also increases bioavailable vascular NO in an eNOS-dependent and cyclooxygenase-1-independent manner. Low-dose aspirin promotes the binding of eNOS to calmodulin. Lysine 609 in the calmodulin autoinhibitory domain of bovine eNOS mediates aspirin-stimulated binding of eNOS to calmodulin and eNOS-derived NO production. HDAC3 inhibits aspirin-stimulated (1) lysine acetylation of eNOS, (2) eNOS enzymatic activity, (3) eNOS-derived NO, and (4) binding of eNOS to calmodulin. Conversely, downregulation of HDAC3 promotes lysine acetylation of eNOS and endothelial NO generation.

Conclusions: Lysine acetylation of eNOS is a posttranslational protein modification supporting low-dose aspirin-induced vasoprotection. HDAC3, by deacetylating aspirin-acetylated eNOS, antagonizes aspirin-stimulated endothelial production of NO.