C2238 atrial natriuretic peptide molecular variant is associated with endothelial damage and dysfunction through natriuretic peptide receptor C signaling

Rationale: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events. Objective: We investigated the mechanisms underlying the vascular effects of C2238-\u3b1ANP. Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-\u3b1ANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-\u3b1ANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-\u3b1ANP reduced endothelial tube formation, as assessed by matrigel. C2238-\u3b1ANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-\u3b1ANP, as evaluated by intracellular cGMP levels. In contrast, C2238-\u3b1ANP, but not T2238-\u3b1ANP, markedly reduced intracellular cAMP levels in an NPR-C-dependent manner. Accordingly, C2238-\u3b1ANP showed higher affinity binding to NPR-C, than T2238-\u3b1ANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-\u3b1ANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-\u3b1ANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirusmediated Akt1 reactivation rescued the detrimental effects of C2238-\u3b1ANP. Overall, these data indicate that C2238-\u3b1ANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-\u3b1ANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results. Conclusions: C2238-\u3b1ANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers