The C2238 Atrial Natriuretic Peptide Molecular Variant is Associated with Endothelial Damage and Dysfunction Through Natriuretic Peptide Receptor C Signaling
Rationale: C2238 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-αANP.
Methods and Results: In vitro, HUVEC were exposed to either wild-type (T2238) or mutant (C2238)-αANP. Cell survival and apoptosis were tested by Trypan blue, annexin V and cleaved caspase-3 assays. C2238-αANP significantly reduced HUVEC survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate NPR-A/B activity with respect to T2238-αANP, as evaluated by intracellular cGMP levels. In contrast, C2238-αANP, but not T2238-αANP, markedly reduced intracellular cAMP levels in a NPR-C-dependent manner. Accordingly, C2238-αANP showed higher affinity binding to NPR-C than T2238-αANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by siRNA rescued survival and tube formation of HUVEC exposed to C2238-αANP. Similar data were obtained in HAEC with NPR-C knock-down. NPR-C activation by C2238-αANP inhibited the PKA/Akt1 pathway and increased ROS. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-αANP. Overall, these data indicate that C2238-αANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/PKA/Akt1 pathway and increased ROS production. Accordingly, C2238-αANP 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-αANP 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.
- natriuretic peptide receptor type C
- C2238 ANP gene variant
- atrial natriuretic peptide
- genetic variation
- endothelial dysfunction
- Received March 6, 2013.
- Revision received March 17, 2013.
- Accepted March 25, 2013.