IRCCS Neuromed, Pozzilli, Italy.
Circ Res. 2013 May 10;112(10):1355-64. doi: 10.1161/CIRCRESAHA.113.301325. Epub 2013 Mar 25.
C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events.
We investigated the mechanisms underlying the vascular effects of C2238-αANP.
In vitro, human umbilical vein endothelial cell 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 human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate natriuretic peptide receptor (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 an 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 small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-αANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-αANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. 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/protein kinase A/Akt1 pathway and increased reactive oxygen species 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.
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.
C2238 心房利钠肽（ANP）的次要等位基因（2238 位胸腺嘧啶被胞嘧啶取代）与心血管事件风险增加相关。
我们研究了 C2238-αANP 对血管作用的机制。
在体外，用野生型（T2238）或突变型（C2238）-αANP 处理人脐静脉内皮细胞。通过台盼蓝、膜联蛋白 V 和 cleaved caspase-3 测定法检测细胞存活率和细胞凋亡。C2238-αANP 显著降低人脐静脉内皮细胞的存活率并增加细胞凋亡。此外，C2238-αANP 通过基质胶评估降低内皮管形成。与 T2238-αANP 相比，C2238-αANP 对细胞内环鸟苷酸（cGMP）水平没有差异调节利钠肽受体（NPR）-A/B 活性。相反，C2238-αANP 以 NPR-C 依赖性方式显著降低细胞内环腺苷酸（cAMP）水平。相应地，C2238-αANP 与 NPR-C 的结合亲和力高于 T2238-αANP。用反义寡核苷酸抑制 NPR-C 或用小干扰 RNA 沉默 NPR-C 基因均可挽救暴露于 C2238-αANP 的人脐静脉内皮细胞的存活和管形成。在具有 NPR-C 敲低的人主动脉内皮细胞中获得了类似的数据。C2238-αANP 通过 NPR-C 激活抑制蛋白激酶 A/Akt1 通路并增加活性氧。用腺病毒介导的 Akt1 再激活挽救了 C2238-αANP 的有害作用。总体而言，这些数据表明，C2238-αANP 通过 NPR-C 依赖性抑制 cAMP/蛋白激酶 A/Akt1 通路和增加活性氧产生来影响内皮细胞完整性。因此，C2238-αANP 导致乙酰胆碱依赖性血管舒张功能障碍，这在外在可通过 NPR-C 药理学抑制得到挽救。最后，携带 C2238 次要等位基因的个体表现出早期内皮功能障碍，这突显了我们研究结果的临床相关性。
C2238-αANP 通过 NPR-C 信号降低内皮细胞存活率并损害内皮功能。针对 NPR-C 的靶向治疗可能是降低 C2238 次要等位基因携带者心血管风险的一种潜在策略。
