Rationale: Aortic valve disease is a cell-mediated process without effective pharmacotherapy. CNP (C-type natriuretic peptide) inhibits myofibrogenesis and osteogenesis of cultured valve interstitial cells and is downregulated in stenotic aortic valves. However, it is unknown whether CNP signaling regulates aortic valve health in vivo.

Objective: The aim of this study is to determine whether a deficient CNP signaling axis in mice causes accelerated progression of aortic valve disease.

Methods and Results: In cultured porcine valve interstitial cells, CNP inhibited pathological differentiation via the guanylate cyclase NPR2 (natriuretic peptide receptor 2) and not the G-protein–coupled clearance receptor NPR3 (natriuretic peptide receptor 3). We used Npr2^+/− and Npr2^+/−;Ldlr^−/− mice and wild-type littermate controls to examine the valvular effects of deficient CNP/NPR2 signaling in vivo, in the context of both moderate and advanced aortic valve disease. Myofibrogenesis in cultured Npr2^+/− fibroblasts was insensitive to CNP treatment, whereas aged Npr2^+/− and Npr2^+/−;Ldlr−/− mice developed cardiac dysfunction and ventricular fibrosis. Aortic valve function was significantly impaired in Npr2^+/− and Npr2^+/−;Ldlr^−/− mice versus wild-type littermates, with increased valve thickening, myofibrogenesis, osteogenesis, proteoglycan synthesis, collagen accumulation, and calcification. 9.4% of mice heterozygous for Npr2 had congenital bicuspid aortic valves, with worse aortic valve function, fibrosis, and calcification than those Npr2^+/− with typical tricuspid aortic valves or all wild-type littermate controls. Moreover, cGK (cGMP-dependent protein kinase) activity was downregulated in Npr2^+/− valves, and CNP triggered synthesis of cGMP and activation of cGK1 (cGMP-dependent protein kinase 1) in cultured porcine valve interstitial cells. Finally, aged Npr2^+/−;Ldlr^−/− mice developed dilatation of the ascending aortic, with greater aneurysmal progression in Npr2^+/− mice with bicuspid aortic valves than those with tricuspid valves.

Conclusions: Our data establish CNP/NPR2 signaling as a novel regulator of aortic valve development and disease and elucidate the therapeutic potential of targeting this pathway to arrest disease progression.

理由：主动脉瓣疾病是一种没有有效药物治疗的细胞介导过程。CNP（C型利钠肽）可抑制培养的瓣膜间质细胞的肌纤维形成和成骨作用，并在狭窄的主动脉瓣膜中下调。但是，尚不清楚CNP信号是否在体内调节主动脉瓣健康。

目的：本研究的目的是确定小鼠中CNP信号轴不足是否会引起主动脉瓣疾病的加速发展。

方法和结果：在培养的猪瓣膜间质细胞中，CNP通过鸟苷酸环化酶NPR2（利钠肽受体2）而非G蛋白偶联清除受体NPR3（利钠肽受体3）抑制病理分化。我们使用NPR2 ^+/-和NPR2 ^{+ / -} ; Ldlr ^{-/ -}小鼠和野生型同窝幼仔对照在中度和晚期主动脉瓣膜疾病的背景下检查体内不足的CNP / NPR2信号传导的瓣膜作用。培养的Npr2 ^{+ / -}成纤维细胞中的肌纤维形成对CNP治疗不敏感，而老年Npr2 ^{+ / -}和Npr2 ^{+ / -} ; Ldlr -/-小鼠发展为心脏功能障碍和心室纤维化。主动脉瓣功能在被显著受损NPR2 ^{+ / -}和NPR2 ^{+ / -} ; Ldlr ^{-/ -}小鼠与野生型同窝仔相比，瓣膜增厚，肌纤维形成，成骨，蛋白聚糖合成，胶原蛋白积累和钙化增加。9.4％的Npr2杂合小鼠具有先天性双尖瓣主动脉瓣，其主动脉瓣功能，纤维化和钙化较Npr2 ^+/-差具有典型的三尖瓣主动脉瓣或所有野生型同窝对照。此外，CGK（cGMP依赖性蛋白激酶）活性下调在NPR2 ^+/-阀和CNP触发的cGMP的合成和在培养的猪阀间质细胞cGK1的活化（cGMP依赖性蛋白激酶1）。最后，老化的Npr2 ^+/- ; LDLR ^{- / -}小鼠产生上升的扩张主动脉，并在更大的动脉瘤进展NPR2 ^+/-与二尖瓣主动脉瓣膜比那些三尖瓣小鼠。

结论：我们的数据建立了CNP / NPR2信号传导作为主动脉瓣发育和疾病的新型调节剂，并阐明了靶向该途径阻止疾病进展的治疗潜力。