Helske Satu, Laine Mika, Kupari Markku, Lommi Jyri, Turto Heikki, Nurmi Laura, Tikkanen Ilkka, Werkkala Kalervo, Lindstedt Ken A, Kovanen Petri T
Wihuri Research Institute, Kalliolinnantie 4, Fin-00140 Helsinki, Finland.
Eur Heart J. 2007 Aug;28(15):1894-903. doi: 10.1093/eurheartj/ehm129. Epub 2007 May 15.
In aortic stenosis (AS), adverse remodelling of the valves may depend on altered local regulation of pro- and antifibrotic systems. We have recently shown that angiotensin-converting enzyme (ACE), which generates profibrotic angiotensin II and inactivates antifibrotic bradykinin (BK), is upregulated in stenotic aortic valves. Here, we analyse the expression of neutral endopeptidase (NEP), another profibrotic and BK-degrading enzyme, and of BK receptors in aortic valves in AS.
Stenotic aortic valves (n = 86) were obtained at valve replacement surgery and control valves (n = 13) at cardiac transplantation. Expression levels of NEP and BK type 1 and 2 receptors (BK-1R and BK-2R) in aortic valves and in isolated valvular myofibroblasts were analysed by real-time PCR and immunohistochemistry, and NEP activity was quantified by autoradiography. NEP, BK-1R, and BK-2R mRNA levels were higher in stenotic than in non-stenotic valves (P < 0.05 for each) and the respective proteins localized to valvular endothelial cells and myofibroblasts. In stenotic valves, the proteolytic activity of NEP was significantly increased (4.5-fold, P < 0.001), and tumour necrosis factor-alpha induced the expression of NEP in cultured myofibroblasts. Finally, treatment of cultured myofibroblasts with an NEP inhibitor (phosphoramidon) downregulated the expression of profibrotic transforming growth factor-beta1, whereas addition of BK decreased the expression of collagens I and III which was reversed by a BK-2R antagonist.
NEP activity is increased in stenotic aortic valves in parallel with increased expression of BK-receptors. The upregulation of NEP and BK-1R have the potential to promote valvular fibrosis and remodelling while the increase in BK-2R may represent a compensatory antifibrotic response. These findings add novel pathogenic insight and raise potential new therapeutic targets in AS.
在主动脉瓣狭窄（AS）中，瓣膜的不良重塑可能取决于促纤维化和抗纤维化系统局部调节的改变。我们最近发现，生成促纤维化血管紧张素II并使抗纤维化缓激肽（BK）失活的血管紧张素转换酶（ACE）在狭窄主动脉瓣中上调。在此，我们分析中性内肽酶（NEP）（另一种促纤维化且降解BK的酶）以及AS患者主动脉瓣中BK受体的表达。
在瓣膜置换手术中获取狭窄主动脉瓣（n = 86），在心脏移植手术中获取对照瓣膜（n = 13）。通过实时PCR和免疫组织化学分析主动脉瓣及分离的瓣膜肌成纤维细胞中NEP、BK 1型和2型受体（BK-1R和BK-2R）的表达水平，并通过放射自显影法定量NEP活性。狭窄瓣膜中NEP、BK-1R和BK-2R的mRNA水平高于非狭窄瓣膜（每项P < 0.05），相应蛋白质定位于瓣膜内皮细胞和肌成纤维细胞。在狭窄瓣膜中，NEP的蛋白水解活性显著增加（4.5倍，P < 0.001），肿瘤坏死因子-α诱导培养的肌成纤维细胞中NEP的表达。最后，用NEP抑制剂（磷酰胺素）处理培养的肌成纤维细胞可下调促纤维化转化生长因子-β1的表达，而添加BK可降低I型和III型胶原的表达，BK-2R拮抗剂可逆转这种降低。
狭窄主动脉瓣中NEP活性增加，同时BK受体表达增加。NEP和BK-1R的上调有可能促进瓣膜纤维化和重塑，而BK-2R的增加可能代表一种代偿性抗纤维化反应。这些发现为AS提供了新的致病机制见解，并提出了潜在的新治疗靶点。
