Théron Alexis, Odelin Gaëlle, Faure Emilie, Avierinos Jean-François, Zaffran Stéphane
Aix Marseille Université, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France; Inserm, U910, Faculté de Médecine, 13385 Marseille, France; AP-HM, Hôpital de la Timone, Département de Chirurgie Cardiaque, 13005 Marseille, France.
Aix Marseille Université, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France; Inserm, U910, Faculté de Médecine, 13385 Marseille, France.
Arch Cardiovasc Dis. 2016 Mar;109(3):188-98. doi: 10.1016/j.acvd.2015.10.002. Epub 2015 Dec 23.
The mechanism involved in the onset of aortic valve (AoV) disease remains unclear despite its poor prognosis and frequency. Recently, we reported that Krox20 (EGR2 in humans) is involved in AoV development and dysfunction.
Analyze Krox20 heterozygous mice (Krox20(+/-)) to discover whether incomplete expression of Krox20 can cause valvular diseases.
Transcriptional levels of Col1a2/COL1A2 and Krox20/EGR2 in AoVs from Krox20(+/-) mice and human patients operated on for severe aortic regurgitation were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Human control valves were obtained from three transplanted patients without AoV disease. Twenty-one heterozygous Krox20(+/-) mice were compared with 35 controls at different ages. Three independent measurements of valve thickness were performed on magnified tissue sections using Image J software. In vivo valve structure and function were evaluated using the high-frequency Vevo(®) 2100 echocardiogram.
qRT-PCR analysis using AoVs from patients with severe aortic regurgitation showed a decrease in EGR2 expression associated with significant downregulation of COL1A2 expression (P<0.05). Similar results were observed in the AoVs of Krox20(+/-) mice. Anatomical examination revealed that incomplete invalidation of Krox20 caused significant thickening of the aortic leaflet compared with controls (145±22 vs. 75±24μm; P=0.01). Within the mutant group, this thickening worsened significantly over time (Krox20(+/-) mice aged>7 vs.<7months: 136±48 vs. 102±41μm; P<0.001). Moreover, the aortic leaflets of embryonic day 18.5 Krox20(+/-) embryos were significantly more thickened than those from controls, suggesting that this disease begins during embryonic development. Echo-Doppler analysis showed a significant increase in AoV dysfunction in heterozygous versus control mice (53% vs. 17%; P<0.001), suggesting a tight relationship between valve architecture and function. Morphometric analysis revealed that the most severe AoV dysfunction was always associated with the most thickened valves. Classic histological analysis revealed that mutant AoVs had extracellular matrix disorganization, with features of human myxomatous degeneration, including excess of proteoglycan deposition in spongiosa and reduction of collagen fibre in fibrosa, but no calcification.
Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.
尽管主动脉瓣（AoV）疾病预后不良且发病率高，但其发病机制仍不清楚。最近，我们报道Krox20（人类中的EGR2）参与主动脉瓣的发育和功能障碍。
分析Krox20杂合小鼠（Krox20(+/-)），以发现Krox20表达不完全是否会导致瓣膜疾病。
通过定量逆转录-聚合酶链反应（qRT-PCR）评估Krox20(+/-)小鼠和因严重主动脉瓣反流接受手术的人类患者的主动脉瓣中Col1a2/COL1A2和Krox20/EGR2的转录水平。人类对照瓣膜取自三名无主动脉瓣疾病的移植患者。将21只杂合Krox20(+/-)小鼠与35只不同年龄的对照小鼠进行比较。使用Image J软件在放大的组织切片上对瓣膜厚度进行三次独立测量。使用高频Vevo(®) 2100超声心动图评估体内瓣膜结构和功能。
对严重主动脉瓣反流患者的主动脉瓣进行qRT-PCR分析显示，EGR2表达降低，同时COL1A2表达显著下调（P<0.05）。在Krox20(+/-)小鼠的主动脉瓣中也观察到了类似结果。解剖学检查显示，与对照组相比，Krox20不完全失活导致主动脉瓣叶显著增厚（145±22 vs. 75±24μm；P=0.01）。在突变组中，这种增厚随时间显著恶化（年龄>7个月的Krox20(+/-)小鼠与<7个月的相比：136±48 vs. 102±41μm；P<0.001）。此外，胚胎第18.5天的Krox20(+/-)胚胎的主动脉瓣叶比对照组的明显更厚，表明这种疾病在胚胎发育期间就开始了。超声多普勒分析显示，杂合小鼠与对照小鼠相比，主动脉瓣功能障碍显著增加（53% vs. 17%；P<0.001），表明瓣膜结构与功能之间存在密切关系。形态计量分析显示，最严重的主动脉瓣功能障碍总是与最厚的瓣膜相关。经典组织学分析显示，突变的主动脉瓣有细胞外基质紊乱，具有人类黏液瘤样变性的特征，包括海绵层蛋白聚糖沉积过多和纤维层胶原纤维减少，但无钙化。
小鼠中Krox20表达降低导致主动脉瓣叶变性和细胞外基质紊乱，引起瓣膜功能障碍。
