title: Krox20 heterozygous mice: A model of aortic regurgitation associated with decreased expression of fibrillar collagen genes.
abstract: BACKGROUND
  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.
  
  
  OBJECTIVE
  Analyze Krox20 heterozygous mice (Krox20(+/-)) to discover whether incomplete expression of Krox20 can cause valvular diseases.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.
authors.affiliation: 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.
authors.forename: Alexis
authors.initials: A
authors.lastname: Théron
authors.affiliation: Aix Marseille Université, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France; Inserm, U910, Faculté de Médecine, 13385 Marseille, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Odelin
authors.affiliation: Aix Marseille Université, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France; Inserm, U910, Faculté de Médecine, 13385 Marseille, France.
authors.forename: Emilie
authors.initials: E
authors.lastname: Faure
authors.affiliation: 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 Cardiologie, 13005 Marseille, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Avierinos
authors.affiliation: Aix Marseille Université, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France; Inserm, U910, Faculté de Médecine, 13385 Marseille, France. Electronic address: stephane.zaffran@univ-amu.fr.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Zaffran
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000000981:diagnostic imaging / Q000378:metabolism* / Q000503:physiopathology; D001022:Aortic Valve Insufficiency / Q000000981:diagnostic imaging / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D016022:Case-Control Studies; D024042:Collagen Type I / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D015536:Down-Regulation; D051767:Early Growth Response Protein 2 / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D015150:Echocardiography, Doppler; D020022:Genetic Predisposition to Disease; D006579:Heterozygote; D006801:Humans; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012720:Severity of Illness Index; D014158:Transcription, Genetic
chemical_list: C000601279:COL1A2 protein, human; D024042:Collagen Type I; C497326:EGR2 protein, human; D051767:Early Growth Response Protein 2; C497327:Egr2 protein, mouse; D012333:RNA, Messenger
keywords: Aortic valve regurgitation; Collagens; Collagènes; Extracellular matrix; Heart valve; Krox20; Matrice extracellulaire; Régurgitation aortique; Souris; Valve cardiaque