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Archives of cardiovascular diseases
Volume 109, n° 3
pages 188-198 (mars 2016)
Doi : 10.1016/j.acvd.2015.10.002
Received : 11 May 2015 ;  accepted : 5 October 2015
Krox20 heterozygous mice: A model of aortic regurgitation associated with decreased expression of fibrillar collagen genes
La souris Krox20 +/−  : un modèle d’insuffisance aortique associée à une diminution des collagènes fibrillaires
 

Alexis Théron a, b, c, 1, Gaëlle Odelin a, b, 1, Emilie Faure a, b, Jean-François Avierinos a, b, d, Stéphane Zaffran a, b,
a Aix Marseille Université, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France 
b Inserm, U910, Faculté de Médecine, 13385 Marseille, France 
c AP–HM, Hôpital de la Timone, Département de Chirurgie Cardiaque, 13005 Marseille, France 
d AP–HM, Hôpital de la Timone, Département de Cardiologie, 13005 Marseille, France 

Corresponding author at: Aix Marseille Université, Inserm, GMGF UMR_S910, Faculté de Médecine, 27, boulevard Jean-Moulin, 13385 Marseille, France.
Summary
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.

Aim

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.

Conclusion

Decreased expression of Krox20 in mice causes degeneration of the aortic leaflets and disorganization of the extracellular matrix, causing valvular dysfunction.

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Résumé
Contexte

Les mécanismes impliqués dans la survenue des valvulopathies aortiques restent à ce jour non élucidés malgré leur fréquence et leur mauvais pronostic. Récemment, nous avons montré que Krox20 (EGR2  chez l’Homme) joue un rôle important dans la valvulogenèse aortique et la survenue de dysfonction valvulaire.

Objectif

Notre objectif était de savoir si la perte d’un allèle de Krox20  pouvait conduire à un défaut valvulaire.

Méthodes

Nous avons mesuré par PCR quantitative en temps réel (QPCR) le niveau d’expression du gène Col1a2/COL1A2  et de Krox20/EGR2  au sein de valves aortiques provenant de souris hétérozygotes Krox20 +/− et de patients opérés d’insuffisance aortique sévère et sur des valves témoins issues de patients transplantés indemnes de valvulopathie aortique. Nous avons comparé un groupe de souris hétérozygotes Krox20 +/− (n =21) à un groupe témoin (n =35) à différents âges de vie. Les échocardiographies ont été réalisées à l’aide d’un échocardiogramme Vevo® 2100. Les coupes histologiques ont été obtenues après inclusion des échantillons de cœurs murins en paraffine et section au microtome. L’épaisseur valvulaire a été mesurée à trois reprises grâce au logiciel Image J software.

Résultats

L’analyse par QPCR révèle une réduction de l’expression du gène EGR2  associée à une diminution significative de COL1A2 (p <0,05) dans les valves aortiques de patients opérés de valvulopathie aortique concordant avec une diminution de Col1a2  au sein de valves aortiques de souris mutantes. L’examen anatomique montre que l’invalidation incomplète de Krox20  conduit à un épaississement valvulaire significatif par rapport au groupe témoin (145±22 vs 75±24μm ; p =0,01). L’épaississement de la valve aortique s’aggrave au cours du temps (âge>7 mois versus<7 mois, 136±48 vs 102±41μm ; p <0,001). De plus, les feuillets valvulaires d’embryons mutants sont significativement plus épais que les feuillets des embryons témoins à E18,5, suggérant une apparition de la dégénérescence valvulaire dès la vie embryonnaire. L’analyse échocardiographique montre une augmentation significative de valvulopathies aortiques chez les souris Krox20 +/− (53 % vs 17 % ; p <0,001), suggérant une relation étroite entre architecture valvulaire et dysfonctionnement. L’analyse morphométrique des échantillons démontre que les valvulopathies les plus sévères sont associées aux épaississements valvulaires les plus importants. Enfin, les feuillets aortiques des souris Krox20 +/− présentent une désorganisation de la matrice extracellulaire évocatrice de dégénérescence myxoïde incluant un excès de protéoglycans dans la spongiosa , une raréfaction des fibres de collagène dans la fibrosa et une absence de calcification.

Conclusion

Nos résultats montrent que la diminution de l’expression de Krox20  chez la souris est à l’origine d’une dégénérescence des feuillets aortiques et d’une désorganisation de la matrice extracellulaire responsable de dysfonction valvulaire.

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Keywords : Aortic valve regurgitation, Krox20 , Heart valve, Extracellular matrix, Collagens

Mots clés : Régurgitation aortique, Krox20 , Valve cardiaque, Souris, Matrice extracellulaire, Collagènes

Abbreviations : AoV, ECM, LV, oim , Postn, qRT-PCR, RNA


Background

Valvular heart disease is the second major cardiac concern after coronary heart disease. Aortic valve (AoV) disease is the most frequent, occurring in 2.5% of the population in industrialized countries [1]. Despite its high incidence and negative prognostic effect, the underlying mechanism of AoV disease remains unknown. AoV architecture changes throughout the life, suggesting environmentally mediated dysregulation in the pathological condition [2]. However, it is now recognized that adult valve disease may originate in anomalies of valve development [3].

Valvulogenesis occurs after the initial stages of cardiogenesis, as a result of endocardial cushion formation and extensive remodelling of the extracellular matrix (ECM). The atrioventricular cushions give rise to the mitral and tricuspid valves, while the aortic and pulmonary valves arise from outflow tract cushions. Cushion formation initiates with expansion of the cardiac jelly, the ECM residing between the myocardium and the endocardium. The essential first step is an endothelial-to-mesenchymal transformation of endocardial cells, induced by signalling factors coming from the myocardium [4]. Semilunar valve development is distinguished from atrioventricular valve formation by the invasion of neural crest-derived cells, which participate in outflow tract septation [3]. The mature valve structure is composed of a highly organized ECM, with three layers rich in elastin (ventricularis), proteoglycans (spongiosa) and fibrillar collagen (fibrosa). The fibrosa is situated on the aortic aspect of the semilunar valves [5, 6]; this layer is composed predominantly of fibrillar collagens types I and III, which provide tensile stiffness. There is a tight relationship between valve architecture and function. Misexpression and disorganization of fibrillar collagen in the ECM are associated with AoV dysfunction, as well as aortic stenosis or regurgitation, caused by impaired valvulogenesis and altered architecture of the aortic leaflet. Collagen accumulation in fibrotic degeneration promotes aortic stenosis by stiffening of the valve during systole. Conversely, collagen degradation in myxomatous degeneration causes leaflet prolapse and aortic regurgitation during diastole. To date, the mechanisms involved in transcriptional regulation of collagen genes remain unknown.

Recently, we demonstrated that the transcription factor Krox20 (called EGR2 in human) is expressed in the semilunar and atrioventricular valve primordia, and that its function is required for cardiac valve development. Indeed, in vivo imaging and histological analysis revealed that complete loss of Krox20 in the mouse results in AoV dysfunction, with a significant thickening and disorganization of the ECM, including excess of proteoglycan and reduction of fibrillar collagens [7].

In this study, we used in vivo imaging to show that Krox20 heterozygous mice also have AoV dysfunction. In addition, histological and transcriptional analyses reveal that AoV anomalies are associated with disorganization of the collagen layer and impaired Col1a2 transcriptional levels.

Methods
Animal experiments

This work was approved by the “comité d’éthique pour l’expérimentation animale” (Marseille Animal Care Committee; Protocol No. 38-09102012) and conformed to Directive 2010/63/EU of the European Parliament. Krox20 lacZ (Krox20 ) mice [8] were intercrossed with the C57BL/6 mice to generate Krox20 +/− heterozygous offspring that were obtained at expected Mendelian ratios. After echocardiography analysis, adult Krox20 +/− mice were sacrificed via intraperitoneal injection of pentobarbital sodium (0.5mL). Heart samples were harvested, fixed in 4% paraformaldehyde and stored in 1X phosphate buffered saline.

Histological analysis

Standard histological procedures were used [7]. Heart tissues from Krox20 +/− and littermate controls were paraffin embedded and cut at 8μm per tissue section. Sections were stained with Masson's trichrome (Sigma, St. Louis, MO, USA), orcein (Sigma), alcian blue (Sigma), Von Kossa (Sigma) and alizarin red (Sigma), according to the manufacturer's instructions.

Quantification of valve anomalies

Regarding quantification of valve thickness, the distal region of the leaflets or leaflets of valves were used over a minimum depth of 100μm using a DM5000 microscope with LAS software (Leica Microsystems, Wetzlar, Germany). Image J software was used to measure the surface and the thickness of the valve. Three independent measurements were taken per leaflet, from 10 different sections, and the values were averaged. At least six animals were used per genotype for statistical analysis.

Echocardiography

In vivo valve structure and function were evaluated using an ultra high-frequency, high resolution ultrasound (Vevo® 2100; VisualSonics, Inc., Toronto, ON, Canada). A total of 35 controls and 21 mutant mice were analysed for our study. The chests of the mice were treated with a chemical hair remover to reduce ultrasound attenuation. Heart rate and core body temperature were continuously monitored. Mice were anaesthetized with 1–2% isofluorane inhalation, and placed on a heated platform to maintain their temperature during the analysis. Two-dimensional imaging was recorded with a 40MHz transducer to capture long- and short-axis projections with guided M-Mode, B-Mode and colour and pulsed-wave Doppler. Doppler interrogation was performed on the semilunar valve outflow in the parasternal long-axis view to assess stenosis and regurgitation using a sample volume toggle to optimize the angle of interrogation. A modified right parasternal long-axis view was required in some cases to ensure ascertainment of the maximum velocity. All measurements were obtained using an angle of interrogation<50°. Aortic regurgitation was defined as valve incompetence with reversal of flow in diastole. Statistical significance was determined using Student's t -test and defined as P <0.05.

Human AoV samples and processing

This study was performed in accordance with institutional guidelines and was approved by the local research ethics committee at the University Hospital of Marseille (No. 2013-A01020-45). Written consent was obtained from all patients involved. Control valves were collected from three transplanted hearts with no valvulopathy. Four abnormal AoVs were collected from patients operated on for severe aortic dysfunction. Ribonucleic acid (RNA) was extracted as described below.

Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

The AoVs and surrounding tissue containing the leaflets, annulus and aortic root were manually dissected from 3-month-old Krox20 mutant mice. After genotyping, samples from six mice of the same genotype were pooled, and RNA was isolated using a NucleoSpin® RNA/Protein Kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) in accordance with the manufacturer's instructions. Human AoV tissues were lysed on TRIzol® (Life Technologies, Carlsbad, CA, USA), and RNA was extracted using an RNeasy® Mini Kit (Qiagen, Hilden, Germany). Reverse transcriptions were performed by using a first strand complementary deoxyribonucleic acid synthesis kit (Roche, Basel, Switzerland) in accordance with the manufacturer's instructions. A LightCycler® 480 SYBR Green I Master mix (Roche) was used for real-time qRT-PCR analysis with a LightCycler® 480 (Roche), following the manufacturer's instructions. The gene-specific primers used in this study have been described previously [7]. Each experiment was performed in triplicate for each genotype. Samples were normalized to endogenous housekeeping gene (TBP or GAPDH genes for mouse or human samples, respectively). Level changes were calculated by the comparative cycle threshold (ΔΔCT) method. Normalized expression levels in the control (Krox20 +/+ ) were set to 1.0 for each gene.

Statistical analysis

All parametric data are expressed as mean±standard error of the mean. Statistics were carried out using Student's t -test to compare variances and the Chi2 test for two independent samples. A P value<0.05 was considered significant.

Results
Analysis of COL1A2/Col1a2 and EGR2/Krox20 expression in human and mouse AoV disease

Molecular mechanisms leading to extracellular matrix (ECM) disturbance, such as collagen fibres in the AoV, are still unclear. We collected explants of diseased human AoV, with no obvious fibrocalcific remodelling, from patients operated on for severe AoV regurgitation (mean age 43.25±12.75years), to determine transcriptional levels of fibrillar collagen genes by real-time qRT-PCR. Control valve tissues were obtained during cardiac transplantation from patients with end-stage heart failure and no AoV disease. Among all the fibrillar collagen genes tested, only COL1A2 was significantly downregulated in diseased AoV compared with controls (P <0.05) (Figure 1A). Interestingly, qRT-PCR showed 50% reduced expression of KROX20/EGR2 , known as a direct regulator of fibrillar collagen [7], in all diseased AoVs compared with controls (Figure 1B). This finding emphasizes that downregulation of KROX20/EGR2 expression leads to severe AoV dysfunction. In order to test whether the haploin sufficiency of Krox20 could be involved in the reduction of collagen fibres, we performed qRT-PCR analysis in Krox20 heterozygous mice at 3months of age. Our data revealed a significant reduction in Krox20 expression in the AoV leaflets of heterozygous mice (P <0.05) (Figure 1C). In addition, the level of Col1a2 transcripts was downregulated in the AoVs of Krox20 +/− mice (Figure 1D), confirming the requirement of Krox20 for the activation of that collagen.



Figure 1


Figure 1. 

Comparison of EGR2 /Krox20 and COL1A2/Col1a2 expression in aortic valves (AoVs) from patients and heterozygous mice. A, B. Ribonucleic acid was extracted from human AoVs to quantify transcriptional levels of (A) COL1A2 and (B) EGR2 by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Control AoVs were obtained after heart transplant from three patients with end-stage heart failure with no evidence of valvular defects. Diseased tissues (Diseased) were isolated from the pathological AoVs of four patients with severe AoV dysfunction at the time of valve replacement. C, D. Real-time qRT-PCR was performed on isolated AoVs to quantify Krox20 and Col1a2 expression in Krox20+/− (n =6) and control (n =6) embryos at 3months of age. Experiments were performed in triplicate and normalized on expression from controls. (*P <0.05, Student's t -test). mRNA: messenger ribonucleic acid.

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AoV dysfunction is more frequent in Krox20 heterozygous mice

AoV function in Krox20 +/− mice was assessed by echocardiography at the ages of 3months, 6months, 9months and>12months (Figure 2, Figure 3, Table 1). AoV diseases were significantly more frequent in heterozygous mice (53%) than in control mice (17%) (Figure 3A; P <0.001). The incidence of AoV disease increased significantly after 7months of age in Krox20 +/− mice (37.5% vs. 62%; P =0.05; Figure 3B), whereas this incidence remained stable in the control group (10% vs. 20%; P =0.067). Eleven mutant mice (11/21; 52%) had AoV dysfunction diagnosed by echocardiography (Figure 2). The left ventricular (LV) end-diastolic diameter and volume were not increased compared with the control group, despite the increase in LV preload: 4.1±0.12 vs. 3.97±0.1mm (P =0.43) and 75±5 vs. 69±17μL (P =0.5). Among these mice, five had an acceleration of aortic velocity (mean Vmax =2925±215 vs. 1233±127mm/s; P <0.0001) and mean transaortic gradient (9.7±1.8 vs. 1.5±0.3mmHg; P <0.0001) (Figure 2C and D). High gradients can be caused by LV outflow obstruction resulting from thickening of the AoV leaflets, rather than an increase in LV stroke volume caused by the severity of the aortic regurgitation. This subgroup showed a significant augmentation of LV concentric hypertrophy (143±12 vs. 98±6mg; P <0.0001), confirming the increase in LV afterload (Figure 2D). AoVs were tricuspid in all mutant mice. There was no mitral or pulmonary valve disease. Among the 35 controls, six (17%) had trivial regurgitation. The AoVs of controls were all tricuspid. There was no mitral or pulmonary valve disease. Overall, these results demonstrate that incomplete deletion of Krox20 leads to AoV dysfunction.



Figure 2




Figure 2. 

Aortic valve (AoV) function in Krox20+/− adult mice. A. Pulse-wave Doppler analysis from the aorta of 3-month-old control mice demonstrated no AoV dysfunction. B. Pulse-wave Doppler record showing mild aortic regurgitation (AR) in Krox20+/− mice. C. Pulse-wave Doppler record of aortic flow displays severe aortic stenosis with measurement of maximum velocity at 3000mm/s. D. Histograms showing measurements of cardiac variables calculated from control (n =35) and mutant (n =11) mice with mild-to-moderate AR (n =6) and severe AR (n =5). Haemodynamic evaluation revealed increased mean velocity and pressure gradients through the aortic valve of Krox20+/− mice with severe AR. Histograms are expressed as mean±standard error of the mean (*P <0.05, Student's t -test). BPM: beats per minute; IVS: interventricular septum; LV: left ventricular; RR: respiratory rate.

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Figure 3


Figure 3. 

Incidence of aortic valve (AoV) dysfunction in Krox20+/− versus control adult mice. A. Percentage of mice in each subgroup of AoV dysfunction. B. Comparison of AoV dysfunction occurrence in mutant mice aged<7months and>7months. AR: aortic regurgitation.

Zoom

Krox20 +/− mice exhibit aortic leaflet thickening

Histological analysis revealed that Krox20 +/− mice had thicker and longer AoV leaflets than control littermates. The valve area was significantly larger in the mutant group (56,076±19,344 vs. 21,762±7476μm2; P =0.005), as were AoV leaflet length (719±101 vs. 380±41μm; P =0.002) and AoV leaflet thickness (145±22 vs. 75±24μm; P =0.01) (Figure 4A–C). Within the 11 mutant mice with AoV dysfunction, the five with the most severe dysfunction exhibited significant thickening of the aortic leaflet (valve area 86,151±4550 vs. 49,400±9400μm2; P =0.04). Morphometric analysis showed that this thickening concerned the integrality of the leaflet in these mice, and was restricted to the free edge of the aortic leaflet in the subgroup of mutant mice with aortic regurgitation, but no excessive LV outflow tract acceleration.



Figure 4


Figure 4. 

Krox20 +/− mice have enlarged aortic valve (AoV) leaflets. A–C. Quantification of the surface, length and thickness of the AoV leaflets from Krox20+/− and control mice. D–F. Quantification of the surface, length and thickness of Krox20+/− AoVs at age<7months and>7months. All measurements were calculated from six mice for each genotype. Histograms are expressed as mean±standard error of the mean (*P <0.05, Student's t -test).

Zoom

At embryonic day 18.5, Krox20 +/− AoV leaflets were more dysmorphic and enlarged compared with those of control mice. Indeed, the valve area (14,190±2300 vs. 11,500±2400μm2; P <0.001), leaflet length (250±33 vs. 221±36μm; P <0.001) and leaflet thickness (88±15 vs. 73±13μm; P <0.001) were significantly increased in mutant mice. Interestingly, Krox20 +/− mice aged>7months exhibited more important thickening of the aortic leaflet than Krox20 +/− mice aged<7months. Valve area (51,500±18,200μm2 vs. 32,030±13,075μm2; P =0.002), leaflet length (708±250 vs. 611±249μm; P <0.7001) and leaflet thickness (136±48 vs. 102±41μm; P <0.001) were significantly increased in Krox20 +/− mice aged>7months (Figure 4D–F). Overall, these data indicate that a 50% reduction in Krox20 expression leads to aortic leaflet thickening. This enlargement is tightly associated with the onset of AoV dysfunction; it begins during the embryonic period and worsens during life.

AoV anomalies are associated with disorganization of the ECM

ECM composition and organization were examined using Masson's Trichrome, alcian blue and orcein stains (Figure 5). Histological analysis demonstrated disorganization of the ECM. This disorganization primarily manifested as a decrease in collagen deposition in the distal thickened region of the AoV leaflets in Krox20 +/− mice, as indicated by diffuse blue staining (Figure 5A and E). No obvious differences were observed in the medial and proximal (hinge) regions of mutant valves. In addition, increased proteoglycans deposition was observed through alcian blue staining of the AoVs from Krox20 +/− mice (Figure 5F). Orcein staining did not reveal major differences between Krox20 heterozygous and control mice (Figure 5C and G). Von Kossa staining was performed to evaluate the present of calcification. At 3months of age, the AoV of Krox20 +/− mice showed no signs of calcification, as indicated by an absence of Von Kossa stains (Figure 5D and H). All together, these findings indicate that a 50% reduction in Krox20 expression contributes to the disorganization of the ECM and enlargement of the AoV leaflets.



Figure 5


Figure 5. 

Histological analysis of the aortic valve (AoV) from adult Krox20+/− mice. Comparison of (A, E) Masson's trichrome, (B, F) alcian blue, (C, G) orcein and (D, H) Von Kossa staining of the AoV from (A–D) Krox20+/+ and (E–H) Krox20+/− at age 3months (mo). (A, E) Masson's trichrome staining reveals reduction of collagen fibres in Krox20+/− mice compared with control mice. (B, F) Comparison of alcian blue staining demonstrates an increase of proteoglycan in Krox20+/− mice. (C, G) Orcein staining shows no major difference between Krox20+/− and control mice. (D, H) Von Kossa staining shows no signs of calcification in Krox20+/− and control AoVs. * Indicates the presence of black melanocytes in control AoV. Scale bars: 100μm.

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Discussion

The mechanism involved in the onset of AoV disease remains unclear, despite its poor prognosis and its frequency. Recently, we reported that complete loss of Krox20 results in alteration of valvular interstitial cell distribution and disorganization of the ECM by downregulation of the expression of fibrillar collagen in the AoV at embryonic and adult stages [7]. In the present study, we showed that the incomplete invalidation of Krox20 in mice was associated with the onset of AoV disease, and led to a similar ECM disorganization as that described in Krox20 −/− mice, despite less severe impairment of expression of fibrillar collagen genes in the AoV. Interestingly, we demonstrated that enlargement of the aortic leaflet in mutant mice increased during life, suggesting a pathological tissue response to environmental stimuli.

Complete expression of Krox20 is required during AoV development

Consistent with what we reported previously in the Krox20 −/− mice model, the incomplete expression of Krox20 leads to dysmorphic and thickened aortic leaflets and valve dysfunction. Histology shows a disorganization of the ECM, with a decrease in fibrillar collagen and an increase in proteoglycan deposition, especially in the free edge of the leaflet. No calcification was observed in the aortic leaflet, ruling out any chondrogenic or osteogenic process at the time of dissection.

This pattern is similar to myxomatous degeneration, frequently described in human mitral valve prolapse, but also in other mouse models, including osteogenesis imperfecta murine (oim ) mice and Periostin (Postn ) mice [9, 10]. The phenotype of the Krox20 +/− AoV appears to be more similar to oim than Postn mice. Indeed, ECM disorders affect the semilunar valve exclusively, and the thickness of the leaflet is limited to the free edge in both. These similarities are probably explained by the same molecular pathogenesis affecting fibrillar collagen. In the present study, we demonstrated a decrease in Col1a2 expression in the AoV leaflet of heterozygous mice (P =0.05), suggesting that complete expression of Krox20 is required for the regulation of fibrillar collagen in the ECM. Consistently, our previous study showed direct regulation of Col1a1 and Col3a1 promoters by Krox20 during embryonic development and adult life [7].

An increase in proteoglycan deposition in the Krox20 +/− AoV could be explained by the same molecular mechanism as in oim mice. Expression of Hapln 1 , Col10a1 and Asporin could be increased in response to fibrillar collagen rarefaction, with intent to protect AoV functions [9]. Another less consistent hypothesis could be an alteration of proteoglycan cleavage because of the loss of an ECM protease, as previously described in ADAMTS5 deficient mice [11].

Taken together, all these data confirm the major role of Krox20 in AoV disease pathogenesis, and the absolute need for its complete expression to maintain ECM homeostasis.

Thickening of the diseased aortic leaflet is a progressive response to mechanical stimuli

The maintenance of aortic leaflet integrity relies on the responses of the ECM to mechanical stimuli. The forces exerted on the leaflet during diastole and systole are flexure, shear stress and tension [12]. Flexure occurs as the valve opens and closes, shear stress results from the passing blood flow and tension occurs during coaptation and full loading.

The ECM composition varies as a function of loading conditions. Sacks and Yoganathan reported that a mitral valve that is exposed to higher pressure overload than a tricuspid valve has an ECM composition with a higher concentration of smooth muscle alpha actin and heat shock protein 47, which reflect cellular stiffness and collagen biosynthesis, respectively [12]. Consistently, Aikawa et al. demonstrated a maturation of fibrillar collagen during postnatal life, while fibroblasts became progressively quiescent [13]. The synthesis of collagen (especially COL1A1 and COL3A1 ) in the ECM is dependent on the duration and magnitude of mechanical stress, emphasizing the plasticity of ECM and its reactive response to environmental stimuli [14].

Fibrillar collagen synthesis is one of several mechanisms of ECM adaptation. Expression of others components of the ECM, such as elastin and proteoglycan, in response to environmental stress is less known. A perturbation of this delicate equilibrium can lead to pathological remodelling of the tissue matrix and compromised valve function, as is also observed calcific AoV disease [3]. Indeed, pathological calcification of human valves is associated with significant alterations that occur in the organization, composition and mechanical properties of the valve ECM [15]. Structural alterations in the valve ECM that define calcified valves affect the biomechanical function of the valve. However, there is growing evidence to suggest that ECM contributes to the progression of this disease through different mechanisms, including transducing haemodynamic forces and resisting cell-generated tensile forces [15]. Considerable increase in the synthesis of type I collagen and slight up-regulation of type III collagen content are normally observed in calcified valves, indicating increased collagen turnover [16]. Another pathological clinical feature associated with AoV disease is fibrosis, which involves myofibroblast infiltration and secretion of ECM proteins, including type I and III collagens [17]. Remarkably, fibrosis is not observed in the AoVs of Krox20 mutant mice [7], underlying the requirement for type I and III collagens for this process. Therefore, absence of calcification in mutant mice suggests that a decrease in Krox20 protects against this process.

In our study, age-related thickening of aortic leaflet in mutant mice reflected the plasticity of the ECM. Moreover, we hypothesize that deposition of proteoglycan in the free edge of the valve could be a protective response of the ECM to mechanical stimuli in the absence of fibrillar collagen. Mechanical stress would contribute to thickening and vice versa.

Overall, our results confirmed the relative responsiveness of the ECM to environmental stimuli. Overabundance of proteoglycan may be a consequence of fibrillar collagen reduction in mutant mice.

Clinical implications in humans

Mutations in KROX20 , so-called EGR2 in humans, have been described previously in patients with Dejerine-Sottas neuropathy and Charcot-Marie-Tooth disease type 1D. However, their association with AoV disease remains the subject of debate because of their low prevalence and the high rate of mortality compromising cardiovascular screening.

Here, we showed that expression of KROX20 (EGR2) as well as COL1A2 is impaired in patients operated on for severe aortic regurgitation, leading to leaflet enlargement and AoV dysfunction. Moreover, we demonstrated that the thickening of pathological leaflet in mutant mice is a dynamic process, evolving with age, which may be supported by external stimuli, such as biomechanical signals. Two conditions may be necessary to lead to AoV disease: genetic defects present at the time of valve morphogenesis, responsible for ECM disorganization; and mechanical stress that can be transduced into a cell-mediated process, promoting the misadaptation of the valves to their local environment.

AoV disease is a major concern in public health, and more than 10,000 AoV replacements with a mechanical valve or a bioprosthesis are performed each year in the USA. Mechanical prostheses need lifelong anticoagulation treatment, with the risk of haemorrhage/thrombosis, while patients with a bioprosthesis are inevitably exposed to the risk of reintervention in 10–15years. Moreover, patients with a mechanical valve or a bioprosthesis are exposed to the risk of endocarditis. These treatments remain very aggressive in the absence of proven pharmacological therapies that could stop the progression of the AoV disease. Description of multiple mice models, such as Krox20 +/− mice, may provide essential information about the pathogenesis and progression of human AoV disease, which could lead to the emergence of new therapeutic opportunities.

Conclusion

In summary, our data confirm the potential role of Krox20 in AoV disease. Impairment in the expression of Krox20 , even if incomplete, leads to the same histological pattern as complete deletion, including AoV thickening by disruption of ECM homeostasis, increased proteoglycan deposition and downregulation of fibrillar collagen genes. Moreover, evidence for a causative effect of environmental stimuli on AoV disease progression is emerging, facilitating novel therapeutic perspectives.

Disclosure of interest

The authors declare that they have no competing interest.


Acknowledgments

We thank Prof. P. Charnay and Dr. P. Topilko for the Krox20 lacZ mice, and N. Eudes for her technical help. The recording of echo-Doppler imaging was performed at the CERIMED, Marseille, France.Sources of funding : this work was supported by grants from the “AFM-Téléthon” (grant number NMH-Decrypt) and the “Fondation pour la Recherche Médicale” (grant number DPC20111123002) to S. Zaffran. G. Odelin received fellowships from the “Fondation pour la Recherche Médicale”, the “Institut National de la Santé et de la Recherche Médicale” and the “Fondation Lefoulon-Delalande”. E. Faure received a postdoctoral fellowship from the “AFM-Téléthon”.

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1  Alexis Théron and Gaëlle Odelin contributed equally to this work.


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