Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice

Abstract

Epidermal growth factor receptor (EGFR) signaling contributes to aortic valve development in mice. Because developmental phenotypes in Egfr-null mice are dependent on genetic background, the hypomorphic Egfr(wa2) allele was made congenic on C57BL/6J (B6) and 129S1/SvImJ (129) backgrounds and used to identify the underlying cellular cause of EGFR-related aortic valve abnormalities. Egfr(wa2/wa2) mice on both genetic backgrounds develop aortic valve hyperplasia. Many B6-Egfr(wa2/wa2) mice die before weaning, and those surviving to 3 mo of age or older develop severe left ventricular hypertrophy and heart failure. The cardiac phenotype was accompanied by significantly thicker aortic cusps and larger transvalvular gradients in B6-Egfr(wa2/wa2) mice compared with heterozygous controls and age-matched Egfr(wa2) homozygous mice on either 129 or B6129F1 backgrounds. Histological analysis revealed cellular changes in B6-Egfr(wa2/wa2) aortic valves underlying elevated pressure gradients and progression to heart failure, including increased cellular proliferation, ectopic cartilage formation, extensive calcification, and inflammatory infiltrate, mimicking changes seen in human calcific aortic stenosis. Despite having congenitally enlarged valves, 129 and B6129F1-Egfr(wa2/wa2) mice have normal lifespans, absence of left ventricular hypertrophy, and normal systolic function. These results show the requirement of EGFR activity for normal valvulogenesis and demonstrate that dominantly acting genetic modifiers curtail pathological changes in congenitally deformed valves. These studies provide a novel model of aortic sclerosis and stenosis and suggest that long-term inhibition of EGFR signaling for cancer therapy may have unexpected consequences on aortic valves in susceptible individuals.

Fig. 1.

Characterization of Egfr^wa2 mice. A: survival curve of B6 littermates (n = 50, P < 0.001). B: gross and histological comparison of hearts from three-mo-old B6 littermates. Normalized heart (C) and lung (D) weights by genotype and genetic background in 3- to 5-mo-old littermates (n = 16, 13, and 17 sibling pairs, respectively; ***P < 0.001).

Fig. 2.

Expression and activity of EGFR. Analysis of total EGFR, phospho-EGFR, and phospho-ERK1/2 heart (A) and liver lysates (B). Left: Westerns blots. Right: quantification by densitometry of signal relative to B6-Egfr^wa2/+ values. All values were normalized to the β-tubulin loading control. Values represent the means from at least three samples. Phospho-EGFR was not detected in heart lysates. Images were imported into Adobe Photoshop and underwent global brightness and contrast adjustment to improve quality. C: Egfr transcript levels in total RNA extracted from hearts of adult Egfr^wa2 littermates. Fold change is relative to B6-Egfr^wa2/+ samples. **P < 0.01.

Fig. 3.

Cardiac phenotypes. A: comparison of mean cardiomyocyte cross-sectional area in postnatal day 1 and 12-wk-old mice (n = 3 and 8 sibling pairs, respectively; 300 cardiomyocytes measured per animal; **P < 0.01). Representative hemotoxylin and eosin stained cardiac cross sections from B6-Egfr^wa2/+ and B6-Egfr^wa2/wa2 sibling pair (×15 and ×100 magnification; bar = 1 mm and 100 μm, respectively). B: quantification of average percent fibrotic area per Masson's Trichrome stained section and representative sections demonstrating interstitial and perivascular fibrosis observed in B6-Egfr^wa2/wa2 hearts (n = 4 sibling pairs analyzed per genetic background; ×100 magnification). *P < 0.05. C: representative short-axis M-mode tracings from 3-mo-old littermates.

Fig. 4.

Aortic valve assessment. A: comparison of mean aortic valve cusp diameter by genotype and genetic background (B6, n = 8 sibling pairs; B6129F1, n = 4 sibling pairs; 129, n = 6 sibling pairs). **P < 0.01. A representative hemotoxylin and eosin stained section of the thickest region of two cusps of the aortic valves is also shown. B: simple linear regression analysis for correlation of peak systolic pressure (PSP) and mean cusp thickness. Solid line is the regression line for all data points (R² = 0.84), whereas the broken line represents the regression line for B6-Egfr^wa2/wa2 mice (R² = 0.77). C: simple linear regression analysis of heart weight and mean cusp thickness by genotype and genetic background. Solid line is the regression line for all data points (R² = 0.51), whereas the broken line represents best fit for B6-Egfr^wa2/wa2 mice (R² = 0.77).

Fig. 5.

Cellular proliferation in aortic valves. Aortic valve sections stained for phospho-ERK1/2 (A) and PCNA (B) [magnification = ×200, scale bar= 100 μm (A) or 50 μm (B)]. Significantly more positive nuclei (arrow) were counted in B6-Egfr^wa2/wa2 aortic valves compared with F1 or 129S1-Egfr^wa2/wa2 aortic valves (*P < 0.05). At least three sections per aortic valve were analyzed; data points represent the mean value.

Fig. 6.

Aortic valve composition. For each stain, the aortic valves of at least 4 littermate pairs per genetic background were examined and scored by two observers blinded to genotype and genetic background. A: Movat's pentachrome stain for ECM composition [magnification = ×100 (left) and ×400 (middle and right), scale bar = 100 μm or 50 μm]. B: osteopontin staining for osteoblasts (arrows, positively staining cells; magnification = ×400, scale bar = 100 μm). C: Alizarin red S staining for calcification (arrow points to positive staining; AZ, Alizarin red S and adjacent; H & E, hematoxylin and eosin stained sections; magnification = ×200 scale bar = 100 μm) and quantification of percent calcified area. At least three sections per aortic valve were analyzed; data points represent the mean value. *P < 0.05. D: MOMA2 staining for inflammatory cells (arrows, red = Cy3-labeled MOMA2-positive cells; blue = DAPI stained nuclei; magnification = ×200, scale bar = 100 μm).