A mouse model for Costello syndrome reveals an Ang II-mediated hypertensive condition - PubMed (original) (raw)

A mouse model for Costello syndrome reveals an Ang II-mediated hypertensive condition

Alberto J Schuhmacher et al. J Clin Invest. 2008 Jun.

Abstract

Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin-Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies.

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Figures

Figure 1

Figure 1. Functional characterization of the H-Ras signaling pathway in H-_Ras_G12V adult brain and E14.5 embryos.

(A) Western blot analysis of the expression levels of wild-type H-Ras+/+ and mutant H-RasG12V proteins. Protein extracts (200 μg) derived from colons of H-Ras+/+, H-Ras+/G12V, H-_Ras_G12V/G12V, and H-_Ras_–/– mice as well as from brain (Br), heart (He), lung (Lu), kidney (Ki), and liver (Li) of H-Ras+/G12V animals were subjected to Western blot analysis. In the case of brain, only one-fourth of the sample was loaded. Migration of the wild-type H-Ras and mutant H-RasG12V proteins is indicated by arrowheads. (B) Protein extracts (40 μg) from the indicated genotypes obtained from 2-month-old adult brains and E14.5 whole embryos were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with antibodies against H-Ras and the nonphosphorylated and phosphorylated forms of Erk1/2, Mek1, and Akt. Levels of active H-Ras protein bound to GTP (H-Ras–GTP) were determined by its ability to interact with the Ras binding domain of c-Raf. GAPDH was used as loading control.

Figure 2

Figure 2. H-Ras signaling in heart and kidneys of H-_Ras_G12V mutant mice.

Protein extracts (40 μg) obtained from (A) heart and kidneys of 2-month-old H-_Ras_–/–, H-Ras+/+, H-Ras+/G12V, and H-_Ras_G12V/G12V mice and from (B) cardiomyocytes and fibroblasts isolated form neonatal hearts of H-Ras+/+ and H-_Ras_G12V/G12V animals were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and blotted with antibodies against H-Ras and the nonphosphorylated and phosphorylated forms of Erk1/2, Mek1, and Akt. GAPDH was used as loading control.

Figure 3

Figure 3. Mammary gland hyperplasia in H-_Ras_G12V virgin female mice.

Red carmine whole-mount staining (A–C) and H&E staining of paraffin sections (D–F) of mammary glands of H-Ras+/+ (A and D), H-Ras+/G12V (B and E), and H-_Ras_G12V/G12V (C and F) mice. Insets show representative detail. Mammary glands of H-_Ras_G12V/G12V mice displayed substantial white fat atrophy, greater ductal-lobulillar development (arrowheads), and ductal ectasia (asterisk). Scale bars: 1 mm (A–C); 10 μm (D–F).

Figure 4

Figure 4. Facial dysmorphia.

(A) Top and side views of heads of H-Ras+/+ and H-_Ras_G12V/G12V mice illustrating the prominent forehead and blunt nose of mutant animals (arrowheads). (B) CT sections of H-Ras+/+ and H-_Ras_G12V/G12V littermates. Top: Coronal projection. Arrowhead indicates choanal atresia. Bottom: Sagittal projection. Arrowhead indicates the shortened and depressed nasal bridge and premaxillar bone.

Figure 5

Figure 5. Heart and kidney defects in H-_Ras_G12V mice.

(A) Formalin-fixed hearts. (B) H&E-stained heart ventricular sections. Interventricular wall (IV), LV, and RV are indicated. (C) Fibrosis in the LV, as shown by Sirius red–stained preparations. (D) H&E-stained aortic valves (arrowheads). (E) H&E-stained aorta media wall (asterisks). (F) Kidney fibrosis, as shown by Sirius red–stained preparations. Scale bars: 1 mm (A and B); 50 μm (D); 100 μm (C, E, and F).

Figure 6

Figure 6. Analysis of the renin–Ang II and ET systems in H-Ras mutant mice.

(A) Concentration of Ang II and ET in the plasma of mice of the indicated genotypes. *P < 0.01, #P < 0.05 versus wild-type. (B) Representative real-time recordings of arterial blood pressure variations upon administration of bosentan (B) and/or losartan (L). Scale bar: 1 min. (C) Quantification of mean arterial pressure upon administration of either bosentan or losartan. Values were taken when the arterial pressure became stabilized after the indicated treatment.

Figure 7

Figure 7. Effect of captopril treatment on heart, aorta, and kidneys of H-_Ras_G12V mutant mice.

H-Ras+/+ and H-_Ras_G12V/G12V mice were left untreated, or treated with captopril, for 12 (A–F) or 24 weeks (G–L). (A and G) Formalin-fixed hearts. (B and H) H&E-stained heart ventricular sections. Interventricular wall, LV, and RV are indicated. (C and I) Fibrosis in the LV, as shown by Sirius red staining. (D and J) H&E-stained aortic valves (arrowheads). (E and K) H&E-stained aorta media wall (asterisks). (F and L) Kidney fibrosis, as shown by Sirius red staining. Scale bars: 1 mm (A, B, G, and H); 50 μm (D and J); 100 μm (C, E, F, I, K, and L).

Figure 8

Figure 8. Effect of captopril treatment on cardiovascular parameters of H-_Ras_G12V mutant mice.

Systolic arterial pressure (A), LV and RV cardiomyocyte area (B), and total heart weight (C) in H-Ras+/+ (white bars) and H-_Ras_G12V (black bars) mice that were left untreated or treated with captopril for 12 or 24 weeks. Untreated mice were controls for the 24-week treatment protocol. *P < 0.01 versus H-Ras+/+.

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