TGF-beta-dependent pathogenesis of mitral valve prolapse in a mouse model of Marfan syndrome

Abstract

Mitral valve prolapse (MVP) is a common human phenotype, yet little is known about the pathogenesis of this condition. MVP can occur in the context of genetic syndromes, including Marfan syndrome (MFS), an autosomal-dominant connective tissue disorder caused by mutations in fibrillin-1. Fibrillin-1 contributes to the regulated activation of the cytokine TGF-beta, and enhanced signaling is a consequence of fibrillin-1 deficiency. We thus hypothesized that increased TGF-beta signaling may contribute to the multisystem pathogenesis of MFS, including the development of myxomatous changes of the atrioventricular valves. Mitral valves from fibrillin-1-deficient mice exhibited postnatally acquired alterations in architecture that correlated both temporally and spatially with increased cell proliferation, decreased apoptosis, and excess TGF-beta activation and signaling. In addition, TGF-beta antagonism in vivo rescued the valve phenotype, suggesting a cause and effect relationship. Expression analyses identified increased expression of numerous TGF-beta-related genes that regulate cell proliferation and survival and plausibly contribute to myxomatous valve disease. These studies validate a novel, genetically engineered murine model of myxomatous changes of the mitral valve and provide critical insight into the pathogenetic mechanism of such changes in MFS and perhaps more common nonsyndromic variants of mitral valve disease.

Figure 1

Histologic and morphometric assessment of mitral valve architecture in P6.5 mice. Fbn1 genotypes are indicated as follows: +/+ (Fbn1^+/+), +/− (Fbn1^C1039G/+), and −/− (Fbn1^{C1039G/C1039G}). (A) Representative mitral valve sections from each genotype at P6.5 showing increased length and thickness in mutant valves as compared to wild-type littermates. Magnification, ×20. Scale bars: 100 μm. (B) Morphometric analysis of mitral valve length during the first week of postnatal life. Fbn1^{C1039G/C1039G} valves were significantly longer by P6.5 when compared with those of Fbn1^+/+ animals (^–;P –; 0.05). (C) Morphometric analysis of mitral valve thickness during the first week of postnatal life. Increased valve thickness in Fbn1^{C1039G/C1039G} versus Fbn1^+/+ mice was significant by P4.5 (*P –; 0.005 vs. Fbn1^+/+), and by P6.5, differences between all genotypes were statistically significant (*P –; 0.005 vs. Fbn1^+/+; ^–;P –; 0.05 vs. Fbn1^C1039G/+). Error bars indicate 95% confidence intervals. (D) Echocardiographic parasternal long axis views of 9-month Fbn1^C1039G/+ and Fbn1^+/+ mouse hearts. The anterior mitral valve leaflet (arrowhead) shows increased length and thickening, as well as systolic prolapse into the LA in fibrillin-1–;deficient mice. AoR, aortic root; AscAo, ascending aorta; LA, left atrium.

Figure 2

TGF-β activity and signaling in mitral valves of fibrillin-1–;deficient mice. Immunohistochemical analysis using antibodies specific to free and active TGF-β (LC1-30), LAP-β1, and P-Smad2 in wild-type (Fbn1^+/+) mice and littermates either heterozygous (Fbn1^+/–;) or homozygous (Fbn1^–;/–;) for mutation C1039G. Mutant valves show increased TGF-β activity and signaling but not increased expression of LAP-β1, indicative of increased TGF-β activation rather than cytokine production. Magnification, ×100. Scale bars: 20 μm.

Figure 3

Phenotypic rescue after TGF-β antagonism. (A) Morphometric analysis of mitral valve length at P6.5 in untreated (control) mice, in mice receiving irrelevant IgG (IgG), and in mice receiving TGF-β–;neutralizing Ab (NeuAb). Leaflet length was significantly shorter in neuAb-treated mice. (B) Morphometric analysis of mitral valve thickness after TGF-β antagonism. Valves from NeuAb-treated mice were significantly less thick than those from IgG-treated counterparts for all genotypes Error bars indicated 95% confidence intervals. *P –; 0.005 vs. IgG; P –; 0.01 vs. IgG.

Figure 4

Potential downstream effectors of the myxomatous phenotype. (A) βIGH3 expression in late embryonic bovine mitral (MV) and tricuspid (TV) valves, as assessed by in situ hybridization. (B) Assessment of cell proliferation and apoptosis in P6.5 murine mitral valves using Ki-67 and TUNEL staining, respectively. Mutant valves showed both increased cell proliferation and decreased apoptosis. Magnification, ×100. Scale bars: 20 μm.

Figure 5

Expression of BMPs 2, 4, and 6 in fibrillin-1–;deficient mitral valves. Fbn1^{C1039G/C1039G} (Fbn1^–;/–;) valves exhibited increased expression of BMPs 2, 4, and 6, as compared to Fbn1^+/+ valves. Magnification, ×100. Scale bars: 20 μm.