Differential activation of valvulogenic, chondrogenic, and osteogenic pathways in mouse models of myxomatous and calcific aortic valve disease.

Studies of human diseased aortic valves have demonstrated increased expression of genetic markers of valve progenitors and osteogenic differentiation associated with pathogenesis. Three potential mouse models of valve disease were examined for cellular pathology, morphology, and induction of valvulogenic, chondrogenic, and osteogenic markers. Osteogenesis imperfecta murine (Oim) mice, with a mutation in Col1a2, have distal leaflet thickening and increased proteoglycan composition characteristic of myxomatous valve disease. Periostin null mice also exhibit dysregulation of the ECM with thickening in the aortic midvalve region, but do not have an overall increase in valve leaflet surface area. Klotho null mice are a model for premature aging and exhibit calcific nodules in the aortic valve hinge-region, but do not exhibit leaflet thickening, ECM disorganization, or inflammation. Oim/oim mice have increased expression of valve progenitor markers Twist1, Col2a1, Mmp13, Sox9 and Hapln1, in addition to increased Col10a1 and Asporin expression, consistent with increased proteoglycan composition. Periostin null aortic valves exhibit relatively normal gene expression with slightly increased expression of Mmp13 and Hapln1. In contrast, Klotho null aortic valves have increased expression of Runx2, consistent with the calcified phenotype, in addition to increased expression of Sox9, Col10a1, and osteopontin. Together these studies demonstrate that oim/oim mice exhibit histological and molecular characteristics of myxomatous valve disease and Klotho null mice are a new model for calcific aortic valve disease.

Wnt signaling in heart valve development and osteogenic gene induction.

Wnt signaling mediated by beta-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of beta-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.

Shared gene expression profiles in developing heart valves and osteoblast progenitor cells.

The atrioventricular (AV) valves of the heart develop from undifferentiated mesenchymal endocardial cushions, which later mature into stratified valves with diversified extracellular matrix (ECM). Because the mature valves express genes associated with osteogenesis and exhibit disease-associated calcification, we hypothesized the existence of shared regulatory pathways active in developing AV valves and in bone progenitor cells. To define gene regulatory programs of valvulogenesis relative to osteoblast progenitors, we undertook Affymetrix gene expression profiling analysis of murine embryonic day (E)12.5 AV endocardial cushions compared with E17.5 AV valves (mitral and tricuspid) and with preosteoblast MC3T3-E1 (subclone4) cells. Overall, MC3T3 cells were significantly more similar to E17.5 valves than to E12.5 cushions, supporting the hypothesis that valve maturation involves the expression of many genes also expressed in osteoblasts. Several transcription factors characteristic of mesenchymal and osteoblast precursor cells, including Twist1, are predominant in E12.5 cushion. Valve maturation is characterized by differential regulation of matrix metalloproteinases and their inhibitors as well as complex collagen gene expression. Among the most highly enriched genes during valvulogenesis were members of the small leucine-rich proteoglycan (SLRP) family including Asporin, a known negative regulator of osteoblast differentiation and mineralization. Together, these data support shared gene expression profiles of the developing valves and osteoblast bone precursor cells in normal valve development and homeostasis with potential functions in calcific valve disease.

Psychometric properties of the Revised Cheek and Buss Shyness Scale.

Although the Revised Cheek and Buss Shyness Scale (RCBS; Cheek, 1983) is widely used, its psychometric properties largely are unknown. In this investigation, we examined the normative data, factor structure, internal consistency, test-retest reliability, and convergent/discriminant validity of the RCBS using a sample of 261 university students. Results provided strong support for the stability of normative data over time, reliability of the measure, and its predicted associations with contemporary measures of shyness, social anxiety, and related constructs. Although support was obtained for a unifactorial conceptualization of shyness, an exploratory factor analysis revealed an alternative 3-factor solution that was supportive of a previously proposed meta-analytic model of shyness (Jones, Briggs, & Smith, 1986) and was consistent with other prominent shyness theories (Buss, 1980; Pilkonis, 1977a, 1977b; Zimbardo, 1977). This factor model was replicable on a holdout sample, and there were some data to support the discriminant validity of factors.