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

Abstract

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.

Fig. 1.

Differential gene expression in atrioventricular (AV) valve development and similarity with a preosteoblast cell line. A: heat map representing Affymetrix gene expression profiling analysis of murine embryonic day (E)12.5 AV endocardial cushions (EC), E17.5 AV valves, and preosteoblast MC3T3-E1 (subclone 4) cells. A total of 3,119 genes with differential expression (>2.0-fold) in E17.5 valve compared with E12.5 EC are shown, and expression in mouse preosteoblast MC3T3 cells is also indicated. Arbitrary gene expression level based on raw intensity values is indicated by color-coded expression value bar. Red indicates increased, blue indicates decreased, and yellow shows unchanged expression. B: Venn diagram showing significant similarity in differentially expressed genes enriched in E17.5 valves and preosteoblast MC3T3 cells. Genes common to E12.5 EC and preosteoblast MC3T3 or E17.5 AV valve and preosteoblast MC3T3 are indicated in Venn diagrams. In E12.5 AV cushion, a total of 662 genes are more strongly expressed, whereas 2,457 genes are predominant in E17.5 AV valve. Importantly, in E12.5 AV cushion, of 662 increased genes, only 106 genes (16%) are shared with MC3T3 cells. In contrast, in E17.5 AV valve, of total 2,457 increased genes, 1,227 genes (49.9%) are shared with MC3T3 cells.

Fig. 2.

Mouse Twist1 is highly expressed in E12.5 AV cushion but is downregulated during AV valve maturation. A: quantitative RT-PCR (qRT-PCR) validation of microarray results for Twist1 in EC at E12.5 and AV valves at E17.5. Normalized Twist1 expression in E17.5 AV valves is set to 1, and then the fold change is shown for E12.5 EC. Osteonectin expression is also shown as a control gene that did not change significantly in E12.5 EC and E17.5 AV valves. The average fold increase is depicted, where n = biological 3×; error bars represent SE. Statistical significance was determined by Student's t-test; *P < 0.05. B–E: in situ hybridization (ISH) is shown on 14-μm paraffin sections of E12.5 and E17.5 mouse hearts with probes specific for mouse Twist1 (B and C) and Osteonectin (D and E). Twist1 is strongly expressed in E12.5 EC cells (B) in contrast to E17.5 mitral valve (MV) leaflets (arrows in C). Osteonectin ISH is shown as control for similar gene expression in both EC (D) and late remodeled MV leaflets (arrows in E). Scale bars, 200 nm.

Fig. 3.

Differential expression of collagen genes during mouse embryonic AV valve development. A: qRT-PCR validation of microarray result for Col9a3, Col1a1, and Col14a1 was performed as in Fig. 2A. Col9a3 expression in E17.5 AV valves is normalized to 1, and then the fold changes are calculated for E12.5 EC. In contrast, Col1a1 and Col14a1 expression in E12.5 EC is normalized to 1, and then the fold changes are calculated for E17.5 AV valve. Statistical significance was determined by Student's t-test; *P < 0.05. B–G: ISH with probes specific for mouse Col9a3 (B and C), Col1a1 (D and E), and Col14a1 (F and G). Strong expression of Col9a3 is detected in E12.5 EC cells (B) compared with its expression in E17.5 MV leaflets (arrows in C). In contrast, strong expression of Col1a1 (E) and Col14a1 (G) is detected in E17.5 MV leaflets compared with their expression in E12.5 EC (D and F, respectively). Scale bars, 200 nm.

Fig. 4.

qRT-PCR validation of microarray results for mouse Asporin (Aspn), Osteoglycin (Ogn), and Matrilin2 (Matn2). qRT-PCR was performed with specific primer sets for Aspn, Ogn, and Matn2 as described in Table 1A. The normalized gene expression in E12.5 cushions is set to 1, and then the fold changes are calculated for E17.5 AV valves. Statistical significance was determined by Student's t-test; *P < 0.05.

Fig. 5.

Expression of mouse Asporin, Osteoglycin, and Matrilin2 in embryonic and adult AV valves: ISH on 14-μm paraffin sections of E12.5, E17.5, and adult mouse hearts with probes specific for Aspn (A–C), Ogn (D–F), and Matn2 (G–I). Strong expression of Aspn (B and C), Ogn (E and F), and Matn2 (H and I) are demonstrated in late embryonic (E17.5) and adult MV leaflets (arrows in B, C, E, F, H, and I), but not in E12.5 EC (A, D, and G). Scale bars, 200 nm.