Here we demonstrate that Twist1 promotes progenitor cell proliferation and affects expression of several ECM proteins, including the target gene Col2a1, during heart valve development in mice. Likewise, increased Twist1 expression is apparent in adult human calcific aortic valve disease. In mice, Tie2Cre-mediated expression of Twist1 promotes cell proliferation and Tbx20 expression during late embryonic heart valve development. Persistent Twist1 expression also results in increased expression of several early ECM markers, including Col2a1, Mmp2 and Mmp13 at this stage (E17.5). In E12.5 EC, Twist1 binds to a conserved Twist1-responsive E-box element present in the Col2a1 intron 1, supporting direct regulation of gene expression by Twist1. Valve remodeling also is affected by increased Twist1 expression as indicated by thickening of AoV and MV leaflets in addition to dysregulation of mature fibrillar collagen deposition and increased periostin expression at E17.5. In human diseased valves with calcification, increased Twist1, Col2, and cell proliferation are detected in overlapping regions. Co-immunofluorescence provides evidence that a subset of cells expressing Twist1 in the diseased valves is actively proliferating. Overall, these data support a role for Twist1 as a direct regulator of cell proliferation and ECM gene expression in embryonic valve progenitor cells during development. In addition, the detection of increased Twist1 expression in adult valve disease provides initial evidence that Twist1 promotes cell proliferation and Col2 expression during human valve pathogenesis.
A significant reduction in cell proliferation is a feature of late embryonic valve development, and normally little or no cell proliferation is apparent in adult VICs (Aikawa et al., 2006
; Hinton et al., 2006
; Lincoln et al., 2004
). In chicken primary EC cells, Twist1 is necessary and sufficient to promote cell proliferation (Shelton and Yutzey, 2008
). In addition, Twist1 promotes Tbx20 expression in avian primary EC explant cultures, and Tbx20 promotes expression of the proto-oncogene N-myc
in EC and cardiomyocytes (Cai et al., 2005
; Shelton and Yutzey, 2007
). Consistent with these data, Tie2Cre-mediated Twist1 overexpression is sufficient to promote cell proliferation and increased Tbx20 expression in E17.5 remodeling valves. However, no significant change is observed in either proliferative indices or Tbx20 expression in postnatal valves overexpressing Twist1. It is possible that Twist1 requires cooperating factors that are absent in postnatal valves in order to promote cell proliferation or that postnatal VICs are refractory to reentering the cell cycle. Together, these findings demonstrate that persistent Twist1 expression is sufficient to prolong the period of cell proliferation in the E17.5 remodeling valve leaflets.
During normal valve development, the primitive, relatively unstructured ECM of the EC remodels into highly organized and stratified ECM of the mature valve leaflets. Valve leaflet remodeling is characterized by the down regulation of early ECM markers and remodeling enzymes and increased expression of mature ECM proteins, including fibrillar collagens and elastin (Chakraborty et al., 2008
; Hinton et al., 2006
). Persistent expression of Twist1 results in increased expression of several early ECM markers, including Col2a1
, and Mmp13.
A direct transcriptional regulatory mechanism is supported by the identification of intronic sequences of the Col2a1
gene that are bound by endogenous Twist1 in E12.5 EC in vivo and are trans-activated by Twist1 in cultured cells. In addition, Twist1 and Col2 protein expression are induced in overlapping regions of human diseased aortic valves. Since Twist1 expression is normally downregulated during the stages when Col1a1
expression is induced, it is likely that fibrillar collagen gene induction occurs by an indirect mechanism (Chakraborty et al., 2008
). Postn expression also is increased with persistent expression of Twist1 in developing mouse valves. In pre-osteoblast cells, Twist1 binds directly to a canonical E-box sequence in the murine Postn
promoter and trans-activates a Postn
reporter gene, supporting a direct regulatory mechanism (Oshima et al., 2002
). Additional candidate Twist1 downstream target genes identified in mouse valves with persistent Twist1 expression and in avian endocardial cushion cells include Tbx20
, but Twist1-responsive regulatory elements have not yet been reported for these genes (Shelton and Yutzey, 2007
). Together, these data support Twist1 as a critical transcriptional activator of multiple ECM and remodeling enzyme genes expressed in the mesenchymal EC.
During valve pathogenesis, VICs are activated, apparent in increased cell proliferation, ECM gene induction, and secretion of matrix remodeling enzymes (Schoen, 2008
). These properties of activated VIC are shared with valve progenitors of the embryonic EC. The detection of increased Twist1 expression and cell proliferation in human aortic valve disease suggests that Twist1 may influence cell proliferation and VIC activation during disease. Increased Twist1 expression occurs in small clusters of cells in regions displaying ECM disorganization, active cell proliferation, and diseased calcified nodules. Based on our studies in mice, it is unlikely that increased expression of Twist1 alone is sufficient to cause calcific aortic valve disease. However, the observed increase in Twist1 expression in human diseased valves could contribute to cell activation in response to injury, allowing for ECM reorganization and increased cell proliferation. In addition, it is unclear whether Twist1 reactivation during aortic valve disease is part of a protective mechanism for valve repair, or if it has pathologic consequences leading to valve calcification. The origins of activated VICs in valve disease have not been identified, but they could arise from a resident stem cell population or alternatively from extra-cardiac origins, such as mesenchymal or hematopoietic stem cells. There is increasing evidence that regulatory programs active during valve development are reactivated in disease and could be a mechanism for valve homeostasis or regeneration. Therefore, Twist1 function in valve progenitor cell proliferation and ECM gene expression may hold future clinical relevance in human valve disease diagnosis and therapies.