Molecular Mechanisms
Controlling the Differentiation of Valvular Interstitial
Cells
Heart valves, which prevent backflow of blood during
cardiac contraction and relaxation, contain two main
cell populations: endothelial cells and valvular
interstitial cells (VICs). VICs are heterogeneous, but
are comprised mainly of fibroblasts, myofibroblasts and
smooth muscle cells. These cells play a key role in
maintaining valve homeostasis by shifting to a higher
ratio of myofibroblasts in response to tissue damage.
Calcific aortic stenosis (CAS), which affects 2-3% of
people over 65, has been suggested to be associated with
osteoblast differentiation of certain cellular
component(s) in the VIC population. However, neither the
cellular component nor the signaling pathways which
contribute to VIC differentiation are known. As
fibroblasts are mesenchymal cells with the potential to
differentiate to multiple cell types, we hypothesize
that valvular fibroblasts are the cellular determinant
for VIC differentiation into myofibroblasts and
osteoblasts. To test this, I will first sort fibroblasts
from the VIC population based on a combination of cell
surface markers using fluorescent-activated cell
sorting. With a relatively pure population of valvular
fibroblasts, I will then study the signaling pathways
which regulate their cell fate. In response to TGF-β1,
VICs have been shown to express myofibroblast makers,
e.g. α-smooth muscle actin (α-SMA), after 2 days and to
acquire some osteoblast phenotypes, such as the
expression of Cbfa1, a transcriptional factor that
initiates osteoblast differentiation, after 7 days. I
will use lineage-specific promoters for myofibroblasts
or osteoblasts fused with fluorescent protein to track
fibroblast differentiation in real-time. After
determining whether the same fibroblasts or different
subsets of fibroblasts give rise to myofibroblasts and
osteoblasts, I will try to define the signaling pathways
activated by TGF-β1 to promote osteoblast
differentiation. This can be done by introducing
specific inhibitors of certain pathway or transfecting
cells with constitutively active (or dominant negative)
signaling molecules. So far, I have made the reporter
constructs of α-SMA-promoter driven Dsred (for
myofibroblasts) and Cbfa1-promoter driven GFP (for
osteoblasts). The long-term goal is to determine the
signaling pathways involved in these differentiation
events so that we can manipulate the properties of
valvular fibroblasts and control the regeneration of
live tissue for valve replacement in vitro.
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