The epicardium retains stem cell-like properties and when given the appropriate cues, it can differentiate into multiple cardiac cell types26-28
. In a zebrafish cardiac injury model, the epicardium is an essential component of the regeneration process29
. In addition to being a source of cardiac16, 26
and vascular cells2, 3
, the epicardium also secretes growth factors essential for myocardial development30, 31
. Therefore, a better understanding of the epicardium during development may help define the signals essential for reactivation of these differentiation processes to improve outcomes of human heart disease.
We show that PDGFRβ provides essential cues for efficient epicardial migration, cVSMC formation, and coronary vessel maturation. Previous expression studies have demonstrated ligands and receptors within the epicardium foreshadowing a requirement for PDGF signal transduction12, 32
. Explant studies in rat and chick proepicardial and epicardial cells have demonstrated that stimulation with PDGF ligands leads to filamentous actin formation and expression of smooth muscle cell markers33, 34
. We now provide in vivo illustration of the role for PDGFRβ in epicardial function and show that disruption of this signaling impacts more than just VSMC proliferation. The heart is the first tissue to demonstrate an absolute requirement for PDGFRβ signaling to promote VSMC differentiation. In most other tissues VSMC differentiation occurs but expansion is disrupted in the absence of PDGFRβ4
It is an established fact that VSMC are a heterogeneous population and that they come from a vast range of embryonic origins. In the chick, lineage-tracing analysis has demonstrated that the majority of cVSMC are derived from the proepicardium3, 35, 36
. Consistent with recent lineage tracing studies16, 26
, we have shown that in the mouse, a majority of VSMC also arise from the epicardium. However, one question concerning the heterogeneity of cVSMC is the origin of the residual cells that are present around the coronary arteries in PDGFRβ epicardial mutant hearts. From the current experiments it is difficult to determine if these cVSMC arise ectopically due to the absence of epicardially-derived VSMC, or if they are a normal subpopulation of cells contributing to the coronary arteries. Both neural crest-derived cells and cells from the secondary heart field can contribute to VSMC in the outflow tract and the coronary arteries37-39
. The possibility that cVSMC are heterogeneous in origin is an important consideration because the two cell populations are likely to express different genes and respond differently under pathological conditions or in response to pharmacologic intervention.
Based on our analysis of the formation of the coronary vessels in PDGFRβ mutant hearts, we propose that PDGFRβ signaling can indirectly help shape the mature coronary vasculature. The observation that the coronary arteries defects in PDGFR
but not PDGFRβ-/-
hearts improve over time suggests that cVSMC may be involved in the coronary vessel remodeling process. We have shown that cVSMC formation was completely disrupted, while epicardial cell migration was reduced but not abrogated in PDGFR
hearts. The most likely explanation for this observation is that presence of PDGFRα can compensate for loss of PDGFRβ in the epicardium but not cVSMC. These two receptors signal through very similar pathways and can bind some but not all of the same ligands40
. We have recently shown that PDGF receptor function in neural crest cells is also partially redundant, therefore ligand availability may be a key factor in determining the contribution of each receptor. The exquisite expression of the PDGFDD ligand in the epicardium32
, which predominantly activates PDGFRβ41
, could favor signaling through the PDGFRβ. Therefore, it is possible that PDGFDD may provide an autocrine signal that induces cytoskeletal rearrangements necessary for EMT. Recently, PDGFDD overexpression has been demonstrated to induce an EMT-like transformation in prostate cancer cells42
Our current data cannot determine the temporal requirement for PDGFRβ signaling. The migration defect we observe may predominantly affect only the cells destined to become cVSMC. In this scenario, cVSMC progenitors might not reach their final destination to receive the differentiation cues from myocardium or endothelium. In support of this possibility, clonal analysis of VSMC in the chick has shown that specification of VSMC occurs prior to the formation of the epicardium and that some VSMC markers are expressed in the proepicardium3
. Because the requirement for PDGFRβ signaling in many tissues is to promote VSMC proliferation6, 7
, another way to explain our results is that PDGFRβ may be required at two stages: within the epicardium for migration and within the epicardial-derived mesenchyme to promote VSMC differentiation or expansion.
A role for PDGFRβ in directing cytoskeletal rearrangements via PI3K has been established in multiple cell types (reviewed by Heldin43
). Here, we have demonstrated that PDGF stimulation of PI3K is also required for cortactin localization to lamellipodia. The failure of cortactin localization is likely due to the loss of PI3K-induced Rac activation44
. The epicardial cells are incapable of controlling directed actin filament growth, and directed migration into the myocardium is inefficient.
In conclusion, we have demonstrated a novel role for the PDGFRβ in epicardial development in vivo and identified PI3K signaling as one of the pathways associated with this process. In the absence of this signaling, the epicardium fails to adopt a motile phenotype leading to a reduction in cVSMC and abnormal coronary vessels. Overall, these findings suggest that PDGF signaling is acting to promote epicardial migration and that modulation of PDGF receptor signaling should be considered when exploring options for therapeutic applications of epicardial-derived cells.