Our previous study revealed that end-stage pediatric cardiac allografts showed significant epicardial fibrosis . These epicardial changes correlated with the restrictive hemodynamics of these grafts assessed for relisting. Morphologic analysis revealed that fibroblastic proliferation and collagen deposition involved epicardial surface and underlying epicardial adipose tissue. These fibroblasts demonstrated nuclear accumulation of β-catenin, a hallmark of canonical Wnt signaling activation. The nuclear translocation of β-catenin is involved in epithelial-mesenchymal transformation, which is an initial and key step in fibrosing processes such as pulmonary fibrosis and myocardial infarction repair.
Patients with pericardial diseases such as constrictive pericarditis or open heart surgery to correct structural defects can develop pericardial adhesions with thicken epicardium. Most of our patients did not have open cardiac surgery or pericardial fibrosis in their native hearts before heart transplantation. Whether Wnt/β-catenin signaling is also activated in these conditions is clinically relevant, but undetermined. Answering this question will help clarify whether Wnt/β-catenin activation is unique to epicardial fibrosis of cardiac allograft or a common repair response to cardiac surgery.
There are 4 β-catenin nuclear partners of TCF/LEF family. In adults, Tcf-1 and Lef-1 are mainly expressed in lymphoid tissues by Northern blotting . Lef-1 could not compensate the loss of Tcf-1 in T-cells as global deletion of Tcf-1 affects T-cell differentiation and maturation . In pediatric heart allografts, both Tcf-1 and Lef-1 were present in epicardial lymphoid infiltrates. In normal hearts, Tcf-1 or Lef-1 can not be detected by Western or Northern blotting . We did not find Tcf-1 and Lef-1 expression in any cells of the epicardium from either native hearts or cardiac allografts except in lymphocytes. Tcf-3 is ubiquitously expressed during early mouse development, but gradually disappears after E7.5 and becomes undetectable by in-situ hybridization after embryonic day (E)10.5 . Similarly, we did not observe Tcf-3 expression in either native hearts or cardiac allografts in this study by immunohistochemistry. Tcf-4 is first expressed at E10.5 by in-situ hybridization and mainly found in di- and mes-encephalon and the intestinal epithelium during mouse development . In adult mice, Tcf-4 has wider tissue expression than other TCF/LEF family members. TCF4 is abundant in brain, bur is also detected in other tissues by Northern blotting. Another investigation has revealed wider and higher expression of Tcf-4 in adult mice with highest levels observed in the liver, an endodermally derived organ . Additionally, TCF4 is detected in intestines and mammary glands by immunohistochemistry . In this study, we found TCF-4, but not other TCF/LEF family members, was expressed in epicardial fibroblasts of failed pediatric heart allografts. This indicates that TCF4 is the only nuclear partner of β-catenin in cardiac fibroblasts in the TCF/LEF family.
Interstitial and perivascular lymphocytic infiltration in the myocardium after transplantation is a major morphologic feature of acute cellular rejection, which is graded dependent on the pattern, distribution, and severity of lymphoid infiltrates . However, purely endocardial lymphoid aggregation is generally considered as so-called “Quilty lesions”, but not rejection. Epicardial lymphoid infiltrates that can be associated with and may mimic acute cellular rejection, are occasionally observed in endomyocardial biopsy for cardiac transplant rejection evaluation, and frequently seen in autopsy cases . However, Quilty lesions exhibit morphologic and immunophenotypic features which are distinguishable from rejection-associated infiltrates . Although we showed the nuclear positivity of β-catenin, TCF4, TCF-1 and LEF-1 in epicardial lymphocytes of explanted pediatric allografts, these markers were also expressed in lymphocytes of benign lymph nodes or tonsils. All failed heart allografts had variable amounts of lymphoid infiltrates in the epicardium. However, we did not observe apparent association between the degree of lymphoid infiltrate and severity of epicardial fibrosis in failed allografts.
In summary, we found that fibroblast in epicardial fibrosis of failed pediatric heart allografts showed nuclear accumulation of β-catenin together with its nuclear partner, TCF4. Nuclear translocation of β-catenin is a cardinal sign of canonical Wnt signaling. This suggests that the canonical Wnt pathway is activated in epicardial fibrosis of failed pediatric heart transplants. Recent advancements in targeting canonical Wnt signaling have produced promising therapeutic options to inhibit its activity. If Wnt activation is proven causal to epicardial fibrosis with an animal model, then Wnt inhibitors can be explored for treatment.