The Roles of S100A4 and the EGF/EGFR Signaling Axis in Pulmonary Hypertension with Right Ventricular Hypertrophy

Abstract

Pulmonary hypertension (PH) is characterized by increased pulmonary arterial pressure caused by the accumulation of mesenchymal-like cells in the pulmonary vasculature. PH can lead to right ventricular hypertrophy (RVH) and, ultimately, heart failure and death. In PH etiology, endothelial-to-mesenchymal transition (EndMT) has emerged as a critical process governing the conversion of endothelial cells into mesenchymal cells, and S100A4, EGF, and EGFR are implicated in EndMT. However, a potential role of S100A4, EGF, and EGFR in PH has to date not been elucidated. We therefore quantified S100A4, EGF, and EGFR in patients suffering from chronic thromboembolic pulmonary hypertension (CTEPH) and idiopathic pulmonary arterial hypertension (iPAH). To determine specificity for unilateral heart disease, the EndMT biomarker signature was further compared between PH patients presenting with RVH and patients suffering from aortic valve stenosis (AVS) with left ventricular hypertrophy. Reduced S100A4 concentrations were found in CTEPH and iPAH patients with RVH. Systemic EGF was increased in CTEPH but not in iPAH, while AVS patients displayed slightly diminished EGF levels. EGFR was downregulated in all patient groups when compared to healthy controls. Longitudinal data analysis revealed no effect of surgical therapies on EndMT markers. Pulmonary thrombo-endarterectomized samples were devoid of S100A4, while S100A4 tissue expression positively correlated with higher grades of Heath-Edwards histopathological lesions of iPAH-derived lung tissue. Histologically, EGFR was not detectable in CTEPH lungs or in iPAH lesions. Together, our data suggest an intricate role for S100A4 and EGF/EGFR in PH with right heart pathology.

Keywords: S100A4; atrial valve stenosis; chronic thromboembolic pulmonary hypertension; endothelial-to-mesenchymal transition; epidermal growth factor; epidermal growth factor receptor; idiopathic pulmonary arterial hypertension; pulmonary hypertension.

Figure 1

Study design. Patients presenting with PH (CTEPH and iPAH) and RVH were enrolled and compared to patients with AVS and LVH. Healthy volunteers served as controls. Serum samples were obtained from all study groups and during patient follow-up visits after surgical interventions. Serum samples were analyzed for S100A4, EGF, and EGFR. Endarterectomized specimens and iPAH lung tissues were used for immunohistochemical assessment of S100A4 and EGFR expressions.

Figure 2

Serum concentrations of S100A4 in (A) CTEPH, (B) iPAH, and (C) AVS patient groups and controls. (D) Comparison of S100A4 levels between AVS, iPAH, and CTEPH patients. Each dot represents one donor. Arithmetic means and standard deviations are indicated by horizontal lines. Patient groups were compared to controls by Mann–Whitney U test. One-way ANOVA with Bonferroni–Holm post hoc test was used to compare 3 groups.

Figure 3

Serum concentrations of EGF in (A) CTEPH, (B) iPAH, and (C) AVS patient groups and controls. (D) Comparison of EGF levels between AVS, iPAH, and CTEPH patients. Each dot represents one donor. Arithmetic means and standard deviations are indicated by horizontal lines. Patient groups were compared to controls by Mann–Whitney U test. One-way ANOVA with Bonferroni–Holm post hoc test was used to compare 3 groups.

Figure 4

Serum concentrations of EGFR in (A) CTEPH, (B) iPAH, and (C) AVS patient groups and controls. (D) Comparison of EGFR levels between AVS, iPAH, and CTEPH patients. Each dot represents one donor. Arithmetic means and standard deviations are indicated by horizontal lines. Patient groups were compared to controls by Mann–Whitney U test. One-way ANOVA with Bonferroni–Holm post hoc test was used to compare 3 groups.

Figure 5

Pre- and post-operative serum concentrations of S100A4, EGF, and EGFR. Pre- and post-surgical S100A4 levels were determined for (A) CTEPH, (B) iPAH, and (C) AVS. EGF concentrations before and after surgery in (D) CTEPH, (E) iPAH, and (F) AVS patients. EGFR levels of (G) CTEPH, (H) iPAH, and (I) AVS patients before surgery and after follow-up. Longitudinal comparisons were performed by Wilcoxon signed-rank test. Each dot represents one patient. Arithmetic means and standard deviations are indicated by horizontal lines.

Figure 6

S100A4 expression in iPAH patients. Immunohistochemical staining of S100A4 in iPAH lung tissues. Representative micrographs of characteristic pulmonary artery changes in small vessels according to Heath–Edwards classification (A) grade I, (B) grade II, (C) grade III, and (D) grade IV are shown. (E) Alveolar macrophages, (F) alveolar epithelium, (G) peribronchiolar fibrous tissue, and (H) tunica intima and tunica media of larger pulmonary vessels positive for S100A4 are shown. Tissues of interest are highlighted by open arrowheads.

Figure 7

EGFR expression in iPAH specimen. Hematoxylin eosin and EGFR staining in (A,B) Heath–Edwards grade I, (C,D) grade II, (E,F) grade III, (G,H) grade IV, (I,J) grade V lesions, (K,L) plexiform lesions. EGFR staining of (M) alveolar pneumocytes and (N) basal epithelium of bronchioles. Scale bars, 100 µm.