Endothelial-to-mesenchymal transition: a precursor to pulmonary arterial remodelling in patients with idiopathic pulmonary fibrosis

Abstract

Background: We have previously reported arterial remodelling in patients with idiopathic pulmonary fibrosis (IPF) and suggested that endothelial-to-mesenchymal transition (EndMT) might be central to these changes. This study aims to provide evidence for active EndMT in IPF patients.

Methods: Lung resections from 13 patients with IPF and 15 normal controls (NCs) were immunostained for EndMT biomarkers: vascular endothelial cadherin (VE-cadherin), neural cadherin (N-cadherin), S100A4 and vimentin. Pulmonary arteries were analysed for EndMT markers by using computer- and microscope-assisted image analysis software Image ProPlus7.0. All the analysis was done with observer blinded to subject and diagnosis.

Results: Increased expression of mesenchymal markers N-cadherin (p<0.0001), vimentin (p<0.0001) and S100A4 (p<0.05) was noted with downregulation of junctional endothelial VE-cadherin (p<0.01) in the intimal layer of the arteries from patients with IPF compared to NCs. Cadherin switch was observed in IPF patients, showing increase in endothelial N-cadherin and decrease in VE-cadherin (p<0.01). There was also VE-cadherin shift from junctions to cytoplasm (p<0.01), effecting endothelial cell integrity in patients with IPF. In IPF, individual mesenchymal markers vimentin and N-cadherin negatively correlated with diffusing capacity of the lungs for carbon monoxide (r'= -0.63, p=0.03 and r'= -0.66, p=0.01). Further, N-cadherin positively correlated with arterial thickness (r'=0.58, p=0.03).

Conclusion: This is the first study to demonstrate active EndMT in size-based classified pulmonary arteries from IPF patients and potential role in driving remodelling changes. The mesenchymal markers had a negative impact on the diffusing capacity of the lungs for carbon monoxide. This work also informs early origins of pulmonary hypertension in patients with IPF.

FIGURE 1

a) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for VE-cadherin measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification). b) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for N-cadherin measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification). c) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for vimentin measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification). d) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for S100A4 measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification).

FIGURE 1

a) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for VE-cadherin measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification). b) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for N-cadherin measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification). c) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for vimentin measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification). d) Slides from normal control (NC) and idiopathic pulmonary fibrosis (IPF) patients of pulmonary arteries immunostained for S100A4 measuring 100–199 µm (20× magnification), 200–399 µm (20× magnification), 400–599 µm (20× magnification) and 600–1000 µm (10× magnification).

FIGURE 2

a) Per cent expression of VE-cadherin and N-cadherin positive cells in intimal layer in idiopathic pulmonary fibrosis (IPF) and normal control (NC) across arterial sizes 100–1000 µm: VE-cadherin junctional positive, VE-cadherin cytoplasmic positive, N-cadherin junctional positive and N-cadherin cytoplasmic positive. b) VE-cadherin and N-cadherin positive cells ratio in IPF and NC: VE-cadherin junctional positive versus N-cadherin junctional positive, and VE-cadherin cytoplasmic positive versus N-cadherin cytoplasmic positive. All data are presented as multiple comparisons with ordinary one-way ANOVA; p≤0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 was considered significant. NS: nonsignificant.

FIGURE 3

N-cadherin expression in normal control (NC) and idiopathic pulmonary fibrosis (IPF): a) total arterial N-cadherin expression across arterial sizes 100–1000 µm, b) 100–199 µm, c) 200–399 µm, d) 400–599 µm and e) 600–1000 µm. All data are presented as multiple comparisons with ordinary one-way ANOVA; p≤0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 was considered significant.

FIGURE 4

Vimentin expression in normal control (NC) and idiopathic pulmonary fibrosis (IPF): a) total arterial vimentin expression across arterial sizes 100–1000 µm, b) 100–199 µm, c) 200–399 µm, d) 400–599 µm and e) 600–1000 µm. All data are presented as multiple comparisons with ordinary one-way ANOVA; p≤0.05 was considered significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 was considered significant.

FIGURE 5

S100A4 expression in normal control (NC) and idiopathic pulmonary fibrosis (IPF): a) total arterial vimentin expression across arterial sizes 100–1000 µm, b) 100–199 µm, c) 200–399 µm, d) 400–599 µm and e) 600–1000 µm. All data are presented as multiple comparisons with ordinary one-way ANOVA; p≤0.05 was considered significant. *p<0.05, ***p<0.001, ****p<0.0001 was considered significant. NS: nonsignificant.

FIGURE 6

Correlations between a) total N-cadherin % expression versus total arterial thickness, b) total vimentin % expression versus total arterial thickness, c) total N-cadherin % expression versus % diffusing capacity of the lung for carbon monoxide (D_LCO) and d) total vimentin % expression versus % D_LCO.

FIGURE 7

a) CD4, CD8 and mast cell chymase (CMA1) staining in arterial layers of normal control (NC) and patients with idiopathic pulmonary fibrosis (IPF). Images were taken at 40× magnification. b) Neutrophil elastase (NE), CD68 and PDGFR-β staining in arterial layers of NC and patients with IPF. Images were taken at 40× magnification, and the insert image was taken at 100× with a bright field.