Activated Endothelial TGFβ1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1

Abstract

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFβ (transforming growth factor-β) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFβ in the pathophysiology of CTEPH is unknown. Objective: To determine whether defective TGFβ signaling in endothelial cells contributes to thrombus nonresolution and fibrosis. Methods and Results: Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFβ1 in platelets (Plt.TGFβ-KO) or TGFβ type II receptors in endothelial cells (End.TGFβRII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGFβ1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFβRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFβ1 levels, endothelial TGFβRI/ALK1 (activin receptor-like kinase), and TGFβRI/ALK5 expression were detected in End.TGFβRII-KO mice, and activated TGFβ signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFβRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFβ1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFβRII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGFβ1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFβRII-KO mice. Conclusions: Endothelial TGFβ1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.