Activated Endothelial TGFβ1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1 Potential Role for Chronic Thromboembolic Pulmonary Hypertension

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGF beta (transforming growth factor-beta) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGF beta in the pathophysiology of CTEPH is unknown. Objective: To determine whether defective TGF beta 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 TGF beta 1 in platelets (Plt.TGF beta-KO) or TGF beta type II receptors in endothelial cells (End.TGF beta RII-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 TGF beta 1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGF beta RII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGF beta 1 levels, endothelial TGF beta RI/ALK1 (activin receptor-like kinase), and TGF beta RI/ALK5 expression were detected in End.TGF beta RII-KO mice, and activated TGF beta signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGF beta RII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGF beta 1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGF beta RII-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 TGF beta 1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGF beta RII-KO mice. Conclusions: Endothelial TGF beta 1 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.