Background and Aim: Angiogenesis is a critical event during venous thrombus remodelling, and endothelial cell-poor, fibrotic thrombi obstruct the pulmonary artery lumen in patients with chronic thromboembolic pulmonary hypertension (CTEPH). The aim of this study was to examine the genetic footprint of endothelial cells from pulmonary thrombofibrotic material and to better understand thrombus non-resolution and fibrosis.

Methods: Cells outgrown from pulmonary endarterectomy specimens expressing endothelial cell markers (CTEPH-ECs) were processed for whole gene microarray analysis. Spatial laser microdissection followed by nCounter gene expression analysis and immunohistochemistry of tissue microarrays were used to confirm expression of candidate proteins in CTEPH tissue. Plasma levels of candidate genes (BIGH3, TAGLN, FSTL3 and STC2) were examined in CTEPH patients. Lentiviral overexpression of BIGH3 in human pulmonary artey endothelial cells (HPAECs) and exogenous administration of recombinant BIGH3 via osmotic pumps to C57BL/6J mice was used to assess the role of BIGH3 for angiogenesis and thrombus resolution.

Results: Out of 26,808 genes examined, 527 were differentially regulated in CTEPH-ECs compared to HPAECs. Biological pathway analysis and RT² PCR profiler analysis confirmed that factors downstream of transforming growth factor beta (TGFβ) such as TGFβ-induced (TGFBI/BIGH3) or transgelin (TAGLN), or involved in TGFβ signalling, such as follistatin-like 3 (FSTL3), were expressed at significantly higher levels in CTEPH-ECs. Spatial laser microdissection followed by nCounter gene expression analysis and immunohistochemistry of tissue microarrays localised potential disease candidates to vessel-rich regions. Whereas circulating levels of TAGLN and FSTL3 were also increased in patients with pulmonary arterial hypertension, BIGH3 plasma levels were specifically elevated only in CTEPH patients and found to decrease after pulmonary endarterectomy. Lentiviral overexpression of BIGH3 in HPAECs induced the expression of TAGLN, STC2 and the transcriptional repressor SNAI2, thus phenocopying gene expression patterns in CTEPH-ECs. Increased plasma concentrations of BIGH3 in C57BL/6J mice was associated with delayed thrombus resolution after IVC ligation.

Conclusions: Our findings strengthen the importance of endothelial alterations in the pathophysiology of CTEPH and suggest that overexpression of BIGH3 in endothelial cells is causally involved in thrombofibrosis.