TMEM100 expressing endothelial cells vascularize pulmonary thrombi in Chronic Thromboembolic Pulmonary Hypertension

Background and Aim: Endothelial cells (ECs) are critical for venous thrombus formation and resolution. Work from us and other groups has shown that thrombi obstructing the pulmonary arteries of patients with Chronic Thromboembolic Pulmonary Hypertension (CTEPH) exhibit impaired angiogenesis. However, little is known about the phenotype and origin of ECs lining vascular structures within the chronic thrombotic material in CTEPH. Recent single gene transcriptome analyses revealed unique expression signatures of ECs and potential markers that distinguish ECs in the lung from those in other organs. The aim of this study was to determine the expression of endothelial genetic markers in CTEPH.

Methods: Primary ECs outgrown from pulmonary endarterectomy specimens (CTEPH-ECs) or commercially available human pulmonary arterial ECs (HPAECs) and human saphenous vein ECs (HSaVECs) were subjected to quantitative real-time PCR and immunocytochemistry. Human pulmonary endarterectomy specimens and whole blood clots were examined using immunohistochemistry.

Results: Our analyses showed that the expression of general endothelial markers (CDH5, VWF) was similar in CTEPH-ECs, HPAECs and HSaVECs. Analysis of markers providing additional information about their potential origin revealed that CTEPH-ECs express high levels of Prostaglandin I2 Synthase (PTGIS), characteristic for pulmonary venous ECs. The expression of Transmembrane Protein 100 (TMEM100), previously identified as lung-specific endothelial gene, and of Activin Receptor-Like Kinase 1 (ALK1), an activator of TMEM100 expression and integral part of the Transforming Growth Factor-beta signaling pathway, also were significantly upregulated in CTEPH-ECs compared to HPAECs and HSaVECs. Immunocytochemistry confirmed that CTEPH-ECs express TMEM100 on a significantly higher level compared to HPAECs and HSaVECs. Strong TMEM100 immunosignals were also detected in cells lining the lumen of vascular structures within the obstructive thrombotic material in CTEPH as well as within ex vivo generated human blood clots pre-mixed with HPAECs. Interestingly, exploratory microarray analysis of CTEPH-ECs revealed a significantly upregulated expression of two micro-RNAs (miR), miR-1234 and miR-3162, reported to be induced by TGFβ signaling and regulating PTGIS and TMEM100 expression, suggesting a possible role of these miRs in controlling gene transcription in CTEPH-ECs that could be specifically targeted.

Conclusions: Our findings show that endothelial cells vascularizing the obstructive thrombotic material in CTEPH exhibit a distinct expression profile with elevated levels of pathophysiologically potentially relevant genetic markers and regulatory miRs. We believe that our data will improve the understanding of endothelial cells and their contribution to pulmonary thrombus non-resolution in CTEPH.