The role of endothelial cell function in regulating fibrinolysis and the development and progression of thromboembolic pulmonary hypertension

Abstract

Pulmonary hypertension (PH) is a rare but disabling disorder, characterised by raised pulmonary artery pressure leading to right ventricular heart failure, progressive disability and death. Chronic thromboembolic pulmonary hypertension (CTEPH), a distinct category of PH, is a condition in which the initiating event is either overt or occult pulmonary embolism (PE). It is believed that the persistence of thrombus is a prerequisite for the development of PH following PE. It was hypothesised that defective fibrinolysis on the surface of the pulmonary endothelium causes progression from PE to CTEPH. Thrombin generation and fibrinolysis were assayed in parallel on endothelial cells using exogenous tissue factor or TNF-α stimulation. Thrombin generation was measured using a modified calibrated automated thrombogram assay (CAT). Fibrinolysis was measured using a turbidometric assay. Assays were established first on human umbilical vein endothelial cells (HUVECs). The study of patient’s endothelium is complicated by the difficulty in accessing tissue. Using a recently developed technique, blood outgrowth endothelial cells (BOECs) were isolated from the mononuclear fraction of peripheral blood from patients with CTEPH and controls. Discrete colonies of BOECs appeared between days 7-21 in culture. Cells were passaged to obtain confluent BOEC monolayers with characteristic endothelial cobblestone morphology. Endothelial identity was confirmed using immunofluorescence with a range of endothelial markers including VWF and VE-Cadherin. BOECs were successfully isolated from patients with CTEPH and healthy controls in order to evaluate thrombin generation and fibrinolysis using the assays developed on HUVECs. No difference in thrombin generation was observed but surprisingly shorter clot lysis times were shown on BOECs from CTEPH patients as compared to controls. In order to determine the cause of the paradoxical increase in fibrinolysis, mRNA expression of a panel of candidate genes was performed using RT-PCR array analysis.

Differences in gene expression were seen between CTEPH and healthy control BOECs. Statistically significant results included an increase in fibroblast growth factor 2 and fibronectin expression with a decrease in VEGF receptor 2 expression. Other genes implicated in the pathogenesis of PH and CTEPH for example endothelin receptor A, matrix metalloproteinase 2 and 9 also showed altered expression and therefore further studies with larger numbers are required to confirm these findings.