HDAC6 is a novel regulator of endothelial-to-mesenchymal transition in venous thrombosis
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Background
Venous thromboembolism (VTE), which encompasses deep vein thrombosis (DVT) and pulmonary embolism (PE), is a frequent disease associated with thrombus formation and vein wall remodeling. Hence, fibrosis might result from endothelial-to-mesenchymal transition (EndMT), characterized by the loss of endothelial markers and the acquisition of mesenchymal markers. In chronic thromboembolic pulmonary hypertension, transforming growth factor (TGFβ), the most potent inducer of EndMT, impairs thrombus resolution. However, the molecular mechanisms implicated in TGFβ signaling in the context of VTE are unknown. We hypothesized that epigenetic processes regulate the TGFβ signaling pathway in endothelial cells promoting EndMT and vascular fibrosis.
Aims
To determine if the histone deacetylase 6 (HDAC6) regulates the TGFβ signaling pathway in endothelial cells promoting EndMT and delays venous thrombosis.
Methods
To study the role of HDAC6 in EndMT, endothelial cells were treated with a pharmacological inhibitor (TCS20b) and incubated with TGFβ and thrombin for 2, 3, and 5 days. Real time PCR and Western blot were performed to analyze endothelial and mesenchymal marker expression and TGFβ signaling. An experimental model of VTE was used to study the role of HDAC6 on thrombus size overtime. Animals were treated or not with a specific HDAC6 inhibitor (tubastatin A) for 7 to 21 days. Analysis of RNAseq data sets publicly available were used to confirm our main results. Within group and treatment differences were analyzed using two-way ANOVA and Tukey’s multiple comparisons.
Results
Expression of the mesenchymal markers, calponin and transgelin, was increased by TGFβ and thrombin. Interestingly these changes were inhibited in presence of TCS20b. TGFβ mediated these effects through ERK1/2 and HDAC6 activation. Inhibition of HDAC6 in vivo reduced thrombus size 7 days after surgery compared to controls. This was associated with reduced expression of the EndMT marker transgelin in endothelial cells compared to the control animals. We found that FN1-EDA expression was associated with EndMT and regulated by HDAC6 in vitro . This marker was also associated with thrombosis in the RNAseq data set that we analyzed and potentially in patients with recurrent DVT.
Conclusion
We found that HDAC6 regulates EndMT in venous thrombosis and impairs thrombus resolution. HDAC6 also regulates expression FN1-EDA that appears to be a strong marker associated with DVT and DVT recurrence. Thus, HDAC6 might represent an attractive therapeutic target for patients with a high risk of recurrent VTE.
