Venous thromboembolism (VTE), which includes deep vein thrombosis (DVT) and pulmonary embolism (PE), is characterized by thrombus formation and remodeling of the vein wall. Fibrotic remodeling can follow an endothelial-to-mesenchymal transition (EndMT), a process marked by loss of endothelial markers and acquisition of mesenchymal markers. Transforming growth factor beta (TGFβ) is identified as a potent inducer of EndMT and has been implicated in impaired thrombus resolution in chronic thromboembolic pulmonary hypertension. However, mechanisms that modulate TGFβ signaling in endothelial cells during VTE were not well defined prior to this work. The authors proposed that epigenetic modulation—specifically by histone deacetylase 6—could regulate TGFβ-driven EndMT and affect thrombus resolution.
The primary aim was to determine whether histone deacetylase 6 (HDAC6) regulates the TGFβ signaling pathway in endothelial cells, promotes EndMT, and thereby delays venous thrombus resolution. The study used pharmacological inhibition in cell models, an experimental animal model of VTE, and analyses of publicly available RNAseq datasets to address these aims.
In vitro: Endothelial cells were exposed to TGFβ and thrombin for 2, 3, and 5 days to induce EndMT. A pharmacological HDAC6 inhibitor, TCS20b, was used to assess the role of HDAC6. Endothelial and mesenchymal marker expression and components of TGFβ signaling were evaluated by real-time PCR and Western blot.
In vivo: An experimental model of VTE was employed to examine the effect of HDAC6 inhibition on thrombus evolution. Animals were treated or not with the specific HDAC6 inhibitor tubastatin A for periods reported between 7 and 21 days. Thrombus size over time and endothelial expression of EndMT markers were analyzed.
Transcriptomic support: Publicly available RNAseq datasets were analyzed to confirm and extend the principal findings, including associations of specific transcripts with thrombosis and potential links to recurrent DVT.
Statistical analyses for within-group and treatment differences used two-way ANOVA followed by Tukey’s multiple comparisons.
Exposure of endothelial cells to TGFβ and thrombin increased expression of mesenchymal markers, notably calponin and transgelin. These TGFβ- and thrombin-induced increases in mesenchymal marker expression were inhibited when cells were treated with the HDAC6 inhibitor TCS20b, indicating a role for HDAC6 in promoting EndMT in this model.
The mechanistic data reported indicate that TGFβ exerts these effects through activation of ERK1/2 and concurrent activation of HDAC6, linking TGFβ/ERK1/2 signaling to HDAC6-dependent modulation of endothelial phenotype.
Pharmacological inhibition of HDAC6 in the animal VTE model reduced thrombus size seven days after surgery compared with control animals. Treatment durations in the study ranged from 7 to 21 days. Reduction in thrombus size was accompanied by decreased expression of the EndMT marker transgelin in endothelial cells from treated animals relative to controls, supporting an in vivo role for HDAC6 in promoting EndMT and impairing thrombus resolution.
The authors identified the splice isoform FN1-EDA as associated with EndMT and regulated by HDAC6 in vitro. Analysis of public RNAseq datasets showed that FN1-EDA expression was associated with thrombosis in the datasets the authors examined, and the report notes a potential association with recurrent DVT in patient data included in those analyses. These findings link HDAC6 activity to regulation of a matrix-associated isoform implicated in thrombosis and EndMT.
Group and treatment differences were evaluated using two-way ANOVA with Tukey’s multiple comparisons testing, as reported in the study methods. The source document reports these analyses were applied to both in vitro and in vivo experimental comparisons.
The study reports that HDAC6 is a regulator of EndMT in the context of venous thrombosis and that HDAC6 activity impairs thrombus resolution in the experimental model. HDAC6 inhibition reduced mesenchymal marker expression in endothelial cells and decreased thrombus size at the early (7-day) time point. The splice variant FN1-EDA emerged as a marker associated with EndMT and thrombosis and is regulated by HDAC6 in vitro, with supporting associations in analyzed RNAseq data and potential links to recurrent DVT in patients.
Taken together, these preclinical findings support consideration of HDAC6 as a potential therapeutic target for patients at high risk of recurrent VTE. The authors declared funding from Fondation du Souffle and Fédération française de cardiologie and reported no competing interests. The original report is a preprint; additional validation in clinical cohorts and further mechanistic studies would be required before therapeutic translation.