D28-23 E2F Transcription Factor 2 (E2F2) Supports Increased Proliferation in Pulmonary Arterial Smooth Muscle Cells in Pulmonary Arterial Hypertension

Rationale

Pulmonary arterial hypertension (PAH) is a devastating deadly disease. The key pathological process driving progression of PAH is the thickening of PA wall occurring due to hyper-proliferation and survival of vascular cells within a PA wall, mainly smooth muscle cells (PASMC), mechanism of which are still need to be elucidated. Developing of anti-remodeling therapies with anti-proliferative effect by targeting cell cycle regulators is a new frontier in the development of novel PAH therapies. Our recent studies suggest that transcriptional coregulator homeodomain-interacting protein kinase-2 (HIPK2) supports pro-proliferative/pro-survival phenotype of PAH PASMC but the mechanisms of HIPK2-mediated regulation of PASMC increased proliferation in PAH remains unknown.

Methods/Results

To identify potential signaling pathways which mediate HIPK2 regulation of hyper-proliferation of PAH PASMC, we performed RNAseq analysis of human PAH PASMCs treated with HIPK2 selective ATP- competitive inhibitor tBID. We identified that HIPK2 inhibition led to gene expression downregulation of ten transcription factors in PAH PASMCs, with top two decreased being MYBL2 and E2F2, but only E2F2 was also decreased on the protein level as confirmed by immunoblot. Importantly, RNAseq, analysis of publicly available dataset GSE144274, and immunoblot analysis demonstrated that E2F2 is upregulated in primary PASMCs from PAH patients compared to non-diseased subjects, both on mRNA and protein level. Notably, since the E2F2 is well known regulator of G1- to S phase cell-cycle progression and binds DNA in a complex with TFDP factors to activate expression of growth-related genes, we also analyzed TFDP1 gene expression and found upregulation of TFDP1 in RNAseq data set and in the publicly available dataset GSE144274 in primary PASMCs from PAH patients compared to non-diseased subjects suggesting that E2F2/TFDP1 complex is activated in PAH PASMCs. We also found that mechanism of E2F2 up-regulation in PAH PASMCs is independent from pro-proliferative Akt kinase and pro-proliferative transcriptional co-activator YAP as was shown by siRNA- and inhibitor-based studies. Importantly, siRNA-mediated downregulation of E2F2 significantly decreased proliferation (BrdU incorporation) in hyperproliferative PAH PASMCs.

Conclusions

E2F2 factor is upregulated in human PAH PASMCs, acts downstream of HIPK2 by YAP- and AKT-independent mechanism and supports pro-proliferative phenotype of PASMCs in human PAH. Further studies are needed to identify the mechanisms of E2F2 activation in PAH PASMCs, evaluate its role in pulmonary vascular remodeling and experimental PH in vivo, and investigate the possibility of targeting E2F2 or its upstream signaling pathways as a novel anti-remodeling therapeutic approach for PAH.

This abstract is funded by: NIH/NHLBI R01HL166932