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A4611 - Targeting Epigenetic Regulators Is More Effective than Targeting Individual Transcription Factors in Normalizing the Persistent Activation of Vascular Cells in Pulmonary Hypertension
Author Block: H. Zhang1, A. Laux2, A. Flockton1, M. Li1, C. Hu2, K. R. Stenmark1; 1University of Colorado Denver, Aurora, CO, United States, 2Craniofacial Biology, University of Colorado Denver, Aurora, CO, United States.
Rationale: Though pulmonary hypertension (PH) can be induced by a variety different stresses/stimuli, strikingly, most forms of PH share many common characteristics including persistent activation of pulmonary vascular cells and vascular remodeling. Extensive research has also established several common cellular phenotypes in PH including hyper-proliferation, apoptosis-resistance and increased production of proinflammatory proteins. Studies have established the critical role of several stress transcription factors (Nf-kb, STAT3, HIF and others) and epigenetic factors such as histone deacetylase (HDAC), histone acetyltransferases (HAT) and histone acetylation reader (BRD) in PH initiation/development. However, the role of these stress TFs and epigenetic regulators in maintaining PH cellular phenotypes are largely unknown. Answering such questions can unravel novel pathways amenable to therapeutic intervention in PH.
Methods: Human pulmonary artery fibroblasts and SMCs were derived from patients with IPAH or from control donors. Cells were assessed for RNA and protein levels of several stress TFs and epigenetic regulators. Cells were treated with HIF1α siRNA, HIF2α siRNA, both HIF1α and HIF2a siRNAs, BAY (Nf-Kb inhibitor), Stattic (STAT3 inhibitor), SAHA and Apicidin (HDACs inhibitor), C646 (histone acetyltransferase p300/CBP inhibitor), and (+)-JQ1 (BRDs inhibitor). Pro-inflammatory genes were evaluated using RT-PCR. Cell proliferation was evaluated using CyQUANT proliferation analysis Kit.
Results: Increased expression of pro-inflammatory genes (e.g. CXCL12/SDF-1, CCL2/MCP-1) in PH-Fibs and PH-SMCs are persistent in vitro, though expression of each gene was far greater in Fibs than SMCs. Furthermore, we detected constitutive activation of multiple stress TFs (Nf-kb, STAT3 and HIF) and increased expression of epigenetic regulators (HDACs, P300 and BRDs). Inhibition of NF-Kb, STAT3, HIF1α or/and HIF2α activity, individually exerted only minimal effects on transcription of inflammatory genes (e.g. CXCL12/SDF-1, CCL2/MCP-1) in PH cells, while inhibition of HDACs, P300/CBP or BDRs activity almost completely decreased the gene expression profile to that of control cells. Importantly, the BRD4 inhibitor JQ1 decreases expression only of genes that are abnormally expressed in PH-vascular cells, while the HDAC and p300/CBP inhibitors alter expression of additional genes. Additionally, we observed that BRD inhibitor JQ1 decreased the proliferation of PH-Fibs and PH-SMCs in a dose dependent manner without cell toxicity, while p300/CBP inhibitor is toxic for cells (SMCs particularly).
Conclusion: Targeting epigenetic regulators, but not individual TF is more effective in normalizing persistently activated PH vascular cell phenotype. JQ1, a clinically available inhibitor of BRDs, exhibits a greater potential for specificity to reverse the persistent activation of pulmonary vascular cells, which could offer new therapeutic approaches.