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A2065 - The Molecular Mechanisms of Persistent Inflammatory Cellular Phenotypes in PH, Roles of Chromatin Structure, Multiple Transcription Factors, and Epigenetic Regulators
Author Block: C. Hu1, H. Zhang2, A. Laux1, A. Flockton2, M. Li2, K. R. Stenmark2; 1Craniofacial Biology, University of Colorado Denver, Aurora, CO, United States, 2University of Colorado Denver, AURORA, CO, United States.
Rationale: Pulmonary artery adventitial inflammation, evidenced by persistent activation of Fibs, increased activation and recruitment of resident/recruited macrophages, markedly increased levels of pro-inflammatory mediators (MCP1, CXCL12, GM-CSF, IL6, and VCAM1), is consistently observed throughout the PH process including late stage (out of proportion) PH. This chronic, non-resolving inflammation is essential in PH disease maintenance. In a companion Abstract (Zhang et al), we present evidence that implicates inflammatory TFs (HIF, STAT3, and NF-KB) and epigenetic regulators (HDAC, HAT, lysine acetylation readers BRDs), in sustaining pro-inflammatory gene expression in PH cells. Further, we show that BRD inhibitors are more potent and more specific in normalizing pro-inflammatory genes in PH cells, than inhibition of individual TF (HIF, STAT3, or NF-KB) or HADC or p300/CBP. Importantly, the Bonnet group reports that BRD inhibitor is effective in reversing established PH in vivo. However, the molecular mechanisms concerning why inhibition of BRDs, a family of general epigenetic readers, only target a specific set of genes in PH cells, are largely unknown. Clinical application for BRD inhibitor in PH, without a sound biological basis, is dangerous and will likely to lead to adverse events. Thus, the goal of this study is to elucidate the mechanisms concerning how TFs, epigenetic regulators, and distinct chromatin interact to control inflammatory gene expression in established PH.
Methods: Human pulmonary artery fibroblasts were derived from PAH patients or from control donors. Chromatin-immunoprecipitation (ChIP) and q-PCR assays were performed, on two pro-inflammatory gene (MCP1 and CXCL12) promoters/enhancers in both control and PH-Fibs to compare 1) the binding of pro-inflammatory TFs (HIF1, NF-KB, STAT3); 2) the chromatin structure changes by probing markers for active enhancer (H3K27Ac, H3K4Me1); 3) the binding of epigenetic regulators BRD4 and P300.
Results: Compared with control Fibs, regulatory regions of MCP1 and CXCL12 genes in PH-Fibs exhibit: 1) increased binding of inflammatory TFs (HIF1, NF-KB, STAT3); 2) more “open” chromatin structure as evidenced by increased levels of active enhancer markers (H3K27Ac and H3K4Me1), at larger/extended regulatory regions; and 3) increased binding of epigenetic regulators BRD4 and P300.
Conclusion: These data support the hypothesis that persistent expression of pro-inflammatory genes in PH-Fibs is maintained by enhancers that exhibit high binding densities of inflammatory TFs and epigenetic regulators (P300 and BRDs). Further experiments are needed to determine the interplay between TFs, chromatin, and epigenetic regulators in the contributing to perpetuation of functional abnormality of cells in chronic PH.