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A4610 - Histone Deacetylase Inhibitors Synergize with Sildenafil to Disrupt Purinergic Signaling and Stop Proliferation of Pulmonary Hypertensive Fibroblasts
Author Block: A. D'Alessandro1, H. Zhang2, M. Li2, J. A. Reisz1, S. Gehrke1, S. Riddle2, M. Akshay2, T. M. Bull2, K. L. Jones1, K. C. El Kasmi2, K. R. Stenmark2; 1Biochemistry and Molecular Genetics, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, United States, 2Developmental Lung Biology and Cardiovascular Pulmonary Research Laboratories, University of Colorado Denver - Anschutz Medical Campus, Aurora, CO, United States.
Rationale Metabolic reprogramming is a key hallmark of pulmonary hypertension (PH) and underlies the hyperproliferative and inflammatory phenotype observed in animal models and clinical patients. While sildenafil treatment disrupts purinergic signaling and mitigates PH, little is known about its effects on metabolism. Indeed, metabolic reprogramming towards glycolysis is observed in all resident cells of the PH vessel wall and recapitulates the Warburg phenotype of proliferating cancer cells, where incomplete glucose oxidation provides carbon backbones for anabolic reactions to sustain cell proliferation.
Objective We hypothesize a synergistic effect between and sildenafil and apicidin (a histone deacetylase-HDAC inhibitor) in reverting the hyperproliferative and metabolic phenotype of human and bovine PH fibroblasts.
Methods RNA sequencing and metabolomics analyses were performed at the steady state level or upon incubation with U-13C-glucose of PH fibroblast from bovine or human pulmonary arteries cultured under normoxia. Metabolomics analyses were performed on plasma of 27 patients suffering from PH – prior to and after treatment with sildenafil. Pharmacological activation of pyruvate kinase M (PKM) isoform 2, or genetic manipulation of upstream regulators of isoform splicing was obtained through silencing of the polypyrimidine-tract binding protein 1 (PTBP1) or transfection of its upstream regulator miR-124. Negative regulation of miR-124 by histone deacetylases was achieved through the administration of two different inhibitors (SAHA or apicidin), alone or in combination of sildenafil.
Results Bovine PH fibroblasts are characterized by transcriptional reprogramming boosting purinergic signaling, which can be targeted by sildenafil. Metabolic reprogramming is observed in human and bovine PH fibroblasts and can only be in part reversed by sildenafil treatment, a finding that was confirmed by metabolomics analyses of plasma of 27 patients following sildenafil administration. Tracing experiments suggested a role for the activation of pentose phosphate pathway and serine anabolism in sustaining nucleoside biosynthesis in bovine and human PH fibroblasts. Pharmacological activation of PKM2 or genetic manipulation of the miR-124/PTBP1 axis, which controls PKM2/PKM1 ratios in cancer and PH, prevented increases in serine and purines observed in untreated PH fibroblasts in comparison to controls. Inhibition of HDAC phenocopied these results and corrected metabolic reprogramming. HDAC inhibitor apicidin was more effective than sildenafil treatment in slowing down cell proliferation of human and bovine PH fibroblasts. Combinatorial treatment with both drugs effectively arrested cell proliferation in human and bovine PH fibroblasts.
Conclusions HDAC inhibitors may be used in combination with sildenafil to disrupt both metabolic reprogramming and purinergic signaling in PH.