Downloaded from https://journals.lww.com/jtrauma by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3n4/ufkX3x2fNokYq91MxH/qukLsurVt4psWJZrttSumP18ldoPnO1w== on 07/14/2018 Valproic acid induces prosurvival transcriptomic changes in swine subjected to traumatic injury and hemorrhagic shock Patrick E. Georgoff, MD, Vahagn C. Nikolian, MD, Gerald Higgins, MD, PhD, Kiril Chtraklin, DVM, Hassan Eidy, Mohamed H. Ghandour, Aaron Williams, MD, Brian Athey, PhD, and Hasan B. Alam, MD, Ann Arbor, Michigan BACKGROUND: Valproic acid (VPA) is a histone deacetylase inhibitor that improves outcomes in large animal models of trauma. However, its protective mechanism of action is not completely understood. We sought to characterize the genetic changes induced by VPA treatment following traumatic injuries. METHODS: Six female Yorkshire swine were subjected to traumatic brain injury (controlled cortical impact), polytrauma (liver and splenic laceration, rib fracture, rectus crush), and hemorrhagic shock (HS, 40% total blood volume). Following 2 hours of HS, animals were randomized to resuscitation with normal saline (NS) or NS + 150 mg/kg of intravenous VPA (n = 3/cohort, 18 samples total). Blood samples were col- lected for isolation of peripheral blood mononuclear cells at three distinct time points: baseline, 6 hours following injuries, and on postinjury day 1. RNA was extracted from peripheral blood mononuclear cells and sequenced. Differential expression analysis (false discovery rate < 0.001 and p value <0.001) and gene set enrichment (Panther Gene Ontology and Ingenuity Pathway Analysis) was used to compare VPA to non–VPA-treated animals. RESULTS: A total of 628 differentially expressed RNA transcripts were identified, 412 of which were used for analysis. There was no difference between treatment groups at baseline. The VPA-induced genetic changes were similar at 6 hours and on postinjury day 1. Upregulated genes were as- sociated with gene expression ( p 2.13E-34), cellular development (1.19E-33), cellular growth and proliferation (1.25E-30), and glucocorticoid receptor signaling (8.6E-21). Downregulated genes were associated with cell cycle checkpoint regulation (3.64E-22), apoptosis signaling (6.54E-21), acute phase response signaling (5.84E-23), and the inflammasome pathway (1.7E-19). CONCLUSION: In injured swine, VPA increases the expression of genes associated with cell survival, proliferation, and differentiation and decreases those associated with cell death and inflammation. These genetic changes could explain the superior clinical outcomes in VPA-treated animals, including smaller brain lesion size and improved neurologic recovery. (J Trauma Acute Care Surg. 2018;84: 642–649. Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.) KEY WORDS: Valproic acid; histone deacetylase inhibitor; genomics; RNA sequencing; trauma; hemorrhage; traumatic brain injury. V alproic acid (VPA) is a nonspecific histone deacetylase (HDAC) inhibitor that was originally approved for the treat- ment of epilepsy in 1978. 1 Valproic acid has since been used to treat non-neurologic diseases. In addition to VPA's antiepileptic effects, including gamma-aminobutyric acidergic potentiation, glutamate and N-methyl-D-aspartate receptor inhibition, and block- age of voltage-gated sodium channels, HDAC inhibitors, like VPA, are thought to selectively increase acetylation of key proteins, including histones. 2 In doing so, VPA augments gene expres- sion through conformational changes in chromatin. 3 These pow- erful epigenetic effects have been shown to be beneficial in a number of pathological conditions, including traumatic injury. 4 In swine subjected to hemorrhage and polytrauma, treatment with a single large dose of VPA decreases mortality by 50% and does so in the absence of isotonic fluid resuscitation. 5,6 Improved out- comes have been validated in animal models of stroke, 7,8 spinal cord injury, 9,10 hemorrhagic shock with sepsis, 11–13 and hemor- rhagic shock with traumatic brain injury (TBI). 14–16 The therapeutic properties of VPA are being studied in a variety of other conditions, most notably cancer. 17 However, VPA's paradoxical action in different disease states underscores our lim- ited understanding of how HDAC inhibitors work. When used as an antineoplastic agent, VPA facilitates cancer cell death, 18 but when used following traumatic injury, VPA enhances cell differ- entiation and proliferation. 4 Studies have also demonstrated the disease and cell-specific effects of HDAC inhibitors like VPA, which target injured and diseased cells but not healthy ones. 19–22 These unique properties may be explained by the differential reg- ulation of key genes and proteins. In swine subjected to TBI and hemorrhagic shock, VPA increases the expression of genes that enhance neurogenesis, including MEF2D, MYT1L, NEUROD1, and PAX6. 23 Similarly, in healthy humans a single dose of intra- venous VPA upregulates proteins in peripheral blood mononu- clear cell (PBMC) that control cellular differentiation and proliferation but downregulate proteins involved in apoptosis. 24 The aim of this study was to better characterize the genetic changes induced by VPA treatment following multiple injuries. To do this, we used RNA sequencing to examine PBMCs Submitted: August 27, 2017, Revised: October 2, 2017, Accepted: November 21, 2017, Published online: December 15, 2017. From the Department of Surgery (P.E.G., V.C.N., K.C., H.E., M.H.G., A.W., H.B.A.), and Department of Computational Medicine & Bioinformatics (G.H., B.A.), Uni- versity of Michigan, Ann Arbor, Michigan Address for reprints: Hasan B. Alam, MD, 2920 Taubman Center/5331 University of Michigan Hospital 1500 E. Medical Center Drive Ann Arbor, MI 48109-5331; email: alamh@med.umich.edu. Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jtrauma.com). DOI: 10.1097/TA.0000000000001763 ORIGINAL ARTICLE 642 J Trauma Acute Care Surg Volume 84, Number 4 Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.