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Background Traumatic brain injury (TBI) and hemorrhagic shock (HS) are the leading causes of trauma-related deaths. These insults disrupt coagulation and endothelial systems. This study investigated whether previously reported differences in lesion size and brain swelling during normal saline (NS), colloids (Hextend [HEX]), and fresh frozen plasma (FFP) resuscitation are associated with differential effects on coagulation and endothelial systems. Methods We subjected 15 Yorkshire swine to TBI and HS (40% blood volume), and kept in HS for 2 hours before resuscitation with NS, HEX, or FFP. Markers of endothelial activation (E-selectin, Intercellular adhesion molecule [ICAM]-1), coagulation activation (prothrombin fragment 1 + 2), and natural anticoagulation (activated protein C [aPC]) were determined in serum and brain whole cell lysates. Results Serum levels of aPC were greater in the NS group (203 ± 30 pg/mL) compared with HEX (77 ± 28 pg/mL; P = .02) and FFP (110 ± 28 pg/mL; P = .09), as was PF 1 + 2 in the brain when compared with FFP (PF 1 + 2, 89 ± 46 vs 37 ± 14 ng/mL; P = .035). Brain E-selectin was greater in the NS group compared with FFP (3.36 ± 0.02 vs 3.31 ± 0.01 ng/mL; P = .029). Circulating ICAM-1 levels were increased in the NS group (151 ± 9 ng/mL) compared with the HEX (100 ± 9 ng/mL; P Conclusion In this clinically realistic large animal model of TBI + HS, NS resuscitation was associated with an early activation of coagulation, natural anticoagulation, and endothelial systems, compared with HEX and FFP.

We have recently demonstrated that in a rodent model of lipopolysaccharide (LPS)-induced shock, an increase in circulating citrullinated histone H3 (Cit H3) is associated with lethality of sepsis, and treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor (HDACI), significantly improves survival. However, the role of Cit H3 in pathogenesis and therapeutics of sepsis are largely unknown. The present study was designed to test whether treatment with HDACI could inhibit cellular Cit H3 production, and inhibition of peptidylarginine deiminase (PAD, an enzyme producing Cit H3) with Cl-amidine (PAD inhibitor) or neutralization of blood Cit H3 with anti-Cit H3 antibody could improve survival in a clinically relevant mouse model of cecal ligation and puncture (CLP)-induced septic shock.Three experiments were carried out. In experiment I, HL-60 neutrophilic cells grown on a coverslip were treated with LPS (100 ng/mL) in the presence or absence of SAHA (5 μmol) for 3 hours, and subjected to immunostaining with anti-Cit H3 antibody to assess effect of SAHA on Cit H3 production under a fluorescence microscope. The ratio of Cit H3 positive cells was calculated as mean values ± SD (n = 3). In experiment II, male C57BL/6J mice were subjected to CLP, and 1 hour later randomly divided into 2 groups for intraperitoneal injection as follows: (1) Dimethyl sulfoxide (DMSO), (2) SAHA (50 mg/kg) in DMSO, and (3) Cl-amidine (80 mg/kg) in DMSO (n = 10/group). In experiment III, male C57BL/6J mice were divided into control and treatment groups, and subjected to CLP. Two hours later, immunoglobulin (Ig)G and Cit H3 antibody (20 mg/kg IV; n = 5/group) were injected into the control and treatment groups, respectively. Survival was monitored for ≤10 days.In experiment I, LPS induced Cit H3 production in the HL-60 cells, and SAHA treatment inhibited H3 citrullination significantly (P.05). In experiment II, all vehicle-injected mice died within 3 days with increased circulating Cit H3 levels, whereas treatment with HDACI or Cl-amidine notably improved long-term survival (P.01). In experiment III, administration of IgG did not improve survival, but a single treatment with Cit H3 specific antibody significantly improved survival (P.014).Inhibition of HDAC or PAD significantly suppresses Cit H3 production in vitro and improves survival in vivo. Neutralization of Cit H3 significantly improves survival in septic mice. Collectively, our findings indicate for the first time that Cit H3 could not only serve as a potential biomarker, but also a novel therapeutic target in sepsis.

Background An overproduction of corticosterone during severe sepsis results in increased apoptosis of immune cells, which may result in relative immunosuppression and an impaired ability to fight infections. We have previously demonstrated that administration of tubastatin A, a selective inhibitor of histone deacetylase-6 (HDAC6), improves survival in a lethal model of cecal ligation and puncture (CLP) in mice. The purpose of this study was to characterize the effects of this treatment on sepsis-induced stress responses and immune function. Methods C57BL/6J mice were subjected to CLP, and 1 hour later given an intraperitoneal injection of either tubastatin A dissolved in dimethyl sulfoxide (DMSO), or DMSO only. Blood samples were collected to measure the levels of circulating corticosterone and adrenocorticotropic hormone (ACTH). Thymus and long bones (femur and tibia) were subjected to hematoxylin and eosin staining, and immunohistochemistry was utilized to detect cleaved-caspase 3 in the splenic follicles as a measure of cellular apoptosis. Results All vehicle-treated CLP animals died within 3 days, and displayed increased corticosterone and decreased ACTH levels compared with the sham-operated group. These animals also developed atrophy of thymic cortex with a marked depletion of thymocytes. Tubastatin A treatment significantly attenuated the stress hormone abnormalities. Treated animals also had significantly lower percentages of thymic atrophy (95.0 ± 5.0 vs 42.5 ± 25.3; P = .0366), bone marrow depletion and atrophy (58.3 ± 6.5 vs 25.0 ± 14.4%; P = .0449), and cellular apoptosis in the splenic follicles (41.2 ± 3.7 vs 28.5 ± 4.3 per 40× field; P = .0354). Conclusion Selective inhibition of HDAC6 in this lethal septic model was associated with a significant blunting of the stress responses, with attenuated thymic and bone marrow atrophy, and decreased splenic apoptosis. Our findings identify a novel mechanism behind the survival advantage seen with tubastatin A treatment.

Background Sepsis has a profound impact on the inflammatory and hemostatic systems. In addition to systemic inflammation, it can produce disseminated intravascular coagulation, microvascular thrombosis, consumptive coagulopathy, and multiple organ failure. We have shown that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor (HDACI), improves survival in a lethal model of cecal ligation and puncture (CLP) in mice, but its effect on coagulation remains unknown. The goal of this study was to quantify the impact of SAHA treatment on coagulopathy in sepsis. Methods C57BL/6J mice were subjected to CLP, and 1 hour later given intraperitoneally either SAHA dissolved in dimethyl sulfoxide (DMSO) or DMSO only. Sham-operated animals were handled in similar manner without CLP. Blood samples were collected by cardiac puncture and evaluated using the TEG 5000 Thrombelastograph Hemostasis Analyzer System. Results Compared with the sham group, all animals in DMSO vehicle group died within 72 hours, and developed coagulopathy that manifested as prolonged initial fibrin formation and fibrin cross-linkage time, and decreased clot formation speed, platelet function, and clot rigidity. SAHA treatment significantly improved survival and was associated with improvement in fibrin cross-linkage and clot formation, as well as platelet function and clot rigidity, without a significant impact on the clot initiation parameters. Conclusion SAHA treatment enhances survival and attenuates sepsis-associated coagulopathy by improving fibrin cross-linkage, rate of clot formation, platelet function, and clot strength. HDACI may represent a novel therapeutic strategy for correcting sepsis-associated coagulopathy.

Traumatic brain injury (TBI) and hemorrhagic shock (HS) are major causes of trauma-related deaths and are especially lethal as a combined insult. Previously, we showed that early administration of fresh frozen plasma (FFP) decreased the size of the brain lesion and associated swelling in a swine model of combined TBI+HS. We have also shown separately that addition of valproic acid (VPA) to the resuscitation protocol attenuates inflammatory markers in the brain as well as the degree of TBI. The current study was performed to determine whether a combined FFP+VPA treatment strategy would exert a synergistic effect.Yorkshire swine (42-50 kg) were instrumented to measure hemodynamic parameters, intracranial pressure, and brain tissue oxygenation. TBI was created through a 20-mm craniotomy using a computer-controlled cortical impactor: 15-mm cylindrical tip impactor at 4 m/s velocity, 100 ms dwell time, and 12-mm penetration depth. The TBI was synchronized with the initiation of volume-controlled hemorrhage (40 ± 5% of total blood volume). After a 2-hour period of shock, animals were randomized to 1 of 3 resuscitation groups (n = 5 per group): (1) 0.9% saline (NS); (2) FFP; and (3) FFP and VPA 300 mg/kg (FFP+VPA). The resuscitative volume for FFP was equivalent to the shed blood, whereas NS was 3 times this volume. VPA treatment was started 1 hour after hemorrhage. Animals were monitored for 6 hours post-resuscitation. At this time the brains were harvested, sectioned into 5-mm slices, and stained with 2,3,5-triphenyltetrazolium chloride to quantify the lesion size (mm(3)) and brain swelling (percent change compared with the uninjured side).The combined TBI+HS model resulted in a highly reproducible brain injury. Lesion size and brain swelling (mean value ± standard error of the mean) in the FFP+VPA group (1,459 ± 218 mm(3) and 13 ± 1%, respectively) were less than the NS group (3,285 ± 131 mm(3) [P.001] and 37 ± 2% [P.001], respectively), and the FFP alone group (2,160 ± 203 mm(3) [P.05] and 22 ± 1% [P.001], respectively).In a large animal model of TBI+HS, early treatment with a combination of FFP and VPA decreases the size of brain lesion and the associated swelling.

Background We demonstrated recently that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, improved survival in a rodent model of lipopolysaccharide (LPS)-induced endotoxic shock. The precise mechanisms, however, have not been well-defined. The aim of this study was to investigate the impact of SAHA treatment on gene expression profiles at an early stage of shock. Methods Male C57BL/6J mice were treated with or without SAHA (50 mg/kg, IP), followed by a lethal dose of LPS (20 mg/kg, IP) and a second dose of SAHA. Lungs of the animals (LPS and SAHA+LPS groups; n = 3 per group) were harvested 3 hours post-LPS insult. Sham mice (no LPS and no SAHA) served as controls. RNA was isolated from the tissues and gene expression was analyzed using Affymatrix microarray (23,000 genes). A lower confidence bound of fold change was determined for comparison of LPS versus SAHA + LPS, and genes with a lower confidence bound of >2 were considered to be differentially expressed. Reverse transcriptase polymerase chain reaction, Western blotting, and tissue staining were performed to verify the key changes. Network graphs were used to determine gene interaction and biologic relevance. Results The expression of many genes known to be involved in septic pathophysiology changed after the LPS insult. Interestingly, a number of genes not implicated previously in the septic response were also altered. SAHA treatment attenuated expression of several key genes involved in inflammation. It also decreased neutrophil infiltration in lungs and histologic evidence of acute lung injury. Further analysis confirmed genes engaged in the cellular and humoral arms of the innate immune system were specifically inhibited by SAHA. Gene network analysis identified numerous molecules for the potential development of targeted therapies. Conclusion Administration of SAHA in a rodent model of LPS shock rapidly modulates gene transcription, with an attenuation of inflammatory mediators derived from both arms (cellular and humoral) of the innate immune system. This may be a novel mechanism responsible for the survival advantage seen with SAHA treatment.

Background Acute lung injury (ALI) is a complication of hemorrhagic shock (HS). Histone deacetylase inhibitors, such as valproic acid (VPA), can improve survival after HS; however, their effects on late organ injury are unknown. Herein, we have investigated the effects of HS and VPA treatment on ALI and circulating cytokines that may serve as biomarkers for the development of organ injury. Methods Anesthetized Wistar-Kyoto rats (250–300 g) underwent 40% blood volume hemorrhage over 10 minutes followed by 30 minutes of unresuscitated shock and were treated with either VPA (300 mg/kg) or vehicle control. Blood samples were obtained at baseline, after shock, and before death (at 1, 4, and 20 hours; n = 3–4/timepoint/group). Serum samples were screened for possible biomarkers using a multiplex electrochemiluminescence detection assay, and results were confirmed using enzyme-linked immunosorbent assay (ELISA). In addition, lung tissue lysate was examined for chemokine and myeloperoxidase (MPO) levels as a marker for neutrophil infiltration and ALI. Lung cytokine-induced neutrophil chemoattractant-1 (CINC-1; a chemokine belonging to the interleukin-8 family that promotes neutrophil chemotaxis) mRNA levels were measured by real-time polymerase chain reaction studies. Results Serum screening revealed that hemorrhage rapidly altered levels of circulating CINC-1. ELISA confirmed that CINC-1 protein was significantly elevated in the serum as early as 4 hours and in the lung at 20 hours after hemorrhage, without any significant changes in CINC-1 mRNA expression. Lung MPO levels were also elevated at both 4 and 20 hours after hemorrhage. VPA treatment attenuated these changes. Conclusion Hemorrhage resulted in the development of ALI, which was prevented with VPA treatment. Circulating CINC-1 levels rose rapidly after hemorrhage, and serum CINC-1 levels correlated with lung CINC-1 and MPO levels. This suggests that circulating CINC-1 levels could be used as an early marker for the subsequent development of organ inflammation and injury.

Circulating proteins may serve as biomarkers for the early diagnosis and treatment of shock. We have recently demonstrated that treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, significantly improves survival in a rodent model of lipopolysaccharide (LPS)-induced septic shock. Preliminary proteomic data showed that LPS-induced shock altered a number of proteins in circulation, including histone H3 (H3) and citrullinated histone H3 (Cit H3). The present study was designed to confirm these findings and to test whether the pro-survival phenotype could be detected by an early alteration in serum biomarkers.Three experiments were performed. In experiment I, Western blotting was performed on serum samples from male C57B1/6J mice (n = 9, 3/group) that belonged to the following groups: (a) LPS (20 mg/kg)-induced septic shock, (b) SAHA-treated septic shock, and (c) sham (no LPS, no SAHA). In experiment II, HL-60 granulocytes were cultured and treated with LPS (100 ng/m1) in the absence or presence of SAHA (10 μmol/L). Sham (no LPS, no SAHA) granulocytes served as controls. The medium and cells were harvested at 3 hours, and proteins were measured with Western blots. In experiment III, a large dose (LD, 35 mg/kg) or small dose (SD, 10 mg/kg) of LPS was injected intraperitoneally into the C57B1/6J mice (n = 10 per group). Blood was collected at 3 hours, and serum proteins were determined by Western blots or enzyme-linked immunosorbent assay (ELISA). All of the Western blots were performed with antibodies against H3, Cit H3, and acetylated H3 (Ac H3). ELISA was performed with antibody against tumor necrosis factor (TNF)-α. Survival rates were recorded over 7 days.In experiment I, intraperitoneal (IP) injection of LPS (20 mg/kg) significantly increased serum levels of H3, which was prevented by SAHA treatment. In experiment II, LPS (100 ng/mL) induced expression and secretion of Cit H3 and H3 proteins in neutrophilic HL-60 cells, which was decreased by SAHA treatment. In experiment III, administration of LPS (LD) caused a rise in serum H3 and Cit H3 but not Ac H3 at 3 hours, and all of these animals died within 23 hours (100% mortality). Decreasing the dose of LPS (SD) significantly reduced the mortality rate (10% mortality) as well as the circulating levels of Cit H3 (non detectable) and H3. An increase in serum TNF-α was found in both LPS (LD) and (SD) groups, but in a non-dose-dependent fashion.Our results reveal for the first time that Cit H3 is released into circulation during the early stages of LPS-induced shock. Moreover, serum levels of Cit H3 are significantly associated with severity of LPS-induced shock. Therefore, Cit H3 could serve as a potential protein biomarker for early diagnosis of septic shock, and for predicting its lethality.

Treatment with histone deacetylases inhibitors (HDACi) such as valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) has been shown to improve survival after lethal insults through mechanisms that are incompletely understood. Cell survival under adverse conditions requires a healthy network of capillaries to ensure adequate oxygen delivery. Angiogenic activation of endothelial cells to migrate and form sprouts is associated with characteristic changes in gene expression profiles. Because HDACi can modulate expression of various genes involved in angiogenic activity, we investigated the effect of these agents on capillary-like sprout formation in this study.Human umbilical vein endothelial cells (HUVECs) were cultured as multicellular spheroids within a type I collagen matrix, which promotes formation of sprouts resembling angiogenesis in vitro. HUVECs were cultured as multicellular spheroids within a type I collagen matrix, which promotes formation of sprouts (in vitro angiogenesis). Cells were cultured under the following conditions: Control (no growth factors); VPA (1 mmol/L); vascular endothelial growth factor (VEGF; 10 ng/mL); VPA + VEGF; SAHA (5 mmol/L), and SAHA + VEGF. After 24 hours of treatment, the length of spheroid sprouting and cell migration was assessed quantitatively. The levels of acetylated histone H3, phosphor-extracellular signal-regulated kinase (ERK)1/2, and β-catenin in HUVECs were measured by Western blotting at 6 hours after treatment.High levels of acetylated histone H3 were detected in VPA and SAHA treated-groups. Compared with the VEGF-alone treated group (2379 ± 147.1 μm), the spheroid sprouting was 1.7 times increased with VPA and VEGF combined treatment (3996 ± 192.5 μm; P.01). Cell migrations did not show a significant difference after addition of VPA, whereas SAHA suppressed migration. Expression of β-catenin was significantly increased by VPA and SAHA treatments. Addition of VPA greatly enhanced expression of phosphor-ERK1/2.Exposure of HUVECs to VPA and SAHA increased the expression of β-catenin and enhanced spheroid sprout formation in vitro. Modulation of HDAC-dependent pathways may offer a novel approach to alter angiogenic processes and provide a useful therapeutic target.

Background We have demonstrated that valproic acid (VPA), a histone deacetylase inhibitor, can improve animal survival after hemorrhagic shock and protect neurons from hypoxia-induced apoptosis. This study investigated whether VPA treatment works through the β-catenin survival pathways. Methods Wistar-Kyoto rats underwent hemorrhagic shock (60% blood loss) followed by treatment with or without VPA (300 mg/kg). Brains were harvested after 1, 6, and 24 hours and analyzed for acetylated histone-H3 at lysine-9 (Ac-H3K9), acetylated and total β-catenin, and Bcl-2 by Western blot. In addition, primary neurons dissociated from E18 rat embryos were exposed to hypoxia (0.5% O 2 ) for 16 hours with or without VPA (1 mmol/L) and analyzed using confocal microscopy. Results After treatment of hemorrhaged animals with VPA, acetylated β-catenin was found in both the cytosol and nucleus, along with Ac-H3K9. Bcl-2 transcript increased after 1 hour followed by an increase in Bcl-2 protein at 6 hours. Confocal imaging demonstrated that after VPA treatment, β-catenin translocated into the nucleus and colocalized with Ac-H3K9. Conclusion VPA treatment acetylates H3K9 and β-catenin and enhances translocation of β-catenin into the nucleus, where it colocalizes with Ac-H3K9 and stimulates the transcription of survival gene bcl-2. This finding suggests that VPA protects cells after severe insult through the β-catenin survival pathway.

Background DNA transcription is regulated in part by acetylation of nuclear histones, controlled by 2 groups of enzymes: histone deacetylases (HDAC) and histone acetyl transferases (HAT). We have shown previously that hemorrhage and resuscitation are associated with HDAC/HAT imbalance, which influences the acetylation status of cardiac histones. The goals of this study were to determine whether: (1) resuscitation after hemorrhage affects histone acetylation in a fluid- and organ-specific fashion; and (2) administration of HDAC inhibitors influences histone acetylation and subsequent gene expression. Methods In the first experiment, rats (n = 6/group) were subjected to volume-controlled hemorrhage and resuscitated with: (1) racemic lactated Ringer’s (DL-LR); (2) L-lactated Ringer’s (L-LR); (3) 7.5% hypertonic saline (HTS); (4) ketone Ringer’s (KR); or (5) pyruvate Ringer’s (PR). Control groups included: (6) no hemorrhage (Sham); and (7) hemorrhage with no resuscitation (NR). In the second experiment (n = 5/group), 3 HDAC inhibitors, valproic acid (VPA), trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), were added to normal saline and used as fluid for resuscitation. At the end of resuscitation, lung and liver tissues were subjected to subcellular protein fractionation and Western blotting to analyze histone acetylation. In addition, cDNA microarrays and RT-PCR were used to measure expression of selected genes. Results Hemorrhage did not change the level of histone acetylation in lungs, whereas resuscitation predominantly hyperacetylated histones. An analysis of histone acetylation on 10 lysine sites showed that L-LR, HTS, and KR resuscitation caused the largest number of changes (7, 6, and 6 respectively). SAHA hyperacetylated 7 sites in liver and affected expression of 57 genes (44 up, 13 down). Conclusions Resuscitation with various fluids, as well as infusion of pharmacologic HDAC inhibitors affects histone acetylation in a fluid- and organ-specific fashion, even when administered post-insult for a limited period of time. Uniquely affected genes are associated with metabolism, cellular growth, proliferation, differentiation, transformation, and cellular signaling.