Histone Cytotoxicity Research Articles

PSLBI-15 Effects of heat shock protein 70 on cell viability and protective capacity of serum against histone cytotoxicity

Abstract Bovine respiratory disease (BRD) is the most significant health problem associated with newly received cattle in the beef industry. Serum concentrations of extracellular histones increase in a variety of diseases and histones are known to be cytotoxic to mammalian cells. Work in our lab indicated that failure to protect against cytotoxic effects of extracellular histones may be a component of severe cases of respiratory disease in cattle. The mechanisms or proteins that neutralize cytotoxic effects of extracellular histones are not fully elucidated, but C-reactive protein and complement activity have been associated with a reduced cytotoxic effect of extracellular histones. Work in the literature suggests that heat shock protein 70 (HSP70) concentrations in the serum is increased in disease and may directly activate complement. Unpublished observations in our lab indicated that HSP70 potentially interacts with histones to exacerbate cytotoxicity leading to our hypothesis that increased concentrations of HSP70 will decrease the protective capacity against histone cytotoxicity. Therefore, the objectives of the current study were to evaluate serum concentrations of HSP70 in weaned bulls entering the feedlot and the effects of increasing HSP70 concentrations on histone cytotoxicity in vitro. Weaned seedstock bulls (n = 130) composed of various breeds were fed using the GrowSafe System at the Tucumcari Bull Test for 122 d. At receiving, blood samples were obtained via coccygeal venipuncture, centrifuged, and serum immediately frozen on dry ice. Serum was analyzed for complement activity (CH50 assay) and protective capacity against extracellular histones using our novel histone toxicity assay. The ratio value of histone toxicity to CH50 was used to separate a subset of bulls as protective (n = 10) and non-protective (n = 10). Concentrations of HSP70 were quantified utilizing a commercially available ELISA kit. Data were analyzed using the GLM procedure of SAS. Serum concentrations of HSP70 were similar (P > 0.10) for protective and non-protective bulls. To test the responses to increased concentrations of HSP70 to mimic disease, protective and non-protective serum was tested against histones alone and in combination with 0.5 or 40 ng/mL recombinant bovine HSP70. Complement activity from protective and non-protective bulls was not influenced (P > 0.10) in the presence and absence of recombinant bovine HSP70. Exogenous HSP70 at 0.5 or 40 ng/mL did not influence protective capacity in non-protective animals (P = 0.95). However, HSP70 at 0.5 ng/mL did not (P = 0.18) reduce protective capacity and reduced protective capacity (P < 0.01) at 40 ng/mL in serum from bulls that were assigned to the protective group. Results indicate that weaned bulls entering the feedlot display varied capacity to protect against histone toxicity and increased concentrations of HSP70 can reduce the protective capacity indicating that circulating HSP70 may play a role in bovine respiratory disease pathogenesis.

Design and characterization of novel activated protein C variants for the proteolysis of cytotoxic extracellular histone H3

BackgroundExtracellular histone H3 is implicated in several pathologies including inflammation, cell death, and organ failure. Neutralization of histone H3 is a strategy that was shown beneficial in various diseases, such as rheumatoid arthritis, myocardial infarction, and sepsis. It was shown that activated protein C (APC) can cleave histone H3, which reduces histone cytotoxicity. However, due to the anticoagulant properties of APC, the use of APC is not optimal for the treatment of histone-mediated cytotoxicity, in view of its associated bleeding side effects. ObjectivesThis study aimed to investigate the detailed molecular interactions between human APC and human histone H3, and subsequently use molecular docking and molecular dynamics simulation methods to identify key interacting residues that mediate the interaction between APC and histone H3 and to generate novel optimized APC variants. MethodsAfter molecular simulations, the designed APC variants 3D2D-APC (Lys37-39Asp and Lys62-63Asp) and 3D2D2A-APC (Lys37-39Asp, Lys62-63Asp, and Arg74-75Ala) were recombinantly expressed and their abilities to function as anticoagulant, to bind histones, and to cleave histones were tested and correlated with their cytoprotective properties. ResultsCompared with wild type-APC, both the 3D2D-APC and 3D2D2A-APC variants showed a significantly decreased anticoagulant activity, increased binding to histone H3, and similar ability to proteolyze histone H3. ConclusionsOur data show that it is possible to rationally design APC variants that may be further developed into therapeutic biologicals to treat histone-mediated disease, by proteolytic reduction of histone-associated cytotoxic properties that do not induce an increased bleeding risk.

Unfractionated Heparin Protects Microcirculation in Endotoxemic Rats by Antagonizing Histones

IntroductionLevels of extracellular histones are highly increased in sepsis and may facilitate microcirculatory dysfunction. Unfractionated heparin (UFH) binds histones and neutralizes their cytotoxicity. We investigated the effect of UFH on microcirculatory dysfunction by interacting with extracellular histones in endotoxemic rats. MethodsTwenty-four Wistar rats were randomly divided into three groups: control, lipopolysaccharide (LPS) group, and LPS + UFH group. In the LPS and LPS + UFH groups, 10 mg/kg LPS was injected to induce endotoxemia, and 100 IU/kg/h UFH was administered intravenously in the LPS + UFH group. The rats underwent midline laparotomy, and then intestinal microcirculation was evaluated using an incident dark field microscope. Circulating histones and microstructures of the rat intestinal microvascular endothelium were also detected. Additionally, the antagonistic effect of UFH on histone-induced cytotoxicity was investigated in human intestinal microvascular endothelial cells. ResultsUFH protected the microcirculation of the intestinal serosa and mucosa in endotoxemic rats, as evidenced by increased total vessel density, perfused vessel density, and proportion of perfused vessels of both the serosa and mucosa, and increased microcirculatory flow index of the mucosa in the LPS + UFH group. UFH treatment decreased the levels of circulating histones and alleviated intestinal microvascular endothelial injuries in endotoxemic rats. Furthermore, UFH inhibited histone cytotoxicity in vitro. ConclusionsUFH attenuated microcirculatory dysfunction in endotoxemic rats by antagonizing extracellular histones, thereby providing a potential therapeutic strategy for sepsis.

Factor VII activating protease (FSAP) inhibits the outcome of ischemic stroke in mouse models.

The outcome of ischemic stroke can be improved by further refinements of thrombolysis and reperfusion strategies. Factor VII activating protease (FSAP) is a circulating serine protease that could be important in this context. Its levels are raised in patients as well as mice after stroke and a single nucleotide polymorphism (SNP) in the coding sequence, which results in an inactive enzyme, is linked to an increased risk of stroke. In vitro, FSAP cleaves fibrinogen to promote fibrinolysis, activates protease-activated receptors, and decreases the cellular cytotoxicity of histones. Based on these facts, we hypothesized that FSAP can be used as a treatment for ischemic stroke. A combination of tissue plasminogen activator (tPA), a thrombolytic drug, and recombinant serine protease domain of FSAP (FSAP-SPD) improved regional cerebral perfusion and neurological outcome and reduced infarct size in a mouse model of thromboembolic stroke. FSAP-SPD also improved stroke outcomes and diminished the negative consequences of co-treatment with tPA in the transient middle cerebral artery occlusion model of stroke without altering cerebral perfusion. The inactive MI-isoform of FSAP had no impact in either model. FSAP enhanced the lysis of blood clots in vitro, but in the tail transection model of hemostasis, FSAP-SPD treatment provoked a faster clotting time indicating that it also has pro-coagulant actions. Thus, apart from enhancing thrombolysis, FSAP has multiple effects on stroke progression and represents a promising novel therapeutic strategy in the treatment of ischemic stroke.

Fibrinogen protects neutrophils from the cytotoxic effects of histones and delays neutrophil extracellular trap formation induced by ionomycin

Neutrophils are pivotal players in immune defence which includes a process of release of histones and DNA as neutrophil extracellular traps (NETs). Histones, while toxic to invading pathogens, also kill host cells, including neutrophils. Bacteria have evolved mechanisms to escape neutrophils, including the secretion of leucocidins (e.g. ionomycin). Live cell video microscopy showed how fibrinogen and fibrin influence NETosis and neutrophil responses to extracellular histones. Histones were rapidly lethal to neutrophils after binding to cells, but formation of fibrinogen/fibrin-histone aggregates prevented cell death. Histone cytotoxicity was also reduced by citrullination by peptidyl arginine deiminase 4, or digestion by serine proteases. Ionomycin and phorbol 12-myristate 13 acetate (PMA) are used to trigger NETosis. Fibrinogen was responsible for a second distinct mechanism of neutrophil protection after treatment with ionomycin. Fibrinogen clustered on the surface of ionomycin-stimulated neutrophils to delay NETosis; and blocking the β integrin receptor, αMβ2, abolished fibrinogen protection. Fibrinogen did not bind to or protect neutrophils stimulated with PMA. Fibrinogen is an acute phase protein that will protect exposed cells from damaging circulating histones or leucocidins; but fibrinogen depletion/consumption, as in trauma or sepsis will reduce protection. It is necessary to consider the role of fibrinogen in NETosis.

Histones, DNA, and Citrullination Promote Neutrophil Extracellular Trap Inflammation by Regulating the Localization and Activation of TLR4

Neutrophil extracellular traps (NETs) promote atherosclerosis by inducing proinflammatory cytokines, but the underlying mechanism remains unknown. NET DNA is immunogenic, but given the cytotoxicity of NET histones, it is unclear how it activates cells without killing them. Here, we show that histones, DNA, citrullination, and fragmentation synergize to drive inflammation below the histone cytotoxicity threshold. At low concentrations, nucleosomes induce cytokines, but high concentrations kill cells before cytokines are produced. The synergy between histones and DNA is critical for sub-lethal signaling and relies on distinct roles for histones and DNA. Histones bind and activate TLR4, whereas DNA recruits TLR4 to histone-containing endosomes. Citrullination is dispensable for NETosis but potentiates histone-mediated signaling. Consistently, chromatin blockade or PAD4 deficiency reduces atherosclerosis. Inflammation is also reduced in infected mice expressing GFP-tagged histones that block TLR4 binding. Thus, chromatin promotes inflammation in sterile disease and infection via synergistic mechanisms that use signals with distinct functions.

PSII-29 Identification of serum proteins that interact with extracellular histones in feedlot cattle

Abstract Bovine respiratory disease complex remains the largest challenge facing the cattle industry. Extracellular histones, which are released during disease, have a pathological role in death associated with acute sepsis or respiratory distress syndrome. Treatment against histone cytotoxicity improves survivability of mice suffering from sepsis and respiratory disease. Previously, we demonstrated calves with severe or lethal cases of bovine respiratory disease had an impaired capacity to protect against histone cytotoxicity. However, the mechanism or component of the serum which modulates histone cytotoxicity is unclear. Therefore, the objective of the current study was to identify components in serum which interact with extracellular histones. To achieve this objective serum proteins that interact with histones were precipitated by the addition and incubation of exogenous histones (2.5μg/μL; 1 h; 23°C). Serum protective capacity was determined pre- and post-precipitation, using our novel in-vitro assay and cell viability was estimated using resazurin. Fluorescent values were analyzed using GLM procedure of SAS and means were separated using PDIFF function. Fluorescent values decreased (P < 0.05) after precipitation, indicating interacting proteins were effectively precipitated. Protein from the precipitate was then separated using SDS-PAGE. Proteomes, excluding IgG and BSA, were analyzed by LC-MS/MS using a label-free data-dependent “shotgun” mass spectrometry approach. Proteins were identified and quantified by searching UniProtKB database (restricted to Bos taurus; 32,967 entries) using Andromeda search engine in MaxQuant. Seven of the top twenty candidate proteins identified were associated with the complement system. Results in the literature suggest that histones play a role in controlling activation of the complement system through binding of complement associated proteins. Results from the current study extend those findings and suggest the complement system may interact with extracellular histones to minimize histone cytotoxicity, indicating aberrant regulation of the complement system or extracellular histones can lead to increased tissue damage during disease.

Individual Impact of Distinct Polysialic Acid Chain Lengths on the Cytotoxicity of Histone H1, H2A, H2B, H3 and H4.

Neutrophils are able to neutralize pathogens by phagocytosis, by the release of antimicrobial components, as well as by the formation of neutrophil extracellular traps (NETs). The latter possibility is a DNA-meshwork mainly consisting of highly concentrated extracellular histones, which are not only toxic for pathogens, but also for endogenous cells triggering several diseases. To reduce the negative outcomes initiated by extracellular histones, different approaches like antibodies against histones, proteases, and the polysaccharide polysialic acid (polySia) were discussed. We examined whether each of the individual histones is a binding partner of polySia, and analyzed their respective cytotoxicity in the presence of this linear homopolymer. Interestingly, all of the histones (H1, H2A, H2B, H3, and H4) seem to interact with α2,8-linked sialic acids. However, we observed strong differences regarding the required chain length of polySia to bind histone H1, H2A, H2B, H3, and H4. Moreover, distinct degrees of polymerization were necessary to act as a cytoprotective agent in the presence of the individual histones. In sum, the outlined results described polySia-based strategies to bind and/or to reduce the cytotoxicity of individual histones using distinct polySia chain length settings.

Circulating Heparan Sulfate Fragments Attenuate Histone-Induced Lung Injury Independently of Histone Binding.

Extracellular histones are cationic damage-associated molecular pattern molecules capable of directly inducing cellular injury via charge-mediated interactions with plasma membranes. Accordingly, histones released into the plasma during critical illness are known to contribute to the onset and propagation of lung injury. Vascular injury (with consequent degradation of the endothelial glycocalyx) simultaneously releases anionic heparan sulfate fragments (hexa- to octasaccharides in size) into the plasma. It is unknown whether this endogenous release of heparan sulfate fragments modulates charge-dependent histone cytotoxicity, or if exogenous heparan sulfate fragments could therapeutically attenuate histone-induced lung injury. Using isothermic calorimetry, we found that extracellular histones only bind to heparan sulfate fragments ≥ 10 saccharides in size, suggesting that glycocalyx-derived heparan sulfate hexa/octasaccharides are incapable of intercepting/neutralizing circulating histones. However, we found that even heparan sulfate fragments incapable of histone binding (e.g., tetrasaccharides) attenuated histone-induced lung injury in vivo, suggesting a direct, size-independent protective effect of heparan sulfate. We found that histones had no effect on human neutrophils ex vivo but exerted toll-like receptor-independent cytotoxicity on human pulmonary microvascular endothelial cells in vitro. This cytotoxicity could be prevented by either the addition of negatively charged (i.e., highly sulfated) heparan sulfate tetrasaccharides (incapable of binding histones) or decasaccharides (capable of binding histones). Taken together, our findings suggest that heparan sulfate oligosaccharides may directly exert pulmonary endothelial-protective effects that attenuate histone-mediated lung injury.

DNA and factor VII-activating protease protect against the cytotoxicity of histones.

Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes; however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize histones are present in plasma. Importantly, factor VII-activating protease (FSAP) is activated upon contact with histones and subsequently proteolyzes histones. We aimed to determine the effect of FSAP on the cytotoxicity of both histones and nucleosomes. Indeed, FSAP protected against histone-induced cytotoxicity of cultured cells in vitro. Upon incubation of serum with histones, endogenous FSAP was activated and degraded histones, which also prevented cytotoxicity. Notably, histones as part of nucleosome complexes were not cytotoxic, whereas DNA digestion restored cytotoxicity. Histones in nucleosomes were inefficiently cleaved by FSAP, which resulted in limited cleavage of histone H3 and removal of the N-terminal tail. The specific isolation of either circulating nucleosomes or free histones from sera of Escherichia coli challenged baboons or patients with meningococcal sepsis revealed that histone H3 was present in the form of nucleosomes, whereas free histone H3 was not detected. All samples showed signs of FSAP activation. Markedly, we observed that all histone H3 in nucleosomes from the patients with sepsis, and most histone H3 from the baboons, was N-terminally truncated, giving rise to a similarly sized protein fragment as through cleavage by FSAP. Taken together, our results suggest that DNA and FSAP jointly limit histone cytotoxicity and that free histone H3 does not circulate in appreciable concentrations in sepsis.

Uterine Histone Secretion Likely Fosters Early Embryo Development So Efforts to Mitigate Histone Cytotoxicity Should Be Cautious.

Uterine Histone Secretion Likely Fosters Early Embryo Development So Efforts to Mitigate Histone Cytotoxicity Should Be Cautious.

Interaction of factor VII activating protease (FSAP) with neutrophil extracellular traps (NETs)

The circulating zymogen form of Factor VII activating protease (FSAP) can be activated by histones and nucleosomes in vivo. These cell-death-associated nuclear factors are also actively extruded into the extracellular space by neutrophils through a process called neutrophil extracellular trap (NET) formation (NETosis). NETs are thought to be involved in host defense, inflammation as well as thrombosis. We have investigated the bidirectional interactions of FSAP and NETs. Phorbol ester-mediated NET formation was marginally stimulated by FSAP. Plasma-derived FSAP as well as exogenous FSAP bound to NETs. There was co-localization of FSAP and NETs in coronary thrombi from patients with acute myocardial infarction. Contrary to our expectations no activation of pro-FSAP by NETs was evident. However, after disintegration of NETs with DNase, a robust activation of pro-FSAP, due to release of histones from nucleosomes, was detected. The released histones were in turn degraded by FSAP. Histone cytotoxicity towards endothelial cells was neutralized by FSAP more potently than by activated protein C (APC). One more consequence of histone degradation was a decrease in nucleosome release from apoptotic neutrophils. Taken together, NETs bind to FSAP, but do not activate pro-FSAP unless histones are released from NETs by DNAse. This activation of FSAP is likely to be important in diminishing the cytotoxic effect of histones, thus limiting the damaging effect of NETosis.

Factor VII-Activating Protease Is a Regulator of Histone Cytotoxicity

Background: During sepsis, ischemia-reperfusion injury and sterile inflammation, levels of circulating histones and nucleosomes have been found to correlate with disease severity. Although the pathophysiological effects of circulating nucleosomes are not fully understood, free histones have been described to mediate cytotoxicity and contribute to disease severity. Several plasma proteins have been identified that modulate these cytotoxic effects by neutralizing or degrading histones. We have previously described that the plasma serine protease Factor VII-activating protease (FSAP) is activated upon contact with late apoptotic or necrotic cells and induces release of nucleosomes from these cells. During this release, histone H1 is cleaved. In addition, it has been described that FSAP is (auto)activated upon contact with histones and subsequently degrades histone H3.Aims: To investigate the regulation of histone-induced cytotoxicity by FSAP. Moreover, we aimed to compare differences in the cytotoxicity of histones and nucleosomes (histone+DNA) and potential differences in their proteolysis by FSAP.Methods: To determine FSAP activation and proteolysis of histones in serum we incubated normal donor or FSAP-deficient serum with 100ug/ml calf thymus histones. Upon its activation FSAP is rapidly bound by plasma inhibitors and we measured complexes of FSAP and its plasma inhibitor alpha 2-antiplasmin as an indirect readout for FSAP activation. To detect FSAP mediated proteolysis of histones we performed immunoblotting for histone H3. Results were corroborated using plasma-purified or recombinant FSAP. To determine histone-mediated cytotoxicity, we measured LDH levels after overnight incubation of HEK293 or ECRF cells with 100ug/ml calf thymus histone that had undergone a pre-incubation step in the presence or absence of healthy donor serum, FSAP-deficient serum or FSAP-depleted serum. Moreover, we tested the cytotoxicity of purified nucleosomes that were pre-incubated in the presence or absence of benzonase to degrade DNA.Results: EndogenousFSAP in serum was activated upon contact with histones, and histone proteolysis was observed. Similarly, histones were proteolyzed upon incubation using active plasma-purified or pre-activated recombinant FSAP, whereas no histone proteolysis was observed using recombinant inactive FSAP. These results indicate that FSAP is responsible for the observed histone degradation. FSAP-mediated histone degradation was able to protect HEK293 and ECRF cells against histone-induced cytotoxicity, showing that the resultant histone breakdown products had lost their cytotoxic effects. Recombinant inactive FSAP did not affect histone-induced cytotoxicity, indicating that the catalytic activity of FSAP is required for the observed protective effect of FSAP on cytotoxicity. When FSAP-deficient or FSAP-depleted serum was used, this led to greatly diminished histone proteolysis and reduced histone-induced cytotoxicity. Using our assay, we found that nucleosomes did not show cytotoxic effects unless they had been pre-incubated with benzonase to release histones. Notably, whereas intact nucleosomes were resistant against degradation by FSAP, FSAP protected against the cytotoxicity induced by histones released from these nucleosomes.Conclusion: FSAP in serum is activated by histones and subsequently proteolyses histones ,resulting in protection of cells from histone-induced cytotoxicity. Histones in the form of nucleosomes did not induce cytotoxicity, however after DNA removal the histones regained cytotoxicity. FSAP efficiently protected against the cytotoxicity of the unbound histones. We predict that the form in which histones are released into the blood is highly relevant in the context of human health and disease. We speculate that when endogenous DNases in serum degrade the DNA in nucleosomes, or alternatively, when highly cytotoxic histones are inadvertently released from dead cells in their free form rather than in the form of nucleosomes, FSAP mediates their efficient degradation to protect against the damaging effects of free histones. Neutralization of histones by FSAP may be an effective therapeutic approach to treat systemic inflammation. DisclosuresZeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Inter-α inhibitor protein and its associated glycosaminoglycans protect against histone-induced injury

AbstractExtracellular histones are mediators of tissue injury and organ dysfunction; therefore they constitute potential therapeutic targets in sepsis, inflammation, and thrombosis. Histone cytotoxicity in vitro decreases in the presence of plasma. Here, we demonstrate that plasma inter-α inhibitor protein (IAIP) neutralizes the cytotoxic effects of histones and decreases histone-induced platelet aggregation. These effects are mediated through the negatively charged glycosaminoglycans (GAGs) chondroitin sulfate and high-molecular-weight hyaluronan (HMW-HA) associated with IAIP. Cell surface anionic glycosaminoglycans heparan sulfate and HA protect the cells against histone-mediated damage in vitro. Surface plasmon resonance showed that both IAIP and HMW-HA directly bind to recombinant histone H4. In vivo neutralization of histones with IAIP and HMW-HA prevented histone-induced thrombocytopenia, bleeding, and lung microvascular thrombosis, decreased neutrophil activation, and averted histone-induced production of inflammatory cytokines and chemokines. IAIP and HMW-HA colocalized with histones in necrotic tissues and areas that displayed neutrophil extracellular traps. Increasing amounts of IAIP-histone complexes detected in the plasma of septic baboons correlated with increase in histones and/or nucleosomes and consumption of plasma IAIP. Our data suggest that IAIP, chondroitin sulfate, and HMW-HA are potential therapeutic agents to protect against histone-induced cytotoxicity, coagulopathy, systemic inflammation, and organ damage during inflammatory conditions such as sepsis and trauma.

Cattle with increased severity of bovine respiratory disease complex exhibit decreased capacity to protect against histone cytotoxicity

Bovine respiratory disease complex (BRDC) is the leading cause of morbidity and mortality in feedlot cattle. Significant inflammation and lesions are often observed in lungs of infected cattle. During acute inflammatory responses, histones contribute to mortality in rodents and humans and serum proteins can protect against histone-induced cytotoxicity. We hypothesized that cattle experiencing chronic or fatal cases of BRDC have reduced ability to protect against cytotoxic effects of histones. Serum samples were collected from 66 bull calves at the time of normal feedlot processing procedures. Animals were retrospectively assigned to groups consisting of calves never treated for BRDC (control [CONT]; n = 10), calves treated with antimicrobials once for BRDC (1T; n = 16), calves treated twice for BRDC (2T; n = 13), calves treated 3 times for BRDC (3T; n = 14), or calves treated 4 times for BRDC (4T; n = 13). Samples were also collected each time animals received antimicrobial treatment; animals within a group were further sorted by calves that recovered and calves that died to test histone cytotoxicity. Bovine kidney cells were cultured in duplicate in 96-well plates and exposed to 0 or 50 μg/mL of total histones for 18 h with 1% serum from each animal. Cell viability was assessed by the addition of resazurin for 6 h followed by fluorescent quantification. Fluorescent values from serum alone were subtracted from values obtained for histone treatment for each animal. Serum from CONT, 1T, and 2T at initial processing all exhibited a similar (P > 0.10) response to histone treatment with fluorescent values of –312 ± 557, –1,059 ± 441, and –975 ± 489, respectively. However, 3T and 4T demonstrated an impaired capacity (P < 0.05) to protect against histones (–2,778 ± 471 and –3,026 ± 489) at initial processing when compared to the other groups. When sorted by mortality within group, calves that were treated twice and recovered (–847 ± 331) demonstrated a greater (P < 0.05) protective capacity than calves that were treated twice and died (–2,264 ± 412), indicating that calves that contract BRDC and ultimately die might have reduced protective capacity against histone cytotoxicity. Results suggest that calves that require multiple treatments for BRDC have reduced ability to protect against cytotoxicity of histones. Understanding the primary mechanism responsible for protecting against histone cytotoxicity could lead to improved identification of animals susceptible to severe cases of BRDC, improved focus and use of available resources, or better treatments for severe cases of BRDC.

Abstract 15058: Disruption of Extracellular Cytotoxic Chromatin by Acute DNase1 Administration Improves Left Ventricular Remodeling after Myocardial Infarction

Rationale: Myocardial infarction (MI) leads to necrosis of multinucleated and polyploid myocytes. This causes uncontrolled release of cellular content like chromatin to the infarct area. Chromatin is mainly comprised of histones which are essential for controlling and packing of DNA but paradoxically are also known to be cytotoxic. This makes free chromatin a toxic DNA polymer creating local high concentrations of hazardous histones. Objective: We hypothesized that chromatin from necrotic cells accumulates in ischemic myocardium, creates local high concentrations of cytotoxic histones, and thereby potentiates ischemic damage to the heart after MI. The endonuclease DNase1 is capable of dispersing extracellular chromatin through linker DNA digestion and could decrease local histone concentrations and cytotoxicity. Methods and Results: After permanent coronary artery ligation in mice we found extracellular histones accumulated within the infarcted myocardium. Histone cytotoxicity towards isolated myocytes was confirmed in vitro. To reduce histone related cytotoxicity in vivo DNase1 was injected within the first 6 hours after induction of MI. DNase1 accumulated in the infarcted region of the heart, effectively disrupted extracellular cytotoxic chromatin and thereby reduced high local histone concentration. Animals acutely treated with DNase1 revealed significantly improved left ventricular remodeling as measured by serial echocardiography up to 28 days after MI (e.g. NaCl vs DNase1, papillary end diastolic area [mm 2 ]: 23.26 ± 2.06 vs 18.90 ± 1.24, n=9 vs 10, p&lt;0,05). Treatment did not influence mortality, infarct size or inflammatory parameters as determined by neutrophil infiltration and RTQ-PCR analysis of characteristic cytokines. However improved myocyte survival was discovered within the infarct region which might account for the protective effects in DNase1 treated animals (NaCl vs DNase1: 3.0 ± 0.7% vs 8.3 ± 2.3%; p&lt;0.05; n=7 vs 8). Conclusions: Targeting extracellular cytotoxic chromatin within the infarcted heart by DNase1 is a promising approach to preserve myocytes from histone induced cell death and to conserve left ventricular function after MI. The efficacy of other chromatin degrading agents is now under investigation.

Reduction of histone cytotoxicity by the Alzheimer β-amyloid peptide precursor

In a search for Alzheimer β-amyloid peptide precursor ligands, Potempska et al. (Arch. Biochem. Biophys. (1993) 304, 448) found that histones bind with high affinity and specificity to the secreted precursor. Because exogenous histones can be cytotoxic, we compared the effects of histones on the viability of cells which produce little β-amyloid peptide precursor (U-937) to those on cells that produce twenty times as much precursor (COS-7). Addition of purified histones caused necrosis of U-937 cells (histone H4, LD 50=1.5 μM). Extracellular A β precursor in the submicromolar range prevented histone-induced U-937 cell necrosis. Cell-surface precursor also reduced histone toxicity: COS-7 cells were less sensitive to the toxic effects of histone H4 (LD 50=5.4 μM). COS-7 cells in which the expression of an APP mRNA-directed ribozyme reduced the synthesis of the protein by up to 80% were more sensitive to histone H4 (LD 50=3.2 μM) than cells that expressed the vector alone. Histone H4 binds to cell-associated A β precursor. Cells expressing the A β precursor-directed ribozyme bound less 125I-labeled histone H4 than those expressing the vector alone. In the limited extracellular space of tissues in vivo, both secreted and cell-surface A β precursor protein may play significant roles in trapping chromatin or histones and removing them from the extracellular milieu.