The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

OBJECTIVE: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. METHODS: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. RESULTS: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-ß regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. CONCLUSIONS: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Antibodies to histone in the pediatric population: a retrospective chart review.

BACKGROUND: Antibodies to histone have been associated in the adult literature with systemic lupus erythematosus(SLE) and drug induced lupus(DILE). Little data is available regarding the spectrum of pathology that antibodies to histone encompass in the pediatric population. Prior studies suggest an association with SLE, juvenile idiopathic arthritis(JIA), uveitis and linear scleroderma. METHODS: Patient charts were reviewed that contained positive anti-histone antibody testing during a consecutive three year period. Patient diagnosis along with the presence of: anti-histone antibody titer, ANA, and the presence of other autoantibodies to SSA, SSB, Sm, RNP, dsDNA and chromatin were obtained. The frequency of SLE, JIA and DILE was further investigated in specific subsets. RESULTS: 139 individual charts were reviewed containing 41 different diagnoses. The most common diagnosis was hypermobility arthralgia with 22 patients. The most frequent rheumatologic diagnosis was JIA(nonsystemic) with 19. 13 patients in this study were diagnosed with SLE and 2 with DILE. 18 patients had other autoantibody production, of these, 11 had SLE or DILE. Only one of 62 patients with a weak antihistone antibody titer(1.0-1.5) was diagnosed with SLE. When strong titers are present(> 2.5), the antihistone antibody test was associated with a greater than 50% incidence of an underlying rheumatologic disease and ten times higher incidence of SLE than a weak titer. In regards to the frequency of SLE, there was a statistically significant difference between weak and moderate titers and between weak and strong titers. CONCLUSION: The presence of anti-histone antibody was observed in a variety of diagnoses in the pediatric population. Overall, the presence of anti-histone antibodies appears to have poor diagnostic utility for any specific condition. However, diagnostic utility for SLE does appear to improve with higher titers, when combined with other autoantibody positivity. Strength of titer did not appear to be a factor for JIA, but was the most frequently observed rheumatologic disease in this study.

H3K36 trimethylation-mediated biological functions in cancer.

Histone modification is an important form of epigenetic regulation. Thereinto, histone methylation is a critical determination of chromatin states, participating in multiple cellular processes. As a conserved histone methylation mark, histone 3 lysine 36 trimethylation (H3K36me3) can mediate multiple transcriptional-related events, such as the regulation of transcriptional activity, transcription elongation, pre-mRNA alternative splicing, and RNA m6A methylation. Additionally, H3K36me3 also contributes to DNA damage repair. Given the crucial function of H3K36me3 in genome regulation, the roles of H3K36me3 and its sole methyltransferase SETD2 in pathogenesis, especially malignancies, have been emphasized in many studies, and it is conceivable that disruption of histone methylation regulatory network composed of "writer", "eraser", "reader", and the mutation of H3K36me3 codes have the capacity of powerfully modulating cancer initiation and development. Here we review H3K36me3-mediated biological processes and summarize the latest findings regarding its role in cancers. We highlight the significance of epigenetic combination therapies in cancers.

Kidney injury induced by elevated histones in community-acquired pneumonia.

Previous studies showed that extracellular histones could damage organs, but the role of extracellular histones in pneumonia patients with acute kidney injury (AKI) is unknown. This study aims to investigate the impact of extracellular histones on patients with community-acquired pneumonia (CAP) developed AKI. Blood samples were obtained within 24 h after admission to hospital from patients who were diagnosed with CAP. According to the discharge diagnosis, the patients were divided into 2 groups (Non-AKI and AKI). In vitro, A549 cells were treated with lipopolysaccharides (LPS) and conditioned media were collected. HK2 cells were exposed to the conditioned media or not. Cells proliferation and apoptosis of HK2 were determined. Clinically, Log2 Histones (OR 3.068; 95% CI 1.544-6.097, P = 0.001) and estimated glomerular filtration rate (eGFR) (OR 0.945; 95% CI 0.914-0.978, P = 0.001) were predictors of AKI in CAP patients. Compared to the lower histones group, patients in the higher histones group were more likely to be admitted to ICU, receive mechanical ventilation, and have a longer length of in-hospital stay. In vitro, A549 cells injured by LPS released extracellular histones, in conditioned media which significantly promoted HK2 cells apoptosis. Extracellular histones was a high risk factor for developing AKI in CAP patients and a predictor of worse short-term outcomes. We also showed that extracellular histones in conditioned media damaged HK2 cells.Trial registration number: KY20181102-03; Date of registration: 20181102.

Polysialic Acid in Human Plasma Can Compensate the Cytotoxicity of Histones.

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.

Protective effect of a newly developed fucose-deficient recombinant antithrombin against histone-induced endothelial damage.

Antithrombin is expected to modulate both prothrombotic and proinflammatory reactions in sepsis; vascular endothelium is the primary target. In the present study, we sought to evaluate the protective effects of a newly developed fucose-deficient recombinant antithrombin. Endothelial cells were treated in vitro with histone H4 to induce cellular damage. Low to high doses of either plasma-derived antithrombin or recombinant thrombomodulin were used as treatment interventions. Morphological change, apoptotic rate, cell viability, cell injury, and syndecan-4 level in the medium were evaluated. Immunofluorescent staining with anti-syndecan-4 was also performed. Both types of antithrombin reduced cellular damage and apoptotic cell death. Both plasma-derived and recombinant antithrombin improved cell viability and reduced cellular injury when administered at a physiological concentration or higher. Syndecan-4 staining became evident after treatment with histone H4, and both antithrombins suppressed the staining intensity at similar levels. The syndecan-4 level in the medium was significantly decreased by both antithrombins. None of the indicators showed a significant difference between plasma-derived and recombinant antithrombin. In conclusion, both recombinant and plasma-derived antithrombin can protect vascular endothelial cells. Recombinant antithrombin may represent a useful new therapeutic agent for sepsis-associated vascular damage.

Presence of Cytotoxic Extracellular Histones in Machine Perfusate of Donation After Circulatory Death Kidneys.

BACKGROUND: Extracellular histones are cytotoxic molecules that are related to cell stress and death. They have been shown to play a crucial role in multiple pathophysiologic processes like sepsis, inflammation, vascular dysfunction, and thrombosis. Their role in organ donation and graft function and survival is still unknown. The aim of this study was to assess whether an association exists between the presence of extracellular histones in machine perfusates and deceased donor kidney viability. METHODS: Machine perfusates of 390 donations after circulatory death kidneys were analyzed for histone concentration, and corresponding graft function and survival were assessed. RESULTS: Extracellular histone concentrations were significantly higher in perfusates of kidneys with posttransplant graft dysfunction (primary nonfunction and delayed graft function) and were an independent risk factor for delayed graft function (odds ratio, 2.152; 95% confidence interval [95% CI], 1.199-3.863) and 1 year graft failure (hazard ratio, 1.386; 95% CI, 1.037-1.853), but not for primary nonfunction (odds ratio, 1.342; 95% CI, 0.900-2.002). One year graft survival was 12% higher in the group with low histone concentrations (P = 0.008) as compared with the group that contained higher histone concentrations. CONCLUSIONS: This study warrants future studies to probe for a possible role of cytotoxic extracellular histones in organ viability and suggests that quantitation of extracellular histones might contribute to assessment of posttransplant graft function and survival.

Role of extracellular histones in the cardiomyopathy of sepsis.

The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca(2+)]i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht-, LRR-, and PYD-domains-containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca(2+)]i, as well as defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.

Recombinant thrombomodulin protects mice against histone-induced lethal thromboembolism.

INTRODUCTION: Recent studies have shown that histones, the chief protein component of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and act as major mediators of the death of an organism. This study was designed to elucidate the cellular and molecular basis of histone-induced lethality and to assess the protective effects of recombinant thrombomodulin (rTM). rTM has been approved for the treatment of disseminated intravascular coagulation (DIC) in Japan, and is currently undergoing a phase III clinical trial in the United States. METHODS: Histone H3 levels in plasma of healthy volunteers and patients with sepsis and DIC were measured using enzyme-linked immunosorbent assay. Male C57BL/6 mice were injected intravenously with purified histones, and pathological examinations were performed. The protective effects of rTM against histone toxicity were analyzed both in vitro and in mice. RESULTS: Histone H3 was not detectable in plasma of healthy volunteers, but significant levels were observed in patients with sepsis and DIC. These levels were higher in non-survivors than in survivors. Extracellular histones triggered platelet aggregation, leading to thrombotic occlusion of pulmonary capillaries and subsequent right-sided heart failure in mice. These mice displayed symptoms of DIC, including thrombocytopenia, prolonged prothrombin time, decreased fibrinogen, fibrin deposition in capillaries, and bleeding. Platelet depletion protected mice from histone-induced death in the first 30 minutes, suggesting that vessel occlusion by platelet-rich thrombi might be responsible for death during the early phase. Furthermore, rTM bound to extracellular histones, suppressed histone-induced platelet aggregation, thrombotic occlusion of pulmonary capillaries, and dilatation of the right ventricle, and rescued mice from lethal thromboembolism. CONCLUSIONS: Extracellular histones cause massive thromboembolism associated with consumptive coagulopathy, which is diagnostically indistinguishable from DIC. rTM binds to histones and neutralizes the prothrombotic action of histones. This may contribute to the effectiveness of rTM against DIC.

Histones induce rapid and profound thrombocytopenia in mice.

Histones are released from dying cells and contribute to antimicrobial defense during infection. However, extracellular histones are a double-edged sword because they also damage host tissue and may cause death. We studied the interactions of histones with platelets. Histones bound to platelets, induced calcium influx, and recruited plasma adhesion proteins such as fibrinogen to induce platelet aggregation. Hereby fibrinogen cross-linked histone-bearing platelets and triggered microaggregation. Fibrinogen interactions with αIIbß3 integrins were not required for this process but were necessary for the formation of large platelet aggregates. Infused histones associated with platelets in vivo and caused a profound thrombocytopenia within minutes after administration. Mice lacking platelets or αIIbß3 integrins were protected from histone-induced death but not from histone-induced tissue damage. Heparin, at high concentrations, prevented histone interactions with platelets and protected mice from histone-induced thrombocytopenia, tissue damage, and death. Heparin and histones are evolutionary maintained. Histones may combine microbicidal with prothrombotic properties to fight invading microbes and maintain hemostasis after injury. Heparin may provide an innate counter mechanism to neutralize histones and diminish collateral tissue damage.

The "stright-jacket" syndrome in chicks. II. Mechanism of development.

In a longitudinal study including measurement of the pressure in the amniotic cavity, amniotomy, and planimetric evaluation of the size of the amniotic sac, we investigated the development of the "strait jacket" syndrome in White Leghorn chicken embryos injected intraamniotically (ia) or paraamniotically on the fourth day of incubation with histone or embryotoxic serum, with the following results. Hyperlordosis and eventration developed as an outcome of tonic contraction of the amnion, which was observed only three hours after ia administration. Contraction of the amnion caused elevation of the intraamniotic pressure, which, 12 hours after ia injection, attained a mean value of 22.4 Pa (2.3 mm H2O). This value was not only significantly higher than the mean for control embryos (3.9 Pa), but it was critically close to the mean fluid pressure in the brain vesicles. Loss of the latter overpressure caused the vesicles to collapse, and the walls shriveled and exencephaly developed. Paraamniotic injection was not followed by either contraction of the amnion, or significant increase in intraamniotic pressure. This did not prevent heart malformations and cranioschisis of various extent. The majority of cardiovascular malformations were probably the hemodynamic consequence of overfilling of the intraembryonic vascular bed, which was one of the early signs of the effect question. Cranial-vault defects can be causally associated with the formation of amnionic adhesion and fusion with the epidermal ectoderm. This observation stresses the significance of the embryonic membranes and the fluid pressures within them for the development of certain congenital deformities and concentrates attention on teratological study of substances that induce protracted contraction of smooth muscle.