Linear ubiquitination modification of NR6A1 by LUBAC inhibits RIPK3 kinase activity and attenuates apoptosis of vascular smooth muscle cells.

Nuclear receptor subfamily 6 group A member 1 (NR6A1) is involved in promoting the apoptotic process of vascular smooth muscle cells (VSMCs) which is a critical process involved in atherosclerosis, but the action mechanism remains to be determined. Therefore, we studied the underlying mechanisms by which NR6A1 accelerated VSMC apoptosis in atherosclerosis. An atherosclerosis model has been established in apolipoprotein E-deficient rats with a high-fat diet for 12 weeks, which was characterized by pathological aortic plaques, increased lipid deposition and collagen content in aortic tissues, and high cholesterol and triglycerides levels in the serum. NR6A1 was experimentally shown to increase at protein level rather than messenger RNA level in atherosclerotic rats. Immunofluorescence exhibited the main location of NR6A1 in the cell nucleus of rat aortic tissues. By performing ectopic expression experiments, NR6A1 was demonstrated to suppress the viability and expedite the apoptosis of VSMCs, corresponding to augmented caspase-3, caspase-8, and caspase-9 activities. It was further unraveled that NR6A1 could activate receptor-interacting serine/threonine-protein kinase 3 (RIPK3) by inducing its phosphorylation. Conversely, RIPK3 inhibitor GSK872 undermined the proapoptotic effect of NR6A1 on VSMCs. The co-immunoprecipitation assay identified that linear ubiquitin chain assembly complex (LUBAC) can be pulled down by NR6A1. Furthermore. LUBAC inhibited the expression of NR6A1 by promoting its linear ubiquitination, thereby dephosphorylating RIPK3 and consequently inhibiting the VSMC apoptosis. Overall, LUBAC-induced linear ubiquitination of NR6A1 can potentially arrest the apoptosis of VSMCs in atherosclerosis by downregulating RIPK3 and attenuating caspase activity. This finding suggests promising athero-protective targets by limiting VSMC apoptosis.

Inhibition of miR-188-5p Suppresses Progression of Experimental Abdominal Aortic Aneurysms.

ABSTRACT: Abdominal aortic aneurysm (AAA) is an aging-related degenerative disease. miR-188-5p was reported to induce cell senescence and play a key role in aging-related disease. Therefore, in this study, we investigated miR-188-5p expression during progression in experimental AAAs. Furthermore, we investigated whether inhibition of miR-188-5p could suppress AAA progression. Experimental AAAs were created in 9-12-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase. Expression of miR-188-5p levels were assessed in aneurysmal and control aortae during the progression of aneurysm. For inhibition experiment, miR-188 inhibiting group mice were injected with AAV2-miR188-5p sponge through tail vein and control group mice were injected with AAV2-CMV-GFP. Influences on experimental AAA progression were assessed by measurements of aortic diameter and histopathologic analysis at sacrifice. Meanwhile, immunohistochemistry and fluorescence in situ hybridization were used to determine the inflammatory cells infiltration and colocalization of miR-188-5p in aortic sections. Expression of miR-188-5p is upregulated during progression of AAA. Importantly, miR-188-5p inhibition treatment prevented enlargement of experimental aneurysms. Meanwhile, miR-188-5p inhibition regimens attenuated medial elastin degradation, smooth muscle cell depletion, and mural angiogenesis and the accumulation of macrophages, T cells, and angiogenesis. Furthermore, colocalization of miR188-5p with CD68 and CD3 was observed, which suggest miR-188-5p was expressed mainly in infiltrated macrophages and T cells. Expression of miR-188-5p is increased in experimental AAAs. Treatment with miR-188-5p inhibition limits experimental AAA progression, with histologic evidence of reduced neovessels and attenuated mural leukocyte infiltration. These findings underscore the potential significance of miR-188-5p in aneurysm pathogenesis and as a target for suppression of AAA disease.

"Ring on Anastomosis" for Avoidance of Acute Proximal Anastomotic Leak after Open Surgical Repair of Abdominal Aortic Aneurysm.

BACKGROUND: Acute proximal anastomotic leak is among severe complications after open surgical repair (OSR) of abdominal aortic aneurysm. We have proposed an approach of "ring on anastomosis" (ROA) as a technical improvement of conventional OSR to reinforce proximal anastomotic section. METHODS: One hundred and nineteen abdominal aortic aneurysm patients admitted to Xiangya Hospital, Central South University were enrolled. Patients were randomly divided into conventional group (n = 54) and ROA group (n = 65). The ring is prepared by cutting out a 2-cm circle from the graft. Operative time, intraoperative blood loss, perioperative mortality, and retroperitoneal hematoma were recorded. Poisson distribution analysis was used between two groups. All methods were carried out in accordance with Declaration of Helsinki. RESULTS: No obvious difference in operative time or intraoperative blood loss was identified [(205.5 ± 6.535) versus (195.6 ± 6.034) minutes, P > 0.05; (756 ± 98.22) versus (673.1 ± 98.93) ml, P > 0.05, respectively]. Two patients in conventional group (2/54, 3.7%) died while no dead case was reported in ROA group (P = 0.047). Three cases in conventional group experienced retroperitoneal hematoma while none was witnessed in ROA group (P = 0.027). An average of 18 months of follow-up was obtained in all patients, and no proximal anastomotic stenosis was reported. CONCLUSIONS: As a technical improvement of conventional OSR, ROA reinforces aorta graft anastomotic section and diminishes anastomotic leak as well as perioperative death without extra cost of time and money.

An Integrated Plasmo-Photoelectronic Nanostructure Biosensor Detects an Infection Biomarker Accompanying Cell Death in Neutrophils.

Bacterial infections leading to sepsis are a major cause of deaths in the intensive care unit. Unfortunately, no effective methods are available to capture the early onset of infectious sepsis near the patient with both speed and sensitivity required for timely clinical treatment. To fill the gap, the authors develop a highly miniaturized (2.5 × 2.5 µm2 ) plasmo-photoelectronic nanostructure device that detected citrullinated histone H3 (CitH3), a biomarker released to the blood circulatory system by neutrophils. Rapidly detecting CitH3 with high sensitivity has the great potential to prevent infections from developing life-threatening septic shock. To this end, the author's device incorporates structurally engineered arrayed hemispherical gold nanoparticles that are functionalized with high-affinity antibodies. A nanoplasmonic resonance shift induces a photoconduction increase in a few-layer molybdenum disulfide (MoS2 ) channel, and it provides the sensor signal. The device achieves label-free detection of serum CitH3 with a 5-log dynamic range from 10-4 to 101 ng mL and a sample-to-answer time <20 min. Using this biosensor, the authors longitudinally measure the dynamic CitH3 profiles of individual living mice in a sepsis model at high resolution over 12 hours. The developed biosensor may be poised for future translation to personalized management of systemic bacterial infections.

Inhibition of Phosphatidylinositol 3-Kinase γ by IPI-549 Attenuates Abdominal Aortic Aneurysm Formation in Mice.

OBJECTIVE: The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signalling pathway plays a pivotal role in abdominal aortic aneurysm (AAA). However, systemic inhibition of this pathway causes serious side effects, thus limiting the clinical use of pan-PI3K inhibitors. In this study, it was hypothesised that the γ subunit of PI3K plays an important role in the PI3K/AKT signalling pathway during AAA, and that specifically targeting PI3Kγ may prevent this process. METHODS: Aortic specimens were collected from AAA patients and organ donors. Furthermore, a classical AAA model in male C57BL/6 mice was created via an intra-aortic porcine pancreatic elastase (PPE) infusion and aortas were collected. A specific PI3Kγ inhibitor, IPI-549, was administered to mice orally. The protein expression level of PI3Kγ was examined by immunohistochemistry and western blotting. The aortic leukocytes were examined by immunohistochemistry and flow cytometry. RESULTS: PI3Kγ protein levels were elevated in the aortas of AAA patients and PPE infused mice. Three color immunofluorescence staining revealed the predominant area of PI3Kγ by T cells and macrophages in aneurysmal aortas. IPI-549 treatment significantly prevented AAA formation in mice. Aortic macrophages, T cells and neo-angiogenesis were significantly reduced in mice treated with IPI-549 compared with vehicle treated PPE infused mice. Flow cytometry analysis also revealed that CD45+ leukocytes and CD45+ F4/80+ macrophages in IPI-549 treated mouse aortas decreased dramatically. Additionally, IPI-549 treatment inhibited the phosphorylation of AKT in experimental aneurysmal lesions. CONCLUSION: Specific inhibition of PI3Kγ limits AAA formation. Targeting PI3Kγ prevents inflammatory cell infiltration through inhibition of AKT phosphorylation in AAA.

Site-specific apparent optimum air temperature for vegetation photosynthesis across the globe.

The apparent optimum air temperature for vegetation photosynthesis (Topt) is a key temperature parameter in terrestrial ecosystem models estimating daily photosynthesis or gross primary production (GPP, g C/m2/day). To date, most models use biome-specific Topt (Topt-biome) parameter values. Given vegetation acclimation and adaptation to local climate, site-specific Topt (Topt-site) is needed to reduce uncertainties in estimating daily GPP across the scales from site to region and the globe. Previous studies have demonstrated using the Enhanced Vegetation Index (EVI) derived from Moderate Resolution Imaging Spectroradiometer (MODIS) images and daytime air temperature data to estimate the Topt-site at the eddy covariance tower sites. This study used MODIS-derived EVI and ERA5 climate data to estimate and generate global Topt-site data products from 2000 to 2019. The Topt-site of individual pixels within a biome has large variation, which clearly cannot be represented accurately by the widely used Topt-biome. Therefore, using this global dataset of Topt-site estimates might significantly affect GPP simulation in current ecosystem models.

PI3Kgamma promotes neutrophil extracellular trap formation by noncanonical pyroptosis in abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is one of the most life-threatening cardiovascular diseases; however, effective drug treatments are still lacking. The formation of neutrophil extracellular traps (NETs) has been shown to be crucial trigger of AAA and identifying upstream regulatory targets is thus key to discovering therapeutic agents for AAA. We revealed that phosphoinositide 3-kinase gamma (PI3Kγ) acted as an upstream regulatory molecule, and that PI3Kγ inhibition reduced NET formation and aortic wall inflammation, thereby markedly ameliorating AAA. However, the mechanism of NET formation regulated by PI3Kγ remains unclear. In this study, we showed that PI3Kγ deficiency inactivated the noncanonical pyroptosis pathway, which suppressed downstream NET formation. In addition, PI3Kγ regulation of noncanonical pyroptosis was dependent on cAMP/protein kinase A (cAMP/PKA) signaling. These results clarify the molecular mechanism and crosstalk between PI3Kγ and NETosis in the development of AAA, potentially facilitating the discovery of therapeutic options for AAA.

D-Dimer Is a Diagnostic Biomarker of Abdominal Aortic Aneurysm in Patients With Peripheral Artery Disease.

Background: Etiology and risk factors of peripheral artery disease (PAD) include age, smoking, and hypertension, etc. , which are shared by an abdominal aortic aneurysm (AAA). Concomitance with AAA in patients with PAD is not rare but is easily overlooked in the clinical situation, though management strategies are altered totally. This study aims to investigate diagnostic biomarkers for the prediction of AAA in patients with PAD. Methods: A total of 684 patients diagnosed with AAA and/or PAD were enrolled and analyzed retrospectively. Each patient with PAD and AAA was gender and age-matched. Demographic data, medical history, and serum laboratory test profiles were obtained. Statistical analysis was performed to determine diagnostic biomarkers of AAA in patients with PAD. Results: Firstly, 320 patients with PAD-only and 320 patients with AAA-only were compared. Levels of bilirubin and D-Dimer were decreased, while the incidence of diabetes mellitus, levels of fibrinogen, and platelet count were increased significantly in patients with PAD-only compared with those in patients with AAA-only (P < 0.001). Next, 364 patients with PAD (44 patients with AAA) and 364 patients with AAA (44 patients with PAD) were compared. Multivariate logistic regression analysis confirmed the differential distribution of bilirubin, D-dimer, fibrinogen, and platelet count between patients with AAA and patients with PAD (P < 0.05). Receiver operator curves (ROC) showed that the area under the curve (AUC) of total bilirubin, direct bilirubin, D-dimer, fibrinogen, and platelet count was 0.6113, 0.5849, 0.7034, 0.6473, and 0.6785, respectively. Finally, to further validate the predictive efficacy of mentioned markers, a multivariable logistics regression analysis was performed between the PAD only group and the PAD with AAA group. The results suggested increased levels of D-dimer in the PAD with AAA group compared to the PAD only group (OR: 2.630, 95% CI:1.639-4.221; P < 0.001). In particular, the Youden index suggested that the cut-off value of D-dimer for predicting AAA in patients with PAD was 0.675 mg/L with a sensitivity of 76.9% and a specificity of 84.9% (AUC = 0.8673; 95% CI, 0.8106-0.9240, P < 0.001). In all 364 patients with PAD, 41.46% patients were diagnosed AAA when D-dimer is >0.675 mg/L, while only 3.55% patients were diagnosed AAA when D-dimer ≤ 0.675 mg/L. Conclusions: PAD and AAA exert different clinical and serum profiles; D-dimer (>0.675 mg/L) is a reliable biomarker for the prediction of AAA in patients with PAD.

Circulating CitH3 Is a Reliable Diagnostic and Prognostic Biomarker of Septic Patients in Acute Pancreatitis.

Purpose: Acute pancreatitis (AP) is an inflammatory disease. AP starts with sterile inflammation and is often complicated with critical local or systemic infection or sepsis in severe cases. Septic AP activates peptidyl arginine deiminase (PAD) and citrullinates histone H3 (CitH3), leading to neutrophil extracellular trap (NET) formation. Investigating the role of NETs and underlying mechanisms in septic AP may facilitate developing diagnostic and therapeutic approaches. In this study, we sought to identify the expression of CitH3 in septic AP patients and to analyze the correlation of CitH3 concentration with NET components as well as clinical outcomes. Methods: Seventy AP patients with or without sepsis (40 septic cases, 30 nonseptic cases) and 30 healthy volunteers were recruited in this study. Concentration of NET components (CitH3 and double-strain DNA) and key enzymes (PAD2/4) were measured. Clinical and laboratory characteristics of patients were recorded and analyzed. Results: Levels of CitH3 were elevated significantly in septic AP patients compared with those in nonseptic AP and healthy volunteers. The area under the curve (AUC, 95% confidence interval) for diagnosing septic AP was 0.93 (0.86-1.003), and the cutoff was 43.05 pg/ml. Among septic AP cases (n = 40), the concentration of CitH3 was significantly increased in those who did not survive or were admitted to the intensive care unit, when compared with that in those who survived or did not require intensive care unit. Association analysis revealed that CitH3 concentration was positively correlated with PAD2, PAD4, dsDNA concentration, and Sequential Organ Failure Assessment scores. Conclusion: CitH3 concentration increased in septic AP patients and was closely correlated with disease severity and clinical outcomes. CitH3 may potentially be a diagnostic and prognostic biomarker of septic AP.

LncRNA SENCR suppresses abdominal aortic aneurysm formation by inhibiting smooth muscle cells apoptosis and extracellular matrix degradation.

Abdominal aortic aneurysm (AAA) is a progressive chronic dilatation of the abdominal aorta without effective medical treatment. This study aims to clarify the potential of long non-coding RNA SENCR as a treatment target in AAA. Angiotensin II (Ang-II) was used to establish AAA model in vitro and in vivo. Reverse transcription quantitative PCR and western blot were performed to measure the expression of SENCR and proteins, respectively. Annexin V-FITC/PI double staining was carried out to detect the apoptotic rate in vascular smooth muscle cells (VSMCs), and cell apoptosis in aortic tissues was determined by TUNEL staining. Besides, hematoxylin and eosin and Elastica van Gieson staining were performed for histological analysis of aortic tissues. SENCR was downregulated in AAA tissues and Ang-II-stimulated VSMCs. Overexpression of SENCR could inhibit Ang-II-induced VSMC apoptosis, while inhibition of SENCR facilitated Ang-II-induced VSMC apoptosis. Moreover, the expression of matrix metalloproteinase (MMP)-2 and MMP-9 in Ang-II-induced VSMCs was reduced following SENCR overexpression, while tissue inhibitor of metalloproteinases 1 (TIMP-1) expression was increased. In vivo, overexpression of SENCR improved the pathological change in aortic tissues and the damage in arterial wall elastic fibers induced by Ang-II, as well as suppressed Ang-II-induced cell apoptosis and extracellular matrix degradation. Overall, SENCR was decreased in AAA. Overexpression of SENCR inhibited AAA formation via inhibition of VSMC apoptosis and extracellular matrix degradation. We provided a reliable evidence for SENCR acting as a potential target for AAA treatment.

Citrullinated Histone H3 Mediates Sepsis-Induced Lung Injury Through Activating Caspase-1 Dependent Inflammasome Pathway.

Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to infection that often results in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). An emerging mechanism of sepsis-induced ARDS involves neutrophils/macrophages undergoing cell death, releasing nuclear histones to cause tissue damage that exacerbates pulmonary injury. While published studies focus on unmodified histones, little is known about the role of citrullinated histone H3 (CitH3) in the pathogenesis of sepsis and ALI. In this study, we found that levels of CitH3 were elevated in the patients with sepsis-induced ARDS and correlated to PaO2/FiO2 in septic patients. Systematic administration of CitH3 peptide in mice provoked Caspase-1 activation in the lung tissue and caused ALI. Neutralization of CitH3 with monoclonal antibody improved survival and attenuated ALI in a mouse sepsis model. Furthermore, we demonstrated that CitH3 induces ALI through activating Caspase-1 dependent inflammasome in bone marrow derived macrophages and bone marrow derived dendritic cells. Our study suggests that CitH3 is an important mediator of inflammation and mortality during sepsis-induced ALI.

The role of autophagy in abdominal aortic aneurysm: protective but dysfunctional.

Autophagy, an evolutionarily conserved mechanism that promotes cell survival by recycling nutrients and degrading long-lived proteins and dysfunctional organelles, is an important defense mechanism, and its attenuation has been well documented in senescence and aging-related diseases. Abdominal aortic aneurysm (AAA), a well-known aging-related disease, has been defined as a chronic degenerative process in the abdominal aortic wall; however, the complete mechanism is unknown, and a clinical treatment is lacking. Accumulating evidence has recently revealed that numerous drugs that can induce autophagy are effective in the treatment of AAA. The purpose of this systematic review was to focus on the cross-talk between autophagy and high-risk factors and the potential pathogenesis of AAA to understand not only the host defense and pathogenesis but also potential treatments.

Inhibition of PAD2 Improves Survival in a Mouse Model of Lethal LPS-Induced Endotoxic Shock.

Endotoxemia induced by lipopolysaccharide (LPS) is an extremely severe syndrome identified by global activation of inflammatory responses. Neutrophil extracellular traps (NETs) play an important role in the development of endotoxemia. Histone hypercitrullination catalyzed by peptidylarginine deiminases (PADs) is a key step of NET formation. We have previously demonstrated that simultaneous inhibition of PAD2 and PAD4 with pan-PAD inhibitors can decrease NETosis and improve survival in a mouse model of LPS-induced endotoxic shock. However, the effects of PAD2 specific inhibition during NETosis and endotoxic shock are poorly understood. Therefore, in the present study, we aimed to investigate the effect of the specific PAD2 or PAD4 inhibitor on LPS-induced endotoxic shock in mice. We found that PAD2 inhibition but not PAD4 inhibition improves survival. Also, the levels of proinflammatory cytokines and NETosis were significantly reduced by PAD2 inhibitor. To our knowledge, this study demonstrates for the first time that PAD2 inhibition can reduce NETosis, decrease inflammatory cytokine production, and protect against endotoxin-induced lethality. Our findings provided a novel therapeutic strategy for the treatment of endotoxic shock.

Spermidine Suppresses Development of Experimental Abdominal Aortic Aneurysms.

Background The protective effects of polyamines on cardiovascular disease have been demonstrated in many studies. However, the roles of spermidine, a natural polyamine, in abdominal aortic aneurysm (AAA) disease have not been studied. In this study, we investigated the influence and potential mechanisms of spermidine treatment on experimental AAA disease. Methods and Results Experimental AAAs were induced in 8- to 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase. Spermidine was administered via drinking water at a concentration of 3 mmol/L. Spermidine treatment prevented experimental AAA formation with preservation of medial elastin and smooth muscle cells. In immunostaining, macrophages, T cells, neutrophils, and neovessels were significantly reduced in aorta of spermidine-treated, as compared with vehicle-treated elastase-infused mice. Additionally, flow cytometric analysis showed that spermidine treatment reduced aortic leukocyte infiltration and circulating inflammatory cells. Furthermore, we demonstrated that spermidine treatment promoted autophagy-related proteins in experimental AAAs using Western blot analysis, immunostaining, and transmission electron microscopic examination. Autophagic function was evaluated for human abdominal aneurysmal and nonaneurysmal adjacent aortae from AAA patients using Western blot analysis and immunohistochemistry. Dysregulated autophagic function, as evidenced by increased SQSTM1/p62 protein and phosphorylated mTOR, was found in aneurysmal, as compared with nonaneurysmal, aortic segments. Conclusions Our results suggest that spermidine supplementation limits experimental AAA formation associated with preserved aortic structural integrity, attenuated aortic inflammatory infiltration, reduced circulating inflammatory monocytes, and increased autophagy-related proteins. These findings suggest that spermidine may be a promising treatment for AAA disease.

Peptidylarginine deiminase 2 has potential as both a biomarker and therapeutic target of sepsis.

Peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes that are involved in a variety of human disorders, including cancer and autoimmune diseases. Although targeting PAD4 has shown no benefit in sepsis, the role of PAD2 remains unknown. Here, we report that PAD2 is engaged in sepsis and sepsis-induced acute lung injury in both human patients and mice. Pad2-/- or selective inhibition of PAD2 by a small molecule inhibitor increased survival and improved overall outcomes in mouse models of sepsis. Pad2 deficiency decreased neutrophil extracellular trap (NET) formation. Importantly, Pad2 deficiency inhibited Caspase-11-dependent pyroptosis in vivo and in vitro. Suppression of PAD2 expression reduced inflammation and increased macrophage bactericidal activity. In contrast to Pad2-/-, Pad4 deficiency enhanced activation of Caspase-11-dependent pyroptosis in BM-derived macrophages and displayed no survival improvement in a mouse sepsis model. Collectively, our findings highlight the potential of PAD2 as an indicative marker and therapeutic target for sepsis.

Citrullinated Histone H3 as a Therapeutic Target for Endotoxic Shock in Mice.

Sepsis results in millions of deaths every year, with acute lung injury (ALI) being one of the leading causes of mortality in septic patients. As neutrophil extracellular traps (NETs) are abundant in sepsis, neutralizing components of NETs may be a useful strategy to improve outcomes of sepsis. Citrullinated histone H3 (CitH3) has been recently shown to be involved in the NET formation. In this study, we demonstrate that CitH3 damages human umbilical vein endothelial cells (HUVECs) and potentiates NET formation through a positive feedback mechanism. We developed a novel CitH3 monoclonal antibody to target peptidylarginine deiminase (PAD) 2 and PAD 4 generated CitH3. In a mouse model of lethal lipopolysaccharide (LPS) induced shock, neutralizing CitH3 with the newly developed anti-CitH3 monoclonal antibody attenuates inflammatory responses, ameliorates ALI, and improves survival. Our study suggests that effectively blocking circulating CitH3 might be a potential therapeutic method for the treatment of endotoxemia.

Inhibition of peptidylarginine deiminase alleviates LPS-induced pulmonary dysfunction and improves survival in a mouse model of lethal endotoxemia.

Immune cell death caused by neutrophil extracellular traps (NETs), referred to as NETosis, can contribute to the pathogenesis of endotoxemia and organ damage. Although the mechanisms by which infection induces NETosis and how that leads to organ dysfunction remain largely unknown, NET formation is often found following citrullination of histone H3 (CitH3) by peptidylarginine deiminase (PAD). We hypothesized that lipopolysaccharide (LPS)-induced activation of PAD and subsequent CitH3-mediated NET formation increases endothelial permeability and pulmonary dysfunction and, therefore, that inhibition of PAD can mitigate damage and improve survival in lethal endotoxemia. Here, we showed that treatment with YW3-56, a PAD2/PAD4 inhibitor, significantly diminished PAD activation, blocked LPS-induced pulmonary vascular leakage, alleviated acute lung injury, and improved survival in a mouse model of lethal LPS-induced endotoxemia. We found CitH3 in the bloodstream 30 min after intraperitoneal injection of LPS (35 mg/kg) into mice. Additionally, CitH3 production was induced in cultured neutrophils exposed to LPS, and NETs derived from these LPS-treated neutrophils increased the permeability of endothelial cells. However, YW3-56 reduced CitH3 production and NET formation by neutrophils following LPS exposure. Moreover, treatment with YW3-56 decreased the levels of circulating CitH3 and abolished neutrophil activation and NET formation in the lungs of mice with endotoxemia. These data suggest a novel mechanism by which PAD-NET-CitH3 can play a pivotal role in pulmonary vascular dysfunction and the pathogenesis of lethal endotoxemia.

Protective Effect of Tubastatin A in CLP-Induced Lethal Sepsis.

We have found earlier that Tubastatin A (TubA), a selective inhibitor of histone deacetylase 6 (HDAC6), improves survival in a mouse model of lethal cecal ligation and puncture (CLP)-induced sepsis. However, the underlying mechanisms have not been fully established. This study sought to test the hypothesis that TubA could affect both lung and splenic functions. C57BL/6J mice were subjected to CLP, and randomized to receive either TubA (70 mg/kg) dissolved in dimethyl sulfoxide (DMSO), or DMSO alone, 1 h following CLP. Sham animals acted as control. Twenty-four hours later, lung tissue was harvested for pathological examination, and splenic tissue was harvested for bacterial colonization. In a parallel study, the spleen was collected 48 h following CLP, and single cell suspension was prepared. Splenocytes then underwent flow cytometry to analyze the immune cell population. RAW264.7 macrophages were treated with lipopolysaccharide (LPS) with or without the presence of TubA (10 µM) at 37 °C for 3 h to assess the effect on macrophage phagocytosis. We found that acute lung injury secondary to lethal sepsis was attenuated by TubA. Treatment with TubA restored the percentage of B lymphocytes, and significantly increased percentages of innate immune cells and macrophages compared to the vehicle-treated CLP group. Moreover, TubA significantly decreased the bacterial load in the spleen, and improved the phagocytic ability of RAW264.7 murine macrophages in vitro. Such findings may help to explain the beneficial effects of TubA treatment in a model of lethal sepsis, as previously reported.

CitH3: a reliable blood biomarker for diagnosis and treatment of endotoxic shock.

Current biomarkers for sepsis are limited by their non-specificity, short half-life, and insensitive response to therapy. Recently, we have demonstrated that citrullinated histone H3(CitH3) is released into the blood from neutrophil extracellular traps(NETs) in response to severe infection, and CitH3 may be a potential biomarker for sepsis. In the present study, we found that NET components were released in mouse models of both lipopolysaccharide(LPS)-induced shock (LPSS) and hemorrhagic shock (HS). To further quantify CitH3 in the NETs, we established a CitH3 specific enzyme-linked immunosorbent assay. Circulating CitH3 was found to be elevated only in LPSS but not in HS. Importantly, blood CitH3 was detected 30 minutes after LPS insult, and remained elevated for 24 hours (period of the highest mortality). Treatment of endotoxic mice with YW3-56, a peptidylarginine deiminase-2/4 inhibitor, significantly diminished levels of CitH3 in the blood. Interleukin-1ß did not respond to LPS early, and interleukin-1ß and interleukin-6 fluctuated although they responded to treatment. Procalcitonin reacted to LPS insult late. Compared to CitH3, these biomarkers were non-specifically induced in LPSS and HS. Collectively, our results demonstrate that YW3-56 protects animals from LPSS, and CitH3 is a reliable biomarker due to its early appearance, specificity, duration, and response to therapeutic intervention.

Lung Protective Effects of Low-Volume Resuscitation and Pharmacologic Treatment of Swine Subjected to Polytrauma and Hemorrhagic Shock.

Hemorrhage is a common cause of death in the battlefield. Valproic acid (VPA) has been associated with improved outcomes in multiple models of trauma, when combined with isotonic fluid resuscitation. However, isotonic fluid administered in this setting is logistically impractical and may be associated with complications. In this study, we sought to evaluate the feasibility and immunologic impact of combining VPA treatment with low-volume hypertonic saline (HTS). In vivo: female Yorkshire swine were subjected to hemorrhage (40% total blood volume) and polytrauma (rib fracture and delayed liver injury). Animals were kept in shock for 30 minutes and resuscitated with (1) normal saline (NS, 3× hemorrhaged volume), (2) HTS (7.5% saline, 4 mL/kg), or (3) HTS + VPA (4 mg/kg; 150 mg/kg; n = 3/cohort). After 18 hours of observation, animals were euthanized and the lungs evaluated for acute injury and expression of myeloperoxidase (MPO) and caveolin-1 (Cav-1). In vitro: human umbilical vein endothelial cells (HUVECs) were exposed to anoxic conditions (5% CO2, 95% N2) for 16 hours in (1) normosmotic, (2) hyperosmotic (400 mOsm), or (3) hyperosmotic + VPA (4 mM) media. Immunohistochemistry and Western blots were performed to determine Cav-1 expression. Lungs from VPA-treated animals demonstrated decreased acute injury, MPO expression, and endothelial expression of Cav-1 when compared to lungs from animals resuscitated with NS or HTS alone. Similarly, HUVECs cultured in hyperosmotic media containing VPA demonstrated decreased expression of Cav-1. This study demonstrates that combined treatment with VPA and HTS is a viable strategy in hemorrhagic shock and polytrauma. Attenuation of lung injury following VPA treatment may be related to modulation of the inflammatory response.