Synthetic Secoisolariciresinol Diglucoside (LGM2605) Prevents Asbestos-Induced Inflammation and Genotoxic Cell Damage in Human Mesothelial Cells.

Although alveolar macrophages play a critical role in malignant transformation of mesothelial cells following asbestos exposure, inflammatory and oxidative processes continue to occur in the mesothelial cells lining the pleura that may contribute to the carcinogenic process. Malignant transformation of mesothelial cells following asbestos exposure occurs over several decades; however, amelioration of DNA damage, inflammation, and cell injury may impede the carcinogenic process. We have shown in an in vitro model of asbestos-induced macrophage activation that synthetic secoisolariciresinol diglucoside (LGM2605), given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage. Therefore, it was hypothesized that LGM2605 could also be effective in reducing asbestos-induced activation and the damage of pleural mesothelial cells. LGM2605 treatment (50 µM) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 µg/cm2). Supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure for reactive oxygen species (ROS) generation, DNA damage (oxidized guanine), inflammasome activation (caspase-1 activity) and associated pro-inflammatory cytokine release (IL-1ß, IL-18, IL-6, TNFα, and HMGB1), and markers of oxidative stress (malondialdehyde (MDA) and 8-iso-prostaglandin F2a (8-iso-PGF2α). Asbestos induced a time-dependent ROS increase that was significantly (p < 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% ± 1.0%. Although levels of inflammasome-activated cytokines, IL-1ß and IL-18, reached 29.2 pg/mL ± 0.7 pg/mL and 43.9 pg/mL ± 0.8 pg/mL, respectively, LGM2605 treatment significantly (p < 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values (3.8 pg/mL ± 0.2 pg/mL and 5.4 pg/mL ± 0.2 pg/mL, respectively). Furthermore, levels of IL-6 and TNFα in asbestos-exposed mesothelial cells were high (289.1 pg/mL ± 2.9 pg/mL and 511.3 pg/mL ± 10.2 pg/mL, respectively), while remaining undetectable with LGM2605 pretreatment. HMGB1 (a key inflammatory mediator and initiator of malignant transformation) release was reduced 75.3% ± 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2α, markers of oxidative cell injury, were significantly (p < 0.001) reduced by 80.5% ± 0.1% and 76.6% ± 0.3%, respectively. LGM2605, given preventively, reduced ROS generation, DNA damage, and inflammasome-activated cytokine release and key inflammatory mediators implicated in asbestos-induced malignant transformation of normal mesothelial cells.

Editorial to the Special Issue: "Dysregulation of Human Molecular and Metabolic Mechanisms Resulting in Oxidative Stress and Damage Generation in the Space Environment".

Commercial space industries are emergent, bolstered by new exciting rocket systems, orbital and landing vehicles, the creation of multi-country orbital platforms, satellite technology, the renewed promise of low Earth orbit (LEO) business opportunities, as well as promised planetary exploration [...].

Late Inflammation Induced by Asbestiform Fibers in Mice Is Ameliorated by a Small Molecule Synthetic Lignan.

Exposure to Libby amphibole (LA) asbestos-like fibers is associated with increased risk of asbestosis, mesothelioma, pulmonary disease, and systemic autoimmune disease. LGM2605 is a small molecule antioxidant and free radical scavenger, with anti-inflammatory effects in various disease models. The current study aimed to determine whether the protective effects of LGM2605 persist during the late inflammatory phase post-LA exposure. Male and female C57BL/6 mice were administered daily LGM2605 (100 mg/kg) via gel cups for 3 days before and 14 days after a 200 µg LA given via intraperitoneal (i.p.) injection. Control mice were given unsupplemented gel cups and an equivalent dose of i.p. saline. On day 14 post-LA treatment, peritoneal lavage was assessed for immune cell influx, cytokine concentrations, oxidative stress biomarkers, and immunoglobulins. During the late inflammatory phase post-LA exposure, we noted an alteration in trafficking of both innate and adaptive immune cells, increased pro-inflammatory cytokine concentrations, induction of immunoglobulin isotype switching, and increased oxidized guanine species. LGM2605 countered these changes similarly among male and female mice, ameliorating late inflammation and altering immune responses in late post-LA exposure. These data support possible efficacy of LGM2605 in the prolonged treatment of LA-associated disease and other inflammatory conditions.

Copper Oxide Nanoparticle-Induced Acute Inflammatory Response and Injury in Murine Lung Is Ameliorated by Synthetic Secoisolariciresinol Diglucoside (LGM2605).

Metal-oxide nanoparticles (MO-NPs), such as the highly bioreactive copper-based nanoparticles (CuO-NPs), are widely used in manufacturing of hundreds of commercial products. Epidemiological studies correlated levels of nanoparticles in ambient air with a significant increase in lung disease. CuO-NPs, specifically, were among the most potent in a set of metal-oxides and carbons studied in parallel regarding DNA damage and cytotoxicity. Despite advances in nanotoxicology research and the characterization of their toxicity, the exact mechanism(s) of toxicity are yet to be defined. We identified chlorination toxicity as a damaging consequence of inflammation and myeloperoxidase (MPO) activation, resulting in macromolecular damage and cell damage/death. We hypothesized that the inhalation of CuO-NPs elicits an inflammatory response resulting in chlorination damage in cells and lung tissues. We further tested the protective action of LGM2605, a synthetic small molecule with known scavenging properties for reactive oxygen species (ROS), but most importantly, for active chlorine species (ACS) and an inhibitor of MPO. CuO-NPs (15 µg/bolus) were instilled intranasally in mice and the kinetics of the inflammatory response in lungs was evaluated 1, 3, and 7 days later. Evaluation of the protective action of LGM2605 was performed at 24 h post-challenge, which was selected as the peak acute inflammatory response to CuO-NP. LGM2605 was given daily via gavage to mice starting 2 days prior to the time of the insult (100 mg/kg). CuO-NPs induced a significant inflammatory influx, inflammasome-relevant cytokine release, and chlorination damage in mouse lungs, which was mitigated by the action of LGM2605. Preventive action of LGM2605 ameliorated the adverse effects of CuO-NP in lung.

Assessment of a Small Molecule Synthetic Lignan in Enhancing Oxidative Balance and Decreasing Lipid Accumulation in Human Retinal Pigment Epithelia.

Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 ß release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 ß release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.

Phase II Trial of Flaxseed to Prevent Acute Complications After Chemoradiation for Lung Cancer.

Background: Thoracic radiotherapy is complicated by acute radiation-induced adverse events such as radiation pneumonitis (RP) and radiation esophagitis (RE). Based on preclinical work and a randomized pilot trial from our laboratory, this single-arm phase II trial investigated administering flaxseed as a radioprotector in patients receiving definitive chemoradiation for nonsmall cell lung cancer (NSCLC). Methods: Between June 2015 and February 2018, 33 patients with locally advanced or metastatic NSCLC with planned definitive chemoradiation were enrolled. Finely-ground Linum usitatissimum L. (Linaceae; flaxseed or linseed) in 40-g packets were provided for daily consumption in any patient-desired formulation 1 week before radiotherapy and throughout radiotherapy as tolerated. The primary outcomes were overall adverse events, with particular focus on Grade ≥3 RP, and flaxseed tolerability. Adverse events were graded according to CTCAE v4.0. Results: Of the 33 patients enrolled, 5 patients (15%) did not receive chemoradiation, 4 (12%) withdrew promptly after enrollment, 4 (12%) did not return a flaxseed consumption log, and 1 patient had irritable bowel syndrome (3%). The remaining 19 patients (57%) had chemoradiation and flaxseed ingestion with a mean completion and standard deviation of the intended flaxseed course of 62% ± 8.3%. Nine (50%) of these 19 patients reported difficulties with flaxseed consumption, citing nausea, constipation, odynophagia, or poor taste or texture. One patient (5%), with unverifiable flaxseed consumption, developed Grade 3 RP. There were no cases of Grade 2 RP. Six patients (32%) developed Grade 2 RE, but no patients developed Grade ≥3 RE. Median overall and progression-free survival were 31 and 12 months, respectively. Conclusions: Despite the low incidence of acute radiation-induced complications reported, significant treatment-related gastrointestinal toxicities and subsequently low flaxseed tolerability inhibit accurate determination of flaxseed effect in patients receiving concurrent thoracic chemoradiation. Thus, further investigations should focus on optimizing flaxseed formulation for improved tolerability and evaluation. ClinicalTrials.gov ID: NCT02475330.

KLF5 Is Induced by FOXO1 and Causes Oxidative Stress and Diabetic Cardiomyopathy.

RATIONALE: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes, accompanied by altered cardiac energetics, impaired mitochondrial function, and oxidative stress. Previous studies indicate that type-1 diabetes is associated with increased cardiac expression of KLF5 (Krüppel-like factor-5) and PPARα (peroxisome proliferator-activated receptor) that regulate cardiac lipid metabolism. OBJECTIVE: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. METHODS AND RESULTS: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and were higher compared with nondiabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 (Forkhead box protein O1) deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacological gain and loss of KLF5 function approaches and AAV (adeno-associated virus)-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 (NADPH oxidase 4) expression. This was accompanied by accumulation of cardiac ceramides. Pharmacological or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation, and improved cardiac function in diabetic mice. CONCLUSIONS: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation, and causes DbCM.

Synthetic Secoisolariciresinol Diglucoside Attenuates Established Pain, Oxidative Stress and Neuroinflammation in a Rodent Model of Painful Radiculopathy.

Painful cervical radiculopathy is characterized by chronic neuroinflammation that lowers endogenous antioxidant responses leading to the development of oxidative stress and pain after neural trauma. Therefore, antioxidants such as secoisolariciresinol diglucoside (SDG), that promote antioxidant signaling and reduce oxidative damage may also provide pain relief. This study investigated if repeated systemic administration of synthetic SDG after a painful root compression reduces the established pain, oxidative stress and spinal glial activation that are typically evident. SDG was administered on days 1-3 after compression and the extent of oxidative damage in the dorsal root ganglia (DRG) and spinal cord was measured at day 7 using the oxidative stress markers 8-hydroxguanosine (8-OHG) and nitrotyrosine. Spinal microglial and astrocytic activation were also separately evaluated at day 7 after compression. In addition to reducing pain, SDG treatment reduced both spinal 8-OHG and nitrotyrosine, as well as peripheral 8-OHG in the DRG. Moreover, SDG selectively reduced glial activation by decreasing the extent of astrocytic but not microglial activation. These findings suggest that synthetic SDG may attenuate existing radicular pain by suppressing the oxidative stress and astrocytic activation that develop after painful injury, possibly identifying it as a potent therapeutic for painful radiculopathies.

Radiation activates myeloperoxidase (MPO) to generate active chlorine species (ACS) via a dephosphorylation mechanism - inhibitory effect of LGM2605.

BACKGROUND: Radiation exposure of tissues is associated with inflammatory cell influx. Myeloperoxidase (MPO) is an enzyme expressed in granulocytes, such as neutrophils (PMN) and macrophages, responsible for active chlorine species (ACS) generation. The present study aimed to: 1) determine whether exposure to γ-irradiation induces MPO-dependent ACS generation in murine PMN; 2) elucidate the mechanism of radiation-induced ACS generation; and 3) evaluate the effect of the synthetic lignan LGM2605, known for ACS scavenging properties. METHODS: MPO-dependent ACS generation was determined by using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF) and a highly potent MPO inhibitor, 4-aminobenzoic acid hydrazide (ABAH), and confirmed in PMN derived from MPO-/- mice. Radiation-induced MPO activation was determined by EPR spectroscopy and computational analysis identified tyrosine, serine, and threonine residues near MPO's active site. RESULTS: γ-radiation increased MPO-dependent ACS generation dose-dependently in human MPO and in wild-type murine PMN, but not in PMN from MPO-/- mice. LGM2605 decreased radiation-induced, MPO-dependent ACS. Protein tyrosine phosphatase (PTP) and protein serine/threonine phosphatase (PSTP) inhibitors decreased the radiation-induced increase in ACS. Peroxidase cycle results demonstrate that tyrosine phosphorylation blocks MPO Compound I formation by preventing catalysis on H2O2 in the active site of MPO. EPR data demonstrate that γ-radiation increased tyrosyl radical species formation in a dose-dependent manner. CONCLUSIONS: We demonstrate that γ-radiation induces MPO-dependent generation of ACS, which is dependent, at least in part, by protein tyrosine and Ser/Thr dephosphorylation and is reduced by LGM2605. This study identified for the first time a novel protein dephosphorylation-dependent mechanism of radiation-induced MPO activation.

Ozone-induced enhancement of airway hyperreactivity in rhesus macaques: Effects of antioxidant treatment.

BACKGROUND: Ozone (O3) inhalation elicits airway inflammation and impairs treatment responsiveness in asthmatic patients. The underlying immune mechanisms have been difficult to study because of the lack of relevant experimental models. Rhesus macaques spontaneously have asthma and have a similar immune system to human subjects. OBJECTIVES: We sought to investigate mucosal immune changes after O3 inhalation in a clinically relevant nonhuman primate asthma model and to study the effects of an antioxidant synthetic lignan (synthetic secoisolariciresinol diglucoside [LGM2605]). METHODS: A cohort of macaques (n = 17) previously characterized with airway hyperreactivity (AHR) to methacholine was assessed (day 1). Macaques were treated (orally) with LGM2605 (25 mg/kg) or placebo twice per day for 7 days, exposed to 0.3 ppm O3 or air for 6 hours (on day 7), and studied 12 hours later (day 8). Lung function, blood and bronchoalveolar lavage (BAL) fluid immune cell profile, and bronchial brushing and blood cell mRNA expression were assessed. RESULTS: O3 induced significant BAL fluid neutrophilia and eosinophilia and increased AHR and expression of IL6 and IL25 mRNA in the airway epithelium together with increased BAL fluid group 2 innate lymphoid cell (ILC2s), CD1c+ myeloid dendritic cell, and CD4+ T-cell counts and diminished surfactant protein D expression. Although LGM2605 attenuated some of the immune and inflammatory changes, it completely abolished O3-induced AHR. CONCLUSION: ILC2s, CD1c+ myeloid dendritic cells, and CD4+ T cells are selectively involved in O3-induced asthma exacerbation. The inflammatory changes were partially prevented by antioxidant pretreatment with LGM2605, which had an unexpectedly disproportionate protective effect on AHR.

An inflammatory pulmonary insult post-traumatic brain injury worsens subsequent spatial learning and neurological outcomes.

BACKGROUND: Severe traumatic brain injury (TBI) patients are at high risk for early aspiration and pneumonia. How pneumonia impacts neurological recovery after TBI is not well characterized. We hypothesized that, independent of the cerebral injury, pneumonia after TBI delays and worsens neurological recovery and cognitive outcomes. METHODS: Fifteen CD1 male mice were randomized to sham craniotomy or severe TBI (controlled cortical impact [CCI] - velocity 6 m/s, depth 1.0 mm) ± intratracheal lipopolysaccharide (LPS-2 mg/kg in 0.1 mL saline) as a pneumonia bioeffector. Neurological functional recovery by Garcia Neurologic Testing (GNT) and body weight loss were recorded daily for 14 days. On Days 6-14, animals underwent Morris Water Maze learning and memory testing with cued trials (platform visible), spatial learning trials (platform invisible, spatial cues present), and probe (memory) trials (platform removed, spatial clues present). Intergroup differences were assessed by the Kruskal-Wallis test with Bonferroni correction (p < 0.05). RESULTS: Weight loss was greatest in the CCI + LPS group (maximum 24% on Day 3 vs. 8% [Sham], 7% [CCI], both on Day 1). GNT was lowest in CCI + LPS during the first week. Morris Water Maze testing demonstrated greater spatial learning impairment in the CCI + LPS group vs. Sham or CCI counterparts. Cued learning and long-term memory were worse in CCI + LPS and CCI as compared to Sham. CONCLUSION: A pneumonia bioeffector insult after TBI worsens weight loss and mortality in a rodent model. Not only is spatial learning impaired, but animals are more debilitated and have worse neurologic performance. Understanding the adverse effects of pneumonia on TBI recovery is the first step d patients.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits Libby amphibole fiber-induced acute inflammation in mice.

BACKGROUND: Exposure to the Libby amphibole (LA) asbestos-like fibers found in Libby, Montana, is associated with inflammatory responses in mice and humans, and an increased risk of developing mesothelioma, asbestosis, pleural disease, and systemic autoimmune disease. Flaxseed-derived secoisolariciresinol diglucoside (SDG) has anti-inflammatory, anti-fibrotic, and antioxidant properties. We have previously identified potent protective properties of SDG against crocidolite asbestos exposure modeled in mice. The current studies aimed to extend those findings by evaluating the immunomodulatory effects of synthetic SDG (LGM2605) on LA-exposed mice. METHODS: Male and female C57BL/6 mice were given LGM2605 via gavage initiated 3 days prior to and continued for 3 days after a single intraperitoneal dose of LA fibers (200 µg) and evaluated on day 3 for inflammatory cell influx in the peritoneal cavity using flow cytometry. RESULTS: LA exposure induced a significant increase (p < 0.0001) in spleen weight and peritoneal influx of white blood cells, all of which were reduced with LGM2605 with similar trends among males and females. Levels of peritoneal PMN cells were significantly (p < 0.0001) elevated post LA exposure, and were significantly (p < 0.0001) blunted by LGM2605. Importantly, LGM2605 significantly ameliorated the LA-induced mobilization of peritoneal B1a B cells. CONCLUSIONS: LGM2605 reduced LA-induced acute inflammation and WBC trafficking supporting its possible use in mitigating downstream LA fiber-associated diseases. SUMMARY: Following acute exposure to Libby amphibole (LA) asbestos-like fibers, synthetic SDG (LGM2605), a small synthetic molecule, significantly reduced the LA-induced increase in spleen weight and peritoneal inflammation in C57BL/6 male and female mice. Our findings highlight that LGM2605 has immunomodulatory properties and may, thus, likely be a chemopreventive agent for LA-induced diseases.

Chemically synthesized Secoisolariciresinol diglucoside (LGM2605) improves mitochondrial function in cardiac myocytes and alleviates septic cardiomyopathy.

Sepsis is the overwhelming systemic immune response to infection, which can result in multiple organ dysfunction and septic shock. Myocardial dysfunction during sepsis is associated with advanced disease and significantly increased in-hospital mortality. Our group has shown that energetic failure and excess reactive oxygen species (ROS) generation constitute major components of myocardial dysfunction in sepsis. Because ROS production is central to cellular metabolic health, we tested if the synthetic anti-oxidant lignan secoisolariciresinol diglucoside (SDG; LGM2605) would alleviate septic cardiac dysfunction and investigated the underlying mechanism. Using the cecal ligation and puncture (CLP) mouse model of peritonitis-induced sepsis, we observed impairment of cardiac function beginning at 4 h post-CLP surgery. Treatment of mice with LGM2605 (100 mg/kg body weight, i.p.) 6 h post-CLP surgery reduced cardiac ROS accumulation and restored cardiac function. Assessment of mitochondrial respiration (Seahorse XF) in primary cardiomyocytes obtained from adult C57BL/6 mice that had undergone CLP and treatment with LGM2605 showed restored basal and maximal respiration, as well as preserved oxygen consumption rate (OCR) associated with spare capacity. Further analyses aiming to identify the cellular mechanisms that may account for improved cardiac function showed that LGM2605 restored mitochondria abundance, increased mitochondrial calcium uptake and preserved mitochondrial membrane potential. In addition to protecting against cardiac dysfunction, daily treatment with LGM2605 and antibiotic ertapenem (70 mg/kg) protected against CLP-associated mortality and reversed hypothermia when compared against mice receiving ertapenem and saline. Therefore, treatment of septic mice with LGM2605 emerges as a novel pharmacological approach that reduces cardiac ROS accumulation, protects cardiac mitochondrial function, alleviates cardiac dysfunction, and improves survival.

LGM2605 Reduces Space Radiation-Induced NLRP3 Inflammasome Activation and Damage in In Vitro Lung Vascular Networks.

Updated measurements of charged particle fluxes during the transit from Earth to Mars as well as on site measurements by Curiosity of Martian surface radiation fluxes identified potential health hazards associated with radiation exposure for human space missions. Designing mitigation strategies of radiation risks to astronauts is critical. We investigated radiation-induced endothelial cell damage and its mitigation by LGM2605, a radioprotector with antioxidant and free radical scavenging properties. We used an in vitro model of lung vascular networks (flow-adapted endothelial cells; FAECs), exposed to gamma rays, low/higher linear energy transfer (LET) protons (3⁻4 or 8⁻10 keV/µm, respectively), and mixed field radiation sources (gamma and protons), given at mission-relevant doses (0.25 gray (Gy)⁻1 Gy). We evaluated endothelial inflammatory phenotype, NLRP3 inflammasome activation, and oxidative cell injury. LGM2605 (100 µM) was added 30 min post radiation exposure and gene expression changes evaluated 24 h later. Radiation induced a robust increase in mRNA levels of antioxidant enzymes post 0.25 Gy and 0.5 Gy gamma radiation, which was significantly decreased by LGM2605. Intercellular cell adhesion molecule-1 (ICAM-1) and NOD-like receptor protein 3 (NLRP3) induction by individual or mixed-field exposures were also significantly blunted by LGM2605. We conclude that LGM2605 is a likely candidate to reduce tissue damage from space-relevant radiation exposure.

Applications of Out of Body Lung Perfusion.

RATIONALE AND OBJECTIVES: Out of body organ perfusion is a concept that has been around for a long time. As technology has evolved, so have the systems available for out of body perfusion making whole organ preservation for extended evaluation, resuscitation, and discovery routine. MATERIALS AND METHODS: Clinical use of ex vivo lung perfusion (EVLP) systems has continued to expand as evidence has accumulated to suggest EVLP transplants experience similar mortality, ICU length of stay, length of mechanical ventilation, hospital length of stay, and rates of primary graft dysfunction as conventional lung transplants. In 2017, more lung transplants were performed than any previous year in the US history. RESULTS: Early success of EVLP has motivated groups to evaluate additional donor types and methods for expanding the donor pool. The ability to keep a lung alive in a physiologically neutral environment opens the ability to better understand organ quality, define pathophysiology in certain disease conditions, and provides a platform for interventions to prevent or repair injury. CONCLUSION: The next several years will usher in significant changes in understanding and interventions focused on lung injury. This manuscript highlights applications of EVLP to clarify how this system can be used for basic and translational research.

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae.

One of the goals of nanomedicine is targeted delivery of therapeutic enzymes to the sub-cellular compartments where their action is needed. Endothelial caveolae-derived endosomes represent an important yet challenging destination for targeting, in part due to smaller size of the entry aperture of caveolae (ca. 30-50 nm). Here, we designed modular, multi-molecular, ferritin-based nanocarriers with uniform size (20 nm diameter) for easy drug-loading and targeted delivery of enzymatic cargo to these specific vesicles. These nanocarriers targeted to caveolar Plasmalemmal Vesicle-Associated Protein (Plvap) deliver superoxide dismutase (SOD) into endosomes in endothelial cells, the specific site of influx of superoxide mediating by such pro-inflammatory signaling as some cytokines and lipopolysaccharide (LPS). Cell studies showed efficient internalization of Plvap-targeted SOD-loaded nanocarriers followed by dissociation from caveolin-containing vesicles and intracellular transport to endosomes. The nanocarriers had a profound protective anti-inflammatory effect in an animal model of LPS-induced inflammation, in agreement with the characteristics of their endothelial uptake and intracellular transport, indicating that these novel, targeted nanocarriers provide an advantageous platform for caveolae-dependent delivery of biotherapeutics.

Oxidative Stress and Space Biology: An Organ-Based Approach.

The environment of space provides many challenges to the human physiology and therefore to extended habitation and exploration[...].

Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605) Reduces Asbestos-Induced Cytotoxicity in an Nrf2-Dependent and -Independent Manner.

Asbestos exposure triggers inflammatory processes associated with oxidative stress and tissue damage linked to malignancy. LGM2605 is the synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant, and anti-inflammatory properties in diverse inflammatory cell and mouse models, including exposure to asbestos fibers. Nuclear factor-E2 related factor 2 (Nrf2) activation and boosting of endogenous tissue defenses were associated with the protective action of LGM2605 from asbestos-induced cellular damage. To elucidate the role of Nrf2 induction by LGM2605 in protection from asbestos-induced cellular damage, we evaluated LGM2605 in asbestos-exposed macrophages from wild-type (WT) and Nrf2 disrupted (Nrf2-/-) mice. Cells were pretreated with LGM2605 (50 µM and 100 µM) and exposed to asbestos fibers (20 µg/cm²) and evaluated 8 h and 24 h later for inflammasome activation, secreted cytokine levels (interleukin-1ß (IL-1ß), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNFα)), cytotoxicity and cell death, nitrosative stress, and Nrf2-regulated enzyme levels. Asbestos exposure induced robust oxidative and nitrosative stress, cell death and cytotoxicity, which were equally mitigated by LGM2605. Inflammasome activation was significantly attenuated in Nrf2-/- macrophages compared to WT, and the protective action of LGM2605 was seen only in WT cells. In conclusion, in a cell model of asbestos-induced toxicity, LGM2605 acts via protective mechanisms that may not involve Nrf2 activation.

Synthetic secoisolariciresinol diglucoside (LGM2605) inhibits myeloperoxidase activity in inflammatory cells.

BACKGROUND: Myeloperoxidase (MPO) generates hypochlorous acid (HOCl) during inflammation and infection. We showed that secoisolariciresinol diglucoside (SDG) scavenges radiation-induced HOCl in physiological solutions. However, the action of SDG and its synthetic version, LGM2605, on MPO-catalyzed generation of HOCl is unknown. The present study evaluated the effect of LGM2605 on human MPO, and murine MPO from macrophages and neutrophils. METHODS: MPO activity was determined fluorometrically using hypochlorite-specific 3'-(p-aminophenyl) fluorescein (APF). The effect of LGM2605 on (a) the peroxidase cycle of MPO was determined using Amplex Red while the effect on (b) the chlorination cycle was determined using a taurine chloramine assay. Using electron paramagnetic resonance (EPR) spectroscopy we determined the effect of LGM2605 on the EPR signals of MPO. Finally, computational docking of SDG was used to identify energetically favorable docking poses to enzyme's active site. RESULTS: LGM2605 inhibited human and murine MPO activity. MPO inhibition was observed in the absence and presence of Cl-. EPR confirmed that LGM2605 suppressed the formation of Compound I, an oxoiron (IV) intermediate [Fe(IV)O] containing a porphyrin π-radical of MPO's catalytic cycle. Computational docking revealed that SDG can act as an inhibitor by binding to the enzyme's active site. CONCLUSIONS: We conclude that LGM2605 inhibits MPO activity by suppressing both the peroxidase and chlorination cycles. EPR analysis demonstrated that LGM2605 inhibits MPO by decreasing the formation of the highly oxidative Compound I. This study identifies a novel mechanism of LGM2605 action as an inhibitor of MPO and indicates that LGM2605 may be a promising attenuator of oxidant-dependent inflammatory tissue damage.

Targeting superoxide dismutase to endothelial caveolae profoundly alleviates inflammation caused by endotoxin.

Inflammatory mediators binding to Toll-Like receptors (TLR) induce an influx of superoxide anion in the ensuing endosomes. In endothelial cells, endosomal surplus of superoxide causes pro-inflammatory activation and TLR4 agonists act preferentially via caveolae-derived endosomes. To test the hypothesis that SOD delivery to caveolae may specifically inhibit this pathological pathway, we conjugated SOD with antibodies (Ab/SOD, size ~10nm) to plasmalemmal vesicle-associated protein (Plvap) that is specifically localized to endothelial caveolae in vivo and compared its effects to non-caveolar target CD31/PECAM-1. Plvap Ab/SOD bound to endothelial cells in culture with much lower efficacy than CD31 Ab/SOD, yet blocked the effects of LPS signaling with higher efficiency than CD31 Ab/SOD. Disruption of cholesterol-rich membrane domains by filipin inhibits Plvap Ab/SOD endocytosis and LPS signaling, implicating the caveolae-dependent pathway(s) in both processes. Both Ab/SOD conjugates targeted to Plvap and CD31 accumulated in the lungs after IV injection in mice, but the former more profoundly inhibited LPS-induced pulmonary inflammation and elevation of plasma level of interferon-beta and -gamma and interleukin-27. Taken together, these results indicate that targeted delivery of SOD to specific cellular compartments may offer effective, mechanistically precise interception of pro-inflammatory signaling mediated by reactive oxygen species.