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Amifostine prevents agonist-induced lung EC cytoskeletal remodeling and adherens junction disruption
Source publication
Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. The present study investigated the effect of pre-treatment with amifostine, a thiol antioxidant compound, on lung endothelial barrier dysfunction induced by Gram-negative bacteria...
Context in source publication
Context 1
... assess effects of amifostine on cytoskeletal rearrangement associated with EC dysfunction induced by H 2 O 2 , LPS, or IL-6, we performed immunofluorescence staining of human pulmonary EC stimulated with these agonists with or without amifostine WR-1065 pre-treatment. In unstimulated cells, F-actin was primarily organized into actin bundles which were similar in cells treated with WR-1065 (4 mM) alone ( Figure 2A). After 30 min of H 2 O 2 (250 μM) stimulation, or 6 hrs of stimulation with LPS (200 ng/ml) or IL-6 (25 ng/ ml combined with IL6-SR, 100ng/ml), F-actin was reorganized into thicker stress fibers in the center of the cells, whereas peripheral actin rim was significantly weakened. ...
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Citations
... It functions as an oxygen and ROS scavenger. We previously showed that amifostine protected mice from lipopolysaccharide (LPS)induced acute lung injury (ALI) and ventilator-induced lung injury (VILI) [21,22]. In the present study, we reasoned that amifostine may inhibit fibrosis in a bleomycininduced murine pulmonary fibrosis model by reducing oxidative stress and protecting mitochondrial dysfunction. ...
... BAL cytokine levels were measured by ProcartaPlex ™ Multiplex Immunoassay (Invitrogen, EPX360-26092-901) with a Luminex ™ 100 instrument according to the manufacture's instructions. The pellets from the first centrifuge were resuspended in PBS and the total and differential cell counts analyzed as previously described [21]. ...
Background
Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disease characterized by irreversible scarring of the lung parenchyma. Despite various interventions aimed at mitigating several different molecular aspects of the disease, only two drugs with limited clinical efficacy have so far been approved for IPF therapy.
Objective
We investigated the therapeutic efficacy of amifostine, a detoxifying drug clinically used for radiation-caused cytotoxicity, in bleomycin-induced murine pulmonary fibrosis.
Methods
C57BL6/J mice were intratracheally instilled with 3 U/kg of bleomycin. Three doses of amifostine (WR-2721, 200 mg/kg) were administered intraperitoneally on days 1, 3, and 5 after the bleomycin challenge. Bronchoalveolar lavage fluid (BALF) was collected on day 7 and day 21 for the assessment of lung inflammation, metabolites, and fibrotic injury. Human fibroblasts were treated in vitro with transforming growth factor beta 1 (TGF-β1), followed by amifostine (WR-1065, 1–4 µg/mL) treatment. The effects of TGF-β1 and amifostine on the mitochondrial production of reactive oxygen species (ROS) were assessed by live cell imaging of MitoSOX. Cellular metabolism was assessed by the extracellular acidification rate (ECAR), the oxygen consumption rate (OCR), and the concentrations of various energy-related metabolites as measured by mass spectrum (MS). Western blot analysis was performed to investigate the effect of amifostine on sirtuin 1 (SIRT1) and adenosine monophosphate activated kinase (AMPK).
Results
Three doses of amifostine significantly attenuated lung inflammation and pulmonary fibrosis. Pretreatment and post-treatment of human fibroblast cells with amifostine blocked TGF-β1-induced mitochondrial ROS production and mitochondrial dysfunction in human fibroblast cells. Further, treatment of fibroblasts with TGF-β1 shifted energy metabolism away from mitochondrial oxidative phosphorylation (OXPHOS) and towards glycolysis, as observed by an altered metabolite profile including a decreased ratio of NAD + /NADH and increased lactate concentration. Treatment with amifostine significantly restored energy metabolism and activated SIRT1, which in turn activated AMPK. The activation of AMPK was required to mediate the effects of amifostine on mitochondrial homeostasis and pulmonary fibrosis. This study provides evidence that repurposing of the clinically used drug amifostine may have therapeutic applications for IPF treatment.
Conclusion
Amifostine inhibits bleomycin-induced pulmonary fibrosis by restoring mitochondrial function and cellular metabolism.
... As discussed above, we have shown that hematopoietic cells and myeloid cells contribute to ~50% of the activation of coagulation in a mouse model of endotoxemia 97,102 . In addition, increased vascular permeability, which occurs during endotoxemia and sepsis 130,131 , will expose perivascular TF to blood and this will also contribute to the activation of coagulation. Clearly, additional studies are needed to resolve the different observations and to determine the cellular sources of TF that activates coagulation following activation of inflammasomes. ...
The coagulation system is a part of the mammalian host defense system. Pathogens and pathogen components, such as bacterial lipopolysaccharide (LPS), induce tissue factor (TF) expression in circulating monocytes that then activates the coagulation protease cascade. Formation of a clot limits dissemination of pathogens, enhances the recruitment of immune cells and facilitates killing of pathogens. However, excessive activation of coagulation can lead to thrombosis. Here, we review studies on the mechanism of LPS induction of TF expression in monocytes and its contribution to thrombosis and disseminated intravascular coagulation. Binding of LPS to Toll-like receptor 4 on monocytes induces a transient TF expression that involves activation of intracellular signaling pathways and binding of various transcription factors, such as c-rel/p65 and c-Fos/c-Jun, to the TF promoter. Inhibition of TF in endotoxemia and sepsis models reduces activation of coagulation and improves survival. Studies with endotoxemic mice showed that hematopoietic cells and myeloid cells play major roles in the activation of coagulation. Monocyte TF expression is also increased after surgery. Activated monocytes release TF-positive extracellular vesicles (EVs) and levels of circulating TF-positive EVs are increased in endotoxemic mice and in patients with sepsis. More recently, it was shown that inflammasomes contribute to the induction of TF expression and activation of coagulation in endotoxemic mice. Taken together, these studies indicate that monocyte TF plays a major role in the LPS-induced activation of coagulation. Selective inhibition of monocyte TF expression may reduce pathologic activation of coagulation in sepsis and other diseases.
... Radiation-induced pneumonitis is characterized by hyperpermeability of the epithelial and alveolar-capillary barrier, resulting in leukocyte infiltration and extravasation of the vascular fluid into the lung tissue [38]. A 7.5-fold increase in the fluorescence intensity of plasma FITC-dextran, suggestive of enhanced lung permeability due to epithelial barrier dysfunction, was found at 12 weeks following irradiation, which was reduced by 50% in mice pretreated with DRDE-30 (Fig. 5A). ...
... Radiation-induced oxidative stress activates various signaling molecules, which are intimately linked to the regulation of innate and adaptive immune functions, endothelial cell permeability, and inflammation [38,41]. Thoracic irradiation resulted in a 40% increase in the level of phosphorylated p38 and an 82% increase in the level of the p65 (active) subunit of NF-κB (Fig. 6D). ...
Background
Radiotherapy of thoracic neoplasms and accidental radiation exposure often results in pneumonitis and fibrosis of lungs. Here, we investigated the potential of amifostine analogs: DRDE-07, DRDE-30, and DRDE-35, in alleviating radiation-induced lung damage.
Methods
C57BL/6 mice were exposed to 13.5 Gy thoracic irradiation, 30 min after intraperitoneal administration of the analogs, and assessed for modulation of the pathological response at 12 and 24 weeks.
Key findings
DRDE-07, DRDE-30 and DRDE-35 increased the survival of irradiated mice from 20% to 30%, 80% and 70% respectively. Reduced parenchymal opacity (X-ray CT) in the lungs of DRDE-30 pre-treated mice corroborated well with the significant decrease in Ashcroft score (p < 0.01). Two-fold increase in SOD and catalase activities (p < 0.05), coupled with a 50% increase in GSH content and a 60% decrease in MDA content (p < 0.05) suggested restoration of the antioxidant defence system. A 20% to 40% decrease in radiation-induced apoptotic and mitotic death in the lung tissue (micronuclei: p < 0.01), resulted in attenuated lung and vascular permeability (FITC-Dextran leakage) by 50% (p < 0.01), and a commensurate reduction (~50%) in leukocyte infiltration in the injured tissue (p < 0.05). DRDE-30 abrogated the activation of pro-inflammatory NF-κB and p38/MAPK signaling cascades, suppressing the release of pro-inflammatory cytokines (IL-1β: p < 0.05; TNF-α: p < 0.05; IL-6: p < 0.05) and up-regulation of CAMs on the endothelial cell surface. Reduction in hydroxyproline content (p < 0.01) and collagen suggested inhibition of lung fibrosis which was associated with attenuation of TGF-β/Smad pathway-mediated-EMT.
Conclusion
DRDE-30 could be a potential prophylactic agent against radiation-induced lung injury.
... Rapid onset and short duration of action can be noticed in the acute form, which is facilitated by the excretion of numerous cytokines including interleukin-1 (IL-1), IL-6, IL-11, IL-8, and tumor necrosis factor-alpha (TNF-α) [1,2]. Nevertheless, in chronic inflammation, persistence of the inflammatory reactions could induce the migration of lymphocytes and macrophages to the damaged tissues [3]. Chronic inflammatory responses have been associated with the progression of various diseases such as asthma, arthritis, and neurodegenerative disorders [4]. ...
Objective:
Oxidative stress-mediated inflammatory events involve in the progress of several diseases such as asthma, cancers, and multiple sclerosis. Auraptene (AU), a natural prenyloxycoumarin, possesses numerous pharmacological activities. Here, the anti-inflammatory effects of AU were investigated in lipoteichoic acid- (LTA-) induced macrophage cells (RAW 264.7).
Methods:
The expression of cyclooxygenase (COX-2), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, c-Jun N-terminal kinase (JNK), heme oxygenase (HO-1), p65, and IκBα were all identified by western blotting assay. The level of nitric oxide (NO) was measured by spectrometer analysis. The nuclear translocation of p65 nuclear factor kappa B (NF-κB) was assessed by the confocal microscopic staining method. Native polyacrylamide gel electrophoresis was performed to perceive the activity of antioxidant enzyme catalase (CAT).
Results:
AU expressively reduced NO production and COX-2, TNF-α, IL-1 β, and iNOS expression in LTA-stimulated cells. AU at higher concentration (10 µM) inhibited ERK and JNK, but not p38 phosphorylation induced by LTA. Moreover, AU blocked IκB and p65 phosphorylation, and p65 nuclear translocation. However, AU pretreatment was not effective on antioxidant HO-1 expression, CAT activity, and reduced glutathione (GSH, a nonenzymatic antioxidant), in LTA-induced RAW 264.7 cells.
Conclusion:
The findings of this study advocate that AU shows anti-inflammatory effects via reducing NF-κB/MAPKs signaling pathways.
... We have shown that c-Abl-mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury 13 . While LPS-induced ROS also modulates endothelial cell permeability 19 , whether mitochondrial fission/mtROS play a role in LPSinduced endothelial dysfunction is unknown. As shown in Fig. 8 These results suggest that LPS-induced barrier disruption is mediated by DRP1 activation and mtROS generation. ...
We have shown that both reactive oxygen species (ROS) and paxillin tyrosine phosphorylation regulate LPS-induced human lung endothelial permeability. Mitochondrial ROS (mtROS) is known to increase endothelial cell (EC) permeability which requires dynamic change in mitochondrial morphology, events that are likely to be regulated by paxillin. Here, we investigated the role of paxillin and its tyrosine phosphorylation in regulating LPS-induced mitochondrial dynamics, mtROS production and human lung microvascular EC (HLMVEC) dysfunction. LPS, in a time-dependent manner, induced higher levels of ROS generation in the mitochondria compared to cytoplasm or nucleus. Down-regulation of paxillin expression with siRNA or ecto-expression of paxillin Y31F or Y118F mutant plasmids attenuated LPS-induced mtROS in HLMVECs. Pre-treatment with MitoTEMPO, a scavenger of mtROS, attenuated LPS-induced mtROS, endothelial permeability and VE-cadherin phosphorylation. Further, LPS-induced mitochondrial fission in HLMVECs was attenuated by both a paxillin siRNA, and paxillin Y31F/Y118F mutant. LPS stimulated phosphorylation of dynamin-related protein (DRP1) at S616, which was also attenuated by paxillin siRNA, and paxillinY31/Y118 mutants. Inhibition of DRP1 phosphorylation by P110 attenuated LPS-induced mtROS and endothelial permeability. LPS challenge of HLMVECs enhanced interaction between paxillin, ERK, and DRP1, and inhibition of ERK1/2 activation with PD98059 blocked mitochondrial fission. Taken together, these results suggest a key role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, mtROS generation and EC barrier dysfunction.
... For the evaluation of lung injury parameters, BAL fluid was collected after intratracheal injection of 1 mL of sterile Hanks balanced salt buffer and total cells and protein content in BAL was measured as described previously. 45 The vascular leak was analyzed by injecting Evans blue dye (30 mg/kg) into the external jugular vein 2 hours before the end of the experiment as described earlier 45 and following perfusion excised lungs were imaged by a Kodak digital camera. ...
... For the evaluation of lung injury parameters, BAL fluid was collected after intratracheal injection of 1 mL of sterile Hanks balanced salt buffer and total cells and protein content in BAL was measured as described previously. 45 The vascular leak was analyzed by injecting Evans blue dye (30 mg/kg) into the external jugular vein 2 hours before the end of the experiment as described earlier 45 and following perfusion excised lungs were imaged by a Kodak digital camera. ...
Suppressors of cytokine signaling (SOCS) provide negative regulation of inflammatory reaction. The role and precise cellular mechanisms of SOCS1 in control of endothelial dysfunction and barrier compromise associated with acute lung injury remain unexplored. Our results show that siRNA‐mediated SOCS1 knockdown augmented lipopolysaccharide (LPS)‐induced pulmonary endothelial cell (EC) permeability and enhanced inflammatory response. Consistent with in vitro data, EC‐specific SOCS1 knockout mice developed more severe lung vascular leak and accumulation of inflammatory cells in bronchoalveolar lavage fluid. SOCS1 overexpression exhibited protective effects against LPS‐induced endothelial permeability and inflammation, which were dependent on microtubule (MT) integrity. Biochemical and image analysis of unstimulated EC showed SOCS1 association with the MT, while challenge with LPS or MT depolymerizing agent colchicine impaired this association. SOCS1 directly interacted with N2 domains of MT‐associated proteins CLIP‐170 and CLASP2. Furthermore, N‐terminal region of SOCS1 was indispensable for these interactions and SOCS1‐ΔN mutant lacking N‐terminal 59 amino acids failed to rescue LPS‐induced endothelial dysfunction. Depletion of endogenous CLIP‐170 or CLASP2 abolished SOCS1 interaction with Toll‐like receptor‐4 and Janus kinase‐2 leading to impairment of SOCS1 inhibitory effects on LPS‐induced inflammation. Altogether, these findings suggest that endothelial barrier protective and anti‐inflammatory effects of SOCS1 are critically dependent on its targeting to the MT.
... Imatinib inhibits TNF-α release by reducing the DNA binding of NFKB [89]. Amifostine is considered a thera-peutic agent of lung inflammation that acts by suppressing IL-6 induced activation of redox sensitive signaling [115]. Ribavirin inhibits the expression of TNF-α, IL-6, and IL-10 in blood lymphocytes by reduced their mRNA levels [110], [111]. ...
div>Recent studies have been demonstrated that host immune imbalance is an important factors leading to acute respiratory distress syndrome (ARDS) in COVID-19 patients. Therefore, discovery of potential drugs and identification of their mechanisms of action for the prevention of immune imbalance in COVID-19 patients are urgently needed. In this study, we proposed a network representation learning-based methodology, termed AIdrug2cov, to discover drug mechanism and anti-inflammatory response for patients with COVID19. In AIdrug2cov, a deep bidirectional Transformer encoder network representation approach is developed to automatically learn lowdimensional vector of heterogeneous network. Using the representation vectors, AIdrug2cov identifies 40 potential targets and 24 high-confidence drugs that bind to tumor necrosis factor(TNF)-α or interleukin(IL)-6 to prevent excessive inflammatory responses in COVID-19 patients. In particular, AIdrug2cov indicated that chloroquine and hydroxychloroquine are able to reduce fatality of COVID-19 patients, and that their mechanisms of action are likely mediated through their inhibition of inflammatory cytokines on top of their antiviral ability, consistent with the findings of clinical studies. In addition, the results in 5 pharmacological application suggested that AIdrug2cov significantly outperforms 5 other state-of-the-art network representation approaches, future demonstrating the availability of AIdrug2cov in drug development field. In summary, AIdrug2cov is practically useful for accelerating COVID-19 therapeutic development. The source code and data can be downloaded from https://github.com/pengsl-lab/AIdrug2cov.git</div
... In our study prophylactic treatment with amifostine and DRDE-07 significantly reduced the MPO and β-glucuronidase activity. Besides, our result also supported by an earlier study of Fu et al. (2009), in which amifostine treatment reduced the lipopolysaccharide-induced MPO activity and neutrophil accumulation in the lung parenchyma cells. 37 Chakrabarti and Patel 38 demonstrated that the MMP-9 synthesis increased under a variety of physiological and pathological conditions which is responsible for epithelial cell detachment from the basement membrane. ...
... Besides, our result also supported by an earlier study of Fu et al. (2009), in which amifostine treatment reduced the lipopolysaccharide-induced MPO activity and neutrophil accumulation in the lung parenchyma cells. 37 Chakrabarti and Patel 38 demonstrated that the MMP-9 synthesis increased under a variety of physiological and pathological conditions which is responsible for epithelial cell detachment from the basement membrane. In Results are mean ± SEM of 5 animals per group. ...
ABSTRACT
Aim: The present study was undertaken to investigate the comparative pulmonary
protective efficacy of Amifostine (S-2[3-aminoprophylamino] ethyl
phosphorothioate) and its analogues DRDE-07 (S-2(2-aminoethylamino)
ethyl phenyl sulfide) against sulfur mustard toxicity in mice. Materials and
Methods: Twenty female mice were divided into four groups: Control, SM
group animals were percutaneously exposed to 16.2 mg/kg. The third and
fourth group of animals received amifostine and DRDE-07 (210 and 250
mg/kg respectively) through the oral route, 30 min before SM exposure.
The clinical symptoms and body weight changes were observed daily and
sacrificed on 7th day. Bronchoalveolar lavage fluid (BALF) and lung tissue
were collected for biochemical and histopathological studies. The following
biochemical endpoints were studied in BALF (total cell count, lactate
dehydrogenase, protein content, β-glucuronidase activity, MMP-2, 9 activity
and FSH) whereas reactive oxygen species (ROS), reduced glutathione
(GSH), lipid peroxidation, superoxide dismutase, catalase and myeloperoxidase
activity was measured in lung tissue. The above biochemical observations
are also supported by histopathology studies. Results: Dermal
exposure to SM significantly reduced body weight. The significant increase
in BALF LDH leakage, protein content, cell number and MMPs activity in
the SM exposed animals suggest disruption of endothelial barrier in the
lung (p<0.05). Significant ROS generation (p<0.05) was observed in lung
tissue of SM group which results in a significant decrease in SOD GSH
and CAT and an increase in MDA (p<0.05). These alterations in BALF as
well in lung tissue due to SM exposure was significantly prevented by the
pretreatment of amifostine and DRDE-07 (p<0.05). The histopathological
observations also support the above results. The above results indicate that
the preventive efficacy of DRDE-07 is better than amifostine. Conclusion:
The percutaneous SM exposure-induced pulmonary damages were significantly
protected by DRDE-07 than amifostine in mice.
Keywords: Amifostine, BALF, Chemical Warfare Agents, DRDE-07, Pulmonary
injury, Oxidative stress, Sulfur mustard.
... The BAL protein concentration was determined by BCATM Protein Assay kit (Thermo Scientific, Pittsburg, PA). BAL inflammatory cell counting was performed using a standard hemacytometer technique ( Fu et al., 2009). As an additional parameter reflecting increased lung vascular leakiness, Evans blue accumulation in the lung tissue was evaluated as described elsewhere . ...
Mechanical ventilation remains an imperative treatment for the patients with acute respiratory distress syndrome, but can also exacerbate lung injury. We have previously described a key role of RhoA GTPase in high cyclic stretch (CS)-induced endothelial cell (EC) barrier dysfunction. However, cellular mechanotransduction complexes remain to be characterized. This study tested a hypothesis that recovery of a vascular EC barrier after pathologic mechanical stress may be accelerated by cell exposure to physiologic CS levels and involves Rap1-dependent rearrangement of endothelial cell junctions. Using biochemical, molecular, and imaging approaches we found that EC pre- or postconditioning at physiologically relevant low-magnitude CS promotes resealing of cell junctions disrupted by pathologic, high-magnitude CS. Cytoskeletal remodeling induced by low CS was dependent on small GTPase Rap1. Protective effects of EC preconditioning at low CS were abolished by pharmacological or molecular inhibition of Rap1 activity. In vivo, using mice exposed to mechanical ventilation, we found that the protective effect of low tidal volume ventilation against lung injury caused by lipopolysaccharides and ventilation at high tidal volume was suppressed in Rap1 knockout mice. Taken together, our results demonstrate a prominent role of Rap1-mediated signaling mechanisms activated by low CS in acceleration of lung vascular EC barrier restoration.
... After intratracheal injection of 1 ml of sterile Hanks balanced salt buffer, BAL was carried out, and total protein and cells content were measured as described previously (69). To analyze vascular leak, Evans blue dye (30 mg/kg) was injected into the external jugular vein 2 hr before the end of the experiment as described elsewhere (69). ...
... After intratracheal injection of 1 ml of sterile Hanks balanced salt buffer, BAL was carried out, and total protein and cells content were measured as described previously (69). To analyze vascular leak, Evans blue dye (30 mg/kg) was injected into the external jugular vein 2 hr before the end of the experiment as described elsewhere (69). ...
Staphylococcus aureus is a major etiological agent of sepsis and induces endothelial cell (EC) barrier dysfunction and inflammation, two major hallmarks of acute lung injury. However, the molecular mechanisms of bacterial pathogen–induced EC barrier disruption are incompletely understood. Here, we investigated the role of microtubules (MT) in the mechanisms of EC barrier compromise caused by heat-killed S. aureus (HKSA). Using a customized monolayer permeability assay in human pulmonary ECs and MT fractionation, we observed that HKSA-induced barrier disruption is accompanied by MT destabilization and increased histone deacetylase-6 (HDAC6) activity resulting from elevated reactive oxygen species (ROS) production. Molecular or pharmacological HDAC6 inhibition rescued barrier function in HKSA-challenged vascular endothelium. The HKSA-induced EC permeability was associated with impaired MT-mediated delivery of cytoplasmic linker–associated protein 2 (CLASP2) to the cell periphery, limiting its interaction with adherens junction proteins. HKSA-induced EC barrier dysfunction was also associated with increased Rho GTPase activity via activation of MT-bound Rho-specific guanine nucleotide exchange factor-H1 (GEF-H1) and was abolished by HDAC6 down-regulation. HKSA activated the NF-κB proinflammatory pathway and increased the expression of intercellular and vascular cell adhesion molecules in ECs, an effect that was also HDAC6-dependent and mediated, at least in part, by a GEF-H1/Rho-dependent mechanism. Of note, HDAC6-knockout mice or HDAC6 inhibitor–treated wild type mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus. Our results indicate that S. aureus–induced, ROS-dependent up-regulation of HDAC6 activity destabilizes MT and thereby activates the GEF-H1/Rho pathway, increasing both EC permeability and inflammation.
