When Place Matters: Shuttling of Enolase-1 Across Cellular Compartments

Enolase is a glycolytic enzyme, which catalyzes the inter-conversion of 2-phosphoglycerate to phosphoenolpyruvate. Altered expression of this enzyme is frequently observed in cancer and accounts for the Warburg effect, an adaptive response of tumor cells to hypoxia. In addition to its catalytic function, ENO-1 exhibits other activities, which strongly depend on its cellular and extracellular localization. For example, the association of ENO-1 with mitochondria membrane was found to be important for the stability of the mitochondrial membrane, and ENO-1 sequestration on the cell surface was crucial for plasmin-mediated pericellular proteolysis. The latter activity of ENO-1 enables many pathogens but also immune and cancer cells to invade the tissue, leading further to infection, inflammation or metastasis formation. The ability of ENO-1 to conduct so many diverse processes is reflected by its contribution to a high number of pathologies, including type 2 diabetes, cardiovascular hypertrophy, fungal and bacterial infections, cancer, systemic lupus erythematosus, hepatic fibrosis, Alzheimer’s disease, rheumatoid arthritis, and systemic sclerosis. These unexpected non-catalytic functions of ENO-1 and their contributions to diseases are the subjects of this review

Time-dependent changes in pulmonary surfactant function and composition in acute respiratory distress syndrome due to pneumonia or aspiration

BACKGROUND: Alterations to pulmonary surfactant composition have been encountered in the Acute Respiratory Distress Syndrome (ARDS). However, only few data are available regarding the time-course and duration of surfactant changes in ARDS patients, although this information may largely influence the optimum design of clinical trials addressing surfactant replacement therapy. We therefore examined the time-course of surfactant changes in 15 patients with direct ARDS (pneumonia, aspiration) over the first 8 days after onset of mechanical ventilation. METHODS: Three consecutive bronchoalveolar lavages (BAL) were performed shortly after intubation (T0), and four days (T1) and eight days (T2) after intubation. Fifteen healthy volunteers served as controls. Phospholipid-to-protein ratio in BAL fluids, phospholipid class profiles, phosphatidylcholine (PC) molecular species, surfactant proteins (SP)-A, -B, -C, -D, and relative content and surface tension properties of large surfactant aggregates (LA) were assessed. RESULTS: At T0, a severe and highly significant reduction in SP-A, SP-B and SP-C, the LA fraction, PC and phosphatidylglycerol (PG) percentages, and dipalmitoylation of PC (DPPC) was encountered. Surface activity of the LA fraction was greatly impaired. Over time, significant improvements were encountered especially in view of LA content, DPPC, PG and SP-A, but minimum surface tension of LA was not fully restored (15 mN/m at T2). A highly significant correlation was observed between PaO(2)/FiO(2 )and minimum surface tension (r = -0.83; p < 0.001), SP-C (r = 0.64; p < 0.001), and DPPC (r = 0.59; p = 0.003). Outcome analysis revealed that non-survivors had even more unfavourable surfactant properties as compared to survivors. CONCLUSION: We concluded that a profound impairment of pulmonary surfactant composition and function occurs in the very early stage of the disease and only gradually resolves over time. These observations may explain why former surfactant replacement studies with a short treatment duration failed to improve outcome and may help to establish optimal composition and duration of surfactant administration in future surfactant replacement studies in acute lung injury

Pulmonary siRNA Delivery with Sophisticated Amphiphilic Poly(Spermine Acrylamides) for the Treatment of Lung Fibrosis

RNA interference (RNAi) is an efficient strategy to post-transcriptionally silence gene expression. While all siRNA drugs on the market target the liver, the lung offers a variety of currently undruggable targets, which can potentially be treated with RNA therapeutics. To achieve this goal, the synthesis of poly(spermine acrylamides) (P(SpAA) is reported herein. Polymers are prepared via polymerization of N-acryloxysuccinimide (NAS) and afterward this active ester is converted into spermine-based pendant groups. Copolymerizations with decylacrylamide are employed to increase the hydrophobicity of the polymers. After deprotection, polymers show excellent siRNA encapsulation to obtain perfectly sized polyplexes at very low polymer/RNA ratios. In vitro 2D and 3D cell culture, ex vivo and in vivo experiments reveal superior properties of amphiphilic spermine-copolymers with respect to delivery of siRNA to lung cells in comparison to commonly used lipid-based transfection agents. In line with the in vitro results, siRNA delivery to human lung explants confirm more efficient gene silencing of protease-activated receptor 2 (PAR2), a G protein-coupled receptor involved in fibrosis. This study reveals the importance of the balance between efficient polyplex formation, cellular uptake, gene knockdown, and toxicity for efficient siRNA delivery in vitro, in vivo, and in fibrotic human lung tissue ex vivo

The Role of Decorin and Biglycan Signaling in Tumorigenesis

The complex and adaptive nature of malignant neoplasm constitute a major challenge for the development of effective anti-oncogenic therapies. Emerging evidence has uncovered the pivotal functions exerted by the small leucine-rich proteoglycans, decorin and biglycan, in affecting tumor growth and progression. In their soluble forms, decorin and biglycan act as powerful signaling molecules. By receptor-mediated signal transduction, both proteoglycans modulate key processes vital for tumor initiation and progression, such as autophagy, inflammation, cell-cycle, apoptosis, and angiogenesis. Despite of their structural homology, these two proteoglycans interact with distinct cell surface receptors and thus modulate distinct signaling pathways that ultimately affect cancer development. In this review, we summarize growing evidence for the complex roles of decorin and biglycan signaling in tumor biology and address potential novel therapeutic implications

Basement membrane product, endostatin, as a link between inflammation, coagulation and vascular permeability in COVID-19 and non-COVID-19 acute respiratory distress syndrome

Background: Immune cell recruitment, endothelial cell barrier disruption, and platelet activation are hallmarks of lung injuries caused by COVID-19 or other insults which can result in acute respiratory distress syndrome (ARDS). Basement membrane (BM) disruption is commonly observed in ARDS, however, the role of newly generated bioactive BM fragments is mostly unknown. Here, we investigate the role of endostatin, a fragment of the BM protein collagen XVIIIα1, on ARDS associated cellular functions such as neutrophil recruitment, endothelial cell barrier integrity, and platelet aggregation in vitro. Methods: In our study we analyzed endostatin in plasma and post-mortem lung specimens of patients with COVID-19 and non-COVID-19 ARDS. Functionally, we investigated the effect of endostatin on neutrophil activation and migration, platelet aggregation, and endothelial barrier function in vitro. Additionally, we performed correlation analysis for endostatin and other critical plasma parameters. Results: We observed increased plasma levels of endostatin in our COVID-19 and non-COVID-19 ARDS cohort. Immunohistochemical staining of ARDS lung sections depicted BM disruption, alongside immunoreactivity for endostatin in proximity to immune cells, endothelial cells, and fibrinous clots. Functionally, endostatin enhanced the activity of neutrophils, and platelets, and the thrombin-induced microvascular barrier disruption. Finally, we showed a positive correlation of endostatin with soluble disease markers VE-Cadherin, c-reactive protein (CRP), fibrinogen, and interleukin (IL)-6 in our COVID-19 cohort. Conclusion: The cumulative effects of endostatin on propagating neutrophil chemotaxis, platelet aggregation, and endothelial cell barrier disruption may suggest endostatin as a link between those cellular events in ARDS pathology

Basement membrane product, endostatin, as a link between inflammation, coagulation and vascular permeability in COVID-19 and non-COVID-19 acute respiratory distress syndrome

BackgroundImmune cell recruitment, endothelial cell barrier disruption, and platelet activation are hallmarks of lung injuries caused by COVID-19 or other insults which can result in acute respiratory distress syndrome (ARDS). Basement membrane (BM) disruption is commonly observed in ARDS, however, the role of newly generated bioactive BM fragments is mostly unknown. Here, we investigate the role of endostatin, a fragment of the BM protein collagen XVIIIα1, on ARDS associated cellular functions such as neutrophil recruitment, endothelial cell barrier integrity, and platelet aggregation in vitro.MethodsIn our study we analyzed endostatin in plasma and post-mortem lung specimens of patients with COVID-19 and non-COVID-19 ARDS. Functionally, we investigated the effect of endostatin on neutrophil activation and migration, platelet aggregation, and endothelial barrier function in vitro. Additionally, we performed correlation analysis for endostatin and other critical plasma parameters.ResultsWe observed increased plasma levels of endostatin in our COVID-19 and non-COVID-19 ARDS cohort. Immunohistochemical staining of ARDS lung sections depicted BM disruption, alongside immunoreactivity for endostatin in proximity to immune cells, endothelial cells, and fibrinous clots. Functionally, endostatin enhanced the activity of neutrophils, and platelets, and the thrombin-induced microvascular barrier disruption. Finally, we showed a positive correlation of endostatin with soluble disease markers VE-Cadherin, c-reactive protein (CRP), fibrinogen, and interleukin (IL)-6 in our COVID-19 cohort.ConclusionThe cumulative effects of endostatin on propagating neutrophil chemotaxis, platelet aggregation, and endothelial cell barrier disruption may suggest endostatin as a link between those cellular events in ARDS pathology

Altered fibrin clot structure and dysregulated fibrinolysis contribute to thrombosis risk in severe COVID-19

The high incidence of thrombotic events suggests a possible role of the contact system pathway in COVID-19 pathology. Here, we demonstrate altered levels of factor XII (FXII) and its activation products in critically ill COVID-19 patients in comparison to patients with severe acute respiratory distress syndrome due to influenza virus (ARDS-influenza). Compatible with this data, we report rapid consumption of FXII in COVID-19, but not in ARDS-influenza, plasma. Interestingly, the lag phase in fibrin formation, triggered by the FXII activator kaolin, was not prolonged in COVID-19 as opposed to ARDS-influenza. Using confocal and electron microscopy, we showed that increased FXII activation rate, in conjunction with elevated fibrinogen levels, triggers formation of fibrinolysis-resistant, compact clots with thin fibers and small pores in COVID-19. Accordingly, clot lysis was markedly impaired in COVID-19 as opposed to ARDS-infleunza subjects. Dysregulatated fibrinolytic system, as evidenced by elevated levels of thrombin-activatable fibrinolysis inhibitor, tissue-plasminogen activator, and plasminogen activator inhibitor-1 in COVID-19 potentiated this effect. Analysis of lung tissue sections revealed wide-spread extra- and intra-vascular compact fibrin deposits in COVID-19 patients. Together, compact fibrin network structure and dysregulated fibrinolysis may collectively contribute to high incidence of thrombotic events in COVID-19

Vaccination with ENO1 DNA Prolongs Survival of Genetically Engineered Mice with Pancreatic Cancer.

BACKGROUND & AIMS:: Pancreatic ductal adenocarcinoma (PDA) is an aggressive tumor, and patients typically present with late-stage disease; rates of 5-year survival after pancreaticoduodenectomy are low. Antibodies against -enolase (ENO1), a glycolytic enzyme, are detected in more than 60% of patients with PDA, and ENO1-specific T cells inhibit the growth of human pancreatic xenograft tumors in mice. We investigated whether an ENO1DNA vaccine elicits anti-tumor immune responses and prolongs survival of mice that spontaneously develop autochthonous, lethal pancreatic carcinomas. METHODS:: We injected and electroporated a plasmid encoding ENO1 (or a control plasmid) into Kras(G12D)/Cre mice (KC) and Kras(G12D)/Trp53 (R172H) /Cre (KPC) mice when they were 4 weeks old (when pancreatic intraepithelial lesions are histologically evident). Anti-tumor humoral and cellular responses were analyzed by histology, immunohistochemistry, ELISAs, flow cytometry, and ELISpot and cytotoxicity assays. Survival was analyzed by Kaplan-Meier analysis. RESULTS:: The ENO1 vaccine induced antibody and a cellular responses and increased survival times by a median 138 days in KC mice and 42 days in KPC mice, compared with mice given the control vector. In histologic analysis, the vaccine appeared to slow tumor progression. The vaccinated mice had increased serum levels of anti-ENO1 immunoglobulin G, which bound the surface of carcinoma cells and induced complement-dependent cytotoxicity. ENO1 vaccination reduced numbers of myeloid-derived suppressor cells and T-regulatory cells, and increased T-helper 1 and 17 responses. CONCLUSIONS:: In a genetic model of pancreatic carcinoma, vaccination with ENO1DNA elicits humoral and cellular immune responses against tumors, delays tumor progression, and significantly extends survival. This vaccination strategy might be developed as a neo-adjuvant therapy for patients with PD

Potential mechanisms underlying the acute lung dysfunction and bacterial extrapulmonary dissemination during Burkholderia cenocepacia respiratory infection

<p>Abstract</p> <p>Background</p> <p><it>Burkholderia cenocepacia</it>, an opportunistic pathogen that causes lung infections in cystic fibrosis (CF) patients, is associated with rapid and usually fatal lung deterioration due to necrotizing pneumonia and sepsis, a condition known as cepacia syndrome. The key bacterial determinants associated with this poor clinical outcome in CF patients are not clear. In this study, the cytotoxicity and procoagulant activity of <it>B. cenocepacia </it>from the ET-12 lineage, that has been linked to the cepacia syndrome, and four clinical isolates recovered from CF patients with mild clinical courses were analysed in both <it>in vitro </it>and <it>in vivo </it>assays.</p> <p>Methods</p> <p><it>B. cenocepacia-</it>infected BEAS-2B epithelial respiratory cells were used to investigate the bacterial cytotoxicity assessed by the flow cytometric detection of cell staining with propidium iodide. Bacteria-induced procoagulant activity in cell cultures was assessed by a colorimetric assay and by the flow cytometric detection of tissue factor (TF)-bearing microparticles in cell culture supernatants. Bronchoalveolar lavage fluids (BALF) from intratracheally infected mice were assessed for bacterial proinflammatory and procoagulant activities as well as for bacterial cytotoxicity, by the detection of released lactate dehydrogenase.</p> <p>Results</p> <p>ET-12 was significantly more cytotoxic to cell cultures but clinical isolates Cl-2, Cl-3 and Cl-4 exhibited also a cytotoxic profile. ET-12 and CI-2 were similarly able to generate a TF-dependent procoagulant environment in cell culture supernatant and to enhance the release of TF-bearing microparticles from infected cells. In the <it>in vivo </it>assay, all bacterial isolates disseminated from the mice lungs, but Cl-2 and Cl-4 exhibited the highest rates of recovery from mice livers. Interestingly, Cl-2 and Cl-4, together with ET-12, exhibited the highest cytotoxicity. All bacteria were similarly capable of generating a procoagulant and inflammatory environment in animal lungs.</p> <p>Conclusion</p> <p><it>B. cenocepacia </it>were shown to exhibit cytotoxic and procoagulant activities potentially implicated in bacterial dissemination into the circulation and acute pulmonary decline detected in susceptible CF patients. Improved understanding of the mechanisms accounting for <it>B. cenocepacia</it>-induced clinical decline has the potential to indicate novel therapeutic strategies to be included in the care <it>B. cenocepacia</it>-infected patients.</p

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants.

In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif) ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs) and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1) in a TLR4- and Toll/interleukin (IL)-1R domain-containing adaptor inducing interferon (IFN)-β (TRIF)-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk)1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions