ABSTRACT
ABSTRACT: Glycophorin A (GPA), a red blood cell (RBC) surface glycoprotein, can maintain peripheral blood leukocyte quiescence through interaction with a sialic acid-binding Ig-like lectin (Siglec-9). Under inflammatory conditions such as sickle cell disease (SCD), the GPA of RBCs undergo structural changes that affect this interaction. Peripheral blood samples from patients with SCD before and after RBC transfusions were probed for neutrophil and monocyte activation markers and analyzed by fluorescence-activated cell sorting (FACS). RBCs were purified and tested by FACS for Siglec-9 binding and GPA expression, and incubated with cultured endothelial cells to evaluate their effect on barrier function. Activated leukocytes from healthy subjects (HS) were coincubated with healthy RBCs (RBCH), GPA-altered RBCs, or GPA-overexpressing (OE) cells and analyzed using FACS. Monocyte CD63 and neutrophil CD66b from patients with SCD at baseline were increased 47% and 27%, respectively, as compared with HS (P = .0017, P = .0162). After transfusion, these markers were suppressed by 22% and 17% (P = .0084, P = .0633). GPA expression in RBCSCD was 38% higher (P = .0291) with decreased Siglec-9 binding compared with RBCH (0.0266). Monocyte CD63 and neutrophil CD66b were suppressed after incubation with RBCH and GPA-OE cells, but not with GPA-altered RBCs. Endothelial barrier dysfunction after lipopolysaccharide challenge was restored fully with exposure to RBCH, but not with RBCSCD, from patients in pain crisis, or with RBCH with altered GPA. Pretransfusion RBCSCD do not effectively maintain the quiescence of leukocytes and endothelium, but quiescence is restored through RBC transfusion, likely by reestablished GPA-Siglec-9 interactions.
Subject(s)
Anemia, Sickle Cell , Vascular Diseases , Humans , Endothelial Cells/metabolism , Glycophorins/metabolism , Erythrocytes/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolismABSTRACT
BACKGROUND: Glioblastoma multiforme (GBM) stands as a formidable challenge in oncology because of its aggressive nature and severely limited treatment options. Despite decades of research, the survival rates for GBM remain effectively stagnant. A defining hallmark of GBM is a highly acidic tumor microenvironment, which is thought to activate pro-tumorigenic pathways. This acidification is the result of altered tumor metabolism favoring aerobic glycolysis, a phenomenon known as the Warburg effect. Low extracellular pH confers radioresistant tumors to glial cells. Notably GPR68, an acid sensing GPCR, is upregulated in radioresistant GBM. Usage of Lorazepam, which has off target agonism of GPR68, is linked to worse clinical outcomes for a variety of cancers. However, the role of tumor microenvironment acidification in GPR68 activation has not been assessed in cancer. Here we interrogate the role of GPR68 specifically in GBM cells using a novel highly specific small molecule inhibitor of GPR68 named Ogremorphin (OGM) to induce the iron mediated cell death pathway: ferroptosis. METHOD: OGM was identified in a non-biased zebrafish embryonic development screen and validated with Morpholino and CRISPR based approaches. Next, A GPI-anchored pH reporter, pHluorin2, was stably expressed in U87 glioblastoma cells to probe extracellular acidification. Cell survival assays, via nuclei counting and cell titer glo, were used to demonstrate sensitivity to GPR68 inhibition in twelve immortalized and PDX GBM lines. To determine GPR68 inhibition's mechanism of cell death we use DAVID pathway analysis of RNAseq. Our major indication, ferroptosis, was then confirmed by western blotting and qRT-PCR of reporter genes including TFRC. This finding was further validated by transmission electron microscopy and liperfluo staining to assess lipid peroxidation. Lastly, we use siRNA and CRISPRi to demonstrate the critical role of ATF4 suppression via GPR68 for GBM survival. RESULTS: We used a pHLourin2 probe to demonstrate how glioblastoma cells acidify their microenvironment to activate the commonly over expressed acid sensing GPCR, GPR68. Using our small molecule inhibitor OGM and genetic means, we show that blocking GPR68 signaling results in robust cell death in all thirteen glioblastoma cell lines tested, irrespective of genetic and phenotypic heterogeneity, or resistance to the mainstay GBM chemotherapeutic temozolomide. We use U87 and U138 glioblastoma cell lines to show how selective induction of ferroptosis occurs in an ATF4-dependent manner. Importantly, OGM was not-acutely toxic to zebrafish and its inhibitory effects were found to spare non-malignant neural cells. CONCLUSION: These results indicate GPR68 emerges as a critical sensor for an autocrine pro-tumorigenic signaling cascade triggered by extracellular acidification in glioblastoma cells. In this context, GPR68 suppresses ATF4, inhibition of GPR68 increases expression of ATF4 which leads to ferroptotic cell death. These findings provide a promising therapeutic approach to selectively induce ferroptosis in glioblastoma cells while sparing healthy neural tissue.
ABSTRACT
The generation of bioactive truncated oxidized phospholipids (Tr-OxPLs) from oxidation of cell-membrane or circulating lipoproteins is a common feature of various pathological states. Scavenger receptor CD36 is involved in lipid transport and acts as a receptor for Tr-OxPLs. Interestingly, Tr-OxPLs and CD36 are involved in endothelial dysfunction-derived acute lung injury, but the precise mechanistic connections remain unexplored. In the present study, we investigated the role of CD36 in mediating pulmonary endothelial cell (EC) dysfunction caused by Tr-OxPLs. Our results demonstrated that the Tr-OxPLs KOdia-PC, Paz-PC, PGPC, PON-PC, POV-PC, and lysophosphocholine caused an acute EC barrier disruption as revealed by measurements of transendothelial electrical resistance and VE-cadherin immunostaining. More importantly, a synthetic amphipathic helical peptide, L37pA, targeting human CD36 strongly attenuated Tr-OxPL-induced EC permeability. L37pA also suppressed Tr-OxPL-induced endothelial inflammatory activation monitored by mRNA expression of inflammatory cytokines/chemokines and adhesion molecules. In addition, L37pA blocked Tr-OxPL-induced NF-κB activation and tyrosine phosphorylation of Src kinase and VE-cadherin. The Src inhibitor SU6656 attenuated KOdia-PC-induced EC permeability and inflammation, but inhibition of the Toll-like receptors (TLRs) TLR1, TLR2, TLR4, and TLR6 had no such protective effects. CD36-knockout mice were more resistant to Tr-OxPL-induced lung injury. Treatment with L37pA was equally effective in ameliorating Tr-OxPL-induced vascular leak and lung inflammation as determined by an Evans blue extravasation assay and total cell and protein content in BAL fluid. Altogether, these results demonstrate an essential role of CD36 in mediating Tr-OxPL-induced EC dysfunction and suggest a strong therapeutic potential of CD36 inhibitory peptides in mitigating lung injury and inflammation.
Subject(s)
Acute Lung Injury , Phospholipids , Animals , Mice , Humans , Phospholipids/metabolism , Acute Lung Injury/pathology , Inflammation , Peptides , Lung/pathologyABSTRACT
BACKGROUND: Blood clots are living tissues that release inflammatory mediators including IL-8/CXCL8 and MCP-1/CCL2. A deeper understanding of blood clots is needed to develop new therapies for prothrombotic disease states and regenerative medicine. OBJECTIVES: To identify a common transcriptional shift in cultured blood clot leukocytes. METHODS: Differential gene expression of whole blood and cultured clots (4 hours at 37 °C) was assessed by RNA sequencing (RNAseq), reverse transcriptase-polymerase chain reaction, proteomics, and histology (23 diverse healthy human donors). Cultured clot serum bioactivity was tested in endothelial barrier functional assays. RESULTS: All cultured clots developed a polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) signature, including up-regulation of OLR1 (mRNA encoding lectin-like oxidized low-density lipoprotein receptor 1 [Lox-1]), IL-8/CXCL8, CXCL2, CCL2, IL10, IL1A, SPP1, TREM1, and DUSP4/MKP. Lipopolysaccharide enhanced PMN-MDSC gene expression and specifically induced a type II interferon response with IL-6 production. Lox-1 was specifically expressed by cultured clot CD15+ neutrophils. Cultured clot neutrophils, but not activated platelets, shed copious amounts of soluble Lox-1 (sLox-1) with a donor-dependent amplitude. sLox-1 shedding was enhanced by phorbol ester and suppressed by heparin and by beta-glycerol phosphate, a phosphatase inhibitor. Cultured clot serum significantly enhanced endothelial cell monolayer barrier function, consistent with a proresolving bioactivity. CONCLUSION: This study suggests that PMN-MDSC activation is part of the innate immune response to coagulation which may have a protective role in inflammation. The cultured blood clot is an innovative thrombus model that can be used to study both sterile and nonsterile inflammatory states and could be used as a personalized medicine tool for drug screening.
Subject(s)
Myeloid-Derived Suppressor Cells , Thrombosis , Humans , Interleukin-8 , Neutrophils , Myeloid-Derived Suppressor Cells/pathology , Blood Coagulation/physiology , Thrombosis/pathologyABSTRACT
BACKGROUND: Impairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. RESULTS: In prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. CONCLUSIONS: To our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.
Subject(s)
Prion Diseases , Prions , Animals , Mice , Blood-Brain Barrier , Astrocytes , Endothelial Cells , Claudin-5 , Interleukin-6 , OccludinABSTRACT
Truncated phospholipid oxidation products (Tr-OxPL) increase in blood circulation with aging; however, their role in the severity of vascular dysfunction and bacterial lung injury in aging groups remains poorly understood. We investigated the effects of six Tr-OxPL species: KOdiA-PC, POVPC, PONPC, PGPC, Paz-PC, and Lyso-PC on endothelial dysfunction and lung inflammation caused by heat-killed Staphylococcus aureus (HKSA) in young (aged 2-4 months) and old (aged 12-18 months) mice, organotypic culture of precisely cut lung slices, and endothelial cells (mLEC) isolated from young and old mice. HKSA and Tr-OxPL combination caused a higher degree of vascular leak, the accumulation of inflammatory cells and protein in bronchoalveolar lavage, and inflammatory gene expression in old mice lungs. HKSA caused a greater magnitude of inflammatory gene activation in cell and ex vivo cultures from old mice, which was further augmented by Tr-OxPLs. L37pA peptide targeting CD36 receptor attenuated Tr-OxPL-induced endothelial cell permeability in young and old mLEC and ameliorated KOdiA-PC-induced vascular leak and lung inflammation in vivo. Finally, CD36 knockout mice showed better resistance to KOdiA-PC-induced lung injury in both age groups. These results demonstrate the aging-dependent vulnerability of pulmonary vasculature to elevated Tr-OxPL, which exacerbates bacterial lung injury. CD36 inhibition is a promising therapeutic approach for improving pneumonia outcomes in aging population.
Subject(s)
Lung Injury , Pneumonia , Animals , Mice , Phospholipids/metabolism , Endothelial Cells/metabolism , Lung Injury/metabolism , Pneumonia/metabolism , AgingABSTRACT
Oxidized phospholipids (OxPLs) are present at basal levels in circulation of healthy individuals, but a substantial increase and changes in composition of OxPLs may rapidly occur during microbial infections, sepsis, and trauma. Specifically, truncated oxidized phospholipids (Tr-OxPLs) exhibit detrimental effects on pulmonary endothelium, yet their role on modulation of lung injury caused by bacterial pathogens remains to be elucidated. This study investigated the effects of Tr-OxPL species: KOdiA-PC, POV-PC, PON-PC, PAz-PC, PGPC, and Lyso-PC on endothelial permeability and inflammatory responses to gram-positive bacterial particles. Results showed that all six tested Tr-OxPLs augmented endothelial barrier disruption caused by heat-killed Staphylococcus aureus (HKSA) as determined by VE-cadherin immunostaining and monitoring transendothelial electrical resistance. In parallel, even moderate elevation of Tr-OxPLs augmented HKSA-induced activation of NF-κB, secretion of IL-6 and IL-8, and protein expression of ICAM-1 and VCAM-1. In the mouse model of acute lung injury caused by intranasal injection of HKSA, intravenous Tr-OxPLs administration augmented HKSA-induced increase in BAL protein content and cell counts, tissue expression of TNFα, KC, IL1ß, and CCL2, and promoted vascular leak monitored by lung infiltration of Evans Blue. These results suggest that elevated Tr-OxPLs act as critical risk factor worsening bacterial pathogen-induced endothelial dysfunction and lung injury.
Subject(s)
Acute Lung Injury , Phospholipids , Animals , Mice , Phospholipids/metabolism , Phospholipids/pharmacology , Endothelium/metabolism , Lung/metabolism , Acute Lung Injury/metabolism , Intercellular Adhesion Molecule-1/metabolism , Oxidation-ReductionABSTRACT
Background: Impairment of the blood-brain barrier (BBB) is considered to be a common feature among neurodegenerative diseases, including Alzheimer's, Parkinson's and prion diseases. In prion disease, increased BBB permeability was reported 40 years ago, yet the mechanisms behind the loss of BBB integrity have never been explored. Recently, we showed that reactive astrocytes associated with prion diseases are neurotoxic. The current work examines the potential link between astrocyte reactivity and BBB breakdown. Results: In prion-infected mice, the loss of BBB integrity and aberrant localization of aquaporin 4 (AQP4), a sign of retraction of astrocytic endfeet from blood vessels, were noticeable prior to disease onset. Gaps in cell-to-cell junctions along blood vessels, together with downregulation of Occludin, Claudin-5 and VE-cadherin, which constitute tight and adherens junctions, suggested that loss of BBB integrity is linked with degeneration of vascular endothelial cells. In contrast to cells isolated from non-infected adult mice, endothelial cells originating from prion-infected mice displayed disease-associated changes, including lower levels of Occludin, Claudin-5 and VE-cadherin expression, impaired tight and adherens junctions, and reduced trans-endothelial electrical resistance (TEER). Endothelial cells isolated from non-infected mice, when co-cultured with reactive astrocytes isolated from prion-infected animals or treated with media conditioned by the reactive astrocytes, developed the disease-associated phenotype observed in the endothelial cells from prion-infected mice. Reactive astrocytes were found to produce high levels of secreted IL-6, and treatment of endothelial monolayers originating from non-infected animals with recombinant IL-6 alone reduced their TEER. Remarkably, treatment with extracellular vesicles produced by normal astrocytes partially reversed the disease phenotype of endothelial cells isolated from prion-infected animals. Conclusions: To our knowledge, the current work is the first to illustrate early BBB breakdown in prion disease and to document that reactive astrocytes associated with prion disease are detrimental to BBB integrity. Moreover, our findings suggest that the harmful effects are linked to proinflammatory factors secreted by reactive astrocytes.
ABSTRACT
Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined. Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes liver X receptor-dependent (LXR-dependent) clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h-/- mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains proinflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h, and non-hematopoietic Ch25h deletion is sufficient to confer lung protection. In patients with acute respiratory distress syndrome, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting on different cell types in the lung communicates distinct, temporal LXR-independent and -dependent signals to regulate inflammatory homeostasis.
Subject(s)
Acute Lung Injury , Hydroxycholesterols , Animals , Mice , Hydroxycholesterols/metabolism , Hydroxycholesterols/pharmacology , Macrophages, Alveolar/metabolism , Acute Lung Injury/chemically inducedABSTRACT
Oxidized phospholipids (OxPLs) are generated by enzymatic or autooxidation of esterified polyunsaturated fatty acids (PUFAs) residues. OxPLs are present in circulation and atherosclerotic plaques where they are thought to induce predominantly proinflammatory and toxic changes in endothelial (ECs) and other cell types. Unexpectedly, we found that low concentrations of OxPLs were not toxic but protected ECs from stress induced by serum deprivation or cytostatic drugs. The protective effect was observed in ECs obtained from different vessels and was monitored using a variety of readouts based on different biological and chemical principles. Analysis of the structure−activity relationship identified oxidized or missing fatty acid residue (OxPLs or Lyso-PLs, respectively) as a prerequisite for the protective action of a PL. Protective OxPLs or Lyso-PLs acquired detergent-like properties and formed in solution aggregates <10 nm in diameter (likely micelles), which were in striking contrast with large aggregates (>1000 nm, likely multilayer liposomes) produced by nonoxidized precursor PLs. Because surfactants, OxPLs, and Lyso-PLs are known to extract membrane cholesterol, we tested if this effect might trigger the protection of endothelial cells. The protective action of OxPLs and Lyso-PLs was inhibited by cotreatment with cholesterol and mimicked by cholesterol-binding beta-cyclodextrin but not inactive α-cyclodextrin. Wide-scale mRNA expression analysis in four types of ECs showed the induction of genes encoding for heat shock proteins (HSPs) and secreted prosurvival peptides and proteins. Inducers of HSPs, chemical chaperones, and pure prosurvival factors mimicked the protective action of OxPLs/Lyso-PLs. We hypothesize that oxidation changes the physicochemical properties of PLs, thus promoting membrane cholesterol redistribution or extraction leading to the expression of intra- and extracellular prosurvival factors.
ABSTRACT
Extracellular DNA-binding proteins such as histones are danger-associated molecular pattern released by the injured tissues in trauma and sepsis settings, which trigger host immune response and vascular dysfunction. Molecular events leading to histone-induced endothelial cell (EC) dysfunction remain poorly understood. This study performed comparative analysis of H1, H2A, H2B, H3, and H4 histone subunits effects on human pulmonary EC permeability and inflammatory response. Analysis of transendothelial electrical resistance and EC monolayer permeability for macromolecues revealed that H3 and H4, but not H1, H2A, or H2B caused dose-dependent EC permeability accompanied by disassembly of adherens junctions. At higher doses, H3 and H4 activated nuclear factor kappa B inflammatory cascade leading to upregulation EC adhesion molecules ICAM1, VCAM1, E-selectin, and release of inflammatory cytokines. Inhibitory receptor analysis showed that toll-like receptor (TLR) 4 but not TLR1/2 or receptor for advanced glycation end inhibition significantly attenuated deleterious effects of H3 and H4 histones. Inhibitor of Rho-kinase was without effect, while inhibition of Src kinase caused partial preservation of cell-cell junctions, H3/H4-induced permeability and inflammation. Deleterious effects of H3/H4 were blocked by heparin. Activation of Epac-Rap1 signaling restored EC barrier properties after histone challenge. Intravenous injection of histones in mice caused elevation of inflammatory markers and increased vascular leak. Post-treatment with pharmacological Epac/Rap1 activator suppressed injurious effects of histones in vitro and in vivo. These results identify H3 and H4 as key histone subunits exhibiting deleterious effects on pulmonary vascular endothelium via TLR4-dependent mechanism. In conclusion, elevation of circulating histones may represent a serious risk of exacerbated acute lung injury (ALI) and multiple organ injury during severe trauma and infection.
Subject(s)
Histones , Inflammation , Animals , Endothelium, Vascular/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Histones/metabolism , Humans , Inflammation/metabolism , Mice , PermeabilityABSTRACT
Extracellular histones released into the circulation following trauma, sepsis, and ARDS may act as potent damage-associated molecular pattern signals leading to multiple organ failure. Endothelial cell (EC) dysfunction caused by extracellular histones has been demonstrated in vitro and in vivo; however, precise mechanistic details of histone-induced EC dysfunction and exacerbation of ongoing inflammation remain poorly understood. This study investigated the role of extracellular histones in exacerbating preexisting endothelial dysfunction and acute lung injury. Histone subunits H3 and H4, but not H1, H2A, or H2B, induced permeability in human pulmonary EC. H3 and H4 at concentrations above 30 µg/mL caused EC inflammation reflected by activation of the NF-κB pathway, transcriptional activation, and release of cytokines and chemokines including IL-6 and IL-8, and increased mRNA and protein expression of EC adhesion molecules VCAM-1 and ICAM-1. Pharmacological inhibitors targeting Toll-like receptor TLR4 but not TLR2/6, blocked histone-induced EC dysfunction. H3 and H4 also strongly augmented EC permeability and inflammation caused by Gram-negative and Gram-positive bacterial particles, endotoxin, and TNFα. Heparin blocked histone-induced augmentation of EC inflammation caused by endotoxin and TNFα. Injection of histone in mouse models of lung injury caused by bacterial wall lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus (HKSA) augmented ALI parameters: increased protein content, cell count, and inflammatory cytokine secretion in bronchoalveolar lavage fluid. Important clinical significance of these findings is in the demonstration that even a modest increase in extracellular histone levels can act as a severe exacerbating factor in conjunction with other EC barrier disruptive or proinflammatory agents.
Subject(s)
Acute Lung Injury , Histones , Acute Lung Injury/metabolism , Animals , Humans , Inflammation/metabolism , Lipopolysaccharides/pharmacology , Mice , Toll-Like Receptor 4/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Traumatic brain injury (TBI) has been associated with the development of indirect acute respiratory distress syndrome (ARDS). However, the causative relationship between TBI and lung injury remains unclear. To explore potential mechanisms linking TBI with the development of ARDS, we characterized the effects of serum factors released following TBI and hemorrhagic shock (HS) in a rat model on the pulmonary endothelial cell (EC) barrier dysfunction, a key feature of ARDS. We found that serum samples from animals exposed to both controlled cortical impact (CCI) and HS, but not from sham-operated rats induced significant barrier dysfunction in human pulmonary artery EC monolayers at 2 days post injury. Thrombin inhibitor and thrombin receptor antagonist attenuated the acute phase of the serum-induced trans-endothelial resistance (TER) decline caused by CCI-HS serum, but not in later time points. However, both the early and late phases of CCI-HS-induced EC permeability were inhibited by heparin. The barrier disruptive effects of CCI-HS serum were also prevented by serum preincubation with heparin-sepharose. Pulmonary EC treated for 3 h with serum from CCI-HS rats demonstrated a significant decline in expression of EC junctional protein, VE-Cadherin, and disassembly of peripheral EC adherens junction complexes monitored by immunostaining with VE-cadherin antibody. These results suggest that exposure to CCI-HS causes early and late-phase barrier disruptive effects in vascular endothelium. While thrombin-PAR1 signaling has been identified as a mechanism of acute EC permeability increase by CCI-HS serum, the factor(s) defining long-term EC barrier disruption in CCI-HS model remains to be determined.
Subject(s)
Brain Injuries, Traumatic , Respiratory Distress Syndrome , Shock, Hemorrhagic , Vascular Diseases , Animals , Brain Injuries, Traumatic/complications , Rats , Shock, Hemorrhagic/complications , ThrombinABSTRACT
TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment in vitro. Both conditioned media of miR-146a-5p-treated macrophages (MÏ) and septic sera of wild-type mice induce a marked EC barrier disruption in vitro, whereas MÏ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the MÏ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.
Subject(s)
Membrane Glycoproteins , MicroRNAs , Respiratory Distress Syndrome , Sepsis , Toll-Like Receptor 7 , Animals , Culture Media, Conditioned , Endothelial Cells/metabolism , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Respiratory Distress Syndrome/immunology , Sepsis/complications , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolismABSTRACT
Earlier studies investigating the pathogenesis of chronic vascular inflammation associated with atherosclerosis described pro-inflammatory and vascular barrier disruptive effects of lipid oxidation products accumulated in the sites of vascular lesion and atherosclerotic plaque. However, accumulating evidence including studies from our group suggests potent barrier protective and anti-inflammatory properties of certain oxidized phospholipids (OxPLs) in the lung vascular endothelium. Among these OxPLs, oxidized 1-palmitoyl-2-arachdonyl-sn-glycero-3-phosphocholine (OxPAPC) causes sustained enhancement of lung endothelial cell (EC) basal barrier properties and protects against vascular permeability induced by a wide variety of agonists ranging from bacterial pathogens and their cell wall components, endotoxins, thrombin, mechanical insults, and inflammatory cytokines. On the other hand, truncated OxPLs cause acute endothelial barrier disruption and potentiate inflammation. It appears that multiple signaling mechanisms triggering cytoskeletal remodeling are involved in OxPLs-mediated regulation of EC barrier. The promising vascular barrier protective and anti-inflammatory properties exhibited by OxPAPC and its particular components that have been established in the cellular and animal models of sepsis and acute lung injury has prompted consideration of OxPAPC as a prototype therapeutic molecule. In this review, we will summarize signaling and cytoskeletal mechanisms involved in OxPLs-mediated damage, rescue, and restoration of endothelial barrier in various pathophysiological settings and discuss a future potential of OxPAPC in treating lung disorders associated with endothelial barrier dysfunction.
Subject(s)
Acute Lung Injury/metabolism , Capillary Permeability/physiology , Endothelium, Vascular/metabolism , Phospholipids/metabolism , Acute Lung Injury/drug therapy , Animals , Capillary Permeability/drug effects , Endothelium, Vascular/drug effects , Humans , Oxidation-Reduction/drug effects , Phosphatidylcholines/metabolism , Phosphatidylcholines/pharmacology , Phosphatidylcholines/therapeutic use , Phospholipids/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
Severe internal trauma results in millions of hospitalizations each year, including thousands of deaths caused by subsequent multiple organ failure. The majority of these deaths occur within the first 24 h, and thus, rapid diagnosis of internal trauma severity is necessary for immediate treatment. For early organ damage identification, diagnosis in point-of-care settings is crucial for rapid triage and treatment. Recent reports suggest that circulating histones may serve as a biomarker for severe organ damage and the risk of multiple organ failure. Here, we report a point-of-care diagnostic system that utilizes the inherent interactions between histones and DNA for the fluorescence-based detection of histones in whole blood. In the assay, histones within the sample are wrapped by DNA, thus preventing an intercalating dye from binding the DNA and fluorescing. To allow for quantitative fluorescent measurements to be made in a point-of-care setting, we integrate a rapid, automated blood separation step into our assay. Furthermore, we eliminate manual reagent additions using a thermally responsive alkane partition (TRAP), thus making the system sample-to-answer. Finally, we demonstrate the assay in a portable fluorescence reader compatible with a point-of-care environment. We report a limit of detection 112 ng/mL in whole blood, suggesting that our device can be used to rapidly diagnose internal trauma severity and the likelihood of multiple organ failure in near-patient settings.
Subject(s)
Histones , Point-of-Care Systems , Biomarkers , HumansABSTRACT
Recent studies suggest an anti-inflammatory protective role for class B scavenger receptor BI (SR-BI) in endotoxin-induced inflammation and sepsis. Other data, including ours, provide evidence for an alternative role of SR-BI, facilitating bacterial and endotoxin uptake and contributing to inflammation and bacterial infection. Enhanced endotoxin susceptibility of SR-BI-deficient mice due to their anti-inflammatory glucocorticoid deficiency complicates the understanding of SR-BI's role in endotoxemia/sepsis, calling for the use of alternative models. In this study, using human SR-BI (hSR-BI) and hSR-BII transgenic mice, we found that SR-BI and, to a lesser extent, its splicing variant SR-BII protect against LPS-induced lung damage. At 20 h after intratracheal LPS instillation, the extent of pulmonary inflammation and vascular leakage was significantly lower in hSR-BI and hSR-BII transgenic mice than in wild-type mice. Higher bronchoalveolar lavage fluid (BALF) inflammatory cell count and protein content and lung tissue neutrophil infiltration found in wild-type mice were associated with markedly (2 to 3 times) increased proinflammatory cytokine production compared to these parameters in transgenic mice following LPS administration. The markedly lower endotoxin levels detected in BALF of transgenic versus wild-type mice and the significantly increased BODIPY-LPS uptake observed in lungs of hSR-BI and hSR-BII mice 20 h after the i.t. LPS injection suggest that hSR-BI- and hSR-BII-mediated enhanced LPS clearance in the airways could represent the mechanism of their protective role against LPS-induced acute lung injury.
Subject(s)
Acute Lung Injury/metabolism , Lysosomal Membrane Proteins/metabolism , Receptors, Scavenger/metabolism , Scavenger Receptors, Class B/metabolism , A549 Cells , Acute Lung Injury/chemically induced , Animals , Bronchoalveolar Lavage Fluid , Cell Line, Tumor , Cytokines/metabolism , Disease Models, Animal , Endotoxemia/metabolism , Humans , Inflammation/immunology , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neutrophils/metabolism , Sepsis/metabolismABSTRACT
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.
Subject(s)
Endothelial Cells/drug effects , Lipopolysaccharides/toxicity , Suppressor of Cytokine Signaling 1 Protein/metabolism , Acute Lung Injury/chemically induced , Acute Lung Injury/metabolism , Animals , Cell Line , Endothelial Cells/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Inflammation/chemically induced , Mice , Mice, Knockout , Suppressor of Cytokine Signaling 1 Protein/geneticsABSTRACT
Proinflammatory cytokines such as IL-6 induce endothelial cell (EC) barrier disruption and trigger an inflammatory response in part by activating the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. The protein suppressor of cytokine signaling-3 (SOCS3) is a negative regulator of JAK-STAT, but its role in modulation of lung EC barrier dysfunction caused by bacterial pathogens has not been investigated. Using human lung ECs and EC-specific SOCS3 knockout mice, we tested the hypothesis that SOCS3 confers microtubule (MT)-mediated protection against endothelial dysfunction. SOCS3 knockdown in cultured ECs or EC-specific SOCS3 knockout in mice resulted in exacerbated lung injury characterized by increased permeability and inflammation in response to IL-6 or heat-killed Staphylococcus aureus (HKSA). Ectopic expression of SOCS3 attenuated HKSA-induced EC dysfunction, and this effect required assembled MTs. SOCS3 was enriched in the MT fractions, and treatment with HKSA disrupted SOCS3-MT association. We discovered that-in addition to its known partners gp130 and JAK2-SOCS3 interacts with MT plus-end binding proteins CLIP-170 and CLASP2 via its N-terminal domain. The resulting SOCS3-CLIP-170/CLASP2 complex was essential for maximal SOCS3 anti-inflammatory effects. Both IL-6 and HKSA promoted MT disassembly and disrupted SOCS3 interaction with CLIP-170 and CLASP2. Moreover, knockdown of CLIP-170 or CLASP2 impaired SOCS3-JAK2 interaction and abolished the anti-inflammatory effects of SOCS3. Together, these findings demonstrate for the first time an interaction between SOCS3 and CLIP-170/CLASP2 and reveal that this interaction is essential to the protective effects of SOCS3 in lung endothelium.