Amphipathic Helical Peptide L37pA Protects against Lung Vascular Endothelial Dysfunction Caused by Truncated Oxidized Phospholipids via Antagonism with CD36 Receptor.

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.

Mechanisms of pulmonary endothelial permeability and inflammation caused by extracellular histone subunits H3 and H4.

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.

TLR7 Mediates Acute Respiratory Distress Syndrome in Sepsis by Sensing Extracellular miR-146a.

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 (Mϕ) and septic sera of wild-type mice induce a marked EC barrier disruption in vitro, whereas Mϕ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the Mϕ 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.

SOCS3-microtubule interaction via CLIP-170 and CLASP2 is critical for modulation of endothelial inflammation and lung injury.

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.

Circulating extracellular histones exacerbate acute lung injury by augmenting pulmonary endothelial dysfunction via TLR4-dependent mechanism.

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.

Microtubule-dependent mechanism of anti-inflammatory effect of SOCS1 in endothelial dysfunction and lung injury.

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.

Class B Scavenger Receptors BI and BII Protect against LPS-Induced Acute Lung Injury in Mice by Mediating LPS.

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.

Deletion of P21-activated kinase-1 induces age-dependent increased visceral adiposity and cardiac dysfunction in female mice.

It is known that there is an age-related progression in diastolic dysfunction, especially prevalent in postmenopausal women, who develop heart failure with preserved ejection fraction (HFpEF, EF > 50%). Mechanisms and therapies are poorly understood, but there are strong correlations between obesity and HFpEF. We have tested the hypothesis that P21-activated kinase-1 (PAK1) preserves cardiac function and adipose tissue homeostasis during aging in female mice. Previous demonstrations in male mice by our lab that PAK1 activity confers cardio-protection against different stresses formed the rationale for this hypothesis. Our studies compared young (3-6 months) and middle-aged (12-15 months) female and male PAK1 knock-out mice (PAK1-/-) and wild-type (WT) equivalent. Female WT mice exhibited increased cardiac PAK1 abundance during aging. By echocardiography, compared to young WT female mice, middle-aged WT female mice showed enlargement of the left atrium as well as thickening of posterior wall and increased left ventricular mass; however, all contraction and relaxation parameters were preserved during aging. Compared to WT controls, middle-aged PAK1-/- female mice demonstrated worsening of cardiac function involving a greater enlargement of the left atrium, ventricular hypertrophy, and diastolic dysfunction. Moreover, with aging PAK1-/- female mice, unlike male PAK1-/- mice, exhibited increased adiposity with increased accumulation of visceral adipose tissue. Our data provide evidence for the significance of PAK1 signaling as an element in the preservation of cardiac function and adipose tissue homeostasis in females during aging.

Staphylococcus aureus-induced endothelial permeability and inflammation are mediated by microtubule destabilization.

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 EC 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 EC, 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 WT mice were partially protected from vascular leakage and inflammation caused by both HKSA or methicillin-resistant S. aureus (MRSA). 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.

Contrasting effects of stored allogeneic red blood cells and their supernatants on permeability and inflammatory responses in human pulmonary endothelial cells.

Transfusion of red blood cells (RBCs) is a common life-saving clinical practice in severely anemic or hemorrhagic patients; however, it may result in serious pathological complications such as transfusion-related acute lung injury. The factors mediating the deleterious effects of RBC transfusion remain unclear. In this study, we tested the effects of washed long-term (RBC-O; >28 days) versus short-term (RBC-F; <14 days) stored RBCs and their supernatants on lung endothelial (EC) permeability under control and inflammatory conditions. RBCs enhanced basal EC barrier function as evidenced by an increase in transendothelial electrical resistance and decrease in permeability for macromolecules. RBCs also attenuated EC hyperpermeability and suppressed secretion of EC adhesion molecule ICAM-1 and proinflammatory cytokine IL-8 in response to LPS or TNF-α. In both settings, RBC-F had slightly higher barrier protective effects as compared with RBC-O. In contrast, supernatants from both RBC-F and RBC-O disrupted the EC barrier. The early phase of EC permeability response caused by RBC supernatants was partially suppressed by antioxidant N-acetyl cysteine and inhibitor of Src kinase family PP2, while addition of heme blocker and inhibition of NOD-like receptor family pyrin domain containing protein 3 (NLRP3), stress MAP kinases, receptor for advanced glycation end-products (RAGE), or Toll-like receptor-4 (TLR4) signaling were without effect. Morphological analysis revealed that RBC supernatants increased LPS- and TNF-α-induced breakdown of intercellular junctions and formation of paracellular gaps. RBC supernatants augmented LPS- and TNF-α-induced EC inflammation reflected by increased production of IL-6, IL-8, and soluble ICAM-1. These findings demonstrate the deleterious effects of RBC supernatants on EC function, which may have a major impact in pathological consequences associated with RBC transfusion.

Elevated truncated oxidized phospholipids as a factor exacerbating ALI in the aging lungs.

As mechanisms controlling redox homeostasis become impaired with aging, exaggerated oxidant stress may cause disproportional oxidation of cell membranes and circulating phospholipids (PLs), leading to the formation of truncated oxidized PL products (Tr-OxPLs), which exhibit deleterious effects. This study investigated the role of elevated Tr-OxPLs as a factor exacerbating inflammation and lung barrier dysfunction in an animal model of aging. Mass spectrometry analysis of Tr-OxPL species in young (2-4 mo) and aging (18-24 mo) mice revealed elevated basal levels of several products [1-palmitoyl-2-(5-oxovaleroyl)- sn-glycero-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl- sn-glycero-phosphocholine, lysophosphocholine, 1-palmitoyl-2-(9-oxo-nonanoyl)- sn-glycero-3-phosphocholine, 1-palmitoyl-2-azelaoyl- sn-glycero-3-phosphocholine, O-1-O-palmitoyl-2-O-(5,8-dioxo-8-hydroxy-6-octenoyl)-l-glycero-3-phosphocholine, and others] in the aged lungs. An intratracheal (i.t.) injection of bacterial LPS caused increased generation of Tr-OxPLs in the lungs but not in the liver, with higher levels detected in the aged group. In addition, OxPLs clearance from the lung tissue after LPS challenge was delayed in the aged group. The impact of Tr-OxPLs on endothelial cell (EC) barrier compromise under inflammatory conditions was further evaluated in the 2-hit cell culture model of acute lung injury (ALI). EC barrier dysfunction caused by cell treatment with a cytokine mixture (CM) was augmented by cotreatment with low-dose Tr-OxPLs, which did not significantly affect endothelial function when added alone. Deleterious effects of Tr-OxPLs on inflamed ECs stimulated with CM were associated with further weakening of cell junctions and more robust EC hyperpermeability. Aged mice injected intratracheally with TNF-α exhibited a more pronounced elevation of cell counts and protein content in bronchoalveolar lavage (BAL) samples. Interestingly, intravenous administration of low POVPC doses-which did not affect BAL parameters alone in young mice exposed to i.t. TNF-α challenge-augmented lung injury to the levels observed in aged mice stimulated with TNF-α alone. Inhibition of Tr-OxPL generation by ectopic expression of PL-specific platelet-activating factor acetylhydrolase 2 (PAFAH2) markedly reduced EC dysfunction induced by CM, whereas PAFAH2 pharmacologic inhibition augmented deleterious effects of cytokines on EC barrier function. Moreover, exacerbating effects of PAFAH2 inhibition on TNF-α-induced lung injury were observed in vivo. These results demonstrate an age-dependent increase in Tr-OxPL production under basal conditions and augmented Tr-OxPL generation upon inflammatory stimulation, suggesting a major role for elevated Tr-OxPLs in more severe ALI and delayed resolution in aging lungs.-Ke, Y., Karki, P., Kim, J., Son, S., Berdyshev, E., Bochkov, V. N., Birukova, A. A., Birukov, K. G. Elevated truncated oxidized phospholipids as a factor exacerbating ALI in the aging lungs.

Anti-Inflammatory Effects of OxPAPC Involve Endothelial Cell-Mediated Generation of LXA4.

RATIONALE: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling. OBJECTIVE: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4). METHODS AND RESULTS: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-α in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-α and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA-induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2-/- (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX. CONCLUSIONS: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium.

Prostaglandin E receptor-4 receptor mediates endothelial barrier-enhancing and anti-inflammatory effects of oxidized phospholipids.

Unlike other agonists that cause transient endothelial cell (EC) response, the products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) oxidation that contain cyclopenthenone groups, which recapitulate prostaglandin-like structure, cause sustained enhancement of the pulmonary EC barrier. The mechanisms that drive the sustained effects by oxidized PAPC (OxPAPC) remain unexplored. On the basis of the structural similarity of isoprostanoid moieties that are present in full-length oxygenated PAPC species, we used an inhibitory approach to perform the screening of prostanoid receptors as potential candidates that mediate OxPAPC effects. Results show that only prostaglandin E receptor-4 (EP4) was involved and mediated the sustained phase of the barrier-enhancing effects of OxPAPC that are associated with the activation of Rac GTPase and its cytoskeletal targets. EC incubation with OxPAPC also induced EP4 mRNA expression in pulmonary ECs and lung tissue. EP4 knockdown using gene-specific small interfering RNA did not affect the rapid phase of OxPAPC-induced EC barrier enhancement or the protective effects against thrombin-induced EC permeability, but abolished the advanced barrier enhancement phase and suppressed the protective effects of OxPAPC against more sustained EC barrier dysfunction and cell inflammatory response caused by TNF-α. Endothelial-specific knockout of the EP4 receptor in mice attenuated the protective effect of intravenous OxPAPC administration in the model of acute lung injury caused by intratracheal injection of LPS. Taken together, these results demonstrate a novel role for prostaglandin receptor EP4 in the mediation of barrier-enhancing and anti-inflammatory effects caused by oxidized phospholipids.-Oskolkova, O., Gawlak, G., Tian, Y., Ke, Y., Sarich, N., Son, S., Andreasson, K., Bochkov, V. N., Birukova, A. A., Birukov, K. G. Prostaglandin E receptor-4 receptor mediates endothelial barrier-enhancing and anti-inflammatory effects of oxidized phospholipids.

Effects of prostaglandin lipid mediators on agonist-induced lung endothelial permeability and inflammation.

Prostaglandins (PG), the products of cyclooxygenase-mediated conversion of arachidonic acid, become upregulated in many situations including allergic response, inflammation, and injury, and exhibit a variety of biological activities. Previous studies described barrier-enhancing and anti-inflammatory effects of PGE2 and PGI2 on vascular endothelial cells (EC). Yet, the effects of other PG members on EC barrier and inflammatory activation have not been systematically analyzed. This study compared effects of PGE2, PGI2, PGF2α, PGA2, PGJ2, and PGD2 on human pulmonary EC. EC permeability was assessed by measurements of transendothelial electrical resistance and cell monolayer permeability for FITC-labeled tracer. Anti-inflammatory effects of PGs were evaluated by analysis of expression of adhesion molecule ICAM1 and secretion of soluble ICAM1 and cytokines by EC. PGE2, PGI2, and PGA2 exhibited the most potent barrier-enhancing effects and most efficient attenuation of thrombin-induced EC permeability and contractile response, whereas PGI2 effectively suppressed thrombin-induced permeability but was less efficient in the attenuation of prolonged EC hyperpermeability caused by interleukin-6 or bacterial wall lipopolysaccharide, LPS. PGD2 showed a modest protective effect on the EC inflammatory response, whereas PGF2α and PGJ2 were without effect on agonist-induced EC barrier dysfunction. In vivo, PGE2, PGI2, and PGA2 attenuated LPS-induced lung inflammation, whereas PGF2α and PGJ2 were without effect. Interestingly, PGD2 exhibited a protective effect in the in vivo model of LPS-induced lung injury. This study provides a comprehensive analysis of barrier-protective and anti-inflammatory effects of different prostaglandins on lung EC in vitro and in vivo and identifies PGE2, PGI2, and PGA2 as prostaglandins with the most potent protective properties.

Effect of sphingosine-1-phosphate on L-type calcium current and Ca(2+) transient in rat ventricular myocytes.

Modulation of Ca(2+) homoeostasis in cardiac myocytes plays a major role in beat-to-beat regulation of heart function. Previous studies suggest that sphingosine-1-phosphate (S1P), a biologically active sphingomyelin metabolite, regulates Ca(2+) handling in cardiac myocytes, but the underlying mechanism is unclear. In the present study, we tested the hypothesis that S1P-induced functional alteration of intracellular Ca(2+) handling includes the L-type calcium channel current (ICa,L) via a signalling pathway involving P21-activated kinase 1 (Pak1). Our results show that, in rat ventricular myocytes, S1P (100 nM) does not affect the basal activity of ICa,L but is able to partially reverse the effect of the ß-adrenergic agonist Isoproterenol (ISO, 100 nM) on ICa,L. S1P (25 nM) also significantly prevents ISO (5 nM)-induced Ca(2+) waves and diastolic Ca(2+) release in these cells. Our further molecular characterisation demonstrates that Pak1 activity is increased in myocytes treated with S1P (25 nM) compared with those myocytes without treatment of S1P. By immunoprecipitation we demonstrate that Pak1 and protein phosphatase 2A (PP2A) are associated in ventricular tissue indicating their functional interaction. Thus the results indicate that S1P attenuates ß-adrenergic stress-induced alteration of intracellular Ca(2+) release and L-type Ca(2+) channel current at least in part via Pak1-PP2A-mediated signalling.

p21-Activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes.

Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca(2+) handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1(-/-)) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca(2+) transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1(-/-) VMs during 15 min of simulated ischemia. However, Pak1(-/-) VMs exhibited an exaggerated increase in [Ca(2+)]i, which resulted in spontaneous Ca(2+) release events and waves. The Ca(2+) overload in Pak1(-/-) VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1(-/-) VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47(phox-/-)) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1(-/-) VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca(2+) overload in Pak1(-/-) VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca(2+) overload under conditions where no significant changes in excitation-contraction coupling are yet evident.

Whole blood coagulation in an ex vivo thrombus is sufficient to induce clot neutrophils to adopt a myeloid-derived suppressor cell signature and shed soluble Lox-1.

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.

Functional integrity of the T-tubular system in cardiomyocytes depends on p21-activated kinase 1.

p21-activated kinase (Pak1), a serine-threonine protein kinase, regulates cytoskeletal dynamics and cell motility. Recent experiments further demonstrate that loss of Pak1 results in exaggerated hypertrophic growth in response to pathophysiological stimuli. Calcium (Ca) signaling plays an important role in the regulation of transcription factors involved in hypertrophic remodeling. Here we aimed to determine the role of Pak1 in cardiac excitation-contraction coupling (ECC). Ca transients were recorded in isolated, ventricular myocytes (VMs) from WT and Pak1(-/-) mice. Pak1(-/-) Ca transients had a decreased amplitude, prolonged rise time and delayed recovery time. Di-8-ANNEPS staining revealed a decreased T-tubular density in Pak1(-/-) VMs that coincided with decreased cell capacitance and increased dis-synchrony of Ca induced Ca release (CICR) at individual release units. These changes were not observed in atrial myocytes of Pak1(-/-) mice where the T-tubular system is only sparsely developed. Experiments in cultured rabbit VMs supported a role of Pak1 in the maintenance of the T-tubular structure. T-tubular density in rabbit VMs significantly decreased within 24h of culture. This was accompanied by a decrease of the Ca transient amplitude and a prolongation of its rise time. However, overexpression of constitutively active Pak1 in VMs attenuated the structural remodeling as well as changes in ECC. The results provide significant support for a prominent role of Pak1 activity not only in the functional regulation of ECC but for the structural maintenance of the T-tubular system whose remodeling is an integral feature of hypertrophic remodeling.

Truncated oxidized phospholipids exacerbate endothelial dysfunction and lung injury caused by bacterial pathogens.

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.