Enhanced Leukocyte Adhesion Research Articles

Combined Radiotherapy and Hyperthermia: A Systematic Review of Immunological Synergies for Amplifying Radiation-Induced Abscopal Effects.

The abscopal effect is a systemic immune response characterized by metastases regression at sites distant from the irradiated lesion. This systematic review aims to explore the immunological mechanisms of action underlying the abscopal effect and to investigate how hyperthermia (HT) can increase the chances of radiotherapy (RT) triggering systemic anti-tumor immune responses. This review is created in accordance with the PRISMA guidelines. HT and RT have both complementary and synergistic immunological effects. Both methods trigger danger signal release, promoting cytokine and chemokine secretion, which increases T-cell infiltration and facilitates cell death. Both treatments upregulate extracellular tumor HSP70, which could amplify DAMP recognition by macrophages and DCs, leading to stronger tumor antigen presentation and CTL-mediated immune responses. Additionally, the combined increase in cell adhesion molecules (VCAM-1, ICAM-1, E-selectin, L-selectin) could enhance leukocyte adhesion to tumors, improving lymphocyte trafficking and boosting systemic anti-tumor effects. Lastly, HT causes vasodilation and improves blood flow, which might exacerbate those distant effects. We suggest the combination of local radiotherapy with fever-range whole-body hyperthermia to optimally enhance the chances of triggering the abscopal effect mediated by the immune system.

Csk controls leukocyte extravasation via local regulation of Src family kinases and cortactin signaling.

C-terminal Src kinase (Csk) targets Src family kinases (SFKs) and thereby inactivates them. We have previously shown that Csk binds to phosphorylated tyrosine 685 of VE-cadherin, an adhesion molecule of major importance for the regulation of endothelial junctions. This tyrosine residue is an SFK target, and its mutation (VE-cadherin-Y685F) inhibits the induction of vascular permeability in various inflammation models. Nevertheless, surprisingly, it increases leukocyte extravasation. Here, we investigated whether endothelial Csk is involved in these effects. We found that the deficiency of Csk in endothelial cells augments SFK activation and the phosphorylation of VE-cadherin-Y685 but had no net effect on vascular leak formation. In contrast, the lack of endothelial Csk enhanced leukocyte adhesion and transmigration in vitro and in vivo. Furthermore, the silencing of Csk increased tyrosine phosphorylation of the SFK substrate cortactin. Importantly, the effects of Csk silencing on the increase in SFK activation, cortactin phosphorylation, and neutrophil diapedesis were all dependent on Y685 of VE-cadherin. Deletion of cortactin, in turn, erased the supporting effect of Csk silencing on leukocyte transmigration. We have previously shown that leukocyte transmigration is regulated by endothelial cortactin in an ICAM-1-dependent manner. In line with this, blocking of ICAM-1 erased the supporting effect of Csk silencing on leukocyte transmigration. Collectively, our results establish a negative feedback loop that depends on the phosphorylation of VE-cadherin-Y685, which recruits Csk, which in turn dampens the activation of SFK and cortactin and thereby the clustering of ICAM-1 and the extravasation of neutrophils.

Abstract 1119: Ultrafine Particle Inhalation Initiates Vascular Inflammation Via Hepatic-derived Xanthine Oxidoreductase-mediated Epigenetic Alterations

Introduction: Poor air quality has become a growing health concern. Ultrafine particles (UFP), a component of air pollution, has been associated with cardiovascular disease/ events, establishing a critical need to understand the cellular mechanisms involved. Using a model UFP we have shown elevated vascular oxidant production, and inflammation. However, the underlying mechanisms were unexplored. Xanthine oxidoreductase (XOR), increases in circulation following various insults, which we recently established is actively derived from hepatocytes. The binding of endothelial cells (EC) and the mechanisms by which EC bound XOR mediates impairment are not fully understood. Bound XOR produces almost exclusively H 2 O 2 , which can across the membrane and influence cellular signaling and gene expression. We hypothesize that UFP inhalation elevates hepatic XOR release into circulation and XOR binds to EC initiating redox signaling pathways leading to epigenetic-mediated inflammatory gene upregulation. Methods & Results: Male (M) and female (F) rats and mice (8 wk) were exposed to sham air or UFP for 3 days. Plasma XOR activity increased 5-fold M and 3-fold F exposed to UFP (both p<0.05). We then determined that UFP activation of PKC (rat liver 2.4-fold, mouse liver 2.3-fold, AML12 cells 1.7-fold, all p<0.05) leads to G9a-dependent upregulation and release of XOR in hepatocytes (Control 2± 0.3 vs PMA 8± 1 vs PMA G9a siRNA 2± 2 μU/ml, p<0.05). In arterioles, UFP arterioles showed greater XOR binding when perfused with Alexaflur tagged XOR (69±6 vs 51±4 AFU, p<0.05) and impaired dilation, which was prevented in hepatocyte XOR knockouts mice (KO) (max dilation sham 62± 3 vs UFP 35± 4 vs UFP KO 50± 4, p<0.05). In culture EC were treated with XOR for 1h (for binding) then washed with PBS and cultured for 24 h (XOR-EC). XOR-EC increased demethylase KDM4a expression (1.7-fold, p<0.05) and inflammatory markers ICAM, VCAM, TNF, and TLR4 (p<0.05), leading to enhanced leukocyte adhesion (~2-fold, p<0.05). KDM4a loss of function (iKDM4a) prevented ICAM expression (XOR-EC 8-fold vs iKDM4a 2-fold, p<0.05) and diminished leukocyte adhesion (p<0.05). Conclusion: These findings suggest that vascular inflammation caused by UFP inhalation is mediated in part by an EC bound XOR- KDM4a pathway.

Loss of soluble guanylyl cyclase in platelets contributes to atherosclerotic plaque formation and vascular inflammation

Variants in genes encoding the soluble guanylyl cyclase (sGC) in platelets are associated with coronary artery disease (CAD) risk. Here, by using histology, flow cytometry and intravital microscopy, we show that functional loss of sGC in platelets of atherosclerosis-prone Ldlr−/− mice contributes to atherosclerotic plaque formation, particularly via increasing in vivo leukocyte adhesion to atherosclerotic lesions. In vitro experiments revealed that supernatant from activated platelets lacking sGC promotes leukocyte adhesion to endothelial cells (ECs) by activating ECs. Profiling of platelet-released cytokines indicated that reduced platelet angiopoietin-1 release by sGC-depleted platelets, which was validated in isolated human platelets from carriers of GUCY1A1 risk alleles, enhances leukocyte adhesion to ECs. Importantly, pharmacological sGC stimulation increased platelet angiopoietin-1 release in vitro and reduced leukocyte recruitment and atherosclerotic plaque formation in atherosclerosis-prone Ldlr−/− mice. Therefore, pharmacological sGC stimulation might represent a potential therapeutic strategy to prevent and treat CAD.

Induction of ICAM1 in Brain Vessels is Implicated in an Early AD Pathogenesis by Modulating Neprilysin.

Intercellular adhesion molecule 1 (ICAM1) is a vessel adhesion protein induced during brain vascular inflammation, which could be closely linked with the development of Alzheimer's disease (AD). This study investigated the effect of ICAM1 on amyloid-degrading enzymes (ADEs) in endothelial cells and their potential involvement in inflammation and AD progression. TNF-α treatment increased ICAM1 in human brain microvascular endothelial cells (HBMVECs) but decreased the neprilysin (NEP) protein level. Knock-down of ICAM1 using siRNA enhanced NEP, which increased the degradation of amyloid-β. In the brains of 4-month-old AD transgenic mice (APPswe/PSEN1dE9), there were significantly higher levels of ICAM1 expression and amyloid deposits but lower levels of NEP and insulin-degrading enzymes (IDE), demonstrating an inverse correlation of ICAM1 with NEP and IDE expression. Further studies demonstrated significantly increased GFAP protein levels in the brain, specifically localized near blood vessels, of both TNF-α-injected and 4-month-old AD transgenic mice. Taken together, the induction of ICAM1 in endothelial cells suppresses NEP expression, accelerating the accumulation of amyloid-β in blood vessels. It also enhances leukocyte adhesion to blood vessels stimulating the migration of leukocytes into the brain, subsequently triggering brain inflammation.

A Multi-Omics Analysis of Metastatic Melanoma Identifies a Germinal Center-Like Tumor Microenvironment in HLA-DR-Positive Tumor Areas.

The emergence of immune checkpoint inhibitors has dramatically changed the therapeutic landscape for patients with advanced melanoma. However, relatively low response rates and a high incidence of severe immune-related adverse events have prompted the search for predictive biomarkers. A positive predictive value has been attributed to the aberrant expression of Human Leukocyte Antigen-DR (HLA-DR) by melanoma cells, but it remains unknown why this is the case. In this study, we have examined the microenvironment of HLA-DR positive metastatic melanoma samples using a multi-omics approach. First, using spatial, single-cell mapping by multiplexed immunohistochemistry, we found that the microenvironment of HLA-DR positive melanoma regions was enriched by professional antigen presenting cells, including classical dendritic cells and macrophages, while a more general cytotoxic T cell exhaustion phenotype was present in these regions. In parallel, transcriptomic analysis on micro dissected tissue from HLA-DR positive and HLA-DR negative areas showed increased IFNγ signaling, enhanced leukocyte adhesion and mononuclear cell proliferation in HLA-DR positive areas. Finally, multiplexed cytokine profiling identified an increased expression of germinal center cytokines CXCL12, CXCL13 and CCL19 in HLA-DR positive metastatic lesions, which, together with IFNγ and IL4 could serve as biomarkers to discriminate tumor samples containing HLA-DR overexpressing tumor cells from HLA-DR negative samples. Overall, this suggests that HLA-DR positive areas in melanoma attract the anti-tumor immune cell infiltration by creating a dystrophic germinal center-like microenvironment where an enhanced antigen presentation leads to an exhausted microenvironment, nevertheless representing a fertile ground for a better efficacy of anti-PD-1 inhibitors due to simultaneous higher levels of PD-1 in the immune cells and PD-L1 in the HLA-DR positive melanoma cells.

TMEM16A Inhibition Preserves Blood-Brain Barrier Integrity After Ischemic Stroke.

The inflammatory response plays a pivotal role in Blood–Brain Barrier (BBB) destruction following ischemic brain injury. Enhanced leukocyte adhesion to vascular endothelial cells is an essential event in the inflammatory process. TMEM16A, a newly discovered protein regulating calcium-activated chloride channels, is widely expressed in eukaryotes. Recent studies have suggested that upregulated expression of TMEM16A is associated with the occurrence and development of many diseases. However, the role of TMEM16A in regulating BBB integrity after ischemic stroke has not been fully investigated. In this study, we found that TMEM16A is mainly expressed in brain endothelial cells and upregulated after ischemic stroke in the mouse brain. Caccinh-A01, an TMEM16A inhibitor that reduced its upregulation, attenuated brain infarct size and neurological deficits after ischemic stroke. ICAM-1 and MPO expression and BBB permeability were decreased after TMEM16A inhibitor administration. In addition, TMEM16A silencing rescued oxygen-glucose deprivation/reoxygenation (OGD/R)-induced transendothelial permeability in vitro accompanied by decreased ICAM-1 expression and leukocyte adhesion. Furthermore, our mechanistic study showed that TMEM16A knockdown alleviated NF-κB activation and nuclear translocation, indicating that TMEM16A knockdown downregulated OGD/R-induced ICAM-1 expression in an NF-κB-dependent manner. Finally, NF-κB inhibitor treatment also alleviated OGD/ R-induced BBB permeability, confirming that activated NF-κB and increased ICAM-1 are essential factors involved in ischemia-induced BBB damage. Thus, our research provides a promising treatment strategy against BBB destruction after ischemic stroke, and TMEM16A may become a potential target for the treatment of ischemic stroke.

Sepsis-induced damage to endothelial glycocalyx (literature review)

Glycocalyx is a gel-like layer covering the surface of vascular endothelial cells. It consists of membrane-attached proteoglycans, glycosaminoglycan chains, glycoproteins, and plasma adhesive proteins. Glycocalyx plays a key role in maintaining vascular homeostasis, controls vascular permeability and the tone of the microvasculature, prevents microvascular thrombosis and regulates leukocyte adhesion. In sepsis and septic shock, damage and shedding of glycocalyx occurs. The degradation of glycocalyx is activated by reactive oxygen species and pro-inflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1β (IL-1β). The inflammation-mediated degradation of glycocalyx leads to vascular hyperpermeability, unregulated vasodilation, microvascular thrombosis, and enhanced leukocyte adhesion. The inflammation-mediated degradation of glycocalyx leads to vascular hyperpermeability, unregulated vasodilation, microvascular thrombosis, and enhanced leukocyte adhesion. Clinical studies have demonstrated a correlation between the levels of glycocalyx components in the blood and organ dysfunction and mortality in sepsis and septic shock. Inflammation-induced damage to glycocalyx can cause a number of specific clinical effects of sepsis, including acute kidney damage, respiratory failure and liver dysfunction. Infusion therapy is an integral part of the treatment of sepsis, but super-aggressive infusion load methods (leading to hypervolemia) may increase the degradation of glycocalyx. Moreover, some markers of glycocalyx degradation, such as circulating levels of syndecan 1 or heparan sulfate, can be used as markers of endothelial dysfunction and sepsis severity.

Shared Features of Endothelial Dysfunction between Sepsis and Its Preceding Risk Factors (Aging and Chronic Disease).

Acute vascular endothelial dysfunction is a central event in the pathogenesis of sepsis, increasing vascular permeability, promoting activation of the coagulation cascade, tissue edema and compromising perfusion of vital organs. Aging and chronic diseases (hypertension, dyslipidaemia, diabetes mellitus, chronic kidney disease, cardiovascular disease, cerebrovascular disease, chronic pulmonary disease, liver disease, or cancer) are recognized risk factors for sepsis. In this article we review the features of endothelial dysfunction shared by sepsis, aging and the chronic conditions preceding this disease. Clinical studies and review articles on endothelial dysfunction in sepsis, aging and chronic diseases available in PubMed were considered. The main features of endothelial dysfunction shared by sepsis, aging and chronic diseases were: (1) increased oxidative stress and systemic inflammation, (2) glycocalyx degradation and shedding, (3) disassembly of intercellular junctions, endothelial cell death, blood-tissue barrier disruption, (4) enhanced leukocyte adhesion and extravasation, (5) induction of a pro-coagulant and anti-fibrinolytic state. In addition, chronic diseases impair the mechanisms of endothelial reparation. In conclusion, sepsis, aging and chronic diseases induce similar features of endothelial dysfunction. The potential contribution of pre-existent endothelial dysfunction to sepsis pathogenesis deserves to be further investigated.

Erratum: A high-salt diet enhances leukocyte adhesion in association with kidney injury in young Dahl salt-sensitive rats.

This corrects the article DOI: 10.1038/hr.2017.31.

A high-salt diet enhances leukocyte adhesion in association with kidney injury in young dahl salt-sensitive rats

Salt-sensitive hypertension is associated with severe organ damage. Generating oxygen radicals is an integral component of salt-induced kidney damage, and activated leukocytes are important in oxygen radical biosynthesis. We hypothesized that a high-salt diet causes the upregulation of immune-related mechanisms, thereby contributing to the susceptibility of Dahl salt-sensitive rats to hypertensive kidney damage. For verifying the hypothesis, we investigated leukocytes adhering to retinal vessels when Dahl salt-sensitive rats were challenged with a high-salt (8% NaCl) diet using acridine orange fluoroscopy and a scanning laser ophthalmoscope. The high-salt diet increased leukocyte adhesion after 3 days and was associated with a significant increase in mRNA biosynthesis of monocyte chemotactic protein-1 and intercellular adhesion molecule-1 (ICAM-1) -related molecules in the kidney. Losartan treatment did not affect increased leukocyte adhesion during the early, pre-hypertensive phase of high salt loading; however, losartan attenuated the adhesion of leukocytes during the hypertensive stage. Moreover, the inhibition of leukocyte adhesion in the pre-hypertensive stage by anti-CD18 antibodies decreased tethering of leukocytes and was associated with the attenuation of functional and morphological kidney damage without affecting blood pressure elevation. In conclusion, a high-salt challenge rapidly increased leukocyte adhesion through the over-expression of ICAM-1. Increased leukocyte adhesion in the pre-hypertensive stage is responsible for subsequent kidney damage in Dahl salt-sensitive rats. Immune system involvement may be a key component that initiates kidney damage in a genetic model of salt-induced hypertension.

Platelet-mediated adhesion facilitates leukocyte sequestration in hypoxia-reoxygenated microvessels.

Leukocyte transendothelial migration and sequestration are two distinct outcomes following leukocyte adhesion to endothelium during ischemia-reperfusion injury, in which platelets may play a pivotal role. In the present study, we established an in vitro hypoxia-reoxygenation model to mimic ischemia-reperfusion injury and found platelet pre-incubation significantly increased leukocyte adhesion to endothelial cells after hyoxia-reoxygenation (over 67%). Blockade of endothelial-cell-expressed adhesion molecules inhibited leukocyte direct adhesion to endothelial cells, while platelet-mediated leukocyte adhesion was suppressed by blockade of platelet-expressed adhesion molecules. Further experiments revealed platelets acted as a bridge to mediate leukocyte adhesion, and platelet-mediated adhesion was the predominant pattern in the presence of platelets. However, platelet pre-incubation significantly suppressed leukocyte transendothelial migration after hypoxia-reoxygenation (over 31%), which could be aggravated by blockade of endothelial-cell-expressed adhesion molecules, but alleviated by blockade of platelet- expressed adhesion molecules. This would indicate that platelet-mediated adhesion disrupted leukocyte transendothelial migration. An in vivo mesenteric ischemia-reperfusion model demonstrated leukocyte transfusion alone caused mild leukocyte adhesion to reperfused vessels and subsequent leukocyte infiltration, while simultaneous leukocyte and platelet transfusion led to massive leukocyte adhesion and sequestration within reperfused microvessels. Our studies revealed platelets enhanced leukocyte adhesion to endothelial cells, but suppressed leukocyte transendothelial migration. Overall, this leads to leukocyte sequestration in hypoxia-reoxygenated microvessels.

Efficacy of Leukadherin-1 in the Prevention of Hyperoxia-Induced Lung Injury in Neonatal Rats.

Lung inflammation plays a key role in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants. The challenge in BPD management is the lack of effective and safe antiinflammatory agents. Leukadherin-1 (LA1) is a novel agonist of the leukocyte surface integrin CD11b/CD18 that enhances leukocyte adhesion to ligands and vascular endothelium and thus reduces leukocyte transendothelial migration and influx to the injury sites. Its functional significance in preventing hyperoxia-induced neonatal lung injury is unknown. We tested the hypothesis that administration of LA1 is beneficial in preventing hyperoxia-induced neonatal lung injury, an experimental model of BPD. Newborn rats were exposed to normoxia (21% O2) or hyperoxia (85% O2) and received twice-daily intraperitoneal injection of LA1 or placebo for 14 days. Hyperoxia exposure in the presence of the placebo resulted in a drastic increase in the influx of neutrophils and macrophages into the alveolar airspaces. This increased leukocyte influx was accompanied by decreased alveolarization and angiogenesis and increased pulmonary vascular remodeling and pulmonary hypertension (PH), the pathological hallmarks of BPD. However, administration of LA1 decreased macrophage infiltration in the lungs during hyperoxia. Furthermore, treatment with LA1 improved alveolarization and angiogenesis and decreased pulmonary vascular remodeling and PH. These data indicate that leukocyte recruitment plays an important role in the experimental model of BPD induced by hyperoxia. Targeting leukocyte trafficking using LA1, an integrin agonist, is beneficial in preventing lung inflammation and protecting alveolar and vascular structures during hyperoxia. Thus, targeting integrin-mediated leukocyte recruitment and inflammation may provide a novel strategy in preventing and treating BPD in preterm infants.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-β2-glycoprotein I (anti-β2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of β2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

12/15‐Lipoxygenase‐Dependent ROS Production Is Required For Diet‐Induced Endothelial Barrier Dysfunction

To understand the mechanisms of 15(S)‐hydroxyeicosatetraenoic acid (15(S)‐HETE)‐induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)‐HETE induced JamA phosphorylation on Y164, Y218 and Y280 involving XO‐mediated ROS production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction disruption, increased vascular permeability and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appear to be critical for its role in 15(S)‐HETE‐induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high‐fat diet (HFD) feeding, WT but not 12/15‐LO‐/‐ mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by Allopurinol. These observations provide novel insights on the role of XO in 12/15‐LO‐induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.

12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction

To understand the mechanisms of 15(S)-HETE-induced endothelial cell (EC) barrier dysfunction, we examined the role of xanthine oxidase (XO). 15(S)-HETE induced junction adhesion molecule A (JamA) phosphorylation on Y164, Y218, and Y280 involving XO-mediated reactive oxygen species production and Src and Pyk2 activation, resulting in its dissociation from occludin, thereby causing tight junction (TJ) disruption, increased vascular permeability, and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on Y164, Y218, and Y280 appears to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from occludin and restored TJ integrity and barrier function. In response to high-fat diet (HFD) feeding, WT, but not 12/15-lipoxygenase (LO)(-/-), mice showed enhanced XO expression and its activity in the artery, which was correlated with increased aortic TJ disruption and barrier permeability with enhanced leukocyte adhesion and these responses were inhibited by allopurinol. These observations provide novel insights on the role of XO in 12/15-LO-induced JamA tyrosine phosphorylation and TJ disruption leading to increased vascular permeability in response to HFD.

Xanthine Oxidase Mediates High Fat Diet-Induced Vascular Permeability via Junction Adhesion Molecule a Tyrosine Phosphorylation and Tight Junction Disruption

Previously we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the 15-LO metabolite of arachidonic acid (AA), affects endothelial cell (EC) barrier function. In this study, we have examined the role of xanthine oxidase (XO) in 15(S)-HETE-induced EC barrier dysfunction. 15(S)-HETE induced JamA phosphorylation on tyrosine residues 164, 218 and 280 involving XO-mediated reactive oxygen species (ROS) production and Src and Pyk2 activation, resulting in its dissociation from Occludin and causing tight junction disruption with increased EC barrier permeability and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on tyrosine residues 164, 218, and 280 appear to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from Occludin and restored TJ integrity and barrier function. Feeding WT mice but not 12/15-LO-/- mice with HFD for 3 months resulted in enhanced XO expression and its activity in the artery, which was correlated with increased aortic EC TJ disruption and its barrier permeability with enhanced leukocyte adhesion. These observations provide additional support for the role of 12/15-LOX in the dysregulation of vascular permeability in response to HFD. DisclosuresNo relevant conflicts of interest to declare.

Palmitate-stimulated monocytes induce adhesion molecule expression in endothelial cells via IL-1 signaling pathway.

Increased intake of saturated fatty acids (SFAs), such as palmitate (Pal), is linked to a higher risk of type 2 diabetes and cardiovascular disease. Although recent studies have investigated the direct effects of SFAs on inflammatory responses in vascular endothelial cells, it remains unknown whether SFAs also induce these responses mediated by circulating cells. In this study, especially focused on adhesion molecules and monocytes, we investigated the indirect effects of Pal on expression and release of ICAM-1 and E-selectin in vascular endothelial cells. Phorbol 12-myristate 13-acetate (PMA)-treated THP-1 (pTHP-1) cells and human monocytes were stimulated with various free fatty acids (FFAs). SFAs, but not unsaturated fatty acids (UFAs), increased interleukin (IL)-1β secretion and decreased IL-1 receptor antagonist (IL-1Ra) secretion, resulting in an increase in the IL-1β/IL-1Ra secretion ratio. UFAs dose-dependently inhibited the increase in IL-1β secretion and decrease in IL-1Ra secretion induced by Pal. Moreover, in human aortic and vein endothelial cells, expression and release of ICAM-1 and E-selectin were induced by treatment with conditioned medium collected from Pal-stimulated pTHP-1 cells and human monocytes, but not by Pal itself. The up-regulated expression and release of adhesion molecules by the conditioned medium were mostly abolished by recombinant human IL-1Ra supplementation. These results suggest that the Pal-induced increase in the ratio of IL-1β/IL-1Ra secretion in monocytes up-regulates endothelial adhesion molecules, which could enhance leukocyte adhesion to endothelium. This study provides further evidence that IL-1β neutralization through receptor antagonism may be useful for preventing the onset and development of cardiovascular disease.

Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation.

Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte-endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution.

210 The Endothelial Transcription Factor ERG Inhibits Vascular Inflammation

The ETS transcription factor ERG is constitutively expressed in endothelial cells (EC) and acts as a master-regulator of endothelial function. ERG drives expression of genes which determine endothelial lineage and...