Cell Surface ICAM-1 Research Articles

Exposure to Secreted Bacterial Factors Promotes HIV-1 Replication in CD4+ T Cells.

Microbial translocation is associated with systemic immune activation in HIV-1 disease. Circulating T cells can encounter microbial products in the bloodstream and lymph nodes, where viral replication takes place. The mechanisms by which bacteria contribute to HIV-associated pathogenesis are not completely deciphered. Here, we examined how bacteria may impact T cell function and viral replication. We established cocultures between a panel of live bacteria and uninfected or HIV-1-infected activated peripheral blood CD4-positive (CD4+) T cells. We show that some bacteria, such as Escherichia coli and Acinetobacter baumannii, sustain lymphocyte activation and enhance HIV-1 replication. Bacteria secrete soluble factors that upregulate CD25 and ICAM-1 cell surface levels and activate NF-κB nuclear translocation. Our data also demonstrate that CD25 polarizes at the virological synapse, suggesting a previously unappreciated role of CD25 during viral replication. These findings highlight how interactions between bacterial factors and T cells may promote T cell activation and HIV-1 replication. IMPORTANCE People living with HIV suffer from chronic immune activation despite effective antiretroviral therapy. Early after infection, HIV-1 actively replicates in the gut, causing the breakage of the intestinal epithelial barrier and microbial translocation. Microbial translocation and chronic immune activation have been proven linked; however, gaps in our knowledge on how bacteria contribute to the development of HIV-related diseases remain. Whether T cells in the peripheral blood react to bacterial products and how this affects viral replication are unknown. We show that some bacteria enriched in people living with HIV activate T cells and favor HIV-1's spread. Bacteria release soluble factors that cause the overexpression of cellular molecules related to their activation state. T cells overexpressing these molecules also replicate HIV-1 more efficiently. These results help us learn more about how HIV-1, T cells, and bacteria interact with each other, as well as the mechanisms behind chronic immune activation.

Differential Inflammatory Responses in Cultured Endothelial Cells Exposed to Two Conjugated Linoleic Acids (CLAs) under a Pro-Inflammatory Condition.

Conjugated linoleic acid (CLA) isomers have been shown to possess anti-atherosclerotic properties, which may be related to the downregulation of inflammatory pathways in different cell types, including endothelial cells (ECs). However, whether different CLA isomers have different actions is not entirely clear, with inconsistent reports to date. Furthermore, in cell culture studies, CLAs have often been used at fairly high concentrations. Whether lower concentrations of CLAs are able to affect EC responses is not clear. The aim of this study was to evaluate the effects of two CLAs (cis-9, trans-11 (CLA9,11) and trans-10, cis-12 (CLA10,12)) on the inflammatory responses of ECs. ECs (EA.hy926 cells) were cultured under standard conditions and exposed to CLAs (1 to 50 μM) for 48 h. Then, the cells were cultured for a further 6 or 24 h with tumour necrosis factor alpha (TNF-α, 1 ng/mL) as an inflammatory stimulant. ECs remained viable after treatments with 1 and 10 μM of each CLA, but not after treatment with 50 μM of CLA10,12. CLAs were incorporated into ECs in a concentration-dependent manner. CLA10,12 increased the levels of ICAM-1, IL-6, and RANTES in the culture medium, while CLA9,11 had null effects. Both CLAs (1 μM) decreased the appearance of NFκB1 mRNA, but only CLA9,11 maintained this downregulation at 10 μM. CLA10,12 had no effect on THP-1 cell adhesion to ECs while significantly decreasing the percentage of ECs expressing ICAM-1 and also levels of ICAM-1 expression per cell when used at 10 µM. Although CLA9,11 did not have any effect on ICAM-1 cell surface expression, it reduced THP-1 cell adhesion to the EA.hy926 cell monolayer at both concentrations. In summary, CLA10,12 showed some pro-inflammatory effects, while CLA9,11 exhibited null or anti-inflammatory effects. The results suggest that each CLA has different effects in ECs under a pro-inflammatory condition, highlighting the need to evaluate the effects of CLA isomers independently.

Melatonin, a Potential Therapeutic Agent for Preeclampsia, Reduces the Extrusion of Toxic Extracellular Vesicles from Preeclamptic Placentae.

Preeclampsia, characterised by maternal endothelial cell activation, is triggered by toxic factors, such as placental extracellular vesicles (EVs) from a dysfunctional placenta. The increased oxidative stress seen in the preeclamptic placenta links to endoplasmic reticulum (ER) stress. The ER regulates protein folding and trafficking. When the ER is stressed, proteins are misfolded, and misfolded proteins are toxic. Misfolded proteins can be exported from cells, via EVs which target to other cells where the misfolded proteins may also be toxic. Melatonin is a hormone and antioxidant produced by the pineal gland and placenta. Levels of melatonin are reduced in preeclampsia. In this study we investigated whether melatonin treatment can change the nature of placental EVs that are released from a preeclamptic placenta. EVs were collected from preeclamptic (n = 6) and normotensive (n = 6) placental explants cultured in the presence or absence of melatonin for 18 h. Misfolded proteins were measured using a fluorescent compound, Thioflavin-T (ThT). Endothelial cells were exposed to placental EVs overnight. Endothelial cell activation was measured by the quantification of cell-surface ICAM-1 using a cell-based ELISA. EVs from preeclamptic placentae carried significantly (p < 0.001) more misfolded proteins than normotensive controls. Incubating preeclamptic placental explants in the presence of melatonin (1 µM and 10 µM) significantly (p < 0.001) reduced the misfolded proteins carried by EVs. Culturing endothelial cells in the presence of preeclamptic EVs significantly increased the expression of ICAM-1. This increased ICAM-1 expression was significantly reduced when the endothelial cells were exposed to preeclamptic EVs cultured in the presence of melatonin. This study demonstrates that melatonin reduces the amount of misfolded proteins carried by EVs from preeclamptic placentae and reduces the ability of these EVs to activate endothelial cells. Our study provides further preclinical support for the use of melatonin as a treatment for preeclampsia.

Abstract 4548: SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity

Abstract CD112R (PVRIG) is a recently identified inhibitory receptor of the PVR gene family, which includes TIGIT, CD96, and CD226, that is expressed on NK cells and T cells. CD112R suppresses immune cell activation through its association with the cell adhesion molecule CD112 (PVRL2), a ligand that it competes for with the activating receptor CD226. CD112 binding to CD112R induces downstream signaling via recruitment of SHP-1, SHP-2, and SHIP-1 phosphatases to the ITIM in the cytoplasmic tail, resulting in dampened effector cell activation. As CD112 is commonly upregulated on cancer cells and is also expressed on tumor infiltrating myeloid cells, it was hypothesized that disruption of the CD112:CD112R interaction would promote anti-tumor immune responses. Phenotypic screening of a panel of antibodies generated against CD112R led to the identification of SRF813, a high affinity, fully human CD112R antibody that blocks the interaction of CD112R with CD112. To explore the therapeutic potential of CD112R blockade, the ability of SRF813 to increase immune cell activation was evaluated in human peripheral blood mononuclear cells. In vitro assays showed that SRF813 treatment enhanced NK cell activation in response to multiple tumor cell lines. This activation was characterized by upregulation of several cell surface markers, including 4-1BB and ICAM-1, in addition to increased cytokine production and degranulation. The activity of SRF813 was highly dependent on the antibody isotype and Fc receptor engagement. Mechanistically, the enhanced immunologic responses observed in NK cells treated with SRF813 could be reversed in the presence of a CD226 blocking antibody, confirming the role of CD112R in regulating CD226 mediated signaling. Blockade of CD112R in mouse syngeneic tumor models using a murine surrogate of SRF813 demonstrated reduced tumor growth with an accompanying increase in TIL activation following treatment. In the CT-26 tumor model, anti-CD112R treatment resulted in complete tumor regression in a subset of treated mice. These mice rapidly rejected tumors upon re-challenge, signifying the development of an immunologic anti-tumor memory response in these animals. Additional depletion studies highlighted that the anti-tumor efficacy was dependent on both CD8 T cells and NK cells. Moreover, the combination of anti-CD112R with PD-1 blockade led to greater inhibition of tumor growth than either treatment alone. Collectively, these preclinical data demonstrated that CD112R is a negative regulator of immune responses and that CD112R blockade can potentiate anti-tumor responses in cancers that express CD112. Citation Format: Marisella Panduro, Roy M. Dornbrook, Kshama A. Doshi, Jing Hua, Jamie Strand, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill, James F. Mohan. SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4548.

Transport of Extracellular Vesicles across the Blood-Brain Barrier: Brain Pharmacokinetics and Effects of Inflammation.

Extracellular vesicles can cross the blood–brain barrier (BBB), but little is known about passage. Here, we used multiple-time regression analysis to examine the ability of 10 exosome populations derived from mouse, human, cancerous, and non-cancerous cell lines to cross the BBB. All crossed the BBB, but rates varied over 10-fold. Lipopolysaccharide (LPS), an activator of the innate immune system, enhanced uptake independently of BBB disruption for six exosomes and decreased uptake for one. Wheatgerm agglutinin (WGA) modulated transport of five exosome populations, suggesting passage by adsorptive transcytosis. Mannose 6-phosphate inhibited uptake of J774A.1, demonstrating that its BBB transporter is the mannose 6-phosphate receptor. Uptake rates, patterns, and effects of LPS or WGA were not predicted by exosome source (mouse vs. human) or cancer status of the cell lines. The cell surface proteins CD46, AVβ6, AVβ3, and ICAM-1 were variably expressed but not predictive of transport rate nor responses to LPS or WGA. A brain-to-blood efflux mechanism variably affected CNS retention and explains how CNS-derived exosomes enter blood. In summary, all exosomes tested here readily crossed the BBB, but at varying rates and by a variety of vesicular-mediated mechanisms involving specific transporters, adsorptive transcytosis, and a brain-to-blood efflux system.

TGF-β regulated leukemia cell susceptibility against NK targeting through the down-regulation of the CD48 expression

Transforming growth factor-β (TGF-β) is known to function as a dual role regulatory cytokine for being either a suppresser or promoter during tumor initiation and progression. In solid tumors, TGF-β secreted from tumor microenvironment acts as a suppresser against host immunity, like natural killer (NK) cells, to favor tumor evasion. However, besides solid tumors, the underlying mechanism of how TGF-β regulates leukemogenesis, tumor progression, immunoediting, and NK function is still not clear in detail. In this study, we found that TGF-β induced leukemia MEG-01 and U937 cells to become less sensitive to NK-92MI targeting by down-regulating CD48, a ligand for NK activating receptor 2B4, but not down-regulating other tumor-associated carbohydrate antigens (TACAs). In CD48-knockdown cells, cells responding to NK-92MI targeting displayed a phenotype of less NK susceptibility and cell conjugation. On the other hand, when NK cells were treated with TGF-β, TGF-β suppressed NK recognition, degranulation, and killing activity in time-dependent manner by regulating ICAM-1 binding capacity instead of affecting expressions of activating and inhibitory receptors. Taken together, both leukemia cells and immune NK cells could be regulated by TGF-β through suppressing leukemia cell surface CD48 to escape from host surveillance and down-regulating NK cell surface ICAM-1 binding activity to impair NK functions, respectively. Our results suggested that TGF-β had effect in leukemia similar to that observed in solid tumors but through different regulatory mechanism.

Endothelial Cell Biomechanical Responses are Dependent on Both Fluid Shear Stress and Tensile Strain.

The goal of this study was to investigate how concurrent shear stress and tensile strain affect endothelial cell biomechanical responses. Human coronary artery endothelial cells were exposed to concurrent pulsatile shear stress and cyclic tensile strain in a programmable shearing and stretching device. Three shear stress-tensile strain conditions were used: (1) pulsatile shear stress at 1Pa and cyclic tensile strain at 7%, simulating normal stress/strain conditions in a healthy coronary artery; (2) shear stress at 3.7Pa and tensile strain at 3%, simulating pathological stress/strain conditions near a stenosis; (3) shear stress at 0.7Pa and tensile strain at 5%, simulating pathological stress/strain conditions in a recirculation zone. Cell morphology was quantified using immunofluorescence microscopy. Cell surface PECAM-1 phosphorylation, ICAM-1 expression, ERK1/2 and NF-κB activation were measured using ELISA or Western blot. Simultaneous stimulation from pulsatile shear stress and cyclic tensile strain induced a significant increase in cell area, compared to that induced by shear stress or tensile strain alone. The combined stimulation caused significant increases in PECAM-1 phosphorylation. The combined stimulation also significantly enhanced EC surface ICAM-1 expression (compared to that under shear stress alone) and transcriptional factor NF-κB activation (compared to that under control conditions). Pulsatile shear stress and cyclic tensile strain could induce increased but not synergistic effect on endothelial cell morphology or activation. The combined mechanical stimulation can be relayed from cell membrane to nucleus. Therefore, to better understand how mechanical conditions affect endothelial cell mechanotransduction and cardiovascular disease development, both shear stress and tensile strain need to be considered.

Merkel cell carcinoma and cellular cytotoxicity: sensitivity to cellular lysis and screening for potential target antigens suitable for antibody-dependent cellular cytotoxicity.

The recent success of checkpoint inhibitors in the treatment of Merkel cell carcinoma (MCC) confirms that MCC tumors can be immunogenic. However, no treatment directly targeting the tumor is available for use in combination with these checkpoint inhibitors to enhance their efficacity. This study was carried out to characterize MCC line sensitivity to cellular lysis and to identify cell surface antigens that could be used for direct targeting of this tumor. For five representative MCC lines, the absence or low expression of MICA, MICB, HLA-I, and ICAM-1 was associated with low level of recognition by NK cells and T lymphocytes. However, expression of HLA-I and ICAM-1 and sensitivity to cellular lysis could be restored or increased after exposure to INFγ. We tested 41 antibodies specific for 41 different antigens using a novel antibody-dependent cellular cytotoxicity (ADCC) screening system for target antigens. Anti-CD326 (EpCAM) was the only antibody capable of inducing ADCC on the five MCC lines tested. Because MCC tumors are often directly accessible, local pharmacologic manipulation to restore HLA class-I and ICAM-1 cell surface expression (and thus sensitivity to cell lysis) can potentially benefit immune therapeutic intervention. In line with this, our observation that ADCC against EpCAM can induce lysis of MCC lines and suggests that therapeutic targeting of this antigen deserves to be explored further.

ICAM-1 upregulation is not required for retinoic acid-induced human eosinophil survival

Active metabolites of vitamin A, retinoic acids (RAs), are known to play critical roles in mucosal immune responses and dramatically inhibit human eosinophil apoptosis, but the detailed mechanisms have not been elucidated. We previously screened for ICAM-1 (CD54) upregulation in RA-stimulated human eosinophils by gene microarray analysis. As ICAM-1 induction and activation were observed to have a role in maintenance of eosinophil survival, we tested the hypothesis that RAs prolong eosinophil survival through ICAM-1 outside-in signaling. Blood-derived isolated eosinophils cultured with 9-cis RA and all-trans RA showed significant upregulation of ICAM-1 mRNA and cell surface expression. TTNPB, a retinoic acid receptor agonist, also induced ICAM-1 expression, while HX630, a retinoid X receptor agonist, did not. Furthermore, an RAR antagonist, HX531, completely inhibited the effect of RAs. Upregulated ICAM-1 was associated with altered kinetics of Akt, ERK, and p38 MAP kinase phosphorylation through ICAM-1 cross-linking, but an ICAM-1-blocking antibody did not affect RA-mediated cell survival. These findings indicate that RAs induce functional ICAM-1 expression through RARs, but the induced ICAM-1 does not contribute to prolongation of eosinophil survival.

JAK2V617F - Positive Endothelial Cells Display Pro-Thrombotic Characteristics

Background: Clinical complications of BCR-ABL1-negative Myeloproliferative Neoplasms (MPNs) include mainly vascular events and on longer term transformation to Myelofibrosis (MF) or acute leukemia. MPNs are also associated with neo-angiogenesis leading to extramedullary hematopoiesis and increased vascularity in the spleen and bone marrow. The most frequent molecular alteration present in hematopoietic cells is the JAK2V617F mutation, which has also been detected in hepatic Endothelial Cells (ECs) of MPN patients with Budd-Chiari syndrome (BCS) and in ECs derived from patients' splenic capillaries. The consequences of the presence of the JAK2V617F mutation into the endothelial lineage are not known although these cells play an important role in thrombosis.Methods: Isogenic JAK2 wild-type (WT) and JAK2V617F mutant induced pluripotent stem cells (iPSC) were derived from an MPN patient. The reprogrammed clonal cells were induced to differentiate toward mesoderm for 3 days and then driven toward the endothelial lineage using Stem-Pro medium, Vascular Endothelial Growth Factor (VEGF) and Forskolin. Enriched CD34+/CD144+ progenitors were then matured in VEGF-supplemented Stem-Pro medium for 5 days before being cultured in the EGM-2 specific EC medium in the presence of the TGF-β inhibitor SB-431542. Cells were harvested and tested for specific surface markers by flow cytometry before expanding them; passages 3 to 6 were used for further characterization and functional studies. Transcriptomic analysis was performed in triplicate from two independent endothelial differentiation experiments using Affymetrix HTA 2.0 gene expression arrays.Results: Phenotypically, the endothelial-like cells obtained after differentiation from both mutant and WT JAK2 iPSC displayed a classical EC morphology and were positive for CD34 and EC markers (CD144, CD309, CD31 and ICAM-1). Such characteristics were conserved for at least 6 rounds of cell passaging. TNF-α treatment increased ICAM-1 cell surface levels and induced the expression of VCAM-1, as observed in other EC models. Our iPS-derived cells were also responsive to VEGF, which induced cellular proliferation and angiogenesis demonstrated by tube formation in Matrigel with both JAK2V617F and WT ECs. As expected, JAK2V617F ECs exhibited higher levels of phosphorylated forms of JAK2, STAT3 and Akt in a serum and growth factor-reduced medium, showing that, as in hematopoietic cells, the presence of the mutation increases basal intracellular signaling. Interestingly, Weibel-Palade bodies detection by confocal microscopy revealed a more important amount of Von Willebrand factor (vWF) in JAK2V617F ECs (2-fold increase), suggesting a higher pro-thrombotic potential for these cells. Basal surface expression of the adhesion molecule ICAM-1 was also clearly increased (1.8-fold) compared to WT ECs suggesting that JAK2V617F ECs may be more prone to recruit activated leukocytes expressing LFA-1, the ICAM-1 receptor. Gene expression microarrays analysis confirmed a significantly higher expression of cell adhesion molecules involved in leukocyte or platelet adhesion in JAK2V617F vs. WT ECs (ICAM-1, VCAM-1, or P-selectin), together with several inflammatory chemokines (IL8, CXCL1, or CXCL5), chemokine receptors (CXCR4 or IL7R) and extracellular matrix modulators (Periostin, Tenascin C, Lysyl Oxidase, or MMP-10) . Finally, Gene Set Enrichment Analysis pointed out gene sets induced in situations of inflammation, response to cytokines and hypoxia. These results could be in agreement with an activated endothelium, leukocyte recruitment and vascular remodeling.Conclusion: Previous discovery of JAK2V617F-positive endothelial cells in the splanchnic vasculature of patients with BCS or MF was of particular interest because of the well-known association between thrombosis and MPN. In this work we first demonstrate that the presence of JAK2V617F doesn't impair endothelial differentiation from stem/progenitor cells. Moreover, JAK2V617F-mutant ECs display features of pro-inflammatory and pro-thrombotic EC activation, which we are currently further characterizing with additional functional studies. Finally, this iPSC model may represent an effective screening tool for molecules antagonizing the pro-thrombotic events in a JAK2V617F context. DisclosuresKiladjian:Novartis: Honoraria, Research Funding; AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Increased levels of HMGB1 in trophoblastic debris may contribute to preeclampsia.

Preeclampsia is triggered by an as yet unknown toxin from the placenta. Antiphospholipid antibodies (aPL), a strong risk factor for preeclampsia, have been shown to induce the production of toxic trophoblastic debris from the placenta. High mobility group box 1 (HMGB1) is a proinflammatory danger signal, and the expression of it has been reported to be increased in preeclampsia. This study examined whether aPL or preeclamptic sera increase the expression of HMGB1 in the syncytiotrophoblast or trophoblastic debris. Trophoblastic debris from normal placental explants that had been cultured with aPL or preeclamptic sera was exposed to endothelial cells. Endothelial cell activation was quantified by cell-surface ICAM-1 expression and U937 monocyte adhesion. The expression of HMGB1 in placental explants and trophoblastic debris that had been treated with aPL or preeclamptic sera was measured by immunohistochemistry and western blotting. The expression of the receptor for advanced glycation end products (RAGE) in endothelial cells was quantified by western blotting. Compared with controls, the expression of HMGB1 in the cytoplasm of the syncytiotrophoblast and trophoblastic debris was increased by treating placental explants with aPL or preeclamptic sera. The increased levels of HMGB1 contributed to endothelial cell activation, mediated in part by the RAGE. Preeclamptic sera and aPL both induced an increase in the cytoplasmic levels of the danger signal HMGB1 in trophoblastic debris. This increased HMGB1 in trophoblastic debris may be one of the toxic factors released from the placenta in preeclampsia.

Trophoblastic debris extruded from hydatidiform molar placentae activates endothelial cells: Possible relevance to the pathogenesis of preeclampsia

IntroductionPreeclampsia is currently thought to be induced by a placental factor that triggers maternal endothelial activation. It is now well known that trophoblastic debris shed from the placenta into the maternal blood is associated with this disease. Hydatidiform mole is a pathological pregnancy characterised by hyperplastic trophoblast with little or no fetal development. Women with molar pregnancies may exhibit symptoms resembling preeclampsia. Deportation of trophoblastic debris occurs in molar pregnancies but, whether trophoblastic debris from molar pregnancies expresses pathogenic signals or activates endothelial cells is unknown. MethodsTrophoblastic debris were collected from either hydatidiform molar or normal first trimester placental explants and then exposed to monolayers of endothelial cell for 24 h. Endothelial cell activation was measured by quantifying cell-surface ICAM-1using ELISA. In addition, the expressions of High mobility group box 1(HMGB1) and heat shock protein 70 (HSP70) on molar placenta were examined by immunohistochemistry and western blotting. Circulating levels of sEndoglin in molar pregnancy was also measured. ResultsExposing trophoblastic debris from molar placentae increased endothelial cell surface ICAM-1 expression compared to endothelial cells exposed to trophoblastic debris from controls. Expression of HSP70 but not HMGB1 was significantly increased in hydatidiform molar placentae. The circulating levels of sEndoglin in hydatidiform molar pregnancy were not increased compared to controls. DiscussionOur results suggest that trophoblastic debris from molar pregnancies induces endothelial cell activation. HSP70 but not HMGB1 expressed on hydatidiform molar placenta may be a pathogenic signal to endothelial cells.

61 Melatonin supplementation prevents endothelial cell activation, possible relevance to preeclampsia

Introduction Preeclampsia, a human pregnancy-specific disease, is a leading cause of maternal and perinatal mortality and morbidity. It is characterised by maternal endothelial cell dysfunction. Although the exact causes of this disease are unclear, it is thought that there is an increase in oxidative stress in the placenta and leading to the release of a toxic factor (s) from the placenta that triggers maternal endothelial cell activation. Endothelial cell activation precedes the clinical signs and symptoms of the maternal syndrome of preeclampsia. Trophoblast debris, which is shed from the placenta into the maternal blood, is hypothesised to be one such trigger. Our previous studies showed that treating placentae with antiphospholipid antibodies (aPL), which increase the risk of women developing preeclampsia 10 fold [1, 2], or preeclamptic sera, results in the production of toxic trophoblastic debris which induces endothelial cell activation [3]. Melatonin is a lipid soluble hormone originally found to be released by the pineal gland but which has been shown to have antioxidant activities and to be produced in the placenta. Given the action of melatonin in regulating oxidative stress and cell death, we investigated whether melatonin supplementation can reverse the effects of preeclamptic sera or aPL on placental oxidative damage and the production of toxic trophoblastic debris. Methods First trimester placental explants were cultured with aPL (20 μ g/ml) or preeclamptic sera (n = 6) in the presence or absence of melatonin (1 μ M, 10 μ M) for 24 h. Trophoblastic debris was collected from the explants and then exposed to monolayers of endothelial cells (HMEC-1) for 24 h and cell surface ICAM-1 expression was measured by cell-based ELISA to determine endothelial cell activation. The expression of nitrotyrosine in placental explants that were treated with aPL or preeclamptic sera was measured by immunohistochemistry or western blotting. Results 1) Trophoblastic debris from placental explants that were treated with aPL or preeclamptic sera (n = 6) significantly (p 2) In contrast, trophoblastic debris from explants that were treated with aPL or preeclamptic sera in the presence of melatonin (n = 6) did not significantly increased the expression of ICAM-1 compared to control-treated endothelial cells 3) The expression of nitrotyrosine was significantly increased in placentae that had been treated with aPL or preeclamptic sera. However, this increase in levels of nitrotyrosine was not found in placental explants where aPL or PE sera were added in the presence of melatonin. Conclusions Our results suggest that there is oxidative damage induced in placentae by either aPL or preeclamptic sera and consequently trophoblastic debris produced by these placentae is more toxic to endothelial cells. Melatonin can reverse this placental oxidative damage and reduced the toxicity of the trophoblastic debris. Since the pathogenesis of preeclampsia is thought to be partially dependent on the maternal response to placental factors such as toxic trophoblastic debris shed from placentae, our results encouraging suggest that melatonin might be a useful tool to alleviate the clinical manifestations of preeclampsia.

Cold-inducible RNA-binding protein causes endothelial dysfunction via activation of Nlrp3 inflammasome.

Cold-inducible RNA-binding protein (CIRP) is a damage-associated molecular pattern (DAMP) molecule which stimulates proinflammatory cytokine release in hemorrhage and sepsis. Under these medical conditions, disruption of endothelial homeostasis and barrier integrity, typically induced by proinflammatory cytokines, is an important factor contributing to morbidity and mortality. However, the role of CIRP in causing endothelial dysfunction has not been investigated. In this study, we show that intravenous injection of recombinant murine CIRP (rmCIRP) in C57BL/6 mice causes lung injury, evidenced by vascular leakage, edema, increased leukocyte infiltration and cytokine production in the lung tissue. The CIRP-induced lung damage is accompanied with endothelial cell (EC) activation marked by upregulation of cell-surface adhesion molecules E-selectin and ICAM-1. Using in vitro primary mouse lung vascular ECs (MLVECs), we demonstrate that rmCIRP treatment directly increases the ICAM-1 protein expression and activates NAD(P)H oxidase in MLVECs. Importantly, CIRP stimulates the assembly and activation of Nlrp3 inflammasome in MLVECs accompanied with caspase-1 activation, IL-1β release and induction of proinflammatory cell death pyroptosis. Finally, our study demonstrates CIRP-induced EC pyroptosis in the lungs of C57BL/6 mice for the first time. Taken together, the released CIRP in shock can directly activate ECs and induce EC pyroptosis to cause lung injury.

A novel anti-inflammatory mechanism of high density lipoprotein through up-regulating annexin A1 in vascular endothelial cells

High density lipoprotein (HDL) as well as annexin A1 have been reported to be associated with cardiovascular protection. However, the correlation between HDL and annexin A1 was still unknown. In this study, HDL increased endothelial annexin A1 and prevented the decrease of annexin A1 in TNF-α-activated endothelial cells in vitro and in vivo, and above effects were attenuated after knockdown of annexin A1. Annexin A1 modulation affected HDL-mediated inhibition of monocyte adhesion to TNF-α-activated endothelium (45.2±13.7% decrease for annexin A1 RNA interference; 78.7±16.3% decrease for anti-Annexin A1 antibody blocking; 11.2±6.9% increase for Ad-ANXA1 transfection). Additionally, HDL up-regulated annexin A1 through scavenger receptor class B type I, involving ERK, p38MAPK, Akt and PKC signaling pathways, and respective inhibitors of these pathways attenuated HDL-induced annexin A1 expression as well as impaired HDL-mediated inhibition of monocyte–endothelial cell adhesion. Apolipoprotein AI also increased annexin A1 and activated similar signaling pathways. Endothelial annexin A1 from apolipoprotein AI knockout mice was decreased in comparison to that from wild type mice. Finally, HDL-induced annexin A1 inhibited cell surface VCAM-1, ICAM-1 and E-selectin, and secretion of MCP-1, IL-8, VCAM-1 and E-selectin, thereby inhibiting monocyte adhesion.

Reduction in the severity of early onset severe preeclampsia during gestation may be associated with changes in endothelial cell activation: A pathological case report

ABSTRACTEarly severe preeclampsia with changes consistent with the Hemolysis elevated liver enzymes low platelet count (HELLP) variant and severe fetal growth restriction rarely resolves prior to delivery. Established clinical disease is preceded by endothelial dysfunction and inflammation. Endothelial activation is reported in vitro to be raised in the presence of necrotic trophoblastic debris which is deported into the maternal circulation in preeclampsia. We report on an early severe preeclamptic patient admitted at 24 weeks gestation. Maternal serum was taken at day 2, 16, 30 of admission and 45 days postpartum. 20% maternal serum or trophoblastic debris from first trimester placental explants that had been cultured with 10% maternal serum was exposed to endothelial cells. Endothelial cell activation was quantified by the cell surface ICAM-1 expression and U937 monocyte adhesion assay. The clinical condition of this patient improved including the blood pressure, liver function, and platelet count by the 3rd day after antihypertensive treatment and remained normal until delivery at 37 weeks. ICAM-1 expression and U937 moncyte adhesion assay of endothelial cells was significantly increased following exposure of the endothelial cells to the maternal serum or trophoblastic debris from placentae treated with maternal serum drawn on day 2. However, ICAM-1 expression and the monocyte adhesion assay were significantly reduced following exposure of endothelial cells to maternal serum or trophoblastic debris from placenta treated with maternal serum drawn on day 16 or 30. Our data suggest unknown factor(s) in the maternal serum triggered endothelial cell activation when the clinical symptoms were present. The improvement in the clinical condition occurred along with the changes in endothelial cell activation.

Extracellular Uridine Triphosphate and Adenosine Triphosphate Attenuate Endothelial Inflammation through miR-22-Mediated ICAM-1 Inhibition

Adenosine and uridine triphosphate (ATP and UTP) can act as extracellular signalling molecules, playing important roles in vascular biology and disease. ATP and UTP acting via the P2Y₂-receptor have, for example, been shown to regulate endothelial dilatation, inflammation and angiogenesis. MicroRNAs (miRNAs), a class of regulatory, short, non-coding RNAs, have been shown to be important regulators of these biological processes. In this study, we used RNA deep-sequencing to explore changes in miRNA expression in the human microvascular endothelial cell line HMEC-1 upon UTP treatment. The expression of miR-22, which we have previously shown to target ICAM-1 mRNA in HMEC-1, increased significantly after stimulation. Up-regulation of miR-22 and down-regulation of cell surface ICAM-1 were confirmed with qRT-PCR and flow cytometry, respectively. siRNA-mediated knockdown of the P2Y₂-receptor abolished the effect of UTP on miR-22 transcription. Leukocyte adhesion was significantly inhibited in HMEC-1 following miR-22 overexpression and treatment with UTP/ATP. In conclusion, extracellular UTP and ATP can attenuate ICAM-1 expression and leukocyte adhesion in endothelial cells through miR-22.

The reduction of circulating levels of IL-6 in pregnant women with preeclampsia by magnesium sulphate and nifedipine: In vitro evidence for potential mechanisms

IntroductionWomen with preeclampsia have elevated levels of inflammatory cytokines including IL-6. IL-6, which is known to activate endothelial cells and induce the production of necrotic trophoblastic debris from the placenta, may be important in the pathogenesis of preeclampsia. MgSO4 is a major therapy for the prevention of seizures in preeclampsia but it has been suggested to also have anti-inflammatory and vasodilatory properties. Methods22 pregnant women with preeclampsia and 68 normotensive controls were recruited and circulating IL-6 levels in these women were measured before MgSO4 and nifedipine treatment and after delivery. In addition, endothelial cells were treated with IL-6 or necrotic trophoblastic debris, generated from first trimester placental explants in the presence or absence of MgSO4 in vitro, and cell-surface ICAM-1 was measured by ELISA. The levels of IL-6 in the culture medium were also measured. Furthermore nitric oxide synthetase activity in endothelial cells that had been treated with IL-6 was measured using l-NAME. ResultsCirculating levels of IL-6 in preeclampsia were reduced significantly following administration of MgSO4. In vitro, MgSO4 reversed the activation of endothelial cells induced by IL-6 but not by necrotic trophoblastic debris. The effect of MgSO4 in reversing the IL-6 induced activation of endothelial cells was not dependent upon nitric oxide synthetase. Treating placental explants with MgSO4 prevented the production of necrotic trophoblastic debris induced by IL-6. Discussionwe demonstrated that IL-6 levels drop following treatment with MgSO4 and nifedipine in vivo, and have identified several mechanisms by which this positive effect on IL-6 may occur in vitro.

The combined effect of sidestream smoke and dynamic shear stress on endothelial cell inflammatory responses

Both cigarette smoke and altered shear stress are risk factors for cardiovascular disease. Sidestream smoke is the major component of secondhand smoke. An in vitro model was developed to investigate the combined effect of sidestream smoke and physiologically relevant dynamic shear stress on endothelial cell inflammatory responses. Human coronary artery endothelial cells were exposed to sidestream smoke and dynamic shear stress (at normal or low level) simultaneously, and endothelial cell surface ICAM-1 and thrombomodulin expression was measured using a solid phase ELISA approach. Endothelial cell associated complement activation was assessed by cell surface C1q, C4d and iC3b deposition. The expression of complement inhibitors (C1 inhibitor and CR1) and C1q receptors (gC1qR and cC1qR) was also measured. The results demonstrated that sidestream smoke enhanced endothelial cell surface ICAM-1 expression and caused cell activation. While under normal pulsatile shear stress, endothelial cell surface ICAM-1 expression reduced to baseline level as thrombomodulin expression increased. Physiological dynamic shear stress also induced a significant increase in endothelial cell associated complement activation, through enhanced gC1qR and cC1qR expression. Subsequently, CR1 expression increased as well. Overall, physiological dynamic shear stress reduced endothelial cell activation by enhancing thrombomodulin expression. Physiological flow also enhanced the expression of endothelial cell surface C1q receptors, gC1qR and cC1qR, to promote C1q activation and initiate the classical pathway complement activation, which could be a potential protective mechanism to clear injured or damaged cells.

Trophoblast debris extruded from preeclamptic placentae activates endothelial cells: A mechanism by which the placenta communicates with the maternal endothelium

IntroductionPreeclampsia is characterized by maternal endothelial dysfunction. While the mechanisms leading to preeclampsia are unclear, a factor(s) from the placenta is responsible for triggering the disease. One placental factor implicated in triggering preeclampsia is trophoblast debris which may transmit pathogenic signals from the placenta to endothelial cells. In this study, we investigated whether trophoblast debris from preeclamptic placentae triggered endothelial cell activation. MethodsTrophoblast debris from preeclamptic or normotensive placentae, or trophoblast debris from normal placental explants that had been cultured with preeclamptic (n = 14) or normotensive sera (n = 14) was exposed to endothelial cells. Activation of the endothelial cells was quantified by cell surface ICAM-1 and U937 adhesion to endothelial cells. The levels of IL-1β, pro-caspase-1 and active caspase-1 in the trophoblast debris were measured. ResultsCompared to controls, the levels of ICAM-1 and U937 adhesion to endothelial cells were significantly increased following exposure of the endothelial cells to trophoblast debris from preeclamptic placentae or placentae treated with preeclamptic sera. The levels IL-1β, pro-caspase-1 and active caspase-1 were significantly increased in both trophoblast debris from preeclamptic placentae and placentae treated with preeclamptic sera. DiscussionThese results provide the first direct evidence that trophoblast debris produced from preeclamptic placentae or placentae treated with preeclamptic sera can activate the endothelium. ConclusionsTrophoblast debris from preeclamptic but not normotensive placentae can induce endothelial cell activation. This may be one mechanism by which the preeclamptic placenta communicates with the maternal endothelium to induce activation of the endothelium.