Cell Surface Expression Of ICAM-1 Research Articles

Abstract 5089: Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates

Abstract Background: Endometrial cancer is the most frequently occurring gynecologic cancer in developed countries and the third leading cause of cancer deaths among women. It remains one of the few malignant tumors with rapidly increasing mortality and morbidity. Patients with recurrent or metastatic endometrial cancer continue to experience poor outcomes. Antibody-drug conjugates (ADCs) represent a class of targeted therapeutic modalities that offer biomarker-based therapeutic options for patients with endometrial cancer. In this study, we proposed to identify a novel molecular target for the preclinical development of an ADC for endometrial cancer-targeted therapy. Methods: Firstly, to determine the level of target expression within endometrial cancer tissue components as well as differences, we performed immunohistochemical staining of human endometrial cancer and paracancerous tissue. Secondly, the expression of the target on the cell membrane surface was quantitated and qualitatively verified using flow cytometry and single-photon confocal microscopy. In the third step, an endocytosis assay was used to verify the endocytosis of the target antibody by EC cells. Confocal imaging can directly observe the endocytosis of the target antibody, and flow cytometry can be analyzed and quantified. Co-localization experiments with antibody fluorescence can verify the specific site where the target antibody is endocytosed into cancer cells. We then designed and characterized two targeted ADCs. Next, the in vitro inhibitory activities of these ADCs against endometrial cancer cell lines and normal cells were evaluated using the CCK8 assay. Finally, we established a cell line-derived xenograft (CDX) model of endometrial cancer to validate the in vivo efficacy and bio-safety of the ADCs. Results: We have determined a correlation between ICAM1 overexpression and EC, and IHC staining of patient tissues showed significantly higher levels of ICAM1 expression in endometrial cancer tissues than in paracancerous tissues. The high cell surface expression of ICAM1 was verified in a panel of various human endometrial cancer cell lines. Moreover, two ICAM1 ADCs were established using clinically-approved ADC linker and payload combinations. It was visually confirmed that ICAM1 ADCs selectively recognize endometrial tumors and efficiently enter endometrial cells via antigen-mediated endocytosis. We compared the anti-tumor activity of ICAM1 ADCs with standard-of-care chemodrugs, and ICAM1 ADCs outperformed chemodrugs in the CDX model of endometrial cancer without toxicity. Conclusion: We identified that ICAM1 is a promising ADC target for endometrial cancer and confirmed the potent anti-tumor activity and biosafety of ICAM1 ADCs in vitro and in vivo, warranting further pre-clinical and clinical investigations. Citation Format: Hanfei Xie, Lu Sun, ShiLi Yao, Peng Guo, YingLi Zhang. Therapeutically targeting endometrial cancer by ICAM1 antibody-drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5089.

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.

Endothelial Activation in Orientia tsutsugamushi Infection Is Mediated by Cytokine Secretion From Infected Monocytes.

Scrub typhus, caused by Orientia tsutsugamushi, is a common systemic infection in Asia. Delay in diagnosis and treatment can lead to vasculitis in the visceral organs and other complications. The mechanisms that drive endothelial activation and the inflammatory response in O. tsutsugamushi infection remain unknown. In addition, the interaction between monocytes and endothelial cells is still unclear. Here we demonstrate that O. tsutsugamushi-infected human dermal microvascular endothelial cells produced moderate levels of chemokines and low levels of IL-6 and IFN-β, but not TNF or IL-1β. Recombinant TNF and cytokine-rich supernatants from infected monocytes markedly enhanced chemokine production in infected endothelial cells. We also show that TNF and monocyte supernatants, but not O. tsutsugamushi infection of endothelial cells per se, upregulated the endothelial cell surface expression of ICAM-1, E-selectin, and tissue factor. This finding was consistent with the inability of O. tsutsugamushi to induce cytokine secretion from endothelial cells. The upregulation of surface molecules after stimulation with monocyte supernatants was significantly reduced by neutralizing anti-TNF antibodies. These results suggest that endothelial cell activation and response are mainly mediated by inflammatory cytokines secreted from monocytes.

Potential Suppressive Effect of Nicotine on the Inflammatory Response in Oral Epithelial Cells: An In Vitro Study.

Smoking is a well-recognized risk factor for oral mucosal and periodontal diseases. Nicotine is an important component of cigarette smoke. This study aims to investigate the impact of nicotine on the viability and inflammatory mediator production of an oral epithelial cell line in the presence of various inflammatory stimuli. Oral epithelial HSC-2 cells were challenged with nicotine (10−8–10−2 M) for 24 h in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (LPS, 1 µg/mL) or tumor necrosis factor (TNF)-α (10−7 M) for 24 h. The cell proliferation/viability was determined by MTT assay. Gene expression of interleukin (IL)-8, intercellular adhesion molecule (ICAM)-1, and β-defensin was assayed by qPCR. The production of IL-8 protein and cell surface expression of ICAM-1 was assessed by ELISA and flow cytometry, respectively. Proliferation/viability of HSC-2 cells was unaffected by nicotine at concentrations up to 10−3 M and inhibited at 10−2 M. Nicotine had no significant effect on the basal expression of IL-8, ICAM-1, and β-defensin. At the same time, it significantly diminished P. gingivalis LPS or the TNF-α-induced expression levels of these factors. Within the limitations of this study, the first evidence was provided in vitro that nicotine probably exerts a suppressive effect on the production of inflammatory mediators and antimicrobial peptides in human oral epithelial cells.

Advancing human induced pluripotent stem cell-derived blood-brain barrier models for studying immune cell interactions.

Human induced pluripotent stem cell (hiPSC)‐derived blood‐brain barrier (BBB) models established to date lack expression of key adhesion molecules involved in immune cell migration across the BBB in vivo. Here, we introduce the extended endothelial cell culture method (EECM), which differentiates hiPSC‐derived endothelial progenitor cells to brain microvascular endothelial cell (BMEC)‐like cells with good barrier properties and mature tight junctions. Importantly, EECM‐BMEC‐like cells exhibited constitutive cell surface expression of ICAM‐1, ICAM‐2, and E‐selectin. Pro‐inflammatory cytokine stimulation increased the cell surface expression of ICAM‐1 and induced cell surface expression of P‐selectin and VCAM‐1. Co‐culture of EECM‐BMEC‐like cells with hiPSC‐derived smooth muscle‐like cells or their conditioned medium further increased the induction of VCAM‐1. Functional expression of endothelial ICAM‐1 and VCAM‐1 was confirmed by T‐cell interaction with EECM‐BMEC‐like cells. Taken together, we introduce the first hiPSC‐derived BBB model that displays an adhesion molecule phenotype that is suitable for the study of immune cell interactions.

Comparing effects of polyunsaturated fatty acids derived from marine and plant sources on endothelial cell inflammation

AbstractHigh consumption of ω-3 long chain polyunsaturated fatty acids (PUFAs) has long-term health benefits. The principal dietary source of these ω-3 PUFAs (eicosapentaenoic acid, EPA and docosapentaenoic acid, DHA) is seafood, particularly oily fish. However current fish stocks are decreasing, indicating a need for alternative sources of bioactive PUFAs. Plant-derived ω-3 PUFAs (alpha-linolenic acid, ALA and stearidonic acid, SDA) may be able to provide land-based sustainable sources, but their functionality has been underexplored.Anti-inflammatory effects of ALA and SDA were compared to EPA and DHA in cultured EA.hy926 endothelial cells. Cells were treated with PUFAs (10, 25 and 50 μM) for 48 hours prior to stimulation with tumour necrosis factor for 24 hours. PUFAs incorporation was measured by gas chromatography and inflammatory responses were measured by ELISA, RT-PCR, western blot and flow cytometry. Adhesion of THP-1 monocytes to EA.hy926 cells was determined using a static adhesion assay.All PUFAs were incorporated into EA.hy926 cells in a dose-dependant manner (10 and 50 μM). Pre-treatment with ALA, SDA, EPA and DHA (50 μM) had differential effects on inflammatory responses in EA.hy926 cells depending on PUFA and response examined.EA.hy926 cells pre-treated with SDA had lower concentrations of soluble ICAM-1 (p < 0.05); however EPA and DHA resulted in greater reduction (p < 0.0001). EPA and DHA pre-treated EA.hy926 cells had significantly lower concentrations of IL-6 (p < 0.0001), IL-8 (p < 0.0001) and MCP-1 (p < 0.05, p < 0.01). ALA pre-treatment did not significantly affect any of the cytokines examined. Lower cell surface expression of ICAM-1 (p < 0.05), was seen for EA.hy926 cells pre-treated with SDA again to a lesser extent than EPA and DHA (p < 0.001, p < 0.0001), with no effect seen after ALA treatment.EA.hy926 cells pre-treated with ALA had significantly higher relative gene expression of NFKB (p < 0.05), as well as a tendency for more phosphorylated NFKBp65 protein (p < 0.06). In contrast, EA.hy926 cells pre-treated with DHA had significantly less phosphorylated NFKB (p < 0.0001). EA.hy926 cells with DHA treatment had significantly higher relative gene expression of PPARa (p < 0.05). SDA and EPA had no effect on expression of either of the genes or proteins examined.Finally pre-treatment with ALA, SDA and DHA all resulted in reduced adhesion of THP-1 monocytes to EA.hy926, however this effect not observed with EPA.Marine derived PUFA, particularly DHA, resulted in potent anti-inflammatory effects within this endothelial cell model. Of the two plant derived PUFAs, SDA treatment lead to some anti-inflammatory effects, which were not seen after treatment with ALA.

Gamma-linolenic and pinolenic acids exert anti-inflammatory effects in cultured human endothelial cells through their elongation products

AbstractIt is recommended that humans consume fatty fish twice a week to increase dietary intake of eicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA) to achieve long-term health benefits. However current stocks of fish are likely insufficient to meet the needs of humans. Plant-derived polyunsaturated fatty acids (PUFAs) gamma-linolenic acid (GLA) and pinolenic acid (PIN) may provide sustainable land-based sources of bioactive fatty acids.Anti-inflammatory effects of GLA and PIN were compared to EPA and DHA in cultured EA.hy926 cells. Cells were treated with PUFAs (10, 25 and 50 μM) for 48 hours prior to stimulation with tumour necrosis factor for 24 hours. Incorporation of PUFA was measured by gas chromatography; inflammatory responses were measured by ELISA and flow cytometry.All fatty acids were incorporated into EA.hy926 cells, after 48 hours, in a dose dependent manner (10 and 50 μM). Pre-treatment with GLA and PIN (50 μM) resulted in significant increases in their elongation products, dihomo-γ-linolenic acid (DGLA) (p < 0.0001) from GLA and eicosatrienoic (ETrA) (p < 0.0001) from PIN.Pre-treatment with GLA, PIN, EPA or DHA (50 μM) had differential effects depending on fatty acid and cytokine examined. Pre-treatment of EA.hy926 cells with both GLA and PIN resulted in a lower concentration of soluble ICAM-1 (p < 0.01); however EPA and DHA showed greater reduction (p < 0.0001). MCP-1 production was significantly lower after treatment with PIN (p < 0.05), again to a lesser extent than EPA and DHA (p < 0.0001). Pre-treatment with EPA and DHA (50 μM) resulted in lower cell surface expression of ICAM-1 (p < 0.001, p < 0.0001), an effect not observed with GLA or PIN.Anti-inflammatory effects of GLA and PIN were possibly due to their elongation products, and therefore silencing of elongase 5 (ELOVL5) was explored. ELOVL5 siRNA significantly inhibited the production of DGLA and ETrA in EA.hy926 cells pre-treated with GLA and PIN (50 μM). Furthermore significant decreases in sICAM-1 and MCP-1 were not seen after pre-treatment with GLA or PIN in ELOVL5 siRNA silenced EA.hy926 cells.Plant PUFAs (GLA and PIN) demonstrate anti-inflammatory effects in this model using endothelial cells, but are less potent than EPA or DHA. Anti-inflammatory effects of GLA and PIN may be due to their elongation metabolites; DGLA and ETrA.

Enhanced angiogenic function in response to fibroblasts from psoriatic arthritis synovium compared to rheumatoid arthritis

IntroductionAngiogenesis is an early event in the pathogenesis of both psoriatic arthritis (PsA) and rheumatoid arthritis (RA); however, there are striking differences in blood vessel morphology and activation between the two arthropathies. The aim of this study was to assess if the PsA and RA joint microenvironments differentially regulate endothelial cell function.MethodsPsA and RA primary synovial fibroblasts (SFC) were isolated from synovial biopsies, grown to confluence, and supernatants harvested and termed ‘conditioned media’ (CM). Human umbilical vein endothelial cells (HUVEC) were cultured with PsA SFC or RA SFC-CM (20%). HUVEC tube formation, migration, and PBMC adhesion were assessed by matrigel tube formation, wound repair, and PBMC adhesion assays. HUVEC cell surface expression of ICAM, VCAM, and E-Selectin was assessed by flow cytometry. Transcriptome analysis of genes promoting angiogenesis was performed by real-time PCR. Finally, a MSD multiplex angiogenic assay was performed on PsA SFC and RA SFC supernatants.ResultsMacroscopic synovitis and vascularity were similar in PsA and RA patients; however, significant differences in vascular morphological pattern were recorded with tortuous, elongated vessels observed in PsA compared to straight regular branching vessels observed in RA. Transcriptome analysis showed strong upregulation of the pro-angiogenic signature in HUVEC primed with PsA SFC-CM compared to RA SFC-CM and basal control. In parallel, paired PsA SFC-CM significantly induced HUVEC tube formation compared to that of RA SFC-CM. Furthermore, PsA SFC-CM induced HUVEC migration was paralleled by a significant induction in VEGFA, PFKFB3, ICAM-1, and MMP3 mRNA expression. A significant increase in PBMC adhesion and cell surface expression of VCAM-1, ICAM-1, and E-Selectin expression was also demonstrated in PsA SFC-CM-primed HUVEC compared to RA SFC-CM. Finally, VEGF, TSLP, Flt-1, and Tie-2 expression was elevated in PsA SFC-CM compared to RA SFC-CM, with no significant difference in other pro-angiogenic mediators including MIP-3, bFGF, PIGF, and MCP-1.ConclusionPsA SFC and RA SFC secreted factors differentially regulate endothelial cell function, with soluble mediators in the PsA joint microenvironment inducing a more pro-angiogenic phenotype compared to the RA.

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.

The main rhinovirus respiratory tract adhesion site (ICAM-1) is upregulated in smokers and patients with chronic airflow limitation (CAL)

BackgroundICAM-1 is a major receptor for ~60% of human rhinoviruses, and non-typeable Haemophilus influenzae, two major pathogens in COPD. Increased cell-surface expression of ICAM-1 in response to tobacco smoke exposure has been suggested. We have investigated epithelial ICAM-1 expression in both the large and small airways, and lung parenchyma in smoking-related chronic airflow limitation (CAL) patients.MethodsWe evaluated epithelial ICAM-1 expression in resected lung tissue: 8 smokers with normal spirometry (NLFS); 29 CAL patients (10 small-airway disease; 9 COPD-smokers; 10 COPD ex-smokers); Controls (NC): 15 normal airway/lung tissues. Immunostaining with anti-ICAM-1 monoclonal antibody was quantified with computerized image analysis. The percent and type of cells expressing ICAM-1 in large and small airway epithelium and parenchyma were enumerated, plus percentage of epithelial goblet and submucosal glands positive for ICAM- 1.ResultsA major increase in ICAM-1 expression in epithelial cells was found in both large (p < 0.006) and small airways (p < 0.004) of CAL subjects compared to NC, with NLFS being intermediate. In the CAL group, both basal and luminal areas stained heavily for ICAM-1, so did goblet cells and sub-mucosal glands, however in either NC or NLFS subjects, only epithelial cell luminal surfaces stained. ICAM-1 expression on alveolar pneumocytes (mainly type II) was slightly increased in CAL and NLFS (p < 0.01). Pack-years of smoking correlated with ICAM-1 expression (r = 0.49; p < 0.03).ConclusionAirway ICAM-1 expression is markedly upregulated in CAL group, which could be crucial in rhinoviral and NTHi infections. The parenchymal ICAM-1 is affected by smoking, with no further enhancement in CAL subjects.

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.

Abstract 135: Endothelial Dysfunction in Acute Graft-versus-host Disease After Allogeneic Hematopoietic Cell Transplantation. In vitro Evidence of the Protective Effect of Defibrotide

Acute Graft-Versus-Host Disease (aGVHD) is the most common early complication after allogeneic Hematopoietic Cell Transplantation (allo-HCT). We demonstrated endothelial dysfunction (ED) in association with allo-HCT. According to this data, aGVHD has been linked to an inflammatory process that may affect the endothelium. To investigate the differential degree of endothelial damage in patients developing or not aGVHD, to identify potential biomarkers, and to explore the protective effect of defibrotide (DF) in this scenario. DF has orphan designation for GVHD prevention. Patients blood samples were collected before allo-HCT, at day 0, and every week till day 28 after HCT. Plasma proteins (sTNFR1, sVCAM-1, VWF and ADAMTS-13) were measured as biomarkers of ED in individual samples from patients developing (GVHD, n=24), or not (NoGVHD, n=13), aGVHD. In in vitro assays, endothelial cells (EC) in culture were exposed to media containing pooled sera from patients to evaluate changes in the: a) expression of VCAM-1 and ICAM-1 on cell surfaces; b) presence of VWF on the extracellular matrix (ECM) and c) reactivity of the ECM towards platelets, under flow. The effect of DF was explored in the in vitro experiments by previous exposure of the EC (for 24h) followed by continuous incubation (100 μg/ml, added every 24h). Levels of sTNFRI, sVCAM-1 and VWF in samples from group GVHD were significantly higher than in NoGVHD (increases of 100, 37 and 150% respectively, at diagnose, p&lt;0.01). ADAMTS-13 activity and VWF levels were inversely related. In in vitro studies, cell surface expression of VCAM-1 and ICAM-1, presence of VWF and platelet adhesion on the ECM in response to GVHD samples were always superior (increases vs NoGVHD of 80, 40, 100 and 21%, respectively, at diagnose). In vitro exposure of EC to DF attenuated signs of endothelial injury reducing significantly (p&lt;0.05) the expression of VCAM-1, ICAM-1 and VWF (reductions of 22, 30 and 30%, respectively) in the GVHD condition. Our results demonstrate endothelial damage in association with aGVHD, as evidenced by elevated plasma levels of several biomarkers. The in vitro approach showed a marked proinflammatory and prothrombotic phenotype in association with aGVHD, which could be significantly prevented by defibrotide.

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.

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.

Endothelial Cells Modulate Platelet Response to Dynamic Shear Stress

The goal of this study was to investigate how endothelial cells affect platelet response to physiologically relevant dynamic shear stress. In a cone and plate shearing device, washed platelets were exposed to constant (0.1, 0.3, 1, and 3 Pa) or dynamic (normal, low and temporally elevated) shear stress waveforms with or without the presence of endothelial cells. Platelet surface CD62P, CD42b and CD41a expression was measured using flow cytometry. Endothelial cell surface ICAM-1 and vWF expression was measured using ELISA, endothelial cell surface PECAM-1 expression was measured using fluorescence microscopy and endothelial cell derived microparticle generation was measured using flow cytometry. Elevated constant shear stress significantly increased platelet surface CD62P expression, and reduced platelet surface CD42b and CD41a expression. However, under dynamic flow conditions, low or elevated shear stress did not cause significant changes in platelets. But with the presence of endothelial cells, dynamic shear stress induced a significant increase in platelet surface CD62P expression and reduced platelet surface CD42b and CD41a expression. A significant increase in soluble vWF and the number of endothelial cell microparticles was observed, suggesting that soluble vWF and endothelial microparticle may be the mediators that induced platelet activation under pathological dynamic flow. These results suggest that pathological dynamic shear stress may not activate platelets directly. Through shear stress stimulated endothelial cells, platelets may become activated. As a protective mechanism, following cell activation, platelet adhesion protein expression is reduced to restrain platelet adhesion, aggregation and thrombus formation.

Molecular mechanisms regulating the synergism between IL-32γ and NOD for the activation of eosinophils

IL-32 is a proinflammatory cytokine associated with infections, autoimmune diseases, and allergic asthma. In the present study, we elucidated the synergistic effect of IL-32γ and NOD ligand on the activation of human eosinophils, principal effector cells for allergic inflammation, and the underlying mechanisms. Specific IL-32-binding protein, PR3, was found to localize on the cell surface and in the cytoplasm of eosinophils. IL-32γ was more capable of activating eosinophils than its isotype variant IL-32α and exhibited synergistic effect with NOD1 ligand iE-DAP and NOD2 ligand MDP on the induction of allergic inflammation-related IL-1β, TNF-α, and chemokines CXCL8, CCL3, and CCL4 (P<0.05). Moreover, IL-32γ and iE-DAP or MDP induced the significant up-regulation of the cell-surface expression of adhesion molecule CD18 and ICAM-1 on eosinophils. Synergism between IL-32γ and NOD ligands was dependent on the activation of intracellular caspase 1, ERKs, p38 MAPK, and NF-κB pathways in eosinophils. The further-enhanced CD18 and ICAM-1 expression and production of cytokines and chemokines were observed in eosinophils cocultured with human bronchial epithelial BEAS-2B cells. Furthermore, combined treatment of IL-32γ and NOD ligand could activate the release of eosinophil extracellular DNA traps, thereby implying the pathogen-defense mechanisms of eosinophils. Together, the above study provides pivotal immunological mechanisms by which bacterial infection-mediated activation of NOD1,2, together with IL-32γ, can synergize the activation of eosinophils interacting with bronchial epithelial cells.