Flow-based Adhesion Assays Research Articles

Single-cell imaging and transcriptomic analyses of firm adhesion between patient-derived cancer and endothelial cells under shear stress

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is a key event during tumour progression and metastasis formation. However, the analysis of its underlying cellular and molecular mechanisms is largely limited by the intrinsic difficulties to study the interactions between circulating cancer cells and endothelial cells in vivo, and in vitro under conditions that mimic the in vivo blood flow. Here, we developed a method to study cell:cell firm adhesion under shear-stress conditions coupled to high-content live-cell imaging, and single-cell RNAseq analysis. As the model system, we used cancer cells freshly isolated from patient-derived xenografts (PDXs) and human primary endothelial cells. Breast cancer is the most common cancer in women worldwide and the leading cause of cancer-related deaths among women. Therefore, we set up protocols for breast cancer PDX tumour dissociation, isolation and purification to obtain freshly isolated PDX-derived human cancer single cell suspension. We then implemented an in vitro assay to study cancer to endothelial cells firm adhesion under shear-stress, using an all–human microfluidic model coupled to time-lapse and live-cell imaging. Finally, we developed a method to successfully retrieve, separate and enrich alive endothelial and cancer cells from the flow-based firm adhesion assay. Most notably, we used retrieved cells for single-cell RNAseq analysis and showed that samples quality, number of cells and transcripts per cell were consistent and optimal for downstream discovery analyses. In conclusion, we developed a workflow method that can provide insights into the mechanisms of cancer adhesion to endothelial cells, and identify new targets for personalized treatments development for the clinic to prevent and/or treat breast cancer metastasis formation.

JAM-A is a multifaceted regulator in hepatic fibrogenesis, supporting LSEC integrity and stellate cell quiescence.

Leukocyte infiltration is a hallmark of hepatic inflammation. The Junctional Adhesion Molecule A (JAM-A) is a crucial regulator of leukocyte extravasation and is upregulated in human viral fibrosis. Reduced shear stress within hepatic sinusoids and the specific phenotype of liver sinusoidal endothelial cells (LSEC) cumulate in differing adhesion characteristics during liver fibrosis. The aim of this study was to define the functional role of cell-specific adhesion molecule JAM-A during hepatic fibrogenesis. Complete, conditional (intestinal epithelial; endothelial) and bone marrow chimeric Jam-a knockout animals and corresponding C57Bl/6 wild-type animals were treated with carbon tetrachloride (CCl4 , 6weeks). For functional analyses of JAM-A, comprehensive in vivo studies, co-culture models and flow-based adhesion assays were performed. Complete and bone marrow-derived Jam-a-/- animals showed aggravated fibrosis with increased non-sinusoidal, perivascular accumulation of CD11b+ F4/80+ monocyte-derived macrophages in contrast to wild-type mice. Despite being associated with disturbed epithelial barrier function, an intestinal epithelial Jam-a knockout did not affect fibrogenesis. In endothelial-specific Jam-a-/- animals, liver fibrosis was aggravated alongside sinusoid capillarization and hepatic stellate cell (HSC) activation. HSC activation is induced via Jam-a-/- LSEC-derived secretion of soluble factors. Sinusoid CD31 expression and hedgehog gene signalling were increased, but leukocyte infiltration and adhesion to LSECs remained unaffected. Our models decipher cell-specific JAM-A to exert crucial functions during hepatic fibrogenesis. JAM-A on bone marrow-derived cells regulates non-sinusoidal vascular immune cell recruitment, while endothelial JAM-A controls liver sinusoid capillarization and HSC quiescence.

Endothelial-Derived Extracellular Vesicles Induce Cerebrovascular Dysfunction in Inflammation.

Blood–brain barrier (BBB) dysfunction is a key hallmark in the pathology of many neuroinflammatory disorders. Extracellular vesicles (EVs) are lipid membrane-enclosed carriers of molecular cargo that are involved in cell-to-cell communication. Circulating endothelial EVs are increased in the plasma of patients with neurological disorders, and immune cell-derived EVs are known to modulate cerebrovascular functions. However, little is known about whether brain endothelial cell (BEC)-derived EVs themselves contribute to BBB dysfunction. Human cerebral microvascular cells (hCMEC/D3) were treated with TNFα and IFNy, and the EVs were isolated and characterised. The effect of EVs on BBB transendothelial resistance (TEER) and leukocyte adhesion in hCMEC/D3 cells was measured by electric substrate cell-substrate impedance sensing and the flow-based T-cell adhesion assay. EV-induced molecular changes in recipient hCMEC/D3 cells were analysed by RT-qPCR and Western blotting. A stimulation of naïve hCMEC/D3 cells with small EVs (sEVs) reduced the TEER and increased the shear-resistant T-cell adhesion. The levels of microRNA-155, VCAM1 and ICAM1 were increased in sEV-treated hCMEC/D3 cells. Blocking the expression of VCAM1, but not of ICAM1, prevented sEV-mediated T-cell adhesion to brain endothelia. These results suggest that sEVs derived from inflamed BECs promote cerebrovascular dysfunction. These findings may provide new insights into the mechanisms involving neuroinflammatory disorders.

Development of a Translatable Ultrasound Molecular Imaging Agent for Inflammation

This study details the development, characterization and non-clinical efficacy of an ultrasound molecular imaging agent intended for molecular imaging of P-selectin in humans. A targeting ligand based on a recently discovered human selectin ligand was manufactured as fusion protein, and activity for human and mouse P- and E-selectin was evaluated by functional immunoassay. The targeting ligand was covalently conjugated to a lipophilic anchor inserted into a phospholipid microbubble shell. Three lots of the targeted microbubble drug product, TS-07-009, were produced, and assays for size distribution, zeta potential and morphology were established. The suitability of TS-07-009 as a molecular imaging agent was evaluated in vitro in a flow-based adhesion assay and in vivo using a canine model of transient myocardial ischemia. Selectivity for P-selectin over E-selectin was observed in both the human and murine systems. Contrast agent adhesion increased with P-selectin concentration in a dynamic adhesion assay. Significant contrast enhancement was observed on ultrasound imaging with TS-07-009 in post-ischemic canine myocardium at 30 or 90 min of re-perfusion. Negligible enhancement was observed in resting (no prior ischemia) hearts or with a control microbubble 90 min after ischemia. The microbubble contrast agent described here exhibits physiochemical properties and in vivo behavior suitable for development as a clinical imaging agent.

Longitudinal Evaluation of a Standardized P-Selectin Flow Adhesion Bioassay: Potential Role for the Assessment and Prediction of Vaso-Occlusive Episodes in Sickle Cell Disease

Vaso-occlusive episodes (VOEs) in sickle cell disease (SCD) are characterized by severe pain and microvascular obstruction precipitated by adhesive interactions among endothelial cells (ECs), red blood cells, white blood cells (WBCs), and platelets. P-selectin contributes to microvascular occlusion by supporting abnormal interactions between blood cells and the vascular endothelium. Inhibition of p-selectin mediated adhesion reverses acute, VOEs in SCD mice. Additionally, SCD mice, deficient in either P- or E-selectin, develop fewer vascular occlusions. These reports led to the development of anti-adhesive therapies targeting p-selectin. For example, Crizanlizumab, an anti-P-selectin antibody, reduced the annual rate of VOEs prospectively and Rivipansel, a pan-selectin inhibitor, decreased the length of active VOEs. Objective measures of VOEs are nonexistent.We developed a standardized, flow-based adhesion assay to measure p-selectin mediated adhesion in SCD. Whole blood (Wb) and isolated WBC samples were perfused (1.0 dynes/cm2, 1.67Hz) through micro-fluidic channels. Adhered cells were enumerated and an adhesion index was calculated (total # of adhered cells/mm2). Blood samples were collected from SCD subjects (n = 35) every 3 weeks for 6 months at baseline and within 24 and 48hrs of a patient-reported VOE. Baseline and VOE states were confirmed by an electronic patient reported outcomes (ePRO) device, as previously described. VOEs were separated into 2 groups: VOEs managed at-home (Home-VOE) or during ER visit and/or hospitalization (Contact-VOE).WB (n = 288; mean=42 ± 48 cells/mm2, median=31 cells/mm2) and WBC (n = 282; mean=163 ± 149 cells/mm2; median= 116 cells/mm2) adhesion to p-selectin varied in SCD subjects (n=35) at baseline. WBC adhesion to P-selectin was significantly (p=0.36) higher during VOE (n=59; 205 ± 179) when compared to baseline (n = 282; mean=163±149 cells/mm2, median=116 cells/mm2). For individual subjects, the geometric mean for WBC adhesion to P-selectin was significantly (p = 0.023) higher for VOEs vs. baseline (VOE= 109, n= 23, 95% confidence interval 94-126 vs. baseline= 148, n = 35, 95% confidence interval 114-192). Additionally, WB adhesion to p-selectin correlated with VOE frequency (time to 2ndVOE; r = -0.71, n = 10, p = 0.02 or time between 1stand 2ndVOEs; r = -0.68, n = 10, p = 0.032).WB adhesion to p-selectin positively correlated with C-reactive protein (CRP; r = 0.12, p = 0.047), reticulocyte % (r = 0.15, p = 0.017), and WBC counts (r = 0.26, p < 0.0001) at baseline. WBC adhesion to p-selectin positively correlated with hematocrit (Hct; r = -0.37, p = 0.027) and reticulocyte % (r = 0.44, p = 0.0058) during VOEs managed at home; WB adhesion to p-selectin positively correlates with CRP (r = 0.44, p = 0.011) and WBC count (r = 0.63, p < 0.0001); WB adhesion to p-selectin positively correlated with WBC count (r = 0.51, p = 0.0021) during contact-VOE.These data confirm the normal range and longitudinal variability of SCD adhesion to p-selectin at baseline and during VOEs. WB adhesion to p-selectin was inversely related to VOE frequency although there was no difference between baseline and VOE samples. These data suggest that WB adhesion to p-selectin may be a better predictor of disease severity and not SCD symptomology. In contrast, WBC adhesion to p-selectin differentiated baseline from VOE samples suggesting this assay may be a more suitable reflection of SCD clinical state. Both WB and WBC adhesion to VCAM-1 correlates with biomarkers of hemolysis and inflammation. P-selectin may be selective for the identification of pro-adhesive phenotypes directly related to hemolytic and inflammatory influencers in SCD. DisclosuresLiu:Functional Fluidics: Equity Ownership. Gao:Functional Fluidics: Equity Ownership. White:functional fluidics: Equity Ownership. Callaghan:Alnylam Pharmaceuticals: Equity Ownership; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Employment, Membership on an entity's Board of Directors or advisory committees; Hema Pharmaceuticals: Honoraria; Grifols: Honoraria; Sancilio Pharmaceuticals Company: Employment; Global Blood Therepeutics: Employment; Roche/Genentech: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Honoraria; Octapharma: Honoraria; Pfizer: Employment, Honoraria, Research Funding; Amgen: Employment. Hines:functional fluidics: Equity Ownership.

Ability of Platelet-Derived Extracellular Vesicles to Promote Neutrophil-Endothelial Cell Interactions

We tested the ability of platelet-derived extracellular vesicles (PEV) to promote adhesion of flowing neutrophils to endothelial cells (EC). PEV were collected from platelets stimulated with collagen-related peptide, and differential centrifugation was used to collect larger vesicles enriched for platelet membrane microvesicles (PMV) or smaller vesicles enriched for platelet exosomes (Pexo). Vesicle binding and resultant activation of neutrophils and EC were assessed by flow cytometry. Flow-based adhesion assays assessed binding of neutrophils directly to deposited vesicles or to EC, after neutrophils or EC had been treated with vesicles. PEV bound efficiently to neutrophils or EC, with resultant upregulation of activation markers. Binding was Ca++-dependent and dominantly mediated by CD62P for neutrophils or by integrins for EC. Deposited PEV supported mainly transient attachments of flowing neutrophils through CD62P and some stable adhesion through CXC-chemokines. Neutrophil adhesion to EC was promoted when either cell was pre-treated with PEV, although the effect was less prominent when EC were pre-activated with tumor necrosis factor-α. The pro-adhesive effects on neutrophils could largely be attributed to the larger PMV rather than Pexo. Thus, surface-bound PEV can capture flowing neutrophils, while PEV also activate neutrophils and EC to promote interactions. PEV may potentiate inflammatory responses after tissue injury.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.

CD151 supports VCAM-1-mediated lymphocyte adhesion to liver endothelium and is upregulated in chronic liver disease and hepatocellular carcinoma.

CD151, a member of the tetraspanin family of receptors, is a lateral organizer and modulator of activity of several families of transmembrane proteins. It has been implicated in the development and progression of several cancers, but its role in chronic inflammatory disease is less well understood. Here we show that CD151 is upregulated by distinct microenvironmental signals in a range of chronic inflammatory liver diseases and in primary liver cancer, in which it supports lymphocyte recruitment. CD151 was highly expressed in endothelial cells of the hepatic sinusoids and neovessels developing in fibrotic septa and tumor margins. Primary cultures of human hepatic sinusoidal endothelial cells (HSECs) expressed CD151 at the cell membrane and in intracellular vesicles. CD151 was upregulated by VEGF and HepG2 conditioned media but not by proinflammatory cytokines. Confocal microscopy confirmed that CD151 colocalized with the endothelial adhesion molecule/immunoglobulin superfamily member, VCAM-1. Functional flow-based adhesion assays with primary human lymphocytes and HSECs demonstrated a 40% reduction of lymphocyte adhesion with CD151 blockade. Inhibition of lymphocyte adhesion was similar between VCAM-1 blockade and a combination of CD151/VCAM-1 blockade, suggesting a collaborative role between the two receptors. These studies demonstrate that CD151 is upregulated within the liver during chronic inflammation, where it supports lymphocyte recruitment via liver endothelium. We propose that CD151 regulates the activity of VCAM-1 during lymphocyte recruitment to the human liver and could be a novel anti-inflammatory target in chronic liver disease and hepatocellular cancer prevention.NEW & NOTEWORTHY Chronic hepatitis is characterized by lymphocyte accumulation in liver tissue, which drives fibrosis and carcinogenesis. Here, we demonstrate for the first time that the tetraspanin CD151 supports lymphocyte adhesion to liver endothelium. We show that CD151 is upregulated in chronic liver disease and hepatocellular carcinoma (HCC) and is regulated on endothelium by tissue remodeling and procarcinogenic factors. These regulatory and functional studies identify CD151 as a potential therapeutic target to treat liver fibrosis and HCC.

Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration.

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow‐based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon‐γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time‐lapse recordings, we demonstrated “intracellular crawling” of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule‐1 and stabilin‐1 and was facilitated by the junctional complexes between HSECs. Conclusion: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294‐309).

Platelet integrin α6β1 controls lung metastasis through direct binding to cancer cell–derived ADAM9

Metastatic dissemination of cancer cells, which accounts for 90% of cancer mortality, is the ultimate hallmark of malignancy. Growing evidence suggests that blood platelets have a predominant role in tumor metastasis; however, the molecular mechanisms involved remain elusive. Here, we demonstrate that genetic deficiency of integrin α6β1 on platelets markedly decreases experimental and spontaneous lung metastasis. In vitro and in vivo assays reveal that human and mouse platelet α6β1 supports platelet adhesion to various types of cancer cells. Using a knockdown approach, we identified ADAM9 as the major counter receptor of α6β1 on both human and mouse tumor cells. Static and flow-based adhesion assays of platelets binding to DC-9, a recombinant protein covering the disintegrin-cysteine domain of ADAM9, demonstrated that this receptor directly binds to platelet α6β1. In vivo studies showed that the interplay between platelet α6β1 and tumor cell-expressed ADAM9 promotes efficient lung metastasis. The integrin α6β1-dependent platelet-tumor cell interaction induces platelet activation and favors the extravasation process of tumor cells. Finally, we demonstrate that a pharmacological approach targeting α6β1 efficiently impairs tumor metastasis through a platelet-dependent mechanism. Our study reveals a mechanism by which platelets promote tumor metastasis and suggests that integrin α6β1 represents a promising target for antimetastatic therapies.

PTU-081 CD151 In Chronic Liver Disease and HCC - A New Player in T Cell Recruitment to The Liver

Introduction Inflammation of the liver drives the onset and progression of chronic liver diseases whilst fostering an environment where the formation of neoplastic tumours such as hepatocellular carcinomas (HCC) can occur. A key step in this process is leukocyte recruitment via hepatic sinusoidal endothelial cells. CD151, a member of the tetraspanin family, has been shown to regulate heterotypic partner proteins involved in leukocyte recruitment and therefore an understanding of the expression and function of CD151 in the liver could identify new organ-specific anti-inflammatory therapies. Methods We used immunohistochemistry, dual colour immunofluorescence co-localisation studies, qRT-PCR and western blotting to determine the cell specific expression of CD151 in pathological control liver, chronic liver diseases and tissue taken from patients with HCC. Cell-based ELISA, qRT-PCR and western blotting were then used to determine the regulation of CD151 expression by hepatic sinusoidal endothelial cells (HSEC) following growth factor and cytokine treatment to mimic inflammatory and tumourigenic environments. Flow-based adhesion assays were used to study the role of CD151 in the adhesion of Jurkat cells, a human T cell line, to HSEC monolayers with subsequent immunofluorescence analysis. Results Increased CD151 protein expression was associated with areas of fibrosis and neovascularisation, particularly in parenchymal liver disease such as alcoholic and non-alcoholic fatty liver disease as well as in HCC. CD151 was highly expressed by HSEC both within liver tissue and in vitro . The expression of CD151 in HSEC was upregulated by stimulation with tumour cell line supernatant or a combination of hepatocyte and vascular endothelial growth factors. We found CD151 molecules clustering around adherent Jurkat cells following capture from flow by HSEC monolayers. Function-blocking antibodies to CD151 significantly reduced adherence of Jurkat under conditions of shear stress. Conclusion We demonstrate for the first time the expression of CD151 on human sinusoidal endothelial cells and that this molecule is expressed in neovessels in chronic liver disease and HCC. We also demonstrate that CD151 is upregulated by pro-tumourigenic factors on HSEC and functionally plays a role in T cell adhesion to HSEC. These findings further our understanding of liver inflammation and lymphocyte recruitment to the organ, in both chronic and malignant disease, and could form the basis of a potential therapeutic target. Disclosure of Interest None Declared

Chemoattractant Signals and Adhesion Molecules Promoting Human Regulatory T Cell Recruitment to Porcine Endothelium.

Human CD4+CD25+Foxp3+ T regulatory cells (huTreg) suppress CD4+ T cell-mediated antipig xenogeneic responses in vitro and might therefore be used to induce xenograft tolerance. The present study investigated the role of the adhesion molecules, their porcine ligands, and the chemoattractant factors that may promote the recruitment of huTreg to porcine aortic endothelial cells (PAEC) and their capacity to regulate antiporcine natural killer (NK) cell responses. Interactions between ex vivo expanded huTreg and PAEC were studied by static chemotaxis assays and flow-based adhesion and transmigration assays. In addition, the suppressive function of huTreg on human antiporcine NK cell responses was analyzed. The TNFα-activated PAEC released factors that induce huTreg chemotaxis, partially inhibited by antihuman CXCR3 blocking antibodies. Coating of PAEC with human CCL17 significantly increased the transmigration of CCR4+ huTreg under physiological shear stress. Under static conditions, transendothelial Treg migration was inhibited by blocking integrin sub-units (CD18, CD49d) on huTreg, or their respective porcine ligands intercellular adhesion molecule 2 (CD102) and vascular cell adhesion molecule 1 (CD106). Finally, huTreg partially suppressed xenogeneic human NK cell adhesion, NK cytotoxicity and degranulation (CD107 expression) against PAEC; however, this inhibition was modest, and there was no significant change in the production of IFNγ. Recruitment of huTreg to porcine endothelium depends on particular chemokine receptors (CXCR3, CCR4) and integrins (CD18 and CD49d) and was increased by CCL17 coating. These results will help to develop new strategies to enhance the recruitment of host huTreg to xenogeneic grafts to regulate cell-mediated xenograft rejection including NK cell responses.

Eltrombopag enhances platelet adhesion by upregulating the expression of glycoprotein VI in patients with chronic immune thrombocytopenic purpura

Eltrombopag, a thrombopoietin receptor agonist, has been approved for the treatment of patients with immune thrombocytopenia because of its abilities to enhance platelet production and reduce hemorrhage. Both platelet count and platelet adhesion are crucial to stop bleeding. Although eltrombopag is known to improve platelet counts, its effects on platelet adhesion are not yet known. This study aimed to assess the efficacy of eltrombopag on platelet production and platelet adhesive affinity. To evaluate the efficacy of low-dose eltrombopag (25 mg) for patients with chronic refractory immune thrombocytopenic purpura (ITP) and to determine the ex vivo platelet adhesion ability before and after treatment with eltrombopag, we conducted an open-label, multicenter study in which 25 Taiwanese patients with chronic ITP were enrolled. During the 6-month evaluation, the starting and maximum doses of eltrombopag were 25 and 50 mg, respectively, to maintain the platelet count of ≥50,000 per μL. Flow-based adhesion assay was used to detect the percentage of platelets adhering to immobilized von Willebrand factor-collagen on microslides. Of the enrolled patients, 48% achieved a platelet count of ≥50,000 per μL. Interestingly, 83% of all responders required 25 mg of eltrombopag daily to achieve the target platelet count. In addition, the percentage of bleeding patients was significantly reduced in both responders and nonresponders by 50% from the baseline level throughout the treatment period. The ex vivo platelet adhesion capacity was elevated after the 6-month eltrombopag treatment in both responders and nonresponders. Furthermore, glycoprotein VI (GPVI) expression was significantly upregulated after treatment with eltrombopag. Low-to-intermediate dose of eltrombopag showed good efficacy to expedite platelet production and augment platelet adhesion. These 2 factors might explain the efficacy of eltrombopag in ameliorating hemorrhage in patients with ITP.

PTH-098 Connecting liver and gut in psc: ccl25 expression is upregulated in colitis, correlates with inflammatory activity and facilitates effector ccr9+ lymphocyte recruitment

<h3>Introduction</h3> Recruitment of mucosal T-cells to the liver in response to aberrantly expressed homing signals drives hepatobiliary inflammation in primary sclerosing cholangitis (PSC). This is exemplified by the ‘gut-homing’ chemokine CCL25, which recruits intestinal CCR9⁺effector cells to the PSC liver. However, previous studies report CCL25 expression as being confined to the small bowel whereas PSC is typically associated with colonic inflammation. Our aim was to determine whether CCL25 and CCR9 are expressed in the inflamed human colon in patients with colitis. <h3>Method</h3> Mucosal biopsies were obtained during surveillance colonoscopy (n = 40) and colonic tissue from patients undergoing surgical resection for colitis (n = 6), or non-colitis-associated cancer (n = 5). CCL25 mRNA was evaluated by qRT-PCR (relative to GUSb) and protein expression confirmed using western blotting and tissue-ELISA. Lymphocyte CCR9 expression was quantified by flow cytometry, and the ability of sorted a4b7⁺CCR9⁺and a4b7⁺CCR9^-T-cells to transmigrate across hepatic sinusoidal endothelium (HSEC) investigated in flow-based adhesion assays. <h3>Results</h3> CCL25 mRNA and protein were absent from normal colon but present in colitis. CCL25 mRNA expression correlated with endoscopic Mayo score (p = 0.001) and tissue levels of TNFa (Spearman’s rho 0.811; p &lt; 0.0001) as indices of inflammatory activity (Figure 1). In colitis, &gt;90% of CD3⁺CD4⁺and &gt;15% of CD8⁺T-cells expressed CCR9 (6% and 2% in normal colon; p = 0.01 and 0.03) and were predominantly CD127⁺effector lymphocytes. a4b7⁺CCR9⁺T-cells showed enhanced adhesion and transendothelial migration across HSEC compared with a4b7⁺CCR9^-T-cells (p &lt; 0.05). <h3>Conclusion</h3> CCL25 is expressed in the inflamed human colon, correlates with inflammatory activity and is associated with high frequencies of CCR9⁺tissue-infiltrating lymphocytes. These findings lend further support to the mucosal lymphocyte homing hypothesis of PSC and show how it can be applied to patients with colitis. <h3>Disclosure of interest</h3> None Declared.

Analyzing the effects of stromal cells on the recruitment of leukocytes from flow.

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro "vascular" models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment. Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy. In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium. Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro “vascular” models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment. Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNFα) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy. In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNFα-stimulated endothelium. Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

GPVI Interaction with Polymerized Fibrin Boosts Thrombin Generation and Thrombus Growth

Background: Fibrin, the end product of the coagulation cascade, consolidates the platelet plug at site of thrombosis: polymerized fibrin supports platelet adhesion under low and high shear rate conditions (Hantgan RR et al., Thromb Haemost 1992) and triggers platelet procoagulant activity (Beguin S et al., Blood 1999). These responses are largely independent of the integrin αIIβ3 and are carried by a yet ill-defined receptor. Platelet glycoprotein VI (GPVI) has a well-established key role in the initiation of thrombosis since it supports collagen-mediated platelet activation but it has recently been recognized to interact with other macromolecules such as fibronectin, vitronectin and laminins. We hypothesized GPVI could be the “missing” platelet receptor of fibrin.Aim of the study: to challenge the hypothesis that glycoprotein VI (GPVI) could be a functional fibrin receptorMethods: Thrombin generation was measured using calibrated automated thrombogram (CAT) in PRP from healthy volunteers, four GPVI-deficient patients and one patient with a fibrinogen deficiency. CAT was also performed on washed platelets mixed with prothrombin complex (FII, FVII, FIX, FX), antithrombin and fibrinogen. GPVI was blocked using the Fab of the monoclonal antibody 9O12. Fibrin polymerization was blocked using the GPRP peptide. GPVI binding to fibrin was measured in vitro using recombinant soluble GPVI (GPVI-Fc). Flow based adhesion assays were performed in capillary chambers coated with polymerized fibrin at variable shear rates and platelet morphological changes analyzed by scanning electron microscopy. The formation of fibrin-platelet thrombi was visualized by perfusing recalcified blood containing A647 fibrinogen in flow chambers (Vena8 Fluoro+ Cellix) coated with collagen and tissue factor. In a second step, the perfusion of hirudinated blood in which platelets were stained by A488-RAM1 allowed to visualize platelet recruitment by fibrin rich clots.Results: Thrombin generation triggered by tissue factor was impaired in the PRP of patients with a GPVI deficiency or in the presence of the Fab 9O12 as indicated by a respective decrease in the peak height of 45 and 25% as compared to controls. This effect was observed regardless the trigger of thrombin generation and required platelet activation. Measuring thrombin generation in a purified system showed that fibrinogen dose-dependently increased the thrombin peak by up to 150% at 3 mg/mL but the Fab 9O12 blunted this effect. Moreover, the Fab 9O12 had no effect on thrombin generation in the PRP of a fibrinogen-deficient patient confirming a GPVI/fibrin(ogen)interplay. Blocking fibrin polymerization by GPRP reduced the thrombin peak in normal PRP, in fibrinogen-supplemented PRP of the fibrinogen-deficient patient and in purified conditions. In contrast GPRP had no effect on the thrombin peak in normal PRP containing the Fab 9O12 and in the PRP of GPVI-deficient patients. The proof that GPVI specifically interacts with fibrin was obtained in a binding assay showing a dose-dependent binding of GPVI-Fc to fibrin polymers that was reversed by the Fab 9O12. Platelets adhered to polymerized fibrin resulting in platelet shape change and exposure of phosphatidylserine. Platelet adhesion on a fibrin network was observed at low (300 s-1) and high (1500 s-1) shear rates with the formation of small contractile thrombi. Adhesion was decreased by 62% for 9O12-treated platelets and by 43% with the blood of GPVI-deficient mice as compared to controls. Importantly, lack of GPVI or its blockade decreased stationary adhesion indicating that GPVI is required to stabilize the interactions between platelets and fibrin. Finally when hirudinated blood was perfused at a shear rate of 1500 s-1 onto preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 93%.Conclusions: Here we show for the first time that GPVI acts as a receptor for polymerized fibrin with two major functions: GPVI interaction with polymerized fibrin triggers (i) a new loop amplifying thrombin generation and (ii) platelet recruitment at the clot surface. These, so far, unrecognized properties of GPVI confer it a key role in the maturation of the thrombus by facilitating its growth and stabilization in addition to its well-known effect in the initiation of thrombus formation. DisclosuresJandrot-Perrus:Acticor Biotech: Other. Gachet:Acticor Biotech: Other.

Monocyte Recruitment to HLA Class I Antibody-Activated Endothelial Cells Is Dependent Upon mTOR.

Aim: A hallmark of antibody (Ab)-mediated rejection is the presence of intravascular monocytes in the allograft. The purpose of this study was to identify the role of the mTOR signaling pathway in HLA class I Ab-mediated monocyte recruitment to endothelial cells (EC). Methods: Static and flow-based (ibidi) adhesion assays were used to assess monocyte (MM6) recruitment to HLA-activated primary human aortic endothelial cells. mTOR inhibition was characterized using pharmacological inhibitors (KU 0063794 and sirolimus) at different time points and doses in endothelial cells and monocytes. Results were verified using siRNA knock-down (ko) of rictor and raptor, components of the mTOR pathway. Cell-based ELISA was performed to quantitate Weibel-Palade Body exocytosis; protein phosphorylation was measured by Western blot and quantitated by densitometry. An established murine cardiac transplant model was used to determine the effect of sirolimus treatment on acute AMR. Briefly, Rag1 ko animals were transplanted with an H-2d mismatched heart transplant and received passive transfer of anti-donor Dd+Kd IgG antibodies. Results: Pretreatment of EC for 24 hours with sirolimus or Ku reduced monocyte adherence to EC by > 80%, whereas 2- hour mTOR inhibition resulted in partial inhibition of monocyte binding. siRNA ko of mTOR using siRNA yielded comparable results significantly decreasing monocyte adherence. siRNA against raptor and rictor revealed that both mTORC1 and mTORC2 are involved in HLA-mediated monocyte recruitment. Inhibition of mTOR had no effect on P-selectin induction. Instead, flow-based adhesion assays revealed that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering and its downstream signaling effects. mTOR inhibition in monocytes using sirolimus and Ku significantly impaired monocyte binding to activated endothelium. A reduction in endothelial damage and infiltrating mononuclear cells was observed in vivo in recipients of heart allografts treated with sirolimus. Conclusion: We found that mTOR blockade prevents monocyte recruitment to HLA-activated EC through mTORC1 and mTORC2 signaling pathways. In EC, mTOR inhibition prevents ICAM-1 clustering and blunts firm adhesion of monocytes. Our results provide novel insights into the mechanisms underlying monocyte recruitment in the setting of AMR. DISCLOSURE:Salehi, S.: Grant/Research Support, Pfizer. Reed, E.: Grant/Research Support, Pfizer.

Therapeutic potential of vascular adhesion protein in primary sclerosing cholangitis

BackgroundVascular adhesion protein 1 (VAP1) is an adhesion molecule that also posseses potent amine oxidase activity; it produces hydrogen peroxide via the deamination of dietary amines. Through this function, VAP1 leads to activation of NFκB in hepatic sinusoidal endothelial cells (HSEC) resulting in expression of mucosal-vascular cell-adhesion molecule 1 (MAdCAM1), a mechanism proposed to contribute to the homing of gut-tropic lymphocytes expressing α4β7 to the liver. In view of the putative role of this pathway in hepatic diseases complicating inflammatory bowel disease, we aimed to quantify circulating or soluble (sVAP1) as well as intrahepatic VAP1 enzyme activity in patients with primary sclerosing cholangitis, and assess the functional consequence of its inhibition on MAdCAM1-dependent lymphocyte recruitment to HSEC. MethodsTotal VAP1 concentration was measured by ELISA. VAP1 amine oxidase activity was quantified in human serum and explanted liver tissue with the amplex red assay. We used flow-based adhesion assays with human HSEC isolated from liver explants, activated through tumour necrosis factor α and methylamine (VAP1 substrate), before and after treatment with VAP1 antibody or semicarbazide (VAP1 enzyme inhibitor). Fluorescence-activated cell sorted peripheral blood lymphocytes expressing α4β7 were perfused over HSEC under flow rates simulating physiological shear (0·05 kPa) and differences in adhesion and transmigration measured. FindingsPatients with primary sclerosing cholangitis had higher circulating median VAP1 enzyme activity (114·5 pmol hydrogen peroxide produced per min/mL serum, IQR 100·6–134·7) than patients with inflammatory bowel disease (60·3, 38·5–73·0; p=0·006), healthy controls (84·0, 77·7–105·7; p=0·02), and individuals with primary biliary cirrhosis (53·9, 33·0–90·9; p=0·006) and autoimmune hepatitis (77·6, 51·0–124·5; p=0·2, Mann-Whitney). Total sVAP1 concentration correlated well with sVAP1 enzyme activity (r2=0·75). Intrahepatic median VAP1 activity was also significantly higher in primary sclerosing cholangitis than in primary biliary cirrhosis (97·6 pmol [IQR 69·5–114·5] vs 24·6 [18·7–27·8], p=0·029) and autoimmune hepatitis (32·3 [23·3–35·6], p=0·028). HSEC pretreatment with semicarbazide and antibody led to a reduction in total α4β7+ lymphocyte adhesion (by 50% and 25%, respectively); however, both antibody and enzyme inhibition independently reduced transmigration by about 50% compared with untreated HSEC. InterpretationWe found that sVAP1 enzyme activity was greater in primary sclerosing cholangitis than in inflammatory bowel disease alone, healthy controls, and other immune-mediated liver diseases. Intrahepatic VAP1 enzyme activity was significantly higher in primary sclerosing cholangitis than in autoimmune hepatitis and primary biliary cirrhosis. Inhibition of VAP1 led to abrogation of α4β7-mediated adhesion to HSEC and is therefore a putative target for therapeutic intervention in primary sclerosing cholangitis. FundingWellcome Trust.

The integrin antagonist, cilengitide, is a weak inhibitor of αIIbβ3 mediated platelet activation and inhibits platelet adhesion under flow

The RGD cyclic pentapetide, cilengitide, is a selective inhibitor of αvβ3 and αvβ5 integrins and was developed for antiangiogenic therapy. Since cilengitide interacts with platelet αIIbβ3 and platelets express αv integrins, the effect of cilengitide on platelet pro-coagulative response and adhesion is of interest. Flow-based adhesion assays were performed to evaluate platelet adhesion and rolling on von Willebrand factor (vWf), on fibrinogen and on human umbilical vein endothelial cells (HUVECs). Flow cytometry was used to detect platelet activation (PAC1) and secretion (CD62P) by cilengitide and light transmission aggregometry was used to detect cilengitide-dependent platelet aggregation. Cilengitide inhibited platelet adhesion to fibrinogen at concentrations above 250 µM [which is the Cmax in human studies] and adhesion to vWf and HUVECs at higher concentrations under physiologic flow conditions. Platelet aggregation was already impaired at cilengitide concentrations >10 µM. Activation of αIIbβ3 integrin was inhibited by 250 µM cilengitide, whereas platelet secretion was unaffected by cilengitide. No evidence of cilengitide-induced platelet activation was found at all tested concentrations (0.01–1500 µM). At higher concentrations, platelet activation was inhibited, predominantly due to αIIbβ3 inhibition.