Nguyet-Thin Luu

EACH STEP DURING TRANSENDOTHELIAL MIGRATION OF FLOWING NEUTROPHILS IS REGULATED BY THE STIMULATORY CONCENTRATION OF TUMOUR NECROSIS FACTOR-ALPHA

Migration of circulating neutrophils occurs in several steps: capture and rolling adhesion are followed by activation of beta 2-integrins and immobilisation, and then neutrophils move over and through the endothelium. However, it is not clear how the underlying mechanisms and completion of each step depend on the concentration of stimulatory cytokines such as tumour necrosis factor-alpha (TNF). We therefore perfused neutrophils over human umbilical vein endothelial cells (HUVEC) which had been cultured with varying concentration of TNF (1-1000 U/ml) for 4 h, and recorded adhesion and migration by videomicroscopy. The number of adherent neutrophils increased with increasing TNF up to 5 U/ml, but changed little at higher concentrations. Interestingly, rolling adhesion at first predominated, but an increasing proportion of adherent cells became immobilised and migrated through the HUVEC monolayer over the complete TNF range. Immobilisation was inhibited by treating neutrophils with antibody against CD18, so that the major change in adhesive behaviour at higher levels of TNF occurred because the surface of the HUVEC presented agent(s) able to activate neutrophil beta 2-integrins. It was also evident that the selectins initiating capture of flowing neutrophils varied with concentration of TNF. At 100 U/ml TNF, both E-selectin and P-selectin supported capture and rolling adhesion, and antibody blockade of both receptors was required to inhibit adhesion. At lower dose (10 U/ml TNF), stable adhesion was blocked by antibody against E-selectin, although short-lived attachments could still be seen which were inhibited by antibody against P-selectin. Expression of sclectins increased with increasing concentration of TNF, judging from surface ELISA and reduction in the velocity of rolling adherent cells. Thus the efficiency of capture, the selectins mediating capture and the proportion of captured cells immobilised and migrating all depend on the concentration of TNF to which endothelial cells are exposed. These results suggest a model in which highly localised and efficient migration of neutrophils is achieved if a concentration gradient of TNF exists around an inflammatory locus.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Modulation of functional responses of endothelial cells linked to angiogenesis and inflammation by shear stress: differential effects of the mechanotransducer CD31.

We investigated the roles of the “mechanotransducer” CD31 in the effects of shear stress on endothelial gene expression and functional responses relevant to angiogenesis and inflammation. Human or murine endothelial cells (hEC or mEC) were exposed to different levels of shear stress, while expression of CD31 was modified using siRNA in the hEC, or mEC from CD31−/− mice. Quantitation of expression of genes linked to inflammation or angiogenesis showed several were sensitive to shear. In a “wound” assay, exposure of endothelial cells (EC) to shear stress tended to align migration with the direction of flow and decrease the rate of closure compared to static cultures. When EC were cultured on filters, shear stress promoted migration away from the luminal surface. EC conditioned by shear stress recruited fewer flowing neutrophils, and showed reduced up‐regulation of E‐selectin after stimulation with tumor necrosis factor‐α (TNF). Use of siRNA against CD31 in the hEC, or testing of mEC from mice lacking CD31, indicated that expression of CD31 was not required for the shear‐induced modification of wound closure. However, shear modulation of response to TNF was less effective in the absence of CD31, while reduction of CD31 reduced shear‐sensitivity in some genes (e.g., eNOS), but not others (e.g., KLF‐2). Thus, CD31 played a role in shear‐sensitivity of some genes and of neutrophil recruitment, but not in modulation of endothelial migration. Different mechanotransducers may mediate different functional effects of shear stress. Hence, identification of the specific pathways may provide targets for therapeutic manipulation of angiogenesis or inflammation. J. Cell. Physiol. 227: 2710–2721, 2012.

Integrin-substrate interactions underlying shear-induced inhibition of the inflammatory response of endothelial cells

Conditioning of endothelial cells by shear stress suppresses their response to inflammatory cytokines. We questioned whether signalling through different integrin-matrix interactions, previously associated with the pathogenic effects of disturbed flow, supported the anti-inflammatory action of steady shear. Primary human endothelial cells were cultured on different substrates and exposed to shear stress (2.0Pa) for varying periods before stimulation with tumour necrosis factor-α (TNF). Shear-conditioning inhibited cytokine-induced recruitment of flowing neutrophils. However, the effect was similar for culture on collagen, laminin or fibronectin, even when seeding was reduced to 2 hours, and shear to 3 hours before TNF treatment (to minimise deposition of endothelial matrix). Nevertheless, in short- or longer-term cultures, reduction in expression of β(1)-integrin (but not β(3)-integrin) using siRNA essentially ablated the effect of shear-conditioning on neutrophil recruitment. Studies of focal adhesion kinase (FAK) phosphorylation, siRNA against FAK and a FAK-inhibitor (PF573228) indicated that FAK activity was an essential component downstream of β(1)-integrin. In addition, MAP-kinase p38 was phosphorylated downstream of FAK and also required for functional modification. Mechanotransduction through β(1)-integrins, FAK and p38 is required for anti-inflammatory effects of steady shear stress. Separation of the pathways which underlie pathological versus protective responses of different patterns of flow is required to enable therapeutic modification or mimicry, respectively.

Comparative Ability of Mesenchymal Stromal Cells from Different Tissues to Limit Neutrophil Recruitment to Inflamed Endothelium

Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton’s jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary.

57 Mesenchymal stem cells inhibit recruitment of flowing neutrophils and lymphocytes by endothelial cells: roles of interleukin-6 and transforming growth factor-

Mesenchymal stem cells (MSC) have been suggested to have protective effects through modulation of immune responses. We examined the hypothesis that MSC can modify the ability of endothelial cells (EC) to recruit neutrophils and lymphocytes. Using flow-based assays, leukocytes were perfused over HUVEC that had been cultured with MSC, either on opposite sides of porous filers or in direct contact for 24 h, and then stimulated with tumour necrosis factor-α (TNF-α) for 4 h or TNF-α and interferon-γ for 24 h. Cytokine-stimulated EC supported neutrophil and lymphocyte recruitment, and a combination of rolling, firm adhesion and transmigration was observed. EC cultured with MSC supported significantly lower total adhesion of both types of leukocytes, and the proportion of adherent leukocytes that migrated was reduced compared to EC monocultures. The effects were dependent on the number of MSC added, but were evident for ratios of MSC to EC <<0.1. Interestingly, antibody neutralisation of interleukin-6 (IL-6) or transforming growth factor (TGF)-β during co-culture largely abolished the inhibitory effects of MSC on recruitment of both types of leukocyte. These antibodies did not modify recruitment to cytokine-treated EC cultured alone. We conclude that MSC can adhere to and integrate with EC monolayers, and then inhibit the inflammatory recruitment of leukocytes. The inhibitory effects may be due to linked production and effects of IL-6 and TGF-ï□¢ in the co-culture milieu.

Abstract 1589: Low shear stress induces the novel tumor endothelial marker CLEC14A that mediates cell migration and vascular development

Experimental evidence using animal models has proven the efficacy of targeting a tumor endothelial marker (TEM) in eradicating large solid tumours. It follows that a well characterised tumor endothelial marker would offer much promise as an anti-cancer target. Despite many efforts by numerous groups, a well defined tumor endothelial marker in man has remained elusive. CLEC14A is a novel endothelial specific C-type lectin that is a member of the endosialin/TEM1 and thrombomodulin family of lectins. Protein sequence alignment of CLEC14A shows conservation from fish to man and endothelial specificity of expression is seen in the embryonic zebrafish. Detailed analysis of CLEC14A expression using human tissue arrays showed that CLEC14A is absent in human adult tissue but highly expressed in endothelium across a wide range of tumour tissues. CLEC14A is a superior tumor endothelial marker to Robo4 in terms of specificity and level of expression on the cell surface. CLEC14A is only expressed by endothelium in the absence of shear stress and this could explain its tumor specific expression due to poor blood flow in ill-formed tumor vessels. Functional studies have shown that CLEC14A is present on filopodia and mediates endothelial cell migration and tube formation. Anti-sera to CLEC14A similarly inhibited these processes suggesting that anti-CLEC14A monoclonal antibodies would have anti-angiogenic activity. The expression profile of CLEC14A, its cell surface localisation and inhibition of pro-angiogenic activities by anti-sera make CLEC14A a uniquely interesting target. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1589.