Platelet microparticle (PMP)-induced angiogenesis plays a key role in tumour metastasis and has been proposed to contribute towards cardiovascular disease by enhancing atherosclerotic plaque vulnerability. However, the mechanisms underlying PMP induced angiogenesis are ill defined. Recent reports demonstrate that PMPs deliver micro-RNAs (miRNAs) to recipient cells, controlling gene expression. We therefore evaluated whether miRNA transfer was a key regulator of PMP-induced angiogenesis. Co-culturing PMPs with human umbilical vein endothelial cells (HUVEC) on extracellular matrix gel induced robust capillary like structure formation. PMP treatment altered the release of angiogenesis modulators from HUVEC, including significantly reducing production of anti-angiogenic thrombospondin-1 (THBS-1). Both functional responses were abrogated by treating PMPs with RNase, suggesting the transfer of PMP-derived RNA was a critical event. PMPs were an abundant source of miRNA Let-7a, which was transferred to HUVEC following co-incubation. Using luciferase reporter assays we have shown that Let-7a directly targets the 3’UTR of the THBS-1 mRNA. HUVEC transfection with a Let-7a anti-sense oligonucleotide reduced the ability of PMPs to inhibit THBS-1 release, and significantly decreased PMP induced in vitro angiogenesis. Antibody neutralisation of THBS-1 reversed the anti-angiogenic effect of let-7a inhibition in PMP treated HUVEC, highlighting Let-7a dependent translational repression of THBS-1 drives angiogenesis. Importantly, plasmid overexpression of Let-7a in HUVEC alone induced robust tubule formation on extracellular matrix gel. These data reveal a new role for Let-7a in promoting angiogenesis and show for the first time PMPs induced angiogenic responses occur through miRNA regulation of HUVEC.

The IL-1 family member cytokine IL-36γ is recognised as key mediator in the immunopathology of psoriasis, hallmarks of which involve the activation of both resident and infiltrating inflammatory myeloid cells and aberrant angiogenesis. This research demonstrates a role for IL-36γ in both myeloid activation and angiogenesis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23 and TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. IL-36γ was also demonstrated to induce endothelial tube formation and branching, in a VEGF-A-dependent manner. Furthermore, IL-36γ-stimulated macrophages potently activated endothelial cells and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, psoriasis monocytes showed increased adherence to both the stimulated and unstimulated endothelium when compared with monocytes from healthy individuals. Collectively, these findings show that IL-36γ has the potential to enhance endothelium directed leucocyte infiltration into the skin and strengthen the IL-23/IL-17 pathway adding to the growing evidence of pathogenetic roles for IL-36γ in psoriatic responses. Our findings also point to a cellular response, which could potentially explain cardiovascular comorbidities in psoriasis in the form of endothelial activation and increased monocyte adherence.

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Interleukin-36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL-36 is now a recognised characteristic of psoriatic skin inflammation. IL-36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL-36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated by IL-36 stimulation, and this is reversed by the presence of the endogenous IL-36 receptor antagonist. IL-36-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36 stimulation of endothelial cells. These results suggest a role for IL-36 in the dermal vascular compartment, and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leucocyte recruitment.

Gestational diabetes mellitus (GDM) is known to be associated with fetal endothelial dysfunction, however, the mechanisms are not fully understood. This study examines the effect of maternal diabetes on fetal endothelial function and gene expression under physiological glucose conditions (5mM). Human umbilical vein endothelial cell (HUVEC) isolated from diabetic mothers (d.HUVEC) grew more slowly than HUVEC isolated from healthy mothers (c.HUVEC) and had delayed doubling time despite increased levels of total vascular endothelial growth factor (VEGF) expression and protein production as determined by real-time PCR and ELISA respectively. Using western blot, the levels of antiproliferative VEGF165b isoform were increased in d.HUVEC relative to c.HUVEC. Successful VEGF165b knockdown by small interfering RNA (siRNA) resulted in increased proliferation of d.HUVEC measured by MTT, compared with negative siRNA control, to similar levels measured in c.HUVEC. In addition, d.HUVEC generated excess levels of ROS as revealed by 2,7 Dichlorodihydrofluorescein Diacetate (DCFH-DA) and Nitrotetrazolium blue (NBT). Using microarray, 102 genes were differentially overexpressed between d.HUVEC versus c.HUVEC (>1.5-fold change; P<0.05). Functional clustering analysis of these differentially expressed genes revealed participation in inflammatory responses (including adhesion) which may be related to pathological outcomes. Of these genes, ICAM-1 was validated as upregulated, confirming microarray results. Additional confirmatory immunofluorescence staining revealed increased protein expression of ICAM-1 compared with c.HUVEC which was reduced by vitamin C treatment (100M). Thus, maternal diabetes induces persistent alterations in fetal endothelial function and gene expression following glucose normalization and antioxidant treatment could help reverse endothelium dysfunction. J. Cell. Physiol. 9999: 2695-2705, 2015. (c) 2015 Wiley Periodicals, Inc.

It is known that cells within the inflammatory background in classical Hodgkin lymphoma (cHL) provide signals essential for the continual survival of the neoplastic Hodgkin and Reed-Sternberg (HRS) cells. However, the mechanisms underlying the recruitment of this inflammatory infiltrate into the involved lymph nodes are less well understood. In this study, we show in vitro that HRS cells secrete lymphotoxin-α (LTα) which acts on endothelial cells to upregulate the expression of adhesion molecules that are important for T cell recruitment. LTα also enhances the expression of hyaluronan which preferentially contributes to the recruitment of CD4(+) CD45RA(+) naïve T cells under in vitro defined flow conditions. Enhanced expression of LTα in HRS cells and tissue stroma; and hyaluronan on endothelial cells are readily detected in involved lymph nodes from cHL patients. Our study also shows that although NF-κB and AP-1 are involved, the cyclooxygenase (COX) pathway is the dominant regulator of LTα production in HRS cells. Using pharmacological inhibitors, our data suggest that activity of COX1, but not of COX2, directly regulates the expression of nuclear c-Fos in HRS cells. Our findings suggest that HRS cell-derived LTα is an important mediator that contributes to T cell recruitment into lesional lymph nodes in cHL.

Abstract Growth and survival of neoplastic Hodgkin and Reed-Sternberg cells (H-RS) cells in classical Hodgkin lymphoma (cHL) is dependent on cell-cell contact and soluble mediator signals derived from the inflammatory infiltrate within the lesion. However, the mechanisms underlying recruitment of the inflammatory infiltrate, particularly T cells, into lesional lymph nodes remain unclarified. In this study, we test the hypothesis that H-RS cells secrete soluble mediators to induce endothelial cell (EC) activation to modulate T cell recruitment in cHL. We examined in-vitro interactions, under static and flow conditions, between freshly isolated T cell subsets and EC treated with conditioned medium (CM) harvested from the H-RS cell line, KM-H2. Both CM-stimulated EC and TNFα-stimulated EC support CD4+ naive and memory T-cell interactions under flow conditions. Both CD4+ naive and memory T cell demonstrated enhanced transmigration across CM-stimulated EC monolayers in response to CXCL12 as compared to TNFα-stimulated or unstimulated EC. Our data show that the upregulation of inducible adhesion molecules on CM-stimulated EC is dependent on the NFκB pathway; but the dominant mediator involved is not TNFα. However, blockade of cyclooxygenase activity in KM-H2 cells partially attenuated the efficacy of the CM in activating EC. These findings suggest that H-RS cells secrete soluble mediators that can modulate endothelial cell function and influence recruitment of T cells into the cHL lesion.

Background: Ischemiaereperfusion or hypoxiaereoxygenation (H-R) injury adversely affects hepatic function following transplantation and major resection; the death of human sinusoidal endothelial cells (SECs) by apoptosis may play a central role in this process. Caspase-3 is an important intracellular protease in the intrinsic and extrinsic pathways of apoptosis.Materials and methods: SECs and EAhy926 cells were exposed to warm hypoxia at 37 degrees C, followed by reoxygenation at 37 degrees C. Activity of caspase-3 was quantified using Western blotting and colorimetric kinase assays.Results: H-R caused a significant increase in caspase-3 activity compared with controls in both cell types.Conclusions: Warm H-R injury causes apoptotic cell death of SECs and immortalized cells, but with differing patterns of caspase activity. (C) 2012 Elsevier Inc. All rights reserved.

AIM : The role of ischaemic preconditioning (IPC) on liver regeneration following surgery remains inconclusive. The aim of the study was to assess the effect of IPC on mediators involved in regeneration produced by human hepatocytes, using an in-vitro hypoxia-reoxygenation model to mimic ischaemia-reperfusion injury (IRI). METHODS: Following extraction from samples obtained from liver resection (n=5), confluent culture flasks of hepatocytes were subjected to IRI (1 hour hypoxia + 1 hour reoxygenation), IPC prior to IRI (10 minutes hypoxia + 10 minutes reoxygenation + 1 hour hypoxia + 1 hour reoxygenation), intermittent clamping (IC, 15 minutes hypoxia + 5 minutes reoxygenation x3 + 1 hour reoxygenation) and compared to controls. The production of various mediators was determined over 48 hours. RESULTS: Interleukin (IL)-8, granulocyte-colony stimulating factor (G-CSF) and IL-1-receptor antagonist (IL-1ra) were produced by hepatocytes. IPC prior to IRI did not influence the production of IL-8, G-CSF and IL-1ra by hepatocytes compared to IRI over the study period. Similarly, IC did not significantly influence the profile of these mediators compared to IRI in this model. CONCLUSION: IPC, as well as IC did not influence the production of pro-regenerative mediators in a hepatocyte model of IRI. The role of IPC in liver regeneration remains to be determined.

Abstract Introduction: Classical Hodgkin lymphoma (CHL) is characterized by the presence of a minority of malignant Hodgkin and Reed-Sternberg cells (H-RS cells) surrounded by an abundant mixed inflammatory infiltrate that includes CD4+ T helper 2 cells, regulatory T cells and CD8+ cytotoxic T cells. The mechanisms exploited by HRS cells to influence T cell recruitment into the involved lymph nodes are still unknown. Objective: The aim of this study is to elucidate whether H-RS cells can induce endothelial cell activation to modulate T cell recruitment in CHL. Procedures: Fresh cell culture supernatant (C/S) from the H-RS cell line, KM-H2, was used to stimulate endothelial cells (EC) in-vitro. Controls were EC incubated in medium alone; or with 10ng/ml TNF-α Up-regulation of adhesion molecule expression was assessed by ELISA and FACS. Using the transwell migration system and the parallel plate flow chamber apparatus respectivel, the ability of these stimulated EC to support T cell interactions in-vitro was assessed under static and defined flow conditions. Signaling pathways involved in C/S induced EC activation were examined by western blotting. Results: KM-H2 C/S-stimulated EC up-regulate ICAM-1, VCAM-1 and E-selectin to levels comparable to those of TNF-α-stimulated EC. Both C/S-stimulated EC and TNF-α stimulated-EC support naïve and memory T-cell interactions under defined flow conditions. Furthermore, both CD4+ naïve and memory T cell demonstrated enhanced transmigration across C/S-stimulated EC monolayers in response to SDF-1α, as compared to TNF-α-stimulated or unstimulated EC. The activation of EC by KM-H2 C/S is dependent on the NFkB pathway. Blocking of p65 nuclear translocation by the specific IκBα phosphorylation inhibitor (BAY11-8075) abrogated the stimulatory effect of C/S on EC. Phosphorylation of ERK, p38 and JNK were also detected in the KM-H2 C/S stimulated EC. Intracytoplasmic expression of TNF-α was detected in KM-H2 cells. The bioactivity assay showed that the TNF-α sensitive cell line, L929 is sensitive to KM-H2 C/S. However, treatment of cells with TNF-α neutralizing antibody did not effectively prevent L929 cell death, nor inhibit the stimulatory effect of KM-H2 C/S on EC. Conclusion: Our data suggest that the malignant Hodgkin and Reed-Sternberg cells secrete soluble mediators that modulate endothelial cell function and influence recruitment of T cells into the cHL lesion. While the C/S induced EC activation is dependent on the NFkB pathway, our data suggest that the dominant mediator involved is not TNF-α. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A4.

AIMS:Although the aetiology of varicose veins remains unknown, recent studies have focused on endothelial cell integrity and function. Among the regulatory factors of vessel tone, synthesises, pro- and anti-inflammatory, adhesion molecules and the transcription factor hypoxia inducible factor-1 alpha (HIF-1alpha), which are responsible for recruiting leukocytes, are very important.METHODS:Investigation in this study focused on the expression of ICAM-1, E-selectin and HIF-1alpha on endothelial cells using immunostaining and RT-PCR in varicose vein specimens compared with controls.RESULTS:Findings of this study showed alterations of the intima, such as focal intimal discontinuity and denudation of endothelium in varicose veins. Based on data derived from immunostaining and RT-PCR, no major differences were identified between ICAM-1 and E-selectin expression in varicose vein specimens compared with controls. In contrast, immunostaining results identified HIF-1alpha expression in five (5/20) varicose vein specimens, whereas no control saphenous vein specimens expressed HIF-1alpha.CONCLUSIONS:These findings could explain other evidence of hypoxia in varicose veins. Finally, results already obtained in this investigation suggest that the process of pathogenesis of varicose veins is not restricted to the role of adhesion molecules.

INTRODUCTION:Recanalisation rates (20-32%) 1-3 years after truncal vein foam sclerotherapy (FS) suggest thrombotic occlusion rather than irreversible vein wall injury. This study examines the injury inflicted by sodium tetradecyl sulphate (STD) foam before and after balloon endothelial denudation (BD). METHODS:In 20 patients undergoing great saphenous vein (GSV) stripping 1.5 cm proximal GSV were harvested (controls). The next 1.5 cm were harvested after in situ BD (n = 10) or no denudation (n = 10). These test segments were filled with 1% or 3% STD foam (5 min), flushed and fixed in formalin. Percentage endothelial cell loss (ECL) and tunica media injury were determined (H&E staining) and collagen structure assessed (transmission electron microscopy, TEM). RESULTS:Controls showed no injury. 1% and 3% STD foam caused 86.3% and 92.2% ECL (p < 0.001 versus controls; 1% versus 3%, p = 0.55). Endothelial cells persisted in all sections. BD increased ECL (1%: 96.9%, 3%: 98.1%, p = 0.01) Tunica media injury (smooth muscle vacuolation) was minimal (8.9% (1% STD) and 12% (3% STD) of its depth) and not enhanced by BD (1%: 8.7%, p = 0.93; 3%: 11.3%; p = 0.86). No collagen disruption occurred (TEM). CONCLUSIONS:Balloon denudation increased ECL but did not facilitate tunica media injury. Equivalent injury was inflicted by 1% and 3% STD.

Homocysteine (Hcy) is implicated in inducing cellular oxidative stress (OXS) which mediates endothelial cell (EC) damage via unknown mechanisms. We have previously shown Hcy induces activation of MAPK JNK and increases EC:leukocyte interactions. However, the JNK activator has yet to be identified. OXS is known to cause activation of the JNK MAP kinase pathway. Objective: to study the effects of antioxidants on Hcy EC damage.Human umbilical vein EC (HUVEC) were cultured for 5‐9 days in medium containing 1mM DL‐Hcy alone, with 5mM N‐acetylcysteine (NAC) or 100µM vitamin C. and JNK kinase assays conducted on cell lysates Reactive oxygen species (ROS) generated in HUVEC were also detected after treatment, by confocal microscopy. Confluent cells after treatment were subjected to flowing neutrophils (1x106 cells/ml) at 1.1dynes and tethered, rolled, fixed and transmigrated neutrophils on HUVEC counted.DL‐Hcy caused activation of JNK (1.5fold) and increased ROS accumulation. NAC reversed these effects. Furthermore, DL‐Hcy significantly increased HUVEC:neutrophil interactions. However, the presence of either NAC or Vitamin C reversed these Hcy mediated effects. These results show that OXS mediates Hcy effects through a MAPK kinase dependent pathway. Antioxidants block Hcy stimulated JNK activation, so preventing HUVEC: neutrophil interactions under flow conditions.Funding: HR UK, ORSAS

Hyperhomocysteinemia (hHcy) upregulates expression of cell adhesion molecules (CAM) on EC which recruits leukocytes and initiates atherosclerotic lesion development. Nitric oxide (NO) is anti‐adhesive for leukocytes.Objectiveto study effects of exogenous NO on hHcy CAM expression in human umbilical vein endothelial cells (HUVEC) and recruitment of leukocytes under flow.HUVEC were cultured for 5‐9 days in medium containing 1mM Hcy or Cysteine (negative control). Some Hcy‐stimulated cells were treated with NO donor (S‐nitrosoglutathione: GSNO: 20µM, 10min) in absence of presence of a cGMP inhibitor (ODQ 40µM, 30min). Confluent cells were subjected to flowing neutrophils (1x106cells/ml) at 1.1dynes. Tethered, rolled, fixed and transmigrated neutrophils on HUVEC were counted. Immunofluorescence antibodies against ICAM‐1, E‐selectin and P‐selectin quantified adhesion molecule expression.Hcy induced expression of ICAM‐1, E‐selectin and P‐selectin (p<0.05) and significantly increased interactions between neutrophils and HUVEC while cysteine had no effect. When Hcy‐treated HUVEC were exposed to GSNO with or without ODQ, numbers of all interactions decreased dramatically but significantly decreased only P‐selectin expression (p<0.05).NO inhibits HUVEC: leukocyte interaction through inhibiting the expression of P‐selectin in a cGMP‐independent manner.Funding: HRUK, ORSAS

Hypercholesterolaemia is implicated as an independent risk factor in the pathogenesis of atherosclerosis. HMG-CoA reductase inhibitors (statins) are prescribed for their lipid-lowering effects but recent evidence suggests they have pleiotropic effects independent of lipid balance regulation that may explain their role in dramatically decreasing cardiovascular mortality and morbidity. The mechanisms responsible are unclear but endothelial cell (EC) dysfunction is critical. To investigate potential anti-inflammatory properties of statins on EC, functional responses of human umbilical vein endothelial cells (HUVEC) and human neutrophils under physiological flow conditions were studied. These interactions were quantified in response to inflammatory mediators following pre-treatment with statin. Histamine stimulation resulted in significant (p<0.001) increases in transient interactions between neutrophils and EC (tethering). These effects were significantly reduced (p<0.001) on pre-treatment with statin. TNF-alpha stimulation resulted in significant (p<0.001) increases in rolling interactions. These effects were significantly (p<0.001) reduced following pre-treatment of EC with statin. Mevalonate pre-treatment of EC significantly reversed the effects of statin pre-treatment on both tethering and rolling (p<0.001). Reductions in surface expression of P- and E-selectin were confirmed by ELISA. EC exposed to histamine demonstrated significantly increased (p<0.01) levels of P-selectin, abrogated (p<0.001) by pre-treatment with statin. EC exposed to TNF-alpha demonstrated a significant increase (p<0.001) in levels of E-selectin, reduced (p<0.05) by pre-treatment with statin.

Nitric oxide (NO) is an established regulator of platelet function, although the processes by which NO modulates platelet adhesion are unclear. We studied the importance of Ca2+ and phosphoinositol-3-kinase (PI3kinase) as targets for NO signalling, in the physiological context of platelet adhesion using adenosine diphosphate (ADP)-stimulated adhesion to immobilised fibrinogen. DPTA-NONOate induced a time and concentration-dependent inhibition of adhesion, and reduced protein tyrosine phosphorylation. The action of NO was cGMP-independent despite activation of the cGMP-signalling cascade, as evidenced by VASP phosphorylation. Furthermore, the cGMP-independent mechanism did not involve PKA. Platelet activation by ADP requires Ca2+ and PI3kinase-dependent signalling pathways. We examined the effect of NO on these pathways using two approaches. Firstly, we dissected the signalling pathways using the P2Y(1)-receptor antagonist A3P5P, and secondly, directly inhibited Ca2+ mobilisation and PI3kinase activity. ADP-induced adhesion was reduced but not abolished by A3P5P, suggesting signalling from P2Y(12) can induce adhesion. NO further reduced adhesion in the presence of A3P5P, indicating that NO inhibited adhesion independently of any effects on Ca2+ mobilisation. Dimethyl bis-(o-aminophenoxy) ethane-tetraacetic acid (BAPTA) and wortmannin both partially inhibited ADP-induced adhesion, but completely abolished adhesion when used in combination, demonstrating that ADP-induced adhesion requires Ca2+ and PI3kinase-regulated pathways. Combination of either dimethyl-BAPTA or wortmannin with DPTA-NONOate enhanced inhibition of both the Ca2+ and PI3kinase-dependent pathways when compared to the levels of inhibition with either agent alone. Thus, we demonstrate that NO inhibits alpha(IIb)beta 3-mediated adhesion, by targeting both Ca2+ and PI3kinase pathways in a cGMP-independent manner.

The endothelial cell (EC) adhesion molecule P-selectin plays a crucial role in early stages of leukocyte recruitment during inflammation. This study investigated effects of simvastatin pre-treatment on human saphenous vein EC (HSVEC) in an in vitro flow model of inflammation. HSVEC were seeded into 35mm dishes (2×105 cells/ml) and incubated overnight at 37°C. Confluent EC were treated with histamine (5×10–4M;18min). Neutrophils were perfused over HSVEC (1×106 cells/ml) in a glycotech flow chamber (1.1dyne cm2). EC: neutrophil interactions were recorded and counted. Histamine stimulation resulted in significant (p<0.001) increases in tethering from an average of 40 interactions (negative control) to 380 following histamine stimulation. The effects of histamine were significantly (p<0.001) reduced to levels comparable to the negative control by pre-treatment of the HSVEC with simvastatin (1μM;6h). Statin effects on histamine stimulated EC were reversed by mevalonate, mean tethering interactions not significantly different from histamine treated EC. These results show the anti-inflammatory effects of simvastatin to be dependent on a reduction in endothelial P-selectin expression in an HMG-CoA reductase dependent manner. Funded by Heart Research UK.