Platelet microparticle delivered microRNA-Let-7a promotes the angiogenic switch.

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 Role of Maternal Gestational Diabetes in Inducing Fetal Endothelial Dysfunction.

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 (5 mM). 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 (100 µM). Thus, maternal diabetes induces persistent alterations in fetal endothelial function and gene expression following glucose normalization and antioxidant treatment could help reverse endothelium dysfunction.

Reed-Sternberg cell-derived lymphotoxin-α activates endothelial cells to enhance T-cell recruitment in classical Hodgkin lymphoma.

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.

Pathogenic mechanisms in varicose vein disease: the role of hypoxia and inflammation.

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.

Simvastatin alters human endothelial cell adhesion molecule expression and inhibits leukocyte adhesion under flow.

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.

cGMP-independent inhibition of integrin alphaIIbbeta3-mediated platelet adhesion and outside-in signalling by nitric oxide.

We examined the influence of S-nitrosoglutathione (GSNO) on alpha(IIb)beta(3) integrin-mediated platelet adhesion to immobilised fibrinogen. GSNO induced a time- and concentration-dependent inhibition of platelet adhesion. Inhibition was cGMP-independent and associated with both reduced platelet spreading and protein tyrosine phosphorylation. To investigate the cGMP-independent effects of NO we evaluated integrin beta(3) phosphorylation. Adhesion to fibrinogen induced rapid phosphorylation of beta(3) on tyrosines 773 and 785, which was reduced by GSNO in a cGMP independent manner. Similar results were observed in suspended platelets indicating that NO-induced effects were independent of spreading-induced signalling. This is the first demonstration that NO directly regulates integrin beta(3) phosphorylation.

Inhibition of ADP-induced platelet adhesion to immobilised fibrinogen by nitric oxide: evidence for cGMP-independent mechanisms.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) and PI3kinase-regulated pathways. Combination of either dimethyl-BAPTA or wortmannin with DPTA-NONOate enhanced inhibition of both the Ca(2+) 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 Ca(2+) and PI3kinase pathways in a cGMP-independent manner.