Ronan P. Murphy

Prospective Evaluation of the Risk Conferred by Factor V Leiden and Thermolabile Methylenetetrahydrofolate Reductase Polymorphisms in Pregnancy

Factor V (FV) Leiden and thermolabile methylenetetrahydrofolate reductase (MTHFR) are 2 common polymorphisms that have been implicated in vascular thrombosis. We determined whether these mutations predicted an adverse outcome in pregnancy. Second, we looked for an interaction between these 2 mutations in patients with recurrent fetal loss or thrombosis in pregnancy. Primigravid subjects at their booking visit to the National Maternity Hospital (Holles Street, Dublin, Ireland) were screened for the polymorphisms. Thermolabile MTHFR and FV Leiden genotypes were detected by either restriction fragment length polymorphism or heteroduplex capillary chromatography. The carrier frequency of FV Leiden in the screened primigravid population was 2.7% (allele frequency 1.36%), all being heterozygous for the mutation. This value was lower than expected from previous studies in European populations. Forty-nine percent of the screened population (289 of 584) were heterozygous for thermolabile MTHFR, and 10.6% were homozygous (62 of 584). The frequency of the 2 polymorphisms was no higher in those who subsequently developed preeclampsia (n=12) or intrauterine growth retardation (n=9), and none of the screened population developed thrombosis. However, the frequency of FV Leiden was higher in patients who subsequently miscarried after the first trimester of pregnancy (allele frequency of 5.5%, P=0.0356). Among those positive for FV Leiden, 3 of 27 miscarried, compared with 24 of 572 of FV Leiden-negative patients (11% versus 4.2%). No interaction was found between the 2 mutations in the control or patient populations. In patients with a prior history of venous thrombosis, the carrier rate of FV Leiden was increased (4 of 33, allele frequency of 7.6%, P=0. 0115). In contrast, the carrier frequency for thermolabile MTHFR was no higher, and there was no interaction between the 2 mutations. Neither mutation occurred at a significantly higher frequency in patients with a prior history of recurrent fetal loss. In conclusion, FV Leiden is a risk factor for thrombosis in pregnancy and possibly for second-trimester miscarriage independent of thermolabile MTHFR. However, prospective analysis suggests that the risk conferred by FV Leiden is low in a primigravid population. The thermolabile MTHFR genotype was not implicated in any adverse outcome.

Downregulation of Blood-Brain Barrier Phenotype by Proinflammatory Cytokines Involves NADPH Oxidase-Dependent ROS Generation: Consequences for Interendothelial Adherens and Tight Junctions

Background and Objectives Blood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization. Methods The present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated. Results In response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766). Conclusion A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events.

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

Thrombomodulin (TM) is a 557-amino acid protein with a broad cell and tissue distribution consistent with its wide-ranging physiological roles. When expressed on the lumenal surface of vascular endothelial cells in both large vessels and capillaries, its primary function is to mediate endothelial thromboresistance. The complete integral membrane-bound protein form displays five distinct functional domains, although shorter soluble (functional) variants comprising the extracellular domains have also been reported in fluids such as serum and urine. TM-mediated binding of thrombin is known to enhance the specificity of the latter serine protease toward both protein C and thrombin activatable fibrinolysis inhibitor (TAFI), increasing their proteolytic activation rate by almost three orders of magnitude with concomitant anticoagulant, antifibrinolytic, and anti-inflammatory benefits to the vascular wall. Recent years have seen an abundance of research into the cellular mechanisms governing endothelial TM production, processing, and regulation (including flow-mediated mechanoregulation)—from transcriptional and posttranscriptional (miRNA) regulation of TM gene expression, to posttranslational processing and release of the expressed protein—facilitating greater exploitation of its therapeutic potential. The goal of the present paper is to comprehensively review the endothelial/TM system from these regulatory perspectives and draw some fresh conclusions. This paper will conclude with a timely examination of the current status of TMs growing therapeutic appeal, from novel strategies to improve the clinical efficacy of recombinant TM analogs for resolution of vascular disorders such as disseminated intravascular coagulation (DIC), to an examination of the complex pleiotropic relationship between statin treatment and TM expression.

Cyclic Strain–Mediated Regulation of Vascular Endothelial Occludin and ZO-1 Influence on Intercellular Tight Junction Assembly and Function

Objective—The vascular endothelium constitutes a highly effective fluid/solute barrier through the regulated apposition of intercellular tight junction complexes. Because endothelium-mediated functions and pathology are driven by hemodynamic forces (cyclic strain and shear stress), we hypothesized a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We, therefore, examined the effects of cyclic strain on the expression, modification, and function of 2 pivotal endothelial tight junction components, occludin and ZO-1. Methods and Results—For these studies, bovine aortic endothelial cells were subjected to physiological levels of equibiaxial cyclic strain (5% strain, 60 cycles/min, 24 hours). In response to strain, both occludin and ZO-1 protein expression increased by 2.3±0.1-fold and 2.0±0.3-fold, respectively, concomitant with a strain-dependent increase in occludin (but not ZO-1) mRNA levels. These changes were accompanied by reduced occludin tyrosine phosphorylation (75.7±8%) and increased ZO-1 serine/threonine phosphorylation (51.7±9% and 82.7±25%, respectively), modifications that could be completely blocked with tyrosine phosphatase and protein kinase C inhibitors (dephostatin and rottlerin, respectively). In addition, there was a significant strain-dependent increase in endothelial occludin/ZO-1 association (2.0±0.1-fold) in parallel with increased localization of both occludin and ZO-1 to the cell–cell border. These events could be completely blocked by dephostatin and rottlerin, and they correlated with a strain-dependent reduction in transendothelial permeability to FITC-dextran. Conclusions—Overall, these findings indicate that cyclic strain modulates both the expression and phosphorylation state of occludin and ZO-1 in vascular endothelial cells, with putative consequences for endothelial tight junction assembly and barrier integrity.

Stabilization of brain microvascular endothelial barrier function by shear stress involves VE-cadherin signaling leading to modulation of pTyr-occludin levels

Blood–brain barrier (BBB) regulation involves the coordinated interaction of intercellular adherens and tight junctions in response to stimuli. One such stimulus, shear stress, has been shown to upregulate brain microvascular endothelial cell (BMvEC) barrier function, although our knowledge of the signaling mechanisms involved is limited. In this article, we examined the hypothesis that VE‐cadherin can transmit shear signals to tight junction occludin with consequences for pTyr‐occludin and barrier function. In initial studies, chronic shear enhanced membrane localization of ZO‐1 and claudin‐5, decreased pTyr‐occludin (in part via a dephostatin‐sensitive mechanism), and reduced BMvEC permeability, with flow reduction in pre‐sheared BMvECs having converse effects. In further studies, VE‐cadherin inhibition (VE‐cad ΔEXD) blocked shear‐induced Rac1 activation, pTyr‐occludin reduction, and barrier upregulation, consistent with an upstream role for VE‐cadherin in transmitting shear signals to tight junctions through Rac1. As VE‐cadherin is known to mediate Rac1 activation via Tiam1 recruitment, we subsequently confirmed that Tiam1 inhibition (Tiam1‐C580) could elicit effects similar to VE‐cad ΔEXD. Finally, the observed attenuation of shear‐induced changes in pTyr‐occludin level and barrier phenotype following Rac1 inhibition (NSC23766, T17N) establishes a downstream role for Rac1 in this pathway. In summary, we describe for the first time in BMvECs a role for VE‐cadherin in the transmission of physiological shear signals to tight junction occludin through engagement of Tiam1/Rac1 leading to barrier stabilization. A downstream role is also strongly indicated for a protein tyrosine phosphatase in pTyr‐occludin modulation. Importantly, these findings suggest an important route of inter‐junctional signaling cross‐talk during BBB response to flow. J. Cell. Physiol. 226: 3053–3063, 2011.

The Urokinase Receptor Interactome

The urokinase receptor (uPAR) was originally identified as the membrane receptor of the serine protease urokinase (uPA), thereby implicated in the plasminogen activation cascade and regulation of pericellular proteolysis. Later on, vitronectin was showed to be another major ligand providing uPAR with a role in cell adhesion. Other unrelated ligands have been subsequently reported including for example factor XII and SRPX2 expanding the functions of uPAR to unexpected biological areas such as the initiation of the coagulation cascade or the regulation of language development. Due to its glycosylphosphatidylinositol (GPI) anchor, uPAR has no intracellular domain and thus exerts its signaling capacity through lateral interactions with other components of the plasma membrane that actually mediate uPAR-induced signals. As yet, a total 42 proteins interacting directly with uPAR can be numbered comprising 9 soluble ligands and 33 lateral partners. The fact that uPAR interacts with members of three major families of membrane receptors i.e. G protein-coupled receptors, receptor tyrosine kinases, and integrins implies that the actual number of components constituting the uPAR interacome is extremely high. For example, 156 factors belong to the integrin adhesome. Moreover, in the light of the wide diversity of the components of the uPAR interactome, uPAR appears to be an essential player of major biological systems including the blood coagulation, complement and plasma kallikrein-kinin cascades. This review describes the soluble ligands and lateral partners of the uPAR interactome, the mechanisms regulating uPAR interactions and their proved and/or potential biological functions.

Influence of basolateral condition on the regulation of brain microvascular endothelial tight junction properties and barrier function

Basolateral condition of the brain microvascular endothelium is believed to influence blood-brain barrier (BBB) phenotype, although the precise transcriptional and post-translational mechanisms involved are poorly defined. In vivo, the basolateral surface of the blood-brain endothelium is bathed in serum-free interstitial fluid and encompassed by astrocytic end-feet. We hypothesized that these conditions impact on BBB function by directly modulating expression and biochemical properties of tight junctions. To investigate this, an in vitro transwell culture model was employed to selectively modify the basolateral environment of bovine brain microvascular endothelial cells (BBMvECs). In the absence of basolateral (but not apical) serum, we observed higher levels of expression, association and plasma membrane localization for the tight junction proteins, occludin and zonula occludens-1 (ZO-1), in parallel with elevated transendothelial electrical resistance (TEER) and reduced (14)[C]-sucrose permeability of BBMvEC monolayers. We further examined the effects of non-contact co-culture with basolateral astrocytes (C6 glioma) on indices of BBMvEC barrier function in both the presence and absence of serum. Astrocyte co-culture with serum led to enhanced occludin protein expression, occludin/ZO-1 association, and ZO-1 membrane localization, in parallel with increased TEER of BBMvEC monolayers. Astrocyte co-culture in the absence of serum (i.e. basolateral conditions most consistent with in vivo BBB physiology) however, gave the highest increases in BBMvEC barrier indices. Thus, we can conclude that factors influencing condition of the basolateral environment of the brain microvasculature can directly, and independently, modify BBB properties by regulating the expression and biochemical properties of the tight junction proteins, occludin and ZO-1.

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in β-catenin expression. β-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6(-/-)), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.

Helicobacter pylori-induced inhibition of vascular endothelial cell functions: a role for VacA-dependent nitric oxide reduction

Epidemiological and clinical studies provide compelling support for a causal relationship between Helicobacter pylori infection and endothelial dysfunction, leading to vascular diseases. However, clear biochemical evidence for this association is limited. In the present study, we have conducted a comprehensive investigation of endothelial injury in bovine aortic endothelial cells (BAECs) induced by H. pylori-conditioned medium (HPCM) prepared from H. pylori 60190 [vacuolating cytotoxin A (Vac(+))]. BAECs were treated with either unconditioned media, HPCM (0-25% vol/vol), or Escherichia coli-conditioned media for 24 h, and cell functions were monitored. Vac(+) HPCM significantly decreased BAEC proliferation, tube formation, and migration (by up to 44%, 65%, and 28%, respectively). Posttreatment, we also observed sporadic zonnula occludens-1 immunolocalization along the cell-cell border, and increased BAEC permeability to FD40 Dextran, indicating barrier reduction. These effects were blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (VacA inhibitor) and were not observed with conditioned media prepared from either VacA-deleted H. pylori or E. coli. The cellular mechanism mediating these events was also considered. Vac(+) HPCM (but not Vac(-)) reduced nitric oxide (NO) by >50%, whereas S-nitroso-N-acetylpenicillamine, an NO donor, recovered all Vac(+) HPCM-dependent effects on cell functions. We further demonstrated that laminar shear stress, an endothelial NO synthase/NO stimulus in vivo, could also recover the Vac(+) HPCM-induced decreases in BAEC functions. This study shows, for the first time, a significant proatherogenic effect of H. pylori-secreted factors on a range of vascular endothelial dysfunction markers. Specifically, the VacA-dependent reduction in endothelial NO is indicated in these events. The atheroprotective impact of laminar shear stress in this context is also evident.

The Urokinase Receptor in the Central Nervous System

The urokinase receptor (uPAR) is a multifunctional glycosylphosphatidylinositol-anchored protein that regulates important processes such as gene expression, cell proliferation, adhesion, migration, invasion, and metastasis. uPAR is an essential component of the plasminogen activation cascade, a protease receptor that binds the urokinase-type plasminogen activator. uPAR is also an adhesion-modulating receptor, and a signalling receptor transmitting signals to the cell through lateral interactions with a wide array of membrane receptors. Altogether, the external ligands and membrane-bound partners of uPAR constitute a rich uPAR interactome. Recently, a new ligand of uPAR has been identified as the SRPX2 protein which is essential in language and cognitive development. SRPX2 is the second identified gene involved in language disorders. However, previous studies revealed cognitive disorders and defects in the development of the GABAergic interneurons in uPAR null mice. In addition, the expression of uPAR correlates with important human diseases such as epilepsy, autism, multiple sclerosis, Alzheimers, AIDS dementia, cerebral malaria, and brain tumours. Therefore, uPAR has unexpectedly become a significant receptor in the central nervous system and made a few steps into philosophy. Language is indeed intimately linked to human culture. This in-depth review presents the structure and the sequences of uPAR that are essential for drug design and the generation of new inhibitors. In addition, we summarize all the inhibitors of uPAR that have been created so far. Finally, we discuss the functions of uPAR in the development, functioning, and pathology of the central nervous system.