Publications by authors named "Lopa Leach"

23 Publications

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Compromised barrier integrity of human feto-placental vessels from gestational diabetic pregnancies is related to downregulation of occludin expression.

Diabetologia 2021 Jan 1;64(1):195-210. Epub 2020 Oct 1.

School of Life Sciences, University of Nottingham, Nottingham, UK.

Aims/hypothesis: Reduced occupancy of junctional occludin is a feature of human placental vessels in the diabetic milieu. However, the functional consequence of this and whether this loss is due to differential expression of occludin splice variants is not known. Our study aimed to investigate the effects of gestational diabetes mellitus (GDM), and its treatment, on endothelial junctional integrity, gene and protein expression of occludin splice variants, and potential regulation of expression by microRNAs (miRNAs).

Methods: Term placentas were obtained from normal pregnancies (n = 21), and pregnancies complicated by GDM where glucose levels were controlled by diet (n = 11) or metformin (n = 6). Gene and microRNA (miRNA) expression were determined by quantitative real-time PCR; protein expression by immunoblotting; endothelial junctional occupancy by fluorescence microscopy and systematic sampling; and paracellular leakage by perfusion of placental microvascular beds with 76 M dextran. Transfection studies of miRNAs that target OCLN were performed in HUVECs, and the trans-endothelial electrical resistance and tracer permeability of the HUVECs were measured.

Results: All three predicted OCLN gene splice variants and two occludin protein isoforms were found in human placental samples. In placental samples from diet-controlled GDM (d-GDM) pregnancies we found a lower percentage of conduit vessels showing occludin immunoreactivity (12%, p < 0.01), decreased levels of the fully functional occludin isoform-A protein (29%), and differential gene expression of OCLN variant 2 (33% decrease), variant 3 (3.3-fold increase). These changes were not seen in samples from the group with metformin-controlled GDM. In d-GDM placentas, increased numbers of conduit microvessels demonstrated extravasation of 76 M dextran (2.0-fold). In d-GDM expression of one of the five potential miRNAs targeting OCLN, miR-181a-5p, expression was 2.1-fold that in normal pregnancies. Experimental overexpression of miR-181a-5p in HUVECs from normal pregnancies resulted in a highly significant downregulation of OCLN variant 1 (69%) and variant 2 (46%) gene expression, with decreased trans-endothelial resistance (78%) and increase in tracer permeability (1.3-fold).

Conclusions/interpretation: Downregulation of expression of OCLN variant 2 and the fully functional occludin isoform-A protein are a feature of placentas in d-GDM pregnancies. These may be behind the loss of junctional occludin and the increased extravasation of exogenous dextran observed. miR-181a-5p was in part responsible for the downregulation of occludin in placentas from d-GDM pregnancies. Induced overexpression of miR-181a-5p compromised the integrity of the endothelial barrier. Our data suggest that, despite good glucose control, the adoption of lifestyle changes alone during a GDM pregnancy may not be enough to prevent an alteration in the expression of occludin and the subsequent functional consequences in placentas and impaired vascular barrier function in offspring. Graphical abstract.
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http://dx.doi.org/10.1007/s00125-020-05290-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716932PMC
January 2021

The haemodynamics of the human placenta in utero.

PLoS Biol 2020 05 28;18(5):e3000676. Epub 2020 May 28.

Sir Peter Mansfield Imaging Centre, School of Physics and Astronomy, University of Nottingham, Nottingham, United Kingdom.

We have used magnetic resonance imaging (MRI) to provide important new insights into the function of the human placenta in utero. We have measured slow net flow and high net oxygenation in the placenta in vivo, which are consistent with efficient delivery of oxygen from mother to fetus. Our experimental evidence substantiates previous hypotheses on the effects of spiral artery remodelling in utero and also indicates rapid venous drainage from the placenta, which is important because this outflow has been largely neglected in the past. Furthermore, beyond Braxton Hicks contractions, which involve the entire uterus, we have identified a new physiological phenomenon, the 'utero-placental pump', by which the placenta and underlying uterine wall contract independently of the rest of the uterus, expelling maternal blood from the intervillous space.
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http://dx.doi.org/10.1371/journal.pbio.3000676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255609PMC
May 2020

Activation of Notch signaling by soluble Dll4 decreases vascular permeability via a cAMP/PKA-dependent pathway.

Am J Physiol Heart Circ Physiol 2019 05 25;316(5):H1065-H1075. Epub 2019 Jan 25.

Cancer Biology, Division of Cancer and Stem Cells, School of Medicine, Queen's Medical Centre, University of Nottingham , Nottingham , United Kingdom.

The Notch ligand delta-like ligand 4 (Dll4), upregulated by VEGF, is a key regulator of vessel morphogenesis and function, controlling tip and stalk cell selection during sprouting angiogenesis. Inhibition of Dll4 results in hypersprouting, nonfunctional, poorly perfused vessels, suggesting a role for Dll4 in the formation of mature, reactive, functional vessels, with low permeability and able to restrict fluid and solute exchange. We tested the hypothesis that Dll4 controls transvascular fluid exchange. A recombinant protein expressing only the extracellular portion of Dll4 [soluble Dll4 (sDll4)] induced Notch signaling in endothelial cells (ECs), resulting in increased expression of vascular-endothelial cadherin, but not the tight junctional protein zonula occludens 1, at intercellular junctions. sDll4 decreased the permeability of FITC-labeled albumin across EC monolayers, and this effect was abrogated by coculture with the γ-secretase inhibitor -[-(3,5-difluorophenacetyl)-l-alanyl]--phenylglycine -butyl ester. One of the known molecular effectors responsible for strengthening EC-EC contacts is PKA, so we tested the effect of modulation of PKA on the sDll4-mediated reduction of permeability. Inhibition of PKA reversed the sDll4-mediated reduction in permeability and reduced expression of the Notch target gene Hey1. Knockdown of PKA reduced sDLL4-mediated vascular-endothelial cadherin junctional expression. sDll4 also caused a significant decrease in the hydraulic conductivity of rat mesenteric microvessels in vivo. This reduction was abolished upon coperfusion with the PKA inhibitor H89 dihydrochloride. These results indicate that Dll4 signaling through Notch activation acts through a cAMP/PKA pathway upon intercellular adherens junctions, but not tight junctions, to regulate endothelial barrier function. Notch signaling reduces vascular permeability through stimulation of cAMP-dependent protein kinase A.
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http://dx.doi.org/10.1152/ajpheart.00610.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580391PMC
May 2019

Impaired vasocontractile responses to adenosine in chorionic vessels of human term placenta from pregnant women with pre-existing and gestational diabetes.

Diab Vasc Dis Res 2018 11 22;15(6):528-540. Epub 2018 Aug 22.

1 School of Life Sciences, University of Nottingham, Medical School, Queen's Medical Centre, Nottingham, UK.

Background: There is clinical and experimental evidence for altered adenosine signalling in the fetoplacental circulation in pregnancies complicated by diabetes, leading to adenosine accumulation in the placenta. However, the consequence for fetoplacental vasocontractility is unclear. This study examined contractility to adenosine of chorionic vessels from type 1 diabetes mellitus, gestational diabetes mellitus and normal pregnancies.

Methods: Chorionic arteries and veins were isolated from human placenta from normal, gestational diabetes mellitus and type 1 diabetes mellitus pregnancies. Isometric tension recording measured responses to adenosine and the thromboxane A analogue U46619 (thromboxane A mediates fetoplacental vasoconstriction to adenosine). Adenosine and thromboxane prostanoid receptor protein expression was determined by immunoblotting.

Results: Adenosine elicited contractions in chorionic arteries and veins which were impaired in both gestational diabetes mellitus and type 1 diabetes mellitus. Contractions to potassium chloride were unchanged. Adenosine A and A receptor protein levels were not different in gestational diabetes mellitus and normal pregnancies. Contractions to U46619 were unaltered in gestational diabetes mellitus arteries and increased in type 1 diabetes mellitus arteries. Overnight storage of vessels restored contractility to adenosine in gestational diabetes mellitus arteries and normalized contraction to U46619 in type 1 diabetes mellitus arteries.

Conclusion: These data are consistent with the concept of aberrant adenosine signalling in diabetes; they show for the first time that this involves impaired adenosine contractility of the fetoplacental vasculature.
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http://dx.doi.org/10.1177/1479164118790904DOI Listing
November 2018

IFPA meeting 2017 workshop report: Clinical placentology, 3D structure-based modeling of placental function, placental bed, and treating placental dysfunction.

Placenta 2018 04 15;64 Suppl 1:S4-S8. Epub 2017 Dec 15.

Center for Reproductive Health, MetroHealth Medical Center, Case Western Reserve University, Cleveland, OH, USA. Electronic address:

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2017 there were four themed workshops, all of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) placental bed; 2) 3D structural modeling; 3) clinical placentology; 4) treatment of placental dysfunction.
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http://dx.doi.org/10.1016/j.placenta.2017.12.011DOI Listing
April 2018

Regulation of human feto-placental endothelial barrier integrity by vascular endothelial growth factors: competitive interplay between VEGF-Aa, VEGF-Ab, PIGF and VE-cadherin.

Clin Sci (Lond) 2017 Dec 23;131(23):2763-2775. Epub 2017 Nov 23.

School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG2 7UH, U.K.

The human placenta nourishes and protects the developing foetus whilst influencing maternal physiology for fetal advantage. It expresses several members of the vascular endothelial growth factor (VEGF) family including the pro-angiogenic/pro-permeability VEGF-Aa isoform, the anti-angiogenic VEGF-Ab, placental growth factor (PIGF) and their receptors, VEGFR1 and VEGFR2. Alterations in the ratio of these factors during gestation and in complicated pregnancies have been reported; however, the impact of this on feto-placental endothelial barrier integrity is unknown. The present study investigated the interplay of these factors on junctional occupancy of VE-cadherin and macromolecular leakage in human endothelial monolayers and the perfused placental microvascular bed. Whilst VEGF-Aa (50 ng/ml) increased endothelial monolayer albumin permeability (<0.0001), equimolar concentrations of VEGF-Ab (>0.05) or PlGF (>0.05) did not. Moreover, VEGF-Ab (100 ng/ml; <0.001) but not PlGF (100 ng/ml; >0.05) inhibited VEGF-Aa-induced permeability when added singly. PlGF abolished the VEGF-Ab-induced reduction in VEGF-Aa-mediated permeability (>0.05); PlGF was found to compete with VEGF-Ab for binding to Flt-1 at equimolar affinity. Junctional occupancy of VE-cadherin matched alterations in permeability. In the perfused microvascular bed, VEGF-Ab did not induce microvascular leakage but inhibited and reversed VEGF-Aa-induced loss of junctional VE-cadherin and tracer leakage. These results indicate that the anti-angiogenic VEGF-Ab isoform does not increase permeability in human placental microvessels or HUVEC primary cells and can interrupt VEGF-Aa-induced permeability. Moreover, the interplay of these isoforms with PIGF (and s-flt1) suggests that the ratio of these three factors may be important in determining the placental and endothelial barrier in normal and complicated pregnancies.
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http://dx.doi.org/10.1042/CS20171252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869853PMC
December 2017

Transendothelial migration of human umbilical mesenchymal stem cells across uterine endothelial monolayers: Junctional dynamics and putative mechanisms.

Placenta 2016 12 21;48:87-98. Epub 2016 Oct 21.

Molecular Cell Biology & Development Group, School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, Nottingham, UK. Electronic address:

Introduction: During pregnancy, fetal stem cells can transfer to the maternal circulation and participate in tissue repair. How they transmigrate across maternal endothelial barriers and whether they can subsequently influence maternal endothelial integrity is not known.

Methods: Mesenchymal stem cells (WJ-MSC) were isolated from Wharton's jelly and their interactions with human uterine microvascular endothelial cell (HUtMEC) monolayers, junctional occupancy and expression/phosphorylation of vascular endothelial (VE)- cadherin and vascular endothelial growth factor (VEGF-A) secretion was studied over 48h by real time, confocal microscopy, immunoblotting and ELISA.

Results: WJ-MSC displayed exploratory behaviour with interrogation of paracellular openings and spreading into the resultant increased gaps followed by closing of the endothelium over the WJ-MSC. 62% of added cells crossed within 22h to sub-endothelial niches. There was a concomitant loss of junctional VE-cadherin in HUtMEC followed by a full return and increased VE-cadherin expression after 22h. During early hours, VE-cadherin showed a transient phosphorylation at Tyrosine (Tyr)-685 when VEGF-A secretion were high. From 16 to 22h, there was increased de-phosphorylation of Tyr-731. Anti-VEGF-A blocked Tyr-685 phosphorylation but not the decrease in P-Tyr731; this partially inhibited WJ-MSC transmigration.

Discussion: Fetal WJ-MSC can traverse uterine endothelial monolayers by mediating a non-destructive paracellular pathway. They can promote junctional stability of uterine endothelium from the sub-endothelial niche. Mechanistically, WJ-MSC induces VEGF-dependent phosphorylation events linked with paracellular permeability and VEGF-independent de-phosphorylation events associated with leukocyte extravasation. Our data also allows consideration of a possible role of fetal MSC in mature functioning of the uterine vasculature needed for optimal utero-placental perfusion.
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http://dx.doi.org/10.1016/j.placenta.2016.10.014DOI Listing
December 2016

An international network (PlaNet) to evaluate a human placental testing platform for chemicals safety testing in pregnancy.

Reprod Toxicol 2016 09 18;64:191-202. Epub 2016 Jun 18.

Molecular Cell Biology & Development, School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, UK. Electronic address:

The human placenta is a critical life-support system that nourishes and protects a rapidly growing fetus; a unique organ, species specific in structure and function. We consider the pressing challenge of providing additional advice on the safety of prescription medicines and environmental exposures in pregnancy and how ex vivo and in vitro human placental models might be advanced to reproducible human placental test systems (HPTSs), refining a weight of evidence to the guidance given around compound risk assessment during pregnancy. The placental pharmacokinetics of xenobiotic transfer, dysregulated placental function in pregnancy-related pathologies and influx/efflux transporter polymorphisms are a few caveats that could be addressed by HPTSs, not the specific focus of current mammalian reproductive toxicology systems. An international consortium, "PlaNet", will bridge academia, industry and regulators to consider screen ability and standardisation issues surrounding these models, with proven reproducibility for introduction into industrial and clinical practice.
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http://dx.doi.org/10.1016/j.reprotox.2016.06.006DOI Listing
September 2016

Temporal studies into attachment, VE-cadherin perturbation, and paracellular migration of human umbilical mesenchymal stem cells across umbilical vein endothelial monolayers.

Stem Cells Dev 2015 Feb 10;24(4):426-36. Epub 2014 Dec 10.

Cardiovascular Research Group, School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham , Nottingham, United Kingdom .

Mesenchymal stem cells from Wharton's jelly of human umbilical cords (WJ-MSC) are a valuable alternate source of stem cells. Their role in situ and whether they can interact and cross intact endothelial monolayers requires elucidation. The aim of this study was to investigate the dynamic interactions between WJ-MSC and human umbilical vein endothelial cells (HUVEC), including attachment, transit times, extravasation pathway, and the effects of WJ-MSC on junctional vascular endothelial (VE)-cadherin. HUVEC were grown to near confluence in endothelial media and to full confluence in mixed media before the addition of PKH26-labelled WJ-MSC. Time lapse fluorescence microscopy showed stem cells undergoing membrane blebbing followed by amoeboid movement on HUVEC monolayers before rounding up and changing shape toward the spindle-shaped morphology during/after transmigration to subendothelial positions. Cells demonstrated a time lag of 60 min before paracellular extravasation, confirmed by confocal microscopy. Forty-six percent of attached cells crossed in the first 2 h. By 16 h, a majority of cells had transmigrated with >96% of cells crossing by 22 h. There were concomitant changes in endothelial junctional VE-cadherin with statistically significant increases in discontinuous staining at 2 h, return to control values at 16 h, even as from 22 h onward HUVEC displayed increased percentage of junctions with continuous staining and upregulation of protein. Our data suggests that WJ-MSC crosses the endothelial barrier through the paracellular pathway and can influence junctional organization of HUVEC with discreet perturbation of VE-cadherin preceding transmigration followed by upregulation once the adluminal side is reached. The latter may reflect a perivascular support function of WJ-MSC in the umbilical cord.
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http://dx.doi.org/10.1089/scd.2014.0207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4313426PMC
February 2015

Transport in the placenta: homogenizing haemodynamics in a disordered medium.

Philos Trans A Math Phys Eng Sci 2011 Nov;369(1954):4162-82

School of Mathematical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

The placenta is an essential component of the life-support system for the developing foetus, enabling nutrients and waste to be exchanged between the foetal and maternal circulations. Maternal blood flows between the densely packed branches of villous trees, within which are foetal vessels. Here, we explore some of the challenges in modelling maternal haemodynamic transport using homogenization approaches. We first show how two measures can be used to estimate the minimum distance over which the distribution of villous branches appears statistically homogeneous. We then analyse a simplified model problem (solute transport by a unidirectional flow past a distribution of point sinks) to assess the accuracy of homogenization approximations as a function of governing parameters (Péclet and Damköhler numbers) and the statistical properties of the sink distribution. The difference between the leading-order homogenization approximation and the exact solute distribution is characterized by large spatial gradients at the scale of individual villi and substantial fluctuations that can be correlated over lengthscales comparable to the whole domain. This study highlights the importance of quantifying errors owing to spatial disorder in multi-scale approximations of physiological systems.
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http://dx.doi.org/10.1098/rsta.2011.0170DOI Listing
November 2011

Consequences of fetal programming for cardiovascular disease in adulthood.

Microcirculation 2011 May;18(4):253-5

This Spotlight Issue of Microcirculation contains six current perspectives on the role of the intrauterine environment, especially maternal nutritional status and maternal diabetes, in influencing fetal growth and cardiovascular health in the offspring in later life. The reviews address issues such as the existence of a commonality of mechanism following both under-nutritional and over-nutritional states in utero; alterations in the placental fetal microcirculation in response to maternal and fetal changes; transmission of metabolic or nutritional perturbations affecting fetal endogenous antioxidant defense pathways; the presence of a disadvantageous microvascular phenotype resulting from perinatal priming; interactions between developmental programming and genetic variation in noncommunicable adult diseases such as hypertension and hypercholesterolemia; and unresolved questions on the independency and causal mechanisms for low birth weight/intrauterine growth restriction and the risk of developing the metabolic syndrome. These timely reviews highlight the accumulating evidence that changes in the intrauterine environment have pronounced effects on vascular function in the offspring whether due to maternal diabetes or altered maternal nutritional status or fetal and perinatal overnutrition.
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http://dx.doi.org/10.1111/j.1549-8719.2011.00097.xDOI Listing
May 2011

Placental vascular dysfunction in diabetic pregnancies: intimations of fetal cardiovascular disease?

Authors:
Lopa Leach

Microcirculation 2011 May;18(4):263-9

Cardiovascular Research Group, School of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Nottingham, UK.

In the human placenta, the angioarchitecture of fetal vessels lying in maternal blood is useful for nutrient uptake, but it makes the synthesis, maturation and functioning of placental vessels vulnerable to any alterations in the fetal and maternal environment. This review discusses how the maternal diabetic milieu, and the resultant fetal hyperglycemia and hyperinsulinemia, may act together to produce an altered placental vascular phenotype, which includes increased angiogenesis, altered junctional maturity, increased vascular endothelial-like growth factor (VEGF), altered VEGF and insulin receptor profiles, and upregulation of genes involved in signal transduction, transcription and mitosis in placental endothelial cells. The placental vascular dysfunction does extend to other fetal vascular beds including endothelial cells from umbilical vessels, where there are reports of elevated basal iNOS activity and altered sensitivity to insulin. There is emerging evidence of epigenetic modulation of fetal endothelial genes in diabetes and long-term vascular consequences of this. Thus, placental vascular dysfunction in diabetes may be contributing to and describing disturbances in the fetal vasculature, which may produce an overt pathological response in later life if challenged with additional cardiovascular stresses.
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http://dx.doi.org/10.1111/j.1549-8719.2011.00091.xDOI Listing
May 2011

Isolation and properties of an in vitro human outer blood-retinal barrier model.

Methods Mol Biol 2011 ;686:401-16

School of Biomedical Sciences, University of Nottingham, Nottingham, UK.

The outer blood-retinal barrier is composed of a monolayer of retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris, which is fenestrated. An in vitro model that includes all these layers within a 3-D architecture confers a clear advantage over traditional monolayer cultures. Cells here, whether endothelial or epithelial, reside in conditions resembling that in vivo and can participate in cell-cell and cell-matrix cross talk. This chapter describes how a human trilayer culture model was generated with RPE (ARPE-19) cells cultured on the epithelial surface of amniotic membrane and with human umbilical vein derived endothelial cells (HUVEC) on the interstitial surface. This model resembles the outer retinal barrier both in restricting transport of small molecules (<4 kDa), possession of occludin-rich tight junctions in the RPE and fenestrated endothelial cells. Techniques used to test the generated trilayer properties are also described and these include imaging of structure and molecular occupancy of tight and adherens junctions, estimation of the barrier efficiency of trilayer by measurement of fluorescein and fluorescein-conjugated tracers under flow, measurement of secreted vascular endothelial growth factor-A and ultrastructural studies, which allow analyses of the fine structure of the tight junctions in the RPE, and the endothelial fenestra.
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http://dx.doi.org/10.1007/978-1-60761-938-3_20DOI Listing
June 2011

Hookworm (Necator americanus) larval enzymes disrupt human vascular endothelium.

Am J Trop Med Hyg 2010 Sep;83(3):549-58

Division of Molecular and Cellular Science, Immune Modulation, Boots Science Building, School of Pharmacy, University of Nottingham, Nottingham, UK.

Knowledge of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would aid the development of effective vaccines against this important pathogen. In this work, the impact of N. americanus exsheathing fluid (EF) and excretory/secretory products (ES) on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Cellular responses were assessed by investigating molecular changes at cell-cell junctions and by determining levels of secreted IL-6, IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. It would appear that a repertoire of larval proteases caused a dose-related increase in endothelial permeability as characterized by a decrease in monolayer resistance with increased permeation of tracer-albumin. These barrier changes were associated with disruption of junctional vascular endothelial cadherin (VE-cadherin) and F-actin and an increase in endothelial secretion of IL-6 and IL-8. Our data suggest that larval proteases play an important role in negotiating the endothelium.
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http://dx.doi.org/10.4269/ajtmh.2010.09-0411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929050PMC
September 2010

Vascular dysfunction in the diabetic placenta: causes and consequences.

J Anat 2009 Jul;215(1):69-76

School of Biomedical Sciences, Centre for Integrated Systems Biology and Medicine, Institute of Clinical Research, Faculty of Medicine and Health Sciences, University of Nottingham, UK.

The development and functioning of the human fetoplacental vascular system are vulnerable to the maternal diabetic milieu. These vessels are in direct continuum with the fetal vascular system and are therefore also vulnerable to fetal endocrine derangements. Increased angiogenesis, altered junctional maturity and molecular occupancy, together with increased leakiness, constitute a well-described phenotype of vessels in the Type 1 diabetic human placenta and can be related to increased levels of placental vascular endothelial growth factor. The causes of these observed changes, whether maternal hyperglycaemia or fetal hyperinsulinaemia, still remain to be shown in the human placenta. Mechanistic studies using different vascular systems have shown high glucose and insulin to have profound vascular effects, with elevations in vascular endothelial growth factor, nitric oxide and protein kinase C being behind alterations in junctional adhesion molecules such as occludin and vascular endothelial-cadherin and vascular leakage of albumin. The role of advanced glycation products and oxidative stress in this vascular pathology is also discussed. The altered molecular mechanisms underlying the vascular changes in the diabetic human placenta may reflect similar consequences of high glucose and hyperinsulinaemia.
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http://dx.doi.org/10.1111/j.1469-7580.2009.01098.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714640PMC
July 2009

Cell adhesion molecules in plants and animals.

Biotechnol Genet Eng Rev 2004 ;21:123-32

Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.

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http://dx.doi.org/10.1080/02648725.2004.10648051DOI Listing
October 2006

Behaviour of human endothelial cells on surface modified NiTi alloy.

Biomaterials 2005 Sep;26(26):5359-67

Christchurch School of Medicine, Lipid and Diabetes Research Group, Canterbury District Health Board, 1st Floor Hagley Hostel, Christchurch Hospital, New Zealand.

Intravascular stents are being designed which utilise the shape memory properties of NiTi alloy. Despite the clinical advantages afforded by these stents their application has been limited by concerns about the large nickel ion content of the alloy. In this study, the surface chemistry of NiTi alloy was modified by mechanical polishing and oxidising heat treatments and subsequently characterised using X-ray photon spectroscopy (XPS). The effect of these surfaces on monolayer formation and barrier integrity of human umbilical vein endothelial cells (HUVEC) was then assessed by confocal imaging of the adherens junctional molecule VE-cadherin, perijunctional actin and permeability to 42kDa dextrans. Dichlorofluoroscein assays were used to measure oxidative stress in the cells. XPS analysis of NiTi revealed its surface to be dominated by TiO(2). However, where oxidation had occurred after mechanical polishing or post polishing heat treatments at 300 and 400 degrees C in air, a significant amount of metallic nickel or nickel oxide species (10.5 and 18.5 at%) remained on the surface. Exposure of HUVECs to these surfaces resulted in increased oxidative stress within the cells, loss of VE-cadherin and F-actin and significantly increased paracellular permeability. These pathological phenomena were not found in cells grown on NiTi which had undergone heat treatment at 600 degrees C. At this temperature thickening of the TiO(2) layer had occurred due to diffusion of titanium ions from the bulk of the alloy, displacing nickel ions to sub-surface areas. This resulted in a significant reduction in nickel ions detectable on the sample surface (4.8 at%). This study proposes that the integrity of human endothelial monolayers on NiTi is dependent upon the surface chemistry of the alloy and that this can be manipulated, using simple oxidising heat treatments.
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http://dx.doi.org/10.1016/j.biomaterials.2005.01.067DOI Listing
September 2005

Reversible disruption of tight junction complexes in the rat blood-brain barrier, following transitory focal astrocyte loss.

Glia 2004 Oct;48(1):1-13

MRC Applied Neuroscience Group, School of Biomedical Sciences, Queen's Medical Centre, University of Nottingham, NG7 2UH, Nottingham, United Kingdom.

Breakdown of the blood-brain barrier is a feature of acute and chronic neurodegenerative changes, yet the relationship between astrocytes and the mature barrier remains unclear. We studied this role of astrocytes in vivo using a gliotoxin and evaluated changes in three vascular tight junction markers. Male Fisher F344 rats given systemic 3-chloropropanediol showed astrocytic loss in the inferior colliculus from 12-24 h until the lesion was repopulated 8-28 days later. Within 6 h of astrocyte loss, microvessels in this area began to demonstrate a loss of the normal paracellular localization of the transmembrane proteins occludin and claudin-5 and cytoplasmic zonula occludens-1, which correlated with focal vascular leak of dextran (10 kDa) and fibrinogen. Platelet endothelial adhesion molecule-1 staining revealed that there was no loss of the endothelial lining. Between 4-8 days, severe downregulation of tight junction protein expression was observed, which subsequently returned over the same time period as astrocytes repopulated the lesion. Unexpectedly, dextran and fibrinogen leak from vessels had ceased at 6 days, well before the return of occludin and claudin-5 to appropriate paracellular domains. Control nonvulnerable cortical tissue showed no change in astrocyte morphology and tight junction expression over the same time course. Our data supports a primary role for astrocytic contact in the expression of occludin, claudin-5, and zonula occludens-1 in the mature brain vasculature in vivo. However, barrier integrity to dextran (10 kDa) and fibrinogen can be restored in the absence of astrocytes and tight junction proteins (occludin, claudin-5, and zonula occludens-1).
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http://dx.doi.org/10.1002/glia.20049DOI Listing
October 2004

The phenotype of the human materno-fetal endothelial barrier: molecular occupancy of paracellular junctions dictate permeability and angiogenic plasticity.

Authors:
Lopa Leach

J Anat 2002 Jun;200(6):599-606

School of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, UK.

In vitro models predict that molecular occupancy of endothelial junctions may regulate both barrier function and angiogenesis. Whether this is true in human vascular beds undergoing physiological angiogenesis has not been shown. This review presents data which demonstrate there are two distinct junctional phenotypes, 'activated' and 'stable', present in the vascular tree of the human placenta taken from two distinct highly angiogenic gestational periods (first and last trimester). Stability is conferred by the presence of occludin in tight junctions and plakoglobin in adherens junctions. Their localization may be influenced by vascular endothelial growth factor and angiopoietins 1 and 2 that have a similar temporal and site-specific differential expression. The junctional phenotypes are reversible, as shown in studies with endothelial cells isolated from placental microvessels and grown in the presence/absence of cAMP-enhancing agents. Reductions in protein levels and loss of junctional localization of adhesion molecules result in increased permeability to macromolecules, whilst up-regulation and re-targeting of these molecules inhibit cell proliferation and increase transendothelial resistance. These studies suggest junctional adhesion molecules can regulate physiological angiogenesis and vascular re-modelling. Moreover, the activated junctional phenotype of placental microvessels allows them to participate in increased growth and proliferation. This junctional immaturity appears to be at the expense of barrier function resulting in sites of maximal materno-fetal solute exchange.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1570749PMC
http://dx.doi.org/10.1046/j.1469-7580.2002.00062.xDOI Listing
June 2002

Vasculogenesis, angiogenesis and the molecular organisation of endothelial junctions in the early human placenta.

J Vasc Res 2002 May-Jun;39(3):246-59

School of Biomedical Sciences, Faculty of Medicine and Health Sciences, University of Nottingham, Queens Medical Centre, UK.

Vasculogenesis and angiogenesis are regulated by the capacity of endothelial cells to adhere to each other and form new tubes. The presence and role of junctional adhesion molecules during physiological vasculogenesis is unknown. Using ultrastructural and immunocytochemical approaches, we compared the junctional phenotype of developing vessels of the first-trimester human placenta with vessels in the last trimester; the latter include newly formed terminal capillaries and the quiescent vascular bed. First-trimester placental vessels contained the adherens junctional molecules, vascular endothelial cadherin and alpha- and beta-catenin but lacked plakoglobin, the component of fully differentiated adherens junctions. Furthermore, these vessels did not contain the transmembrane tight junctional molecules occludin and claudin-1 and -2. This profile reflects the phenotype of terminal capillaries but differs from large vessels of the full-term placenta. Electron microscopic studies revealed that endothelial tight junctions are present in the first-trimester placenta. Thus, occludin and claudin-1 appear to play no part in the formation of endothelial tight junctions, but are a later requirement. In the early placenta, the predominant growth factor appears to be vascular endothelial growth factor (VEGF), whilst at term, angiopoietin-1 was present in large vessels, with intense angiopoietin-2 immunofluorescence (and VEGF) located in terminal villous capillaries. Thus, endothelial junctions in the human placenta possess two distinct molecular phenotypes, i.e. stable or dynamic, dependent on maturity and plasticity. These distinct phenotypes may be influenced by the angiopoietins/VEGF present in the placenta.
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http://dx.doi.org/10.1159/000063690DOI Listing
August 2002