Lopa Leach

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

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).

Vasculogenesis, Angiogenesis and the Molecular Organisation of Endothelial Junctions in the Early Human Placenta

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 α- and β-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.

Fine structure of the paracellular junctions of terminal villous capillaries in the perfused human placeta

SummarySelected lobules of human term placentae were extracorporeally perfused for a recovery period of 20 min, fixed by perfusion and mordanted with ferrocyanide prior to processing for transmission electron microscopy. The lateral membranes of the endothelial cells of the terminal villous capillaries were found to be separated by paracellular clefts of mean width 15.6 nm. At tight junctional regions (1–4 sites per cleft) the two membranes approached each other more closely and frequently appeared to fuse. However, tilting of the sections in the electron microscope stage showed that the membranes were separated by a gap of mean width 4.1 nm in at least 94% of tight junctional profiles. When individual tight junctions were studied by a combination of serial sectioning and goniometric tilting, they were seen to widen abruptly within a distance of three to seven consecutive thin sections, indicating they were not continuous throughout the axial length of the capillaries. The wide regions of the clefts usually showed linkers, strands of glycocalyx-like material spanning the gap. Linkers may contribute to cell adhesion and possibly form part of a filter within the tortuous paracellular pathway provided by the discontinuous network of tight junctional strands. Human term placental capillaries appear to resemble closely other continuous non-brain capillaries.

Distinct Patterns of Microvascular Endothelial Cell Morphology Are Determined by Extracellular Matrix Composition

Endothelial interactions with the extracellular matrix (ECM) play important roles in angiogenesis but whether specific ECM signals can determine specific cellular morphologies is unclear. The authors compared in vitro ECM-induced morphological responses of the phenotypically distinct human placental microvascular endothelial cells (HPMECs) with large vessel endothelial cells (HUVECs). HPMECs showed distinct patterns of reorganization in response to collagen-I or collagen-IV (monolayer disruption, sprouting, migration) and Matrigel or laminin-A (intussusception, cord formation, tubulogenesis), and an intermediate response to fibrin; whereas HUVECs responded similarly to collagen-1 and Matrigel (elongation, lattice formation, vacuolation) and showed little response to fibrin. Although the extent of collagen and Matrigel responses of HPMECs were increased by serum, acidic or basic fibroblast growth factor (aFGF, bFGF), or vascular endothelial growth factor (VEGF), and varied with matrix protein concentration, the basic patterns were matrix specific, and were independent of fibronectin. The collagen responses correlated with disruption of adherens and tight junctions and the formation of filopodial protrusions. Matrigel responses were associated with up-regulated junctional localization of VE-cadherin, and tubulogenesis developed mainly through paracellular remodeling rather than intracellular vacuolation. Overall, these findings suggest that distinct ECM interactions stimulate specific morphological responses. These signals may regulate morphological behaviour in the angiogenesis cycle, switching endothelial cells between migratory and vasculogenic phenotypes.

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

Please cite this paper as: Leach (2011). Placental Vascular Dysfunction in Diabetic Pregnancies: Intimations of Fetal Cardiovascular Disease? Microcirculation 18(4), 263–269.

Not trophoblast alone: A review of the contribution of the fetal microvasculature to transplacental exchange

The fetal microcirculation of the term human placenta offers an interesting microvascular model. A perfused placenta can be used for integrated studies of vascular permeability-structure relationships. The organization of the paracellular pathway in human placental microvessels closely resembles not only that of the guinea-pig placenta, but also that seen in typical continuous non-cerebral capillaries such as those of the myocardium. This uniformity of organization has allowed the development of a model of the organization of endothelial junctional complexes that allows testable predictions about the relationship between junctional organization and microvascular permeability. The key features of this model are: (1) molecular size restriction may be determined by a fibre matrix based on cadherin arrays in the zonula adhaerens. (2) The zonula occludens (tight junction) is discontinuous and so cannot act as a molecular sieve for solutes. It may serve as a shutter that limits the proportion of the paracellular cleft available for permeation. The main implication for placental function is that the human placental microcirculation is relatively tight and is an important restriction to diffusive permeation of the maternal-fetal barrier by large molecules.

Establishment of a human in vitro model of the outer blood-retinal barrier

The outer blood–retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruchs membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruchs membrane leads to the neovascular form of age‐related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE‐19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein‐derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE‐19. Both human umbilical vein‐derived endothelial and ARPE‐19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein‐derived endothelial cells in the trilayer became fenestrated when co‐cultured with the ARPE‐19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE‐19 cells. These cells also revealed VE‐cadherin and ZO‐1 at cell–cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO‐1, were localized to cell–cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein‐conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten‐fold reduction) whilst transfer of a 4 kDa FITC‐conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional‐like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE‐19 and human umbilical vein‐derived endothelial cells can be co‐cultured on the amniotic membrane and that the resultant cross‐talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer‐retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.

Effects of gestational diabetes on junctional adhesion molecules in human term placental vasculature.

Aims/hypothesis. The aim of this study was to investigate whether gestational diabetes mellitus, which occurs in the microvascular remodelling phase of placental development, causes alterations in surface expression of tight and adherens junctional molecules involved in endothelial barrier function and angiogenesis. Methods. Term placenta, delivered by elective Caesarian section, from normal pregnancy (n = 5) and those complicated by gestational diabetes (n = 5) were perfusion-fixed and analysed by indirect immunofluorescence and confocal scanning microscopy. Using systematic random sampling, the surface expression of endothelial junctional proteins and the relative incidences of immunostained vessels were compared between the two study groups. Total vessel lengths were measured by stereological techniques. Results. The adherens junctional molecules, vascular-endothelial cadherin and β-catenin, and the tight junctional molecules, occludin and zonula occludens-1 were localised to paracellular clefts in both study groups. The diabetic placentae showed pronounced reductions in the intensity of immunofluorescence and in the number of immuno-positive vessels. A corresponding statistically significant increase (from 19 % to 56 %) in the percentage of vessels showing junctional anti-phosphotyrosine immunoreactivity was found. The differences observed represented real changes in the absolute lengths of immunostained regions along the vessels. The stereological measurements failed to detect any statistically significant change in the combined length of fetal vessels in gestational diabetic placenta. Conclusion/interpretation. Our results suggest that even short duration diabetic insult, alters the surface expression of placental junctional proteins. This alteration could be mediated by the tyrosine-phosphorylation pathway. The changes suggest impaired barrier function rather than accelerated vascular growth. [Diabetologia (2000) 43: 1185–1196]

A Mathematical Model of Intervillous Blood Flow in the Human Placentone

We present a mathematical model for maternal blood flow in a placental circulatory unit (a placentone), describing flow of maternal blood via Darcys law and steady advective transport of a dissolved nutrient. The method of images and computational integration along streamlines are employed to find flow and solute concentration distributions, which are illustrated for a range of governing system parameters. The model shows how the calibre of the basal vessels can be a dominant determinant of the maternal blood flow rate through the placentone, given a driving pressure difference between the spiral arteries and decidual veins. The model supports the hypothesis that basal veins are located on the periphery of the placentone in order to optimise delivery of nutrients and suggests the existence of an optimal volume fraction of villous tissue.

Immunocytochemical localization of neurosecretory amines and peptides in the free-living nematode,Goodeyus ulmi

SummaryMammalian antibodies to the neuroamines, serotonin and γ-amino-butyric acid (GABA) and to the neuropeptides, adrenocorticotrophic hormone (ACTH) and FMRF-amide evoked a response toGoodeyus ulmi, a free-living nematode. Serotonin-like immunoreactivity was found in cell bodies in the nerve ring and in the ventral nerve cord in all developmental stages. Neurons in the vulva, implicated in egg-laying, were immunoreactive to anti-serotonin inG. ulmi females, while in males serotonergic nerve fibres was found in the spicular region. Immunoreactivity to ACTH was also seen to differ depending on the developmental stage ofG. ulmi, being present only in the ventral cord from the late L3 stage. Anti-GABA immunoreactivity was localized in two cell bodies near the amphids in all life stages and FMRF-amide immunoreactivity was seen in the nerve ring in all developmental stages. No reactivity was found with antibodies to vasointestinal peptide and somatostatin-14.