Biomedical subjects

J A Firth

Publications and source records attributed to J A Firth.

At least 19 recordsLinked to original sources

PubMed2003

Characterization of cationic amino acid transporters and expression of endothelial nitric oxide synthase in human placental microvascular endothelial cells.

We investigated the expression and activity of arginine transporters and endothelial nitric oxide synthase (eNOS) in human placental microvascular endothelial cells (HPMEC). Using RT-PCR amplification products for eNOS, CAT1, CAT2A, CAT2B, CAT4, 4F2hc (CD98), rBAT and the light chains y+LAT1, y+LAT2, and b0+T1 were detected in HPMEC, but not B0+. Immunohistochemistry and Western blotting confirmed the presence of 4F2hc and CAT1 protein in HPMEC. 4F2hc-light chain dimers were indicated by a shift in molecular mass detected under nonreducing conditions. L-Arginine transport into HPMEC was independent of Na+ or Cl- and was inhibited by the neutral amino acid glutamine, but not by cystine. The Ki for glutamine inhibition was greater in the absence of Na+. Kinetic analysis supported a two-transporter model attributed to system y+L and system y+. Expression of eNOS in HPMEC was detectable by immunohistochemistry and ELISA but not by Western blotting. Activity of eNOS in HPMEC, measured over 48 h, either as the basal production of nitric oxide (NO) or as the accumulation of intracellular cGMP was not detectable. We conclude that HPMEC transport cationic amino acids by systems y+ and y+L and that basal eNOS expression and activity in these cells is low.

Amino Acid Transport Systems, Basic↗

PubMed2002

Endothelial barriers: from hypothetical pores to membrane proteins.

The anatomical counterpart of the physiologically defined small pore system of capillary endothelia has proved difficult to establish. In non-brain continuous capillaries, the contributions of caveolar and transmembrane pathways are likely to be small and paracellular clefts are probably the dominant routes. Analogy with epithelial paracellular pathways suggests that tight junctions may be the most restrictive elements. However, structural features of tight junction-based models are incompatible with physiological data; it is more likely that the tight junction acts as a shutter limiting the available cleft area. Proposed molecular sieves elsewhere in the paracellular pathway include the glycocalyx and the cadherin-based complexes of the adherens junctions. The molecular architecture of tight junctions and adherens junctions is moderately well defined in terms of molecular species, and there are differences at both sites between the endothelial and epithelial spectra of protein expression. However, definition of the size-restricting pore remains elusive and may require structural biology approaches to the spatial arrangements and interactions of the membrane molecular complexes surrounding the endothelial paracellular clefts.

Adherens Junctions↗

PubMed2001

Cyclic AMP and acidic fibroblast growth factor have opposing effects on tight and adherens junctions in microvascular endothelial cells in vitro.

Endothelial adherens junctions (AJ) and tight junctions (TJ) are important determinants of vascular permeability and cell morphology. Here, we investigate their regulation, in primary human placental microvascular endothelial cell (HPMEC) cultures, by either aFGF plus heparin (ECGS) or elevated cAMP. The proliferation of HPMEC was weakly stimulated by ECGS, while cAMP was inhibitory. ECGS had little effect on transendothelial resistance (TER), but increased macromolecular permeability, whereas cAMP induced a twofold increase in TER and reduced macromolecular permeability. Ultrastructurally, ECGS-treated HPMEC exhibited an "activated" phenotype typified by proliferating cells, with poorly organized cell-cell junctions, whereas cAMP-treated cells appeared quiescent and markedly flattened with extended paracellular junctions, resembling endothelium in situ. The expression and localization of junctional molecules, F-actin, and junctional phosphotyrosine were examined by confocal microscopy and immunoblotting. Junctional molecules in ECGS-treated cells were less organized at lateral membranes than in control cells, whereas in cAMP-treated cells, they were highly localized at continuous contacts. These differences correlated with the intensity of junctional phosphotyrosine, being lowest with cAMP treatment. In the AJ of ECGS-treated and control cells, beta-catenin predominated but in cAMP-treated cells, gamma-catenin/plakoglobin was enriched. In addition, cAMP upregulated junctional expression of VE-cadherin and PECAM-1 and increased the levels of the TJ molecules occludin and ZO-1. The expression levels of junctional components, and their tyrosine phosphorylation, play an important role in dynamic regulation of endothelial cell-cell junctions.

Actins↗

PubMed2001

Phenotype of the endothelium in the human term placenta.

The placental endothelium contributes to regulating transplacental exchange and maintaining the immunological maternofetal barrier. We characterized the endothelial phenotype in human normal term placentae with a panel of antibodies to endothelial antigens using a standardized immunofluorescence method. Placental endothelium strongly expressed vWF, PAL-E, H-antigen, thrombomodulin, PECAM-1, CD34, CD36, ICAM-1, CD44, thy-1, A10/33-1, VE-cadherin, caveolin-1 and HLA-G, whereas occludin, claudin-1, eNOS, angiotensin converting enzyme (ACE), ICAM-2, endoglin and integrin-alphathetabeta(3)were weakly expressed. PGI(2)synthase, tissue factor, E-selectin and VCAM-1 were not detected. Some antigens were heterogenously expressed along the vascular tree or within individual villi. Expression of ACE, eNOS, vWF, P-selectin, E-selectin, integrin alpha(v)beta(3)and endoglin was stronger in the maternal decidual vessels, while PECAM-1, CD44, thy-1 and caveolin-1 expression was stronger in fetal vessels. Some endothelial markers were present in trophoblasts and stroma. Endothelial proliferation was apparent in mature intermediate and terminal villi. There was limited inflammatory response to TNFalpha in explants, characterized by upregulation of vWF, P-selectin, PECAM-1 and CD44, downregulation of thrombomodulin, but no increase in ICAM-1 expression, nor induction of E-selectin, VCAM-1 or tissue factor. These patterns of heterogeneity, proliferative activity and inflammatory activation may underlie the specific physiological roles of the placental endothelium.

Adult↗

PubMed2000

Endothelial glycoconjugates: a comparative lectin study of the brain, retina and myocardium.

There is evidence that the endothelial cell (EC) glycocalyx is a significant determinant of vascular permeability, acting as a charge-size filter to permeant molecules. We have therefore examined its oligosaccharide composition in 3 classes of microvessel with differing permeabilities. EC in rat brain, retina and myocardium were labelled with a panel of lectins and subjected to a semiquantitative analysis. Surprisingly, no substantial differences were evident for any lectin labelling between the 3 microvessel types despite their marked morphophysiological diversity. In particular, all showed substantial sialic acid expression, with Maackia amurensis (MAA) labelling sialic acid in an alpha2-3 linkage to beta-galactose and Sambucus nigra (SNA) recognising sialic acid in an alpha2-6 linkage to beta-galactose. Arachis hypogaea (PNA) binding after neuraminidase digestion indicated the presence of Gal beta1-3GalNAc attached to terminal sialic acid. The results therefore show that the sequences NeuNAc alpha2-3Gal beta1-3GalNAc and NeuNAc alpha2-6Gal beta1-3GalNAc are strongly expressed in the 3 microvessel types irrespective of their permeability properties. This homogeneity suggests that these lectin ligands may be involved in a common set of EC functions, e.g. cell:cell and cell:matrix interactions. However, we cannot rule out the possibility that glycocalyx differences may exist between vessels in the paracellular cleft which may alter its filtration properties.

Animals↗

PubMed1997

Differential distribution of an endothelial barrier antigen between the pial and cortical microvessels of the rat.

An endothelial barrier antigen (EBA), reported to be a marker for endothelial cells (EC) displaying blood-brain barrier (BBB) characteristics, was probed with a monoclonal antibody in pial and cortical microvessels in rat brain. In contrast to the uniform labelling of EC in cortical vessels, pial microvessels showed a heterogeneity in EBA expression. Most pial vessels consisted of a mixture of EBA positive and EBA negative cells whereas a smaller number of vessels were either completely negative or uniformly positive. Significantly, in vessels showing incomplete expression it was typically EC furthest from the brain surface that did not express EBA. Although the function of EBA is unknown, the variable expression in pial microvascular EC may be related to their incomplete barrier characteristics.

Animals↗

PubMed1997

Development of endothelial paracellular clefts and their tight junctions in the pial microvessels of the rat.

The microvessels of the pia mater lack an investment with astrocyte processes but nonetheless have a high transendothelial electrical resistance which has caused them to be regarded as part of the blood-brain barrier. This high resistance is known to be acquired in the perinatal period. The aim of our study was to relate the known physiological changes with differentiation of the endothelial paracellular clefts and especially of their tight junctions which provide the basis for the high transendothelial resistance of blood-brain barrier vessels. Tight junctions of endothelial cell paracellular clefts in pial microvessels were examined by transmission electron microscopy using goniometric tilting to reveal and measure membrane separations at tight junctions in fetal, postnatal and adult rats. These tight junctional membrane separations narrowed over the period (E16: 6.3 nm, D1: 6.4 nm, D7: 5.4 nm) and differentiated into two groups by the adult stage: one with a membrane separation of 2.8 nm and the staining characteristics of non-brain endothelial junctions, and the other with no detectable membrane separation and the staining characteristics of blood-brain barrier endothelial junctions. This patchy and incomplete differentiation of pial tight junctions into a blood-brain barrier-like form could result either from non-uniform exposure to inductive signals or to local variation in responsiveness to such agents. Although these changes in junction organization may be related to the known increase in pial transendothelial resistance in the perinatal period, we have not yet identified any sharply defined structural change which coincides with this physiological event.

Animals↗

PubMed1996

Ontogeny of four blood-brain barrier markers: an immunocytochemical comparison of pial and cerebral cortical microvessels.

Pial and cortical microvessels possess many blood-brain barrier (BBB) properties in common, including impermeability to electron dense tracers, high transendothelial electrical resistance and specialised endothelial cell ultrastructural features. To compare pial and cortical microvessels further, a developmental, immunocytochemical study was undertaken of 4 BBB markers in the rat: OX-47, EBA, GLUT-1 and s-laminin. The appearance of the markers was monitored from embryonic d 16, to postnatal and adult stages. Each of the 4 markers appeared simultaneously in both pial and cortical vessels. GLUT-1 and OX-47 were present in endothelial cells of the BBB from E 16 to the adult. EBA and s-laminin appeared from postnatal d 7 through to the adult. Pial microvessels lack the ensheathment of astrocytes which may be involved in the induction and/or maintenance of BBB markers in the cortex. It is possible that astrocyte-derived factors diffusing from the brain surface are responsible for induction of BBB properties in the pial microvessels.

Animals↗

PubMed1995

Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels.

The microvessels of the human placenta resemble those of skeletal muscle, both in endothelial cell junctional organization and in the single passage extraction of radiolabeled tracers. Addition of histamine (100 microM) to the fetal perfusate in an isolated term lobule resulted in a rapid and sustained rise (40-80%) over a 30-min perfusion period in the single circulation extraction values of 57Co-labeled cyanocobalamin and of 51Cr-labeled EDTA, but not of 22Na. Extraction values for 125I-albumin were not increased in histamine-perfused vessels nor was any focal leakage of label observed in serial cryostat sections of lobules perfused with rhodamine-conjugated albumin. There was no electron microscopic evidence of transendothelial channels in the microvascular bed; no gaps were seen at the paracellular cleft regions or in neighboring cytoplasm in any microvessels. Tilting of sections on a goniometric stage showed a significant increase in the separation between adjoining endothelial membrane leaflets at tight junctional regions (from 4.1 to 6.1 nm) although the dimensions of the wide zones remained unchanged. Placental microvessels contain the endothelial adhesion molecules PECAM-1 and VE-cadherin in the wide regions of paracellular clefts: PECAM-1 is also localized on the luminal membrane. Histamine-stimulated microvessels showed an altered staining pattern for both of these molecules, at the expense of the cleft regions. These changes in adhesion molecule distribution and tight junctional membrane separation may be parts of a series of events which leads to increased permeability during inflammatory events.

Antigens, CD↗

PubMed1995

Actin cytoskeletal isoforms in human endothelial cells in vitro: alteration with cell passage.

The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but not beta-actin or alpha-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction of alpha-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behaviour occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that in prolonged passage, both alpha-smooth muscle actin and beta-actin were expressed, whereas HPMEC at P4 showed a lower level of beta-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirements for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons of in vitro and in vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.

Actins↗

PubMed1995

Advances in understanding permeability in fetal capillaries of the human placenta: a review of organization of the endothelial paracellular clefts and their junctional complexes.

A review is presented of the evidence that the capillaries of the fetal-placental circulations of man and the guinea-pig are typical members of class of continuous non-brain capillaries. Their permeability is similar to that of muscle capillaries, and there is much evidence that the main permeation route for hydrophilic solutes in capillaries of this class is through the paracellular clefts of the endothelium. The properties of this paracellular route are based on discontinuous tight junctions which may serve to restrict the fraction of the paracellular cleft available to solutes, and on a molecular sieve which may be based on arrays of adhesion molecules in the zonula adhaerens of the junctional complex. The characteristics of this paracellular route and of overall microvascular permeability in these types of placenta make it clear that fetal capillaries form a significant component of the total transplacental permeability restriction.

Animals↗

PubMed1995

Have declining clinical necropsy rates reduced the contribution of necropsy to medical research?

AIMS: To examine trends in necropsy based research output for a period of 27 years during which there has been a progressive decline in clinical necropsy rates. METHODS: The numbers of necropsy based research papers published between 1966 and 1993 were determined using the CD-Plus Medline computed literature database. RESULTS: The number of necropsy based research papers containing necropsy or a synonym in the title increased by 220% between 1966 and 1993. When papers including necropsy or a synonym in the abstract, but not in the title, were included, the proportion of all indexed papers increased from 0.35% in 1975, when abstracts were first included, to 0.53% in 1993. Analysis of the subject material indicated that necropsy based research has constantly reflected trends and advances in clinical medicine. Neuroscience related research represented the largest subject category which may reflect the difficulties in obtaining human tissue from sources other than necropsy. CONCLUSIONS: The modern necropsy continues to provide valuable information for all clinical and laboratory based disciplines. The decline in clinical necropsy rates would not yet appear to have undermined the contribution of the necropsy to research.

Autopsy↗

PubMed1994

Isolation of endothelial cells from human term placental villi using immunomagnetic beads.

Chorionic villi excised from freshly delivered human term placentae and small endothelial cell aggregates were released from them by the sequential use of collagenase and trypsin. The endothelial cells were further isolated by rosetting with magnetic polystyrene beads which were coated with QB END/40, the endothelial-specific monoclonal antibody (mAb) to thrombomodulin. Cell rosettes were plated on gelatin coated Petri dishes. The cells initially grew as discrete colonies but reached confluence within 7 days. The monolayers were sub-cultured five times, and grew to confluence each time. All the cells were immunoreactive to the endothelial markers von Willebrand factor, QB-End/40 and Ulex europaeus-1 lectin. They did not show immunoreactivity to trophoblast markers (mAbs ED341 and ED235). The isolated cells could also incorporate acetylated low-density lipoprotein. Most of the cells possessed an elongated morphology, though some were slightly spread and polygonal in shape. The cell monolayers did not resemble the typical cobblestone appearance of endothelial cells isolated from large vessels. Ultrastructurally, most of the cells resembled placental microvascular cells in shape and frequency of caveolae; undifferentiated cell-cell contacts and extracellular matrix material was observed. Human placental microvascular endothelial cells may offer an in vitro model which complements the use of the perfused term placental lobule in studies of microvascular permeability.

Endothelium, Vascular↗

PubMed1993

Adenosine triphosphate-lead histochemical reactions in ependymal epithelia of murine brains do not represent calcium transport adenosine triphosphatase.

The strong enzyme histochemical reactions for adenosine triphosphatase (ATPase) seen in ependymal tanycytes after incubation in calcium-containing media have previously been reported as calcium transport ATPase. Investigation of these reactions showed that: (1) any nucleoside triphosphate can serve as a substrate; (2) diphosphates and monophosphates cannot replace triphosphates; this includes p-nitrophenyl phosphate which is readily hydrolysed by plasma membrane transport ATPases; (3) strong localization occurs in the presence of millimolar concentrations of either calcium or magnesium ions; there is no absolute requirement for calcium ions; (4) they are not inhibited by sulphydryl inhibitors or calmodulin antagonists; (5) lead phosphate precipitates are localized almost entirely on the external face of tanycyte plasma membranes. In addition, the technique gives strong localization to vessels in the choroid plexus but not to the choroidal epithelium. Immunohistochemistry with a primary antibody raised against Ca2+, Mg2(+)-ATPase stains the choroidal epithelium but not the vessels or the ependymal tanycytes. These results are inconsistent with identification of the reaction as calcium transport ATPase but support characterization as an ecto-ATPase.

Adenosine Triphosphatases↗

PubMed1993

Junctions between pericytes and the endothelium in rat myocardial capillaries: a morphometric and immunogold study.

Pericytes are cells of mesodermal origin which are closely associated with the microvasculature. Despite numerous studies little is known about their function. We have studied the relationship between pericytes and the endothelium in rat myocardial capillaries employing ultrastructural and immunogold techniques. 14% of the subendothelial cell membrane is covered by comparatively small pericytic cell processes. About half of these processes are completely embedded in basement membrane material, whereas the remaining half forms closer contacts with the endothelium. These contacts are devoid of anti-laminin immunogold label, a marker for basement membranes. A small fraction of these contacts has been identified as tight junctions resembling those seen between endothelial cells in capillaries of the same tissue. The remaining majority of junctions reveals a cleft of approximately 18 nm between the apposed membranes in which a succession of cleft-spanning structures can often be detected. It was also found that pericytic processes are preferentially located close to interendothelial junctions. We suggest that the high frequency of intimate junctions between pericytes and the endothelium and the preferential localisation near paracellular clefts may have functional significance.

Animals↗

PubMed1993

Permeability of the fetal villous microvasculature in the isolated perfused term human placenta.

1. Capillary permeability-surface area (PS) products for the low molecular weight radioactive tracers, 22Na, 51Cr-EDTA (relative molecular mass 357) and 57Co-cyanocobalamin (relative molecular mass 1353) were measured in the fetal circulation of isolated dually perfused lobules of normal term human placentae using the single circulation, multiple-tracer dilution technique. 2. In lobules perfused with M199 medium, containing dextran and 5 g l-1 bovine albumin, the extractions of all three tracers decreased as the flow was increased over the range of 2-8 ml min-1, and PS products for 51Cr-EDTA and 57Co-cyanocobalamin, but not for 22Na, reached constant values at flows above 0.1 ml min-1 g-1. 3. Flow-independent PS products in the presence of albumin were 0.025 +/- 0.002 ml min-1 g-1 (mean +/- S.E.M., n = 25) for 57Co-cyanocobalamin and 0.057 +/- 0.003 ml min-1 g-1 (n = 25) for 51Cr-EDTA. The ratio of PS values (51Cr-EDTA/57Co-cyanocobalamin) was 2.28, while the ratio of the corresponding free diffusion coefficients was 1.79, indicating substantial restriction to the diffusion of the 57Co-cyanocobalamin. 4. In another series of lobules perfused in the absence of albumin, extraction values for all three test tracers were constant over the same flow range. Values at high flow rates were therefore about twice those measured in the presence of albumin, and PS products for all three tracers failed to reach diffusion-limited values. 6. Lobules perfused with and without albumin were fixed using a glutaraldehyde fixative containing 1% Alcian Blue dye. An ultrastructural examination of the endothelium showed no significant changes in cell or cleft morphology, or in the glycocalyx, in the absence of albumin which might account for the observed permeability change. 7. These data are the first physiological measurements specifically characterizing fetal microvascular permeability in the human placenta. The results suggest that permeability resembles that found in skeletal muscle and, as such, the endothelium presents a significant barrier to the diffusion of large solutes. The observed 'protein effect' indicates that albumin can interact with elements of the solute pathway to increase its restrictiveness.

Alcian Blue↗

PubMed1993

Immunohistochemical localization of adherens junction components in blood-brain barrier microvessels of the rat.

The morphology and molecular composition of intercellular adherens junctions have most frequently been described in epithelial cells and the fascia adhaerens of the intercalated disc. A group of cytoplasmic molecules is known to be associated with adherens junctions. The intercellular bond is mediated by cadherins which bridge the cells by homophilic binding. Recently, endothelial cells have also been shown to form intercellular junctions of the adherens-type. However, they are morphologically less distinct and little is known about their molecular components. In this study we report the localization of some adherens junction components in intact microvessels of the blood-brain barrier in the rat. We used antibodies raised against alpha-actinin, vinculin, zyxin, cadherin (antipan-cadherin antibody) and A-CAM (N-cadherin) in immunohistochemical experiments at light and electron microscopical levels. Microvessel walls reacted positively for all antigens throughout postnatal development. All antigens were localised, though not necessarily exclusively, to interendothelial junctions. At the ultrastructural level, pan-cadherin reactivity was present throughout the entire length of the cleft. These results could mean that in blood-brain barrier endothelial cells the complex tight junction is embedded in an adherens junction which occupies the entire length of the cleft.

Animals↗

PubMed1993

Immunoelectron characterisation of the inter-endothelial junctions of human term placenta.

The molecular constituents of the paracellular clefts in human placental microvessels were investigated using antibodies against PECAM-1, pan-cadherin, A-CAM (N-cadherin), cadherin-5 and two types of integrins (those recognised by antibodies to the beta 1 chain and alpha v beta 3). Ultrastructural localisation of these molecules in ultrathin frozen sections of human term placentae was attempted using colloidal gold immunocytochemistry, after establishing their presence by indirect immunofluorescence. At the light microscopical level, the endothelial paracellular clefts were found to be immunoreactive to the antibodies against PECAM-1, cadherin-5 and pan-cadherin, but not the integrins. The latter showed diffuse distribution in the endothelium and in the abluminal interstitial space. PECAM-1 and pan-cadherin were also seen in the cytoplasm and luminal surface of the endothelium. Immunoelectron studies revealed that the cadherins and PECAM-1 were present in the wide regions of the paracellular clefts, but not in tight junctional regions. Using immunocytochemistry, these wide junctional areas were found to be associated with the cytoskeletal linking molecules vinculin and alpha-actinin. These regions may therefore contain adherens-type junctions. Cadherin-5, localised by two different monoclonal antibodies, 7B4 and TEA, was the only antigen which was cleft-specific, the others also being seen in the cytoplasm of the microvascular endothelium. Cadherin-5 and pan-cadherin were co-localised in the same wide junction, but were usually seen to occupy different microdomains of, and different wide zones of, the same cleft. The cell adhesion molecules localised in the paracellular wide junctions of the human placental microvessels may play a role in maintaining the intercellular spacing between endothelial cells, and may be part of a paracellular "fibre matrix" with permeability-restricting properties.

Actinin↗