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PubMed2000

The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability.

We review here our work on the molecular and functional organization of endothelial cell-to-cell junctions. The first part of the review is dedicated to VE-cadherin, characterized by our group few years ago. This protein is a member of the large family of transmembrane adhesion proteins called cadherins. It is endothelial cell specific and plays a major role in the organization of adherens junctions. Inactivation of VE-cadherin gene or in vivo truncation of its cytoplasmic tail leads to a lethal phenotype due to the lack of correct organization of the vasculature in the embryo. We found that the defect was due to apoptosis of endothelial cells, which became unresponsive to the survival signal induced by vascular endothelial cell growth factor. Our data indicate that VE-cadherin may act as a scaffolding protein able to associate vascular endothelial cell growth factor receptor and to promote its signaling. In the second part of the review we consider another protein more recently discovered by us and called junctional adhesion molecule (JAM). This protein is a small immunoglobulin which is located at tight junctions in the endothelium and in epithelial cells. Evidence is discussed indicating that JAM takes part in the organization of tight junctions and modulates leukocyte extravasation through endothelial intercellular junctions in vitro and in vivo. The general role of tight junctions in endothelial cells is also discussed.

Adherens Junctions↗

PubMed1995

Pubertal development of intersertoli cell junctions in the testis of corriedale ram lambs.

The ultrastructure of the tight junctional complex of pubertal ram lamb Sertoli cells was studied in immersion-fixed samples and related to clinical data and a light microscopical classification of the degree of spermatogenesis attained in the corresponding seminiferous tubule. Although the process followed the general mammalian developmental trend for tight junction complex formation, 2 unusual ultrastructural features were detected: the presence of an active Golgi complex during the early stage of tight junction formation and the transitory presence of ribosomes on both faces of the ectoplasmic cisternae bordering the developing junctions. The significance of these findings is discussed.

Animals↗

PubMed2004

Trimethylated chitosan as polymeric absorption enhancer for improved peroral delivery of peptide drugs.

The absorption enhancing effects of chitosan and its derivatives have been intensively studied in recent years. It has been shown that these compounds are potent absorption enhancers. Chitosan is only soluble in acidic environments and is therefore incapable of enhancing absorption in the small intestine, the main absorption area in the gastrointestinal tract. Special emphasis has been placed on the absorption enhancing properties of N-trimethyl chitosan chloride (TMC), a partially quaternised derivative of chitosan, due to its solubility in neutral and basic environments. TMC is prepared by the reductive methylation of chitosan. The degree of quaternisation can be altered by increasing the number of reaction steps or by increasing the reaction time. Although the molecular weight of the polymer increases with addition of the methyl groups, a net decrease in the molecular weight is observed due to a decrease in the chain length of the polymer. TMC, like chitosan, possesses mucoadhesive properties. In vitro studies performed on Caco-2 cell monolayers showed a pronounced reduction in the transepithelial electrical resistance (TEER). TMC is also able to increase the permeation of hydrophilic compounds such as [14C]-mannitol and [14C] polyethylene glycol 4000 ([14C] PEG 4000, MW4000) across the cell monolayers. It was also shown that the degree of quaternisation of the polymer plays an important role on its absorption enhancing properties, especially in neutral environments where chitosan is ineffective as an absorption enhancer. The reduction in TEER is an indication of the opening of the tight junctions located between epithelial cells. Opening of the tight junctions will result in enhancement of absorption via the paracellular route. Confocal laser scanning microscopy confirmed transport of large hydrophilic compounds via the paracellular route as well as the mechanism of action of the polymer in which redistribution of the cytoskeletal F-actin is provoked, which leads to the opening of the tight junctions. Various in vivo studies in different animal models confirmed the ability of TMC to increase the absorption of the peptide drugs buserelin and octreotide after intraduodenal or -jejunal administration. However, TMC has always been administered as a solution in these studies. The impracticality of administering a solution, as well as the fact that most peptides are unstable in the presence of water, have led to the need for a solid oral dosage form with which TMC can be administered together with peptide drugs. Recent studies have focused on the development and in vivo evaluation of solid oral dosage forms.

Adjuvants, Pharmaceutic↗

PubMed2001

Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability.

Tight junctions regulate paracellular conductance and ionic selectivity. These properties vary among epithelia but the molecular basis of this variation remains unknown. To test whether members of the claudin family of tight junction proteins influence paracellular ionic selectivity, we expressed human claudin-4 in cultured MDCK cells using an inducible promoter. Overexpression increased the complexity of tight junction strands visible by freeze-fracture microscopy without affecting the levels of claudin-1, -2, or -3, occludin, or ZO-1. A decrease in conductance correlated directly with the kinetics of claudin-4 induction. Dilution potentials revealed that the decrease in paracellular conductance resulted from a selective decrease in Na(+) permeability without a significant effect on Cl(-) permeability. Flux for an uncharged solute, mannitol, and the rank order of permeabilities for the alkali metal cations were unchanged. A paracellular site for these effects was supported by the lack of apical/basal directionality of the dilution potentials, the linearity of current-voltage relationships, and the lack of influence of inhibitors of major transcellular transporters. These results provide, to our knowledge, the first direct demonstration of the ability of a claudin to influence paracellular ion selectivity and support a role for the claudins in creating selective channels through the tight-junction barrier.

Animals↗

PubMed1990

Corneal endothelial junctions and the effect of ouabain.

Paired rabbit corneas were perfused in vitro for endothelial permeability (Pac) determination with glutathione bicarbonate Ringer's solution (GBR) and GBR plus ouabain (10(-4) M). Results indicated no difference in Pac between the two groups (3.39 vs 3.67, respectively) despite significantly greater stromal swelling in the group perfused with ouabain. Freeze-fracture microscopy of similarly perfused corneas revealed intact tight junctional complexes in both groups, although the tight junctional complex of perfused corneas appeared less organized than that of freshly enucleated, nonperfused controls. Gap junctions were abundant as observed in freeze-fracture replicas of GBR-perfused endothelium, and appeared to be decreased or absent in ouabain-perfused endothelium. These results indicate that corneal endothelial tight junctions are unaffected by perfusion with ouabain, whereas gap junctions appear to be lost. The permeability and freeze-fracture data reaffirms the importance of tight junctions as permeability barriers and indicates that gap junctions are not of primary importance for maintenance or control of the corneal endothelial barrier.

Animals↗

PubMed1993

Morphological and histochemical changes in intercellular junctional complexes in epithelial cells of mouse small intestine upon X-irradiation: changes of ruthenium red permeability and calcium content.

Changes of calcium-content and permeability of tight junction following X-irradiation were investigated in mouse intestinal epithelial cells by electron microscopy. In the control animals the lower parts of tight junctional area as well as the other junctional elements and the intercellular space are labeled by pyroantimonate precipitates, which contain calcium as revealed by electron spectroscopy and electron energy loss spectrometry. X-irradiation, parallel with morphological changes, lead to rapid decrease of pyroantimonate precipitable calcium content and increase of the permeability of tight junctions indicated by the penetration of ruthenium red into the intercellular space. These changes were readily reversible following 0,5 Gy doses of irradiation however, they persisted up to 24 hours following 5 Gy irradiation. We conclude that irradiation at the applied doses can transiently destabilize the tight junctions in the epithelial layer of the small intestine, presumably through a calcium dependent mechanism.

Animals↗

PubMed1977

Intercellular junctions in the central nervous system of insects.

The intercellular junctional complexes in the central nervous system (CNS) from a variety of insect species have been examined by thin-sectioning and freeze-fracturing techniques. Of particular concern has been the fine-structural basis of the blood-brain barrier observed to be present in the outer perineurial layer around the avascular insect CNS. The basis of this has been found in the form of tight junctions (zonulae occludentes) present both in sections and in replicas of the perineurium. In the latter, they appear as one or two simple linear ridges, lying parallel to the outer surface, which occasionally display overlapping. The complex geometry of the interdigitating perineurial cells apparently permits such a relatively simple series of ridges to function as a barrier, since tracers are found not to penetrate beyond this level into the underlying nervous tissue. Such evidence is supported by microprobe X-ray analysis of lanthanum-incubated tissues, the perineurium compared with the glia-ensheathed axons showing the presence and absence of lanthanum, respectively. Possible physiological mechanisms that could operate 'in vitro' to maintain the blood-brain barrier are also considered. Other intercellular junctions such as desmosomes, septate junctions and gap junctions are found in the perineurial layer too, the last exhibiting EF particle plaques and PF pits. Glia-glia junctions also occur in some insect species; they include desmosomes, inverted gap junctions and occasional tight junctions. Septate, gap and tight junctions are also found on the membranes of tracheoles penetrating the CNS. Short, ridge-like elaborations and other particle arrays are found on the PF on the axon surfaces and the significance of these structures is discussed.

Animals↗

PubMed2001

Use of surface plasmon resonance for real-time analysis of the interaction of ZO-1 and occludin.

Surface plasmon resonance (SPR) spectroscopy was applied to study in real time, the interaction between the tight junction proteins ZO-1 and occludin. To imitate the morphology of tight junctions, a cytosolic tail of mouse occludin was immobilised at the sensor and guanylate kinase-like domain (Guk) was allowed to pass over the modified chip surface. The Guk domain of ZO-1 (residues 644-812) was found to bind to the cytoplasmic, carboxy-terminal region of occludin (residues 378-521). This interaction was systematically characterised with respect to the concentrations of both proteins and the binding conditions. Under the given experimental conditions, association and dissociation showed saturation kinetics, with affinity in micromolar range: k(a) = 4.14 +/- 0.52 x 10(3) M(-1) s(-1), k(d) = 3.04 +/- 0.38 x 10(-3) s(-1), K(D) = 639 +/- 51 nM. The results support the hypothesis that the Guk domain of ZO-1 is involved in the recruitment of the transmembrane protein occludin at tight junctions by interacting with the cytosolic carboxy-terminal sequence of occludin, located far from the cell membrane. We demonstrate the use of SPR spectroscopy as an effective approach for characterisation of the interactions of junction proteins.

Amino Acid Sequence↗

PubMed2000

Multiple domains of occludin are involved in the regulation of paracellular permeability.

Tight junctions form selective paracellular diffusion barriers that regulate the diffusion of solutes across epithelia and constitute intramembrane diffusion barriers that prevent the intermixing of apical and basolateral lipids in the extracytoplasmic leaflet of the plasma membrane. In MDCK cells, previous expression experiments demonstrated that occludin, a tight junction protein with four transmembrane domains, is critically involved in both of these tight junction functions and that its COOH-terminal cytoplasmic domain is of functional importance. By expressing mutant and chimeric occludin that exert a dominant negative effect on selective paracellular diffusion, we now demonstrate that the extracytoplasmic domains and at least one of the transmembrane domains are also critically involved in selective paracellular permeability. Multiple domains of occludin are thus important for the regulation of paracellular permeability. Expression of chimeras containing at least one transmembrane domain of occludin also resulted in an enhanced intracellular accumulation of claudin-4, another transmembrane protein of tight junctions, suggesting that the two proteins may cooperate in the regulation of paracellular permeability.

Animals↗

PubMed1981

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system.

Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6--7 nm particles solely near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions), contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia. The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neutral lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.

Animals↗

PubMed2005

Epithelial transport and barrier function in occludin-deficient mice.

BACKGROUND AND AIMS: This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model. METHODS: Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion. RESULTS: In occludin+/+ mice, Re was 23+/-5 Omega cm2 in jejunum, 66+/-5 Omega cm2 in distal colon and 33+/-6 Omega cm2 in gastric corpus and was not altered in heterozygotic occludin+/- or homozygotic occludin-/- mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6+/-1.0 and 0.3+/-0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin-/- mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8+/-0.8 kOmega cm2 in urinary bladder and 33+/-6 Omega cm2 in stomach and not altered in occludin-/- mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin-/- mice. CONCLUSION: Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation.

Animals↗

PubMedDate not supplied

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 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.

Angiopoietin-1↗

PubMed1997

Interendothelial junctions in normal human Schlemm's canal respond to changes in pressure.

PURPOSE: To determine if changes in the structure and complexity of junctions between endothelial cells lining Schlemm's canal (SC) occur in normal human eyes with changes in perfusion pressure. METHODS: Twelve normal human eyes were either perfusion-fixed (at 15 or 45 mm Hg) or immersion-fixed (0 mm Hg) in modified Karnovsky's fluid. 'Outflow facility was measured continually during the perfusion fixation. The intercellular junctions of the endothelial cells of SC were ultrastructurally examined in thin sections, including serial sections and freeze-fracture replicas. Morphometric data on the number of junctional strands per total length of tight junction were documented and categorized by the number of strands (one, two, or three or more). The length of endothelial cell overlap was measured on thin sections. RESULTS: In freeze-fracture replicas, perfusion-fixed eyes demonstrated less complex junctions. At 15 mm Hg, 18.06% of the total junctional length was represented by three or more strands; at 45 mm Hg, this percentage decreased to 8.59%. In immersion-fixed eyes, 24.17% of the total junctional length was represented by three or more strands. These differences were statistically significant (P < 0.0012). In sections, the amount of endothelial cell overlap, and thus the length of paracellular pathway, was reduced in perfusion-fixed versus immersion-fixed eyes (P < 0.02). Extensive serial sectioning demonstrated that giant vacuoles were formed, either by individual endothelial cells or by two or more adjacent endothelial cells. CONCLUSIONS: When compared with specimens fixed at zero pressure, overlap between endothelial cells of SC is reduced significantly when this cell layer is under conditions of flow similar to those encountered in vivo. The tight junctions between cells of the inner wall of SC become less complex with increasing pressure. Our data suggest that the paracellular pathway into SC in the normal eye is sensitive to modulation within a range of physiologically relevant pressures.

Aged↗

PubMed1984

The gastric mucosal barrier: structure of intercellular junctions in the dog.

The canine gastric mucosa consists of two regions, the surface mucous cells and gland area cells including parietal, chief, and mucous-containing cells. We have used quantitative freeze-fracture methods in conjunction with thin-section extracellular tracers to document and correlate tight junction morphology with epithelial permeability. The number of strands in the tight junction complexes of the surface cells and gland cells is the same, but differences in strand arrangement exist. The surface cells have an interwoven tight junction configuration which is impermeable to extracellular tracers. The gland cell junctions are regularly arranged and often permeable to extracellular lanthanum. The possibility that the observed difference in permeability between the tight junctions of the surface mucous cells and those of the gland cells is related to their structural configuration is discussed.

Animals↗

PubMed1977

Ultrastructural changes of intercellular junctions in rat ascites hepatoma cells with calcium depletion.

To analyse the effect of ethylenediamine tetraacetate (EDTA) on tumour cell adhesiveness, fine structure of intercellular junctions of rat ascites hepatoma cells AH136B and AH7974 (both forming cell islands in vivo) was first compared. The close contact of the apical portion of both cell islands was composed of tight junctions with a narrow gap. The close contact of the inner portion of AH136B cell islands was largely by simple apposition, while that of AH7974 cell islands had many intermediate junctions and desmosomes. Treatment with EDTA (2 mM) induced morphological alteration of simple apposition, intermediate junctions and desmosomes, but tight junctions remained intact. The effect of EDTA on such junctional complexes seemed to be partially reversible on readministration of Ca ions. Changes in desmosomes, as confirmed on AH7974 cells, were initiated by disappearance of the central disc of electron-dense materials, followed by marked opening of intercellular space and disappearance of endoplasmic laminar plaque. These results suggest that Ca ions may be concerned with maintaining the integrity of junctional complexes other than tight junctions.

Animals↗

PubMed2005

Effects of fermentation products of pro- and prebiotics on trans-epithelial electrical resistance in an in vitro model of the colon.

Evidence from in vivo and in vitro studies suggests that the consumption of pro- and prebiotics may inhibit colon carcinogenesis; however, the mechanisms involved have, thus far, proved elusive. There are some indications from animal studies that the effects are being exerted during the promotion stage of carcinogenesis. One feature of the promotion stage of colorectal cancer is the disruption of tight junctions, leading to a loss of integrity across the intestinal barrier. We have used the Caco-2 human adenocarcinoma cell line as a model for the intestinal epithelia. Trans-epithelial electrical resistance measurements indicate Caco-2 monolayer integrity, and we recorded changes to this integrity following exposure to the fermentation products of selected probiotics and prebiotics, in the form of nondigestible oligosaccharides (NDOs). Our results indicate that NDOs themselves exert varying, but generally minor, effects upon the strength of the tight junctions, whereas the fermentation products of probiotics and NDOs tend to raise tight junction integrity above that of the controls. This effect was bacterial species and oligosaccharide specific. Bifidobacterium Bb 12 was particularly effective, as were the fermentation products of Raftiline and Raftilose. We further investigated the ability of Raftilose fermentations to protect against the negative effects of deoxycholic acid (DCA) upon tight junction integrity. We found protection to be species dependent and dependent upon the presence of the fermentation products in the media at the same time as or after exposure to the DCA. Results suggest that the Raftilose fermentation products may prevent disruption of the intestinal epithelial barrier function during damage by tumor promoters.

Anticarcinogenic Agents↗

PubMed1992

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

Selected 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.

Capillaries↗

PubMed1991

Vasopressin and A23187 stimulate phosphorylation of myosin light chain-1 in isolated rat hepatocytes.

Vasopressin (VP) and other hormones that elevate intracellular Ca2+ concentration also increase tight junctional permeability in the liver cell. Data derived from study of other tissues suggest that microfilaments are instrumental in regulating tight junctional permeability. By analogy to microfilament contraction in smooth muscles, it is likely that the transduction pathway for these hormones involves Ca(2+)-stimulated complex formation between calmodulin and myosin light chain (MLC) kinase with activation of this latter enzyme. MLC kinase then phosphorylates MLC, which, in the presence of actin, exerts adenosinetriphosphatase activity and produces microfilament contraction. This transduction pathway in the hepatocyte remains speculative. To demonstrate the likelihood of this pathway, we stimulated isolated hepatocytes with 10(-8) M VP and assayed MLC phosphorylation. We did this by immunoprecipitation of myosin from homogenates of liver cells prelabeled with [32P]-orthophosphate. We used a polyclonal antibody raised in rabbits against rat liver cell myosin. Our data demonstrate that VP is a potent stimulator of MLC phosphorylation. Maximal rises in intracellular Ca2+ and maximal MLC phosphorylation occur within 40 s of VP administration. The dose-response curve for MLC phosphorylation by VP is similar to that for tight junctional permeabilization in perfused liver with maximal effect at about 10(-8) M VP. The calcium ionophore A23187 also stimulated MLC phosphorylation. MLC phosphorylation, therefore, is at least coincident with, and probably responsible for, tight junctional permeabilization caused by elevation of intracellular Ca2+ in the liver cell.

Animals↗