The pathogenesis of cerebrovascular lesions in hypertensive rats

In this study we investigated the pathogenesis of hypertensive cerebrovascular lesions by light microscopy, immunohistochemistry, scanning electron microscopy, and transmission electron microscopy. The brains of rats with experimentally induced hypertension exhibited severe edema and intracerebral hemorrhage. Light microscopy of the arteries showed severe medial lesions and the deposition of fibrinoid substance in the intima. Immunohistochemistry showed that intercellular adhesion molecule (ICAM)-1, platelet-endothelial cell adhesion molecule (PECAM)-1, interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha endothelial cell expression was upregulated. Scanning electron microscopy of these arteries revealed the adhesion of neutrophils, monocytes, and a few platelets to endothelial cells, and their invasion of endothelial cell junctions and opened junctions. Transmission electron microscopy showed neutrophil and monocyte adhesion to the endothelial cells and neutrophil and monocyte invasion of endothelial cell junctions, intimal deposition of fibrinoid substance, and severe medial cell injury. Intravenously injected horseradish peroxidase insulated from endothelial cell junctions and, via pinocytotic vesicles, into the subendothelial space. These findings suggest that hypertension activates endothelial cells to increase the expression of adhesion molecules and cytokines, and induces neutrophil and monocyte adhesion and migration, resulting in endothelial cell injury and increased permeability of endothelial cells, which results in hypertensive arterial disease.     

Permeability of ovarian follicles and capillaries in mice

The permeability of ovarian capillaries and follicles in prepubertal and sexually mature (proestrus and metestrus) randomly bred Swiss Albino female mice (SCH:ARS HA ICR strain) was studied by intravenous injection of either ferritin or horseradish peroxidase (HRP), followed by examination with light and electron microscopes. The study revealed that capillaries in the interstitial and perifollicular regions were provided with a continuous endothelium that had constant permeability characteristics irrespective of sexual maturity or phase of the estrous cycle. Horseradish peroxidase left the capillaries primarily through interendothelial cell junctions and was present in all follicles within 30 seconds after administration of the tracer. Ferritin, on the other hand, was absent from endothelial cell junctions, and left the capillaries, at a slower rate than HRP, via cytoplasmic vesicular transport. Both tracers were found in the granulosa cells but rarely in the oocytes. The tracers reached the oocyte through the intercellular spaces between granulosa cells. These findings demonstrate that the follicular apparatus of the mouse is permeable to ferritin and HRP, and that follicular regions such as the basal lamina of the follicle and the zona pellucida do not stop or retard the passage of either tracer.     

Fine structural studies on ependymal paracellular and capillary transcellular permeability in the subcommissural organ of the guinea pig

Summary Morphological investigations on the permeability of intercellular junctions between ependymal cells and between capillary endothelial cells in the subcommissural organ (SCO) of the guinea pig have been carried out using freeze-fracturing and tracer experiments with horseradish peroxidase (HRP). The ependymal junction reveals a moderately developed network of tight junctional strands surrounding the tall ependymal cell. The apical portion of this junctional network tends to form nearly complete strands, whereas the basal portion usually shows irregular, fragmented strands often arranged in hairpin-like structures. The passage of intraventricularly infused HRP is blocked, leaving unstained areas, at the level of membrane fusions. At the same time the lateral intercellular space below the junction is densely stained, probably due to invasion from the basal side through adjacent ordinary ependymal junctions. The SCO capillary endothelium shows a high distribution density of pinocytotic vesicles. Vesicular transport of intravascularly injected HRP is observed, but no HRP penetration occurs through the endothelial junction. The active participation of vesicles in tracer movement is shown in preparations fixed before administration of HRP. Extravasation of this tracer is indicated to some degree in the SCO capillary, but permeability here appears to be comparable to that of ordinary brain capillaries. Accordingly, the SCO ependymal tight junction seems to form an effective barrier not to blood plasma or similar materials but to apically secreted substances, preventing them from spreading back into SCO intercellular spaces.     

Culture and characterization of microvascular endothelial cells derived from human brain

Primary cultures of human cerebral endothelial cells were established from microvessels isolated from cortical fragments removed at surgery for seizure disorder and from brains at autopsy. A uniform population of cells growing in close association to each other formed confluent monolayers by 7 to 10 days in culture. They contained factor VIII/Von Willebrand antigen, the most specific marker for cells of endothelial origin, and showed lectin-binding sites for Ulex europaeus agglutinin characteristic of human endothelium. Cultured cells formed thin, continuous monolayers, contained few pinocytotic vesicles, and were joined together by tight junctional complexes. More than 99% of the intercellular junctions restricted the transendothelial passage of horseradish peroxidase. Monolayers of human brain microvessel endothelial cells thus resemble cerebral endothelium in vivo and should provide a useful in vitro model for studies of the biology of these cells and their role in the pathogenesis of certain human central nervous system diseases associated with abnormal blood-brain barrier function.     

Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat).

The progression from ischemic injury to pannecrosis that occurs in the rat brain several hours after occluding a large artery may be partly attributable to a worsening of the circulation through the microvessels. The objective of this study was to quantitate selected structural changes involving astrocytes and endothelial cells within an area of focal brain ischemia created by the occlusion of a middle cerebral artery. The magnitude of these structural changes was correlated with alterations in the patency to a circulating macromolecule through the microvessels (< or = 15 mu in diameter) located within the territory of the occluded artery. One hundred eighty-five adult male Wistar rats had the right middle cerebral artery occluded after threading a nylon monofilament through the external carotid artery. Experiments were terminated by either cardiovascular perfusion or decapitation and immersion fixation at intervals ranging between 30 minutes and 7 days after the arterial occlusion. Randomly selected animals from each experimental subgroup were injected intravenously with horseradish peroxidase (molecular weight 44 kd) approximately 20 minutes before death. The progressive decline in the area fraction comprised by the vessels filled with horseradish peroxidase was preceded at 30 to 60 minutes by an increase in the surface area occupied (on a cross-section of a microvessel) by endothelial cells (both nucleus and cytoplasm). This was followed by an increase of 23.7% in the mean diameter of astrocytes nuclei and a decrease of approximately 35% in lumenal surface of the microvessels. These observations suggest that the occlusion of a large cerebral artery causes prompt swelling of endothelial cells and astrocytes; both of these early biological responses may interfere with erythrocyte circulation and oxygen delivery, which (after the arterial occlusion) are entirely dependent on the circulation provided by the collateral arterial connections. Through its interference with microvascular patency and oxygen delivery, cell swelling may influence the rate at which neurons become necrotic. In this model of brain infarct the number of necrotic neurons peaks approximately 72 hours after middle cerebral artery occlusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Junctional complexes in the preimplantation rabbit embryo.

The morphology and development of junctional complexes between blastomeres of the preimplantation rabbit embryo were investigated using several approaches. Electron microscopic examination of embryos stained en bloc with uranyl acetate, and the study of junction permeability using horseradish peroxidase and lanthanum nitrate provided information on structure, intermembrane spacing and permeability of the junctional complexes. In addition, the freeze fracture technique was used with day 5 and day 6 blastocysts, since the large size of these embryos facilitated use of this method. These experiments showed that although rudimentary junctions were present between blastomeres of the early cleavage stages, effective tight junctions were not present until the blastocyst stage. Electron microscopic examination of thin sections revealed apical foci of membrane approximation or "fusion" between trophoblast cells by day 4. Freeze fracturing revealed a lattice of interconnecting ridges (on the A face) and grooves (on the B face) in the apical region between trophoblast cells of the day 5 blastocyst. This lattice formed a continuous band along the apical margin of each cell, and therefore constituted a zonula occludens. The zonula occludens of the day 5 blastocyst averages 2-3 ridges per lattice, while day 6 blastocysts had lattices that averaged 5-6 ridges. Also seen in the freeze fracture replicas from the day 5 and day 6 blastocysts were local accumulations of intramembranous particles on the A face. These particles were often observed in aggregates similar to those of previously described gap junctions. It could not be determined whether these small regions of particles were true gap junctions or a possible primitive form of gap junction because the complementary pitted surfaces (B face pits) were not demonstrated.     

Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile

Background: Interendothelial tight junctions and gap junctions have been described in large blood vessels and in cultures of endothelium derived from large blood vessels. Transfer of microinjected smallmolecular weight tracers between adjacent endothelial cells also has been demonstrated indicating the presence of gap junctional interendothelial communication. Similar transfer of tracers is evident between microvessel endothelial cells in culture and in microvessels in situ. However, gap junctions have not been detectable by electron microscopy of intact capillary systems. This may be due to limited sampling available in diffuse capillary systems and a small area of overlap between adjacent endothelial membranes. Methods: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze substitution. Other samples were freeze-fractured and replicated for examination of endothelial junctional components. Results: A novel tight-gap junctional complex between rete capillary endothelial cells is described. In freeze-fracture replicas of the membrane P face, rows of gap junction subunits are flanked on either side by linear depressions representing grooves previously occupied by tight junctional strands that partition to the E face. In thin sections, the junctions appear in profile as short lengths of closely apposed membranes characteristic of gap junctions. Conclusions: The tight junctional components imply a barrier to paracellular transport across the capillary wall between the endothelial cells. The gap junctional component may provide a mechanism for communication between endothelial cells along the length of the vessel wall. © 1995 Wiley-Liss, Inc.     

Alterations in rat jejunal permeability to a macromolecular tracer during a hyperosmotic load

The effects of intraluminal hyperosmolality on the intestinal permeability of a macromolecular tracer, horseradish peroxidase, was assessed in rats by light and electron microscopic cytochemistry. After 60 minutes of an intraluminal hypertonic perfusion of 600 mOsM mannitol, horseradish peroxidase was demonstrable in the intercellular spaces between adjacent absorptive epithelial cells of jejunal villi, along the microvillar brush border, in numerous pinocytotic vesicles, in multivesicular bodies, as well as in large lysosomes. Horseradish peroxidase was frequently found extending from the luminal surface of the cell through the tight junctional region and into more basal portions of the intercellular space. In occasional cells, fragments of the interdigitating plasma membranes of two adjacent absorptive cells appeared to bud off into the cytoplasm of one cell. In contrast, after 60 minutes of isotonic perfusion, horseradish peroxidase was confined to the microvillar brush border, a few pinocytotic vesicles, and occasional multivesicular bodies. These experiments suggest that the passage of macromolecules across the jejunal epithelium of adult rats is enhanced under conditions of luminal hyperosmotic stress. This may be due to an alteration in the functional integrity of the tight junctional macromolecular barrier or to an enhanced rate of pinocytosis.     

Functional differences between sinusoidal endothelial cells and interlobular or central vein endothelium in rat liver

The fine topological relationship between sinus-lining endothelial cells (SLE) and vessel-lining endothelial cells (VLE) at the opening portion of sinusoids into central or interlobular veins of rat liver was studied by a comparison of morphological and functional properties of both types of cells. Three minutes after intravenous injection of formalin-denatured albumin conjugated with horseradish peroxidase (HRP-FDA), liver was perfused with fixative. Chopped sections of the liver (50 micron thick) were incubated in diaminobenzidine-H2O2 medium, followed by processing for electron microscopy. The HRP-FDA was localized in endocytotic vesicles and vacuoles of the SLE and Kupffer cells but not of the VLE lining interlobular or central veins or interlobular arteries. In the opening portion of the sinusoids into these veins, the attenuated cytoplasmic extensions of the SLE containing positive vesicles were in direct contact with squamous process of the VLE having no positive vesicles. The contact was mediated by overlapping junctions. No intermediate cell type between the SLE and VLE in this region or other portions was noted. The results indicate that the habitat of the SLE is exactly isolated from that of the VLE in rat liver and at the transitional portion from sinusoids to veins or arteries they are directly connected with each other by overlapping junctions.     

Structure of the sinus-lining cells in the popliteal lymph node of the rabbit

The structure of the sinus walls in the popliteal lymph node of the rabbit was studied with the electron microscope. In the marginal sinus, the endothelial cells are connected by gap junctions, puncta adherentia, and surface specializations characterized by focal approximation of the adjoining membranes without fusion. They possess large numbers of simple and compound uncoated invaginations of the plasma membrane that are closed by a diaphragm with a central thickening. The tissue strands that straddle the lumen of the sinus consist of a fibrous core containing both collagen and elastic fibers, surrounded by endothelial cells identical to those composing the outer sinus wall. Cortical sinuses that run independently of the trabeculae were identified by exploiting the fact that their endothelial cells accumulate lymph-borne ferritin, and their lumen is outlined by horseradish peroxidase administered intravenously. They are lined by a flattened, continuous endothelium and lack luminal strands. The walls of the medullary sinuses consist of endothelial cells and macrophages. The endothelial cells are interconnected by specialized junctions and contain fewer plasmalemmal vesicles than in the cortex; furthermore, dense granules are present in their cytoplasm. Macrophages adhere to the surface of the endothelial cells; typically, none of the junctional specializations that characterize the interface between endothelial cells connect endothelial cells to macrophages. However, at points along the contact region with the endothelium, the plasmalemma of the macrophage is decorated by an attachment plaque of fluffy cytoplasmic material. Sinus endothelial cells slowly accumulate lymph-borne ferritin like vascular endothelial cells elsewhere in the body, whereas macrophages contain both ferritin and engulfed erythrocytes.     

Experimental Aortic Intimal Thickening: II. Endothelialization and Permeability

Experimental aortic intimal thickening has been induced in rabbits by two types of injury, suture placement and electrocautery. Scanning electron microscopy showed that endothelialization of the suture plaque was completed at about 10 days following injury. New endothelial cells had no particular orientation or were oriented at right angles to the adjacent normal aortic endothelium. Realignment parallel with the aortic axis had occurred by 21 days after induction of the lesion. Orientation patterns of new endothelial cells over irregularly shaped cautery-induced intimal thickening were difficult to ascertain. Aortic permeability studies were accomplished by using the tracers horseradish peroxidase (HRP) and ferritin. Several naturally occurring intimal thickenings in normal aortas had greater permeability for HRP than did adjacent normal intima. An enhanced penetration of both tracers was observed in mature intimal lesions produced by both experimental procedures compared to adjacent morphologically normal aortic intima. HRP molecules entered the thickened aortic intima in increased amounts through interendothelial junctions and by endothelial pinocytotic vesicles; ferritin molecules were seen only in pinocytotic vesicles. Increased penetration of HRP was observed for as long as 27 weeks after injury, while that of ferritin was observed only for 3 weeks. The enhanced permeability of the thickened intima as compared to normal for these two tracers of considerably different sizes strongly suggests an increased permeability of endothelium overlying intimal thickening for naturally circulating macromolecules.     

Intercellular junctions between specialized ependymal cells in the subcommissural organ of the rat

Summary The permeability of intercellular junctions in specialized ependymal cells in the rat subcommissural organ (SCO) has been studied ultrastructurally by freeze-fracturing and tracer experiments with horseradish peroxidase (HRP). In addition to normal smooth membrane, areas which could be classified as a leaky tight junction are found within the ependymal junctional region. This consists of only one or two relatively continuous strands but with interruptions in the apical portion. Some strands are perpendicular to the apical membrane surface and often form hairpin-like bends in the basal portion of the junction. The junctional region also shows areas with no strands but only a rippled membrane structure which may be equivalent to very close appositions without fusion of adjacent ependymal cell membranes. The relative proportions of normal smooth membrane, strands and rippled structure in the junctional region is approximately 346 including two parts overlapping of the strands and rippled structure. Intraventricularly infused HRP passes through many junctions but is occasionally stopped, leaving unstained intercellular spaces of various lengths between membrane fusions of tight junctions. Even when it is stopped, the intercellular space below the junction is densely stained by the enzyme. Orthogonal arrays of intramembrane particles are found to be distributed on the basal and lateral cell membranes below the junctional region in the SCO ependyma.     

Scanning and transmission electron microscopic studies of microvascular pathology in the osmotically impaired blood-brain barrier

Summary The present investigation focused on the structural events occurring in endothelial cells lining the lumina of brain microvessels in rats subjected to a single intracarotid injection of hypertonic 1.8m l (+) arabinose solution with or without intravenous injection of horseradish peroxidase. Blood vessels from cerebral cortex and thalamus were evaluated by transmission and scanning electron microscopy. After short-term exposure (10–12 min) there was widespread flooding of peroxidase into the brain neuropil of the ipsilateral hemisphere. Peroxidase tracer was frequently observed within vesiculo-tubular profiles, and occasionally within widened interendothelial junctional clefts. Partially fragmented, necrotic endothelial cells appeared to be in the process of desquamation. Individual endothelial cells appeared to be shrunken with widened interendothelial spaces. Some healthy endothelial cells appeared to be involved in repair processes, manifested by the extension of thin cellular processes towards the area of vessel injury. Other pathological alterations included a conspicuous increase in the number of endothelial cell microvilli, large crater-like invaginations of the endothelial plasma membranes and muscular blood vessels in the process of spasm. We also observed a platelet reaction with or without endothelial cell necrosis and attached microthrombi in some arterial segments.     

Morphologic and functional changes of the aortic intima during experimental hypertension.

The morphology and permeability to horseradish peroxidase of the rat aortic intima have been investigated in three experimental models of hypertension having different values of plasma renin content and plasma aldosterone level. During hypertension the aortic endothelium shows three main changes: 1) increased arithmetic mean thickness, with prominent rough endoplasmic reticulum and polyribosomes; 2) the appearance of actin microfilament bundles; and 3) increased permeability to horseradish peroxidase. These changes are not present in all models, do not appear to depend on hypertension per se, and are independent of each other. The subendothelial layer of hypertensive animals shows an increased thickness that appears to be correlated with an increase of endothelial cell volume. Our results suggest that: 1) the aortic intima reacts differently to different types of hypertension, and 2) factors other than hypertension per se play a role in the development of vascular changes observed in animals with elevated blood pressure.     

Regional Variation in the Permeability of Rat Thoracic Aorta

The permeability and ultrastructure of the intercostal orifices and interostial regions of rat thoracic aorta have been compared in the present study. The ostial regions consist of two different portions, an annulus and an inflow tract, the latter being continuous with the intercostal artery. The wall of the inflow tract has a sharp bend, demarcating inner and outer portions. Faint blueing of the lateral portions of aortic annuli is visible from 3½ to 4½ hours after injection of Evans blue dye and is no longer apparent at 5½ hours, when there is diffuse discoloration of the thoracic aorta. Only the annuli are labeled by horseradish peroxidase (HRP) for 1½ to 3½ minutes after intravenous injection. There is more extensive and progressively heavier labeling of annuli and inner halves of the inflow tracts between 5 to 12 minutes. HRP is transported across aortic endothelium within micropinocytotic vesicles. Intercellular endothelial clefts do not appear to be directly permeated from the lumen through their junctions for a period of at least 3½ minutes. The present observations preclude an assessment of the penetration of the junctions by HRP at later intervals. No appreciable differences are observed in the mode of transport of peroxidase through the more permeable endothelium of the annulus and less permeable endothelium of inflow tract and interostial regions. Observations made on tissues exposed to colloidal lanthanum, and on specimens stained en bloc with uranyl acetate and subjected to a combination of rotation-tilting, fail to demonstrate appreciable differences in the ultrastructure of the endothelial cell junctions of intercostal orifices and interostial regions. Since morphologic differences do not seem to account for the greater permeability of annular regions, the role of hemodynamic forces is considered. The relationship between regional variation in permeability and the development of atherosclerotic lesions is also discussed     

Light and scanning electron microscopic and immunohistochemical studies on permeability of hypertensive rat mesenteric arteries

Experimental hypertensive rats were intravenously injected with carbon and iron as tracers, and their mesenteric arteries exhibiting hypertensive arterial lesions were observed by light and scanning electron microscopy and immunohistochemistry. Early arterial lesions showing intense medial damages, deposition of fibrinoid substance consisting of fibrin in the intima and/or media, and granulation tissue in the adventitia were characterized by marked insudation of intravenously injected tracers. Scanning electron microscopy demonstrated numerous leukocytes and platelets adhering to endothelial surface, opened endothelial cell junctions, and desquamation of these cells. Immunohistochemistry revealed laminin and low stainability of fibronectin in the subendothelium. Advanced lesions showed deposition of a large amount of fibrinoid substance and no insudation of tracers in the intima, but scanning electron microscopy manifested opening of endothelial cell junctions, desquamation of endothelial cells, and adherence of leukocytes and platelets. Immunohistochemistry revealed fibronectin in the intima and laminin just beneath the endothelium. In the healed lesions disclosing fibrocellular intimal thickening, there was no insudation of tracers. Scanning electron microscopy showed opened endothelial cell junctions, endothelial cell defects, and adherence of leukocytes and platelets. There were fibronectin in the intima and laminin beneath the endothelium. It was suggested that the opening of endothelial cells junctions and desquamation of endothelial cells would be necessary for the arterial increased permeability in hypertensive rats, and that fibrin-fibronectin complex, fibronectin-acid mucopolysaccharide complex, and basement membrane would together inhibit the increased permeability in the mesenteric arteries of hypertensive rats in spite of endothelial cell injuries and their defects.     

Analysis of absorptive cell occluding junction structure-function relationships in a state of enhanced junctional permeability

Junctional strand count, the number of individual junctional strands which intersect a perpendicular, has been widely used as a morphological estimate of the ability of an occluding junction to resist passive transjunctional molecular flow. Such junctional analysis, which requires the study of freeze fracture replicas, has been useful in studies of occluding junction structure function relationships in unperturbed epithelia and in intestinal epithelia under conditions which lead to increases in junctional resistance. It is unclear if the above junctional structure-function correlates also exist in intestinal or in other epithelia under conditions which result in a state of enhanced junctional permeability. To gain further insight into occluding junction structure-function relationships under such conditions, we utilized an in vivo hypertonic perfusion model previously shown to result in the transfer of luminal macromolecules into the paracellular space of small intestinal villus epithelium. After a 1-hour perfusion with either 600 mOsmoles or 750 mOsmoles mannitol solutions, the macromolecule horseradish peroxidase diffusely filled the paracellular spaces of the upper half of villi. However, analysis of thin sections showed that only junctions at the tip of villi were leaking this tracer. Freeze fracture analysis revealed occluding junction structural abnormalities most marked at the villus tip thus corresponding to the site of transjunctional horseradish peroxidase leak. The most frequent abnormality noted was loss of strand-strand crosslinking and dilation of the interstrand compartments. At such sites, wide unobstructed channels could be traced through much of the junction. However, the apical junctional strand was never noted to display unequivocal discontinuities. Subsequent in vitro studies of perfused tissues revealed that dose dependent decreases in transepithelial resistance and junctional charge selectivity were induced by hypertonic mannitol perfusions. These studies indicate that: paracellular localization of a luminally applied tracer may result from a transjunctional leak at a distant site with subsequent lateral diffusion of the marker; traditional junctional strand count-function relationships may not hold in states which lead to the rearrangement of junctional architecture; and, at least the apical junctional strand may become permeable to macromolecules without the introduction of morphologically detectable strand discontinuities as assessed by routine freeze fracture techniques.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cerebral endothelial cell morphology by extracellular calcium

Cerebral endothelial cells interconnected by tight and adherens junctions constitute the structural basis of the blood-brain barrier. Extracellular calcium ions have been reported to play an important role in the formation and maintenance of the junctional complex. However, little is known about the action of calcium depletion on the structural characteristics of cerebral endothelial cells. Using atomic force microscopy we analyzed the effect of calcium depletion and readdition on the shape and size of living brain endothelial cells. It was found that the removal of extracellular calcium from confluent cell cultures induced the dissociation of the cells from each other accompanied by an increase in their height. After readdition of calcium a gradual recovery was observed until total confluency was regained. We have also demonstrated that Rho-kinase plays an important role in the calcium-depletion-induced disassembly of endothelial tight and adherens junctions. The Rho-kinase inhibitor Y27632 could prevent the morphological changes induced by a lack of calcium as well. Our results suggest that calcium depletion induces Rho-kinase-dependent cytoskeletal changes that may be partly responsible for the disassembly of the junctional complex.     

LIGHT AND SCANNING ELECTRON MICROSCOPIC AND IMMUNOHISTOCHEMICAL STUDIES ON PERMEABILITY OF HYPERTENSIVE RAT MESENTERIC ARTERIES

Experimental hypertensive rats were intravenously injected with carbon and iron as tracers, and their mesenteric arteries exhibiting hypertensive arterial lesions were observed by light and scanning electron microscopy and immunohistochemistry. Early arterial lesions showing entense medial damages, deposition of fibrinoid substance consisting of fibrin in the intima and/or media, and granulation tissue in the adventitia were characterized by marked insudation of intravenously injected tracers. Scanning electron microscopy demonstrated numerous leukocytes and platelets adhering to endothelial surface, opened endothelial cell junctions, and desquamation of these cells. Immunohistochemistry revealed laminin and low stainability of fibronectin in the subendothelium. Advanced lesions showed deposition of a large amount of fibrinoid substance and no insudation of tracers in the intima, but scanning electron microscopy manifested opening of endothelial cell junctions, desquamation of endothelial cells, and adherence of leukocytes and platelets. Immunohistochemistry revealed fibronectin in the intima and laminin just beneath the endothelium. In the healed lesions disclosing fibrocellular intimal thickening, there was no insudation of tracers. Scanning electron microscopy showed opened endothelial cell junctions, endothelial cell defects, and adherence of leukocytes and platelets. There were fibronectin in the intima and laminin beneath the endothelium. It was suggested that the opening of endothelial ceils junctions and desquamation of endothelial cells would be necessary for the arterial increased permeability in hypertensive rats, and that fibrin-fibronectin complex, fibronectin-acid mucopolysaccharide complex, and basement membrane would together inhibit the increased permeability in the mesenteric arteries of hypertensive rats in spite of endothelial cell injuries and their defects.     

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

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.