Vesicles In Endothelial Cells Research Articles

Membrane model of endothelial cells and leukocytes. A proposal for the origin of a cortical stress.

Previous models of the erythrocyte membrane have been based on the assumption that the resting curvature of the membrane is either flat or has a small curvature relative to the overall cell dimension. In contrast, several recent experimental observations, both in leukocytes and in endothelial cells, suggest that local regions of the membrane may have high membrane curvature in the resting state. The resting curvature may be of the order of plasmalemmal vesicles in endothelial cells or surface membrane folds on leukocytes. A tension is required to unfold the membrane with strain energy which depends largely on mean curvature. It is proposed that the tendency of endothelial or leukocyte membranes to wrinkle in the unstressed state may provide a restoring force, i.e. a cortical tension.

The localization of lipopolysaccharide in an endotoxemic rat liver and its relation to sinusoidal thrombogenesis: Light and electron microscopic studies

The distribution of lipopolysaccharide (LPS) and sequential thrombus formation in the liver was investigated by immunohistochemical and cytochemical techniques in endotoxemic rats, using horseshoe crab factor C, a specific ligand for biologically active LPS, a monoclonal antibody against it, and rabbit anti-rat fibrinogen IgG. One hour after the intravenous administration of LPS (5 mg/kg), LPS was localized in the secondary lysosomes of Kupffer cells and in the vesicles of endothelial cells, mainly at the peripheries of the hepatic lobules. Small necrotic foci of hepatic tissue were scattered close to the LPS-containing Kupffer cells, and were frequently associated with infiltration of neutrophils and deposition of fibrin. Three hours after the administration of LPS, the immunohistochemical reaction of LPS became stronger and was mostly confined to Kupffer cells. Strands of polymerized fibrin were frequently observed on both the surface of the LPS-containing Kupffer cells and on endothelial cells. These findings suggest that the activation of the coagulation cascade in plasma is first initiated, even though only transiently, by hepatic necrosis which is probably caused by LPS-activated leukocytes, and then by the procoagulant activity expressed on the surface of both Kupffer cells and endothelial cells. Fibrinogen-related antigens were also immuno-ultrastructurally detected in the lysosomes of Kupffer cells three hours after the injection of LPS, which suggested that the Kupffer cells phagocytozed and degraded fibrin. Therefore Kupffer cells in endotoxemia may closely participate in both the sinusoidal thrombogenesis and degradation of fibrin.

Permeability for horseradish peroxidase in strial capillaries in each turn of cochlea.

The capillary permeability of the stria vascularis in each turn of cochlea was examined at intervals of 10 and 30 min and 1 and 2 h after an injection of horseradish peroxidase (HRP), using light and electron microscopic techniques. Ten minutes after HRP was administered, 18% of examined vessels showed HRP leakage. Thereafter, the level of HRP leakage increased with time. One hour after the injection, HRP had leaked from almost all vessels of the upper three turns. Two hours after the injection, the tracer in the vessel lumen became sparse with very thin staining or nonstaining, and a large amount of HRP was observed outside the vessels. It was noted that the capillary permeability to HRP at the basal turn was different from that of the upper three turns. This seems to be the result of the different pinocytic activity in the capillaries of these two regions. When the tracer disappeared from the vessels (2 h after the HRP injection), labelled vesicles in endothelial cells were frequently visible near the outside surface, whereas very few were visible near the inner surface. It is speculated, therefore, that HRP which has leaked can be reabsorbed back into the blood circulation via a similar micropinocytosis down the concentration gradient.

Pression hydrostatique et pathologie neurochirurgicale

Hydrostatic pressure is a force applied by a liquid on the surface of an immersed body. Inside the circulatory system it depends on the weight of the blood column between the heart and a given level. In neurosurgical patients, the hydrostatic pressure plays an important role in cerebral perfusion, transcapillary fluid movements and venous air embolism. The mean arterial pressure (P̄a), the CSF pressure (CSFP) and the pressure in the jugular vein (JVP) are the hydrostatic determiners of the cerebral perfusion pressure (CPP). The hydrostatic pressure gradient associated with head raising decreases P̄a, cerebral venous pressure and JVP, decreases or increases intracranial pressure (ICP) and decreases CPP. The consequences of the resulting hypoperfusion depend on the status of autoregulation. When the skull is open, the pressure under the cerebral retractors determines the transmural pressure and cerebral perfusion. The transcapillary fluid movements depend on a permeability coefficient, a hydrostatic pressure gradient and an osmotic pressure gradient. In case of a rupture of blood brain barrier (BBB), the increase of P̄a and the decrease of ICP (craniotomies) favour the development of vasogenic oedema. When the P̄a stands higher than the upper limit of autoregulation, the hydrostatic capillary pressure increases. It results in a vasodilation, an increase of cerebral blood flow and oedema, the lesion of BBB being initiated by an increase of the amount and the activity of the pinocytotic vesicles in endothelial cells. The syndrome of BBB rupture at a normal CPP is the consequence of an increase in hydrostatic pressure in the dilated capillary territory and can make more difficult the surgical treatment of arterio-venous malformations. Finally, the occurrence of venous air embolism depends on a negative pressure gradient between the head and the right heart, favouring the penetration of air through a venous opening. This gradient is maximal in the conventional sitting position, however air embolism has been described in other neurosurgical postures. The compression of the jugular veins increases the cerebral venous pressure above the atmospheric pressure and has therefore been advocated for decreasing the risk of venous air embolism.

Cellular localization of endothelial alkaline phosphatase reaction product and enzyme protein in the myocardium.

Myocardial capillary endothelial cells, arteriolar endothelial cells, and the arterial adventitia show positive alkaline phosphatase (AP) enzyme reaction and immunoreactivity in both rat and human hearts. In guinea pigs, however, capillary endothelial staining is discontinuous and arterial adventitia is negative. The ultrastructural correlate of discontinuous capillary staining is a pronounced labeling of pericytes in guinea pig heart and relatively weak endothelial staining. In rat and human heart, enzyme reaction products are localized mainly on plasma membranes and cytotic vesicles of endothelial cells. Comparison of two strains of rat reveals a more dense deposition of enzyme reaction product along the luminal and particularly along the abluminal plasma membrane of Sprague-Dawley rats than of Wistar rats. Quantitative analysis of immunogold labeled anti-AP antibody density confirms the pronounced polarity of capillary endothelial cell labeling in Sprague-Dawley rats. More than 80% of total endothelial AP protein in Sprague-Dawley rats is localized over the abluminal plasma membrane and basal lamina, as compared with less than 30% in Wistar rats. Moreover, the total endothelial cell labeling is almost sixfold higher in Sprague-Dawley than in Wistar rats. Total endothelial labeling and proportion of labeling on the abluminal endothelial plasma membrane in human hearts is intermediate between the two strains of rat. The strain and species differences in enzyme distribution could provide important information concerning enzyme function.

Epidermal growth factor receptor at endothelial cell and pericyte interdigitation in human granulation tissue

Angiogenic immature capillaries in human granulation tissue possess many cytoplasmic interdigitations between endothelial cells and pericytes (CIDEP). Epidermal growth factor (EGF) is a potent mitogenic polypeptide which accelerates angiogenesis in vivo and in vitro. We have recently demonstrated in immature capillaries that EGF is present at the CIDEP and proposed that the CIDEP may be involved in a signaling pathway for EGF (Wakui et al., 1990c. Microvasc. Res. 40, 285–291), This study follows that previous report. In the present study, I have investigated the ultrastructural localization of the EGF receptor (EGF-r) at CIDEP. Immunoreactivity for the EGF-r in immature capillaries was located strictly at the CIDEP in large numbers, at the coated pits and vesicles of endothelial cells, and at some lysosome-like structures of the endothelial cells and the pericytes. On the other hand, immunoreactivity for EGF-r was absent in mature capillaries. At the CIDEP, EGF-r immunoreactivity was present at the cell membrane and a few cytoplasmic elements at the tip of pericyte cytoplasmic projections, but it was completely absent at the corresponding endothelial membrane indentation. The present results support our proposed hypothesis that the CIDEP in immature capillaries act as a pathway for EGF transportation from the endothelial cell to the pericyte by a receptor-mediated process.

Amitriptyline-induced morphological alterations of the rat blood-brain barrier

Amitriptyline is known to increase the permeability of the blood-brain barrier but the morphological basis of this increase has not been studied. As catecholamines can influence pinocytosis in dog peripheral blood vessels, the effect of amitriptyline on the pinocytotic activity of blood brain microvessels was studied. Amitriptyline, 34 mg · kg −1 i.p., was injected to rats and the parietal cortex of control and treated animals was prepared for ultrastructural study. Pinocytotic vesicles in endothelial cells were quantified. Amitriptyline significantly increased the density of pinocytotic vesicles in capillary endothelial cells. No other morphological changes occurred after amitriptyline treatment. We conclude that the increase in blood-brain barrier permeability due to amitriptyline may be ascribed at least in part to an increase of pinocytotic activity in brain capillary endothelial cells.

Distribution of endogenous albumin across the rat aortic wall as revealed by quantitative immunocytochemistry.

Endogenous albumin was revealed over thin sections of rat aortic wall, with high resolution and specificity, by applying the protein A-gold immunocytochemical technique. Gold particles, revealing albumin antigenic sites, were observed over plasmalemmal vesicles in endothelial cells and over the interstitial space throughout the thickness of the aortic wall. The distribution of the labeling in the interstitial space varied from region to region and was associated with the collagen fibers, following the orientation of the bundles. The morphometric evaluation of this labeling demonstrated a first peak in labeling intensity in the intima followed by a steep decrease with low levels in the media, and an increasing gradient towards the adventitia. In the subendothelium, a moderate labeling was observed at the base of the endothelial cells of both aortic and capillary endothelia, followed by a decreasing gradient. Ratios between the labeling density in the intima as well as in the adventitia and that in the capillary lumen (plasma albumin) revealed different concentrations of albumin in these compartments. Endogenous albumin, under steady-state conditions, is thus unevenly distributed over the interstitial spaces across the rat aortic wall, and appears associated along the collagen fibers.

The Cytoplasmic Surface Structures of Uncoated Vesicles in Various Tissues of Rat as Revealed by Quick-Freeze, Deep-Etching Replicas

We examined the cytoplasmic surface structures of the plasmalemmal vesicles in various tissues using quick-freeze, deep-etching replicas, and identified three types of cytoplasmic surface specializations. In addition to the baskets of clathrin-coated vesicles, stripe-surfaced and bumpy-surfaced vesicles could be identified. The striped patterns were clearly observed on the vesicles in endothelial cells and fibroblasts of various tissues, while in epithelial cells such as hepatocytes, renal proximal tubule cells, and superficial cells of the urinary bladder, instead of the striped patterns, rough-surfaced vesicles could be identified. These differences in 'uncoated' vesicles may suggest that there is the tissue specificity of the cytoplasmic surface structures of plasmalemmal vesicles.

Degenerative Changes in Lymphatic Endothelium of Jirds Infected with Brugia pahangi

The quantitative changes of cytoplasmic vesicles and vacuoles in lymphatic endothelial cells of the mongolian jirds associated with Brugia pahangi infections were observed by transmission electron microscopy. The present study revealed a decrease in the proportion of cytoplasm occupied by vesicles and in the number of cytoplasmic vesicles in endothelial cells from lymphatic vessels harboring B. pahangi at 3, 4, and 10 mo after infection (3.55, 3.36, and 2.55 vesicles/micron 2, respectively) when compared with cells from uninfected control vessels (7.03 vesicles/micron 2). On the contrary, there was an increase in the area of vacuoles in endothelial cells of jirds at 3, 4, and 10 mo postinfection. The mean +/- SD diameter of vesicles in cells from lymphatic vessels at 10 mo after infection was significantly smaller (78.6 +/- 5.6 nm) compared to vesicles in uninfected vessels (87.5 +/- 9.7 nm).

Theoretical modeling of fluid transport through endothelial junctions.

This paper will review some recent theoretical models of the fluid transport through an endothelial cell layer from the standpoint of hydrodynamic theories based on ultrastructural information. These theories attempt to use realistic models of the observed morphology of endothelial cell junctions and realistic estimates of the forces, pressures, and flows that can take place in narrow slits and porous media. The classical system of transendothelial pathways consisting of a small pore system, medium size pores and a few large pores for the passage of macromolecules is well supported by experimental data on filtration measured macroscopically. Excellent summaries of this theory and data are available (eg. Curry, 1984; Michel, 1984; Taylor and Granger,1984).1,2,3 However, the possible ultrastructural features of endothelial cells which correspond to the various sizes of pores continue to be refined by electron microscopic and other studies. One of the most striking developments is the demonstration that most vesicles of endothelial cells are attached to cell walls.4,5 This has led to a concept of a lesser role of vesicular transport and a greater emphasis on the filtration through the endothelial cell junctions.6,7 In these more recent theories, the classical system of pores is replaced by a system of narrow slits which realistically represent the endothelial cleft and tight junctions, some wider tortuous gaps between protein junctional strands, and transiently open junctions which are associated with the replacement phase of dying endothelial cells.8

Electron microscopic evaluation of brain edema in rabbits with galactosamine-induced fulminant hepatic failure: ultrastructure and integrity of the blood-brain barrier.

Brain edema is a major complication of fulminant hepatic failure and is responsible for death in a large percentage of patients. We previously demonstrated the progressive accumulation of water in grey matter areas of the brain in the rabbit with galactosamine-induced fulminant hepatic failure. We now report the electron microscopic morphology of the brain in the same model of acute hepatic failure following the intravenous injection of horseradish peroxidase, an intravascular tracer which forms an electron-dense reaction product. Rabbits with both mild and severe encephalopathy had normal blood pressures and blood gases at the time of study. Fixation of brain tissue was obtained by whole-body perfusion. Marked swelling of the cytoplasm, perineuronal and perivascular processes of astrocytes were noted in cortical gray, but not white, matter areas; the other cellular components of the brain had normal morphology. Capillary endothelial cells were normal, and there was no evidence of horseradish peroxidase in endothelial cell vesicles, basement membranes or the brain parenchyma, suggesting that the blood-brain barrier was impermeable to large molecules. Histologic evidence of brain edema is seen in this model, with swelling of astrocytes as the primary manifestation of the accumulation of water. Damage to astrocytes or inhibition of their function may contribute to the pathogenesis of hepatic encephalopathy in this model.

Morphological effects of nitrogen dioxide on the rat lung.

Morphological studies of the rat lung exposed to 20 ppm NO2 for 20 hr (experiment 1), to 0.5 ppm for 19 months (experiment 2), and to 10 ppm for 14 days (experiment 3) were conducted. Changes in the mast cells of the tracheas and main bronchi of rats exposed to 0.5 ppm (experiment 4) were also observed. In the alveolus, cytoplasmic blebbing occurred in a small number of type I cells immediately after exposure to 20 ppm NO2 for 20 hr, and remarkable vacuolar change was observed 3 days after 10 ppm exposure. Exposure to 0.5 ppm did not cause degeneration. Swelling and hyperplasia of type II cells were observed. The cells gradually became flat and began a transition from type II to type I cells. These intermediate-type cells were noticed in experiments 1 and 2, but no intermediate-type cells were found in experiment 3. In each experiment, pinocytotic vesicles of endothelial cells in capillaries, followed by interstitial edema in the alveolar walls, were observed. In addition to these changes, desquamation of endothelium and widening of the endothelial junction of endothelial cells occurred in experiment 3. The early changes observed in the animals exposed to 0.5 ppm NO2 were the numerical and histochemical changes of mast cells in the trachea and main bronchus.

Morphological Effects of Nitrogen Dioxide on the Rat Lung

Morphological studies of the rat lung exposed to 20 ppm NO2 for 20 hr (experiment 1), to 0.5 ppm for 19 months (experiment 2), and to 10 ppm for 14 days (experiment 3) were conducted. Changes in the mast cells of the tracheas and main bronchi of rats exposed to 0.5 ppm (experiment 4) were also observed. In the alveolus, cytoplasmic blebbing occurred in a small number of type I cells immediately after exposure to 20 ppm NO2 for 20 hr, and remarkable vacuolar change was observed 3 days after 10 ppm exposure. Exposure to 0.5 ppm did not cause degeneration. Swelling and hyperplasia of type II cells were observed. The cells gradually became flat and began a transition from type II to type I cells. These intermediate-type cells were noticed in experiments 1 and 2, but no intermediate-type cells were found in experiment 3. In each experiment, pinocytotic vesicles of endothelial cells in capillaries, followed by interstitial edema in the alveolar walls, were observed. In addition to these changes, desquamation of endothelium and widening of the endothelial junction of endothelial cells occurred in experiment 3. The early changes observed in the animals exposed to 0.5 ppm NO2 were the numerical and histochemical changes of mast cells in the trachea and main bronchus.

Luminal vesicles in endothelial cells of retinal and iridial vessels of the rat

Endothelial vesicles on the luminal front of retinal and iridial vessels were studied by perfusion fixation after intravenous injection of peroxidase, and by perfusion fixation of glutaraldehyde with tannic acid or ruthenium red. Many luminal vesicles contained peroxidase reaction product after intravenous injection of peroxidase, but were virtually empty after perfusion fixation. Both tannic acid and ruthenium red delineated the majority of luminal vesicles, singular or in clusters, surface-associated or "free" in the cytoplasm. Only a few free vesicles were unlabeled by the dye. No transendothelial channels were seen. The results suggest that most of the vesicles on the luminal front of endothelial cells are continuous with the cell surface at the time of fixation. The function of these vesicles is not fully understood; some appear to be involved in endocytosis of macromolecules from the vascular lumen.

Surface-associated vesicles in retinal arterioles and venules.

The surface-associated vesicles in retinal arterioles and venules were studied after fixation in glutaraldehyde-tannic acid or after intravitreal injection of peroxidase or lactoperoxidase. The vesicles were concentrated along the abluminal (basal) surface of the endothelial cells and along the plasma membranes of smooth muscle cells in arterioles and of pericytes in post-capillary venules. They were rarely encountered in the deeper regions of these cells. In perpendicular sections through the cell surface the majority of vesicles were in continuity with the plasma membrane whereas in tangential sections, they appeared to lie "free" in the cytoplasm. All such vesicles were labeled after exposure to tannic acid or to the heme-proteins. Peroxidase-reaction product was never seen in the lumen of the vessels. These observations suggest that the surface vesicles in endothelial cells, smooth muscle cells and pericytes are invaginations of the plasma membrane and are thus not involved in the transcytosis or endocytosis of proteins. The vesicles in the latter two cell types may be involved in some aspect of contractility rather than pinocytosis.

Ultrastructure of cerebellar capillary hemangioblastoma. V. Large pinocytic vacuolar bodies (megalopinocytic vesicles) in endothelial cells.

Large pinocytic vacuolar bodies (megalopinocytic vesicles) containing electron-dense granulo-fibrillary material, not previously described in micro-vascular endothelium of brain tumors, were observed in endothelial cells of all five cases of cerebellar hemangioblastoma studied ultrastructurally. They were present in 23% of a total of 132 capillary profiles studied. Some were prominent and aggregated to occupy a large portion of the endothelial cytoplasm. Unlike the ordinary pinocytic vesicles in endothelial cells, they were distributed predominantly in the vicinity of the nucleus and surrounded by abundant organelles. They were irregular and usually several times larger than macropinocytic vesicles. The larger vacuolar bodies were often surrounded by bundles of microfilaments which often anchored on their limiting membrane. They coexisted frequently with Weibel-Palade bodies and occasionally fused with them. Convergence of coated vesicles and micropinocytic vesicles with the vacuolar bodies was present. However, there was no direct contact between the vacuolar bodies and Golgi apparatus, rough and smooth endoplasmic reticulum and mitochondria. The vacuolar bodies were closely associated with pericytic foot processes. It was suggested that they were formed by invagination of the abluminal cytoplasmic membrane with engulfed extracellular material and migrated internally. Discharge of their contents into the vascular lumen and interendothelial space was observed. Some had a disrupted membrane with a suggestion of release of contents into the cytoplasmic matrix. Their function is unknown, but they may serve as a specific vehicle of transport or digestive mechanism in microvascular endothelium under certain pathophysiological conditions, such as neoplasm, to meet the increasing metabolic demands.

Plasma membrane-associated vesicles in retinal capillaries of the rat.

The structure and function of abluminal vesicles in endothelial cells of rat retinal capillaries was examined using glutaraldehyde-tannic acid fixation and the hemeproteins--horseradish peroxidase, microperoxidase, and lactoperoxidase--as tracers. Numerous vesicles, delimited by a tannic acid-positive membrane, were distributed along the abluminal front. Other vesicles were arranged in clusters and chains or tubule-like structures. Such vesicles were not found in the vicinity of the capillary lumen. When the retina was exposed to hemeproteins, either in vitro or after intravitreal injection, the abluminal vesicles became labeled with tracer reaction product. Apparently "free" vesicles and tubules seen in tangential sections through the basal lamina were also labeled, suggesting that they were in continuity with the plasma membrane in another plane of section. No enzyme reaction product was present in the capillary lumen. Peroxidase-positive multivesicular bodies were observed, suggesting that some protein was endocytosed and directed to lysosomes where it was presumably degraded. The results suggest that abluminal endothelial vesicles represent pits or invaginations of the plasma membrane and, as such, are not involved in the transendothelial transport of protein from the perivascular space to the capillary lumen. Tannic acid treatment revealed a population of similar vesicles associated with the plasma membrane of pericytes. After exposure to hemeproteins, enzyme reaction product was localized in these vesicles and in a few multivesicular bodies. The results suggest that the majority of these vesicles are in continuity with the plasma membrane and are not involved in endocytosis.

Alteration of the ultrastructure of the pig liver after a continuous perfusion of six hours in vitro

Livers of mini LEWE pigs were perfused continuously for 6 h on a novel perfusion system through the portal vein (laminar flow from 200 to 300 ml/min, pressure 270 Pa) and through the A. hepatica (pulsating flow from 100 to 200 ml/min, pressure: 10.6/5.5 kPa). The following perfusion media were used for determining the best conservation conditions at 10 to 12 degrees C: Pig blood/Krebs-Henseleit bicarbonate buffer 1:1; Pig blood/Ringer lactate solution 1:1; Pig plasma; Collins C 2 perfusion solution. After perfusion with Collins C 2 solution the hepatocytes show swelling and dilation of the granular endoplasmic reticulum; after perfusion with the other solutions the changes of the hepatocytes are negligible. The alterations of the sinusoidal wall cells depend on the perfusion pressure and flow and the composition of the solution. After continuous perfusion the sinusoidal walls are characterized by vesicles of endothelial cells, dilatation of the Disse space and fragmentation of hepatic microvilli.

Cytochemical studies of adenylate cyclase in the choroid plexus and brain vessels of rat and mouse.

Adenylate cyclase (AC) activity was studied in the choroid plexus and brain vessels of rat and mouse using a recently improved cytochemical method (Palkama A, Kaufman HE, Uusitalo R, Uusitalo H, Virtanen J, Huhtaniitty S: J Histochem Cytochem 29:898, 1981). For comparison, the vasculature of skeletal muscle was also examined. Specimens were fixed for 5 min in 1% paraformaldehyde or 1% paraformaldehyde and 0.1% glutaraldehyde supplemented with activators of AC: isoproterenol and 5'-guanylylimidodiphosphate or forskolin. These activators were also present in the "activating solution" and in the incubation medium. For control purposes no activators were used. Various capturing agents were introduced into incubation media: SrCl2, Pb citrate, NdCl3, and CeCl3. Satisfactory results were obtained with both fixatives, both activators, and the capturing agents SrCl2 and Pb citrate. In rat choroid plexus AC activity was localized in the basal and lateral plasmalemma of epithelial cells, in the plasmalemma of cells interposed between epithelium and capillaries and occasionally in pinocytotic vesicles of endothelial cells. In micro-blood vessels of rat brain, the reaction appeared in the luminal or in both luminal and abluminal plasmalemma of endothelial cells and in smooth muscle cells. In skeletal muscle microvessels, reaction product was mainly located in numerous pits and pinocytotic vesicles associated with luminal plasmalemma of endothelial cells. Some reaction in rat cortex was occasionally observed in Golgi saccules of neurons and in postsynaptic densities. In the mouse choroid plexus and brain vessels the reaction was notably less intense, suggesting the existence of species differences in AC activity as revealed by cytochemical technique.