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N Simionescu

Publications and source records attributed to N Simionescu.

At least 55 records · Page 3Linked to original sources
PubMed1987

Development of intracellular lipid deposits in the lipid-laden cells of atherosclerotic lesions. A cytochemical and ultrastructural study.

In atherosclerotic lesions of rabbits fed a cholesterol-rich diet, the lipid deposits of foam cells derived from monocytes, smooth muscle and endothelial cells were studied by physical, cytochemical and ultrastructural methods. Beginning with the third week of diet, the lipid material that could be visualized at the light microscope level by Oil red O and Nile red staining was progressively accumulated in the intimal cells of the atherosclerotic lesions. In the early stages of foam cell formation, the deposits occurred especially as intracytoplasmic non-membrane bound lipid inclusions (lipid droplets). In polarizing microscopy these appeared as a mixture of iso-, and anisotropic material. The latter were birefringent and showed an axial symmetry with a black cross image, suggesting that the lipids were in a liquid crystalline state. In chemically-fixed specimens, the content of lipid inclusions was preserved in various degrees. In freeze-fractured preparations they displayed a layered onion-like arrangement with smooth cleavage faces surrounding an amorphous core. Upon incubation with filipin, that specifically binds to 3 beta-hydroxysterols, the peripheral layers of the inclusions were labeled, revealing the existence of unesterified cholesterol. In the advanced stages of foam cell formation, lipids were additionally accumulated in the lysosomal compartment as polymorphic multilamellar structures concentrically arranged, with cleavage faces devoid of intralamellar particles. The presence of acid phosphatase showed that these features were modified lysosomes and were tentatively named lysosomal lipid bodies. In the latest stages examined cholesterol crystals developed within lysosomal lipid bodies usually enclosed in multilamellar structures. This lipid coat may represent the place of crystal formation and presumably acts as barrier for the turnover of the crystalline cholesterol, thus impeding plaque regression.

Acid Phosphatase↗
PubMed1987

Prelesional events in atherogenesis. Colocalization of apolipoprotein B, unesterified cholesterol and extracellular phospholipid liposomes in the aorta of hyperlipidemic rabbit.

The appearance and accumulation of apolipoprotein B and unesterified cholesterol in the lesion-prone areas of the aorta in rabbits with diet-induced hyperlipidemia were investigated by histo-, and cytochemical techniques. Apolipoprotein B was detected by an indirect immunoperoxidase procedure both in the light and electron microscopy. Unesterified cholesterol was revealed using filipin and tomatine as specific probes. In the prelesional stages of atherogenesis, before the appearance of any structurally detectable lesions, as demonstrated by bright-field and fluorescence microscopy, apolipoprotein B and free cholesterol accumulated progressively in the extracellular matrix of the subendothelial space. At ultrastructural level, extracellular phospholipid liposomes, unesterified cholesterol and apolipoprotein B concomitantly appeared and accumulated focally in the same areas. Apolipoprotein B was preferentially located on the outer surface of the free cholesterol-containing phospholipid lamellae of the extracellular liposomes. In the lesional stages leading to fatty streak formation, the extracellular liposomes, apolipoprotein B and unesterified cholesterol had also topographically a superimposed localization pattern. Intracellular apolipoprotein B and unesterified cholesterol were also colocalized in some intimal lipid-laden cells. In the prelesional stages of hyperlipidemia the prevalent localization of apolipoprotein B around individual unesterified cholesterol-rich extracellular phospholipid liposomes, progressively accumulating in the subendothelial space, suggests their possible origin from serum-derived lipoproteins.

Animals↗
PubMed1987

Cell surface chemistry of arterial endothelium and blood monocytes in the normolipidemic rabbit.

Chemical mapping of the luminal surface of normal rabbit aortic and coronary endothelium was investigated cytochemically to establish a baseline for further comparison with the biochemical changes possibly induced by the experimental hypercholesterolemia. Morphometric analysis showed that in the aortic endothelium the plasma membrane exposes a large number of uniformly-distributed positively-charged groups of high pKa, and a heterogeneous pattern of dense anionic groups of low pKa. Among the latter, only a third was represented by neuraminidase-cleavable sialic acids. These are constituted by various classes of N-, and O-substituted sialyl residues in glycoconjugates, most frequent being those non-O-acetylated at C8 or C9. Among the oligosaccharides detected with lectins, very abundant were the glycoconjugates containing mannosyl and subterminal galactosyl, whereas N-acetyl-glucosamine, terminal galactosyl and N-acetyl-galactosaminyl moieties were rather poorly represented. The density of the latter two markedly increased after its unmasking by neuraminidase treatment. Coated pits contained both anionic and cationic sites, but only few sialic acids and saccharide residues in significantly lower amounts than plasma membrane. The membrane of plasmalemmal vesicles displayed a high number of cationic sites and mannosyl residues, but very few anionic groups, sialyl residues, and galactosyl and N-acetyl-galactosaminyl moieties. Coronary endothelium displayed a chemical pattern similar to aorta, with some differences, especially in the frequency of some oligosaccharides. Vena cava was low in acidic groups but rather rich in galactose. Plasmalemmal vesicles were only occasionally labeled by the probes used. Monocyte surface exhibited a high density of anionic sites, and binding sites for wheat germ agglutinin and Ricinus communis agglutinin. No mononuclear cells were observed adhering to endothelial surface.

Acetylgalactosamine↗
PubMed1987

Prelesional events in atherogenesis. Changes induced by hypercholesterolemia in the cell surface chemistry of arterial endothelium and blood monocytes, in rabbit.

We investigated the modifications that diet-induced hypercholesterolemia, in rabbit, can produce in the cell surface charge and chemistry of arterial endothelium (E) and blood monocytes (M). Weekly, up to 8 weeks, after blood samples were taken for lipid analysis and blood cell preparation, the vasculature was washed free of blood and the endothelial luminal surface (ES) exposed to cytochemical probes for detecting charged groups, sialoconjugates and oligosaccharides. After fixation in situ, specimens collected from lesion-prone regions (aortic arch and coronary artery) and vena cava, were processed for electron microscopy. Morphometric analysis of tracer distribution on endothelium of nonlesional and lesional areas occurring in various stages of structural alterations, showed a remarkable resistance of the cell coat to very high level of serum cholesterol. In nonlesional zones the E surface charge and glycoconjugates were not significantly changed. In lesional areas, including those with forming fatty streaks, while cationic sites, galactosyl-, and N-acetyl-galactosaminyl residues were not altered whereas mannosyl moieties increased in density. A reduction in anionic groups and sialoconjugates appeared only after advanced extracellular and intracellular accumulation of lipoprotein-derived material and stromal proliferation developed in the intima. Moreover, these ES changes were usually restricted to the relatively rare E cells heavily loaded with lipid inclusions. The modulations were generally paralleled by comparable variations in the M surface. Regardless the extent of surface charge reduction, monocytes continued to migrate and foam cells to egress from the vessel wall. The results suggest that the onset and progression of early intimal lesions are not preceded but followed by significant restricted alterations in cell surface charge and glycoconjugates of arterial endothelium and monocytes.

Animals↗
PubMed1986

Study of the endogenous lectins of the human platelet plasma membrane.

Plasma membranes isolated from human blood platelets and human intact platelets, either fresh or previously fixed, were investigated for their possible content in intrinsic sugar-binding proteins (lectins) which can specifically bind monosaccharides such as fucose, mannose, and galactose. Light and heavy fractions of platelet plasma membrane and intact platelets fixed with 2% formaldehyde and suspended in Tyrode's buffer pH 7.0 were incubated with either L-(1-3H)-fucose, D-(1-3H)-mannose or D-(1-3H)-galactose. Intact fresh platelets were incubated either with L-(1-3H)-fucose, D-(1-3H)-mannose, D-(1-3H)-galactose, D-(1-3H)-glucose and N-acetyl-D-(1-3H)-glucosamine or with the same monosaccharides radiolabeled with (14C). Samples of radiolabeled plasma membranes and intact platelets were electrophoresed on polyacrylamide gels, and prepared for spectrometry. Results indicate that isolated human platelet plasma membranes contain sugar-binding proteins, especially for L-fucose, for which the binding values in the light fraction are about 4 times greater than for D-galactose. For the heavy fraction, the binding value for L-fucose is about 12 and 89 times higher than for D-mannose and D-galactose, respectively. Similar to plasma membrane fractions, fixed intact human platelets bind predominantly L-fucose, followed in decreasing binding values by D-mannose and D-galactose. Unlike these, fresh human platelets bind mainly D-galactose, followed in affinity values by D-glucose, L-fucose and D-mannose and to a lesser extent by N-acetyl-D-glucosamine. The binding of radiolabeled sugars to isolated platelet plasma membranes and to the previously fixed intact platelets suggests that, an endogenous lectin-like activity is expressed by the platelet membrane.

Acetylglucosamine↗
PubMed1986

Specific binding sites for albumin restricted to plasmalemmal vesicles of continuous capillary endothelium: receptor-mediated transcytosis.

The interaction of homologous and heterologous albumin-gold complex (Alb-Au) with capillary endothelium was investigated in the mouse lung, heart, and diaphragm. Perfusion of the tracer in situ for from 3 to 35 min was followed by washing with phosphate-buffered saline, fixation by perfusion, and processing for electron microscopy. From the earliest time examined, one and sometimes two rows of densely packed particles bound to some restricted plasma membrane microdomains that appeared as uncoated pits, and to plasmalemmal vesicles open on the luminal front. Morphometric analysis, using various albumin-gold concentrations, showed that the binding is saturable at a very low concentration of the ligand and short exposure. After 5 min, tracer-carrying vesicles appeared on the abluminal front, discharging their content into the subendothelial space. As a function of tracer concentration 1-10% of plasmalemmal vesicles contained Alb-Au particles in fluid phase; from 5 min on, multivesicular bodies were labeled by the tracer. Plasma membrane, coated pits, and coated vesicles were not significantly marked at any time interval. Heparin or high ionic strength did not displace the bound Alb-Au from vesicle membrane. No binding was obtained when Alb-Au was competed in situ with albumin or was injected in vivo. Gold complexes with fibrinogen, fibronectin, glucose oxidase, or polyethyleneglycol did not give a labeling comparable to that of albumin. These results suggest that on the capillary endothelia examined, the Alb-Au is adsorbed on specific binding sites restricted to uncoated pits and plasmalemmal vesicles. The tracer is transported in transcytotic vesicles across endothelium by receptor-mediated transcytosis, and to a lesser extent is taken up by pinocytotic vesicles. The existence of albumin receptors on these continuous capillary endothelia may provide a specific mechanism for the transport of albumin and other molecules carried by this protein.

Animals↗
PubMed1986

Further evidence for the distribution and nature of histamine receptors on microvascular endothelium.

The localization of histamine receptors and their classes on the microvascular endothelium was assessed by using the electron-opaque conjugate histamine-ferritin (HF). In order to further check the specific binding of this conjugate, two compounds chemically similar to histamine but biologically inactive, tele-methylhistamine (t-MH) and 4-pyridylethylamine (PEA), and a specific H2 histamine receptor antagonist, SK&F 93479, were used. Glutaraldehyde-activated ferritin was covalently coupled with either histamine, as a biologically active HF conjugate, or with tele-methylhistamine, as a biologically inactive conjugate tele-methylhistamine-ferritin (t-MHF). The purity of the conjugates was determined by thin-layer chromatography. The HF conjugate (25 mg protein/100 g body weight) was perfused into RAP mice and bipolar microvascular fields of the diaphragm were fixed and processed for electron microscopy. The HF conjugate decorated restricted domains of the luminal aspect of the microvascular endothelium. The specific density of HF binding, recorded as the average number of binding sites per square micrometer of the luminal endothelial surface, was relatively high in venules (18.46 +/- 5.73) and lower in arterioles (12.89 +/- 6.13) and capillaries (9.51 +/- 4.20). The binding specificity of the HF conjugate was assessed through six groups of experiments: perfusion before HF conjugate with either histamine, tele-methylhistamine or 4-pyridylethylamine, perfusion with t-MHF conjugate only, or administration before t-MHF conjugate of either histamine or tele-methylhistamine. The statistical significance of the data was analyzed by using the "F" distribution test and the "Wilcoxon-Mann-Whitney rank-sum test". Both tele-methylhistamine and 4-pyridylethylamine as well as histamine, showed a higher competition on arterioles and venules than on capillaries. Neither tele-methylhistamine nor histamine inhibited the binding of t-MHF conjugate on the endothelium. Experiments using SK&F 93479 as a very potent H2 histamine receptor antagonist perfused before HF administration, suggested the existence of H2 histamine receptors at a higher concentration in venules (47.03%) and arterioles (38.80%) than in capillaries (14.16%). These findings demonstrate that HF conjugate binds specifically to the histamine receptors of microvascular endothelium, which are characteristically frequent and predominantly of H2 type in venules.

Animals↗
PubMed1986

Changes in the organization of membrane lipids during human platelet activation. Study by fluorescent and freeze-fracture cytochemistry.

Modifications in the membrane lipid organization of human platelets activated with different agents (adenosine 5'-diphosphate, thrombin, collagen type I, and monosaccharides such as fucose, mannose, and galactose) were analyzed in vitro by using three lipid markers. Cholesterol was detected upon interaction with filipin, the anionic phospholipids were reacted with polymyxin B, and alterations in the degree of lipid packing were evaluated with the lipophilic fluorescent probe merocyanine 540, which reportedly inserts into bilayer domains whose lipids are more disordered. Filipin-sterol complexes and polymyxin B-anionic phospholipid complexes form characteristic membrane deformations which were examined in freeze-fracture preparations, whereas the merocyanine 540 binding to platelet membrane was recorded by fluorescent microscopy. In contrast to the resting cells, thrombin-stimulated platelets displayed an uneven distribution of filipin-sterol complexes which occurred in much higher density on the cell body than on pseudopods: on the latter, apparently cholesterol-free domains were very common. Unlike the non-stimulated cells, the platelets aggregated with the various agents employed showed characteristic polymixin B-anionic phospholipid complexes deformations of plasmalemma suggesting the appearance in uneven concentration of anionic phospholipids in the outer membrane leaflet. Incubation with merocyanine 540 did not result in staining of resting platelets when these were maintained in plasma, but a slight fluorescence was observed when platelets were kept in Tyrode buffer. However, platelets stimulated with thrombin, collagen type I, and monosaccharides bound very heavily the fluorescent dye; platelets aggregated with adenosine-5'-diphosphate bound only small amounts of merocyanine 540. The results showed that, during activation by different agents, modifications in lipid membrane organization include alterations in cholesterol and anionic phospholipid distribution, transbilayer movement of anionic phospholipids accompanied by more disordered membrane.

Animals↗
PubMed1986

Prelesional events in atherogenesis. Accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit.

Biochemical, physiologic, and ultrastructural modifications which appear in the aortic intima and atrioventricular valves before monocyte diapedesis and foam cell formation were investigated in rabbits fed a cholesterol-rich diet. In the first 2 weeks of the diet, while plasma beta-VLDL cholesterol was increased up to 15-fold, the intima showed an enhanced uptake and deposition of dietary 3H-cholesterol, 125I-beta-VLDL, and the fluorescent beta-VLDL-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine conjugate. beta-VLDL-gold complex perfused in situ was transcytosed across endothelium by plasmalemmal vesicles. Concomitantly, within the intima, a progressive accumulation of extracellular densely packed uni- or multilamellar vesicles took place. These commonly occurred in cell-free subendothelial spaces and were not associated with any sign of cytolysis. In freeze-fracture preparations, these vesicles appeared as smooth surfaces, suggesting the absence of translamellar proteins. Upon incubation with filipin, these extracellular liposomes (EL) displayed characteristic approximately 20 nm filipin-sterol complexes, revealing the presence of preparations unesterified cholesterol in the phospholipid lamellas. EL deposition was paralleled by proliferation of basal lamina-like material, microfibrils, and proteoglycans, and continued to increase during foam cell formation. For the entire period of our experiments, the endothelium was morphologically intact, and no platelet involvement was detected. The results show that an early prelesional ultrastructural change in lesion-prone aortic and valvular areas is the accumulation of extracellular phospholipid liposomes rich in unesterified cholesterol.

Animals↗
PubMed1985

Interactions of endogenous lipoproteins with capillary endothelium in spontaneously hyperlipoproteinemic rats.

In spontaneously hyperlipoproteinemic old Sprague-Dawley rats, endogenous lipoproteins (LP) in the size range of 15 to 40 nm were directly visualized within the blood vessels due to specimen mordanting with tannic acid. LP morphometric analysis at the level of the endothelium of diaphragm capillaries revealed that particles of the dimensions of low-density lipoproteins, high-density lipoproteins (HDL1), and very low density lipoproteins occur in endothelial structures involved in receptor-mediated endocytosis coated pits-vesicles, endosomes, lysosomes) and transcytosis (plasmalemmal vesicles and transendothelial channels). No such particles could be detected in the intercellular junctions. Intravenously injected cationized ferritin (CF) of pI 8.4 bound uniformly to LP forming an CF-LP complex. Examined at 5, 20, and 60 min after CF administration, the CF-LP complex was found to be taken up by endothelium only by endocytosis (adsorptive via coated pits-vesicles, and fluid phase through a fraction of plasmalemmal vesicles). CF-LP complexes are progressively accumulated within lysosomes. These findings reveal the importance of the net surface charge of macromolecular complexes for their intracellular sorting and fate.

Animals↗
PubMed1985

Anionized and cationized hemeundecapeptides as probes for cell surface charge and permeability studies: differentiated labeling of endothelial plasmalemmal vesicles.

To obtain small membrane markers easily accessible to the charged groups of the cell surface, we prepared, from hemeundecapeptide (HUP), three derivatives that maintain the peroxidatic activity: the anionized hemeundecapeptide, Mr 1,963, estimated diameter 1.68 nm, pl 3.5, for the detection of basic groups; and both a cationized hemeundecapeptide containing predominantly tertiary amino groups, Mr 2,215, estimated diameter 1.75 nm, pl 9.0, and a cationized hemeundecapeptide containing only primary amino groups, Mr 2,271, estimated diameter 1.75 nm, pl 10.6, for labeling acidic residues. The markers were perfused in situ in mice to label the luminal surface of fenestrated endothelium of pancreatic capillaries. Specimens were processed through the cytochemical reaction for peroxidatic activity and examined by electron microscopy. The anionized HUP and HUP (pl 4.85) marked the plasmalemma proper, the coated pits, and the membrane and diaphragms of plasmalemmal vesicles and transendothelial channels. The cationized HUP containing predominantly tertiary amino groups (pl 9.0) decorated all cell surface components with the exception of plasmalemmal vesicles and channels; the latter were, however, labeled by the cationized HUP containing only primary groups (pl 10.6), which suggests that these structures contain on their luminal surface very weak acidic residues of high pKa values. The fact that the membrane of plasmalemmal vesicles can discriminate against permeant cationic macromolecules only up to a pl of approximately 9.0 indicates that in the electrostatic restriction there is a charge limit. In the case of fenestrated capillary endothelium, the upper charge limit seems to be a pl of approximately 9.0. In these vessels, the charge discrimination is effective for molecules as small as 2 nm.

Animals↗
PubMed1985

Differentiated microdomains of the luminal plasmalemma of murine muscle capillaries: segmental variations in young and old animals.

We investigated the luminal surface of the continuous endothelium of the microvasculature of the murine heart and diaphragm to find out whether it has differentiated microdomains. The probes were ferritin molecules, cationized to pI's 6.8, 7.15, 7.6, 8.0 and 8.4, which were introduced by retrograde or anterograde perfusion through the aorta or vena cava after the blood was removed from the vasculature. The pattern of labeling was analyzed by electron microscopy and assessed quantitatively by morphometry in arterioles, capillaries, and venules identified in bipolar microvascular fields in the diaphragm. The results showed that the plasmalemma proper was heavily but discontinuously labeled by all cationized ferritins (CF) used, the labeling being less extensive on the venular endothelium. CF had access as individual molecules to a fraction of the vesicular population opened on the luminal front of the endothelium. Plasmalemmal vesicle labeling increased from approximately 10 to approximately 25% as the pI decreased from 8.4 to 6.8. Vesicle labeling also increased with CF concentration in the perfusate. All CF binding sites were removed by pronase and papain. Heparinase and heparitinase caused only a slight reduction in CF labeling. Neuraminidase decreased the extent and density of labeling, especially on the plasmalemma proper of the venular endothelium; this decrease was particularly pronounced in old animals.

Aging↗
PubMed1984

Partial chemical characterization of the anionic sites in the basal lamina of fenestrated capillaries.

The distribution of anionic sites in the basal laminae of the blood capillaries of the murine pancreas was studied in specimens fixed in ruthenium red (RR)-glutaraldehyde mixtures. The sites appeared as discrete, small (6 to 18 nm) particles distributed throughout the three laminae but concentrated primarily in the lamina rara externa, in which--spaced 80-100 nm apart--they formed a planar, partially ordered lattice comparable to that revealed by cationized ferritin in previous studies (M. Simionescu, N. Simionescu, and G. E. Palade, 1982, J. Cell Biol. 95, 425-434). The chemical nature of the anionic sites was explored by incubating fresh tissue specimens in solutions of selected enzymes before fixation in RR-glutaraldehyde mixtures. Pronase P and papain removed completely the anionic sites and left behind an extensively degraded and disorganized basal lamina. Trypsin caused the removal of anionic sites only, did not degrade the rest of the basal lamina, but detached it completely from the endothelium. Chondroitinase ABC reduced slightly the size and the surface density of RR-stainable particles, and detached focally the rest of the basal lamina from the endothelium and pericytes. Crude heparinase caused a nearly complete removal of anionic sites, and pure heparitinase gave comparable but less extensive results. Similar effects were recorded on the basal laminae of smooth muscle fibers and pancreatic acini and ducts. The results indicate that the anionic sites of all basal laminae examined are contributed primarily by heparin sulfate proteoglycans and trace amounts of chondroitin sulfate proteoglycans.

Animals↗
PubMed1983

Changes concerning the external structure of peritoneal macrophages, due to the DQ-12 standard dust.

The cytotoxic activity of the DQ-12 standard dust on the external structure of peritoneal macrophages, after intraperitoneal introduction, was investigated, vs nontreated macrophages, and kept in in vitro cultures for 3, 6, and 24 hr. The alterations related to the external structure were investigated by scanning electron microscopy (SEM), clearly showing that cell damage can take place when the dust contacts the external surface of the cell, as already shown by the work of Johnson and Davies (paper presented at the International Workshop on the in Vitro Effects of Mineral Dusts, South Wales, U.K., 1979) and Miller (Environ. Res. 20, 162-182, 1979).

Animals↗
PubMed1983

Visualization of the binding, endocytosis, and transcytosis of low-density lipoprotein in the arterial endothelium in situ.

We investigated the interaction and transport of low-density lipoprotein (LDL) through the arterial endothelium in rat aorta and coronary artery, by perfusing in situ native, untagged human, and rat LDL. The latter was rendered electron-opaque after it interacted with the endothelial cell and was subsequently fixed within tissue. We achieved LDL electron-opacity by an improved fixation procedure using 3,3'-diaminobenzidine, and mordanting with tannic acid. The unequivocal identification of LDL was implemented by reacting immunocytochemically the perfused LDL with anti LDL-horseradish peroxidase conjugate. Results indicate that LDL is taken up and internalized through two parallel compartmented routes. (a) A relatively small amount of LDL is taken up by endocytosis via: (i) a receptor-mediated process (adsorptive endocytosis) that involved coated pits/vesicles, and endosomes, and, probably, (ii) a receptor-independent process (fluid endocytosis) carried out by a fraction of plasmalemmal vesicles. Both mechanisms bringing LDL to lysosomes supply cholesterol to the endothelial cell itself. (b) Most circulating LDL is transported across the endothelial cell by transcytosis via plasmalemmal vesicles which deliver LDL to the other cells of the vessel wall. Endocytosis is not enhanced by increasing LDL concentration, but the receptor-mediated internalization decreases at low temperature. Transcytosis is less modified by low temperature but is remarkably augmented at high concentration of LDL. While the endocytosis of homologous (rat) LDL is markedly more pronounced than that of heterologous (human) LDL, both types of LDL are similarly transported by transcytosis. These results indicate that the arterial endothelium possesses a dual mechanism for handling circulating LDL: by a high affinity process, endocytosis secures the endothelial cells' need for cholesterol; by a low-affinity nonsaturable uptake process, transcytosis supplies cholesterol to the other cells of the vascular wall, and can monitor an excessive accumulation of plasma LDL. Since in most of our experiments we used LDL concentrations above those found in normal rats, we presume that at low LDL concentrations saturable high-affinity uptake would be enhanced in relation to nonsaturable pathways.

Animals↗
PubMed1983

Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.

Animals↗