Maturation of myocardial capillaries in the fetal and neonatal rat: an ultrastructural study with a morphometric analysis of the vesicle populations

The ultrastructural morphology of the cellular and extracellular components of the developing myocardial capillary wall--from the 16-day-gestation fetus of the rat to the 21-day neonate--was examined. A morphometric analysis of plasmalemmal vesicles and of coated vesicles and pits of capillary endothelial cells was performed during the same developmental period. As the lateral extensions of the capillary endothelial cells change from irregular to regular in their thickness during development, there is an increase in the number of plasmalemmal vesicles and a progression from clusters of plasmalemmal vesicles to a uniform distribution in the endothelial cell. The ratio of vesicles which are open to the luminal front, which are "free" in the cytoplasm, or which are open to the abluminal front of the endothelial cell was consistent throughout development. The numerical density of plasmalemmal vesicles demonstrates a gradual and significant increase. In contrast, the numbers of coated vesicles and pits are variable within a very narrow range, and no pattern of increase or decrease is discernible during development. Similarly, there is no change in interendothelial cell junctions, which consist of occluding and primitive adhesive junctional types, during development. The lamina densa of the basal lamina gradually develops from discontinuous, patchy densities along the abluminal surface of the endothelial cells to a continuous and distinct layer by 21 days gestation. The presence of the proteoglycan species in the developing basal lamina was assessed with the cationic dye ruthenium red (RR), and the appearance of RR-marked proteoglycans was found to parallel the appearance of lamina densa material. found to parallel the appearance of lamina densa material. The RR sites appear discontinuously in patches; and later, the RR sites appear in a continuous and regular planar lattice in the lamina rara interna and externa at 21 days gestation. A complete array of RR-stainable anionic sites outside a continuous lamina densa near birth indicates that the basal laminae of developing capillaries in the heart are morphologically, and in part biochemically, mature by the end of the first neonatal week. Our results show that the endothelial cells and the subtending basal lamina of myocardial capillaries gradually mature morphologically during the final days of gestation and the first neonatal week.(ABSTRACT TRUNCATED AT 400 WORDS)     

The blood-brain barrier in a reptile, Anolis carolinensis

An electron microscopic study was made of the ultrastructure and permeability of the capillaries in the cerebral hemispheres of the lizard, Anolis carolinensis. The brain of Anolis is vascularized by a loop-type pattern consisting exclusively of arteriovenous capillary loops. The ultrastructure of the endothelium and the arrangement of the various layers from the capillary lumen to the central nervous tissue is similar to that of mammals. The endothelial cells form a continuous layer around the lumen and are joined by tight interendothelial junctions. The basal lamina of the endothelium is also continuous and encloses pericyte processes. The cells of the nervous tissue rest directly on the basal lamina of the capillary and are separated from each other by a 200 Å space. Intravenously injected horseradish peroxidase (MW 40,000) and ferritin (MW 500,000) were used to study the permeability of the capillaries. The entry of horseradish peroxidase and ferritin into the intercellular spaces of the brain is restricted by the tightness of the interendothelial junctions. No vesicular transport of either tracer occurs; however, ferritin does enter the endothelial cells in vacuoles. No tracer molecules are present in the basal lamina, pericytes, or nervous tissue. The different responses of the endothelial cell to the tracers used in this study suggest that endocytotic activities of endothelial cells involve different processes. Vacuoles formed by marginal folds, vacuoles formed by endothelial surface projections or deep invaginations of the plasma membrane, 600–800 Å vesicles, and coated vesicles all seem to differ in the nature of the substances which they endocytose.     

Myocardial capillaries in the fetal and the neonatal rat: a morphometric analysis of the maturing myocardial capillary bed

Developing myocardial capillaries from 16-day-gestation fetus to adult undergo several morphological changes including a thinning of the lateral extensions of the capillary endothelial cells, the formation of a basal lamina, and an increase in the number of plasmalemmal vesicles. A decrease in the extracellular space, an increase in the number of capillaries, and a decrease in the capillary diameter were also observed during the developmental period. In view of these ultrastructural changes, a morphometric analysis was made on the developing myocardial wall to demonstrate specific quantitative changes. The volumes which were occupied by capillary endothelial cells, capillary lumina, extracellular space, and myocardial myocytes within a reference volume of myocardium were measured; and we found that 8% of the reference myocardial volume was occupied by capillary endothelial cells, 85% was occupied by myocardial myocytes, 4% was occupied by capillary lumina, and, except for a significant change in extracellular space at 16 days gestation, 3% was occupied by extracellular space. Each volume ratio was found to be nearly constant throughout the studied period. In contrast to this constancy in the volume ratios, other parameters which were measured demonstrated significant changes during the developmental period studied. These overall changes include a 135% increase in capillary density, a 63% increase in luminal surface area of capillary endothelial cells, a 24% decrease in capillary diameter, a 12% decrease in diffusion distance, and a 35% decrease in the diameter of the erythrocyte population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic research on the capillary permeability of the primary portal plexus in rats in the prenatal period

Permeability of portal capillaries to intravascularly injected ionic lanthanum, ferritin and horse-radish peroxidase has been examined in rats on the 18th fetal day, and on days 1 and 9 of postnatal life. For several minutes, tracer molecules pass through the capillary wall and reach the median eminence. In the case of immature capillaries, the materials pass freely through the endothelial cells, and to a lesser extent are transferred via occasional plasmalemmal vesicles and fenestrae. As the maturation of capillaries proceeds their permeability via plasmalemmal vesicles and fenestrae increases considerably due to a gradual rise in the number of these structures. The plasmalemma of differentiated endothelial cells becomes impermeable to all the tracers. Only ionic lanthanum appears to penetrate through transendothelial channels and intercellular junctions between adjacent endothelial cells.     

Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.     

Increased capillary permeability in muscularis layer of rat intestine caused by kidney extract.

Kidney extract and synthetic angiotensin II were injected into bilaterally nephrectomized rats in dosages capable of raising the mean arterial pressure by about 20 mmHg. Changes in ultrastructure and permeability for ferritin molecules were then examined in capillaries located in muscularis layer of the intestinal walls. Kidney extract with a high renin content was obtained from the renal cortex of rats by means of stepwise centrifugation methods. Animals injected with saline served as controls. In rats receiving kidney extract tissue edema was observed in the spaces around the blood and lymphatic capillaries. In these spaces ferritin molecules accumulated in high concentration indicating plasma protein leakage. Ferritin molecules within the endothelium were restricted within plasmalemmal vesicles, but were not found within interendothelial junctions or within the cytoplasmic matrix. Morphometric analysis of vesicular transport in the endothelial cells revealed a significant increase in labeling rate for the vesicles with ferritin molecules. These results suggest that the kidney extract contains substance(s) which increase capillary permeability for plasma proteins at least via increased vesicular transport, resulting in tissue edema.     

Transendothelial transport of serum albumin: a quantitative immunocytochemical study

The steady-state distribution of endogenous albumin in mouse diaphragm was determined by quantitative postembedding protein A-gold immunocytochemistry using a specific anti-mouse albumin antibody. Labeling density was recorded over vascular lumen, endothelium, junctions, and subendothelial space. At equilibrium, the volume density of interstitial albumin was 18% of that in circulation. Despite this large difference in albumin concentration between capillary lumen and interstitium, plasmalemmal vesicles labeling was uniformly distributed across the endothelial profile. 68% of the junctions displayed labeling for albumin, which was however low and confined to the luminal and abluminal sides. The scarce labeling of the endothelial cell surface did not confirm the fiber matrix theory. The kinetics of albumin transcytosis was evaluated by injecting radioiodinated and DNP-tagged BSA. At 3, 10, 30, and 60 min, and 3, 5, and 24 h circulation time, blood radioactivity was measured and diaphragms were fixed and embedded. Anti-DNP antibodies were used to map the tracer in aforementioned compartments. A linear relationship between blood radioactivity and vascular labeling density was found, with a detection sensitivity approaching 1 gold particle per DNP-BSA molecule. Tracer presence over endothelial vesicles reached rapidly (10 min) a saturation value; initially localized near the luminal front, it evolved towards a uniform distribution across endothelium during the first hour. An hour was also needed to reach the saturation limit within the subendothelial space. Labeling of the junctions increased slowly, out of phase with the inferred transendothelial albumin fluxes. This suggests that they play little, if any, role in albumin transcytosis, which rather seems to proceed through the vesicular way.     

Correlated endothelial caveolin overexpression and increased transcytosis in experimental diabetes.

We investigated the mechanism by which diabetes renders the capillary endothelium more permeable to macromolecules in the lungs of short-term diabetic rats. We used quantitative immunocytochemistry (ICC) to comparatively assess the permeability of alveolar capillaries to serum albumin in diabetic and normoglycemic animals. The effect of diabetes on the population of endothelial caveolae was evaluated by morphometry and by ICC and immunochemical quantification of the amount of caveolin in the whole cell or associated with the purified endothelial plasma membrane. A net increase in the amount of serum albumin taken up by the plasmalemmal vesicles of alveolar endothelial cells and transported to the interstitium was documented in diabetic animals. Interendothelial junctions were not permeated by albumin molecules. The alveolar endothelial cells of hyperglycemic rats contain more caveolae (1.3-fold), accounting for a larger (1.5-fold) fraction of the endothelial volume than those of normal animals. The hypertrophy of the caveolar compartment is accompanied by overexpression of endothelial caveolin 1. Although the aggregated thickness of the endothelial and alveolar epithelium basement membranes increases in diabetes (1.3-fold), the porosity of this structure appears to be unchanged. Capillary hyperpermeability to plasma macromolecules recorded in the early phase of diabetes is explained by an intensification of transendothelial vesicular transport and not by the destabilization of the interendothelial junctions.     

Freeze-fracture of capillary endothelium in rat brain

Summary The rat brain capillary was studied with freeze-fracture technique. The attached plasmalemmal vesicles were quite few in number on the luminal front and sometimes numerous on the contraluminal side. The fracture appearance of some tight junctions showed interconnecting ridges on face A and complementary furrows devoid of particles on face B, comparable to the common tight junction in the normal epithelia. Other tight junctions revealed a preferential disposition of quasicontinuous rows of particles on shallow furrows of face B, resembling the tight junctional strands of capillary endothelium in non-cerebral tissues. Either behavior is probably due to the difference in the fracture plane around the single fibril. In addition, the tight junctional strand could surround the perimeter of the endothelial cell completely although the exposed strand of tight junction was limited in length.     

Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

Cell-cell junctions of dermal microvascular endothelial cells contain tight and adherens junction proteins in spatial proximity

Endothelial cell-cell contacts control the vascular permeability, thereby regulating the flow of solutes, macromolecules, and leukocytes between blood vessels and interstitial space. Because of specific needs, the endothelial permeability differs significantly between the tight blood-brain barrier endothelium and the more permeable endothelial lining of the non-brain microvasculature. Most likely, such differences are due to a differing architecture of the respective interendothelial cell contacts. However, while the molecules and junctional complexes of macrovascular endothelial cells and the blood-brain barrier endothelium are fairly well characterized, much less is known about the organization of intercellular contacts of microvascular endothelium. Toward this end, we developed a combined cross-linking and immunoprecipitation protocol which enabled us to map nearest neighbor interactions of junctional proteins in the human dermal microvascular endothelial cell line HMEC-1. We show that proteins typically located in tight or adherens junctions of epithelial cells are in the proximity in HMEC-1 cells. This contrasts with the separation of the different types of junctions observed in polarized epithelial cells and "tight" endothelial layers of the blood-brain barrier and argues for a need of the specific junctional contacts in microvascular endothelium possibly required to support an efficient transendothelial migration of leukocytes.     

Differentiated microdomains on the luminal surface of the capillary endothelium. I. Preferential distribution of anionic sites

Cationized ferritin (CF), introduced systemically in vivo or by perfusion in situ, binds preferentially to certain microdomains of the luminal plasmalemma of fenestrated capillaries (mouse pancreas and jejunum). The density and affinity of binding decrease in the following order: fenestral diaphragms greater than coated pits greater than plasmalemma proper. CF binds neither to the membrane of plasmalemmal vesicles and transendothelial channels nor to the corresponding stomatal diaphragms. The distribution pattern is the same when glutaraldehyde fixation precedes the administration of the tracer by perfusion, provided fixation is followed by quenching of residual free aldehyde groups. A much smaller cationic probe (alcian blue) perfused together with the fixative reveals a similar distribution pattern. The functional implications of the association of these microdomains with structures involved in capillary permeability are discussed.     

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.     

PERMEABILITY OF MUSCLE CAPILLARIES TO EXOGENOUS MYOGLOBIN

Whale skeletal muscle myoglobin (mol wt 17,800; molecular dimensions 25 x 34 x 42 Å) was used as a probe molecule for the pore systems of muscle capillaries. Diaphragms of Wistar-Furth rats were fixed in situ at intervals up to 4 h after the intravenous injection of the tracer, and myoglobin was localized in the tissue by a peroxidase reaction. Gel filtration of plasma samples proved that myoglobin molecules remained in circulation in native monomeric form. At 30–35 s postinjection, the tracer marked ~75% of the plasmalemmal vesicles on the blood front of the endothelium, 15% of those located inside and none of those on the tissue front. At 45 s, the labeling of vesicles in the inner group reached 60% but remained nil for those on the tissue front. Marked vesicles appeared on the latter past 45 s and their frequency increased to ~80% by 60–75 s, concomitantly with the appearance of myoglobin in the pericapillary spaces. Significant regional heterogeneity in initial labeling was found in the different segments of the endothelium (i.e., perinuclear cytoplasm, organelle region, cell periphery, and parajunctional zone). Up to 60 s, the intercellular junctions and spaces of the endothelium were free of myoglobin reaction product; thereafter, the latter was detected in the distal part of the intercellular spaces in concentration generally equal to or lower than that prevailing in the adjacent pericapillary space. The findings indicate that myoglobin molecules cross the endothelium of muscle capillaries primarily via plasmalemmal vesicles. Since a molecule of this size is supposed to exit through both pore systems, our results confirm the earlier conclusion that the plasmalemmal vesicles represent the large pore system; in addition, they suggest that the same structures are, at least in part, the structural equivalent of the small pore system of this type of capillaries.     

Pitavastatin Strengthens the Barrier Integrity in Primary Cultures of Rat Brain Endothelial Cells

Statins have a neuroprotective effect in neurological diseases, a pleiotropic effect possibly related to blood–brain barrier (BBB) function. We investigated the effect of pitavastatin on barrier functions of an in vitro BBB model with primary cultures of rat brain capillary endothelial cells (RBEC). Pitavastatin increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), at a concentration of 10^?8 M, and decreased the endothelial permeability for sodium fluorescein through the RBEC monolayer. The increase in TEER was significantly reduced in the presence of isoprenoid geranylgeranyl pyrophosphate, whereas farnesyl pyrophosphate had no effect on TEER. Our immunocytochemical and Western blot analyses revealed that treatment with pitavastatin enhanced the expression of claudin-5, a main functional protein of TJs. Our data indicate that pitavastatin strengthens the barrier integrity in primary cultures of RBEC. The BBB-stabilizing effect of pitavastatin may be mediated partly through inhibition of the mevalonate pathway and subsequent up-regulation of claudin-5 expression.     

Formation and regression of capillary sprouts in corpora lutea of immature superstimulated golden hamsters

The ultrastructure of developing and regressing capillary sprouts was studied in corpora lutea of immature golden hamsters between days 4 and 7 after the application of serum gonadotrophin of pregnant mares (PMSG). Horseradish peroxidase (HRP), an endothelial tracer, was localized by ultrahistochemistry. The vascular permeability of HRP was quantified by an enzyme assay in ovarian homogenates. Sprouting endothelial cells looked activated. They showed micropinocytotic vesicles in a high endothelium surrounded by basal laminae. Early capillary growth was at its maximum on day 4 after PMSG. Advanced capillary growth was seen on days 4 and 5 after PMSG. The vascular lumina were formed by dilatation of the interendothelial space. Regression of capillary sprouts started on day 5, was most intense on day 6 and negligible on day 7. Two processes of regression were observed. One led to a complete destruction, the other to an incomplete one. Vascular permeability decreased between days 5 and 6 after PMSG. It is concluded that the corpus luteum can be viewed as a physiological model of angiogenesis.     

Ultrastructural observations at pineal gland capillaries in four rodent species.

The fine structure of the capillaries of the pineal glands of the rat, mouse, chinchilla, and ground squirrel were investigated. The pineal endothelial cells in the rat, mouse and ground squirrel were often composed of attenuated cytoplasmic portions which contained numerous fenestrations, in contrast to pineal capillaries in the chinchilla which were lined by thick non-fenestrated endothelial cells. Marked morphological differences were also apparent in terms of the types of vesicles within the cytoplasm and abutting on the cell surface of pineal endothelial cells from the various species investigated. The interendothelial junctions exhibited remarkable species differences with the chinchilla pineal possessing typical tight endothelial junctions while those in the rat, mouse and ground squirrel lacked such endothelial cell associations. Generally, capillary lining cells in the chinchilla pineal resembled similar cells within the brain, while endothelial cells in pineal glands of rat, mouse and ground squirrel were more typical of those found in other endocrine organs. Species differences in the structure of the pineal capillaries may represent physiological differences as well.     

The role of liver endothelium in the binding and uptake of ceruloplasmin: studies with colloidal gold probe

To determine the mode of uptake of ceruloplasmin (CP) by liver, the protein was labeled with colloidal gold and infused into the portal vein. In cold almost all probes bound to the sinusoidal endothelium, and at 37 degrees C internalization via a system of coated pits and vesicles occurred. Only rarely did the probe appear to bypass the endothelium, moving to the abluminal side through the gaps between endothelial cells. In the endothelial cytoplasm, the probe was seen in coated vesicles, endosomes, tubules, and large vesicles which may have formed by fusion of endosomes and tubules. Moreover, externalization of the probe to the abluminal side was noted, and this also occurred via a system of coated vesicles. The findings suggest that the uptake of CP in the liver may be primarily a transendothelial phenomenon (transcytosis).     

The blood-brain barrier in vitro: Ten years of research on microvessels isolated from the brain

The cerebral microvessels represent a distinctive compartment in the brain with unique physiological, biochemical and morphological properties. The barrier which separates the general circulation from the central nervous system is a complex mechanism that has a relative impermeability to a number of blood-borne substances, but—at the same time—operates with several specific carriers for the various compounds needed in the brain. Morphologically, the presence of tight interendothelial junctions, the paucity of pinocytotic vesicles in the endothelium and the absence of fenestrations are the hallmarks of this barrier.     

Surface charges associated with fenestrated brain capillaries. II. In vivo studies on the role of molecular charge in endothelial permeability

We report on the effect of the net charge of a tracer (ferritin) on its permeability in fenestrated capillaries of the brain. Our experiments show that the charge of this tracer actually influences its interaction with the endothelium. Three phases of tracer-endothelial interaction could be discriminated. Anionic and slightly cationic derivatives (pH 4.5-7.8) do not show any affinity to the luminal endothelial membrane. Ferritin derivatives with a pI value between 7.8 and 9.3 result in the labeling of the fenestrae without coating additional luminal plasmalemmal structures (i.e., coated pits and plasmalemmal vesicles). Tracers with a high positive net charge (pI greater than 9.3) led to their endocytotic uptake and extravasation by some transcytotic mechanism. Extravasated cationic ferritin accumulates in the endothelial basement membrane and binds to striated collagen fibrils. It is suggested that the pericapillary collagen fibrils of fenestrated brain capillaries act as a charge filter with respect to macromolecules.