Endothelial Fenestrae Research Articles

Serotonin stimulates a Ca 2+ permanent nonspecific cation channel in hepatic endothelial cells

The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.

The retinal pigment epithelium induces fenestration of endothelial cells in vivo.

Rodent photoreceptor dystrophies are characterized by late stage ingrowth of retinal blood vessels into the retinal pigment epithelium (RPE) where they proliferate. Some of these vessels develop the fenestrated phenotype of the choriocapillaris (CC). To determine if development of fenestrae in these endothelial cells is a function of the duration of time the endothelial cell had been encapsulated by the RPE, we did an ultrastructural morphometric study of these vessels in urethane induced photoreceptor degeneration in Long-Evans rats. Retinas of animals aged 20, 24, 40 and 56 weeks were studied. The fraction of vessel profiles within the RPE that had fenestrated endothelial cells increased from 10% to 90% between 20 to 56 weeks. The average number of fenestrae per vessel increased approximately 25 fold between 20 and 24 weeks but stabilized after that, despite a decrease in the number of vessels present at 56 weeks. A large number of degenerated retinal vessel profiles were seen in the RPE at 40 weeks. These facts support the idea that the presence of the RPE induces endothelial cell fenestrae, and also show that a complex process of remodelling including proliferation and degeneration is occurring in these vessels. Analogies between the basic cell biology of neovascularization occurring in these rodent models and that of proliferative diabetic retinopathy and age-related macular degeneration are discussed.

Fine structure of the glomerular basement membrane of the rat kidney visualized by high-resolution scanning electron microscopy

The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5- to 9-nm-thick fibrils, had pores 11-30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10-24 nm wide) than those near the filtration slits (16-32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.

Altered pathogenicity in the liver induced by a mouse hepatitis virus type 3 thermosensitive mutant

Intraperitoneal inoculation into sensitive BALB/c mice of D85, a thermosensitive (ts) mutant, provokes acute hepatitis followed by recovery of the mice. The ts mutant was able to replicate in the liver. However, the maximal viral titre was obtained 2 days later than was the case with the wild-type (wt) MHV 3 infection; the viral antigens remained localized within small foci and no invasion of the entire liver was observed. The hepatocytes infected with D85 showed strong steatosis similar to that induced by wt virus, but the other lesions induced by MHV 3 (closing of endothelial cell fenestrae and hepatocytolysis) were not seen. An important feature noticed with the D85 mutant concerned the establishment, in the surviving animals, of persistent infection: this phenomenon was demonstrated by the decrease of viral titre in the liver, viral RNA detection, and the fact that viral antigens gradually decreased until the 3rd month post-infection.

Structure and permeability differ in subepithelial villus and peyer's patch follicle capillaries

Intravenously injected sheep antilaminin immunoglobulin G conjugated with horseradish peroxidase uniformly labeled the basement membrane of mouse villus epithelium but not that of Peyer's patch follicle domes, although laminin is abundant in both. This suggested differences in the permeability of capillaries underlying these epithelia. Therefore, the fine structure of mouse subepithelial villus and follicle capillaries and their permeability to selected macromolecules was compared. Fenestrae, abundant in villus capillaries, were extremely rare in dome capillaries as assessed by electron microscopy of tissue sections and freeze-fracture replicas. Two minutes after IV-injected horseradish peroxidase and 9 minutes after IV-injected hemoglobin, reaction product decorated the pericapillary space of 97% and 95%, respectively, of capillary profiles in the upper half of villi but only 28% and 2%, respectively, of capillary profiles in the upper half of patch domes. Reaction product was intense surrounding most villus capillaries but, when present, was faint in dome capillary adventitia. These results indicate that most subepithelial capillaries in mouse Peyer's patch domes, unlike those in villi, generally lack endothelial fenestrae, and the dome capillary network is less permeable to some macromolecules than that of the villus.

Structure and Permeability Differ in Subepithelial Villus and Peyer’s Patch Follicle Capillaries

Intravenously injected sheep antilaminin immunoglobulin G conjugated with horseradish peroxidase uniformly labeled the basement membrane of mouse villus epithelium but not that of Peyer's patch follicle domes, although laminin is abundant in both. This suggested differences in the permeability of capillaries underlying these epithelia. Therefore, the fine structure of mouse subepithelial villus and follicle capillaries and their permeability to selected macromolecules was compared. Fenestrae, abundant in villus capillaries, were extremely rare in dome capillaries as assessed by electron microscopy of tissue sections and freeze-fracture replicas. Two minutes after IV-injected horseradish peroxidase and 9 minutes after IV-injected hemoglobin, reaction product decorated the pericapillary space of 97% and 95%, respectively, of capillary profiles in the upper half of villi but only 28% and 2%, respectively, of capillary profiles in the upper half of patch domes. Reaction product was intense surrounding most villus capillaries but, when present, was faint in dome capillary adventitia. These results indicate that most subepithelial capillaries in mouse Peyer's patch domes, unlike those in villi, generally lack endothelial fenestrae, and the dome capillary network is less permeable to some macromolecules than that of the villus.

Functional variations in liver tissue during the implantation process of metastatic tumour cells

We have examined several properties of sinusoidal cells in the unaffected tissue of micrometastasis-containing livers. Tumour cells from either B16 melanoma (B16F10) or Lewis lung carcinoma (LLC) were injected intrasplenically in syngeneic mice and sacrificed on the 7th day. Light and scanning electron microscopy (SEM) showed tumour cells in hepatic veins and sinusoids in close contact with endothelial walls and macrophages. Following quantitative analysis of SEM images from sinusoidal walls it was found that endothelial fenestrae from B16F10 or LLC-colonized livers were diffusely reduced both in size and density/microns 2 throughout the sinusoid wall, although especially affected zone 3 segments. Following the intrasplenic injection of 1 microns fluorescent latex particles 1 h prior to sacrifice of the mice a significant reduction of the latex particle uptake by sinusoidal cells was detected in B16F10-colonized livers (27% of controls) which was in contrast to the significant increase in LLC-colonized mice (180% of controls). Despite the focal character of the tumour cell implantation process, hepatic sinusoidal cells reacted diffusely to metastatic cells. However, over liver acini, endothelial cell changes were mainly expressed in zone 3 while phagocytic properties mainly varied in zone 1 and depending on the tumour type. Although the significance of these sinusoidal changes on metastatic development is unclear, data suggests that "soil" conditions in the liver are different before and after being metastasized by tumour cells.

Fine structure and function of kupffer cells

Kupffer cells are macrophages that are attached to the luminal surface or inserted in the endothelial lining of hepatic sinusoids. In this site, Kupffer cells play a key role in host defense by removing foreign, toxic and infective substances from the portal blood and by releasing beneficial mediators. Under some conditions, toxic and vasoactive substances also are released from Kupffer cells which are thought to play a role in a variety of liver diseases. Many of these activities may be modulated by the levels of gut derived endotoxin normally present in the portal blood. The ultrastructural aspects of Kupffer cell structure function in situ are best studied using perfused-fixed livers. In fixed livers, transmission and scanning electron microscopy reveal Kupffer cells during health to be irregular in shape with their exposed surfaces presenting numerous microvilli, filopodia, and lamellopodia. Long filopodia penetrate endothelial fenestrae to secure Kupffer cells to the sinusoid lining. Specific membrane invaginations known as worm-like bodies or vermiform processes are seen in the cytoplasm of Kupffer cells as are numerous endocytotic vesicles and lysosomes which vary in density, shape and size. Sometimes, annulate lamellae connected to the rough endoplasmic reticulum also are found. The principal endocytic mechanisms of Kupffer cells are phagocytosis of particulates and cells, and bristle-coated micropinocytosis for fluid-phase endocytosis of smaller substances. Many of these events are mediated by specific receptors. In some species, Kupffer cells can be distinguished from other sinusoidal lining cells and monocytes by specific cytoplasmic staining or monoclonal antibodies. Kupffer cells have been shown to be of monocytic origin as well as having the capacity for self-replication.

Ultrastructural changes in odontoblasts and pulp capillaries following cavity preparation in rat molars.

Responses of odontoblasts and pulp capillaries to cavity preparation were investigated in the upper first molar teeth of rats, using light and transmission electron microscopy. At 100 days of age, the blood vessels of the pulp formed a subodontoblastic network consisting of continuous capillaries at a short distance from the odontoblast layer. Cavity preparation caused the displacement of some odontoblasts into the dentinal tubules, while others were separated from the predentin by rapid inflammatory exudation after drilling. The subodontoblastic capillary network under the injured dentin was shifted inwards together with the separated odontoblasts. The endothelium of the shifted capillaries showed a remarkable increase of pinocytotic vesicles, an event thought to be closely related to the formation of the exudative lesion. By one day after cavity preparation, most of the damaged odontoblasts had degenerated. Many cells with high nucleus/cytoplasm (N/C) ratios and prominent nucleoli accumulated around the subodontoblastic capillaries, some of which had many endothelial fenestrae facing these cells. These cells were suggestive of newly differentiating odontoblasts receiving nutritional supply from the capillaries. Three days after cavity preparation, newly differentiating odontoblasts took the place of the degenerated odontoblasts. They began to produce reparative dentin by five days after cavity preparation. Capillaries were located beneath the newly differentiating odontoblasts, but endothelial fenestrae gradually decreased in number. During the active reparative dentin formation, capillaries remained closely beneath the new odontoblast layer. Although the rate of reparative dentin deposition was not significantly lower than that in the primary dentin formation, one could not recognize an invasion of capillaries into the odontoblast layer nor a remarkable increase of endothelial fenestrae, both of which are common in active primary dentin formation. The results suggest that the function of capillaries differs between primary and reparative dentin formation.

Rat kidney glomerular basement membrane visualized in situ by embedment‐free sectioning and subsequent platinum‐carbon replication

Following the removal of polyethylene glycol (PEG) from thin sections, and viewing through the endothelial fenestrae and/or the interpedicel spaces, the rat renal glomerular basement membrane in situ was revealed to consist of meshworks and to be electron-transparent when examined at right angles to the plane of the membrane. By subsequent platinum replication of the embedment-free sections, the lamina densa of the basement membrane appeared as a veil composed of rather closely packed particles. The architecture of the split diaphragm and the surface morphology of the endothelial cell membrane were also clearly revealed. The present results indicate that the PEG method, with or without replication, can provide valuable information on basement membrane morphology.

Ultrastructure and quantitative composition of isolated endothelial cells of rat liver

Isolated endothelial cells (n 110; 350-540 microns3 in volume) of the sinusoid of rat liver were found to differ from in situ endothelial cells by their rounded shape with the nucleus usually in the centre and fenestrae of the sieve plates across the entire cytoplasm. Their volume density is 0.2814 (SE 0.0085). Some of them tend to form complex, ramified structures as well as clefts and vacuoles up to 1 micron in size. Dense bodies are other structures quite often seen in endothelial cells (Vv 0.0189, SE 0.0085). Mitochondria, on the other hand, are extremely rare (Vv 0.0023, SE 0.0009). Both the preservation and relocation of sieve structure after isolation may probably be due to the cytoskeleton which in vivo causes changes of size and number of endothelial cell fenestrae under physiological and pathological conditions.

Permeability of Regenerating and Atrophic Choriocapillaris in the Rabbit

When rabbits receive intravenous injections of sodium iodate, large expanses of the retinal pigment epithelium are destroyed. The adjacent capillary bed, the choriocapillaris, atrophies in response to the loss of the pigment epithelium and then regenerates. This provides a model of the permeability of regenerating and atrophic choriocapillaris, which we studied using intravenously injected horseradish peroxidase and catalase. Regenerating capillaries were permeable to peroxidase but not catalase. The permeability to peroxidase was probably due to endothelial fenestrations, since catalase (which is larger than peroxidase and does not penetrate endothelial fenestrae) was retarded at interendothelial junctional complexes, indicating that they were intact. Atrophic choriocapillaries were impermeable to catalase but displayed a heterogeneous permeability to peroxidase. This was correlated with the presence or absence of fenestrae; capillary profiles lacking fenestrae retained peroxidase in their lumina, whereas if fenestrae were present the tracer penetrated into the pericapillary space. The observations indicate that: (1) the permeability of the regenerating choriocapillaris is qualitatively similar to the mature choriocapillaris, and (2) the atrophic choriocapillaris undergoes changes in permeability that are primarily correlated with the loss of endothelial fenestrae. The observations provide a functional correlate - change in permeability - for structural changes in choriocapillaris endothelium (thickening, loss of fenestrae) in response to destruction of the retinal pigment epithelium, which has been postulated to exert a trophic effect on these capillaries.

Endothelial fenestrae in the rat fetal cerebrum

Ultrastructural analysis of endothelial cells during forebrain ontogenesis in the rat was carried out with special reference to endothelial fenestrae. Before the stage of neuroblast formation, i.e., at embryonic day 11 (E11) or E12, blood vessels were present only in the perineural mesenchymal tissue and possessed endothelial fenestrae in most of their cytoplasm. From E13 to E16, blood vessels invaded into the neural parenchyma, where the migrating zone was formed just outside the matrix layer. Peri- and intraneural endothelial cells during this period showed some peculiar ultrastructural changes characterized by cytoplasmic thickening, pseudopodial protrusions and the formation of large vesicles. In association with these changes, the fenestrae rapidly decreased in frequency, and had disappeared completely by E17. These results indicate that non-fenestrated endothelial cells are established in the developing rat cerebrum by E17 onwards.

Fine structural correlates of vascular permeability of chromaffin cell transplants in CNS pain modulatory regions

Adrenal medullary tissue, bovine chromaffin cells, and PC12 cells were transplanted into the pain modulatory regions of the rat midbrain periaqueductal gray (PAG) or dorsal spinal cord. Fine structural studies of vascular permeability of these grafts revealed that in all three cases, the capillary endothelium of the graft vasculature was attenuated and fenestrated, unlike that of the surrounding host CNS tissue. The intravascular injection of the protein marker, horseradish peroxidase (HRP), enters the grafted tissue parenchyma and is found in the extracellular space of the surrounding host CNS. In contrast, control gelfoam transplants, which become vascularized, do not contain vessels with fenestrated endothelium and do not leak HRP. Since cell suspension implants do not contain endothelial cells, the vasculature of the grafts must be derived from the host. However, as their morphological characteristics are similar to those of the in situ adrenal medulla, it appears that the tissue environment of the graft influences the permeability properties of the vascular bed. The increased permeability to HRP is apparently permanent and most likely is due to the passage through endothelial cell fenestrae.

In vitro modulation of endothelial fenestrae: opposing effects of retinoic acid and transforming growth factor beta.

Cultured endothelial cells isolated from fenestrated capillaries express many properties characteristic of their in vivo differentiated phenotype, including the formation of a limited number of fenestrae. In this study, we have investigated whether physiological factors that control cell differentiation might regulate the surface density of fenestrae in capillary endothelial cells. We have found that treatment of the cultures with retinoic acid (10 microM) induces a more than threefold increase in the surface density of endothelial fenestrae, whereas transforming growth factor beta (TGF beta) (2 ng ml-1) causes a sevenfold decrease in the surface density of these structures. These results show that the expression of endothelial fenestrae is susceptible to bidirectional modulation by physiological signals, and suggest that retinoids and TGF beta may participate in the regulation of fenestral density of capillary endothelium in vivo.

Transmission and scanning electron microscopy investigations on the structure of the ultrafilter of glomeruli in human acute renal failure.

In 4 cases of human acute renal failure (ARF) with oligo-anuria, normuria and polyuria the following findings were made. (1) The diameter of endothelial cell fenestrae is on average larger than that of the control values, but not significantly. (2) In ARF the ratio of the endothelial cell fenestral area to the total endothelial area is not significantly lower than in control kidneys. (3) In ARF the average density of fenestrae per unit of area is not significantly lower than in control kidneys. (4) The structure of the podocytes does not differ from that of control kidneys.

Morphological changes of the villous microvascular architecture and intestinal growth in rats with streptozotocin-induced diabetes.

Using intact tissues and vascular corrosion casts, morphological changes in the small intestine of streptozotocin-induced diabetic rats present 1 month after onset were investigated using scanning and transmission electron microscopy. Although there were increases in the height of the villi in the jejunum and not in the ileum, characteristic changes in surface morphology in the diabetic rats were observed only in the ileum. Use of a vascular corrosion cast technique clearly demonstrated the prominent thick marginal vessels of the intestinal villi of the control rats. In the diabetic rats, the central arterioles in the villi were dilated both in the jejunum and the ileum. Marginal vessels were small in the ileum but prominent in the jejunum. Using transmission electron microscopy, decreases in diameter and the number of endothelial fenestra were also evident in the marginal vessels of the ileal villi in the diabetic rats. Thus in rats made diabetic, fine structural alterations of the marginal vessels are induced in the villi of the distal ileum, in addition to quantitative changes in the jejunal villi, presumably related to hyperphagia. The marginal vessels seem to be involved in the regulation of microcirculation of the intestinal villi in the rat.

The uptake of αFcR-gold by human neutrophil granulocytes

The types of capillaries in rabbit oral mucosa and major salivary glands have been identified, and their permeability to macromolecules, ferritin and thorotrast has been studied. The capillary network of the oral mucosa consisted of continuous and fenestrated capillaries. The fenestrated capillaries constituted a small proportion of the capillary bed in the oral mucosa of rabbit and were predominant in the parotid and submaxillary glands.The transport of ferritin and thorotrast across the endothelial cells occurred through pinocytotic vesicles, the markers being found occasionally in both dense bodies and multivesicular bodies. The marginal folds found projecting on the luminal surface of the endothelial cells may have a collaborating role in pinocytosis. Moreover, a concentration gradient was apparent along the fenestrated endothelial barrier where the bridging diaphragms separate the high density on the vascular lumen from the scattered particles in the extravascular connective tissue. Where the marker particles were noted apparently traversing endothelial fenestrae, the bridging diaphragms were not clearly identified. The basement lamina surrounding the endothelial cells acted as a temporary barrier to thorotrast but did not restrict the passage of ferritin.

Increase in the number of fenestrae in mouse endothelial liver cells by altering the cytoskeleton with cytochalasin B.

Endothelial cells of the hepatic sinusoid isolated from mice livers and maintained in culture display typical fenestrae grouped in sieve plates. Treatment with cytochalasin B led to no significant change in the mean diameter of the fenestrae but to an increase in their number and in the porosity of the cells (percentage of the cellular surface opened by the fenestrae) which attained up to 300% of that of the controls. Scanning electron microscopic observations of Triton-extracted cells revealed that these modifications were related to an alteration of the cytoskeleton. The effect of cytochalasin B could be reversed; 3 hr after removal of the drug, the cells recovered their original aspect with sieve plates scattered over their surface. These observations demonstrate that endothelial fenestrae are inducible structures and that the cytoskeleton seems to be involved in their formation.

Structural changes in endothelial cells of developing rat liver in the transition from fetal to postnatal life

Endothelial fenestrae in the microcirculatory walls of fetal (18th and 21st days), newborn (1st and 5th days), and adult rat livers have been studied by an interactive analysis of scanning electron microscope images. Our results show that liver endothelial cells contain different fenestration patterns depending on both their specific location in the liver acinus and the developing period. Portal vessels have a continuous endothelium in both fetal and postnatal livers as in the adult liver. Endothelial cells in central veins change from highly fenestrated in the fetal and neonatal livers to continuous in the adult liver. The number of fenestrae per square micrometer of endothelium is similar along the sinusoidal network of fetal liver, but increases in the zone 3 sinusoids of newborn liver through the adult liver, where it has tripled the number in the zone 1 sinusoids. Porosity values in sinusoidal endothelium progressively decrease in the fetal to postnatal transition due to the disappearance of large fenestrae (greater than 250 nm) which accompanies the residual hemopoietic activity. While we do not known which factors specifically regulate these fenestration patterns, their configuration in fetal liver, before hepatic tissue has assumed its heterogeneous functioning postnatally, is worthy of note.