Phorbol ester induces diaphragmed fenestrae in large vessel endothelium in vitro

The endothelium of visceral capillaries is fenestrated, whereas that of large vessels is not. We show here that the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate (PMA), triggers formation of diaphragmed fenestrae in endothelial cells cultured from the calf pulmonary artery and the human umbilical vein. This demonstrates that large vessel endothelial cells have the potential to express, in response to exogenous signals, a structural specialization uniquely associated with the endothelium of visceral capillaries in vivo.     

Morphological behavior of cultured bovine adrenal medulla capillary endothelial cells.

Bovine adrenal medulla capillary endothelial cells were isolated and cloned, and their morphological behaviors in vitro were examined. In the culture of primary or early passage, one type of colony formed intracellular lumina both on the dish and in the three dimensional collagen gel. Another type proliferated well and showed morphology ranging from slender-shape to cobblestone shape, and were easily cloned. Cloned cells which showed slender-shapes formed tubular network on plastic dish after addition of PMA, OAG or vanadate, and these cells also formed multicellular tubules in the three dimensional collagen gel. However, the formation of diaphragmed fenestrae by these slender-shape clones was rare. One clone which showed cobblestone shape formed diaphragmed fenestrae, when cultured on collagen gel for more than one month. Isolated colonies or clones showed heterogeneity of cell shape, angiogenic behaviors and fenestrae formation.     

Ultrastructure and formation of diaphragmed fenestrae in cultured endothelial cells of bovine adrenal medulla

Summary The ultrastructure of diaphragmed fenestrae and the process of their de novo formation were examined in cultured endothelial cells cloned from fenestrated capillaries of bovine adrenal medulla. One clone frequently formed many diaphragmed fenestrae in highly attenuated regions of endothelium during 1–1.5 months of culture on reconstituted collagen gel. Stereo views of thick sections showed round or oval clusters of geometrically arranged fenestrae, each with a central knob. The number of diaphragmed fenestra per cluster was 50±13/m². The inner diameter of the fenestrae was 50–60 nm. In the cells having small clusters of fenestrae, plasmalemmal vesicles, each having a thin diaphragm with a central knob, were accumulated (56±18/m²) and arranged geometrically, mostly on the basal plasmalemma. At the border between the cytoplasm and the eluster of fenestrae, plasmalemmal vesicles of the basal plasmalemma fused with the opposing apical plamalemma. A model for the process of fenestrae formation in vitro is proposed.     

Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro

Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418     

Rat liver endothelial cells isolated by anti-CD31 immunomagnetic separation lack fenestrae and sieve plates

The gold standard for the identification of sinusoidal endothelial cells (SEC) is the presence of fenestrae organized in sieve plates, which is characteristic of SEC in vivo. One of the methods currently in use to isolate SEC is immunomagnetic sorting for CD31. However, there is evidence to suggest that CD31 is not present on the surface of differentiated SEC. The present study used scanning electron microscopy to image rat hepatic endothelial cells isolated by anti-CD31 and immunomagnetic sorting and cells isolated by gradient centrifugation and centrifugal elutriation. Cells isolated by elutriation had well-developed fenestrae and sieve plates, whereas cells isolated by anti-CD31 and immunomagnetic sorting had significantly fewer fenestrae organized in sieve plates. In conclusion, cells isolated by anti-CD31 and immunomagnetic sorting lacked the hallmark features of SEC.     

The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium.

Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).     

Extracellular matrix specificity for the differentiation of capillary endothelial cells

Capillary endothelial cells in a fenestrated vasculature contain openings in attenuated areas of the cytoplasm which are often covered with one or two diaphragms. However, due to endothelial cell dedifferentiation upon culturing, such indicative membrane structure are often not expressed. The expression of a more differentiated phenotype can be regulated by the extracellular matrix to which the cells attach. In addition, during angiogenesis endothelial cell migration enables their interaction with other cell types and matrices which may directly affect endothelial cell growth and function. We report the ability to modify the expression of diaphragmed fenestrations and transcapillary channels in capillary endothelial cells by specific extracellular matrices. When the number of membrane openings is measured, growth of the cells on Madin--Darby canine kidney cell matrix results in the greatest number while other matrices or isolated matrix components are only partially active. Production of such a biologically active matrix not only allows an avenue to identify fenestrae-associated proteins but also provides an easy culture method to more closely mimic the in vivo phenotype of endothelial cells.     

The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.     

Fenestration patterns in endothelial cells of rat liver sinusoids

Endothelial fenestrae of both zone 1 and zone 3 acinar liver sinusoids have been studied in rats by an interactive analysis of scanning electron microscopical images. Two fenestration patterns have been recognized in the endothelial cells on the basis of local variation in size, distribution and clustering of pores in each acinar zone. Our data indicate that both the number of fenestrae per square micrometer of endothelial surface and the mean diameter of fenestrae are significantly larger in zone 3 than in zone 1. The number of sieve plates is about 1.74 times larger in zone 3 than in zone 1, and the number of fenestrae per plate in zone 3 is nearly twice that in zone 1. Two different classes of fenestrae have been considered: clustered pores, which prevail in zone 3 and have a mean diameter smaller than the other pores, and free pores, which prevail in zone 1 and are bigger.     

PV1 is a key structural component for the formation of the stomatal and fenestral diaphragms

PV1 is an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of caveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. Multiple PV1 homodimers are found within each stomatal and fenestral diaphragm. We investigated the function of PV1 within these diaphragms and their regulation and found that treatment of endothelial cells in culture with phorbol myristate acetate (PMA) led to upregulation of PV1. This correlated with de novo formation of stomatal diaphragms of caveolae and transendothelial channels as well as fenestrae upon PMA treatment. The newly formed diaphragms could be labeled with anti-PV1 antibodies. The upregulation of PV1 and formation of stomatal and fenestral diaphragms by PMA was endothelium specific and was the highest in microvascular endothelial cells compared with their large vessel counterparts. By using a siRNA approach, PV1 mRNA silencing prevented the de novo formation of the diaphragms of caveolae as well as fenestrae and transendothelial channels. Overexpression of PV1 in endothelial cells as well as in cell types that do not harbor caveolar diaphragms in situ induced de novo formation of caveolar stomatal diaphragms. Lastly, PV1 upregulation by PMA required the activation of Erk1/2 MAP kinase pathway and was protein kinase C independent. Taken together, these data show that PV1 is a key structural component, necessary for the biogenesis of the stomatal and fenestral diaphragms.     

Glomerular endothelium: a porous sieve and formidable barrier

The glomerular capillary endothelium is highly specialized to support the selective filtration of massive volumes of plasma. Filtration is driven by Starling forces acting across the glomerular capillary wall, and depends on its large surface area and extremely high water permeability. Glomerular endothelial cells are extremely flat and perforated by dense arrays of trans-cellular pores, the fenestrae. This phenotype is critical for the high glomerular water permeability and depends on podocyte-derived VEGF, as well as TGF-beta. Endothelial cell-derived PDGFB, in turn, is necessary for the establishment of mesangial cells, which sculpt the glomerular loop structure that underlies the large filtration surface area. In pre-eclampsia, inhibition of the VEGF- and TGF-beta signaling pathways leads to endothelial swelling and loss of fenestrae, reducing the glomerular filtration rate. Similarly, in the thrombotic microangiopathies, glomerular endothelial cell injury coupled with inappropriate VWF activation leads to intracapillary platelet aggregation and loss of the flat, fenestrated phenotype, thus reducing the glomerular filtration rate. Normally, a remarkably small fraction of albumin and other large plasma proteins passes across the glomerular capillary wall despite the massive filtration of water and small solutes. An elaborate glycocalyx, which covers glomerular endothelial cells and their fenestrae forms an impressive barrier that, together with other components of the glomerular capillary wall, prevents loss of plasma proteins into the urine. Indeed, microalbuminuria is a marker for endothelial glycocalyx disruption, and most forms of glomerular endothelial cell injury including pre-eclampsia and thrombotic microangiopaties can cause proteinuria.     

Characterization and differential expression of an endothelial cell-specific surface antigen in continuous and sinusoidal endothelial, in skin vascular lesions and in vitro

Continuous and sinusoidal endothelial cells display marked morphological and functional heterogeneity as to their plasmalemmal vesicle content, to the kind of intercellular junctional complexes, to the existence and kind of fenestrae and gaps, to the existence and character of their basement membrane, to their ability for phagocytosis and to other functional parameters. Monoclonal antibody 1F10, raised against human umbilical vein endothelial cells (HUVE cells), reflects these differences in recognizing--without any nonendothelial side reactions--an endothelial cell surface antigen, abundantly expressed in continuous endothelia, low and inconsistently expressed in liver sinusoidal and dermal lymphatic endothelia and absent from splenic sinusoidal endothelial cells. In differentiated skin vascular tumors, 1F10 antigen is expressed in normal amounts while it is only low and inconsistently expressed in the dedifferentiated endothelial cells of Kaposi's sarcoma and hemangiosarcoma. HUVE cells in culture, in contrast to their in situ ancestors, express variable amounts of 1F10 antigen. When endothelial cell-conditioned medium (ECC medium) is supplied to HUVE cells in culture, no 1F10 antigen is expressed, while supplementation with fresh serum-containing medium (FSC medium) or cytokines, such as bFGF, suffices to maintain 1F10 expression in 10-70% of the cells. From this we conclude that developmental regulation, environmental influences and cytokine supply contribute to the differentiation and maintenance of the 1F10+ and 1F10-endothelial cell phenotypes, both in vivo and in vitro.     

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.     

Actin‐spectrin scaffold supports open fenestrae in liver sinusoidal endothelial cells

Fenestrae are open transmembrane pores that are a structural hallmark of healthy liver sinusoidal endothelial cells (LSECs). Their key role is the transport of solutes and macromolecular complexes between the sinusoidal lumen and the space of Disse. To date, the biochemical nature of the cytoskeleton elements that surround the fenestrae and sieve plates in LSECs remain largely elusive. Herein, we took advantage of the latest developments in atomic force imaging and super‐resolution fluorescence nanoscopy to define the organization of the supramolecular complex(es) that surround the fenestrae. Our data revealed that spectrin, together with actin, lines the inner cell membrane and provided direct structural support to the membrane‐bound pores. We conclusively demonstrated that diamide and iodoacetic acid (IAA) affect fenestrae number by destabilizing the LSEC actin‐spectrin scaffold. Furthermore, IAA induces rapid and repeatable switching between the open vs closed state of the fenestrae, indicating that the spectrin‐actin complex could play an important role in controlling the pore number. Our results suggest that spectrin functions as a key regulator in the structural preservation of the fenestrae, and as such, it might serve as a molecular target for altering transendothelial permeability.     

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

Background  

Liver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.     

AN ELECTRON MICROSCOPIC STUDY OF THE PASSAGE OF COLLOIDAL PARTICLES FROM THE BLOOD VESSELS OF THE CILIARY PROCESSES AND CHOROID PLEXUS OF THE RABBIT

The thinnest areas of the capillaries of the choroid plexus and ciliary processes in the eye of the rabbit are characterized by the presence of fenestrae. When various colloidal particles opaque to the electron beam (thorotrast, gold sol, and saccharated iron oxide) were injected into the blood stream, none were found in fenestrae or in areas that might suggest their having passed through fenestrae. The passage of marker particles from the lumen to the surrounding connective tissue does take place on occasion in the areas of thicker walls in the capillaries and venules rather than in the attenuated and fenestrated endothelial walls. The pathway taken by these markers may be either through the cytoplasm of the endothelial cells via membrane-bounded vesicles and vacuoles or through the intercellular spaces of the vessels. An altered aqueous humor (cloudy and plasmoid) was produced by endotoxin injection or by making a draining fistula in rabbit cornea. Both methods gave rise to the same changes in the blood vessels of the ciliary processes. Under such conditions of inflammation the passage of colloidal particles through the thicker walls of the capillaries and venules was greatly increased and occurred primarily as an intercellular passage between the endothelial cells. The attenuated and fenestrated areas of the endothelium of the small capillaries remained unchanged with no particles passing through them. These results on the altered vessels of the ciliary processes parallel those of Majno and Palade (26) on the rat cremaster muscle.     

Mechanism of naked DNA clearance after intravenous injection

BACKGROUND: Injection of naked DNA has been viewed as a safer alternative to current gene delivery systems; however, the rate of clearance from the circulation has been a constant barrier in developing these methods. Naked DNA after intravenous (i.v.) injection will be taken up by the liver and depredated by serum nucleases. MATERIALS AND METHODS: Our study examines the mechanisms involved in clearance of naked DNA by each compartment, the blood and the liver, in an in vivo mouse model. Confocal microscopy and transmission electron microscopy were employed to identify the type of cells taking up DNA and the barrier to DNA access to hepatocytes, respectively. RESULTS: Our data showed the liver could take up over 50% of 5 microg perfused pDNA, with a maximum 25 microg of pDNA during a single pass, and a slower clearance rate compared to that of liver uptake. It was demonstrated that naked DNA is primarily taken up by the liver endothelial cells and this endothelial barrier to transfection could be overcome by manually massaging the liver, which enlarges the fenestrae. CONCLUSIONS: This study clarifies the mechanism by which naked DNA is eliminated from the circulation after i.v. injection, focusing on the role of both the liver and blood compartments in vivo (i.e. mouse). With this knowledge, we can more clearly understand the mechanism of naked DNA clearance and develop more efficient strategies for DNA transfer in vivo.     

Ultrastructural changes in the histohematic barrier of the liver in the early period after endotoxin administration

Investigation of the changes of the mouse liver cells revealed early reaction of endothelial sinusoid cells and Kupffer cells after endotoxin application. During first 60 min. after endotoxin injection there were certain bulge of nuclear zone, changes of nuclear shape, appearance of microfilaments in the cytoplasm of endothelial cells. An increase in the number of the pores and fenestrae grouped in sieve plates was discovered in the endothelial cells. Their luminal surface formed the bubbles. Such changes of the endothelial cells can be described as their activation. Reaction of the endothelial cells of hepatic sinusoids and activation of the Kupffer cells were revealed at the same time.     

Twenty-four-hour changes in pinealocytes,capillary endothelial cells and pericapillary and intercellular spaces in the pineal gland of the mouse

Summary Semiquantitative electron-microscopic observations on the pineal gland of dd-mice were carried out to determine whether 24-h rhythms exist in pinealocytes, pericapillary and intercellular spaces and capillary endothelial cells. Nuclear and cytoplasmic areas of pinealocytes and the area of condensed chromatin in pinealocytes showed inversely related circadian rhythms; the former two increased, whereas the latter decreased, during the light period. The extent of pericapillary and wide intercellular spaces exhibited 24-h changes, with an increase and decrease occurring during the light period and the dark period, respectively. The cross-sectional area of endothelial cells decreased and the number of fenestrae increased during the light period; this was reversed during the dark period. The results suggest that the increase in the nuclear and cytoplasmic areas of pinealocytes, the area of pericapillary and wide intercellular spaces and the number of fenestrae, and the decrease in the area of condensed chromatin and endothelial cells during the light period may be related to an increase in synthetic activity of pinealocytes in the mouse.