白蛋白对内皮细胞单层屏障功能的增强作用

我们建立了体外灌流内皮细胞单层研究血管能透性的方法，可以测定内皮单层的滤过系数（Ｋｆ）和渗透压筢射系数（σ），研究了白蛋白对内皮单层通透屏障功能的影响。不同浓度白蛋白溶液（１，５，１０，２０ｍｇ／ｍｌ）灌流单纯的滤膜时，其流量变化不大，灌流膜上致密生长的内皮细胞单层时，流量和Ｋｆ值随白蛋白浓度的增加而降低。同时，蛋白清除率也不断减小，σ增大，说明白蛋白通过与内皮细胞的作用使内皮单层对水和大分子物质     

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano‐sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage‐derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage‐derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome‐mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage‐derived exosomes were also shown to effectively suppress collagen‐induced activation of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking.     

Ocular cell monolayers cultured on biodegradable substrates.

The aim of this study was to culture retinal pigment epithelial (RPE) and corneal endothelial cells on biodegradable substrates for future use in monolayer transplantation in the eye. The biodegradable polymers, poly-l-lactic (PLLA) and poly-dl-lactic-co-glycolic acid (85:15) (PLGA) (both of molecular weight 105 kd) were the biomaterials used. All materials were seeded with either pig/human retinal pigment epithelial cells or rabbit corneal endothelial cells and were maintained in tissue culture conditions. Upon confluency, the cell density was calculated and cell viability determined. All monolayers were stained with phalloidin-rhodamine for F-actin and antibodies to the tight junction (zonula occludens) protein, ZO1, to demonstrate the presence of tight junctions. The final cell density of human RPE monolayers on PLLA films was 2950 cells/mm(2) (+/-185). The final cell density of pig RPE on PLLA and PLGA film was 2350 cells/mm(2) (+/-152 and 178, respectively). Rabbit corneal endothelial cells had a final cell density of 2650 cells/mm(2) (+/-164). F-actin staining revealed a circumferential ring of actin filaments in all of the cells grown on substrates. ZO(1) immunohistochemistry demonstrated staining along the lateral cell borders of all cell types. The successful culture of retinal pigment epithelial and corneal endothelial monolayers on these substrates may have potential for transplanting cell monolayers in the eye to improve vision.     

Interferon-beta inhibits activated leukocyte migration through human brain microvascular endothelial cell monolayer.

Perivascular leukocyte infiltration into the central nervous system is characteristic of multiple sclerosis (MS) pathology. Interferon-beta (IFN-beta) has shown efficacy in the treatment of patients with MS, but the relevant mechanisms remain incompletely understood. In this study the effects of IFN-beta on leukocyte transendothelial migration were investigated using cells relevant to MS pathogenesis, namely human brain microvascular endothelial cells (HB-MVEC). Activated, but not resting leukocytes exhibited a high transendothelial migration capacity. HB-MVEC prestimulated with tumor necrosis factor (TNF) and IFN-gamma significantly promoted leukocyte transendothelial migration. IFN-beta inhibited the activated leukocyte transendothelial migration on TNF/IFN-gamma-activated HB-MVEC in a dose-dependent manner. A matrix metalloproteinase (MMP) inhibitor and monoclonal antibodies to lymphocyte function antigen-1 (LFA-1) or intercellular adhesion molecule-1 (ICAM-1), but not to very late antigen-4 or to vascular cell adhesion molecule-1 significantly inhibited the transendothelial migration of stimulated leukocytes, suggesting that this phenomenon involves the LFA-1/ICAM-1 interaction and MMP. However IFN-beta did not interfere with the binding of leukocytes to HB-MVEC unless IFN-beta was preincubated with leukocytes or added to HB-MVEC at the time of stimulation. Furthermore IFN-beta did not modulate the expression of adhesion molecules on either stimulated leukocytes or activated HB-MVEC, but partially reduced TNF and interleukin-1 production from stimulated leukocytes during coculture with HB-MVEC. Interestingly, in the presence of IFN-beta, a significant down-regulation of MMP-9 release from stimulated leukocytes was found, especially for the activated form of MMP-9. These results indicate that inhibition of leukocyte transendothelial migration is an important mechanism accounting for the beneficial effects of IFN-beta in the treatment MS patients.     

Penetration of endothelial cell monolayers by Borrelia burgdorferi.

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.     

Platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress blood-retinal barrier breakdown

Platelet microthrombi are present in the diabetic retinal vasculature of humans and rodents; however, the mechanisms and consequences of their presence have not been defined. The current study demonstrates that platelet containing microthrombi accumulate in the retinal vasculature of the rat within 2 weeks of experimental diabetes, a timepoint at which leukocyte-mediated endothelial cell injury and death are known to occur. Platelet accumulation increased with the duration of diabetes, and crossover experiments revealed that maximal platelet accumulation required both diabetic platelets and a diabetic endothelium. Platelet accumulation also coincided with the expression of Fas and FasL in the diabetic retina. When endothelial cell apoptosis was inhibited with an anti-FasL neutralizing antibody, platelet accumulation was effectively suppressed. When platelets were depleted from the systemic circulation with an anti-platelet antibody, blood-retinal barrier breakdown worsened in the diabetic animals. These findings suggest that platelet accumulation in the diabetic retinal vasculature is secondary to endothelial cell death and serves, in part, to suppress blood-retinal barrier breakdown.     

Liquid flow through monolayers of cultured Madin-Darby canine kidney cells.

The rate of liquid flow per unit area (Jv/A) through Madin-Darby canine kidney cell monolayers has been studied at temperatures between 0 and 38 degrees C and at transmonolayer hydrostatic pressures between 14 and 44 cmH2O. Jv/A decreased exponentially with time during application of a constant pressure to the free surface of each monolayer. This behaviour resembles the sealing of cultured vascular endothelium. For monolayers sealed between 33-38 degrees C and 30-33 cmH2O, the mean (+/- S.E.M.) half-time (t1/2) of sealing was 228 (+/- 88) s (n = 6). The decrease in Jv/A during sealing can be expressed as a fraction of the initial Jv/A. Between 33-38 degrees C and 30-33 cmH2O, the mean (+/- S.E.M.) sealing fraction was 0.58 (+/- 0.06; n = 6). Mean sealing t1/2 was longer at lower temperatures, and longer for glutaraldehyde-fixed than for unfixed monolayers, but did not vary with transmonolayer pressure. Sealing fraction was not affected by variations in temperature or transmonolayer pressure, or by glutaraldehyde fixation of monolayers. It is argued that sealing is a physical, rather than a biological, phenomenon and that monolayers have non-linear mechanical properties.     

Human Merkel cell regeneration in skin derived from cultured keratinocyte grafts.

Human Merkel cell regeneration in epidermis derived from cultured keratinocyte autografts was studied from 6 days to 6 years after transplantation. Cultured keratinocyte sheets derived from skin of the sole, axilla, groin, or scalp were transplanted to full-thickness wounds in 20 pediatric patients treated for massive burns or giant congenital nevi. Normal age- and site-matched skin as well as meshed split-thickness autografts from the same patients served as controls. Merkel cells were identified by immunohistochemistry using antibodies to cytokeratins #8 and #18. Cultured keratinocytes in vitro expressed no neuroendocrine markers, but nonspecific, simple-epithelial cytokeratin expression was observed in about 20% of cells. After transplantation, Merkel cells were identified only in cultured grafts derived from sole skin and appeared in the epidermis as early as 21 days postgrafting. Dermal Merkel cells were rarely observed, but their appearance invariably succeeded that of intraepidermal Merkel cells. Regenerated Merkel cells were never innervated, and their emergence was unrelated either spatially or temporally to epidermal reinnervation. In skin bridges of meshed split-thickness grafts, Merkel cells survived after degeneration of associated neurites, but no Merkel cells appeared within re-epithelialized interstices. Among the neuroendocrine markers tested, Merkel cells in cultured grafts, meshed skin grafts or normal pediatric skin expressed only neuron-specific enolase. They failed to stain for calcitonin, chromogranin A, Leu-7, synaptophysin, bombesin, or vasoactive intestinal polypeptide by immunohistochemistry. These findings suggest that: (a) Merkel cells derive from keratinocyte precursors which undergo neuroendocrine differentiation in the epidermis; (b) that keratinocyte stem cells are capable of undergoing Merkel cell differentiation postnatally; (c) that postnatal Merkel cell differentiation may be body-site dependent; and (d) that Merkel cell development and maintenance is independent of neural induction.     

Immunohistochemical localization of blood-retinal barrier breakdown in human diabetics.

Localization of the site of blood-retinal barrier breakdown in diabetes has been controversial. It has been particularly difficult to make assessments in clinical material where the use of tracer materials may not be practical. In this study, immunohistochemical staining for albumin was performed on paraffin-embedded eyes from patients with no known ocular disease and those with various stages of diabetic retinopathy. No extravascular albumin was detected in the retina or retinal pigment epithelium (RPE) of normal nondiabetics or diabetics with no ocular findings, but it was detected in 12.5% of mildly affected diabetics, 20% of background diabetic retinopathy cases, and 89% of proliferative diabetic retinopathy cases. The inner retinal vasculature appeared to be the primary site of leakage in diabetics because all cases demonstrating extravascular albumin-positivity expressed it in the inner retina. It usually permeated the vessel walls and spread along the inner surface of the retina. Some of these cases also contained albumin in the outer retina and RPE, suggesting that additional leakage also may occur through the RPE. A case of cytomegalovirus (CMV) retinitis showed albumin staining predominantly in the inner retina, whereas a rhegmatogenous retinal detachment showed only outer retina staining. These data suggest immunohistochemical staining for albumin may be a useful technique for localizing blood-retinal barrier breakdown.     

Perturbation of the blood-retinal barrier after enzyme perfusion. A cytochemical study

The retina is protected from circulating molecules by a blood-retinal barrier. This is comprised of the impermeable apical-lateral junctions of the retinal pigment epithelium and intraretinal blood vessels lined by endothelia that have impermeable junctions and vesicles that do not transport material from the luminal to abluminal front. This study examined the effect of enzyme digestion upon the restrictive properties of the retinal capillary endothelium. Rats were perfused first with enzymes and then by hemoglobin that was visualized by ultrastructural cytochemical methods. After perfusion of buffer alone or buffers containing neuraminidase or heparinase, the cytochemical reaction product was confined to the capillary lumina and to endothelial cell vesicles facing the luminal front. In contrast, after heparitinase or pronase perfusion, reaction product filled the extravascular spaces. Chains of endothelial cell vesicles and patent transendothelial channels were often encountered. Endothelial cell junctions did not appear to be affected by enzyme treatment. These findings indicate that a cell-surface heparan sulfate proteoglycan (or a nonidentified protein removed by proteolysis) is a key molecule required for the maintenance of the blood-retinal barrier.     

Fibronectin fragments released from phorbol ester-stimulated pulmonary artery endothelial cell monolayers promote neutrophil chemotaxis.

We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.     

Sequential migration of neutrophils across monolayers of endothelial and epithelial cells

In the course of granulocyte-dominated lung inflammation, granulocytes migrate across the endothelium and epithelium of the lung and cause severe tissue damage. To study this process in more detail, we developed a bilayer transmigration model composed of primary human endothelial and lung epithelial cells, simultaneously cultured on opposite sides of Transwell filters. Electron microscopical analysis showed that the morphology of the cells and the expression of junctional proteins remained unaltered and that matrix components were deposited onto the filter. Intriguingly, neutrophil migration was more efficient across the bilayers than across single epithelial monolayers and did not differ from migration across single endothelial monolayers. Coculture experiments showed that endothelial cells stimulated epithelial cells to release IL-6 and that epithelial cells enhanced release of IL-8 from endothelial cells. Together these data reveal bidirectional signaling and enhanced neutrophil migration in a transmigration model of primary human epithelial and endothelial cells.     

Lymphocyte traffic through chronic inflammatory lesions: differential migration versus differential retention.

Afferent lymphatics draining granulomas and efferent lymphatics from normal lymph nodes were cannulated in sheep. Cells collected from these lymphatics were radiolabelled in vitro with 111In (afferent lymph cells) and 51Cr (efferent lymphocytes) and both labelled cells were returned to the animal simultaneously by i.v. injection. The reappearance of these labelled cells in lymph, and the amount of 111In and 51Cr in normal or antigenically stimulated lymph nodes, cutaneous inflammatory sites (FCA-granulomas, NLT- and BCG-induced lesions) and blood was determined 24 hr later. As previously reported, labelled afferent cells preferentially migrated from the blood back through the granuloma into afferent lymph, and efferent lymphocytes back into efferent lymph. Forty per cent as many 111In- as 51Cr-labelled cells ;appeared in efferent lymph. This was caused by the greater migration of 51Cr-labelled cells appeared in efferent lymph. This was caused by the greater migration of 51Cr- than 111In-labelled cells out of the blood into the node. Neither cell type was selectively retained in the node, and 28% of the labelled cells that entered the node migrated on into efferent lymph in 24 hr. Similarly, there was no selective retention of either cell type in the granuloma, and equal amounts of 111In the 51Cr appeared in afferent lymph. The ratio 111In/51Cr in the blood suggested that in the lymph node the two labelled cell populations were extracted equally, while in the granuloma selectively at the level of the vascular endothelium resulted in the preferential extraction of 111In-labelled (afferent lymph) cells.     

Cocaine-induced increase in the permeability function of human vascular endothelial cell monolayers.

The effects of cocaine on endothelial cell macromolecular transport, electrical resistance, and morphology were assessed. In confluent endothelial monolayers grown on microporus filters, cocaine (0.01 to 1 mmol/L) induced a rapid concentration-dependent increase in permeability to peroxidase and low density lipoprotein. Along with increased transport, the cocaine effect was paralleled by a decrease in transendothelial electrical resistance. Alterations in membrane resistance were fully reversible following washout of the drug, providing evidence that cocaine does not cause permanent injury to the integrity of the monolayer. Cocaines major metabolites, benzoylecgonine and ecgonine methyl ester, had minimal effect on electrical resistance properties, whereas monolayer impedance was markedly depressed by the novel cocaine/alcohol metabolite, cocaine ethyl ester (cocaethylene). Morphologic studies of cocaine-treated endothelial cells revealed a marked disruption of F-actin and the formation of intercellular gaps; no evidence of cell lysis and/or detachment was noted. Forskolin, a potent activator of adenylate cyclase known to promote the endothelial cell barrier function, impaired cocaine-induced changes in electrical resistance and morphology. Cocaine, however, had no effect on resting levels of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) in confluent endothelial monolayers. In summary, the results indicate that cocaine directly induces structural defects in the endothelial cell barrier which enhance the transport of macromolecular tracers, the mechanism does not appear to involve intracellular cAMP.     

Lymphocyte migration in malignant disease.

Studies of normal lymphocyte migratory properties in small animals and more recently in man using 111indium oxine labelling techniques are helping the clinician to understand the biology of tumours involving cells of the immune system. However, not enough is known about subset migration to interpret all the various patterns of dissemination observed in lymphoma patients. Such studies are important since they introduce new possibilities for therapy including the use of labelled cells to home a source a radiation preferentially to affected tissue.     

Engineering high-density endothelial cell monolayers on soft substrates

This study demonstrates that a confluent monolayer of endothelial cells (ECs) can be tissue engineered on a soft substrate with a cell density and morphology that approximates in vivo conditions. We achieved formation of a confluent EC monolayer on polydimethylsiloxane (PDMS) elastomer by microcontact printing of fibronectin (FN) in a square lattice array of 3 μm diameter circular islands at a 6 μm pitch. Uniform coatings of FN or serum proteins on PDMS or on tissue-culture-treated polystyrene failed to support the equivalent EC density and/or confluence. The ECs on the FN micropatterned PDMS achieved a density of 1,536 ± 247 cells mm^?2, close to the 3,215 ± 336 cells mm^?2 observed in vivo from porcine pulmonary artery and significantly higher (2- to 5-fold) than EC density on other materials. The probable mechanism for enhanced EC adhesion, growth and density is increased focal adhesion (FA) formation between the ECs and the substrate. After 14 days culture, the micropatterned FN surface increased the average number of FAs per cell to 35 ± 10, compared to 7 ± 6 for ECs on PDMS uniformly coated with FN. Thus, microscale patterning of FN into FA-sized, circular islands on PDMS elastomer promotes the formation of EC monolayers with in vivo-like cell density and morphology.     

Polymerizable monolayers derived from crown ether surfactants

Starting from dibenzo-18-crown-6, 1,10-diaza-18-crown-6 and 1,7-diaza-15-crown-5, several crown ether surfactants were synthesized. They all form stable monolayers on water. The methacryloyloxy derivative of 1,10-bis(3-dodecyloxy-2-hydroxypropyl)-1,10-diaza-18-crown-6, 2c , could be polymerized in the monolayer by UV irradiation. This polymerizable crown ether surfactant also forms synthetic vesicles by sonication in water.     

Effect of lymphocytic infiltration on the blood-retinal barrier in experimental autoimmune uveoretinitis.

Using an experimental model of autoimmune uveoretinitis, we have examined the relationship of T cell infiltration in the retina to blood-retinal barrier (BRB) breakdown. Sensitive quantitative in vivo techniques were used to examine BRB permeability to sucrose, a low mol. wt non-transported solute. Electron microscopy was also used to localize extravasated horseradish peroxidase, a macromolecular visual tracer, from the retinal vasculature and to identify the route by which any leakage was occurring. No increase in BRB permeability was found prior to lymphocytic infiltration. By day 10 of the disease inflammatory cells could be seen within the structurally intact retina, which was shortly followed by an increase in the permeability of the BRB to sucrose. Only later in the disease process, when damage to the photoreceptor layer became apparent, did extravasation of the macromolecule HRP occur. At no stage of the disease process was there any detectable damage to inter-endothelial tight junctions. The size-dependancy of tracer extravasation in the initial stages of the disease is indicative of a paracellular route being responsible for the increase in BRB permeability. In later stages of the disease some evidence of horseradish peroxidase filled 'vesicle-like' profiles was observed. We suggest that the devastating complication of BRB breakdown in ocular inflammation is a direct consequence of lymphocytic infiltration.