Transcytosis Of Albumin Research Articles

Albumin endocytosis via megalin in astrocytes is caveola‐ and Dab‐1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.

Independent two-photon measurements of albumin GSC give low values

Independent two-photon measurements of albumin GSC give low values

Megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid

We have previously shown that the uptake and transcytosis of albumin in astrocytes promote the synthesis of the neurotrophic factor oleic acid. Although the mechanism by which albumin induces oleic acid synthesis is well known, the mechanism of albumin uptake in astrocytes remains unknown. In this work, we found that astrocytes express megalin, an endocytic receptor for multiple ligands including albumin. In addition, when the activity of megalin is blocked by specific antibodies or by silencing megalin with specific siRNA, albumin binding and internalization is strongly reduced indicating that megalin is required for albumin binding and internalization in the astrocyte. Since the uptake of albumin in astrocytes aims at synthesizing the neurotrophic factor oleic acid, we tested the ability of megalin-silenced astrocytes to synthesize and release oleic acid in the presence of albumin. Our results showed that the amount of oleic acid found in the extracellular medium of megalin-silenced astrocytes was strongly reduced as compared with their controls. Together, the results of this work indicate that megalin is a receptor for albumin in astrocytes and is required for the synthesis of the neurotrophic factor oleic acid. Consequently, the possible involvement of albumin in the holoprosencephalic syndrome observed in megalin-deficient mice is suggested.

Albumin-bound paclitaxel: a next-generation taxane

Taxanes are standard treatment for metastatic breast cancer; however, the solvents used as vehicles in these formulations cause severe toxicities. The FDA recently approved a solvent-free formulation of paclitaxel for the treatment of metastatic breast cancer that utilises 130-nanometer albumin-bound (nab™) technology (Abraxane®; nab-paclitaxel) to circumvent the requirement for solvents. nab-Paclitaxel utilises the natural properties of albumin to reversibly bind paclitaxel, transport it across the endothelial cell and concentrate it in areas of tumour. The proposed mechanism of drug delivery involves, in part, glycoprotein 60-mediated endothelial cell transcytosis of paclitaxel-bound albumin and accumulation in the area of tumour by albumin binding to SPARC (secreted protein, acidic and rich in cysteine). Clinical studies have shown that nab-paclitaxel is significantly more effective than paclitaxel formulated as Cremophor® EL (CrEL, Taxol®, CrEL-paclitaxel), with almost double the response rate, increased time to disease progression and increased survival in second-line patients. The absence of CrEL from the formulation is associated with decreased neutropenia and rapid improvement of peripheral neuropathy with nab-paclitaxel, compared with CrEL-paclitaxel. For these reasons, nab-paclitaxel can be administered using higher doses of paclitaxel than that achievable with CrEL-paclitaxel, with shorter infusion duration and without the requirement for corticosteroid and antihistamine premedication to reduce the risk of solvent-mediated hypersensitivity reactions. Taken together, these studies have demonstrated that nab technology has increased the therapeutic index of paclitaxel compared with the conventional, solvent-based formulation.

Immunogold study of effects of prenatal exposure to lipopolysaccharide and/or valproic acid on the rat blood-brain barrier vessels

The involvement of blood microvessels, representing the anatomic site of the blood-brain barrier (BBB), in brain damage induced by prenatal exposure to lipopolysaccharide (LPS) and/or valproic acid (VPA) was studied in four-week-old rats. The immunogold procedure was applied for localization at the ultrastructural level of endogenous albumin and glucose transporter (GLUT-1) in three brain regions: cerebral cortex, cerebellum and hippocampus. Four groups of rats were used: (1) untreated control, (2) prenatally VPA-treated, (3) prenatally LPS-treated, and (4) prenatally LPS- and VPA-treated. The functional state of the BBB was evaluated as follows: (a) by its tightness, i.e., permeability to blood-borne albumin, and (b) by the expression of GLUT-1 in the endothelial cells (ECs). Using morphometry, the labelling density for GLUT-1 was recorded over luminal and abluminal plasma membranes of the ECs, also providing information on their functional polarity. No extensive increase of vascular permeability and/or any considerable dysfunction of the BBB in experimental groups nos. 2 and 3 were observed, although in solitary vascular profiles, increased endocytosis or even transcytosis of albumin by ECs was noted. In experimental group no. 4, some vascular profiles showed scanty leakage (microleakage), manifested by the presence of immunosignals for albumin in the perivascular area. Although some fluctuations in the expression of GLUT-1 occurred in all experimental groups, especially in group no. 3, a most pronounced and significant diminution of the labelling density, in all three regions of the brain, was observed in group no. 4. This finding suggests the synergistic action of prenatally applied LPS and VPA that affects specific transport functions of glucose in the microvascular endothelium. The diminished or disturbed supply of glucose to selected brain regions can be one of the factors leading to previously observed behavioral disturbances in similarly treated rats.

Integrated control of lung fluid balance

This review summarizes the highlights of the EB2004 symposium that dealt with the integrated aspects of the lung fluid balance. It is apparent that maintenance of lung fluid balance requires the proper functioning of vascular endothelial and alveolar epithelial barriers. Under physiological conditions, the transcytotic pathway requiring repeated fission-fusion events of the caveolar membrane with other caveolae solely transports albumin. Caveolin-1, which forms caveolae, and albumin-binding proteins play a central role in signaling the transcytosis of albumin. Signals responsible for increasing endothelial permeability in lung microvessels in response to inflammatory mediators were also described. These studies in gene knockout mouse models revealed the importance of Ca(2+) signaling via store-operated transient receptor channel 4 and the activation of endothelial myosin light chain kinase isoform in mediating the increase in microvessel permeability. Increases in the cytosolic Ca(2+) in situ in microvessel endothelia can occur by mitochondria-dependent as well as mitochondria-independent pathways (such as the endoplasmic reticulum). Both these pathways, by triggering endothelial cell activation, may result in lung microvascular injury. The resolution of alveolar edema, requiring clearance of fluid from the air space, is another area of intense investigation in animal models. Although beta-adrenergic agonists can activate alveolar fluid clearance, signaling pathways regulating these events in intact alveoli remain to be established. Development of mouse models in which the function of regulatory proteins (identified in cell culture studies) can be systematically analyzed will provide a better and more integrated picture of lung fluid balance. In vivo veritas!

Role of Src-induced Dynamin-2 Phosphorylation in Caveolae-mediated Endocytosis in Endothelial Cells

Albumin transcytosis, a determinant of transendothelial permeability, is mediated by the release of caveolae from the plasma membrane. We addressed the role of Src phosphorylation of the GTPase dynamin-2 in the mechanism of caveolae release and albumin transport. Studies were made in microvascular endothelial cells in which the uptake of cholera toxin subunit B, a marker of caveolae, and (125)I-albumin was used to assess caveolae-mediated endocytosis. Albumin binding to the 60-kDa cell surface albumin-binding protein, gp60, induced Src activation (phosphorylation on Tyr(416)) within 1 min and resulted in Src-dependent tyrosine phosphorylation of dynamin-2, which increased its association with caveolin-1, the caveolae scaffold protein. Expression of kinase-defective Src mutant interfered with the association between dynamin-2, which caveolin-1 and prevented the uptake of albumin. Expression of non-Src-phosphorylatable dynamin (Y231F/Y597F) resulted in reduced association with caveolin-1, and in contrast to WT-dynamin-2, the mutant failed to translocate to the caveolin-rich membrane fraction. The Y231F/Y597F dynamin-2 mutant expression also resulted in impaired albumin and cholera toxin subunit B uptake and reduced transendothelial albumin transport. Thus, Src-mediated phosphorylation of dynamin-2 is an essential requirement for scission of caveolae and the resultant transendothelial transport of albumin.

Albumin transcytosis from the pleural space.

Occurrence of transcytosis in pleural mesothelium was verified by measuring removal of labeled macromolecules from pleural liquid in experiments without and with nocodazole. To this end, we injected 0.3 ml of Ringer-albumin with 750 microg of albumin-Texas red or with 600 microg of dextran 70-Texas red in the right pleural space of anesthetized rabbits, and after 3 h we measured pleural liquid volume, labeled macromolecule concentration, and, hence, labeled macromolecule quantity in the liquid of this space. Labeled albumin left was 318 +/- 28 microg in control and 419 +/- 17 microg in nocodazole experiments (means +/- SE); hence, whereas ventilation was similar its removal was greater (P < 0.01) in control experiments. Labeled dextran left was 283 +/- 10 microg in control and 381 +/- 21 microg in nocodazole experiments; hence, whereas ventilation was similar its removal was greater (P < 0.01) in control experiments. These findings indicate occurrence of transcytosis from the pleural space. Liquid removed by transcytosis was 0.05 ml/h. This amount times unlabeled albumin concentration under physiological conditions (10 mg/ml) times lumen-vesicle partition coefficient for albumin (0.78) provides fluid-phase albumin transcytosis: approximately 203 microg. h(-1) kg(-2/3). Transcytosis might contribute a relevant part of protein and liquid removal from the pleural space.

Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey.

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.

Albumin uptake and transcytosis in endothelial cells in vivo induced by albumin-binding protein.

The 60-kDa endothelial cell surface albumin-binding glycoprotein (gp60) is postulated to be a docking site for albumin that mediates the uptake of albumin and its transport in cultured microvessel endothelial cells. In the present study, we used an isolated Krebs-perfused rat lung preparation to address the in vivo role of gp60 in mediating albumin uptake and transport. Addition of primary anti-gp60 antibody to the perfusate followed by the secondary antibody to cross-link gp60 increased the vessel wall (125)I-albumin permeability-surface area (PS) product 2.5-fold without affecting the capillary filtration coefficient (K(f,c;) a measure of liquid permeability). In contrast, EDTA (5 mM), which induces interendothelial gap formation, produced parallel increases in both K(f,c) and (125)I-albumin PS product. Increasing perfusate albumin concentration to >1 g/100 ml (EC(50) 1.2 g/100 ml) was sufficient to block (125)I-albumin PS product, indicating that the perfusate albumin competed with tracer albumin for transendothelial albumin transport. Cross-linking of gp60 in lungs perfused with saturating concentration of albumin resulted in a greater increase in (125)I-albumin PS product, indicating that gp60 function was capable of being modulated. These results show that activation of gp60 in pulmonary microvessels induces albumin uptake and its transport through a non-hydraulic pathway that fits with a model of albumin permeability via the transcellular pathway.

Transcytosis of albumin in astrocytes activates the sterol regulatory element-binding protein-1, which promotes the synthesis of the neurotrophic factor oleic acid.

We have recently reported that albumin, a serum protein present in the developing brain, stimulates the synthesis of oleic acid by astrocytes, which promotes neuronal differentiation. In this work, we gain insight into the mechanism by which albumin induces the synthesis of this neurotrophic factor. Our results show that astrocytes internalize albumin in vesicle-like structures by receptor-mediated endocytosis. Albumin uptake was followed by transcytosis, including passage through the endoplasmic reticulum, which was required to induce the synthesis of oleic acid. Oleic acid synthesis is feedback-regulated by the sterol regulatory element-binding protein-1, which induces the transcription of stearoyl-CoA 9-desaturase, the key rate-limiting enzyme for oleic acid synthesis. In our research, the presence of albumin activated the sterol regulatory element-binding protein-1 and increased stearoyl-CoA 9-desaturase mRNA. Moreover, when the activity of sterol regulatory element-binding protein-1 was inhibited by overexpression of a truncated form of this protein, albumin did not affect stearoyl-CoA 9-desaturase mRNA, indicating that the effect of albumin is mediated by this transcription factor. The effect of albumin was abolished when traffic to the endoplasmic reticulum was prevented or when albumin was accompanied with oleic acid. In conclusion, our results suggest that the transcytosis of albumin includes passage through the endoplasmic reticulum, where oleic acid is sequestrated, initiating the signal cascade leading to an increase in its own synthesis.

Evidence for the role of alveolar epithelial gp60 in active transalveolar albumin transport in the rat lung.

1. Transcytosis of albumin, involving the 60 kDa albumin-binding glycoprotein, gp60, was studied in cultured type II alveolar epithelial cells obtained from rat lungs. 2. Type II cells internalized the interfacial fluorescent dye RH 414, which marks for plasmalemma vesicles. Fluorescent forms of albumin and anti-gp60 antibody colocalized in the same plasmalemma vesicles. 3. Antibody (100 microg ml(-1)) cross-linking of gp60 for brief periods (15 min) markedly stimulated vesicular uptake of fluorescently tagged albumin. The caveolar disrupting agent, filipin (10 nM), abolished the stimulated internalization of albumin. 4. The vast majority of plasmalemmal vesicles carrying albumin also immunostained for caveolin-1; however, lysosomes did not stain for caveolin-1. Filipin depleted the epithelial cells of the caveolin-1-positive, albumin-transporting plasmalemma vesicles. 5. Prolonged (> 1 h) stimulation of type II cells with cross-linking anti-gp60 antibody produced loss of cell-surface gp60 and abolished endocytic albumin uptake. 6. Transalveolar transport of albumin was also studied in the isogravimetric rat lung preparation perfused at 37 degrees C. (125)I-labelled albumin was instilled into distal airspaces of lungs, and the resulting (125)I-labelled albumin efflux into the vascular perfusate was determined. 7. Unlabelled albumin (studied over a range of 0-10 g (100 instilled ml)(-1)) inhibited 40 % of the transport of labelled albumin ((5.7 +/- 0.4) x 10(5) counts (instilled ml)(-1)) with an IC(50) value of 0.34 g (100 ml)(-1). 8. Filipin blocked the displacement-sensitive component of (125)I-labelled albumin transport, but had no effect on the transport of the paracellular tracer (3)[H]mannitol. 9. Displacement-sensitive (125)I-labelled albumin transport had a significantly greater Q(10) (27-37 degrees C) than the non-displaceable component. 10. Cross-linking of gp60 by antibody instillation stimulated only the displacement-sensitive (125)I-labelled albumin transalveolar transport in intact rat lungs. 11. To estimate the transport capacity of the displacement-sensitive system, the percentage of instilled (125)I-labelled albumin counts remaining in lung tissue was compared in lungs treated with instillates containing either 0.05 g (100 ml)(-1) unlabelled albumin or 5 g (100 ml)(-1) unlabelled albumin. Approximately 25 % of instilled (125)I-labelled albumin was cleared from the lung preparations per hour by the displacement-sensitive transport pathway. This component was blocked by filipin.

Endothelial vesicles in the blood-brain barrier: are they related to permeability?

1. Macromolecules cross capillary walls via large vascular pores that are thought to be formed by plasmalemmal vesicles. Early hypotheses suggested that vesicles transferred plasma constituents across the endothelial wall either by a "shuttle" mechanism or by fusing to form transient patent channels for diffusion. Recent evidence shows that the transcytotic pathway involves both movement of vesicles within the cell and a series of fusions and fissions of the vesicular and cellular membranes. 2. The transfer of macromolecules across the capillary wall is highly specific and is mediated by receptors incorporated into specific membrane domains. Therefore, despite their morphological similarity, endothelial vesicles from heterogeneous populations in which the predominant receptor proteins incorporated in their membranes define the functions of individual vesicles. 3. Blood-brain barrier capillaries have very low permeabilities to most hydrophilic molecules. Their low permeability to macromolecules has been presumed to be due to an inhibition of the transcytotic mechanism, resulting in a low density of endothelial vesicles. 4. A comparison of vesicular densities and protein permeabilities in a number of vascular beds shows only a very weak correlation, therefore vesicle numbers alone cannot be used to predict permeability to macromolecules. 5. Blood-brain barrier capillaries are fully capable of transcytosing specific proteins, for example, insulin and transferrin, although the details are still somewhat controversial. 6. It has recently been shown that the albumin binding protein gp60 (also known as albondin), which facilitates the transcytosis of native albumin in other vascular beds, is virtually absent in brain capillaries. 7. It seems likely that the low blood-brain barrier permeability to macromolecules may be due to a low level of expression of specific receptors, rather than to an inhibition of the transcytosis mechanism.

Gp60 activation mediates albumin transcytosis in endothelial cells by tyrosine kinase-dependent pathway.

We investigated the function of gp60, an endothelial cell membrane 60-kDa albumin-binding protein localized in caveolae, and the mechanism of its activation in regulating endothelial permeability of albumin. Gp60 organization on the bovine pulmonary microvessel endothelial cell (BPMVEC) surface was punctate as shown by immunofluorescence using an anti-gp60 antibody (Ab) conjugated with bisfunctional, N-hydroxysuccinimidyl fluorophore (Cy3). Addition of a secondary Ab to anti-gp60 Ab-treated BPMVEC induced cross-linking of gp60 as evident by increased size of fluorescent particles and cell surface gp60 clustering. Gp60 cross-linking also produced 2-3-fold increases in the endothelial cell uptake and the luminal to abluminal permeability of 125I-albumin as well as the fluid-phase tracer, horseradish peroxidase. The increased transendothelial permeability of macromolecules was the result of transcytosis as it was not associated with an increase in the paracellular pathway. Incubation of anti-gp60 Ab with BPMVEC at 37 degrees C caused internalization of gp60, and thereby reduced the uptake of the macromolecules. Activation of gp60 by either albumin (the gp60 ligand) or gp60 cross-linking induced the phosphorylation of both gp60 and caveolin-1 (the major structural caveolar protein) on tyrosine residues. Gp60 activation also phosphorylated the Src family tyrosine kinases pp60(c-Src) and Fyn. The activated pp60(c-Src) and Fyn co-immunoprecipitated with caveolin-1 in BPMVEC membrane. Protein tyrosine kinase (PTK) inhibitors, herbimycin A and genistein, prevented gp60-activated macromolecule uptake and transcytosis in a concentration-dependent manner, indicating the functional significance of the PTK pathway in activating albumin transcytosis. These findings indicate that activation of gp60 stimulates the Src PTK signaling pathway, and thus regulates the transcytosis of albumin across the endothelial cell monolayer.

Transport of insulin and albumin by the microvascular endothelium of the rete mirabile.

Vascular permeability for albumin and insulin in the continuous capillary network of the rete mirabile of the eel swimbladder was evaluated by ultrastructural immunocytochemistry and countercurrent perfusion experiments. Upon perfusion of the rete capillaries with a buffer solution containing albumin and insulin, these serum proteins were revealed at the electron microscope level, by the Protein A-gold immunocytochemical technique on a post-embedding step. For the simultaneous detection of both proteins, the double labeling technique with different sized gold particles was used. Furthermore, labeling was performed with the mixture of anti-albumin and anti-insulin anti-bodies. The labelings obtained were morphometrically evaluated and demonstrate that: (1) serum proteins such as albumin and insulin are transported by the endothelial cells through their plasmalemmal vesicular system; (2) insulin is transported preferentially to albumin; and (3) this transport involves different populations of plasmalemmal vesicles. Measurements of diffusion permeability coefficients have confirmed the preferential transport of insulin, its coefficient being higher than that of albumin. Conversely, when compared to that of insulin or sucrose, which are assumed to be markers of the paracellular diffusion, it was found to be much lower, indicating that transcytosis through the vesicular system is less efficient than diffusion along the intercellular junctions. These results indicate that transcytosis of insulin and albumin occurs via different sets of plasmalemmal vesicles, probably through receptor-mediated mechanisms, and that the overall rate of transport across the rete capillaries, with respect to paracellular diffusion, is higher for insulin than for albumin.

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.

Fibrin contact increases endothelial permeability to albumin.

We studied the effects of contact of bovine pulmonary artery endothelial cell monolayers with fibrin on the endothelial barrier function. Fibrin formed by clotting purified fibrinogen (0.5 to 3.0 mg/ml) with alpha-thrombin (1 U/ml) was added to endothelial monolayers and permeability measurements were made after fibrin removal. Fibrin incubation for 3 hours resulted in 2- to 5-fold increases in transendothelial 125I-albumin permeability. Permeability returned to baseline value within 3 hours after fibrin removal. Direct contact with fibrin was necessary for the response, since fibrin separated from the endothelium did not increase permeability. Contact with agarose (2 mg/ml) or fibrinogen (0.5 to 3.0 mg/ml) also did not increase endothelial permeability. Transmission electron microscopic examination indicated normal appearance of interendothelial junctions at a time when albumin permeability was increased and no overt evidence of endothelial injury. Incubation of fibrin with endothelial monolayers at 4 degrees C prevented the increase in albumin permeability. We examined the possibility that increased albumin transcytosis was responsible for fibrin's effect using 14C-sucrose (Mr = 342D), a lipid insoluble tracer. Fibrin increased sucrose flux by 1.5-fold compared to 2- to 5-fold increases in albumin flux. The results indicate that fibrin contact with the endothelial cell increases endothelial permeability. The effect of fibrin may involve activation of temperature-sensitive bulk phase transcytosis of albumin.

Gp60 is an albumin-binding glycoprotein expressed by continuous endothelium involved in albumin transcytosis

Albumin reduces capillary permeability and acts as a carrier for various small molecules. Recently, we identified a 60-kDa sialoglycoprotein (gp60) on the surface of cultured rat microvascular endothelial cells (MEC) that binds albumin and antiglycophorin serum (alpha-gp). We verified that alpha-gp recognizes the albumin-binding gp60 by affinity, purifying proteins from MEC extracts using immobilized albumin. gp60 was immunoblotted with alpha-gp only when the MEC extract was reacted with albumin and not in controls. We immunoprecipitated gp60 from biosynthetically radiolabeled MEC lysates and from extracts containing endothelial surface proteins of isolated rat hearts that were radioiodinated in situ. gp60 was immunoblotted selectively in rat tissue microvascular beds lined with continuous endothelium (heart, lung, diaphragm, fat, skeletal muscle, mesentery, and duodenal muscularis but not cortical brain) and not those exclusively lined with fenestrated or sinusoidal endothelium (adrenal, pancreas, liver, and small intestinal mucosa). MEC isolated from rat heart, lung, and epididymal fat pad expressed gp60 and bound albumin, whereas various nonendothelial cells and brain-derived MEC did not. gp60 is an albumin-binding glycoprotein expressed specifically on the surface of continuous endothelium that binds albumin apparently not only to initiate its transcytosis via plasmalemmal vesicles but also to increase capillary permselectivity.

Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I-labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcytosis. The transport of 125I-BSA tracer across BPAEC grown on gelatin- and fibronectin-coated filters (0.8 microns pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I-BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10(-7) M. In contrast, gelatin added to the medium altered neither 125I-BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I-BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I-BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I-BSA binding nor transport. The reduction of 125I-BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin-dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.

Transcytosis of albumin in capillary endothelium.

We have used a variety of immunocytochemical procedures to localize albumin in transit through the capillary endothelium of the murine myocardium and thereby identify endothelial cell structures involved in albumin efflux. The most informative results were obtained with a protocol that included (a) removal of endogenous albumin by perfusion of the heart with PBS supplemented with 14 mM glucose, (b) perfusion of the heart vasculature with exogenous (bovine) albumin for various short time periods, (c) fixation of the vessels by formaldehyde-glutaraldehyde mixtures, (d) processing of fixed myocardium specimens through L. R. White embedding followed by sectioning, or direct thin frozen sectioning, and (e) reacting the surface of such specimens with antialbumin antibodies followed by gold-labeled secondary antibodies. The results indicate that (a) monomeric albumin binds (with low affinity) to the luminal surface of the capillary endothelium, (b) it is restricted in transit through the endothelium to plasmalemmal vesicles, and (c) it appears in the pericapillary spaces less than 15 s after the beginning of its perfusion. No albumin concentration gradients, centered with their maxima on the exits from intercellular spaces, were detected at any time points, including the shortest ones (15 and 30 s) investigated. Additional information comparing monomeric vs. polymeric albumin transcytosis was obtained using albumin-gold complexes. The results are discussed in terms of vesicular transport of albumin across the endothelium and the relations of this type of transport to the postulated pore systems of the physiological literature.