ep60 is an albumin-binding glycoprotein expressed
`by continuous endothelium involved in albumin transcytosis
`
`JAN E. SCHNITZER
`Department of Medicine and Pathology, Division of Cellular and Molecular Medicine and Institute for
`Biomedical Engineering, University of California, San Diego, School of Medicine,
`La Jolla, California 92093-0651
`
`Schnitzer, Jan E. gp60 is an albumin-binding glycoprotein
`expressed by continuous endothelium involved in albumin
`transcytosis. Am. J. Physiol. 262 (Heart Circ. Physiol. 31):
`H246-H254, 1992.—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 {a-gp). Weverified that a-
`gp recognizes the albumin-binding gp60 by affinity, purifying
`proteins from MECextracts using immobilized albumin. gp60
`was immunoblotted with a-gp only when the MEC extract was
`reacted with albumin and not in controls. We immunoprecipi-
`tated gp60 from biosynthetically radiolabeled MEC lysates and
`from extracts containing endothelial surface proteins of iso-
`lated 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 plasmalem-
`malvesicles but also to increase capillary permselectivity.
`rat; biological
`transport; capillary permeability; membrane
`receptors; membraneproteins; sialoglycoproteins; receptor-me-
`diated transport; vesicular transport; plasmalemmal vesicles;
`glycocalyx
`
`endothelium (10, 15, 19, 22), and its binding within
`transport pathways, such as plasmalemmalvesicles and
`the introit of intercellular junctions, apparently forms a
`molecularfilter within these pathways (3) that can elec-
`trostatically (20) and sterically (3, 21) restrict the trans-
`port of water, small solutes, and macromolecules across
`the microvascular wall (6, 11-14, 19). In addition, albu-
`min is transcytosed across vascular endothelium via plas-
`malemmalvesicles (10, 15, 19) and acts as a carrier for
`small ligands bound to it, such as fatty acids (5). This
`apparent receptor-mediated transcytosis of albumin ap-
`pears to occur selectively in certain tissues with vascular
`beds lined with continuous endothelium (10, 28) andis
`influenced greatly by the ligands bound to albumin(5).
`Thebinding of albumin to cultured microvascular en-
`dothelium has been quantitated and immunolocalized to
`the surface of cultured rat microvascular endothelial
`monolayers (22). Specific albumin binding was shownto
`be saturable, reversible, competible, dependent on cell
`type and cell numberandto have a negative cooperativity
`in nature (22). Albumin binding wassensitive to pronase
`but not to trypsin digestion of the cell surface and was
`inhibited significantly by the presence of Limax flavus
`(LFA), Ricinus communis (RCA), and Triticum vulgare
`(wheat germ; WGA) agglutinins but not several other
`lectins (23). Recently, a group of rat endothelial plas-
`malemmalsialoglycoproteins has been identified both in
`situ and in culture (25). One of these sialoglycoproteins,
`called gp60, was identified as an albumin-bindingprotein
`because it 7) interacts with albumin conjugated to beads
`A PRIMARY FUNCTIONof the attenuated layer of vascular
`endothelium is to act as a barrier to the transvascular
`(23); 2) binds RCA, LFA, and WGAbutnototherlectins
`(23, 25, 26); and 3) is sensitive to pronase andsialidase
`transport of plasma molecules in many tissues. The
`but not to trypsin digestion (23, 25). Another laboratory
`selectivity of the endothelial barrier varies in different
`has also identified gp60 as one of three major albumin-
`vascular beds and is strongly dependent on the structure
`binding proteins (8, 9). The structural and functional
`and type of endothelium lining the microvasculature
`relationship of these albumin-binding proteins to one
`(29). Several pathwaysexist for the transport of plasma
`another is unknown. Further characterization of gp60
`molecules across continuous endothelium: /) intercellu-
`showed that it apparently contains O-linked but not N-
`lar junctions are highly regulated structures that form
`linked glycans (25) and mayalso be antigenically related
`the paracellular pathway for the passive, pressure-driven
`to another sialoglycoprotein, namely glycophorin (26).
`filtration of water and small solutes; 2) plasmalemmal
`Polyclonal antiserum (a-gp)
`raised against purified
`vesicles transcytose plasma macromolecules, apparently
`mouse glycophorin gp3 recognized a 60-kDa WGA-and
`by shuttling their contents adsorbed from blood from the
`RCA-binding sialoglycoprotein on the surfade of cultured
`luminal to antiluminal aspect of the endothelium (16);
`rat microvascular endothelial cells (26). This apparent
`and 3) transendothelial channels, which may form tran-
`recognition of gp60 by a-gp wasinhibited in the presence
`siently by the fusion of two or more plasmalemmal
`of murine glycophorins.
`vesicles, may provide a direct conduit for the exchange
`In this study, the specific interaction of gp60 with
`of both small and large plasma molecules (31). Recently,
`albumin and with a-gp serum is demonstrated. Then, a-
`it has become clear that capillary permeability in many
`gp is used as a probe for gp60. Because albumin binding
`vascular beds is also dependent on the interaction of
`and transcytosis via plasmalemmalvesicles. are not ob-
`these transendothelial transport pathways with the mul-
`served in the endothelium lining the vasculature of many
`tifunctional plasma protein, albumin (3, 6, 11-14, 19).
`tissues, tissue-specific expression of gp60 should be ex-
`Albumin binds to the luminal glycocalyx of continuous
`H246
`0363-6135/92 $2.00 Copyright © 1992 the American Physiological Society
`
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`

`ENDOTHELIAL ALBUMIN-BINDING GLYCOPROTEIN 6r60
`
`H247
`
`pected if gp60 does indeed function as a physiologically
`significant. albumin-binding protein. Therefore various
`rat tissue and cell extracts were tested using a-gp for the
`presence of gp60. In addition, microvascular endothelial
`cells have been isolated from several organs and grown
`in culture’ to compare both gp60 expression and albumin
`surface binding. These experiments demonstrate J) the
`' specific expression of gp60 only by those cultured. éendo-
`'
`thelial cells that bind albumin and not. various other
`cells, 2) the presence of gp60 on the surface of vascular
`endothelium both in situ and in culture, and 2) the
`selective distribution of gp60 in rat tissues with. vascular
`beds lined with continuous endothelium that transcytosé
`albumin via plasmalemmal vesicles.
`
`METHODS
`
`Isolation. and growth of endothelial cells in culture. Male
`albino rats (Sprague-Dawley, 200-250'g). were. anesthetized with
`ether. The heart, lungs, and cortical brain were surgically
`removed from. three to five rats, were submerged in, cold Dul-
`beceo’s modified Eagle’s medium (DMEM) supplemented with
`15-20% fetal calf serum (FCS)
`(DMEM+), and each was
`minced in a vial using small sterile ‘surgical scissors. For the
`heart, only myocardium was used aftér caréful excision of the
`epi: and endocardium, For the lung, only peripheral regions of
`the lobes were used. Both of these excision procedures. are
`designed to- eliminate obvious large arterial and venous seg-
`ments of the vasculature and to increase the probability for
`isolation of microvascular endothelium. After centrifugation
`for 5. minat 1,600 g at: 4°C, the pellet was resuspended in 2 mg/
`ml of collagenase (from. Clostridium histolyticum,
`type II,
`Sigma) in DMEM-+at 37°C usingfive times the tissue volume,
`After a 1-h incubation, the suspension was poured over a sterile
`Nitex monosereen cloth (no. 3-112-40xx; Tetko, Briar Cliff
`Manor, NY) to filter out tissue clumps. The filtrate was cen-
`trifuged for 5 min at 1,000 g, and the pellet was resuspended in
`DMEM-+containing 20 ug/ml! of heparin, 100 U/ml of penicil-
`lin G, 100 g/ml of streptomycin sulfate, and 10% bovine aortic
`endothelium-conditioned media. The cells were plated onto T-
`25 flasks (Corning, Corning, NY) for culture at 37°C with 5%
`COs in air. After several days when confluency was reached,
`the cells were examined and sorted on a fluorescent-activated
`cell sorter using uptake of acetylated low-density lipoprotein
`labeled: with.
`.1,1’-dioctadecy!-3,3,3',3’-tetramethylindocarbo-
`cyanine perchlorate (Dil-AcLDL)as in Refs. 22 and. 30. Madin-
`Darby kidney cells (MDCK) cells were used as a negative
`control. Micrevascular endothelial cells that exhibited uptake
`of Dil-AcLDL.-and the mixture of other cells in the heart and
`lung. preparations that did not internalize Dil-AcLDL were
`replated separately and grown in-culture using the above media
`(without heparin). After 24 h, this medium was replacedforall
`future culturing with DMEM+ for the noriendothelialcells and
`with DMEM-+with retinal-derived growth factor for the endo-
`thelial cells.. Microvascular endothelial cells isolated from. rat
`epididymalfat pads (RFC) were grown and plated as previously
`described (22).
`Immunoblotting of total cell lysate from cultured cells. After
`washing the confluent cell monolayers, their proteins were
`solubilized directly with cold solubilization buffer (SB} contain-
`ing 0.17 M tris(hydroxymethyljaminomethane (Tris) -HCl (pH
`6.8), 3% (wt/vol) sodium dodecy! sulfate (SDS), 1.2%. (vol/vol)
`8-mercaptoethanol, 2 M urea, and 3 mM EDTA in. double-
`distilled watér as described previously (25). After incubation in
`boiling water for 10 min, a lysate volume equivalent of 10° cells
`was processed for SDS-polyacrylamide gel. electrophoresis
`(PAGE), and the separated proteins were electrotransferred
`onto Immobilon filters (Millipore, Bedford, MA) as in Refs. 25
`
`and 26. Strips of these filters were immunoblotted with rabbit
`serum, and any bound immunoglobulin G (IgG) was detected
`using anti-rabbit IgG antibodies conjugated to alkaline phos-
`phatase as in Ref. 26.
`Immunoprecipitation- of gp60 radiolabeled biosynthetically
`usinig tritiated sugars: About-5 x 10° RFC.cells were plated onto
`two T-78 flasks. After 1.5.h, 1.0 mCi of [2,6-°H]mannose (45
`Ci/mmol; Amersham, Arlington Heights, IL) mixed in 3 ml of
`DMEM-+was added ‘to one flask ‘of cells or 1.0 mCi of [6H]
`galactose (20 Ci/mmol; ICN, Costa Mesa, CA) combined with
`1 mCi of [6H] glucosamine (27 Ci/mmol; ICN) mixed in 3 ml
`of DMEM+ was added to another flask of cells. After 3. days
`when the cell. monelayer reached full confluency, the cells were
`washed with 10 ml of DMEM (8 times, 1 min) at. 4°C, lysed
`with 5% Triton X-100 and 1% SDS in. phosphate-buffered
`saline (PBS), and finally scraped from the flask. After a 10-
`min spin at 4°C. (1,000 g), the supernatant was used for im-
`munoprecipitations overnight with o-gp as described in Ref.
`25. The precipitates were analyzed by SDS-PAGE and were
`visualized by fluorography as per Ref. 1.
`immunoprecipitation of gp60 radioiodinated in situ. The
`endothelial luminal surface proteins of the heart vasculature
`were. radioiodinated in situ-using Na"T, and microspheres were
`covalently coated with lactoperoxidase and glucose oxidase as
`described previously (25). Proteins were extracted from 200 mg
`of heart tissue by mincing the tissue in 500 ul of 5% Triton X--
`100 and 1% SDS in PBS at 4°C. The lysate was centrifuged at
`13,000 g for 1 min at 4°C, and 100 ul-of the supernatant (tissue
`extract} was mixed overnight at.4°C with 380 ul of PBS and 20
`pl of rabbit antiserum. The ensuing immune complexes were
`then precipitated with protein A conjugated to Sepharose beads
`as in Ref. 25. The proteins bound to the beads were solubilized
`directly with 150 yl of cold SB, kept in boiling water for 10
`min, and analyzed by SDS-PAGE followed by autoradiography
`as described previously (25).
`Immunoblotting of tissue extracts. Male albino rats (Sprague-
`Dawley, 200-250 g) were anesthetized with an. intraperitoneal
`injection of ketamine (100 mg/kg) and xylazine (33 mg/kg).
`The chest was opened through a median sternotomy. Through
`a needle inserted into thé left ventriele and after the right
`atrium was cut: for outflow purposes, ‘the vasculature was pér-
`fused with DMEM {Irvine Scientific, Irvine, CA) at a mean
`pressure of 60 mmHgfirst for 5 min at. 37°C and then for 10
`min at 10°C. The heart, liver, cortical brain, epididymal fat
`pad, kidney, adrenals, pancreas, duotlenum, diaphragm, and
`gastrocnemius skeletal muscle were excised, and 300 mg {weight
`wet) of each tissue was minced in 1 ml of SB at 4°C. For the
`duodenum, the muscosa of the intestinal wall was separated
`from the muscularis by scraping under visual
`inspection
`through a dissecting microscope. In somecases, only lung tissue
`was excised after perfusion through the right ventricle at a
`mean pressure of 20: mmHg with left atrial outflow. The lysates
`were centrifuged at 13,000 g for 1 min at 4°C, and the super-
`natants (tisstie extracts) were processed equivalently for pre-
`parative SDS-PAGE and immunoblotting as described above.
`Tn the lung and fat. pad preparations,:the centrifugation step
`did not pellet all of the tissue debris, and a top layer of fatty
`material was present and had to be carefully aspirated. before
`the solubie material could be removed: In a few preparations,
`before tissue excision, the vasculature was perfused for 3 min
`directly with a protease inhibitor cocktail containing 400 pe/
`ml benzamidine, 10 »g/tml leupeptin, 10 ng/ml pepstatin A, 65
`pg/ml aprotinin, 10 pg/ml chymostatin, 100 ug/ml O-phenan-
`throline, and 350 «g/ml phenylmethylsulfonyl fluoride. The
`presence. of protease inhibitors did not alter the observed re-
`sults.
`Immunoblotting of gp60 after interaction with tmmobilized
`albumin. A 1% Triton. X-100-soluble fraction from the RFC
`célls was precipitated overnight in 90% ethanol at: —20°C, The
`
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`

`H248
`
`ENDOTHELIAL, ALBUMIN-BINDING GLYCOPROTEIN GPé60
`
`pellet was washed once using 70% ethanol and then resus-
`pended in PBS. The suspension was centrifuged at 13,000.g for
`5 min, and the supernatant was used as the cell extract. Strips
`of Immobilon or nitrocellulose filters (1x 0.25 in.) were incu-
`bated for 4 h at.room temperature in 7) PBS alone, 2) 10 mg/
`mil of albumin in PBS, or 3) 10 mg/m) of transferrin in PBS.
`After air drying for at least.1-h, all strips were quenched for 1
`h with blocking solution (6% Blotto and:0.1% Nonidet P-40 in
`PBS) and then incubated overnight at.raam temperature.in 0,5
`ml ofa 1:50-dilution of the RFC cell extract in blocking solution.
`Some of the albumin strips and transferrin strips were also
`incubated in blocking solution withoutcell extract.as additional
`controls. All strips wére: washed. three times for 5 min with
`0.1% Nonidet P-40 in PBS. and then cut into small pieces.
`Proteins were eluted using 100 ml of SB in.a boiling water bath
`for 15 min, separated by SDS-PAGE on a mini-gel apparatus
`(Bio-Rad), and then electrotransferred onto Immobilon. filters
`at 40 V for 1.5 h. The filters were immunoblotted with a-gp as
`described above.
`.
`
`RESULTS
`
`gp60 interacts with both oa-gp and albumin. Recently,
`we have shown that a-gp does interact with an RCA-
`and WGA-binding 60-kDa sialoglycoprotein (26). present
`on the surface of ciltured microvascular endothelium
`derived from RFC. Other work (23) has implicated an
`RCA- and WGA-binding 60-kDa glycoprotein as an al-
`bumin-binding protein on the RFC cell surface. Because
`these data strongly suggest that o-gp is interacting with
`the albumin-binding protein gp60,.we attempted to in-
`hibit albumin bindingto the surface:of cultured RFC cell
`monolayers using an IgG. fraction of a-gp isolated with
`immobilized protein A as in Ref. 26. The cell monolayers
`were first exposed for 10 min to the IgG fraction of a-gp
`(ap to 100 pg/ml), Then “I-albumin was added to
`achieve a final concentration: of 2 we/ml, and the usual
`binding assay was performed (22, 23). Although this IgG
`fraction did interact with the 60-kDa protein by immu-
`noblotting of RFC cell lysates (26), it did not affect.
`albumin binding to the RFC cell surface (data not,
`shown), suggesting different binding sites for the anti-
`body and albumin.’ Therefore it became necessary first
`to ensure that. «-gp is indeed interacting with the 60-kDa
`''The observation that a-gp, even at high-concentrations, does not
`interfere with albumin binding'to the cell surface suggests that albumin
`and a-gp may interact. with gp60 at different binding sites within the
`molecule. This postulation is supported by our recent, investigation,
`attempting to define the epitope recognized by a-gp that indicates that
`c-gp reacts with a-peptide region located apparently in:the endedomain
`ofthe protein (unpublished observations). Conversely, albumin binding
`to the endothelial cell surface is expected to be via the ectodomain of
`gp60..An alternate explanation is supported by ourimmunofluorescence
`studies. on permeabilized and. nonpérmeabilizéd RFC cells, which
`showed only mild labeling of the cell surface with a-gp (data not
`shown). Bevause the antibody was raised against an antigen denatured
`by SDS-PAGE,
`it:
`is not surprising that o-gp. apparently interacts
`poorly with the native form of gp60 seen on thé cell surtace. The
`epitope may be masked in its native state-and/or require some degree
`of gp80 denaturation before antibody recognition. Furthermore, since
`our results indicate that the epitope liés-in the endodomain of gpé60, it
`may, like glycophorin, interact with cytoskeletal proteins; this inter-
`action may interfere with antibody binding to the permeabilized cells.
`Unfortunately, the poor immunofluorescence staining with a-gp pre-
`cludes more exact. immunolocalization studies at the electron micro-
`scopic level.on either cultured cells or tissue. These immunolocalization.
`studies must be performed witha new antibody that’ recognizes.gp60
`undér more native conditions.
`
`albumin-binding glycoprotein gp60 and not just. another
`surface glycoprotein of similar apparent. molecular mass.
`Albumin was adsorbed to filters and then air dried to
`immobilize the protein to the filters. After blocking the
`filters, they were exposed to an RFC cell extract contain-
`ing gp60 and then washed. Proteins were eluted from the
`strips, separated by SDS-PAGE, electrotransferred to
`filters, and then immunoblotted. with e-gp, Controls
`included 7) exposing the cell extract.to filters alone or
`to transferrin immobilized. to filters and 2) testing the
`material desorbed from the transferrin and albumin fil-
`ters that had not been exposed to the cell extract. Figure
`1 shows that «-gp detected a single 60-kDa protein only
`in eluates. of the albumin-adsorbedstrips.interacted with
`the RFC cell extract. The eluates from all of the control
`strips were negative. These results indicate that 7) gp60
`interacts preferentially with albumin, 2) a-gp does in-
`deed. recognize gp60 present in the RFC cell extracts,
`and 3) a-gp can new be used with confidence as a probe
`for the albumin-binding glycoprotein gp60.
`Endothelial expression. of gp60:
`immunoprecipitation
`with-a-gp of endothelial glycopreteins radiolabeled biasyn-
`thetically in culture. Although it. is clear that. gp60 is
`located on the surface of vascular endothelium in culture
`(23, 25), the biosynthetic origin of gp60 is uncertain.
`Figure 2 shows that gp60 can be immunoprecipitated
`specifically with a-gp from lysates of RFC cells that had
`been biosynthetically radiolabeled with tritiated sugars.
`gp60 was radiolabeled successfully with a mixture of[6-
`"H|galactose and [6-"H]glucosamine but not with [2,6-
`*‘H]mannose alone. To ensure that: gp60 was indeed ex-
`pressed during each radiolabeling procedure, both. radi-
`olabeled celi lysates were subjected to SDS-PAGE, and
`the separated proteins were electrotransferred onto. fil-
`ters that were immunoblotted with a-gp. An equivalent
`signal for gp60 was detected in both cases (data net
`shown). These results indicate that gp60 is indeed ex-
`
`Filters adsorbed with:
`te Fone ok
`Albumin
`Transferrin -
`-- +
`-
`+
`+
`PBS alone ee *
`Filters. exposed:
`to:
`Cell extract
`
`GPo0—
`
`Serum:
`
`IM NI
`
`[IM IM IM IM
`
`Fig. 1. Specific interaction of 60-kDa glyceprotein (gp60) with albu-
`min immobilized on filters and. subsequent detection by immunoblot-
`ting with antiserum against glycoprotein (a-gp). Filters were incubated
`with albumin in phosphate-buffered saline (PBS); transferrin in PBS,
`or PBS aloné (as indiéatéed for each lane above) ahd then air dried to
`imuiobilize the proteins onto filters. As-indicated, ‘strips were then
`either exposed or not exposed to.rat epidymal fat pad (RFC)cell extract
`in a. blocking. solution. Proteins were-eluted from. each strip, separated
`by SDS-PAGE, and-electretransferredonto: Immebilon filters. Strips
`of these filters containing eluted proteins are shown above and, were
`immunoblotted either with nonimmune (NI) and/or immune o-gp (IM)
`serum. Bound ‘immunoglobulin G (IgG) was detected using anti-rabbit
`IgG antibodies conjugated to alkaline phosphatase as described previ-
`ously. (25). Gp60 was deteeted only after interaction of cell extract with
`albumin filters aid not in varictis controls.
`
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`ENDOTHELIAL ALRUMIN-BINDING GLYCOPROTEIN GPré0
`
`H249
`
`IMMUNOPRECIPITATION
`WITH ANTI-GLYCOPHORIN SERUM
`— G/G—-—Man—
`ee
`
`66 -
`
`gpbd- 45 -
`
`NI
`
`IM NI
`
`IM
`
`NUMBER
`CELL
`
`Fig. 2. Biosynthetically radiolabeled glycoproteins of RFC cells im-
`munoprecipitated with e-gp serum. RFC cells. were radiolabeled with
`either [*H] mannose (Man) or both ["H]galactose and [*H]glucosamine
`iG/G)-as-descibed in METHODS. Lysates from thesé.célls were-subjected
`to immunoprecipitation with either nonimmune (NI). or immune (IM)
`a-gp rabbit serum. Immunoprecipitates were separated by SDS-PAGE
`ona 5~15% gradient gel‘and visualized by fluorography. Only ¢p60 was
`detected specifically. with.a-gp, The band found at'45 kDa is considered
`tobe nonspecific since: it. was detected both in nonimmune serum and
`toa lesser extent in immune serum.
`
`pressed by vascularendothelium in culture. Furthermore,
`the lack of incorporation of [2,6--H]mannose into gp60
`is consistent with our previous findings based on direct
`and sequential lectin affinity chromatography (25) that
`suggest that rat.gp60 contains O-linked glycans but ap-
`parently not N-linked glycans.
`Endothelial cell isolation from rat tissues. Fluorescence-
`activated. cell sorting was used to isolate microvascular
`endothelial cells from a mixture of cells derived from a
`collagenase treatment of cortical brain, heart, and lung
`tissues (See METHODS). The specific ability of endothelial
`cells to internalize Dil-AcLDL was used. to isolate endo-
`thelial cells from nonendothelial cells (22, 30). Those
`cells that lacked uptake of Dil-AcLDL were also saved
`from the heart and lung preparations. After sorting, all
`cells were replated, grown in culture for several passages,
`and checked periodically for Dil-AcLDL uptake using
`either the fluorescence-activated cell sorter or simple
`fluoresence microscopy of the cells grown om slides as
`described in Ref. 22. Figure 3 shows the fluorescence
`ptofile of Dil-AcLDL.uptake ofa typical cell preparation.
`The cells initially isolated from tissue have a bimodal
`cell distribution with considerable variation in céll size
`and fluorescence intensity. Most of the cells have fluo-
`rescence intensity far greater than the MDCKcells (neg-
`ative control) and appear to internalize Dil-AcLDE. A
`second group of cells has a fluorescence intensity more
`comparable to the MDCKcells, especially when cell size
`is considered, The top 20% of the cells with the greatest.
`fluorescence intensity and ‘the bottom 20% of the cells
`with the lowest fluorescence intensity were collected
`separately using the cell sorter. These cells after growth
`in culture were examined for Dil-AcLDL uptake once
`again and, as shown in Fig. 3B,the endothelial cells that
`were positive on the first. sort are now unimodal in
`distribution with considerably greater fluorescence in-
`tensity than the negative control. Meanwhile, the other
`isolated cells shown in Fig. 3C, which should be nonen-
`dothelial in origin, also appeared to have a unimodal cell
`distribution; however, in this case, their fluorescence was
`equivalent to the negative control. Many of the endothe-
`lial cell preparations. were also checked periodically by
`immunofluorescence as described in Ref. 22 and in each
`case stained positively for other endothelial markers,
`such as dangiotensin-converting enzyme or factor VIII
`(data not’ shown). This approach has allowedusto isolate
`
`MDCK
`
`MDCK
`
`Cell Size
`
`Fluorescence Intensity
`Fig. 3. Profile of fluorescence-activated cell sort of cultured cells de-
`rived from a rat heart preparation. Cells were isolated:from 4 rat, hearts
`and grown in culture until confluency (see METHODS). These cells were
`mcubated with 1,1’ -dioctadecyl-3,3,3’ ,3’tetramethylindocarbocyanine
`perchlorate-labeled acetylated.
`low-density Jipoprotein. (Dil-AeLDL)
`and processed for flucrescence-activated cell sorting as described. pre-
`viously (20, 30). Cell size and fluorescence intensity profiles of cells are
`shown as a function of éell number. Profiles of initial population of
`heart-derived (IHD)cells are given in.A. MDCK cells were also exam-
`ined a8. a negative control. Twenty percent of cells with highest and
`lowest: fluorescetice intensity were isolated separately using cell sorter
`and then grown in culture. After 3 passages, these 2 cell populations
`were reexamined for Dil-AcLDL uptake, and théir profiles. are shown
`in B frat heart endothelial (RHE) cells are derived from those cells
`with highest fluorescence intensity of Dil-AcLDL-positive cells] and C
`{heart mixture (HM). of cells grown from bottom 20% of cells with
`lowest flourescence]. When total fluorescence of RHE cells. was com-
`pared with that of MDCKcells, >98% of RHE cells internalized more
`Dil-AcLDL than any control cells, On the other hand, HM cells
`exhibited same degree of fluorescence as MDCK cells.
`[To get. a
`population of cells that lacked Dil-AcLDL uptake (1.e., were devoid of
`endothelial cells}, we found it: necessary to passage cells in culture at
`least: twice.]
`‘
`
`and grow in culture endothelial cells from the rat heart,
`lung, and brain and nonendothelial cells from. the heart
`and lung.
`.
`Immunoblotting lysates of cultured cells. The proteins
`from the organ-derived cells isolated and grown in cul-
`ture (as described above) were solubilized, separated by
`SDS-PAGE, electrotransferred to filters, and then im-
`munoblotted with a-gp and nenimmune serum. Proteins
`from RFC cells acted as a positive control. Figure 4
`shows that gp60 was present only in the lysates of
`endothelial cells isolated from the heart and lung but not
`from the cortical brain.’ For the heart and lung prepa-
`tations, only the Dil-AcLDL-positive endothelial cells
`expressed gp60, whereas the mixture of nonendothelial
`cells (probably consisting of fibroblasts, pericytes, and/
`or smooth muscle cells) neither internalized Dil-AcLDL
`nor appeared to-express gp60.. These results indicate that
`under the conditions used here to isolate and grow these
`cells, microvascular endothelia from the heart: and lung
`but not the brain express gp60. in culture, whereas non-
`endothelial.cells from the heart ‘and lung do not, We
`have also checked by immunoblotting ‘several specific
`®. pp60 can also be-irnamunoblotted specifically with e-gp using lysates
`from other cultured endothelium derived from.sheep pulmonary artery,
`bovine aorta, fetal bovine heart (ATCC), microvessels of pig atrium,
`and human umbilical vein (unpublished observations).
`
`Abraxis EX2013
`Cipla Ltd. v. Abraxis Bioscience, LLC
`IPR2018-00162; IPR2018-00163; IPR2018-00164
`Page 4 of 10
`
`

`

`¥1250
`
`ENDOTHELIAL ALBUMIN-BINDING GLYCOPROTEIN cpé0
`
`Tissues -
`Cells -
`
`Dil-AcLDL:
`
`
`IM IM
`iM IM NI
`IM NI
`NIE IM NI
`Serum:
`Fig. A. Differential expression of .gp60 in rat microvascular endothelial
`cells isolated from different tissues. RFC.cells along with microvascular
`endothelial cells isolated frém rat brain (RBE), heart (RHE), and lung
`(RLE) were processed for immunoblotting overnight with a. 1:500
`dilution of either nonimmune. {NI} or immune (IM): a-gp serum. In
`addition, a mixture of nonendothelial cells isolated from rat. heart
`(AM). and lung (LM) were also.immunoblotted with a-gp..In each-case,
`a lysate equivalent of ~10° cells was loaded onto a preparative gel. Both
`endothelial and nonendothelial cells were passaged 3 times in culture
`after. their initial isolation: and separation by fluorescence-activated
`cell sorting. Heart and Iung endothelial cells both internalized Dil-
`AcLDL and expressed gp60, whereas nonendothelial cells did: neither.
`. Gp60-was not detected in lysates of cultured brain endothelium.
`
`nonendothelial cell types grown in culture. Normal rat
`kidney fibroblasts. [from American Type Culture Collec-
`tion (ATCC); NRK-49F] that bind albumin poorly (22)
`and rat aortic smooth. .musclé: cells (from ATCC; A10)
`were negative for gp60 expression (data not shown).
`Therefore, in the cultured celis tested so far, the expres-
`sion of gp60 appears. to be specific for certain endothelial
`célis.
`Selective. albumin binding .to rat cultured. endothelial
`cells. Because the data given above indicate that gp60
`expréssion is limited to oily cértain endothelialcells [rat
`heart endothelial (RHE), RFC, and rat lung endothelial
`(RLE): but not rat brain endothelial (RBE) cells], the
`binding of albumin to these cells should vary in accord-
`ance with gp60 expression if gp60 does indeed play a role
`in albumin binding. Rat serum albumin (RSA) was ra-
`diviodinated, andthe binding of ‘*I-RSA to the surface
`of these cultured cells was compared using 0.1 mg/ml of
`1257.RSA as described previously (22). Because the RFC
`cells bind RSA and were used originally to characterize
`the kinetics of albumin binding to cultured microvascular
`endothelium. (22), they acted as a positive control. As
`shown in Fig. 5,I-RSA binding was ~10-15 times
`preater for the RLE, RHE, and RFC cells than for the
`RBEcells. This small amount. of binding of albumin to
`the RBEcells is comparable to that: observed previously
`for rat fibroblasts (22). This selective albumin binding
`to certain cultured endothelialcells agrees well with the
`above observation that the RLE, RHE, and RFC cells
`expressed gp60 but the RBEcells did not. In addition,
`the increase. of 40-50% in “*I-RSA binding observed for
`the RLE and RHE cells over the RFC célls. correlates
`well with the greater expression of gp60 by these cells
`(see Fig. 4).
`Presence of gp60 on the surface of microvascular endo-
`thelium in situ. Most of our previous work has focused
`on using in vitro systems to study molecular interactions
`at. the surface of the vascular endothelium. Recently, we
`have examined the extent of phenotypic drift, caused by
`cell culture by performing intravascular radioiodinations
`of endothelial surface proteins to compare those proteins
`identified in situ with those in culture (25). Here we
`
`30
`
`RBE
`RLE
`RHE
`RFC
`Fig, 5. Differential albumin binding to cultured rat endothelial cells.
`RFC cells along with microvascular endothelial cells isolated from rat
`brain (RBE), heart (RHE), and lung (RLE) were examined for their
`ability to bind radioiodinated rat serum albumin (RSA) using 0.1 meg/
`mi of *I-RSA in an in-vitro assay described previously (20). For each
`endothelial cell
`type, mean value of binding observed from 4 cell
`monolayers in dishes is expressed as ng/10* cells, with error bars
`denoting caleulated SD values. Data were also normalized relative to
`RFC cells and are-expressed as a percentage, which is written above or
`within columns representing each cell type.
`
`125
`
`I-RSABOUND(ng/million‘cells)
`97
`
`205 -
`
`116
`
`NI
`IM:
`Fig. 6. Intravascular radjoiodinated:surface proteins of heart. vascular
`endothelium irmunoprecipitated with «-gp serum, separated by SDS-
`PAGE, and visualized by au