gp60 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 920930651
`
`Schnitzer Jan E gp60 is an albumin binding glycoprotein
`in albumin
`expressed by continuous
`endothelium involved
`transcytosis Am J Physiol
`262 Heart Circ Physiol 31
`H246 H254 1992Albumin reduces capillary permeability
`and acts as a carrier for various small molecules Recently we
`identified a 60kDa sialoglycoprotein gp60 on the surface of
`cultured rat microvascular endothelial cells MEG that binds
`albumin and antiglycophorin serum agp We verified that a
`gp recognizes the albumin binding gp60 by affinity purifying
`proteins from MEC extracts using immobilized albumin gp60
`was immunoblotted with agp 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
`beds
`tissue microvascular
`in rat
`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
`liver and small
`or sinusoidal endothelium adrenal pancreas
`intestinal mucosa MEC isolated from rat heart
`lung and
`epididymal fat pad expressed gp60 and bound albumin whereas
`various nonendothelial cells and brain derived MEG 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
`mal vesicles but also to increase capillary permselectivity
`capillary permeability membrane
`receptors membrane proteins sialoglycoproteins receptor me
`transport plasmalemmal vesicles
`diated transport
`glycocalyx
`
`rat biological
`
`transport
`
`vesicular
`
`A PRIMARY FUNCTION of the attenuated layer of vascular
`endothelium is to act as a barrier to the transvascular
`in many tissues The
`transport of plasma molecules
`selectivity of the endothelial barrier varies in different
`vascular beds and is strongly dependent on the structure
`and type of endothelium lining the microvasculature
`29 Several pathways exist for the transport of plasma
`molecules across continuous endothelium 1 intercellu
`form
`lar junctions are highly regulated structures that
`the paracellular pathway for the passive pressure driven
`filtration of water and small solutes 2 plasmalemmal
`vesicles transcytose plasma macromolecules apparently
`by shuttling their contents adsorbed from blood from the
`to antiluminal aspect of the endothelium 16
`luminal
`and 3 transendothelial channels which may form tran
`two or more plasmalemmal
`siently by the fusion of
`vesicles may provide a direct conduit for the exchange
`of both small and large plasma molecules 31 Recently
`it has become clear that capillary permeability in many
`vascular beds is also dependent on the interaction of
`these transendothelial transport pathways with the mul
`tifunctional plasma protein albumin 3 6 1114 19
`Albumin binds to the luminal glycocalyx of continuous
`
`H246
`
`The binding 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 shown to
`be saturable reversible competible dependent on cell
`type and cell number and to have a negative cooperativity
`in nature 22 Albumin binding was sensitive 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
`malemmal sialoglycoproteins
`situ and in culture 25 One of these sialoglycoproteins
`called gp60 was identified as an albumin binding protein
`because it 1 interacts with albumin conjugated to beads
`23 2 binds RCA LFA and WGA but notuther lectins
`23 25 26 and 3 is sensitive to pronase and sialidase
`but not to trypsin digestion 23 25 Another laboratory
`has also identified gp60 as one of three major albumin
`binding proteins 8 9 The structural and functional
`these albumin binding proteins to one
`relationship of
`is unknown Further characterization
`another
`of gp60
`it apparently contains 0 linked but not N
`showed that
`linked glycans 25 and may also be antigenically related
`to another sialoglycoprotein namely glycophorin 26
`Polyclonal antiserum agp raised against purified
`mouse glycophorin gp3 recognized a 60kDa WGA and
`RCA binding sialoglycoprotein on the Urfa of cultured
`endothelial cells 26 This apparent
`rat microvascular
`recognition of gp60 by agp was inhibited in the presence
`of murine glycophorins
`In this study the specific interaction of gp60 with
`albumin and with agp serum is demonstrated Then a
`gp is used as a probe for gp60 Because albumin binding
`and transcytosis via plasmalemmal vesicles are not ob
`served in the endothelium lining the vasculature of many
`tissues tissue specific expression of gp60 should be ex
`0363613592 $200 Copyright © 1992 the American Physiological Society
`
`endothelium 10 15 19 22 and its binding within
`transport pathways such as plasmalemmal vesicles and
`the introit of intercellular junctions apparently forms a
`molecular filter within these pathways 3 that can elec
`trostatically 20 and sterically 3 21 restrict the trans
`port of water small solutes and macromolecules
`the microvascular wall 6 1114 19 In addition albu
`min is transcytosed across vascular endothelium via plas
`malemmal vesicles 10 15 19 and acts as a carrier for
`such as fatty acids 5 This
`ligands bound to it
`small
`receptor mediated transcytosis of albumin ap
`apparent
`pears to occur selectively in certain tissues with vascular
`beds lined with continuous endothelium 10 28 and is
`
`across
`
`influenced greatly by the ligands bound to albumin 5
`
`has been identified both in
`
`Abraxis EX2013
`Actavis LLC v Abraxis Bioscience LLC
`1PR201701101 1PR201701103 1PR201701104
`
`

`

`ENDOTHELIAL
`
`ALBUMIN BINDING GLYCOPROTEIN GE60
`
`H247
`
`pected if gp60 does indeed function as a physiologically
`significant albumin binding protein Therefore various
`rat tissue and cell extracts were tested using agp 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 1 the
`specific expression of gp60 only by those cultured endo
`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 3 the
`selective distribution of gp60 in rat tissues with vascular
`beds lined with continuous endothelium that transcytose
`albumin via plasmalemmal vesicles
`
`METHODS
`
`cells
`
`in culture Male
`Isolation and growth of endothelial
`albino rats SpragueDawley 200250 g were anesthetized with
`ether The heart
`lungs and cortical brain were surgically
`removed from three to five rats were submerged in cold Dul
`beccos modified Eagles medium DMEM supplemented with
`1520 fetal calf serum FCS DMEM+ and each was
`minced in a vial using small sterile surgical scissors For the
`heart only myocardium was used after careful excision of the
`epi and endocardiurn 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
`and to increase the probability for
`ments of the vasculature
`endothelium After centrifugation
`isolation of rnicrovascular
`for 5 min at 1000 g at 4°C the pellet was resuspended in 2 mg
`from Clostridium histolyticum ne II
`ml of collagenase
`Sigma in DMEM+ at 37°C using five times the tissue volume
`After a 1h incubation the suspension was poured over a sterile
`Nitex monoscreen cloth no 311240xx Tetko Briar Cliff
`Manor NY to filter out
`tissue clumps The filtrate was cen
`trifuged for 5 min at 1000 g and the pellet was resuspended in
`DMEM+ containing 20 Agm1 of heparin 100 1Jm1 of penicil
`lin G 100 agm1 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
`CO2 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 11 clioctadecy13333tetramethylindocarbo
`cyanine perchlorate DiIAcLDL as in Refs 22 and 30 Madin
`Darby kidney cells MDCK cells were used as a negative
`endothelial cells that exhibited uptake
`control Microvascular
`of DiIAcLDL and the mixture of other cells in the heart and
`internalize DiIAcLDL were
`lung preparations that did not
`replated separately and grown in culture using the above media
`without heparin After 24 h this medium was replaced for all
`future culturing with DMEM+ for the noriendothelial cells and
`with DMEM+ with retinal derived growth factor for the endo
`thelial cells Microvascular endothelial cells isolated from rat
`epididymal fat pads RFC were grown and plated as previously
`described 22
`
`lysate from cultured cells After
`Immunoblotting of total cell
`cell monolayers their proteins were
`washing the confluent
`solubilized directly with cold solubilization buffer SB contain
`ing 017 M trishydroxymethyDarninomethane Tris HC1 pH
`68 3 wtvol sodium dodecyl sulfate SDS 12 volvol
`2 M urea and 3 niM EDTA in double
`distilled water as described previously 25 After incubation in
`boiling water for 10 min a lysate volume equivalent of 106 cells
`was processed for SDSpoiyacrylamide
`gel electrophoresis
`PAGE and the separated proteins were electrotransferred
`onto Immobilon filters Millipore Bedford MA as in Refs 25
`
`fimercaptoethanol
`
`covalently
`
`and 26 Strips of these filters were immunoblotted with rabbit
`serum and any bound immunoglobulin G IgG was detected
`conjugated to alkaline phos
`IgG antibodies
`using anti rabbit
`phatase as in Ref 26
`Immunoprecipitation of gp60 radiolabeled
`biosynthetically
`using tritiated sugars About 5 x 106 RFC cells were plated onto
`two T75 flasks After 15 h 10 mCi of 263Hrnannose
`45
`Cimmol Amersham Arlington Heights IL mixed in 3 ml of
`DMEM+ was added to one flask of cells or 10 mCi of 6311
`galactose 20 Cimmol ICN Costa Mesa CA combined with
`1 mCi of 6311glucosamine 27 Cimmol ICN mixed in 3 ml
`of DMEM+ was added to another flask of cells After 3 days
`when the cell monolayer reached full confluency the cells were
`washed with 10 ml of DMEM 3 times 1 mm at 4°C lysed
`with 5 Triton X100 and 1 SDS in phosphate buffered
`saline PBS and finally scraped from the flask After a 10
`min spin at 4°C 1000 g the supernatant was used for MI
`munoprecipitations overnight with agp as described in Ref
`25 The precipitates were analyzed by SDSPAGE and were
`visualized by fluorography as per Ref 1
`in situ The
`Immunoprecipitation of gp60 radioiodinated
`endothelial
`luminal surface proteins of the heart vasculature
`were radioiodinated in situ using Na1 and microspheres were
`and glucose oxidase as
`coated with lactoperoxidase
`described previously 25 Proteins were extracted from 200 mg
`of heart tissue by mincing the tissue in 500 pl of 5 Triton X
`100 and 1 SDS in PBS at 4°C The lysate was centrifuged at
`13000 g for 1 min at 4°C and 100 pl of the supernatant
`tissue
`extract was mixed overnight at 4°C with 380 al of PBS and 20
`al 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 td of cold SB kept
`for 10
`in boiling water
`min and analyzed by SDSPAGE followed by autoradiography
`as described previously 25
`Imniunoblotting of tissue extracts Male albino rats Sprague
`Dawley 200250 g were anesthetized with an intraperitoneal
`injection of ketamine 100 mgkg and xylazine 33 mgkg
`The chest was opened through a median sternotomy Through
`a needle inserted into the left ventricle and after the right
`atrium was cut
`for outflow purposes the vasculature was per
`fused with DMEM Irvine Scientific Irvine CA at a mean
`pressure of 60 mmHg first
`for 5 min at 37°C and then for 10
`min at 10°C The heart liver cortical brain epididyrnal fat
`duodenum diaphragm and
`pad kidney adrenals pancreas
`skeletal muscle were excised and $00 mg weight
`gastrocnemius
`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
`under visual
`from the muscularis by scraping
`inspection
`through a dissecting microscope In some cases only lung tissue
`was excised after perfusion through the right ventricle
`at a
`mean pressure of 20 mmHg with left atrial outflow The lysates
`at 13000 g for 1 mm at 4°C and the super
`were centrifuged
`natants tissue extracts were processed equivalently for pre
`parative SDSPAGE and immunoblotting as described above
`In the lung and fat pad preparationsIthe centrifugation step
`did nor pellet all of the tissue debris and a top layer of fatty
`material was present and had to be carefully aspirated before
`the soluble 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 41
`ml benzamidine 10 agm1 leupeptin 10 agrtil pepstatin A 65
`agnil aprotinin 10 Agm1 chymostatin 100 agirril Ophenan
`throline and 350 agm1 phenylmethylsulfonyl
`fluoride The
`presence of protease inhibitors did not alter the observed re
`sults
`Immunoblotting of gp60 after interaction with immobilized
`albumin A 1 Triton X3 00 soluble fraction from the RFC
`in 90 ethanol at 20°C The
`
`cells was precipitated overnight
`
`

`

`H248
`
`ENDOTH ELIAL ALBUMIN BINDING GLYCOPROTEIN GP60
`
`filters
`
`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 SDSPAGE electrotransferred to
`and then immunoblotted with agp Controls
`included 1 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 agp detected a single 60kDa protein only
`in eluates of the albumin adsorbed strips interacted with
`the RFC cell extract The eluates from all of the control
`strips were negative These results indicate that 1 gp60
`interacts preferentially with albumin 2 agp does in
`in the RFC cell extracts
`deed recognize gp60 present
`and 3 agp can now be used with confidence as a probe
`for the albumin binding glycoprotein gp60
`Endothelial expression of gp60 immunoprecipitation
`with agp of endothelial glycoproteins radiolabeled biosyn
`thetically in culture Although it
`that gp60 is
`is clear
`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 immunopreeipitated
`specifically with agp from lysates of RFC cells that had
`been biosynthetically radiolabeled with tritiated sugars
`gp60 was radiolabeled successfully with a mixture of 6
`311galactose and 63IIglucosamine but not with 26
`3IImannose alone To ensure that gp60 was indeed ex
`pressed during each radiolabeling procedure both radi
`lysates were subjected to SDSPAGE and
`olabeled cell
`the separated proteins were electrotransferred onto fil
`ters that were immunoblotted with agp An equivalent
`signal for gp60 was detected in both cases data not
`shown These results indicate that gp60 is indeed ex
`
`Filters
`
`adsorbed with
`Albumin
`Transferrin
`PBS alone
`to
`Filters exposed
`Cell extract
`
`+
`
`+
`
`+
`
`+
`
`+
`
`+
`
`+
`
`GP60+
`
`pellet was washed once using 70 ethanol and then resus
`pended in PBS The suspension was centrifuged at 13000 g for
`5 min and the supernatant was used as the cell extract Strips
`of Immobilon or nitrocellulose filters 1 X 025 in were incu
`bated for 4 h at room temperature in 1 PBS alone 2 10 mg
`ml of albumin in PBS or 3 10 mgml of transferrin in PBS
`h with blocking solution 5 Blotto and 01 Nonidet P40 in
`After air drying for at least 1 h all strips were quenched for 1
`
`PBS and then incubated overnight at room temperature in 05
`ml of a 150 dilution of the RFC cell extract in blocking solution
`Some of the albumin strips and transferrin strips were also
`incubated in blocking solution without cell extract as additional
`controls All strips were washed three times for 5 min with
`01 Nonidet P40 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 SDSPAGE on a minigel apparatus
`BioRad and then electrotransferred onto Immobilon filters
`at 40 V for 15 h The filters were immunoblotted with agp as
`described above
`
`RESULTS
`
`gp60 interacts with both agp and albumin Recently
`we have shown that agp does interact with an RCA
`and WGAbinding 60kDa sialoglycoprotein 26 present
`on the surface of cultured microvascular
`endothelium
`derived from RFC Other work 23 has implicated an
`RCA and WGAbinding 60kDa glycoprotein as an al
`bumin binding protein on the RFC cell surface Because
`these data strongly suggest that agp is interacting with
`the albumin binding protein gp6O we attempted to in
`hibit albumin binding to the surface of cultured RFC cell
`rnonolayers using an IgG fraction of agp isolated with
`immobilized protein A as in Ref 26 The cell monolayers
`were first exposed for 10 mm to the IgG fraction of agp
`tgm1 Then 19 albumin was added to
`up to 100
`a final concentration of 2 geml and the usual
`achieve
`binding assay was performed 22 23 Although this IgG
`fraction did interact with the 60kDa protein by immu
`lysates 26 it did not affect
`noblotting of RFC cell
`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 agp is indeed interacting with the 60kDa
`1 The observation
`that agp even at high concentrations does not
`interfere with albumin binding to the cell surface suggests that albumin
`and agp 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 agp that indicates that
`agp reacts with a peptide region located apparently in the endodomain
`of the protein unpublished observations Conversely albumin binding
`to the endothelial cell surface is expected to be via the ectodornain of
`gp00 An alternate explanation is supported by our immunofluorescence
`on permeabilized and nonpermeabilized RFC cells which
`studies
`showed only mild labeling of the cell surface with agp data not
`shown Because the antibody was raised against an antigen denatured
`by SDSPAGE it
`is not surprising that agp apparently interacts
`poorly with the native form of gp60 seen on the cell surface The
`epitope may be masked in its native state andor require some degree
`of gp60 denaturation before antibody recognition Furthermore since
`our results indicate that the epitope lies in the endodomain of gp60 it
`may like glycophorin interact with cytoskeletal
`proteins this inter
`action may interfere with antibody binding to the permeabifized
`cells
`staining with agp pre
`Unfortunately
`immunofluorescence
`the poor
`cludes more exact
`immunolocalization studies at the electron micro
`scopic level on either cultured cells or tissue These immunolocalization
`studies must be performed with a new antibody that recognizes 060
`under more native conditions
`
`Serum
`
`IM IM IM iM
`
`IM NJ
`interaction of 60k1
`Fig 1 Specific
`0 with albu
`min immobilized on filters and subsequent detection by immunoblot
`ting with antiserum against glycoprotein agp Filteis were incubated
`with albumin in phosphate buffered saline PBS transferrin in PBS
`or PBS alone as indicated for each lane above and then air dried to
`immobilize 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 eltited from each strip separated
`by SDSPAGE and electrotransferred onto Immobilon filters Strips
`containing eluted proteins are shown above and were
`immunoblotted either with nonimmune NI andor immune agp IM
`serum Bound immunoglobulin C IgG was detected using anti rabbit
`IgG antibodies
`conjugated to alkaline phosphatase as described previ
`ously 25 Gp60 was detected only after interaction of cell extract with
`albumin filters and not in various controls
`
`of these filters
`
`

`

`ENDOTHELIAL
`
`ALBUMIN BINDING GLYCOPROTEIN GP60
`
`H249
`
`IMMUNOPRECIPITATION
`MORI RUM
`WITH ANTI
`tL
`Mn re
`C
`
`66
`gP60
`
`45
`
`INS
`
`Evl
`
`N I
`NI
`Fig 2 Biosynthetically radiolabeled glycoproteins of RFC cells irn
`munoprecipitated with agp serum RFC cells were radiolabeled with
`either 1Hinannose Man or both 3Hgalactose and 3111giticosamine
`GG as descibed in METHODS Lysates from these cells were subjected
`to immunoprecipitation with either nonimmune NI or immune IM
`agp rabbit serum Immunoprecipitates were separated by SDSPAGE
`on a 515 gradient gel and visualized by fluorography Only gp60 was
`detected specifically with agp The band found at 45 kDa is considered
`since it was detected both in nonimmune serum and
`to be nonspecific
`to a lesser extent in immune serum
`
`pressed by vascular endothelium in culture Furthermore
`the lack of incorporation of 263111mannose
`into gp60
`is consistent with our previous findings based on direct
`lectin affinity chromatography 25 that
`and sequential
`suggest that rat gp60 contains 0linked 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 DiIAcLDL was used to isolate endo
`thelial cells from nonendothelial cells 22 30 Those
`lacked uptake of DiIAcLDL were also saved
`cells that
`from the heart and lung preparations After sorting all
`cells were replated grown in culture for several passages
`and checked periodically for DiIAcLDL uptake using
`either the fluorescence activated cell sorter or simple
`fluoresence microscopy of the cells grown on slides as
`in Ref 22 Figure 3 shows the fluorescence
`described
`profile of DiIAcLDL uptake of a typical cell preparation
`The cells initially isolated from tissue have a bimodal
`cell distribution with considerable variation in cell size
`and fluorescence intensity Most of the cells have fluo
`rescence intensity far greater than the MDCK cells neg
`to internalize DiIAcLDL A
`ative control and appear
`second group of cells has a fluorescence intensity more
`comparable to the MDCK cells especially when cell size
`is considered The top 20 of the cells with the greatest
`intensity and the bottom 20 of the cells
`fluorescence
`with the lowest
`fluorescence intensity were collected
`separately using the cell sorter These cells after growth
`in culture were examined for DiIAcLDL uptake once
`again and as shown in Fig 3B the endothelial cells that
`on the first sort are now unimodal
`in
`were positive
`fluorescence in
`distribution with considerably
`greater
`tensity than the negative control Meanwhile the other
`isolated cells shown in Fig 3C which should be nonen
`in origin also appeared to have a unimodal cell
`dothelial
`in this case their fluorescence was
`distribution however
`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
`enzyme or factor VIII
`such as angiotensinconverting
`data not shown This approach has allowed us to isolate
`
`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
`see METHODS These cells were
`and grown in culture until confluency
`incubated with 11 dioctadecy13333 tetramethylindocarbocyanine
`lipoprotein DiIAeLDL
`low density
`perchloratelabeled acetylated
`and processed for fluorescence activated cell sorting as described pre
`viously 20 30 Cell size and fluorescence intensity profiles of cells are
`shown as a function of cell number Profiles of initial
`population of
`heart derived IHD cells are given in A MDCK cells were also exam
`ined as a negative control Twenty percent of cells with highest and
`fluorescence intensity were isolated separately using cell sorter
`lowest
`and then grown in culture After 3 passages these 2 cell populations
`for DiIAcLDL uptake and their profiles are shown
`were reexamined
`in B rat heart endothelial RHE cells are derived from those cells
`fluorescence intensity of DilAcLDLpositive cells and C
`with highest
`heart mixture HM of cells grown from bottom 20 of cells with
`fluorescence of RHE cells was com
`flourescence When total
`pared with that of MDCK cells >98 of RHE cells internalized more
`DiIAcLDL than any control cells On the other hand HM cells
`as MDCK cells To get a
`exhibited same degree of
`population of cells that lacked DiIAcLDL uptake ie were devoid of
`endothelial cells we found it necessary to passage
`least twice
`
`lowest
`
`fluorescence
`
`cells in culture at
`
`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
`SDSPAGE electrotransferred to filters
`and then im
`munoblotted with agp and nonimmune 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
`rations only the DiIAcLDLpositive endothelial cells
`expressed gp60 whereas the mixture of nonendothelial
`cells probably consisting of fibroblasts pericytes and
`or smooth muscle cells neither internalized DiIAcLDL
`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
`
`gp60 can also be immunoblotted specifically with agp 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
`
`

`

`H250
`
`ENDOTHELIAL
`
`ALBUMIN BINDING GLYCOPROTEIN GP60
`
`Tissues
`
`Cells
`
`DilAcLIL
`
`GP60
`
`Im
`
`NI
`
`IM Ni
`
`ivi
`
`IM
`
`NI
`
`IM IM
`
`from rat heart
`
`Serum
`NI
`Fig 4 Differential expression of gp60 in rat mit coy ascular endothelial
`cells isolated from different tissues RFC cells along with microvascular
`endothelial cells isolated from rat brain RBE heart RIIF and lung
`RLE were processed for
`immunoblotting overnight with a 1500
`dilution of either nonimmune NI or immune 1M egp serum In
`addition a mixture of nonendothelial cells isolated
`BM and lung LM were also immimoblotted with agp In each case
`a lysate equivalent of 106 cells was loaded onto a preparative gel Both
`endothelial and nonendothelial
`cells were passaged 3 times in culture
`isolation and separation
`by fluorescence activated
`after their
`cell sorting Heart and lung endothelial cells both internalized DiI
`AcLDL and expressed gp60 whereas nonendothelial cells did neither
`Gp60 was not detected in lysates of cultured brain endothelium
`
`initial
`
`types grown in culture Normal rat
`nonendothelial
`cell
`kidney fibroblasts from American Type Culture Collec
`tion ATCC NRK49F that bind albumin poorly 22
`and rat aortic smooth muscle cells from ATCC A10
`were negative for gp60 expression data not shown
`Therefore in the cultured cells tested so far the expres
`sion of gp60 appears to be specific for certain endothelial
`cells
`Selective albumin binding to rat cultured endothelial
`cells Because the data given above indicate that gp60
`expression is limited to only certain endothelial cells 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
`dioiodinated and the binding of 25I RSA to the surface
`of these cultured cells was compared using 01 mgm1 of
`I 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 1015 times
`for the RLE RHE and RFC cells than for the
`greater
`RBE cells This small amount of binding of albumin to
`the RBE cells is comparable to that observed previously
`for rat fibroblasts 22 This selective albumin binding
`to certain cultured endothelial cells agrees well with the
`the RLE RHE and RFC cells
`above observation that
`expressed gp60 but the RBE cells did not In addition
`the increase of 4050 in 19 RSA binding observed for
`the RLE and RHE cells over the RFC cells 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
`
`114
`
`egyzrez
`
`RHE
`RFC
`RBE
`RLE
`Fig 5 Differential albumin binding to cultured rat endothelial cells
`RFC cells along with microvascular endothelial cells isolated from rat
`brain RBF heart RBE and lung RLE were examined for their
`ability to bind radioiodinated rat serum albumin RSA using 01 mg
`in an in vitro assay described previously 20 For each
`ml
`type mean value of binding observed from 4 cell
`endothelial cell
`monolayers in dishes is expressed as ng106 cells with error bars
`denoting calculated SD values Data were also normalized relative to
`RFC cells and are expressed as a percentage which is written above or
`wit bin columns representing each cell
`type
`
`205
`
`116
`
`97
`
`66
`
`4$
`
`29
`
`IM
`NI
`Fig 6 Intravascular radioiodinated surface proteins of heart vascular
`endothelium immunoprecipitated with agp serum separated by SDS
`PAGE and visualized
`by autoradiography Heart extracts with 19
`luminal surface proteins 24 were subjected to
`labeled endothelial
`immunoprecipitation with either nonimmnine NI or immune IM
`agp serum Because it appears that this type of radioiodination in situ
`is limited to luminal surface of endothelium 25 these results indicate
`that a go recognizes intravascularly radioiodinated gp60 that is located
`on luminal surface of heart microvascular endothelium in situ Molec
`ular mass standards used were myosin 205 kDa ozgalactosidase 117
`kDa phosphorylase B 97 kDa bovine albumin 66 kDa egg albumin
`45 kDa and carbonic anhydrase 29 kDa
`
`show that gp60 is indeed a molecular component of the
`endothelial glycocalyx in situ by using agp to immuno
`gp60 from heart extracts con
`precipitate radiolabeled
`taining microvascular endothelial proteins that have
`been radioiodinated in situ As described previously 25
`the luminal surface proteins of the myocardial microvas
`cular endothelium were radioiodinated by intravascular
`perfusion of Na125I and small microspheres conjugated
`with both lactoperoxidase and glucose oxidase As shown
`
`

`

`ENDOTHELIAL
`
`ALBUMIN BINDING GLYCOPROTEIN GP60
`
`H251
`
`Table 1 Tissue expression of albumin binding proteins comparison with albumin binding
`and transport by vascular endothelium
`
`Vascular
`Bed
`
`Permeable
`to Albumin
`
`Albumin in
`Plasmalernmal Vesicle
`
`Albumin Binding Protein°
`
`gp60
`
`gp30
`
`gp18
`
`Albumin
`
`Effects
`
`++++
`++++
`
`tr++
`++
`++
`++
`++
`
`ND
`ND
`
`ND
`ND
`
`++
`
`ND
`ND
`ND
`ND
`
`ND
`
`ND
`ND
`ND
`
`ND
`
`Continuous endothelium
`Heart
`Lung
`Brain
`Fat
`Skeletal muscle
`
`Diaphragm
`Mesentery
`Duodenal musculature
`Fenestrated endothelium
`Adrenal
`Pancreas
`Duo