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Human Endothelial Cells Express Integrin Receptors on the Luminal Aspect of Their Membrane By Grazia Conforti, Carmen Dominguez-Jimenez, Adriana Zanetti, Michael A. Gimbrone Jr, Ottavio Cremona, Pier Carlo Marchisio, and Elisabetta Dejana Endothelial cells (EC) form a dynamic interface between blood and the rest of the body. EC surface properties promoting adhesion of reactive plasma proteins and/or circulating cells might be of pivotal importance for the homeostasis of blood and tissues. EC express multiple integrin receptors that promote their attachment to the subendothelialmatrix proteins. Among these receptors, 0 4 3 3 is of particular relevance on EC, since it is abundantly expressed and can bind many different matrix and plasma proteins. It is still unknown whether integrin receptors are selectively located to the basal side of EC membrane or may also be exposed on the cell surface in contact with blood. This issue was addressed using different experimental approaches. First, selective surface radioiodination using lactoperoxidase (LP0)-latex beads and immunoprecipitation analysis were performed. We found that cultured EC, similarly to human skin fibroblasts (HSF), expose a& on both their apical (free) and basal (substratum-attached)surfaces. qp1, mp1, and This held also for other integrins such as Immunoprecipitationdata were verified by morphological techniques. Immunofluorescenceand immunogold-staining of EC with a&, as well as with p1 subfamily antibodies, showed a diffuse and granular distribution of these integrins on EC surface. ~ y p 3and integrins were also detected on the apical membrane of EC at higher magnification by scanning electron microscopy (SEMI. Finally, data obtained on cultured EC were confirmed in vivo on immunogold-labeled ultrathin cryosections of human vessels by transmission electron microscopy (TEM). Data indicate, that in addition to their role in promoting EC attachment t o extracellular matrix proteins, integrin receptors of EC can be exposed to bloodstream and eventually be available for binding of plasma proteins and circulating cells. o 1992by The American Society of Hematology. V surface suitable for the binding of adhesive plasma proteins. In the present study, we addressed this issue by studying the distribution of integrins on EC membrane, and in particular that of the vitronectin (vn) receptor ~433.This protein is one of the major integrins expressed by cultured EC. Its major ligand is vn, but it can also promote EC adhesion to von Willebrand factor,z0,21 fibrinogen,22.23thromb o s p ~ n d i nand , ~ ~thrombin.z We show by multiple experimental approaches, that EC, similarly to “nonpolarized” cells such as human skin fibroblasts (HSF), expose a&, as well as other integrins, on both their abluminal and luminal surface. The distribution of integrins on endothelial blood interface might represent a relevant mechanism for localization of plasma proteins on their surface. ASCULAR ENDOTHELIUM is a simple squamous epithelial lining that faces blood with the luminal surface and tissues of the body with the abluminal surface. In view of its apparently simple structure, the endothelium is devoid of the highly specialized and distinctive morphology of apical and basal membrane domains shown by many epithelia. However, there is evidence that endothelial cells (EC) express specialized functional and structural microdomains differentially distributed on the apical and basal membrane.’ Endothelial luminal surface can modulate important processes such as binding and activation/ inactivation of many elements of the coagulation and fibrinolytic binding of hormones or lipoproteins,6 and expression of adhesive receptors for leukocytes and tumor EC surface and reactivity can be modulated by different stimuli, including thrombin,I0 histamine,“ or inflammatory ~ytolcines.~~J~ Some studies indicate that E C can express an apical/basal asymmetry of structure and function. Muller and GimbroneI4 could directly show an asymmetric distribution of glycoproteins on the EC luminal and abluminal membrane. EC express adhesive receptors for matrix proteins that belong to the “integrin” family of membrane glycoprotein~.’~-’~ These receptors have several structural and functional homologies and consist of two noncovalently linked subunits. Integrins located to the basal side of the membrane recognize and bind different components of the basement membrane extracellular matrix with their ectodomain. Conversely, their cytoplasmic domain links a chain of proteins involved in F-actin microfilament assembly and cytoskeleton organization.’g Considering their major role in promoting matrix adhesion, it is natural to assume that these receptors are localized to the basal aspect of the cells. However, it is not yet known whether integrins could also be exposed on the apical part of EC membrane. This is particularly relevant since EC are in constant contact with blood and might expose a reactive Blood, Vol80, No 2 (July 15), 1992: pp 437-446 From the Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; the Vascular Research Division, Department of Pathology, Brigham & Women’sHospital, Harvard Medical School, Boston, MA; and the Dipartimento di S c i e w Biomediche e Oncolo@, Universith di Torino, Torino, Italy. Submitted August 1,1991; accepted March 20,1992. Supported by C.N.R., Italian National Research Council (P.F. Biotecnologie e Biostrumentazione, P.F. Tecnologie Biomediche e Sanitarie e Progetto Speciale “Peptidi Bwattivi”), by Associuzwne Italiana per la Ricerca sul Cancro (AIRC), and by NATO Research Grant No. CT-042918% Address reprint requests to Dr Grazia Conforti, Istituto di Ricerche Farmacologiche Mario Negri; via Eritrea 62,20157, Milano, Italy. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C.section I734 solely to indicate this fact. 0 1992 by The American Society of Hematology. 0006-497119218002-0018$3.00/0 437 438 CONFORTI ET AL MATERIALS AND METHODS Materials The following reagents were used and are listed below with their source: carboxylate-modified polystyrene latex beads (0.7 pm in diameter) (Polysciences,Wamngton, PA); lactoperoxidase (LPO) (Calbiochem, San Diego CA); poly-L-lysine hydrobromide, glucose oxidase (GO) type VI1 fromdspergillur niger, phenylmethylsulfonyl fluoride (PMSF), leupeptin, trypsin inhibitorfrom soybean (SBTI), protein G-Sepharose 4B, and insulin-transferrin-sodium selenite media supplement for cell culture (Sigma Chemical, St Louis, MO); carrier-free Na151 (New England Nuclear, Boston, MA); Triton X-114 (TX-114) and glutaraldehyde (Fluka Biochemicals, Hauppague, NY);Trasylol (Bayer, Leverkusen, Germany); protein A-Sepharose CL-4B (Pharmacia LKB Biotechnology, Uppsala, Sweden); gold particles conjugated to goat anti-rabbit or antimouse IgGs (5 and 20 nm in diameter) and silver-enhancing kit (Bio Cell Research Laboratory, Cardiff, UK); paraformaldehyde and sucrose (Merck-Schuchardt, Darmstadt, Germany); mowiol 4-88 (Hoechst, Frankfurt/Main, Germany); 14C-methylated protein molecular weight standards (Amersham International, Buckinghamshire, UK); all electrophoretic reagents (Bio-Rad, Richmond, CA); culture reagents (GIBCO, Paisley, UK); secondary antibodies used in immunofluorescence experiments (Dakopatts, Glostrup, Denmark). Human plasma vn and fibronectin (fn) were prepared as previously described.% Antibodies Anti-glycoprotein (Gp)IIIa (anti+,) rabbit serum was prepared in this laboratory as describedz6; anti-ps (cytoplasmic domain) rabbit serum and affinity-purifiedrabbit IgGs anti-a,,p,pswere a gift Dr E. Ruoslahti (La Jolla Cancer Research Foundation, La Jolla, CA); anti-human plasma angiotensin-converting enzyme (ACE) rabbit IgGs were donated by Dr J.J. Lanzillo (Tufts University, Boston, MA), and anti-a5 (cytoplasmicdomain) rabbit serum was donated by Dr G. Tarone (University of Torino, Torino, Italy). Monoclonal antibodies (MoAbs) and the scientists who kindly provided them are listed below: affinity purified LM 142 and LM 609, directed, respectively, to the a subunit and to an a/p complex-dependentepitope of the vn receptor, Dr D.A. Cheresh (Research Institute of Scripps Clinic, La Jolla, CA); VIPI-2 (ascitic fluid), anti-human platelet GpIIIa (p3), Dr W. Knapp (University of Vienna, Vienna, Austria); B1E5 (sovranatant) anti-VLA5 a subunit and AIIB2 (sovranatant) directed to the VLA p subunit, Dr C. Damsky (Universityof California, San Francisco, CA); A1A5 (ascitic fluid), directed to the VLA p subunit, Dr M.E. Hemler (Dana Farber Cancer Institute, Boston, MA); P1E6 (sovranatant), anti-VLA2 a subunit, Dr E. Wayner (Fred Hutchinson Cancer Center, Seattle, WA); TS2/7 (sovranatant) anti-VLAl a subunit and HP2/1 (sovranatant) anti-VLA4 a subunit (both cleaved and uncleaved a chain are recognized), Dr Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain); 5143 (mouse serum) anti-VLA3 a subunit, Dr L.J. Old (Memorial Sloan-Kettering Cancer Center, New York, NY); MT78 (affinity-purified IgGs) anti-VLA6 a subunit, Dr Eberhard Klein (Universityof Ulm, Ulm, Germany). Cell Cultures Human umbilical vein endothelial cells (HWEC) were isolated from normal term umbilical cord veins by collagenase perfusion as previously d e s ~ r i b e d . ’Cells ~ ~ ~were ~ grown on tissue culture dishes coated with gelatin (0.5%) in 20% newborn calf serum (NCS)medium 199 (M199) supplemented with endothelial cell growth supplement (ECGS, prepared from bovine brain) (50 pg/mL), heparin (100 pg/mL), and penicillin (100 U/mL)/streptomycin (100 pg/mL) (pedstrep) and used between the second and third passage. Human saphena vein EC were kindly provided by Dr C. de Castellarnau (Fundacio d’Investigacio Sant Pau, Barcelona, Spain). Cells were isolated from specimens of normal veins removed during coronary bypass surgery from patients who were in overall good health. Briefly, after the vessel was opened and rinsed with phosphate-buffered saline (PBS), pH 7.2, EC were isolated by incubation with trypsin-EDTA solution and removed by gently scraping. Three hours after cell seeding, debris and possible contaminating smooth muscle cells were removed by vigorous washing. Cells were characterized as EC by their “cobblestone”like morphology and by the presence of von Willebrand factor as described.n Cells were cultured in M199, 20% human serum, supplemented with ECGS (50 pg/mL), heparin (100 pg/mL), and pen/strep, on a 0.5% gelatin coating and used at the sixth passage. Bovine microvascular EC from adrenal cortexB were kindly donated by Dr R. Montesano (University of Geneva, Geneva, Switzerland). Cells were cultured in minimum essential medium15% NCS containing penlstrep and used at the tenth passage. HSF were obtained from forearm skin biopsies following the procedure described by Simpson and S t ~ l b e r g ?grown ~ in 20% NCS-M199, pen/strep, and used between the eighth and tenth passage. Selective Radiowdination of Apical, Basal, and Total Cell Surfaces LPO-beads were used to selectively label the apical (A), basal (B), and total (T) surfacesof cells. The entire procedure followed a previously described detailed method.14This technique is based on the fact that radiolabeling with LPO-latexbeads is restricted to the externally disposed membrane proteins. When cells are labeled in monolayer,LPO-beadsdo not have access to the basal (substratumattached) cell surface, because of the beads’ large size. To selectively radioiodinate the basal surface of living EC, we inverted the H W E C monolayer on poly-L-lysine-coated coverslips. Briefly, tissue culture plastic coverslips were precoated with poly-L-lysine (1 mg/mL in Hanks’ balanced salt solution [HBSS] for 5 minutes at room temperature), followed by several washes in distilled water, and air-dried. Confluent HUVEC were washed three times in HBSS at 3TC, then a poly-L-lysine-coatedcoverslip was gently placed down onto the monolayer; several seconds later it was lifted up using a fine forceps. Confluent sheet of HUVEC remained attached by their apical surfaces with their original basal surfaces now in contact with the surrounding medium. The inverted monolayers were immediately placed in cold Dulbecco’s PBS (DPBS) containing calcium and magnesium and protease inhibitors (0.3 mg/mL SBTI and 1.4 KIU Trasylol). This inversion technique was used on primary cultures of HUVEC grown on uncoated culture dishes. Total cell surface labeling was performed on viable populations of nonenzymatically resuspended cells using 30 pmol/L EDTA in HBSS without calcium or magnesium and removed by gentle scraping.Resuspended cells were transferred to a culture dish well, mixed with LPO-latex suspension, and allowed to settle on ice (under these conditions, some LPO-latex would be under the cells). Confluent monolayers of H W E C and HSF were used for radioiodinationof the apical surface. The culture plates containing the A-, B-, or T-cell preparations and LPO-latex (1:400 dilution from LPO-latex stock)14 were centrifuged (3,100 rpm, 4°C for 5 minutes) in microtiter plate carriers in a Minifuge T (Heraeus, Osteroole, Germany). This brought the beads down in intimate contact with the cell surface,where they were adherent. Radioiodi- LUMINAL INTEGRINS IN HUMAN ENDOTHELIAL CELLS nation was performed using 300 pCi/mL carrier-free NaIzI and 12 pU glucose oxidase (G0)lmL in DPBS-20 mmol/L dextrose as previously described.I4 In some experiments, HUVEC were seeded on vn- and fn-coated wells in MI99 supplemented with media supplement ( 1 9 dilution). After 3 hours, at maximum cell spreading, monolayers were selectively radioiodinated on the apical surface as described above. ¶tion of Samplesfor Immunoprecipitation and SDS-PAGE Radioiodinated samples were lysed by scraping with 1% TX-I 14 in IO mmollL Tris. 150 mmol/L NaCI, pH 7.4. containing protease inhibitors (1 mmollL PMSF, IO pglmL leupeptin, 20 KlUlmL Trasylol) and spinningout LPO-latex beads and insoluble material in a microfuge for 5 minutes at 4°C. The resulting supernatant was spun again in a microfuge for 25 minutes at 4°C. and aliquots used for trichloroacetic acid (TCA) precipitation analysi~.’~ Total cell lysate samples were mixed directly with 4x sodium dodecyl sulfate (SDS)-sample buffer containing 2.5% 2-mercaptoethanol and fractionated by SDS-polyacrylamidegel electrophoresis (PAGE) or kept frozen (-20T) until used. Labeled cell lysates were immunoprecipitated with the following polyclonal antibodies or MoAbs: anti-GPllla (40 pLlmL), anti-ps (40 pLlmL), anti-ACE (80 pglmL). antias (30 pLlmL), and normal rabbit serum (40 pL/mL) coupled to protein A-Sepharose CL-4B (50 pL); LM 142 (20 KglmL), BIE5 (100 pL/mL), and an irrelevant MoAb (20 pg/mL) were coupled to protein G-Sepharose4B (50 pL): TS2/7 (200 pLlmL), PIE6 (200 pL/mL), 5143 (4 pLlmL), HP2/1 (200 pL/mL), MT78 (4 pLlmL), and an irrelevant MoAb (4 pglmL) were coupled to protein A-Sepharose CL-4B (50 pL) that was previously incubated with rabbit anti-mouse lgGs (5 KglmL). Immunoprecipitation was performed as previously described.” lmmunocomplexeswere subjected to SDS-PAGE and the electrophoresed gels were fixed, dried, and analyzed by autoradiography. In some experiments, quantitative data on Iz1-aJ33 or 1151asf3~ were achieved by removing the gel that contained radiolabeled integrins (the position of the radiolabeled chains was determined by placing an x-ray film on the dried gel) and counting them in a gamma counter. Immunofluorescence Immunofluorescence tests were performed essentially as described.ZLBriefly, cells were washed twice with DPBS and fixed with 3% paraformaldehyde and 2% sucrose in PBS (15 minutes, room temperature), washed in Tris-buffered saline (TBS)-0.2% bovine serum albumin (BSA). and then incubated with a p3 MoAb (VIPI-2) for 45 minutes at 3PC without any previous detergent permeabilization. After rinsing in TBS-0.2% BSA, rhodaminetagged rabbit anti-mouse IgGs were applied for 30 minutes at 3PC in TBS-I% BSA. Coverslips were then mounted in mowiol 4-88. Routine observationswere performed in a Zeiss Axiophot photomicroscope equipped for epifluorescence and plan-apochromatic lenses (Carl Zeiss, Oberkochen, Germany). Fluorescence images were recorded on Kodak T-Max 400 films exposed at 1,OOO IS0 and developed in T-Max Developer (Kodak Ltd, Hemel, UK) for IO minutes at 20°C. Light Microscopy EC (human umbilical vein, human saphena, and bovine capillary) were grown to confluency in 96-well plates. After a DPBS wash, cells were fixed for 15 minutes at room temperature (3% paraformaldehyde and 2% sucrose in PBS) then washed three times with DPBS, and DPBS-I% BSA treated for 20 minutes at 3PC, followed by two washes with TBS-03% BSA, pH 7.2. The 439 specific antibody (Ab) was then added in the same buffer for 40 minutes at 3PC, followed by two washes with TBS-0.5% BSA, pH 7.2, and two washes with TBS-0.5% BSA, pH 8.2. Finally, immunogold particles (20 nm diameter) conjugated to goat anti-rabbit or anti-mouse lgGs (k70 dilution) were added for45 minutes at room temperature. After several washes, beads were fixed by 2% glutaraldehyde in PBS for 20 minutes at room temperature and revealed by silver enhancement. Gold particles are not visible in the light microscope, unless they are grown in situ using silverenhancing. The final signal (brownlblack) gives a precise localization that can be observed under a common light microscope. This last step was performed following the manufacturer’s procedure. Pictures were taken in a Zeiss IM35 photomicroscope on Kodak plus-X pan 125 films. Immunoelectron Microscopy Scanningelecmnmicmscopy. Cells were plated on 0.5% gelatinprecoated glass coverslips to reach confluency overnight. After two washes in DPBS followed by two saturating incubations with DPBS-I% BSA (IO minutes each), cells were labeled, prior to fixation, with 100 pglmL of anti%& affinity-purifiedantibodies3’ or the MoAb AIIB2 in DPBS-I% BSA-0.01% NaN3 for 40 minutes at 3PC. After thorough rinsing with DPBS. cells were fixed in 3% paraformaldeyde and 2% sucrose in PBS, rinsed with TBS-0.2% BSA, pH 8.2, and finally incubated with gold particles conjugated with goat anti-rabbit IgGs. After dehydration with ethanol, specimens were COz critical-point dried and coated with carbon, then examined in a Cambridge Stereoscan 360 scanning Mr x 10-3 200 A B A B A B - 100 92.5 - Fig 1. Selactlve rodlolodinationof the apical (A) and b a u l (B) EC surface. The iodlnatlon p m e m of three independent experiments h compared. Cells were radiolabeled as described in Materials and Methods. Before cell extraction, the A and B surfaces of EC monolayers were treated with 30 pmol/L EDTA for 10 minutes at 37% to remove any adsorbed protein from cell culture serum. Each lane was obtained by a pool of cells from two 10”. wells for the A surface and from four 2zmZ wells for the B surface. Each set of AB surface-labeled cells was performedfrom a different batch of cells. Each lane contains 300,000cmp of TCA-precipitablematerial. Cell extracts were mixed with 4 x sample buffer and fractionated in a gel gradient 5% to 12% acrylamlde-SDS under reducing conditions. CONFORTI ET AL 440 C B A EC EC A B 1 .. HSF A T B A EC HSF A T B EC B A A 0 . ) - R R NR R Fig 2 Selective radlo1adin.tlon of a p k l (A), bmal (E), and total (1)cell surface, followed by extradon and lmmunopreclpltatlonanalysis using A b . (A) Each lane contains 200.000 cpm of TCA-precipitable cell lysate. Note that the asymmetric distribution of apical and basal surface proteins includes both integral and membrane-associated membrane proteins, because apical and basal layers were processed for SDS-PAGE analysis without subjecting them to EDTA washing"; 5% to 12% acrylamlde gel gradient was used and samples were run under reducing conditions (2.5-hour exposure). (B) 150,000 cpm of TCA-precipitable HUVEC and HSF lysates were immunoprecipitatedby anti-Gpllla rabbit serum (40rLlmL), followed by SDS-PAGE fractionation (7.5% gel) under reducing (R) and nonreducing (NR) conditions. (C) 300,000 cpm of TCA-precipitablecell lysate from A and B surface of EC were immunoprecipitatedby three successive cycles using 40 r L / mL of anti-Gp 111a serum (anti-&) each time. Lanes 1, 2, and 3 show the immunoprecipitated products obtained in each cycle, respectively. This sequential immunoprecipltationanalysis shows that anti-Gpllla serum was used at concentrationable to produce a complete depletion of the antigen a t the first cycle for all samples (B and C). 14C-labeiedmolecular mass markers are on the left lane of each gel (M,200,000 myosin; M, 100.000-92.500 phosphorylase b; M, 69,000 BSA; M, 46,000 ovalbumin; M, 30,000 carbonic anhydrase; M, 14,300 lysozyme). electron microscope (SEM) equipped with a solid-statebackscattering device and a tungsten cathode (Cambridge Instruments, Cambridge, UK). Tmnsmivion electron microscopy. Samples of normal blood vessels from leg peripheral circulation (essentially femoral artery and vein branches) obtained from a vascular surgery department, were fixed in 3% paraformaldehyde and 2% sucrose in PBS for 10 minutes at room temperature, followed by thorough rinsing for 20 minutes in PBS. The technique was essentially that reported by T o k u y a s ~ . ~Briefly, U~ each sample was frozen in liquid Nz after 2 hours of infiltration in 2.3 mol/L sucrose. Ultrathin cryosections were obtained with a Reichert-Jung FC4D cryocutting device mounted on a Ultracut E ultramicrotome (C. Reichert, Wien, Austria). The cutting temperature was -110°C (sample) and - 1 W C (blade). Sections were collected on Ni grids coated with a 15 nm Formvarcarbon film (Polaron Equipment Ltd. Watsord, UK) and incubated with the primary Ab (anti-Gpllla rabbit serum),26 followed by incubation with 5-nm gold particles conjugated with goat anti-rabbit IgGs. Finally, cryosections were embedded in LR-White" and observed i n a Philips EM410 electron microscope (Philips, Eindhoven, The Netherlands). Mr x 10-3 ACE VN-rec NI -200 100 92.5 69 46 RESULTS Detection of Integrin Receptors by Radioiodination of the Apical Surface of EC LPO-latex beads were used for selective radioiodination Of the apical and basal domains, as well as that of the whole surface of HUVEC. This technique14 is based on the fact that LPO-latex beads adhere to the cell surface and radiolabeling is restricted to beadcontacted stretches of the Cell membrane* As previously the resulting radioiodination patterns of the apical and basal HUVEC I,-.A B A B A B 3. meoc& a d ACE d i d b d o m on a p k l and haul H ~ surface am shown. Labeled cell extract was prepared as described in Fig 1; 300.000 cpm of TCA-precipitable material was lmmunoprecipitated, respectively, by anti-Gpllla serum, anti-ACE IgGs, or normal as dmribed in Materials rabbit sarum. lmmunocomplexes and Methods were fractionated by 7.5% SDS-PAGE under reducing conditions and revealed by autoradiography. C 441 LUMINAL INTEGRINS IN HUMAN ENDOTHELIAL CELLS Fig 4. Intogrin characteriza- - .-. -- 1 - tion on H W E C apkal surface by immunoprecipitation analysis. Selective radioiodination of HUVEC apical surface and cell lysate was obtained as described in Materials and Methods:200.000 cpm of TCA-precipitable material for each sample was coupled to integrin Ab,. Immunocomplexes were run in a 7.5% gel under nonreduclng(NR) or reducing (R) conditions and revealed by autoradiography. (A) Anti-B, (rabbtt serum); anti- (LM 142); a n t i g (BlE5); NI (irrelevant MoAb). (B) Anti-a, (TS2/7); anti-a2 (PlE6); a n t i s (J143); anti(HP2/1); a n t i g (BlE5); anti- (MT78);NI (irrelevant MoAb). 14C-labeled molecular mass markers are on the right side of each gel (see legend to Fig 2). --- B Mr Mix al a 2 a3 a4 a 5 a6 NI . .. 200 200 100 92.5 100 92.5 69 69 46 46 NR membranes were distinct (Fig 1). such as specific bands were markedly enriched on one surface domain and absent or weak on the opposite side. The radioiodination pattern of the apical/basal membrane was comparable in different independent experiments as shown in Fig 1. Some difference was found comparing cells that had been washed with EDTA after labeling (Fig 1) with cells that had not been subjected to EDTA washing (Fig 2A), this can be explained by absorption of serum-derived proteins. By immunoprecipitation analysis, a,p3was found both on the apical and basal domains of HUVEC. N o obvious differenceswere observed comparing the M,of basal and apical a,p3 under reducing and nonrcducing conditions (Fig 2B). This receptor was also present on the apical surface of “nonpolarized” cells such as HSF, and showed the same Mr as that of HUVEC (Fig 2B). The results reported in Fig 2 were obtained using p3 polyclonal Abs; comparable results were obtained with the a,MoAbLM 142 (see also Fig 4A). For comparison, the membrane distribution of ACE was analyzed. This molecule has been described as distributed in a polarized fashion.I4 Using cell extract from the same radioiodinated material, was detected by immunoprecipitation analysis both on the apical and basal side of HUVEC, while ACE was only found on the apical surface running at 175 Kd (Fig 3). To know whether other integrins were also exposed on HUVEC apical membrane, immunoprecipitation was performed with Abs directed to different integrin chains. Mainly a&, asplrand a& were located to the luminal HUVEC surface as shown by immunoprecipitation analysis with MoAbs to a2, as, and as,while small amounts of a& were detectable. In contrast, a1 and cq MoAbs did not precipitate any detectable material. Since the a, MoAb (LM142) coprecipitated a single band corresponding to f33 in M, (Fig 4A), we assumed that a,p~and a,Bs integrins were not detectably exposed on HUVEC apical surface; ps was also undetectable on this surface (Fig 4A) using a polyclonal anti-ps chain Ab. A n t i e MoAb coprecipitated R an additional band running at 185 Kd, which is currently under investigation. To establish whether inclusion of ~ $ 3 or as& in adhesion plaques (on the basal aspect of the cell membrane) would prevent their recovery on the apical surface, selective apical iodination of cells seeded either on vn or fn was performed. Previous immunofluorescence data26showed that and aspIare clustered in adhesion plaques when cells are adherent on vn or on fn, respectively. Immunoprecipitation data using anti-GpIIIa rabbit serum and anti-as (cytoplasmic domain) rabbit serum, show that a,p3 and asplwerc detected on the apical surface of the cells irrespectiveof the substrata used. For instance, the radioactivity associated to a,p3 complex (see Materials and Methods) was 2,012 cpm on vn and 1,732 cpm on fn, respectively; the radioactivity associated to a& complex was 1,492 cpm on fn and 2,052 cpm on vn, respectively. Topographyof Integins Light micmscop. As shown in Fig 5, immunofluorescence staining of HUVEC, using the BJ MoAb (VIPI-2), Fig 5. Immunofluorescencedetection of a,& on the apical surface of a nonpermeabilized HUVEC. The signal generated by the Ab is in small diffuse granules. Bar = 5 pm. CONFORTI ET AL 442 showed a diffuse and granular distribution on HUVEC surface. A comparable pattern was obtained using a, (LM 142) and ~ $ (LM 3 609) MoAbs, whereas no staining was observed with irrelevant MoAbs (not shown). We used immunogold labeling of EC monolayers, followed by analysis with light microscopy, as a routine method for the detection of integrinson many EC types. As shown in Fig 6, this system allowed us to identify a , $ 3 receptor on the apical membranc of bovine microvascular EC (Fig 6a) using an anti-GpIIIa polyclonal Ab, and of HUVEC (Fig 6b) and human saphena EC (Fig 6c)using the : : 0 . ~ MoAb LM 609. For human saphena, the topographic distribution of a2 (Fig 6e) and the as (Fig 69 chains, using P1E6 and B1E5 MoAbs, respectively, are also shown. A marked staining was obtained when cells were labeled with $, MoAb A1A5 (Fig 6g). Some background could be obscrved on HUVEC (Fig 6d) labeled with an irrelevant MoAb or with saphena EC (Fig 6h) labeled with nonimmunc rabbit serum. Electron microscopy. The fine location of a,& to the apical domain, was also detected by SEM immunocytochemistry. As shown in Fig 7a and b, a polyclonal a , $ 3 Ab -c . .,.., L. Fig 6. Immunogold-labeling and sihrw-enhancing of EC, analysis by lQht microscopy. Confluent EC monolayers from different sources (a. bovine adrenal cortex microvascular EC; b and d, HUVEC; e, e, 1, g, and h, saphena EC) were fixed and incubated with integrln Ab8 (seeMaterials and Methods). (a) AntiGpllla serum (1:lOO); (b end c) anti*@, (LM 609-50 Ng/mL); (d) irrelevant MoAb (50 pg/mL); (e) anti-a2 (PlE6-undiluted); (1) anti-a, (ElES-undiluted); (g) anti-@, (AlA5-1:50); (h) i m l e vant MoAb (undiluted). Ear = 20 pm. LUMINAL INTEGRINS IN HUMAN ENDOTHELIAL CELLS Fig7. SEM lmmunogoldcytochemical loccllltatlonof Integrim on HUVEC and HSF. a, c, e, and g represent secondaryelectron micrographa, while b, d, f, and h are the corrmponding backscattering pattems produced by gold particles. Affinity-purified lgGa anti%& were used in a and b (HUVEC) and in c and d (HSF); anti-p, MoAb waa used in g and h, and nonimmune rabbit lgGs in e and f (HUVEC). Bar = 1pm. . t . 443 -- t 1. , 1 produced small gold particle clusters on HUVEC apical membrane. This pattern was similar to that observed on HSF (Fig 7c and d). Occasionally, some staining with Ab was observed on the cell matrix (Fig 7a and b). This was attributed to footprints of cells detached during washing procedures. No gold particles were observed with a nonimmune serum eithcr on HUVEC (Fig 7c and f) or on HSF (not shown). Comparable results were obtained using MoAb LM 609 to the a 4 3 3 complex (not shown). The topography of 6, chain was also studied with the same technical approach using MoAb AIIBZ. Figure 7g and h shows the pattern of distribution of 6, chain on HUVEC apical membrane. The data reported above suggest that integrins are exposed on the nonadhering apical domain of cultured EC, but do not provide information as to whether this is also the case in vascular endothelia in situ. To address this issue, we also studied the distribution of 4 3 3 in ultracryosectionsof intact human vessels. Figure 8 shows one example (from human femoral artery branch EC) of the location of a 4 3 3 CONFORTI ET AL 444 Fig 8. E M lmmunogoldcytochemical localization of a,& on an ukracryosectlon of a human femoral artery branch endothelium. Ftve-nanometer gold partlcles coupled to the secondary antibody detected bound @, Abs (anti-Gpllla rabbit serum) on the luminal surface of the endothelium either as single particles (small arrowheads) or in small clusters (largearrowhead). *Web bel-Palade body. Bar = 0.2 pm. obtained in "EM. Five-nanometer gold particles coupled to goat anti-rabbit IgGs detect on the apical surface of EC occasionally in small aggregates. The density of gold particles is consistent with the density of gold particles detected by SEM in cultured cells at lower magnification. DISCUSSION This report provides evidence, obtained by different experimental procedures, including biochemical and morphological techniques, that EC can expose integrin receptors and, in particular, u,,f3~on their apical surface. We first performed a selective iodination of HUVEC apical membrane by LPO-beads followed by immunoprecipitationwith Abs directed to different integrin chains. This technique showed that u&. in contrast to other HUVEC membrane proteins, was not topographically polarized, but was exposed on both sides of the HUVEC membrane. No obvious qualitative difference was found in such receptor extracted from both surfaces. The luminal localization of ~ $ 3 in HUVEC appeared to be a fcature common to the other major integrins expressed by cultured HUVEC, ie, u ~ f 3 ~15,f 3 1 ,and u&. These immunochemical observations were validated by direct topographical identificationof wf33 and PI integrins at the lcvcl of resolution of light and electron microscopy (SEM) on HUVEC apical membrane. A nonpolarized cell type such as HSF presented a comparable distribution of ~ $ 3 on the luminal surface. The apical localization of ~ $ 3 and ~ 5 f 3 did l not seem to be affected by the substratum on which the cells have been seeded (vn or fn). Because ~ $ 3 and ~ 5 f 3 1 are clustered in adhesion plaques on vn and fn. these data indicate that clustering of the basal receptors does not significantly modify their apical distribution. Integrin exposure at the apical aspect of the membrane was not a specific property of HUVEC, because endothelium cultured from saphena vein and adrenal gland microvessels also presentc? these characteristics. In addition, when tissue sections of human vessels were studied, a& was detected on the luminal surface of the endothelium in situ, thus indicating that this type of distribution was not artifactually related to the culture conditions of EC. The biological role of integrin molecules on EC surface in contact with blood remains to be established. In this study, we focused our attention on ~ $ 3 . This integrin is of particular interest, since it can bind different plasma proteins able to modulate thrombosis and immune reactions includingvn, von Willebrand factor, fibrinogen, thrombospondin, and thrombin.'-L5 The possibility that the luminal membrane of the vascular endothelial lining might bind and expose vn has important implications. In addition to its potential adhesive properties for bloodstream circulating cells, vn may link and modulate the activity of several biologically active plasma proteins such as thrombin/ antithrombin 111:53 pla~minogen?~ plasminogen activator i n h i b i t ~ r ,and ~ ~ .the ~ ~terminal components of the complement system.4" We have previously r e p ~ r t e d ~that l . ~ ~fibrinogen could bind to EC in monolayers and that this is inhibited by f33 antibodies. Preissner et alSRshowed that vn could bind to confluent EC in addition to the extracellularmatrix. Furthermore, it was recently found43that SruphyrocoCcus uuwus attachment to EC was mediated by fibrinogen bound from plasma onto the endothelial apical surface. Even if these data are suggestive, direct evidence that ~ $ 3 or the other integrins on EC luminal side can bind soluble plasma ligands is still lacking. In platelets, GpIIb-IIIa can promote cell adhesion to fibrinogen-coated surfaces, but it cannot bind the soluble form of this protein unless the platelets are activated. It remains to be established whether integrins on EC luminal side, in a way comparable to platelets, need cell activation to bind appropriate soluble ligands. Monoclonal antibodies to platelet GpIIb-IIIa are currently under study as antithrombotic agents. One of these, 7E3,44is particularly promising. This antibody recognizes ~ f 3 and 3 GpIIb-IIIa. From the results of this study, it is therefore conceivable that EC, as well as platelets, react with this antibody in vivo. In in situ staining of human tissues, ~ $ 3 is present in large vessel endothelium, but is less expressed in the microcirculation.Is In preliminary studies (L. Ruco et al, unpublished results), we found a similar pattern of distribution for 7E3. 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