cellular prion protein is expressed on endothelial cells and is released during apoptosis on...

C E L L P H Y S I O L O G Y

Cellular prion protein is expressed on endothelial cellsand is released during apoptosis on membrane

microparticles found in human plasma

Jan Simak, Karel Holada, Felice D’Agnillo, Jan Janota, and Jaroslav G. Vostal

BACKGROUND: Blood and plasma of animals experi-mentally infected with transmissible spongiform enceph-alopathies (TSEs) can transmit TSE infection by transfu-sion. A conformational isoform of prion protein (PrPsc)is believed to be the TSE-infectious agent that propa-gates by converting the cellular prion protein (PrPc) toadditional molecules of PrPsc. In orally infected ani-mals, PrPsc accumulates in intestinal endothelial cells.In blood, two thirds of PrPc resides in plasma, but itssource is not known.STUDY DESIGN AND METHODS: The expression ofPrPc in cultured human umbilical vein endothelial cells(HUVECs) was studied using flow cytometry, immunob-lotting, and RT-PCR. Flow cytometry was used to char-acterize endothelial membrane microparticles (MPs) incell culture supernatants and in normal human plasma.RESULTS: HUVECs and bovine aorta endothelial cellsexpress PrPc. The number of surface PrPc moleculesper cell in HUVECs was 58,000 ± 2,800. The inductionof apoptosis in HUVECs led to a marked release ofmembrane MPs (60,000-80,000 MPs/103 cells) that ex-pressed PrPc and other endothelial antigens. The pres-ence of endothelial cell-derived MPs expressing PrPcwas demonstrated in platelet-free human plasma.CONCLUSION: Endothelial cell apoptosis is associatedwith the release of PrPc-positive MPs. These MPs con-tribute to the PrPc pool in plasma and may have a rolein disseminating TSE infectivity in blood.

The cellular prion protein (PrPc) is a glyco-sylphosphatidylinositol-anchored cell mem-brane sialoglycoprotein with a molecularweight of 33 to 35 kDa. The physiologic role of

PrPc is largely unknown, but it plays an essential role inthe neurodegenerative diseases known as transmissiblespongiform encephalopathies (TSEs).1 TSEs are found inanimals and humans and include scrapie in sheep andbovine spongiform encephalopathy in cattle, which mayhave been transmitted to humans as a new variant ofCJD.1 TSEs have a long latent incubation period, which

ABBREVIATIONS: CPT = camptothecin; FSC = forward scatter;

HUVEC(s) = human umbilical vein endothelial cell(s); MP(s) =

microparticle(s); PrPc = cellular prion protein; PrPsc = confor-

mational isoform of prion protein; SSC = side scatter; TSE(s) =

transmissible spongiform encephalopathy(s); ZVAD = Z-Val-

Ala-Asp-fluoromethyl ketone.

From the Laboratory of Cellular Hematology, Center for Bio-

logics Evaluation and Research, Food and Drug Administra-

tion, Bethesda, Maryland.

Address reprint requests to: Jaroslav G. Vostal, MD, PhD,

Division of Hematology, Center for Biologics Evaluation and

Research, FDA, Building 29, Room 323, HFM-335, 8800 Rock-

ville Pike, Bethesda, MD 20892; e-mail: [email protected].

Supported in part by an appointment of Jan Simak to the

Research Participation Program at the Center for Biologics

Evaluation and Research administered by the Oak Ridge Insti-

tute for Science and Education through an interagency agree-

ment between the U.S. Department of Energy and the U.S.

Food and Drug Administration. Travel expenses of Jan Simak

were covered by grant #306/99/0649 of the Grant Agency of

the Czech Republic. Jan Janota was supported by the Institute

for the Care of Mother and Child, Prague, and by the Ministry

of Health, Project CEZ MZOL 340000001, Czech Republic.

The views of the authors represent scientific opinion and

should not be construed as the opinion or policy of the U.S.

FDA.

Received for publication July 25, 2001; revision received

October 31, 2001, and accepted November, 2001.

TRANSFUSION 2002;42:334-342.

334 TRANSFUSION Volume 42, March 2002

can last decades in humans. However, the clinical courseprogresses rapidly, and there is no effective therapy. Thepathophysiology of these diseases has been explained bythe prion hypothesis, which postulates that a conforma-tionally altered isoform of prion protein (PrPsc) is the TSEinfectious agent. It propagates by serving as a template toconvert host PrPc to additional molecules of PrPsc.1 Un-like PrPc, PrPsc is enriched in �-sheet structures, is par-tially resistant to protease digestion, and is insoluble innondenaturing detergents.1,2 Expression of PrPc in hosttissues plays an essential role in propagation of TSE in-fectivity. PrPc knockout mice (PrPc0/0) do not propagateTSE infectivity and are completely resistant to TSE infec-tion.3 PrPc has been described as a protein with ubiqui-tous distribution,4 but the cells functionally compro-mised in TSE lie within the central nervous system. Afterperipheral inoculation, TSE invasion to the brain pro-ceeds via peripheral tissues expressing PrPc, most likelythe peripheral nervous system.5 Depending on the routeof inoculation, the host species, and the TSE strain, acritical step during the incubation period seems to beamplification of infectivity in some nonneural tissues.Growing evidence suggests that the host’s lymphoreticu-lar system, particularly follicular dendritic cells, plays acrucial role in the propagation of TSE infectivity beforeinvasion of the central nervous system.6

The role of blood and blood vessels in propagationand transport of TSE infectivity remains unclear.7 Infec-tivity in blood was demonstrated in TSE-infected miceand hamsters. A recent report of bovine spongiform en-cephalopathy transmission by a blood transfusion froman infected sheep donor while in the preclinical stage ofthe disease highlighted serious concerns about the pos-sibility of TSE transmission by blood transfusion in hu-mans.8 Although there has not been any evidence of this,there is concern that humans who have been infectedwith a variant of CJD but remain asymptomatic mayspread the disease by blood donation. The expression ofPrPc on different blood cells and platelets is highly vari-able among mammalian species, which makes it difficultto extrapolate rodent and sheep TSE models to hu-mans.9,10 Human blood cells such as lymphocytes,monocytes, and platelets express substantial levels ofPrPc;11-14 however, only one-third of the PrPc in humanblood is cell associated, and the remaining two-thirds ispresent in plasma.15 In a rodent model of TSE, infectivityin plasma was mostly absent in the preclinical phase butrose sharply during the symptomatic stage of the dis-ease.16 The source of plasma PrPc and plasma TSE infec-tivity is not known, but endothelial cells may be a goodcandidate. Abundant expression of PrPc in the capillaryendothelial cells in the intestinal villi of the digestive tracthas been reported.17 These findings correlate well with astudy demonstrating PrPsc detection in the capillary en-dothelial cells of the small intestine of lemurs after oral

infection with bovine spongiform encephalopathy.18 Inaddition, PrPsc accumulates in the intestinal nervous sys-tem in scrapie-infected sheep, suggesting a possibletransfer of infectivity from endothelial cells to peripheralnerves.19 Endothelial cells, in addition to lymphoid tis-sues and peripheral nerves, may participate in the gut–brain axis of TSE infectivity progression.

In this study, we characterize PrPc expression on cul-tured endothelial cells and investigate the PrPc releasefrom endothelial cells in the form of membrane micro-particles (MPs). Finally, we demonstrate the presence ofendothelial MPs expressing PrPc in human plasma.

MATERIALS AND METHODS

Antibodies and competing peptidesMoAb 1562 (Chemicon, Temecula, CA) is directed againstthe sequence MKHM in human PrPc (PrPc 109-112).MoAb 6H4 (Prionics AG, Zurich, Switzerland) recognizesthe sequence DYEDRYYRE (PrPc 144-152) in humanPrPc. MoAb FH11 against N-terminal epitope of sheepPrPc 23-85 and MoAb DF7 against linear epitope in theregion of cow PrPc 140-180 were from the TSE ResourceCenter (Institute for Animal Health, Compton, UK; do-nated by C.R. Birkett, PhD). MoAbs were conjugated toFITC using a kit (QuickTag FITC-conjugation kit, Boeh-ringer Mannheim, Indianapolis, IN). The effective fluo-rescein-to-protein ratio was determined by flow cytom-etry, as described previously.9 Competing synthetic pep-tides PrP 102-114 (PSKPKTNMKHMAG) for MoAb 1562 orPrP 142-154 (GSDYEDRYYRENM) for MoAb 6H4 weresynthesized and used for estimation of nonspecific bind-ing. IgG1 isotype controls and MoAb were purchased (PE-and FITC-conjugated IgG1 isotype controls, peridininchlorophyl protein-conjugated MoAb to CD45 [CloneTU116] and MoAb to human CD51/61 [FITC-conjugated,Clone 23C60] were from BD PharMingen, San Diego, CA;MoAb to human CD31 [FITC-conjugated, Clone 5.6E] andMoAb to human CD41 [FITC-conjugated, Clone P2] werefrom Immunotech, Marseille, France; and MoAb to hu-man CD105 [PE-conjugated, Clone N1-3A1] was from An-cell/Alexis, San Diego, CA).

Endothelial cell cultureHuman umbilical vein endothelial cells (HUVECs) andbovine aorta endothelial cells were purchased (Clonetics,San Diego, CA) and were cultured in endothelial cellgrowth media (EGM-2, Clonetics) containing 2-percentFBS and supplements. Cells of second passage were usedin the experiments. Cells were seeded at a density of10,000 cells per cm2 in polystyrene six-well plates (Fal-con) or 60-mm dishes (Becton Dickinson Labware,Franklin Lakes, NJ) and were maintained at 37�C underan atmosphere of 5-percent CO2 and 95-percent room

CELLULAR PRION PROTEIN ON ENDOTHELIAL CELLS

Volume 42, March 2002 TRANSFUSION 335

air. Cells reached confluence at 48 hours in culture andwere harvested in 20 mM HEPES buffer with 100 mMNaCl, 0.5-percent BSA, and 10 mM EDTA, pH 7.4. Afterharvest, cells were centrifuged (300 � g for 5 min),washed with HBSS with 0.35-percent BSA (HBSS-BSA),and used for analysis.

Induction of apoptosis in HUVEC cultureConfluent HUVECs after 48 hours in culture were washedwith HBSS and were incubated for 24 hours at 37�C withvarious agonists in medium: 0.1-percent DMSO (vehiclefor camptothecin [CPT]); CPT (Sigma, St. Louis, MO) 5�M; CPT with caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone (ZVAD, Calbiochem, San Diego, CA)50 �M or ZVAD 50 �M alone. After incubation, mediumand cells were harvested for analysis.

Immunolabeling of endothelial cellsApproximately 105 cells were resuspended in 50 �L ofHBSS-BSA and incubated with saturating concentrationof FITC- or PE-conjugated MoAb. For MoAbs 1562 and6H4 to PrPc, incubation was performed with or without200 �M of competing peptide. After 20 minutes at roomtemperature, the suspension of labeled cells was dilutedwith 2 mL of HBSS-BSA and was centrifuged (300 � g for5 min). Sedimented cells were resuspended in 0.5 mL ofHBSS-BSA and analyzed with flow cytometry.

Annexin V labeling of MPsOne hundred �L of cell culture supernatant was incu-bated with 4 �L of annexin V-FITC (BD PharMingen) for20 minutes at room temperature with or without 50 mMEDTA to estimate the nonspecific binding. Fifty �L oflabeled MPs was added to tubes (TruCount, Becton Dick-inson, San Diego, CA) containing standard beads in 450�L of HBSS-BSA. Samples were immediately analyzed us-ing flow cytometry.

Immunolabeling of annexin V-binding MPs inHUVEC culture mediumCell culture supernatant containing MPs was centrifugedat 110 � g for 5 minutes, and the sediment was dis-carded. Supernatant was further centrifuged at 19,800 �

g for 5 minutes at 10�C, and sedimented MPs were resus-pended in an original volume of HBSS-BSA. Aliquots of 50�L were incubated for 20 minutes at room temperaturewith 2 �L of annexin V-PE (BD Pharmingen) and a satu-rating concentration of FITC-conjugated MoAb. After in-cubation and washing with 1 mL of HBSS-BSA (centri-fuged at 19,800 � g for 5 min at 10�C), samples werediluted with 500 �L of HBSS-BSA and were analyzed byflow cytometry.

Flow cytometry of endothelial cellsCell samples were analyzed by a flow cytometer(FACScan, Becton Dickinson) equipped with software

(CELLQUEST) with forward scatter (FSC) and side scatter(SSC) in linear-mode setting. Populations of intact cellswere gated according to their light-scattering character-istics to exclude debris, and 10,000 gated particles wereanalyzed for each sample. Standard beads (QuantumTM24, Flow Cytometry Standards, San Juan, PR) were runeach day as a separate sample to standardize fluores-cence readings. For PrPc quantitation on cells, a calibra-tion regression line was constructed, and the number ofMoAb 1562-FITC molecules bound per cell was calcu-lated. The number of nonspecifically bound antibodymolecules determined in the presence of competing syn-thetic peptide was subtracted from the total binding foreach sample, as described previously.9 The apoptotic sta-tus of cells was confirmed by flow cytometry using a kit(Annexin V-FITC Apoptosis Detection) for annexin Vand/or propidium iodide cell labeling and by using a kit(Apo-Direct) for terminal deoxynucleotidyltransferasedUTP-FITC nick end labeling according to manufactur-er’s instructions (BD Pharmingen).

Flow cytometric analysis of HUVECmembrane MPsMPs were analyzed in a separate protocol using a modi-fied method by Combes et al.20 The size distribution ofMPs was approximately 0.3 to 3.0 �m, as determined bycomparing FSC values of MP and the polystyrene beads0.3 and 3 �m in diameter from Sigma. For MP quantita-tion, beads (TruCount, Becton Dickinson) were used ineach sample as an internal standard. These beads weregated in an SSC versus FSC logarithmic plot, and analysiswas stopped when 5000 beads were counted. AnnexinV-FITC-positive MPs were gated in an SSC versus fluo-rescence logarithmic plot, and MP count released by 103

cells was calculated for each sample. Antigen expressionon MPs was analyzed by double labeling with annexinV-PE and FITC-conjugated MoAbs. The annexin V-PE-positive MPs were gated, and their binding of FITC-l a b e l e d M o A b s w a s e v a l u a t e d i n S S C v e r s u sFITC�fluorescence logarithmic plots.

Flow cytometry assay of endothelial MPs inhuman bloodVenous blood from healthy blood donors (n = 6) wascollected into a one-tenth volume of 3.8-percent sodiumcitrate at the Department of Transfusion Medicine, Na-tional Institutes of Health (Bethesda, MD). Platelet-poorplasma was obtained by centrifugation of blood for 15minutes at 2700 � g at 10�C. Collected plasma was cen-trifuged again for 5 minutes at 2700 � g to remove con-taminating platelets. Aliquots of 1.5 mL of platelet-freeplasma were centrifuged for 5 minutes at 19,800 � g at10�C to collect MPs. Sedimented MPs were resuspendedin 1 mL of HBSS without calcium, sedimented by cen-

SIMAK ET AL.


trifugation, resuspended in 50 �L of HBSS-BSA, andpooled. Aliquots of 50 �L were incubated for 20 minutesat room temperature with saturating concentrations ofPE-conjugated MoAb to CD105 and FITC-conjugatedMoAb 1562 in the presence or absence of 200 �M of com-peting synthetic peptide PrP 102-114. To distinguishplatelet, WBC, and endothelial MP populations inplasma, triple labeling was performed in a separate MPsample using PE-conjugated MoAb to CD105, FITC-conjugated MoAb to CD41, and peridinin chlorophyl pro-tein-conjugated MoAb to CD45. After incubation andwashing with 1 mL HBSS-BSA, samples were resus-pended with 500 �L of HBSS-BSA and analyzed by flowcytometry. The total count of 100,000 particles was ana-lyzed in each sample, and the results are presented inSSC versus fluorescence logarithmic plots.

RT-PCR of PrPc in HUVECsTotal RNA was isolated (RNAzol B, Tel-Test Inc., Friends-wood, TX) from HUVECs after 48 hours in culture. PCRamplification was carried out from total RNA after reversetranscription using a kit (ProSTAR First Strand RT-PCRKit, Stratagene, La Jolla, CA). Primers for RT-PCR of hu-man prion protein (forward primer 5�-GTGCACGACTGC-GTCAAT-3�, reverse primer 5�-CCTTCCTCATCCCACTAT-CAGG-3�; expected product size 243 bp) and human �

actin (forward primer 5�-CTACAATGAGCTGCGTGTGG-3�, reverse primer 5�-ATAGCAACGTACATGGCTGG-3�; ex-pected product size 138 bp) were synthesized by usingsequences published by Diomede et al.21 PCR amplifica-tion was performed with 10 minutes of initial denatur-ation at 94�C, 30 seconds of annealing at 54�C, and 1minute at 72�C, followed by 35 cycles of 30 seconds at94�C, 20 seconds at 55�C, 1 minute at 72�C, and a finalextension of 7 minutes at 72�C. The same PCR protocolwas used for both prion and � actin. Subsequently, analiquot of each amplified component underwent electro-phoresis through a 1.5-percent agarose gel. SeparatedPCR fragments were stained with ethidium bromide.

Deglycosylation of PrPc in HUVEC lysate by usingN-glycosidase F and immunoblottingHUVECs (3 � 107 cells/mL) in Tris-EDTA buffer (20 mMTris, 154 mM NaCl, 5 mM EDTA, pH 7.4) containing pro-tease inhibitors (1 mM PMSF, 100 �M leupeptin, 100 �Mpepstatin) were lyzed by 1-percent SDS and were heatedat 100�C for 10 minutes. Lysate was sonicated to breakdown DNA and was diluted 10 times with 0.5-percentTriton X-100 in Tris-EDTA containing protease inhibitors.Aliquots of diluted lysate were incubated with or without2 U per mL of N-glycosidase F (Sigma) for 16 hours at37�C. Proteins were precipitated by ice-cold methanoland were sedimented at 19,800 � g for 15 minutes, and

the supernatant was discarded. Pellets were resuspendedin SDS sample buffer (Novex/Invitrogen, Carlsbad, CA),heated at 85�C for 10 minutes, and analyzed on 4- to12-percent precast gels with MES buffer (NuPAGE,Novex-Invitrogen). Western blots prepared according tomanufacturer’s instructions were developed with MoAbs1562 (1:5,000), 6H4 (1:10,000), and DF7 (1:1,000), and the

Fig. 1. Expression of PrPc on HUVECs. (A) Flow cytometric

analysis of PrPc surface expression on HUVECs. Harvested

HUVECs were incubated with FITC-conjugated MoAb 1562

with (dashed line) or without (thick line) 200 �M of the

competing peptide PrP 102-114. Binding of IgG2a isotype

control (IgG-IC) corresponded to the fluorescence of nonla-

beled cells (filled area). (B) Demonstration of PrPc mRNA in

HUVECs by RT-PCR. Total RNA isolated from HUVECs was

reverse transcribed, and PCR was performed as described in

the text. Electrophoresis on a 1.5-percent agarose gel yielded

the expected size of PCR product (243 bp) (Lane 1). No prod-

uct was detected after PCR of the RNA sample where the re-

verse transcription step was omitted (Lane 2). Lane S con-

tains a 100-bp ladder spanning a region from 100 to 1000

bp. (C) Electrophoretic pattern of endothelial PrPc. Aliquots

of diluted SDS lysate of HUVECs were incubated with buffer

only (Lanes 2 and 3) or with 2 U per mL of N-glycosidase F

(Lanes 4 and 5) for 16 hours at 37�C. Nonreduced samples

were prepared from methanol precipitates, and electropho-

retic analysis was performed on 4- to 12-percent NuPAGE

gels. Lysate of human platelets (Lane 1) and protein stan-

dards (Lane S) were analyzed for comparison. Western blots

were developed with MoAb 1562 (left) or MoAb 6H4 (right),

and the bands were visualized using alkaline phosphatase-

conjugated secondary antibody.



bands were visualized with an alkaline phosphatase-conjugated goat F(ab)2 antimouse IgGs (G + L) (Bio-source, Camarillo, CA) and a 5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium phosphatasesubstrate (KPL, Gaithersburg, MD).

RESULTSPrPc is expressed on the surface of cultured human andbovine endothelial cells. Flow cytometric analysis afterimmunolabeling with MoAb 1562 demonstrated that HU-VECs (Fig. 1A) are clearly positive for surface PrPc expres-sion. The actual number of PrPc molecules per cell inHUVECs after 48 hours in culture (approx. 95% conflu-ence) was 58,000 � 2,800, assuming that one molecule ofMoAb 1562 bound to the cell represents one molecule ofPrPc. Similar results were obtained with MoAb 6H4,whereas MoAb FH11 showed lower binding (data notshown), suggesting that part of HUVEC surface PrPc maybe truncated at the N terminus or aggregated in the vi-cinity of octapeptide repeat.1 MoAb 6H4 detected PrPc onthe surface of bovine aorta endothelial cells at a levelcomparable to HUVECs (data not shown).

Demonstration of PrPc mRNA in HUVECby RT-PCRRT-PCR analysis confirmed the presence of PrPc mRNAin HUVECs (Fig. 1B). Electrophoresis yielded the ex-pected size of the PCR product (243 bp). No product wasdetected after PCR of the RNA sample where the reversetranscription step was omitted. In addition, we alsotested another pair of primers for PrPc mRNA analysisthat was used by Dodelet and Cashman12 and generateda PCR product of the same size as they did (approx. 430bp) (data not shown).

Electrophoretic pattern of intact anddeglycosylated endothelial PrPcHUVEC PrPc was further characterized bySDS-PAGE and immunoblotting. OnWestern blots, endothelial PrPc appearedas a diffuse band with apparent molecularweight of 35 to 42 kDa, similar to plateletPrPc (Fig. 1C). Comparable results wereobtained using MoAb 6H4 and MoAb1562. The specificity of binding of anti-bodies to PrPc on blots was demonstratedby their inhibition with competing pep-tides (data not shown). Deglycosylation ofPrPc in HUVEC lysate by N-glycosidase Fresulted in a major band of an apparentmolecular weight of approximately 26kDa, corresponding to the reported mo-lecular weight of deglycosylated humanbrain PrPc.1

PrPc is present on HUVEC MPs released byproapoptotic stimulationWe investigated the release of MPs from HUVECs afterproapoptotic stimulation of the cell culture. The apop-totic process was confirmed by a significant increase ofannexin V-FITC binding and terminal deoxynucleotidyl-transferase dUTP-FITC nick end labeling of the cells (datanot shown). We stimulated HUVEC cultures with 5 �MCPT, a topoisomerase inhibitor that promotes apopto-sis.22 After a 24-hour stimulation with CPT, cells releasedanucleated membrane MPs into the culture medium. TheMPs binding annexin V were counted (Fig. 2). Their sizewas estimated by flow cytometry to be approximately 0.3to 3.0 �m. Interestingly, a small but significant overnightincrease in the MPs count occurred in the medium ofunstimulated cell culture, which probably representsspontaneous apoptosis of cultured HUVECs. A release ofMPs from HUVECs after CPT treatment (Fig. 3) as well asthe spontaneous release of MPs from unstimulated cellswas prevented by 50 �M ZVAD, an inhibitor of caspases.

Double-labeling flow cytometry showed that certainpopulations of annexin-V binding endothelial MPs ex-pressed PrPc and other endothelial antigens CD105,CD31, and CD51/61 (Fig. 4). Binding of the PrPc MoAb1562 to MPs was inhibited by the competing peptide,thus demonstrating specificity. A similar pattern of anti-gen expression was observed on MPs released by CPTtreatment (Fig. 5) and the MPs released overnight fromunstimulated cells. CD105 (endoglin), CD31 (PECAM-1),and CD51/61 are all abundant on HUVECs, with morethan 100,000 copies per cell,23 whereas we found only40,000 to 60,000 PrPc molecules per cell. Thus, with theexception of CD51/61, which was found on MPs in low

Fig. 2. Flow cytometric quantitation of endothelial membrane annexin V-bind-

ing MPs. Aliquots of medium from cell culture after a 24-hour incubation with

0.1-percent DMSO (the CPT vehicle) or with 5 �M CPT were incubated with an-

nexin V-FITC with or without 50 mM EDTA. Then 50-�L aliquots of the incuba-

tion mixture were added to a TruCount tube containing 450 �L of HBSS-BSA

and were analyzed by flow cytometry. TruCount beads were gated in a SSC ver-

sus FSC plot, and the analysis was stopped when 5000 beads were counted. An-

nexin V-FITC-positive MPs were gated in a SSC versus fluorescence plot, and

counts of MPs per 103 cells were calculated. Results obtained by this assay are

presented in Figure 3.

SIMAK ET AL.


levels, our experiments indicate that analyzed antigensare expressed on MPs in proportions similar to that foundon intact cells.

Endothelial cell MPs expressing PrPc are presentin human bloodWe used MoAb to CD105 to identify endothelial MPs inhuman blood obtained from healthy donors. TripleMoAb labeling experiments demonstrated the presenceof three distinct populations of MPs in human plasmabased on the presence of cell-specific antigens (data notshown). The largest population of platelet-derived MPswas recognized as CD41+CD105�CD45�. A popula-tion of MPs CD105+CD41�CD45� was identified asMPs of endothelial origin, and a population of MPsCD45+CD41�CD105� was identified as MPs fromWBCs. We demonstrated the coexpression of PrPc andCD105 on a population of MPs in human blood (Fig. 5).Binding of MoAb 1562 to a subpopulation of CD105+ MPswas prevented by competing peptide PrP 102-114. Theseresults indicate the presence of endothelial MPs express-ing PrPc in human platelet-free plasma.

DISCUSSION

Studies on tissue specificity of PrPc expression are mainlybased on immunohistochemistry methods,24 Northern orWestern blotting,25,26 in situ hybridization,27 or green

fluorescent protein reporter gene expression.17 Some im-munomicroscopy studies reported the presence of PrPcon endothelial cells,17,24,28 but quantitative analysis of thecell surface molecules using flow cytometry was per-formed only on blood cells.11-15,29 In our study, we foundsurface expression of PrPc on HUVECs in a density ofapproximately 60,000 molecules per cell. Similar expres-sion of PrPc was found on bovine aorta endothelial cells.There are no quantitative data available to compare den-sity of PrPc expression on other cultured cell lines.

We cannot exclude a possibility of adsorption ofsome bovine PrPc from FCS in the medium to endothelialcells in culture. However, in the case of HUVECs, it wasnot a significant amount because MoAb 1562 does notreact with bovine PrPc. In addition, generally, the adsorp-

Fig. 3. Release of annexin V-binding MPs from HUVECs after

proapoptotic stimulation. Confluent HUVECs were incubated

with 0.1-percent DMSO (CPT vehicle), 5 �M CPT, or 5 �M

CPT with 50 �M ZVAD. After a 24-hour incubation, superna-

tant from cell culture was harvested, and annexin V-FITC-

positive MPs were counted. Means of four independent ex-

periments � SD are presented; *p < 0.01 vs. DMSO

(ANOVA).

Fig. 4. Expression of PrPc and other endothelial antigens on

annexin V-binding HUVEC MPs. MPs in medium were har-

vested after a 24-hour incubation of HUVECs with 5 �M CPT

and double labeled with annexin V-PE and a saturating con-

centration of an FITC-conjugated MoAb. (A) IgG1-IC, (B)

MoAb 1562 to human PrPc, (C) MoAb 1562 with 200 �M

competing peptide PrP 102-114, (D) MoAb to CD51/61, (E)

MoAb to CD31, and (F) MoAb to CD105. After incubation

and washing, samples were analyzed by flow cytometry. The

annexin V-PE-positive MPs were gated, and their binding of

FITC-labeled MoAbs was evaluated in fluorescence (FITC)

versus SSC plots. Results are representative of six experi-

ments.



tion of PrPc from plasma to blood cells is not a commonphenomenon, as we were not able to detect any PrPc byflow cytometry (MoAb 6H4) on bovine RBCs, granulo-cytes, and only a very little amount of PrPc on bovineplatelets.10

The SDS electrophoretic analysis of intact and degly-cosylated HUVEC PrPc showed an electrophoretic pat-tern similar to that reported for human brain PrPc.1,30

The major band of apparent molecular weight of 26 kDain deglycosylated samples (Fig. 1C, Lanes 4 and 5) isprobably a full-size deglycosylated PrPc peptide. Faintadditional bands representing either partial deglycosyla-tion or truncated forms of PrPc were more clearly stainedby MoAb 6H4 (epitope PrPc144-152). The major 14-kDadegradation band is most likely a C-terminal fragment, asit was strongly detected by MoAb 6H4 (Fig. 1C) and MoAbDF7 (data not shown), but not with MoAb 1562, whichrecognizes an epitope near the N-terminal (PrPc 109-112).

To find a possible origin of PrPc in blood plasma, westudied the release of membrane MPs from apoptoticHUVECs. We focused on a proapoptotic stimulation be-cause it is well known that apoptosis is associated with aloss of phospholipid membrane asymmetry, phosphati-dylserine exposure, and cell membrane vesiculation.31 Inaddition, apoptotic cell death was shown to occur duringTSE neurodegeneration, and PrPc peptides were shownto induce apoptosis in cell cultures, including endothelialcells.32,33 A release of cell membrane MPs from apoptoticcells has also been documented,34,35 but there are veryfew reports on the release of membrane MPs from cul-tured endothelial cells.20,36,37 Moreover, no data are avail-able about MP release from endothelial cells after pro-apoptotic stimulation. MPs of endothelial origin wereidentified in normal human blood,20,38 and increasedcounts of MPs carrying endothelial antigens were docu-

mented in blood of patients with lupusanticoagulant,20 acute coronary syn-drome,39 or thrombotic thrombocyto-penic purpura.40 In our experiments,proapoptotic stimulation of HUVECsinduced a marked release of MPs.Combes et al.20 demonstrated release ofMPs from HUVECs after overnightstimulation with TNF-� and also after a24-hour stimulation with IL-1�, throm-bin, or PMA. However, the reportedcounts of annexin V-binding MPs re-leased after cell stimulation were verylow, with a maximum of approximately250 MPs per 103 cells, corresponding toa 2.5-fold increase as compared withcontrols. In our study, we were able toinduce the release of approximately80,000 MPs per 103 cells after 24 hoursof proapoptotic stimulation with CPT.

When this number is compared with the MP count inculture treated with ZVAD alone (Fig. 3), where sponta-neous apoptosis should be inhibited, there is an approxi-mately 10-fold increase in the MP count related to theproapoptotic stimulation of HUVECs.

Based on the highest expression of CD105 out of theantigens we studied on HUVEC MPs (Fig. 4), we chosethis relatively specific endothelial marker to distinguishendothelial MPs in blood. The MoAb to CD105 recog-nized a distinct population of MPs in human plasma anddid not react with MPs positive for platelet antigen CD41or WBC antigen CD45. We assumed that CD105-positiveMPs are likely of endothelial origin. The presence of en-dothelial MPs in blood of healthy individuals is probablya result of physiologic turnover of endothelial cells, andthe count of endothelial MPs may be increased undervarious pathologic conditions where apoptosis of endo-thelium is enhanced.

The presence of PrPc on endothelial MPs in blood(Fig. 5) opens a new area of speculation about the role ofendothelial cells in TSE transmission and pathogenesis.Vascular endothelial cells are one of the first tissues tocome into contact with the TSE agent after oral or IV TSEinoculation, which are the most epidemiologically rel-evant routes in humans. Although the presence of PrPschas been demonstrated in the intestinal endothelial cellsafter oral bovine spongiform encephalopathy infection inlemurs,18 the role of endothelial cells in TSE pathogenesisremains to be elucidated. A similarly intriguing questionis the origin of plasma PrPc and plasma TSE infectivity. Itis not known whether TSE infectivity in plasma is asso-ciated with soluble proteins or with cell membrane MPs.Brown et al.16 found in a rodent model of TSE that plasmainfectivity was substantially reduced but was not elimi-nated by centrifugation for 30 minutes at 17,000 � g. The

Fig. 5. Expression of PrPc on endothelial MPs in human blood. MPs were sedi-

mented from platelet-free plasma, washed, and labeled with PE-conjugated MoAb

to CD105 and FITC-conjugated MoAb 1562 to PrPc. The total count of 100,000

particles was analyzed by flow cytometry in each sample, and the results are pre-

sented in SSC versus fluorescence logarithmic plots. CD105+ MPs were gated in

R1, and their binding of MoAb 1562 in the presence or absence of 200 �M com-

peting peptide PrP 102-114 is shown. Results are representative of six experi-

ments.

SIMAK ET AL.


endothelial MPs that we studied (>0.3 �m) would be sedi-mented by this centrifugation. The infectivity remainingin plasma supernatant was not likely to be associatedwith cell membrane MPs. Human platelets have beenshown to release a soluble form of PrPc.41 It is possiblethat endothelial cells also release soluble PrPc. We nowshow that endothelial cells can release PrPc-expressingMPs in culture and that normal human plasma containsPrPc-positive MPs of endothelial origin. In plasma, theendothelial cell membrane MPs account for approxi-mately 25 percent of all detected MPs expressing platelet,WBC, or endothelial cell antigens. Depending on sam-pling and storage procedures, there are approximatelytwice as many platelet MPs positive for PrPc as comparedwith MPs of endothelial origin (data not shown). How-ever, in contrast to platelets, RBCs, and marrow cells,endothelial cells were shown to accumulate PrPsc afterTSE infection. This makes endothelial MPs in blood par-ticularly important with respect to a possible role intransmission of TSE infectivity. The marked release ofMPs from HUVECs was observed after proapoptoticstimulation, which is a mechanism that may be particu-larly important in TSE diseases. Recently, it has been re-ported that a prion peptide induces apoptosis of endo-thelial cells in culture.28 After oral TSE infection, gutendothelial cells may accumulate PrPsc and undergo ap-optosis with a release of PrPsc-positive MPs. Similarly,PrPsc that accumulates in the cental nervous system mayinduce apoptosis of brain endothelial cells. PrPsc-positive MPs of endothelial origin may spread the TSEinfectivity in circulation and may be a possible source ofthe infectivity in plasma. Our findings warrant futurestudies on a possible role of apoptotic endothelial cells intransport and propagation of TSE infectivity in blood.

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cellular prion protein is expressed on endothelial cells and is released during apoptosis on...

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