blood-brain barrier genomics.pdf

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BloodBrain Barrier Genomics

Jian Yi Li, Ruben J. Boado, and *William M. Pardridge

Department of Medicine, UCLA School of Medicine, Los Angeles, California, U.S.A.

Summary: The bloodbrain barrier (BBB) is formed by thebrain microvascular endothelium, and the unique transportproperties of the BBB are derived from tissue-specific geneexpression within this cell. The current studies developed agene microarray approach specific for the BBB by purifyingthe initial mRNA from isolated rat brain capillaries to generatetester cDNA. A polymerase chain reactionbased subtraction

cloning method, suppression subtractive hybridization (SSH),was used, and the BBB cDNA was subtracted with drivercDNA produced from mRNA isolated from rat liver and kid-ney. Screening 5% of the subtracted tester cDNA resulted inidentification of 50 gene products and more than 80% of thosewere selectively expressed at the BBB; these included novel

gene sequences not found in existing databases, ESTs, andknown genes that were not known to be selectively expressedat the BBB. Genes in the latter category include tissue plas-minogen activator, insulin-like growth factor-2, PC-3 geneproduct, myelin basic protein, regulator of G protein signaling5, utrophin, IB, connexin-45, the class I major histocompat-ibility complex, the rat homologue of the transcription factors

hbrm or EZH1, and organic anion transporting polypeptide type2. Knowledge of tissue-specific gene expression at the BBBcould lead to new targets for brain drug delivery and couldelucidate mechanisms of brain pathology at the microvascularlevel. Key Words: Bloodbrain barrierEndotheliumGeneexpressionTransferrin receptorBiologic transport.

The transport of solutes and drugs into the brain is

regulated by transport systems present at the bloodbrain

barrier (BBB). The BBB is formed at the luminal and

abluminal membranes of the brain capillary endothelium

(Brightman et al., 1970). The capillary endothelial cells

of the vertebrate brain express epithelial-like high resis-

tance tight junctions that fuse the plasma membranes of

neighboring capillary endothelial cells in brain. The tight

junctions of the BBB eliminate the porous pathways of

solute diffusion across the endothelial cell in brain,

which exist in capillary beds perfusing nonbrain organs.

Transport systems expressed at the BBB may mediate

either the influx of solutes or drugs from blood to brain

or the active efflux of solutes or drugs from brain to

blood (Pardridge, 1998).

Novel genes encoding BBB transporters can be iden-

tified with standard genomic approaches based on gene

microarrays. However, the sensitivity of gene microar-

rays is approximately 10-4 (Schena et al., 1995). The

volume of the brain capillary endothelium forming the

BBB in vivo is


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1991). Brain capillaries were free of adjoining brain tissue(Fig. 1A). The yield of poly A+ RNA from bovine and ratbrain capillaries was 12 g per single bovine brain corticalshell and 3.2 g from the pooled cerebral hemispheres of 21 ratbrains, respectively.

Suppression subtractive hybridizationSuppression subtractive hybridization was performed using

the PCR-Select cDNA subtraction system according to themanufacturers instructions (Clontech, Palo Alto, CA, U.S.A.).The rat brain capillary poly A+ RNA was used to produce 2.5g of tester cDNA, and the subtraction procedure was com-pleted using either rat liver or kidney mRNA to generate drivercDNA. The reaction was followed with [32P]-dCTP. The testeror driver cDNA was digested with RsaI to obtain shorter, blunt-

end molecules, and 2 tester populations were created as eitheradaptor 1 or adaptor 2R and were independently ligated to 350ng of the tester cDNA. The 2 populations of adaptor-ligatedtester cDNA were independently hybridized (at 98C for 1.5minutes and 68C for 8 hours) to the driver cDNA to enrich fordifferentially expressed sequences, and hybridized a secondtime (at 68C for 16 hours) to generate a polymerase chain

reaction (PCR) template. A first-run PCR using PCR primer 1amplifies differentially expressed sequences and was per-formed for 30 cycles (denaturation, 94C for 30 seconds; an-nealing, 66C for 30 seconds; extension, 72C 1.5 minutes). Asecond-run PCR was performed for 15 cycles (denaturation,94C for 30 seconds; annealing, 68C for 30 seconds; exten-sion, 72C 1.5 minutes), using nested PCR primers 1 and 2R.This second-run PCR further enriches for differentially ex-pressed sequences and suppresses the background (Diatchenkoet al., 1996).

Subtracted cDNA screeningThe SSH-PCR products were cloned into the pCR2.1 vector

and a cDNA library was prepared in E. coli INVF cells.Positive clones were identified by differential hybridization.

The cDNA library was plated on Luria-Bertani medium/ampicillin (LB/amp) plates, and white colonies were pickedand individually grown in 200 L LB/amp medium in 96-wellplates for 24 hours at 37C. Colonies were individually blottedonto GeneScreen Plus membrane using a 96-well dot-blot sys-tem (Bio-Rad, Richmond, CA, U.S.A.). Membranes were hy-bridized with [32P]-labeled subtracted or unsubtracted rat braincapillary tester cDNA (Wang et al., 1998), and film autoradi-ography was performed with Biomax MS film (Kodak, Roch-ester, NY, U.S.A.) for 18 hours at 23C. cDNA was purifiedwith Qiaquick PCR purification kit (Qiagen, Santa Clarita, CA,U.S.A.) and labeled as previously described (Boado et al.,1999). Clones showing a strong hybridization signal with thesubtracted probe compared with the unsubtracted one were se-lected for DNA sequencing and Northern blot analysis afterrelease of the pCR2.1 insert with EcoRI. Northern blotting was

performed as described previously (Boado et al., 1999).

DNA sequence analysisDNA sequencing of isolated clones was performed in both

directions at Biotech Core (Mountain View, CA, U.S.A.) usingM13 forward and reverse primers (Boado et al., 1999). Simi-larities with other genes in GenBank were investigated usingthe BLAST program (NCBI, NIH). Plasmid DNA was ampli-fied and purified from positive clones using the Qiafilter MaxiKit (Qiagen, Valencia, CA, U.S.A.).

In situ hybridizationThe cDNA fragment obtained from SSH was released from

the pCR2.1 vector by double digestion with SalI and XbaI and

cloned into the pSPT19 vector at the same restriction endo-nuclease sites. Antisense and sense RNA probes were gener-ated using SalI and XbaI linearized pSPT19- plasmid and T7and SP6 polymerases, respectively, and digoxigenin-11-uridinetriphosphate (DIG-11-UTP), as part of the DIG RNA Labelingkit (Roche Molecular Biochemicals, Indianapolis, IN, U.S.A.).Freshly isolated rat brain capillaries were cytocentrifuged toglass slides and fixed with 10% formalin before storage at70C. The slides were hybridized with 12 ng digoxigenin(DIG)-labeled probes in 30 L of hybridization solutionovernight at 42C, and control sections were hybridized withan equal amount of sense cRNA according to the manufactur-ers instructions.

FIG. 1. (A) Light micrograph of freshly isolated rat brain capillar-ies showing the microvessels are free of adjoining brain tissue.Capillaries were stained with ortho-toluidine blue. Magnificationbar = 104 m. (B) The subtraction efficiency is demonstrated bypolymerase chain reaction (PCR) amplification of the cDNA forG3PDH. Lanes 1 to 5 are subtracted tester G3PDH PCR prod-ucts at 13, 18, 23, 28, and 33 cycles, respectively. Lane 6 is DNAmarkers 1.4, 1.1, 0.87, 0.60, 0.31, 0.28, 0.23, and 0.19 kb. Lanes7 to 11 are unsubtracted tester G3PDH PCR products at 13, 18,23, 28, and 33 cycles, respectively. (C) Agarose gel electropho-resis of the rat bloodbrain barrier IGF-2 (clone LK6) insert afterEcoRI digestion showing the size to be 0.5 kb (lane 1). Molecularweight DNA size standards are shown in lane 2.

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J Cereb Blood Flow Metab, Vol. 21, No. 1, 2001


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RESULTS

The cDNA products of the first and second cycle PCR

ranged in size from 0.2 to 1.4 kb and the majority of the

PCR products had a size ranging from 0.3 to 0.7 kb

(Table 1). An initial small library was prepared with only

kidney-derived driver cDNA, and three clones were iso-

lated and designated K1-K3 (Table 1). The larger library

was generated from rat liver and kidney-derived driver

cDNA, and the first 50 clones isolated were designated

LK1-LK50 (Table 1). The efficiency of the subtraction

procedure was analyzed by PCR amplification of cDNA

for G3PDH as shown in Fig. 1B. Using the subtracted

tester cDNA, no G3PDH PCR product was identified

TABLE 1. Summary of isolated clones

Clone name Identity Accession #Insert

size (kb)Nucleotidessequenced

mRNAsize (kb)

Northerncategory

Originof clone

Nucleotide #(% identity)

K1 Oatp2 U88036 0.8 800 4.1 2 rat 25733230 (98)K2 Novel 0.6 572 2.6 1 K3 Ets-1 L20681 0.15 98 5.6 2 rat 23172391 (100)LK1 EST AI072079 0.3 252 3.2 2 rat embryo 445521 (97)LK2 EST AI103051 0.2 177 4.4 5 rat brain 286406 (100)LK3 Novel 0.8 424 2.6 1 LK4 Novel 0.4, 0.5 800 3.9 5 LK5 TPA M23697 0.4 400 2.5 2 rat 15501882 (99)LK6 IGF-2 X14834 0.5 600 3.3, 1.5 2 rat 27173170 (100)LK7 Flt-1 D28498 0.5 500 6.4 2 rat 21912474 (97)

LK8 CP E X51406 0.5 500 2.1 4 rat 475882 (99)LK9 EST AI231826 0.8 400 4.1 2 rat heart 165525 (99)LK10 Flt-1 D88689 0.65 500 7.8, 4.6 2 mouse 54995652 (87)LK11 CP E X51406 0.9 646 ND nd rat 10151660 (99)LK12 Novel 0.7 577 2.6 1 LK13 Novel 0.5 500 2.4 1 LK14 Novel 0.65 600 ND nd LK15 Novel 0.8 448 2.4 1 LK16 PC-3 M60921 0.8, 0.45 474 2.5 2 rat 18592332 (99)LK17 EST AI598434 0.4 432 5.6, 1.3 2 rat embryo 63448 (99)LK18 Flt-1 D88689 0.7 461 ND nd mouse 54995652 (86)LK19 Vinculin L18880 0.4 418 6.0, 1.4 5 mouse 28413213 (96)LK20 Novel 0.2 159 5.0 2 LK21 MBP K00512 0.4 393 2.1 3 rat 308652 (98)LK22 CP E X51406 0.45 455 ND nd rat 475883 (99)LK23 Rgs5 NM_009063 0.28 249 4.0, 1.9 2 mouse 3 gaps 135 nt (91)LK24 Utrophin AJ002967 0.35 337 9.5, 5.0 2 rat 87219009 (99)

LK25 S100 S53527 1.0 575 1.5 3 rat 340913 (96)LK26 Novel 0.45 435 4.6 4 LK27 IB X63594 0.15 250 1.8 2 rat 8341035 (100)LK28 MBP K00512 0.4 385 nd nd rat 308652 (99)LK29 Connexin 45 X63100 0.7 567 2.0 2 mouse 14321947 (94)LK30 EST AI231826 1.2 1000 4.2 2 rat heart 165622 (99)LK31 EST AA817814 0.3 272 3.0 3 rat muscle 36282 (100)LK32 MBP K00512 0.4 393 nd nd rat 308652 (99)LK33 Novel 0.35 331 4.6 4 LK34 TfR M58040 0.28, 0.15 460 6.6, 5.0 2 rat 33493413 (100)LK35 Novel 0.4 397 4.0 2 LK36 MHC I L40364 0.45 431 2.9, 1.8 2 rat 33415 (99)LK37 hbrm X72889 0.2 153 6.0, 2.1 4 human 57365813 (96)LK38 Novel 0.6 491 5.8 2 LK39 Novel 0.7 570 ND nd LK40 CP E X51406 0.5 448 ND nd rat 475882 (100)LK41 Ezh1 NM_007970 0.6 605 4.5 4 mouse 32243584 (80)

LK42 Oatp2 U88036 0.7 555 ND nd rat 16482161 (99)LK43 Flt-1 D88689 0.75 611 ND nd mouse 53115733 (87)LK44 Novel 0.45 408 5.1, 3.5 2 LK45 Novel 0.7 568 ND nd LK46 Vector none ND none nd LK47 CP E X51406 0.5 448 ND nd rat 475882 (99)LK48 EST AI598315 0.45 402 9.5, 5.6 1 rat embryo 34622 (99)LK49 Novel 0.7 484 ND nd LK50 CP E X51406 0.5 445 ND nd rat 475882 (99)

Northern category: 1, gene expression is specific to the BBB and is not detected in brain or peripheral tissues; 2, gene is expressed in brain onlyat BBB and in peripheral tissues; 3, gene is expressed only at BBB and in whole brain, but not in peripheral tissues; 4, gene is expressed in brain,at the BBB, and in peripheral tissues; 5, gene expression at the BBB is not detectable. CP E, carboxypeptidase E; MBP, myelin basic protein; TfR,transferrin receptor; MHC, major histocompatibility complex; nd, not done; BBB, bloodbrain barrier.

BLOODBRAIN BARRIER GENOMICS 63



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until 33 cycles of PCR (lane 5, Fig. 1B). Conversely, the

G3PDH cDNA was identified in PCR of the unsub-

tracted tester cDNA as early as 18 cycles (lane 8, Fig.

1B). Products of the secondary PCR were cloned into the

pCR 2.1 vector for transformation of INVF. Compari-

son of the subtracted and unsubtracted Southern dot blots

of the 96-well microarrays identified several gene prod-ucts that were enriched at the BBB and the sizes of these

inserts were determined by agarose gel electrophoresis

after release with EcoRI (Fig. 1C).

Northern blot analysis was performed on all clones,

except where sequence analysis indicated there was

overlap with other clones, and the results of the Northern

blots are shown in Figs. 2 to 5. Each blot was also probed

with cDNA for two commonly expressed genes, actin

and 4F2hc, which encode the heavy chain of amino acid

transporter heterodimers (Boado et al., 1999). In situ hy-

bridization results for four selected clones are shown in

Fig. 6 for antisense and sense RNA. Based on the North-

ern blotting results, the clones were classified into 1 of 5categories of differential gene expression, as shown in

Fig. 7. Northern blot analysis with clones LK2, a rat

EST, LK4, a novel gene, and LK19, vinculin (Table 1)

showed the corresponding transcripts were underex-

pressed at the BBB (Fig. 7).

DISCUSSION

Novel genes selectively expressed at the

bloodbrain barrier

Northern blotting results with clones K2, which en-

coded a novel sequence, and LK48, a rat EST, showed

FIG. 2. Northern blotting. (left) Lanes 1 to 6: C6 rat glioma cells,total rat brain, rat heart, rat kidney, rat lung, and rat liver, respec-tively. Film exposure time: 1 day for 4F2hc, K1, and K2; 2 days forLK5; 5 days for LK1, LK4, LK6, LK7, and LK9. (right) Lanes 1 to3: bovine brain capillaries, rat brain capillaries, and total rat brain,respectively. Film exposure time: 3 hours for K2; 4 hours for K1; 24hours for actin and LK6; 2 days for LK5; 5 days for LK1, LK4, LK7,and LK9. Two micrograms poly A+ RNA was applied to all lanes.

FIG. 3. Northern blotting. (left) Lanes 1 to 6: C6 rat glioma cells,total rat brain, rat heart, rat kidney, rat lung, and rat liver, respec-tively. Film exposure time: 1 day for 4F2hc and LK21; 5 days forK3, LK8, and LK16; 6 days for LK2 and LK19; 11 days for LK10.(right) Lanes 1 to 3: bovine brain capillaries, rat brain capillaries,and total rat brain, respectively. Film exposure time: 1 day foractin and LK21; 5 days for LK2, K3, LK8, and LK16; 6 days forLK19; 11 days for LK10. Two micrograms poly A+ RNA wasapplied to all lanes, except only 0.5 g poly A+ RNA was appliedtolanes1 to2 ontherightpanelforthebovine (lane 1)or rat (lane2) brain capillaries.

FIG. 4. Northern blotting. (left) Lanes 1 to 6: C6 rat glioma cells,total rat brain, rat heart, rat kidney, rat lung, and rat liver, respec-tively. Film exposure time: 1 day for 4F2hc; 3 days for LK31; 5days for LK15 and LK23; 6 days for LK17, LK25, LK30, and LK35.(right) Lanes 1 to 3: bovine brain capillaries, rat brain capillaries,and total rat brain, respectively. Film exposure time: 1 day foractin, LK30, and LK31; 5 days for LK15 and LK23; 6 days forLK17, LK25, and LK35. Two micrograms poly A+ RNA was ap-plied to all lanes.

J. Y. LI ET AL.64



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that the transcripts corresponding to these clones were

only expressed at the BBB in brain and were not

expressed in peripheral tissues (Figs. 2 and 5B, respec-

tively). Clone LK48 hybridized to a 5.6 kb major

transcript and a 9.5 kb minor transcript in rat brain cap-

illaries. The clone LK48 mRNA was not detected in rat

peripheral tissues and was only detected at trace amounts

in whole rat brain, which may be derived from the cap-

illary fraction in the brain homogenate (Fig. 5B).

The sequence of K2 was also novel and not found in

any databases (Table 1). This clone was used to screen a

rat brain capillary cDNA library in the pSPORT vector,

which had been described previously (Boado et al.,

1999). A 2.6 kb full length cDNA was identified and

sequenced and named BBB-specific anion transporter

type 1 (BSAT1), because of distant sequence similarity

with a liver-specific anion transporter (Abe et al., 1999).

The full sequence for BSAT1 encompassed the se-

quences of 8 other clones found in the initial BBB library

(LK3, LK12, LK13, LK14, LK15, LK39, LK45, and

LK49). Therefore, the BSAT1 clones represented 16% of

the initial 50 clones identified from the liver and kidney

subtracted library. This suggests that the mRNA for

BSAT1 is highly enriched at the BBB; this was con-

firmed by Northern blot analysis as shown in Fig. 2 for

clone K2. These Northern blot analysis studies were

performed with a BSAT1 partial cDNA encoding for

the 3-UTR and there was no crosshybridization be-tween this clone and mRNA generated from bovine brain

capillaries as shown in Fig. 2. The failure to detect

BSAT1 in the bovine brain capillary preparation sug-

gests that the 3-UTR of the BSAT1 mRNA is not con-

served across species.

Several clones hybridized to transcripts that were ex-

pressed in brain only at the BBB, and this mRNA was

also differentially expressed in some peripheral tissues

(Fig. 7). Three clones (LK1, LK17, and LK30) were

found in the rat EST database (Table 1). Clone LK1

FIG. 6. In situ hybridization. Magnification for LK30, myelin basicprotein, and oatp2 is the same. Magnification bars = 34 and 85m for LK30 and IGF-2, respectively.

FIG. 5. Northern blotting. Lanes 1 to7: C6 rat glioma cells, rat brain capil-laries, total rat brain, rat heart, rat kid-ney, rat lung, and rat liver, respec-tively. Film exposure time: 1 day foractin, 4F2hc, LK24, LK27, LK36,LK38, LK44, and LK48; 5 days forLK20, LK26, LK29, LK33, LK34, andLK41; 6 days for LK37. Two micro-grams poly A+ RNA was applied to alllanes. Panels A to D represent fourdifferent Northern blots.




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hybridized to a 3.2 kb mRNA that was selectively ex-

pressed in brain at the BBB and was also expressed in rat

lung (Fig. 2). Clone LK17 hybridized to a 5.6 kb mRNA

that was selectively expressed in brain at the BBB and

was also expressed in rat lung (Fig. 4). Clone LK30

hybridized to a 4.2 kb mRNA that was selectively ex-

pressed in brain at the BBB and was also expressed in

trace amounts in rat heart (Fig. 4). In situ hybridization

with sense or antisense RNA derived from clone LK30 is

shown in Fig. 6 and reveals continuous immunostaining

of the rat brain microvasculature, which is indicative of

an endothelial origin of the mRNA. The trace signal in

whole rat brain could represent LK30 mRNA derived

from the rat brain microvasculature. Four additional

clones (LK20, LK35, LK38, and LK44) represented

novel sequences not found in the EST or Genbank data-

bases (Table 1), and the mRNA corresponding to this

cDNA was selectively expressed at the BBB in brain, in

parallel with selective expression in peripheral tissues

(Figs. 4 to 5). Clone LK20 hybridized to a 5.0 kb mRNA

that was selectively expressed at the BBB, compared

with whole rat brain or rat peripheral tissues (Fig. 5A).

Clone LK35 hybridized to a 4.0 kb mRNA that was

selectively expressed in brain at the BBB and was also

expressed in rat heart (Fig. 4). Clone LK38 hybridized toa 5.8 kb mRNA that was selectively expressed in brain at

the BBB and was also expressed at lower levels in rat

heart (Fig. 5D). Part of the sequence of clone LK44 is

84% identical with nucleotides 129320 of an EST iden-

tified in human aortic endothelium exposed to tumor

necrosis factor- (TNF-) (Adams et al., 1995). North-

ern blot analysis with LK44 showed a selective expres-

sion of 5.1 and 3.5 kb transcripts at the rat BBB that were

present at levels many fold greater than in total rat brain

or rat peripheral tissues (Fig. 5B). The finding of a

TNF- inducible gene product at the BBB parallels pre-

vious studies showing that the receptors for TNF-,

designated TNFR1 and TNFR2, are both expressed at the

BBB (Nadeau and Rivest, 1999).

Known genes selectively expressed at the

bloodbrain barrier

Figure 7 lists 11 known genes that are selectively ex-

pressed in brain at the BBB, with parallel expression in

some peripheral tissues. These genes include tissue plas-

minogen activator (clone LK5); insulin-like growth fac-

tor (IGF)-2 (clone LK6); the vascular endothelial growth

factor receptor, flt-1 (clones LK 7, LK10, LK18, and

LK43); the PC-3 gene product (clone LK16); the regu-

lator of G protein signaling (Rgs)-5 (clone LK23); utro-

phin (clone LK24); IB (clone LK27); connexin-45

(clone LK29); the transferrin receptor (clone LK34); the

class I major histocompatibility complex (clone LK36);

and organic anion transporting polypeptide type 2 or

oatp2 (clones K1 and LK42). The tissue-specific expres-

sion of these genes at the BBB is shown in the Northern

blots in Figs. 2 to 5, and the sequence information on

each clone is shown in Table 1.

Each of these gene products may play an important

role in brain function. Tissue plasminogen activator(TPA) mediates neurite outgrowth and learning in brain

(Seeds et al., 1999). Therefore, BBB-derived TPA may

play a role in neuronal migration and synaptic connec-

tions. The mRNA for IGF-2 is highest in adult rat brain

compared with any other tissue (Murphy et al., 1987).

However, ISH showed IGF-2 transcript only at the cho-

roid plexus in brain (Hynes et al., 1988), and this led to

the hypothesis that choroid plexus is the site of origin of

IGF-2 production in brain. However, the current results

suggest that an additional source of IGF-2 in the brain is

FIG. 7. Summary of clones. Clonesshown in black ovals correspond togenes that are expressed only at theBBB, and gene expression is not de-tectable in either total rat brain or inrat peripheral tissues. Clones shownin white ovals represent genes thatare expressed only in brain at the

BBB, but are also expressed in somerat peripheral tissues. Clones shownin curved white rectangles representgenes that are expressed only inbrain and at the BBB, but gene ex-pression in peripheral tissues is notdetectable. Clones shown in whitesquares represent genes expressedwidely in brain, at the BBB, and in pe-ripheral tissues. Clones shown inblack squares represent genes thatare not detectable at the BBB. Nu-meric superscripts indicate the num-ber of clones out of the 50 clonesscreened for the liver and kidney sub-tracted library that were detected forthe same gene product.

J. Y. LI ET AL.66



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local production at the brain microvasculature. In situ

hybridization studies demonstrated continuous immuno-

staining of isolated rat brain capillaries with the antisense

probe (Fig. 6), which is indicative of an endothelial ori-

gin of the microvascular IGF-2 mRNA.

There are at least three receptors for vascular endo-

thelial growth factor and these are designated as flt-1,flk-2/kdr, and flt-4 (Stacker et al., 1999). In whole body

autoradiography, the organ with the highest binding of

radiolabeled vascular endothelial growth factor is the

brain and this binding in brain was restricted to both the

choroid plexus and to the microvasculature (Jakeman et

al., 1992). The current studies suggest that the flt-1 iso-

form may be the predominant vascular endothelial

growth factor receptor at the brain microvasculature un-

der normal conditions. Rgs5 acts as a GTPase activating

protein for subunits of heterotrimeric G proteins (Chen et

al., 1999), and these proteins play a role in the regulation

of caveolin and endothelial cell transcytosis (Schnitzer et

al., 1995). Utrophin is also called dystrophin related pro-tein and is a 395 amino acid protein that is 73% identical

to the cytoskeletal protein, dystrophin (Galvagni and

Oliviero, 2000).

The transferrin receptor mRNA (clone LK34) is selec-

tively localized at the BBB, and Northern blot analysis

demonstrated expression of 5.0 and 6.6 kb transcripts in

isolated rat brain capillaries as shown in Fig. 5D. The

expression of the 6.6 kb transferrin receptor transcript

was specific for rat brain capillaries and the 5.0 kb tran-

script was also found in total rat brain, although at re-

duced levels compared with isolated rat brain capillaries.

The 5.0 kb transferrin receptor transcript was also de-

tected in C6 glial cells and in rat heart, and there was no

detectable transcript in rat liver. Recent studies have

identified a second form of the transferrin receptor,

which is encoded by mRNA of 2.9 and 2.5 kb and is

specific for liver (Kawabata et al., 1999).

Clones K1 and LK42 corresponded to oatp2 and

Northern blot analysis suggested the expression of this

gene in the brain is confined to the BBB (Fig. 2). The K1

clone was also used in ISH and showed continuous im-

munostaining of isolated rat brain capillaries, indicating

an endothelial origin of the oatp2 transcript (Fig. 6). The

functional role of oatp2 at the BBB is not clear, but this

protein may participate as an active efflux system at theBBB. A principal substrate of oatp2 is estrone sulfate

(Noe et al., 1997). However, estrone sulfate does not

cross the BBB in vivo (Steingold et al., 1986), which

suggests that oatp2 may function as an active efflux sys-

tem at the BBB.

Genes were selectively expressed in brain and at the

BBB, but not in peripheral tissues. Clone LK21, myelin

basic protein, clone LK25, the S100 calcium binding

protein, and clone LK31, a rat EST, all hybridized to

mRNA found only in brain and at the BBB, but not in rat

peripheral tissues (Figs. 3 and 4). The level of the tran-

script for myelin basic protein in whole rat brain was

comparable to the level of myelin basic protein mRNA in

isolated rat brain capillaries as shown in Fig. 3. (The

amount of mRNA from rat brain applied to lane 3 (2.0

g) of the right panel in Fig. 3 was 4-fold greater than

the amount of rat brain capillary mRNA applied to lane1 or 2 (0.5 g) of the right panel of Fig. 3.) The finding

of gene expression for myelin basic protein at the BBB

was unexpected. The expression of the gene for myelin

basic protein at the BBB is of interest, because both

myelin and the BBB evolved in parallel in all vertebrates.

To further identify the site of origin of the myelin basic

protein transcript at the rat brain microvasculature, ISH

studies were performed and are shown in Fig. 6. The ISH

shows continuous immunostaining of the brain mi-

crovessels, which is indicative of an endothelial origin of

the transcript encoding for myelin basic protein. Promi-

nent hybridization was also found in precapillary arteri-

oles. The function of microvascular myelin basic proteinis currently unknown. The earliest neuropathologic le-

sion in the brain of multiple sclerosis is a perivascular

cuffing of lymphocytes around brain microvessels (Ad-

ams, 1977), and myelin basic protein is an autoantigen in

multiple sclerosis (Bornstein et al., 1987). The current

work also shows selective expression of the class I major

histocompatibility complex at the BBB (clone LK 36),

which suggests that antigen presentation takes place at

the brain microvasculature. This extends previous work

showing abundant class II major histocompatibility com-

plex at the human brain microvasculature in multiple

sclerosis (Pardridge et al., 1989).

Genes widely expressed in peripheral tissues, in

brain, and at the bloodbrain barrier

Carboxypeptidase E (clones LK8, LK11, LK22,

LK40, LK47, and LK50), the transcription factors, hbrm

(clone LK37) and EZH1 (clone LK41), and two clones of

novel sequence, LK26 and LK33, were widely expressed

at the BBB, in brain, and in peripheral tissues, based on

Northern blot analysis (Figs. 2 to 5). The hbrm gene

product is the human homologue of yeast SW12/NSF2

protein and is an activator of transcription factors

(Trouche et al., 1997). The finding that several transcrip-

tion factors such as PC-3, IB, hbrm, or EZH1, are se-lectively expressed at the BBB suggests these proteins

may regulate cell division at the brain microvasculature

in states of angiogenesis.

In summary, these studies describe the initial results of

a BBB genomics program and the numerous gene prod-

ucts that are selectively expressed at the BBB compared

with whole brain. The initial library was prepared from

tester cDNA derived from rat brain capillary poly A+

mRNA after subtraction with driver cDNA derived from

rat liver and rat kidney poly A+ mRNA. Screening just




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5% of the subtracted tester cDNA resulted in identifica-

tion of 50 gene products and over 80% of these were

selectively expressed at the BBB. Numerous ESTs or

genes with novel sequences of unknown function were

selectively expressed at the BBB and the availability of

these partial cDNA will enable cloning of the full-length

gene products for subsequent elucidation of the functionof these genes.

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