doi: 10.1161/01.STR.0000247887.61831.74
(Stroke. 2007;38:686.)
© 2007 American Heart Association, Inc.
Genomics of Ischemia: Introduction |
Blood-Brain Barrier Genomics
From the Department of Medicine, University of California at Los Angeles, Calif.
Correspondence to William M. Pardridge, MD, Department of Medicine, University of California at Los Angeles, Warren Hall 13-164, 900 Veteran Ave, Los Angeles, CA 90024. E-mail wpardridge{at}mednet.ucla.edu
Abstract
Blood-brain barrier (BBB) genomics begins with the isolation of capillaries from fresh animal or human brain and is followed on the same day with the purification of capillary-derived RNA. The identification of microvascular-enriched genes from a whole brain gene microarray is unlikely because the brain capillary endothelial volume is <0.1% of total brain. Libraries of partial cDNAs corresponding to genes that are selectively expressed at the BBB are generated with polymerase chain reaction-based approaches such as subtractive suppressive hybridization. The availability of these partial cDNAs, in conjunction with production of animal or human BBB cDNA libraries, enables the cloning of the full-length cDNAs and a functional analysis of the BBB-enriched genes. The development of BBB genomics technologies enables the acquisition of a large body of new knowledge about the BBB and the brain microvasculature.
Key Words: biological transport • endothelium • microvasculature
The blood-brain barrier (BBB) is formed by epithelial-like high-resistance tight junctions that cement together the capillary endothelia comprising the capillaries of the brain and spinal cord.1 Owing to the presence of these tight junctions, there is no paracellular pathway for free solute exchange between blood and brain interstitial space. There is minimal pinocytosis in the brain capillary endothelium; therefore, there is minimal transcellular pathway for free solute exchange between blood and brain. Owing to the presence of the BBB, molecules in the circulation gain access to brain only via 1 of 2 pathways: (1) lipid-mediated transport of lipid-soluble small molecules with a molecular weight <400 Da and (2) catalyzed transport.2 Catalyzed transport is mediated via endogenous transport systems within the brain capillary endothelium. These BBB endogenous transport systems may be grouped into 1 of 3 families of transporters: (1) carrier-mediated transport for small-molecule nutrients, thyroid hormones, and vitamins; (2) receptor-mediated transport for endogenous large-molecule peptides such as insulin or transferrin; and (3) active eflux transporters such as p-glycoprotein and many other active efflux transport systems within the BBB.3 These endogenous transporters are conduits for drug delivery to the brain. As new technologies are developed in the future to enable the delivery of small- or large-molecule therapeutics to brain, these technologies will most likely access the brain via the endogenous BBB transport systems.4
The BBB is the interface between blood and brain and plays an important role in the pathogenesis of many brain diseases, which have a microvascular component. The brain capillary is composed of 4 cell types: the capillary endothelium, the capillary pericyte, the astrocyte foot process, and nerve endings terminating on the capillary surface. In addition, the smooth muscle cell is found in the precapillary arteriole and plays an important role in regulation of the brain microvasculature. Although the endothelial cell is the principal barrier cell controlling cerebral microvascular permeability, the pericyte, the astrocyte, and the neuron innervating the microvasculature all play important roles in the regulation of brain microvascular function. The pericyte shares the basal lamina with the capillary endothelium. Although pericytes in tissue culture express 
-actin and have contractile function, capillary pericytes in brain do not express -actin and have no contractile function under normal conditions.5 Pericytes may play a role in antigen presentation because the class II DR antigen is highly expressed in capillary pericytes in microvessels isolated from multiple sclerosis brain.6 Capillary pericytes may also play a principal role in the "enzymatic BBB" because capillary pericytes express a number of peptidases.7 Astrocytes extend foot processes that invest >90% of the surface of the brain microvasculature.8 The astrocyte foot process is separated from the luminal membrane of the capillary endothelium only by the basal lamina. Much attention is paid to the intercellular cross-talk at the synapse. An equally high level of intercellular cross-talk occurs at the perivascular space bounded by the capillary endothelium, the capillary pericyte, and the capillary astrocyte foot process.
BBB Genomics Versus Brain Genomics
The generation of new knowledge about the BBB and the brain microvasculature is critical because the BBB and brain microvasculature play an important role not only in brain drug delivery but also in brain pathology. Moreover, the chronic underdevelopment of BBB research within the neurosciences has caused the acquisition of new knowledge about the brain microvasculature to lag behind that of the neuron and glial cell. The shortest pathway to the generation of new knowledge about BBB function is the application of gene microarray technologies to the brain microvasculature, a field called BBB genomics.9 BBB genomics is separate from the field of brain genomics because most BBB-specific genes may not be detected in a whole-brain genomics program. This is because there is <1 µL/g brain of capillary endothelial cytoplasm. Therefore, there is only 1 µL of brain capillary endothelial volume in an entire rat brain and <5 mL of capillary endothelial volume in the 1200-g human brain. The volume of the capillary endothelium in brain is 10–3 parts of brain. The sensitivity of most gene microarrays is 10–4 parts.10 Therefore, only BBB-enriched genes that are expressed at very high levels at the BBB will be detected in a whole-brain gene microarray.
This chapter emphasizes that BBB genomics starts with the isolation of brain capillaries and brain capillary-derived RNA. Capillaries can be produced from animal or human brain with a mechanical homogenization procedure, which takes 
3 hours to isolate purified capillaries from total brain. Because the intent is to isolate capillary-derived RNA, the application of human BBB genomics starts with the isolation of fresh human brain from a neurosurgical preparation. Sufficient capillary-derived polyA+RNA can be isolated from as little as 5 to 7 g of brain tissue, and the capillaries can be purified and separated from the adjoining brain tissues.11 Panel A of the Figure shows a light micrograph of capillaries isolated from a 5- to 7-g specimen of human glioblastoma multiforme. The first human BBB genomics study was performed with capillaries isolated from cerebral dysplasia brain, which was removed for the treatment of intractable epilepsy.11
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Subtractive Suppressive Hybridization
After the isolation of RNA derived from freshly isolated human or animal brain capillaries, the decision must be made as to which genomics methodology will be used to identify tissue-specific gene expression at the BBB. Serial analysis of gene expression (SAGE) can be used.12 SAGE does not lead to the generation of any partial cDNAs corresponding to the BBB-enriched genes. An alternative methodology is subtractive suppressive hybridization (SSH), which allows for the isolation of partial cDNAs corresponding to the BBB-enriched genes.13 The production of a library of partial cDNAs corresponding to BBB-enriched genes is considered advantageous in that these cDNAs can then be used to screen human or animal BBB cDNA libraries to produce the full-length cDNA. The isolation of the full-length cDNA allows for functional studies of the target gene, which is desirable because so many novel genes of unknown function are isolated in a BBB genomics program. The production of a human BBB cDNA library has been described, and the human BBB LAT1 full-length cDNA was cloned from this library.14
In the SSH approach, the brain capillary-derived polyA+RNA is used to produce tester cDNA.13 In parallel, RNA from a control organ is used to synthesize driver cDNA, and the driver cDNA enables the subtraction of widely expressed genes from the BBB pool of genes. The efficiency of the SSH subtraction procedure is demonstrated in the Figure, panel B, which shows the abundance of the mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a common housekeeping gene, in both the subtracted and unsubtracted tester cDNA pool. The GAPDH transcript can be detected with as few as 18 cycles of polymerase chain reaction (PCR) in the unsubtracted tester cDNA. However, after subtraction of the human BBB tester cDNA pool with the SSH procedure with the use of driver cDNA obtained from pooled human liver and kidney RNA, there is no detectable GAPDH transcript in the subtracted BBB tester cDNA pool, even at 33 cycles of PCR (Figure, panel B). Thus, the SSH procedure subtracts commonly expressed genes and enriches the library in genes selectively expressed at the BBB. The brain capillary tester cDNA library is hybridized to the tester cDNA that has been subtracted with the liver/kidney driver cDNA. Double-stranded DNA is produced from tester or from driver polyA+RNA, which is digested with the restriction enzyme RsaI to obtain shorter blunt-end molecules; 2 tester populations are created with 2 different adapter molecules that are independently ligated to the tester cDNA. The 2 populations of adapter-ligated tester cDNA are then independently hybridized to the driver cDNA to enrich differentially expressed genes and hybridized a second time to generate a PCR template. A full-strand PCR amplifies differentially expressed sequences and is performed for 30 cycles. A second run of PCR is performed for 15 cycles with the use of nested PCR primers, and this second PCR run further enriches genes that are differentially expressed at the BBB and suppresses the background of commonly expressed genes. The SSH PCR products are cloned into a vector such as pCR2.1, and a cDNA library is produced in bacteria. Positive clones are identified by differential hybridization. Escherichia coli is transformed, and randomly selected bacterial colonies are cultured overnight in a 96-well plate and are subjected to a Southern blot dot hybridization with 32P-labeled subtracted and unsubtracted tester cDNA followed by film autoradiography, as shown in the Figure, panels C and D. The clones presenting a strong hybridization signal with the subtracted tester cDNA probe (Figure, panel C), relative to the signal generated with an unsubtracted tester cDNA probe (Figure, panel D), are selected for DNA sequencing and Northern analysis.15 Alternatively, real-time PCR may be used instead of Northern blotting to confirm enrichment of gene expression at the BBB. The 96-well gene microarray shown in the Figure, panels C and D, demonstrates that 40% of the clones in the subtracted tester cDNA are selectively enriched at the BBB compared with other tissues. The high enrichment of the BBB-specific genes with the SSH procedure makes possible the identification of large numbers of genes that are selectively expressed at the BBB.
BBB Genomics and Genes of Known Function
After DNA sequencing and analysis with the existing databases, the BBB-enriched genes may be classified as genes of known or unknown function. Previous work screened <5% of the subtracted BBB cDNA library for either the rat BBB15,16 or the human BBB.11 The genes of known function that have been identified in these initial studies are listed in the Table
. Approximately 15% of the BBB-enriched genes of known function that are identified with the SSH procedure are genes that encode for membrane transporters (Table). Carrier-mediated transport systems that have been identified include the CAT1 cationic amino acid transporter and the monocarboxylic transporter type 1 (MCT1). The receptor-mediated transport systems that have been identified include the transferrin receptor (TfR), and active eflux transporter systems that have been identified include BBB-specific anion transporter type 1 (BSAT1) and organic anion transporting polypeptide type 2 (oatp2). The Na/K-ATPase 2 and the FXYD5 proteins may function in concert with active eflux transporter systems at the BBB.
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Genes of known function that are found at the BBB include myelin-related genes, such as myelin basic protein (MBP), myelin protein zero-like 1 protein (MPZL1), and proteolipid protein-1, also known as lipophilin (PLP1) (Table
). The finding of myelin-related proteins such as MBP at the BBB was a surprising finding. The earliest neuropathological lesion in multiple sclerosis is the perivascular cuffing of lymphocytes.17 The chemoattractants causing homing of circulating lymphocytes to focal regions of brain in multiple sclerosis may originate at the brain microvasculature. It is not known why certain genes that are associated with myelin function are enriched at the brain microvasculature. In situ hybridization studies demonstrate that the microvascular cell expressing the MBP gene is the capillary endothelium.15
Amyloid-related genes are found at the brain microvasculature and include amyloid precursor-like protein type 2 (APLP2), sperm membrane protein related to A4 amyloid protein (YWK-II), integral membrane protein type 2a (ITM2a), and serine protease inhibitor 4 (SPI4), also known as protease nexin-1 (PN1) (Table). The finding of amyloid-related genes that are selectively expressed at the brain microvasculature corroborates early electron microscopic results showing that amyloid plaques in Alzheimer disease arise from the basal lamina surface of brain microvessels.18 A disease analogous to Alzheimer disease is familial Danish dementia, which is associated with progressive neurodegeneration, dementia, and amyloid angiopathy. The amyloid plaque of familial Danish dementia is formed by a 4-kDa peptide designated Adan.19 The Adan peptide is produced by point mutation in the BRI gene, which encodes a transmembrane type 2 protein, which is also called the ITM2b protein, which has a 39% amino acid identity with human ITM2a membrane protein, which is found at the human BBB.11
The gene encoding for tissue plasminogen activator (tPA) is selectively expressed in brain at the BBB (Table). tPA mediates neurite outgrowth and learning in the brain.20 Therefore, BBB-derived tPA may play a role in neuronal migration and synaptic connections. Certain neuropeptides that play trophic roles in development are also selectively expressed at the BBB. The mRNA encoding for insulinlike growth factor (IGF)-2 is highest in adult rat brain compared with other tissues in the rat.21 However, in situ hybridization, with the use of film autoradiography, shows that the IGF-2 transcript is detectable in brain only at the choroid plexus,22 and this led to the hypothesis that the choroid plexus is the site of origin of IGF-2 production in brain. However, IGF-2 produced and secreted at the choroid plexus cannot diffuse into the vast proper of brain parenchyma because factors secreted into cerebrospinal fluid are rapidly exported to the general circulation. In contrast, trophic factors secreted at the microvasculature of brain distribute to all cells in the brain because every neuron is virtually perfused by its own blood vessel. IGF-2 is selectively expressed in brain at the microvasculature (Table). Prior in situ hybridization studies examining the expression of the IGF-2 gene in brain could not detect the microvascular IGF-2 transcript22 because film autoradiography was used. Film autoradiography lacks the resolution to detect gene products that are selectively expressed in brain at the microvasculature. Multiple other neuronal trophic factors, other than IGF-2, are secreted directly to brain after synthesis at the brain microvasculature (Table).
BBB Genomics and Genes of Unknown Function
In a BBB genomics program, approximately half of the BBB-enriched genes that were discovered proved to be genes of unknown function.11,15,16 The advantage of the SSH procedure is that this methodology allows for the generation of partial cDNAs encoding the BBB-enriched gene. This cDNA can then be used to screen an animal or human BBB cDNA library to isolate the full-length cDNA encoding the BBB-enriched gene. The availability of the full-length cDNA allows for expression and determination of the function of the BBB-enriched gene. For example, in the early stages of the rat BBB genomics program, one gene product was initially identified only as clone K2.15 This clone was selected for functional identification because Northern blot analysis with RNA from C6 rat glioma cells, rat brain capillaries, total rat brain, rat heart, rat kidney, rat lung, and rat liver showed that the K2 gene product was only expressed at the BBB among these organs of the body. The clone K2 partial cDNA was sequenced, but this cDNA encoded for 3'-untranslated region (UTR) sequence, and there is generally a lack of conservation of 3'-UTR sequence across species. Therefore, a search for homology between a 3'-UTR sequence of the partial cDNA and databases may not provide insight into the function of the isolated novel gene. Cloning of the full-length cDNA corresponding to clone K2 led to the isolation of a 2.6-kb cDNA (Genbank AF 306546). Sequence analysis predicted that this cDNA encoded for a 12-transmembrane domain transporter protein. The open reading frame of the transport protein had a distant homology with organic anion transporters, such as oatp2, and clone K2 was named BBB-specific anion transporter type 1, or BSAT1.15 BSAT1 has since been renamed oatp14 because it is a member of the oatp gene family, which is the solute carrier number 21 family of membrane transporters. BSAT1 is an unusual gene product in that its expression in the body is confined to the BBB. Moreover, the abundance of the BSAT1 mRNA in brain-capillary endothelium is as high as any known transcript that is expressed at the BBB.
The application of genomics methodology to the BBB and the brain microvasculature can rapidly lead to the identification of hundreds of genes of known or unknown function that are selectively expressed in brain at the BBB. A BBB genomics program is the shortest pathway to the generation of new knowledge about the BBB and the brain microvasculature. It is unlikely that significant information on the BBB can be derived from a whole-brain genomics program because of the very small volume of the capillary endothelium in brain. It is important that BBB genomics programs start with the isolation of brain capillary-derived RNA. It is also important that the RNA be derived from capillaries freshly isolated from animal or human brain. Many BBB-specific genes are downregulated when brain capillary endothelial cells are grown in cell culture,23 and therefore cultured endothelium is not generally considered a useful source of BBB-specific gene products. Finally, the application of BBB genomics methodology to disease-specific human brain specimens can elucidate the changes in microvascular gene expression in the brain that are concomitant with the development of specific neurological diseases.
Acknowledgments
The author is indebted to Drs Ruben J. Boado, Eric V. Shusta, and Jian Yi Li for many valuable discussions.
Disclosures
None.
Received May 16, 2006; revision received August 8, 2006; accepted August 22, 2006.
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