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Published as doi: 10.1096/fj.05-4944fje.
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(The FASEB Journal. 2006;20:1703-1705.)
© 2006 FASEB

Microarray analysis of blood microvessels from PDGF-B and PDGF-Rß mutant mice identifies novel markers for brain pericytes

Cecilia Bondjers*, Liqun He{dagger}, Minoru Takemoto{dagger}, Jenny Norlin{dagger}, Noomi Asker*, Mats Hellström{dagger}, Per Lindahl* and Christer Betsholtz{dagger},1

{dagger} Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; and

* Department of Medical Biochemistry, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden

1Correspondence: Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77, Stockholm, Sweden. E-mail: christer.betsholtz{at}ki.se

ABSTRACT

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. Pericyte deficiency has been implicated in the pathogenesis of microvascular abnormalities associated with diabetes and tumors. However, the unambiguous identification of pericytes is still a problem because of cellular heterogeneity and few available molecular markers. Here we describe an approach to identify pericyte markers based on transcription profiling of pericyte-deficient brain microvessels isolated from platelet-derived growth factor (PDGF-B)–/– and PDGF beta receptor (PDGFRß)–/– mouse mutants. The approach was validated by the identification of known pericyte markers among the most down-regulated genes in PDGF-B–/– and PDGFRß–/– microvessels. Of candidates for novel pericyte markers, we selected ATP-sensitive potassium-channel Kir6.1 (also known as Kcnj8) and sulfonylurea receptor 2, (SUR2, also known as Abcc9), both part of the same channel complex, as well as delta homologue 1 (DLK1) for in situ hybridization, which demonstrated their specific expression in brain pericytes of mouse embryos. We also show that Kir6.1 is highly expressed in pericytes in brain but undetectable in pericytes in skin and heart. The three new brain pericyte markers are signaling molecules implicated in ion transport and intercellular signaling, potentially opening new windows on pericyte function in brain microvessels.—Bondjers, C., He, L., Takemoto, M., Norlin, J., Asker, N., Hellström, M., Lindahl, P., Betsholtz, C. Microarray analysis of blood microvessels from PDGF-B and PDGF-Rß mutant mice identifies novel markers for brain pericytes.


Key Words: vascular stability • Kir6.1 • SUR2 • DLK1 • RGS5

ANGIOGENESIS, THE COLLECTIVE term for the sprouting, splitting, growth, and remodeling of blood vessels, is a central process in vertebrate embryonic development as well as in many pathological conditions such as chronic inflammation, diabetes complications, and cancer (1 , 2) . Both the endothelial cells and the mural cells (vascular smooth muscle cells (VSMC)/pericytes), the two constituent cell types of the microvessel wall, are actively involved in the angiogenic process (3) . The precise role of the pericytes in the angiogenic process and in microvessel maturation and function is, however, largely unclear. In diabetic retinal microangiopathy, pericyte loss from microvessels is associated with the formation of microaneurysms, local edema, and microhemorrhaging (4 , 5) , suggesting that pericytes may regulate the stability and/or contractility of the microvessels. In mice lacking PDGF-B (platelet-derived growth factor-B) or the PDGF receptor beta (PDGFRß) (6 , 7) , pericyte recruitment to angiogenic vessels fails, leading to endothelial hyperplasia, impaired endothelial differentiation, increased vascular leakage, and formation of rupturing microaneurysms (8 9 10) . A role for pericytes in microvascular stabilization is also suggested from studies on angiogenesis in the retina and in tumors, in which recruitment of pericytes/VSMC positive for {alpha}-smooth muscle actin (SMA) appears to protect against vessel regression (11 , 12) . Recently, several additional studies have implicated a role for pericytes in stabilizing tumor vessels (13 14 15 16 17 18) , suggesting the possibility of pericyte targeting as part of antiangiogenic tumor treatment.

The molecular mechanisms by which pericytes mediate stabilization of the endothelial tube are unknown. Possibly, pericyte-derived extracellular matrix (ECM) and basement membrane components engage endothelial matrix receptors. Pericytes may also regulate endothelial differentiation and function through direct cell-cell contacts in specialized junctions, such as gap junctions and peg-socket contacts (19 , 20) . Further, pericytes may regulate endothelial growth, survival, or differentiation by secretion or activation of paracrine growth factors such as angiopoietin 1 (Ang1) (21 22 23) , transforming growth factor beta (TGF-ß) (24 25 26) , and vascular endothelial growth factor (VEGF)-A (27 28 29) .

Studies of pericyte origin and function in developmental or pathological angiogenesis are hampered by the shortage of markers for these cells. Markers commonly used to identify pericytes include SMA, desmin, and NG2 (30 31 32) , but none of these is specific for pericytes or labels all pericytes. For example, SMA is not expressed (at readily detectable levels) by normal brain pericytes. SMA and desmin are also not expressed by pericytes in most instances of mouse fetal angiogenesis (8) . NG2 is more broadly expressed in the pericyte population than SMA and desmin (33 ,34) , but is also expressed by additional cell types, such as oligodendrocytes. SMA, desmin, and NG2 are cytoskeletal and membrane components that so far have failed to provide clues to pericyte-specific signaling or function. PDGFRß is expressed by developing pericytes and pericyte precursors, and is down-regulated in the pericytes of mature quiescent vessels (8 ,18) . A similar developmental pattern of expression has been described for regulator of G-protein signaling (RGS)-5 (18 ,35 36 37) . PDGFRß and RGS5 are both involved in intracellular signaling; hence, the identification of events downstream of PDGFRß, or up- or downstream of RGS5 might provide additional information about the signaling pathways that regulate pericyte functions. Other markers that have been used to identify and quantify pericytes include the transgenic reporters desmin-lacZ (38) and XlacZ4 (39) .

The aim of the present study was to develop a method for pericyte marker identification taking advantage of transcription profiling techniques and pericyte-deficient mouse models. Using whole heads of PDGF-B-deficient mouse embryos and a limited array of mouse cDNAs, we previously identified RGS5 as a marker for certain pericyte populations (35) . Here, we refined our approach by profiling freshly isolated microvascular fragments from PDGF-B–/– and PDGFRß–/– mice using a microarray enriched for vascular-expressed genes. Our approach was validated by the identification of most known pericyte markers. Among the candidates for novel pericyte markers, we selected three for in situ hybridization analysis and confirmed their specific expression in brain pericytes of mouse embryos and their loss in PDGF-B–/– tissue. These novel markers are predicted to be involved in ion transport and intercellular signaling.

MATERIALS AND METHODS

Animals
Mice carrying a targeted disruption of the PDGF-B or PDGFRß genes were bred as hybrids of C57Bl6 and 129SV (6 ,7) . The morning of the vaginal plug was counted as embryonic day (E) 0.5.

Microarray experiments
We spotted a collection of sequenced cDNA clones isolated from mouse kidney glomerulus libraries together with a selected set of chosen cDNAs. The details of this cDNA collection will be presented elsewhere (40) . Briefly, the clone inserts were amplified by polymerase chain reaction (PCR), resuspended in 50% DMSO, and printed on gamma amino propyl silane-coated UltraGAPS slides (Corning Inc., London, UK) using a Microgrid II chip printer (BioRobotics, Cambridge, MA, USA). In total, the chips were printed with a set of 17280 mouse glomerulus cDNA clones representing >6000 different genes together with a uni-gene set of 1344 selected mouse expressed sequence tag (EST) clones (Research Genetics, Invitrogen, La Jolla, CA, USA) and 10 different Arabidopsis Thaliana PCR products (Stratagene, San Diego, CA, USA), altogether in triplicate, generating arrays of >55,000 cDNA spots.

Isolation of microvascular fragments
E17.5 mouse embryos were collected and placed in ice-cold PBS. Brains were dissected out, minced into 1 mm (3) pieces, and digested with 5 mg Collagenase A (Roche Diagnostics GmbH, Mannheim, Germany) dissolved in Hanks’ balanced salt solution (HBSS, Invitrogen AB, Lidingö, Sweden) including 1% BSA and 100U DNase at 37°C for 15 min with gentle agitation. The tissue was then gently pressed through a 100 µm cell strainer (Falcon, BD Biosciences, Stockholm, Sweden). Cells were washed out from the strainer in 2 ml of HBSS/1%BSA/100U DNase, pelleted at 200 g for 5 min, suspended in 1.5 ml HBSS/1% BSA/100U DNase, and again pelleted and resuspended. Rat anti-PECAM (BD PharMingen, San Diego, CA, USA) antibody (Ab) -coated magnetic beads (Dynabeads M-450, sheep anti-Rat IgG, Dynal A.S., Oslo, Norway) were added, and after incubation at 4°C for 30 min with gentle agitation, microvascular fragments were isolated with a magnetic particle concentrator (MPC, Dynal) and washed three times with HBSS/1% BSA. The purity of the isolated microvascular fragment was confirmed by lacZ staining of transgenic marker endothelial cells (41) or pericytes (39) .

RNA recovery and T7 RNA amplification
Total RNA was extracted from microvascular fragments isolated from PDGFB–/–, PDGFB±, PDGFRß–/–, and wild-type (WT) embryos using Sigma GenElute Mammalian Total RNA Kit (Sigma Aldrich Sweden AB, Stockholm, Sweden). RNA quantity was assessed with Ribo-Green RNA Quantitation Kit (Molecular Probes Europe BV, Leiden, The Netherlands) in a fluorometer (TD-360; Turner Designs Inc, Sunnyvale, CA, USA) according to the manufacturer’s instructions. T7 RNA (aRNA) amplification was performed in two cycles as described (42) . After the second round of amplification, aRNA was eluted from the column with 50 µl of water.

Target labeling, microarray hybridization, and scanning
Four µg of aRNA was primed with 5 µg of random hexamer (Promega, Madison, WI, USA) primer and labeled in a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP (Amersham AB, Uppsala, Sweden) according to standard protocols (http://cmgm.stanford.edu/pbrown). To allow for standardization of results, all hybridizations were done in competition with common reference samples. The common reference was made as a mixture of amplified RNA from 13 different sources including mouse brain, heart, thymus, lung, liver, spleen, aorta, kidney, skeletal muscle, testis, adult mouse glomeruli, postnatal day 5 glomeruli, and streptozotocin-induced diabetic mouse glomeruli. RNA samples were amplified separately, pooled, aliquoted in small tubes, and stored at –80 C. The differentially labeled targets were combined, mixed with 10 µg yeast tRNA, 10 µg poly(A)+ RNA, vacuum-dried, and resuspended in 120 µl of DIGeasy hybridization buffer (Roche Diagnostics GmbH, Mannheim, Germany). The hybridization mix was placed at 100°C for 2 min, 37°C for 20 min, and 50°C for 3 min before being added to the chip. Hybridization was performed at 42°C in a GeneTAC HybStation (Genomic Solutions, Ann Arbor, MI, USA) for 12–18 h. After hybridization, the slides were washed in 2 x SSC and 0.1% sodium dodecyl sulfate at room temperature for 5 min, 1 x SSC for 5 min, and finally 0.1 x SSC for 5 min. The slides were scanned using a GenePix 4000B microarray scanner (Axon Instruments, Westbury, NY, USA) at laser intensity and photomultiplier tube settings, giving the best dynamic range for each chip in respective channel. The hybridizations were technically repeated six times for PDGFRß–/– and control embryos, and six times for the PDGF-B–/– and control embryos

Data preparation
Image segmentation and spot quantification was performed using ImaGene software (Biodiscovery, Marina Del Rey, CA, USA). After median local background subtraction, the log-transformed ratios were normalized for signal intensity variation and processed using Bioconductor limma package (43) . Each spot was tested for differential expression at the 5% significance level. Multiple test correction was done using the false discovery rate method (44) .

Immunohistochemistry
Tissues were handled as described (45) , sectioned, and incubated with PBLEC (PBS pH 6.8, 1% Triton X-100, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.1 mM MnCl2). Biotin-conjugated BS1-B4 lectin from Bandeiraea simplicifolia (Sigma, L-2140) was used to label endothelial cells. Lectin binding was detected with streptavidin-conjugated horseradish peroxidase (DAKO, D0397) using standard protocols.

In situ hybridization
Nonradioactive in situ hybridization was performed on 14 µm thick sections with digoxygenin-labeled RNA probes (Boehringer Mannheim) visualized by alkaline phosphatase-conjugated antidigoxygenin antibodies, as described previously (9 ,46) . RGS5, Kir6.1, DLK1, and SUR2 Expressed Sequence Tags were obtained from Incyte Genomics (St. Louis, MO, USA), subcloned in a pBluescript KS+ vector, and used to generate sense and antisense digoxygenin-labeled probes according to standard protocols. PDGF-B and -Rß sense and antisense probes were generated as described (9) .

RESULTS

Isolation of microvascular fragments
Microvascular fragments were isolated from E17.5 mouse embryonic brain using a combination of mechanical and enzymatic tissue digestion and magnetic Dynabeads coated with anti-PECAM1 (CD31) antibodies. PECAM1 is expressed in brain vessels and capillaries during development (47 ,48) . We developed a protocol with most of the preparative steps at 0–4°C and only limited exposure at 37°C (see Materials and Methods) in order to minimize deviation from the original mRNA profile as a consequence of the preparation (49) . The purity and cellular content of the isolated brain microvessels were assessed in parallel preparations from Tie2-lacZ transgenic mice that express lacZ (cytoplasmic) in endothelial cells (50) and XlacZ4 mice that express lacZ (nuclear) in brain pericytes (39) (Fig. 1 ). After collagenase digestion, vessel fragments could be visualized within the crude cell suspension (Fig 1A ). Individual Tie2-lacZ-positive fragments showed attached Tie2-lacZ negative cells (Fig 2 B), most of which appear to be pericytes as judged by the XlacZ4 marker (Fig 1C ). The microvascular fragments obtained after magnetic isolation were estimated to be ~90% pure, as judged by the combination of Tie2-lacZ and XlacZ4 staining (Fig 1C and data not shown). Based on light microscopic appearance and yield, we assumed a similar purity of the fragments isolated from PDGF-B and PDGFRß mutants.


Figure 1
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  		Figure 1. Isolation of microvascular fragments. A) Homogenate of brain tissue after mechanical and enzymatic digestion. Tie2-lacZ-positive vascular fragments are visible (arrow) but constitute a minority of the cells and tissue fragments, which are mostly neuronal (a lacZ negative tissue fragment is indicated by the arrowhead. B) A Tie2-lacZ-positive vascular fragment at higher magnification. Note the presence of attached lacZ-negative cells. C) Staining of XlacZ4-positive pericytes (arrows) in a vascular fragment with attached Dynabeads.


Figure 2
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  		Figure 2. Gene expression in brain pericytes. In situ hybridization on sections from E17.5 wild-type embryos using probes for RGS5 (A, B) Kir6.1 (C, D), DLK (E, F) and SUR2 (G, H). The in situ mRNA signal (blue) is shown on sections from the brain in which the endothelial cells are labeled using BS1-B4 lectin (brown). Note that the cells positive for any of the four mRNAs are closely apposed to, but distinct from, the endothelium (arrows). Most of the endothelial abluminal surface is free from coverage by the mRNA-positive cells (arrowheads). Thus, the distribution and density of the cells positive for RGS5, Kir6.1, DLK, and SUR2 is compatible with expression in pericytes.

Identification of down-regulated genes in PDGF-B and PDGFRß null microvascular fragments
The most pronounced pericyte deficiency in PDGF-B and PDGFRß knockout embryos is seen in the central nervous system (CNS), where the pericyte population is reduced by >95% (8 , 49) . We prepared RNA from brain microvascular fragments isolated from E17.5 embryos. At this age most PDGF-B and PDGFRß null mutant embryos are still alive and have not yet developed extensive hemorrhaging or edema (6 , 7) . The yield of microvascular fragment RNA from younger embryos was considerably lower, demanding additional cycles of RNA amplification before microarray analysis, hence increasing the risk for deviation from the original mRNA expression profile.

Brain microvessels preparations from E17.5 PDGF-B–/– embryos and littermate ± controls, as well as from PDGFRß–/– embryos with littermate +/+ controls, were used to prepare labeled targets for microarray hybridization. PDGF-B–/– and PDGFRß–/– embryos display an indistinguishable phenotype (6 7 8) , including a similar degree of pericyte deficiency. We searched for changes in the microvessel transcript profile that were common for the two mutants.

Our analysis provided a list of 142 genes that were >2-fold down-regulated (P < 0.05) in both PDGF-B–/– and PDGFRß–/– brain microvessels compared to controls (Supplemental Table 1). Seven of the 142 genes encode known markers for pericytes and/or VSMC (boldface in Supplemental Table 1), including RGS5 (35 36 37 , 51 ) and NG2, (32) , myosin light chain kinase (52) , caldesmon (53) , parathyroid hormone receptor (54) , glutamyl aminopeptidase (55) , and connexin 45 (56) . PDGFRß was not identified by our analysis because it was not present on the chip.

Kir6.1-, SUR2B-, and DLK are expressed in CNS pericytes
Of the most down-regulated genes in knockout tissue, we selected ATP-sensitive potassium-channel Kir6.1 (also called Kcnj8) and sulfonylurea receptor 2, (SUR2B, also called Abcc9), which are both part of the same channel complex, together with the putative Notch receptor ligand delta homologue 1 (DLK1) for expression analysis by in situ hybridization in E14.5 mouse embryos. At this point the development of the vasculature is already quite advanced in most organs, and the pericyte distribution has been mapped using several different markers (8 , 9 , 35 , 39) . In the E17.5 mouse CNS, antisense RNA probes against Kir6.1, SUR2B, and DLK1 hybridized with a pattern reminiscent of the distribution of pericytes (Fig 2C-H ). In all cases, the positive cells were solitary and closely attached to the abluminal surface of BS1-B4 lectin-positive microvessels. No hybridization signal was obtained with any of the sense probes, which were used as negative controls. The vascular expression patterns of Kir6.1, SUR2, and DLK1 in the CNS were all similar to that of RGS5 (Fig. 2A, B ) and PDGFRß (data not shown).

Although the in situ localization of Kir6.1, SUR2, and DLK mRNA is compatible with predominant expression in pericytes, using currently available protocols, it is difficult to support this conclusion by double labeling with established pericyte markers. As an alternative confirmation of the pericyte expression of Kir6.1, SUR2, and DLK1, we therefore determined the expression pattern of these genes in E17.5 PDGF-B–/– embryos (Fig. 3 ). As expected for a pericyte expression pattern, the number of positive cells for each mRNA was dramatically reduced in the PDGF-B–/– CNS (Fig. 3D, F, H ) compared with the ± control (Fig. 3C, E, G ). A similar result was obtained using an RGS5 probe (Fig. 3A, B ) and a PDGFRß probe (data not shown and (9 , 35) . Not all Kir6.1, SUR2, or DLK1-positive cells were lost in the PDGF-B–/– brain tissue, as would have been expected if the expression of these genes were under the strict control of PDGF-B/Rß signaling. A small population of positive cells, with the location of pericytes, was observed in all cases (Fig 3B , arrow and data not shown). Residual sites of expression in PDGF-B–/– embryos were also observed outside of the CNS (data not shown), which in some cases were compatible with vascular mural cell populations known to persist in PDGF-B or PDGFRß-deficient mice (8) .


Figure 3
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  		Figure 3. Expression patterns in PDGF-B–/– embryos. In situ hybridization on sections from E17.5 PDGF-B ± and PDGF-B–/– embryos using probes for RGS5 (A, B) Kir6.1 (C, D), DLK (E, F), and SUR2 (G, H). The severe reduction in the density of pericytes in PDGF-B–/– brain correlates with the loss of RGS5 expression seen in PDGFB–/– mice, confirming the pericyte-specific expression of RGS5 in the brain (A, B). This loss of expression can also be seen with Kir6.1, DLK, and SUR2B (D, F, H), further implicating their pericyte-specific expression. The microvessels are still present (arrowheads) and occasional in situ positive pericytes are seen (B, arrow).

Pericytic expression of Kir6.1 is largely restricted to the CNS
Relatively broad expression patterns have been reported for SUR2 and DLK1 (57 , 58) . Although we found that brain pericytes constituted a dominant site of expression for these two genes in the mouse embryo, other sites of expression were evident too, such as cardiomyocytes expression of SUR2 and pulmonary epithelial expression of DLK1 (data not shown). The expression of Kir 6.1 was, however, highly restricted. Outside of the CNS, we found significant but rather weak expression in the VSMC surrounding the aorta (Fig. 4 E, F) and its larger branches (data not shown) and in kidney glomerular mesangial cells (data not shown), but not generally in pericytes. Meningeal vessels, which contains abundant pericytes positive for PDGFRß, NG2, XlacZ4 desmin, and SMA (8 , 9 , 49) , lacked Kir6.1-positive cells (Fig. 4A ). Also the skin and heart lacked Kir6.1-positive cells (Fig. 4A, C ), whereas both sites contain abundant PDGFRß-positive pericytes (Fig. 4B, D ). Thus, Kir6.1 appears to be a selective marker for brain pericytes.


Figure 4
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  		Figure 4. Comparison of Kir6.1 and PDGFRß expression. Kir6.1 mRNA expression (blue in panels A, C, E) was restricted to pericytes on brain microvessels (A, C), the VSMC of large arteries such as the aorta (E, F). Kir6.1 was not expressed in the meningeal vascular plexus (M) and neighboring skin (S) in the developing head (A). Kir6.1 was also not expressed in pericytes in the heart (C). The Kir6.1 in situ hybridization was combined with BS1-B4 staining (brown) to visualize the endothelium. PDGFRß mRNA expression (blue in panels B, D) was detected in pericytes in the brain, but also in the meningeal plexus (M), skin (S), and heart (D).

DISCUSSION

We have developed a method for transcription profiling of brain pericytes utilizing the pericyte-deficient state of PDGF-B and PDGFRß null mice and microarray analysis. In a limited scale experiment and using a crude tissue source, we have demonstrated that RGS5 is a marker for developing pericytes (35) . This provided a novel pericyte marker, but also an opening into research on pericyte G-protein signaling (36) . We have now refined our approach and report on the identification of additional pericyte markers implicated in signaling events.

Our current list of candidates for pericyte-specific mRNAs encompasses 142 gene transcripts. Seven of these are markers previously reported for vascular mural cells (Supplemental Table 1). The experimental setup used in this study thus represents a significant improvement compared to our previous study, which detected three known mural cell markers among 54 candidates (35) . This improvement likely has several reasons. First, in the present study the labeled targets were prepared from isolated brain microvascular fragments, as compared to whole heads in the previous study. We estimate that 10–20% of the cells in the vascular fragments are pericytes, whereas in the whole heads, the pericyte population represents <1% of the cells. Thus, the proportion of the labeled mRNA target that represents the pericytes has been increased by more than 10-fold. This likely improves the sensitivity of the hybridization to the pericyte-specific genes on the arrays. Second, the presently used microarray has broader vascular gene coverage. About 85% of the spotted 17280 glomerular ESTs, representing {approx}6000 different genes, are derived from microvascular cells (see Materials and Methods, and ref 40 ). Third, the use of two different knockouts mutants, which both display a primary pericyte deficiency, may exclude genes that are altered as a result of other putative changes in the mutants, e.g., PDGF-B-mediated effects via PDGFR{alpha} or PDGF-D-mediated effects via PDGFRß (59) . Likewise, possible neighborhood effects of the targeted mutations at the PDGF-B and PDGFRß loci, respectively (60) , would be excluded.

Our approach was validated by the identification of seven already known pericyte and VSMC markers at the top of the list of differentially expressed genes. In fact, the list contains the majority of the known pericyte markers (61) . SMA and desmin are noticeable exceptions, but both are weakly expressed by brain pericytes before birth (8) . The three genes Kir6.1, SUR2, and DLK1 were selected for validation by in situ hybridization because of the strong differential expression in wild-type and mutant vessels, and because they appeared particularly interesting from a signaling perspective. However, they are also potentially useful as markers particularly Kir6.1, which is expressed specifically in brain pericytes.

At present we can only speculate about the role of Kir6.1, SUR2, and DLK1 in pericyte development and function. Kir6.1 and a splice isoform of SUR2, SUR2B, together form a hetero-octameric ATP-sensitive potassium channel (KATP), where four Kir6.1 subunits constitute a pore-forming unit and four SUR2B subunits form a modulator unit of the channel (62) . These subunits belong to families where different members are expressed in different tissues, leading to specific KATP channels in each tissue (58) . In pancreatic ß-cells an ATP-sensitive KATP channel composed of Kir6.2 and SUR1 is closed in response to sulfonylurea hypoglycemic drugs, such as glibenclamide, leading to the stimulation of insulin secretion (63) . A channel with electrophysiological properties similar to the one composed of Kir6.1 and SUR2B has been found in VSMC, where it is supposed to play a role in the regulation of contraction (58) . Since the Kir6.1/SUR2B channel is sensitive to ADP rather than ATP, it might play a role in vasodilatation in response to hypoxia and ischemia (64 65 66) . The expression of Kir6.1 and SUR2 transcripts in brain pericytes might therefore suggest a link between metabolism and pericyte function. In diabetes mellitus, one of the first signs of microvascular damage in the retina, is loss of pericytes (67) . If high levels of glucose promote pericyte contraction, one might speculate that chronic hyperglycemia could bring pericytes into a state of exhaustion or fatigue.

DLK1 is a protein containing six epidermal growth factor (EGF) repeats in the extracellular domain, a single transmembrane domain, and a short intracellular tail. DLK1 has been shown to be involved in the differentiation and proliferation process of various cell types (68) . DLK1 has been described by several groups under different names such as fetal antigen 1 (FA1) (69) , preadipocyte factor-1 (Pref-1) (57) , zona glomerulosa-specific factor (ZOG) (70) , and adrenal-specific mRNA (71) . DLK1 is one of several ligands for Notch receptors, and as such it might have important functions in organ development (72) . The expression of DLK1 has been reported in various tissues, but not before in VSMC or pericytes, and its function in these cells remains to be established. Although DLK1-deficient mice show perinatal lethality, vascular defects have not been reported (57) .

In summary, we have combined techniques for fresh tissue isolation from genetic mouse mutant with microarray analysis in order to identify new markers for pericytes in vivo. The validity and power of our approach is illustrated by the fact that the top 6 and 8/14 top differentially expressed genes are validated mural cell markers (Supplemental Table 1). It is likely that further studies of the transcripts/proteins listed in Supplemental Table 1 will lead to the identification of additional mural cell markers, but it may also uncover genes that are regulated by PDGF-B/PDGFRß signaling, as well as endothelial genes regulated by the presence of pericytes. In two recent publications, the dynamics of the pericyte transcriptome was addressed in vitro (73 , 74) . One of the studies (73) identified transcriptional changes accompanying the differentiation of immature mesenchymal 10T1/2 cells into pericyte/VSMC-like cells. Genes that are up-regulated in conjunction with pericyte differentiation in vitro may include pericyte-specific markers. Of 113 up-regulated genes identified in differentiating 10T1/2 cells, only 5 were also identified as down-regulated in PDGF-B and PDGFRß knockout vessels; F2r, Vegfa, Adcy7, Spp1, and D4Wsu53e (see Supplemental Table 1, numbers 20, 24, 26 50, and 61 for full names and aliases). Of these, only Vegfa has previously been reported to be expressed in pericytes (29) . The divergence of these results, and the lack of identification of many of the known and widely used pericyte markers by transcript profiling of differentiating 10T1/2 cells, point to the importance of in vivo studies for the identification of relevant cellular markers.

ACKNOWLEDGMENTS

We thank Helen Hjelm and Monica Elmestam for technical assistance and Phil Soriano for providing us with PDGFRß mutant mice. This study was supported by grants from Novo Nordisk Foundation, Swedish Cancer Foundation, the Association for International Cancer Research (AICR), IngaBritt and Arne Lundberg, Knut and Alice Wallenberg, and Torsten and Ragnar Söderberg Foundations, as well as the European Community (FP6 Lymphangiogenomics), the Swedish Diabetes Foundation and Society for Medical Research, King Gustav V Jubilee Clinic Cancer Research Foundation, Assar Gabrielsson’s Foundation for Clinical Research, Fredrik and Ingrid Thurings Foundation, the Foundation for Lars Hiertas Memory, Svenska Försäkringsföreningen, and Barndiabetesfonden.

Received for publication December 7, 2005. Accepted for publication March 31, 2006.

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