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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-4944fjev1 20/10/1703    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bondjers, C. Articles by Betsholtz, C. Search for Related Content PubMed PubMed Citation Articles by Bondjers, C. Articles by Betsholtz, C.

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

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

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

Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, 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

SPECIFIC AIMS

Normal blood microvessels are lined by pericytes, which contribute to microvessel development and stability through mechanisms that are poorly understood. In this study, we aimed to identify genes that are differentially expressed in microvascular fragments obtained from normal and pericyte-deficient mouse mutants.

PRINCIPAL FINDINGS

1. Genes that are differentially expressed between microvascular fragments from wild-type and pericyte deficient platelet-derived growth factor (PDGF)-B/Rßbeta; mutant mouse embryos correspond to known pericyte markers
We used microarray analysis to compare the transcriptomes of brain microvascular fragments isolated from wild-type, PDGF-B (platelet-derived growth factor-B)–/–, and PDGF receptor-beta (PDGFRßbeta;)–/– E17.5 embryos. At this age, the brain pericyte population is known to be reduced by >95% in the two mutants. This analysis provided a list of 142 gene transcripts that were >2-fold down-regulated in both mutants compared to the littermate controls (Fig. 1 ). Among these we found eight known markers for pericytes and vascular smooth muscle cells (VSMC): RGS5, NG2, myosin light chain kinase, glutamyl aminopeptidase, parathyroid hormone receptor, caldesmon, and connexin 45 (for a complete list, see full-length article online).

View larger version (35K): [in this window] [in a new window]   Figure 1. Schematic diagram.

2. Kir 6.1, SUR2B, and DLK are expressed in central nervous system (CNS) pericytes
Of the most down-regulated genes in knockout tissue, we selected ATP-sensitive potassium-channel Kir 6.1 (also called Kcnj8), sulfonylurea receptor 2, (SUR2B, also called Abcc9) and delta-homologue 1 (DLK1) for further analysis. In the E14.5 mouse CNS, Kir 6.1, SUR2B, and DLK1 showed an expression pattern typical of developing brain pericytes (Fig. 2 ). In all cases, the positive cells were solitary and closely attached to the abluminal surface of isolectin B4-positive microvessels. The vascular expression patterns of Kir6.1, SUR2 and DLK1 in the CNS were all similar to that of RGS5 (Fig. 2 A,B) and PDGFRßbeta; (data not shown). In PDGF-B–/– CNS, the number of cells in situ positive for Kir6.1, SUR2, and DLK1 was severely reduced, giving further evidence for the pericyte-specific expression (Fig. 3 ).

View larger version (113K): [in this window] [in a new window]   Figure 2. Gene expression in brain pericytes. In situ hybridization on sections from E14.5 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 isolectin B4 (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.

View larger version (111K): [in this window] [in a new window]   Figure 3. Expression patterns in PDGF-B–/– embryos. 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).

3. Pericytic expression of Kir 6.1 is largely restricted to the CNS
Whereas SUR2 and DLK1 are expressed in both pericytes and other cell types outside of the CNS, Kir6.1 expression was largely restricted to pericytes within the CNS. In contrast to the pericyte marker PDGFRßbeta;, Kir6.1 was not expressed at detectable levels in pericytes residing in the meningeal vascular plexus, heart, or skin. Weak expression of Kir6.1 mRNA was noticed in VSMC surrounding large arteries, such as the aorta, and in kidney mesangial cells.

CONCLUSIONS AND SIGNIFICANCE

We have developed a method for transcription profiling of brain pericytes utilizing the pericyte-deficient state of PDGF-B–/– and PDGFRßbeta;–/– mice and microarray analysis. The validity and power of this method is illustrated by the fact that the top 6 and 8/14 top differentially expressed genes are validated mural cell markers.

A list of genes down-regulated in microvascular fragments from PDGF-B–/– and PDGFRßbeta;–/– contain the majority of the known pericyte markers. SMA and desmin are noticeable exceptions, but both are very weakly expressed by brain pericytes before birth.

Among the candidates for novel pericyte markers, we selected three for in situ hybridization analysis Kir6.1, SUR2, and DLK1 because of the strong differential expression in wild-type and mutant vessels, and because they appeared particularly interesting from a signaling perspective. Our analyses confirmed the specific expression of Kir6.1, SUR2, and DLK1 in embryonic brain pericytes, and the loss of their expression in PDGF-B–/– tissue.

Whereas the three novel pericyte markers are predicted to be involved in ion transport and intercellular signaling, we can only speculate about their role in pericyte development and function. Kir6.1 form together with a splice isoform of SUR2, SUR2B, a hetero-octameric ATP-sensitive potassium channel (K_ATP). A related channel in pancreatic ßbeta; cells, which is composed of Kir6.2 and SUR1, responds to sulfonylurea drugs, leading to stimulation of insulin secretion. 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. The expression of Kir6.1 and SUR2 transcripts in brain pericytes might therefore suggest a link between metabolism and pericyte function.

DLK1 is one of several ligands for Notch receptors, and as such it might have important functions in organ development. The expression of DLK1 has been reported in various tissues, but not previously in VSMC or pericytes. Although DLK1-deficient mice show perinatal lethality, specific vascular defects have not been described.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4944fje

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This Article Abstract Full Text Full Text (PDF) Supplemental Data All Versions of this Article: fj.05-4944fjev1 20/10/1703    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bondjers, C. Articles by Betsholtz, C. Search for Related Content PubMed PubMed Citation Articles by Bondjers, C. Articles by Betsholtz, C.