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. 2010 Nov 25;468(7323):562-6.
doi: 10.1038/nature09513. Epub 2010 Oct 13.

Pericytes are required for blood-brain barrier integrity during embryogenesis

Richard Daneman  1 , Lu Zhou, Amanuel A Kebede, Ben A Barres
Affiliations

Pericytes are required for blood-brain barrier integrity during embryogenesis

Richard Daneman et al. Nature. .

Abstract

Vascular endothelial cells in the central nervous system (CNS) form a barrier that restricts the movement of molecules and ions between the blood and the brain. This blood-brain barrier (BBB) is crucial to ensure proper neuronal function and protect the CNS from injury and disease. Transplantation studies have demonstrated that the BBB is not intrinsic to the endothelial cells, but is induced by interactions with the neural cells. Owing to the close spatial relationship between astrocytes and endothelial cells, it has been hypothesized that astrocytes induce this critical barrier postnatally, but the timing of BBB formation has been controversial. Here we demonstrate that the barrier is formed during embryogenesis as endothelial cells invade the CNS and pericytes are recruited to the nascent vessels, over a week before astrocyte generation. Analysing mice with null and hypomorphic alleles of Pdgfrb, which have defects in pericyte generation, we demonstrate that pericytes are necessary for the formation of the BBB, and that absolute pericyte coverage determines relative vascular permeability. We demonstrate that pericytes regulate functional aspects of the BBB, including the formation of tight junctions and vesicle trafficking in CNS endothelial cells. Pericytes do not induce BBB-specific gene expression in CNS endothelial cells, but inhibit the expression of molecules that increase vascular permeability and CNS immune cell infiltration. These data indicate that pericyte-endothelial cell interactions are critical to regulate the BBB during development, and disruption of these interactions may lead to BBB dysfunction and neuroinflammation during CNS injury and disease.

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Figures

Figure 1
Figure 1. Time course of cell generation and BBB development in the rat cerebral cortex
a–g, Sections of rat cerebral cortex at indicated ages were stained for endothelial cells with Bandeiraea simplicifolia lectin I (BSL) (green, a–f) and nuclei with DAPI (blue, a, f (left), g), pericytes with anti-PDGFR-β (red, b; white arrows point to pericytes), oligodendrocyte progenitors with anti-PDGFR-α (red, c), astrocytes with anti-aquaporin 4 (red, d), anti-occludin (red, e; yellow arrows indicate tight-junction strands), anti-Glut1 (red, f (right)), and anti-Pgp (red, g). Scale bars represent 100 µm (a–d, f, g) and 20 µm (e). h–j, Rats aged E15 (left), E21 (middle) and adults (right) were given a trans-cardiac perfusion of biotin, and liver (h), muscle (i) and brain (j) tissue sections were stained with streptavidin (green) and DAPI (blue). Scale bar represents 100 µm.
Figure 2
Figure 2. Pericytes are required for BBB formation
a, b, E18 Pdgfrb−/− mice (b) and littermate controls (a) were given a trans-cardiac perfusion of biotin, and tissue sections were stained with streptavidin (green; white arrows indicate tracer in vessels). Scale bars represent 200 µm (upper panel) and 100 µm (lower panel). c, E18 Pdgfrb−/− mice and littermate controls were given a trans-cardiac perfusion of 3 kDa or 70 kDa biotinylated dextran, tissue sections stained with streptavidin-Alexa 488, fluorescence was quantified in ImageJ and permeability relative to control was graphed. *P < 0.05 by Student’s t-test. d–f, Neonatal mouse cerebral cortex from PdgfrbF7/− (f), PdgfrbF7/F7 (e) and littermate controls (d) were stained with BSL (green, d–f (bottom)) and for pericytes with anti-desmin (purple, d–f). Scale bar represents 100 µm. g, Pericyte coverage of CNS vessels in PdgfrbF7/−, PdgfrbF7/F7 and littermate control mice was quantified by analysing per cent length of BSL+ vessels opposed to desmin+ pericytes. h–j, P5 PdgfrbF7− mice (h), PdgfrbF7/F7 mice (i) and littermate controls (j) were given an intraperitoneal injection of Evan’s blue dye, and their brains were dissected the following day after PBS perfusion. k, Neonatal PdgfrbF7/−, PdgfrbF7/F7 and littermate controls were given a trans-cardiac perfusion of biotin and leakage was quantified in tissue sections with streptavidin-Alexa-488 (y axis) and graphed versus pericyte coverage (x axis; values from panel g). All error bars represent s.e.m.
Figure 3
Figure 3. Pericytes regulate structural aspects of the BBB
A, B, Electron microscopy images of CNS vessels from E18 Pdgfrb−/− mice (B) and littermate controls (A) including whole endothelial cell cross-sections (a), cytoplasm (b; yellow arrows indicate cytoplasmic vessels), tight junctions (c; yellow arrows indicate altered junction alignment; yellow arrowheads indicate junctions dipping into parenchyma), and after perfusion with biotin followed by staining with streptavidin–HRP (d, e; white arrows indicate uptake of tracer). Scale bars represent 2 µm (a), 0.5 µm (b, c) and 0.2 µm (d, e). C, Quantification of the number of vesicles per endothelial cross-section for Pdgfrb−/− mice and littermates. D, Angles of tight junctions (TJs) for Pdgfrb−/− mice and littermate controls were classified as parallel to the lumen (0°), perpendicular to the lumen (90°) or in between (45°). *P < 0.05 by Student’s t-test. E, F, Cerebral cortex of E18 Pdgfrb−/− mice (F) and littermate controls (E) were stained with BSL (green) and anti-occludin (red). Scale bars represent 20 µm. G, H, Purified murine brain endothelial cells were cultured alone (G) or with a feeding layer of purified brain pericytes (H) and stained with DAPI (blue) and anti-claudin 5 (left, red) or anti-occludin (right, red; yellow arrows indicate cell borders). Scale bars represent 100 µm (left) and 50 µm (right). I, Per cent length of sealed claudin 5 and occludin junctions in endothelial cells cultured alone or with pericyte feeder layers. **P < 0.01 by Student’s t-test. J, TEER measurements for purified murine brain endothelial cells cultured alone or with a feeding layer of purified brain pericytes. *P < 0.05 by Student’s t-test. All error bars represent s.e.m.
Figure 4
Figure 4. Vascular expression of LAMs in Pdgfrb−/− mice
a, b, Western blots of brain lysates from E18 Pdgfrb−/− (KO) and littermate controls, probing occludin, claudin 5, Icam1, Alcam, Lgals3, β-actin and PDGFR-β. a, Representative blots; b, quantification; *P < 0.05 by Student’s t-test. c–e, Cerebral cortex of E18 Pdgfrb−/− mice (d) and littermate controls (c) were stained with anti-Icam1 (purple) and BSL (green, bottom; white arrows indicate Icam1+ vessels), and per cent Icam1+ vascular length was quantified (e). Scale bar represents 250 µm. **P < 0.005 by Student t-test. f–h, Purified murine brain endothelial cells cultured alone (f) or with a feeding layer of purified brain pericytes (g) and stained for DAPI (blue) and anti-Icam1 (green), and proportion of Icam1+ cells was quantified (h). Scale bar represents 200 µm. **P < 0.005 by Student t-test. i–k, Five-week-old PdgfrbF7/F7 mice (j) and littermate controls (i) were stained with anti-Gr1 (red) and BSL (green, bottom), and number of Gr1+ cells per sagittal section was counted (k). Scale bar represents 250 µm. *P < 0.05 by Students t-test. All error bars represent s.e.m.
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