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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01080

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Endothelium-specific ablation of PDGFB leads to pericyte loss and glomerular, cardiac and placental abnormalities

¹ Department of Medical Biochemistry, Göteborg University, PO Box 440, SE 405 30 Göteborg, Sweden
² Department of Genetics and Pathology, Rudbeck Laboratory, SE 751 85 Uppsala, Sweden
³ Department of Experimental Pathology, Lund University, SE 221 85 Lund, Sweden
⁴ Max Planck Institute for Biochemistry, Department of Molecular Medicine, Am Klopferspitz 18a, D-82152 Martinsried, Germany

View larger version (34K): [in a new window]   Fig. 1. Endothelium-specific Pdgfb knockout strategy. (A) Outline of the conditional (flox) and recombined (lox) alleles. (B) Breeding scheme used to generate endothelium-specific Pdgfb knockouts and various littermate controls. Abbreviations for genotypes used in subsequent figures are shown in parentheses.

View larger version (67K): [in a new window]   Fig. 3. Glomerular abnormalities in endothelium-specific Pdgfb knockouts. Hematoxylin/Erythrosin (H/E) staining (A-F) and -smooth muscle actin immunostaining (G-J) of E18.5 kidney glomeruli of different genotypes. Normal glomeruli containing a mesangial cell core (outlined by arrowheads, G) were seen in wild-type (A) and control (C,E,G) kidneys, whereas the Pdgfb–/– (B) and some of the lox/– (D,J) glomeruli completely lacked mesangial cells. In these cases, the glomerular capillary tufts become replaced by single dilated capillary loops. Most lox/– glomeruli show a reduced mesangial core (outlined by arrowheads, H), leading to a reduction in tuft complexity and dilation of the remaining capillary loops (F,H). (K) Morphometric analysis of glomerular capillary dilation; the relative distribution of capillary diameters in glomeruli of the same genotype is shown. The capillary diameters of the lox/– animals differ significantly from those of all other groups (P<0.005). Note that the range of values, but not their distribution, is similar for lox/– and –/– (k/o). There is no difference between controls (sum of flox/+, flox/–, lox/+) and wild type (P>0.05). Measured diameters have been corrected to the closest 5 µm to yield an illustrative figure. Scale bar: 10 µm.

View larger version (108K): [in a new window]   Fig. 2. Tie1-Cre-mediated lacZ expression in R26R mice. By crossing Tie1-Cre and R26R reporter mice, the expression and efficiency of the Cre recombinase could be studied. These crossings also contained the Pdgfb conditional and null alleles. Sections are stained for ß-gal (blue) and are counterstained with Erythrosin (pink). (A) An overview section of the kidney cortex shows a lacZ expression pattern compatible with vascular expression. g, glomerulus; a, artery; cap, capillary. (B,C) Glomerular endothelial expression is evident at higher magnification. (D,E) Peritubular capillary endothelium is largely but not homogenously ß-gal-positive (arrowheads). (F-L) In the heart and its large vascular connections, the endocardium, the cardiac valves and endothelium are ß-gal positive, albeit not at 100% (arrow in J indicates an unstained endothelial cell). Deep in the myocardium, the capillaries are the only ß-gal-positive structures (H). The endothelium of the large vessels, here illustrated by the aortic arch (F), show a chimeric Cre expression (K,L; boxed regions in F at higher magnification). (M) The placenta vessels that are lined by a ß-gal-stained endothelium (green, arrowheads) could be classified as fetal by their content of large immature erythrocytes. (N) Diagrammatic representation of M, showing maternal sinuses (red) and fetal vessels (blue). Scale bars: 10 µm in A-E,H-L; 100 µm in F,G,M.

View larger version (51K): [in a new window]   Fig. 4. Glomerular abnormalities in adult endothelium-specific Pdgfb knockouts. H/E staining of 3-week-old kidneys revealed an increased glomerulus diameter (A,B) and capillary loop diameter (C) in lox/– mice compared with controls. Capillary diameters of the lox/– animals differ significantly from those of the control group (P<0.005). The relative distribution of the maximal capillary diameter in the control and endothelium-specific Pdgfb knockout groups is shown. Measured diameters have been corrected to the closest 1 µm to yield an illustrative figure. PAS staining of adult kidneys did not reveal signs of pathology at the age of 6 months (D,F) or >1 year (E,G). Glomerular cell count and matrix volume were comparable in the various genotypes, Scale bar: 10 µm). (H,I) Urine albumin levels were increased in lox/– mice that were more than a year old (P<0.05), but not in 6-month-old lox/– mice (P>0.05). Glomerular PDGFB protein levels (J,K) were reduced in lox/– mice and the reduction was significant (P<0.005) in mice that were more than a year old. Bars represent levels (±s.d.) of albumin secretion (H,I) and PDGFB protein (J,K).

View larger version (80K): [in a new window]   Fig. 5. Cardiac and placental abnormalities in endothelium-specific Pdgfb knockouts. H/E staining shows cardiac (A-D) and placental (E-H) abnormalities in E18.5 lox/– embryos similar to those seen in Pdgfb–/– embryos. Arrowheads (B,D) indicate the thin myocardium typical for –/– and lox/– animals. The placenta vessels in G and H that could be classified as either maternal or fetal by their erythrocyte content are outlined in I and J, respectively, to visualize their dilation in the lox/– animal (maternal sinuses are colored green, fetal vessels are colored purple). Scale bar in G: 10 µm.

View larger version (126K): [in a new window]   Fig. 6. Reduced PC recruitment to brain capillaries in endothelium-specific Pdgfb knockouts. Whole-mount ß-gal staining of PCs in the midbrain. (A-D) Ventricular view of entire midbrain hemispheres; (E-H) Distribution of PCs (nuclear staining) in the subventricular zone (SVZ) plexus of the hemisphere. Black arrowheads indicate extensively dilated vessels; red arrowheads indicate focal hemorrhages. Note the dramatic PC deficiency in Pdgfb–/– tissue (B,F,J) and the intermediate PC densities in lox/– tissue (C,D,G,H,K,L). (I-L) Sections (100 µm thick) of midbrain hemispheres, with the meningeal surface to the left and the ventricular surface to the right. The distribution of ß-gal-positive PCs along the radial vessels (rad) is seen. In the lox/– embryos (K,L) many radial branches completely lack ß-gal-positive PC coverage (green arrowheads), as in the –/– embryos (J), whereas other branches show normal coverage (white arrowheads).

View larger version (107K): [in a new window]   Fig. 7. Reduced microvessel density, increased microvessel diameter, microhemorrhage and focal astrogliosis in adult endothelium-specific Pdgfb knockouts. (A-D) Double staining for ß-gal-positive PC nuclei (blue) and PECAM1 (brown) in the striatum (A,B) and cerebellum (C,D) of 3-week-old control (flox/flox; A,C) and lox/– (B,D) mice. Note the reduced number of PCs associated with the microvessels in the lox/– brain and the numerous pathological microvascular profiles seen in areas of complete PC loss (B, arrows). Remaining PCs in lox/– striatum correlate with more normal profiles, as judged by their even diameter and straight outline (B, arrowheads). A dramatic reduction of microvascular density is seen in lox/– cerebellum (D). Microhemorrhage, visible by macroscopic inspection in vibratome sections of P12 mice (E), was analyzed further by confocal microscopy using isolectin B4 coupled to FITC (green) and anti-GFAP antibodies coupled to rhodamin (red). The regions around the microhemorrhages (G,H; H represents the boxed region in G at higher magnification) showed strong GFAP expression in reactive astrocytes (arrows), as well as isolectin B4 staining of microglia (arrowheads). Regions in the lox/– brain without signs of microhemorrhage lacked signs of reactive gliosis (F). pl, pial surface; wm, white matter. Scale bars in A,C: 100 µm.

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