Defective endothelial-pericyte interactions in <i>Ccn2</i> mutants.

<p>(A, B) Co-immunofluorescence staining for desmin and PECAM in E18.5 dermis from (A) WT and (B) <i>Ccn2−/−</i> mice analyzed by confocal microscopy. (C) Quantification of vessel coverage by pericytes in E18.5 dermis; asterisk, p<0.05. (D, E) Co-immunofluorescence staining for NG2 and PECAM in E16.5 lung from (D) WT and (E) <i>Ccn2−/−</i> mice analyzed by confocal microscopy. (F) Quantification of vessel coverage by pericytes in E16.5 lung; asterisk, p<0.05. (G,H) Confocal analysis of NG2 and PECAM immunostaining in (G) WT and (H) <i>Ccn2−/−</i> E16.5 dermis. Pericytes are elongated around the microvessel in (G), whereas in mutants (H), pericytes (arrows) are associated with the endothelium, but are rounder, and fewer of them have elongated along the endothelial surface. (I–L) Confocal sections through E16.5 dermis analyzed for desmin (green) and PECAM (red) immunofluorescence. (I,J) WT desmin positive pericytes appear elongated and cover most of the surface of the microvessels. (K.L) <i>Ccn2−/−</i> desmin-positive pericytes have a rounder appearance and show less extensive coverage of the surface of the endothelium.</p

Endothelial basement membrane defects in <i>Ccn2</i> mutants.

<p>Electron microcopic images of endothelial basement membranes in dermal capillaries of E16.5 (A) WT and (B) <i>Ccn2−/−</i> littermates. Arrows demarcate the plasma membrane (bottom arrow) and top of the interstitial matrix (top arrow). (C,D) Confocal images of dermis of E16.5 WT (C) and <i>Ccn2−/−</i> (D) mice analyzed by immunofluorescence for fibronectin (FN) and PECAM. Arrows identify an arteriole. The arteriole in (C) is surrounded by several layers of FN. The arteriole in (D) is incompletely invested with FN. (E,F), Lower magnification confocal images through (E) WT and (F) <i>Ccn2−/−</i> E16.5 dermis, illustrating less fibronectin throughout the dermis in mutants. (G,H) Confocal images of dermis of E16.5 (G) WT and (H) <i>Ccn2−/−</i> mice analyzed by immunofluorescence for ColIV (Col4α2) and PECAM. Arrows identify an arteriole. ColIV coverage of the mutant vasculature is incomplete. (I,J) Confocal images of lungs of E16.5 (I) WT and (J) <i>Ccn2−/−</i> mice analyzed by immunofluorescence for ColIV and PECAM. Most of the vascular elements in the WT lung are surrounded by ColIV. Coverage is incomplete in the <i>Ccn2</i> mutant lung. Arrows in (J) identify vessels lacking coverage by ColIV. (K) CCN2 induces expression of FN and ColIV in HUVECS. HUVECs were infected with Ad-CCN2-GFP or Ad-control. Lysates were collected at the indicated time points post-infection. Levels of FN are elevated 8 hours after infection, concomitant with accumulation of CCN2. There appeared to be an increase in FN levels at 12 hours in the presence of CCN2 in the blot shown, but this was not seen in every experiment and the result did not reach statistical significance at this time point. Similarly, there was a trend towards increased expression of Col IV at 12 hr, but this increase did not reach statistical significance (p = 0.065). The experiment was repeated three times. A representative blot is shown. Quantification of levels of FN and Col IV are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030562#pone.0030562.s007" target="_blank">Figure S6</a>.</p

Expression of <i>Ccn2</i> in developing vasculature.

<p>(A) β-galactosidase activity in <i>Ccn2-lacZ</i> transgenic mice reveals <i>Ccn2</i> promoter activity throughout the vasculature in E16.5 embryos. (B) <i>Ccn2-lacZ</i> expression in dermal microvessels at E13.5. (C–E) EGFP fluorescence in CCN2-EGFP BAC transgenic mice demonstrates CCN2 expression in the endothelium of arterial elements (C and E), venous elements (C), and developing capillary networks (D). Arrowheads in (C) and (E) demarcate arterial element. Arrow in (C) identifies endothelial cells of a venous element. Arrowhead in (E) highlights EGFP expression in mural cells in the arterial element. Arrow in (E) highlights expression in endothelial cells in the arterial element. (F) Immunofluorescence and (G,H) immunohistochemical staining with an αCCN2 antibody on paraffin sections through dermis, demonstrating CCN2 expression in endothelial cells. Arrows in (F) highlight endothelial cells in E18.5 microvasculature. Specificity of the αCCN2 antibody is demonstrated by the absence of reactivity in the <i>Ccn2−/−</i> section (H). Arrows in (G) and (H) demarcate abluminal surface of the endothelium. Asterisks in (G) and (H) identify blood cells within the vessels. αCCN2 staining in (G) shows punctate intracellular expression, presumably with the Golgi, in addition to the surface expression marked by the arrow.</p

Vascular abnormalities in <i>Ccn2</i> mutant embryos.

<p>(A) E18.5 WT and (B) <i>Ccn2−/−</i> littermate, showing vessel dilation throughout the mutant embryo. (C, D) H&E-stained paraffin sections through the lumbar dorsal dermis of (C) E18.5 WT and (D) <i>Ccn2−/−</i> littermate. Arrowheads point to vessels. Bars highlight the enlarged distance between the hypodermal and epidermal layers in the mutant, indicative of local edema. (E,F) Hematoxylin and eosin-stained sections through E16.5 WT (E) and <i>Ccn2−/−</i> (F) descending aorta at thoracic level. Smooth muscle cells in the tunica media are spindle-shaped and arranged in layers in the WT embryo, but are more cuboidal and disorganized in the <i>Ccn2−/−</i> littermate. (G,H) Higher magnification images through aorta at lumbar level in E16.5 (G) WT and (H) <i>Ccn2−/−</i> littermate showing spindle-shaped smooth muscle cells (arrowheads) in WT that have a cuboidal shape in the mutant. (I,J) Confocal images of PECAM-stained dorsal dermal vasculature in (I) WT and (J) <i>Ccn2−/−</i> littermates. Arrows demarcate arterial elements; arrowheads demarcate venous elements; asterisks identify capillary beds. (K,L) Higher magnification confocal images of (K) WT and (L) <i>Ccn2−/−</i> dorsal dermal capillary beds, showing increased capillary density in the mutant. (M,N) High magnification confocal image of (M) WT and (N) <i>Ccn2−/−</i> dorsal dermal capillaries, showing numerous abluminal protrusions (arrows in (N)) on the mutant capillary. (O,P) Electron micrographs of newborn (P0) (O) WT and (P) <i>Ccn2−/−</i> dermal capillaries, showing abluminal and luminal (arrows in (P)) protrusions.</p

CCN2 potentiates PDGF-B signaling.

<p>(A) rCCN2 induces PDGF-B expression in HUVEC cells. Right panel, representative Western blot. Left panel, Quantification of relative expression levels of PDGF-B in cells treated with or without rCCN2 from three separate experiments. *, p<0.02. (B) Adenovirally expressed CCN2 induces PDGF-B expression in HUVECs compared to transfection with an empty adenoviral control. The extent of PDGF-B induction correlated with levels of CCN2 expression. As reported previously, a higher molecular weight isoform of CCN2, presumably a result of post-translational modification <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030562#pone.0030562-Brigstock1" target="_blank">[1]</a>, is detected 4 and 8 hours post-infection. Relative level of PDGF-B expression was assessed using ImageJ software. The experiment was repeated three times, with similar results each time. The induction of PDGF-B in the presence of CCN2 was statistically significant for each time point; p<0.05. A representative Western blot is shown. (C) Effects of rPDGF-B, and/or pcDNA3-CCN2-HA expression on activation of PDGF pathways in MOVAS cells. PDGF-B stimulated activation of Stat3, ERK, and Akt, whereas CCN2-HA on its own had no effect. However, combined treatment with PDGF-B and CCN2-HA led to prolonged Akt activation (arrows). Relative levels of pAKT expression were assessed using ImageJ software. All experiments were performed in triplicate and repeated three times, with similar results each time. The increase in pAKT levels in the presence of CCN2 was statistically significant at each time point; p<0.05. A representative Western blot is shown.</p

Massive accumulation phenotype in lungs of aged <i>Gpr116</i>^-/- mice.

<p>A. Bright field image of the inflated lung from <i>Gpr116</i> WT, heterozygous and knockout littermates. B. Weights of whole lungs over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). C. Bright field images of heart from <i>Gpr116</i> WT, heterozygous and knockout littermates. D. Weights of the heart (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). E. Bright field images of the spleen from <i>Gpr116</i> WT, heterozygous and knockout littermates. F. Weights of the spleen (left) over total body weight from <i>Gpr116</i> WT, heterozygous and knockout littermates (n≥5 mice per genotype). G. BALF collected from <i>Gpr116</i> WT, heterozygous and knockout littermates (The picture shown is representative of 3 mice for each genotype). H. Quantification of saturated phosphatydilcholine in BALF by ELISA (n = 3 mice per genotype). I. Quantification of protein content in BALF by BCA assay (n = 3 mice per genotype). J. Surfactant proteins detection in BALF by western blot. Molecular weights are indicated on the right. (n = 2 mice per genotype). K. Bright field images of the lung, after hematoxylin and eosin staining. The black arrowheads indicate alveolar macrophages (the image is representative of 4 mice for each genotype). L. Electron microscopy view of <i>Gpr116</i> wildtype and knockout lungs (n = 2 mice for each genotype). M. Confocal images of lung sections stained with ADRP (white) and nuclear stain (Hoechst, blue). Note that a red autofluorescent signal appears in knockout lungs. (the image shown is representative of 2 mice for each genotype). N. Confocal images of lung sections stained with nuclear marker Hoechst (blue) to show autofluorescent cells accumulated in the alveolar space, either in the green or red channel (the image is representative of 3 mice for each genotype). O. Autofluorescence emission spectrum of macrophages in the old knockout lung, upon 405 nm excitation (the image is representative of 2 mice). P. Detection of autofluorescent cells from <i>Gpr116</i> knockout lung by FACS (n = 2 mice per genotype)</p

Blood brain barrier breakdown in <i>Gpr116</i>^-/- mice.

<p>A. Whole brain images taken after 1kDa cadaverine perfusion (left) and associated quantification of extravasated cadaverine (right) in aged <i>Gpr116</i> WT, heterozygous and knockout mice (n≥5 mice for each genotype). B. Whole brain images taken 70 kDa tetramethylrhodamine dextran perfusion (left) and quantification of extravasated tracer (right) in <i>Gpr116</i> WT and heterozygous and <i>Gpr116</i> ECKO mice (n = 3 for wild type and ECKO, n = 2 for <i>PDGF-B</i>^{<i>ret/ret</i>}, n = 1 for uninjected control). C. Confocal images of cerebral cortex from aged <i>Gpr116</i> WT, heterozygous and knockout mice. Astrocytes (GFAP) appear in green, endothelial cells (CD31) in red (the images are representative of 4 mice per genotype) and associated quantification of perivascular associated astrocytes in aged <i>Gpr116</i> WT, heterozygous and knockout mice (n = 4 mice for each genotype, 2 sections at least quantified per genotype). D. Whole brain fluorescence images taken after Alexa 555-cadaverine circulation (upper) and quantification of extravasated cadaverine (lower) in 1.5-month-old <i>Gpr116</i> knockout (n = 3 mice per genotype). E. Whole brain fluorescent images taken after cadaverine circulation (upper) and associated quantification of extravasated cadaverine (lower) in 2-months-old <i>Gpr116</i> AEC KO (n = 6 mice per genotype). F. Whole brain fluorescent images taken after cadaverine circulation (upper) and quantification of extravasated cadaverine (lower) in 2-months-old <i>Gpr116</i> ECKO (n = 7 mice per genotype)</p

Retinal vascular patterning in <i>Gpr116</i>^-/- mice.

<p>A. Vascular network in P4 retinas. Dashed line indicates the limits of the retina (the picture shown is representative of at least 5 mice for each genotype). B. Quantification of the retinal vascular outgrowth at P4 (n = 5 for WT, n = 12 for heterozygotes and n = 6 for knockout). C. Vascular patterning in P7 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. Isolectin (red), CD31 (green) and Erg (grey) were used to visualize endothelium, and NG2 (green) and ASMA (red) to detect mural cells (the images shown are representative of 3 mice for each genotype). D. Vascular patterning in P7 retinas from <i>Gpr116</i> ECKO and littermates controls. Isolectin (red) is used to visualize endothelium, and NG2 (green) and smooth muscle actin α (ASMA, blue) to detect mural cells (the images show are representative of 2 mice per genotype). E. Isolectin (red) and FITC-dextran (green) distribution in P21 retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates. CD31 (green) is used to stain the endothelium, and nuclei are stained with Hoechst (blue) (the images shown are representative of 3 mice per genotype). F. Monolayers formed by isolated endothelial cells from <i>Gpr116</i> WT, heterozygous and knockout brain. Endothelial cells (CD31) and nuclei (Hoechst) are indicated in green and blue, respectively (the pictures shown are representative of 3 mice for each genotype)</p

Normalized pathological angiogenesis in <i>Gpr116</i>^-/- retinas.

<p>A. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (the images shown are representative of 5 mice per genotype). B. Confocal images of post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype). C. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P12 (n = 5 mice at least per genotype). D. Quantification of the avascular area on the post-OIR retinas from <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (n≥7 mice at least per genotype). E. Confocal images of post-OIR tufts (blue arrows) in <i>Gpr116</i> WT, heterozygous and knockout littermates at P17 (the images shown are representative of 5 mice per genotype)</p

Vascular expression and genetic ablation of the <i>Gpr116</i> gene in mouse.

<p>A. <i>Gpr116</i> mRNA expression in the published organ-specific EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref045" target="_blank">45</a>]. B. <i>Gpr116</i> mRNA expression assessed by qRT-PCR in EC from 3-weeks-old and 3-months-old ROSA^mT/mG x Tie2-Cre mice. Results are normalized by brain EC expression. Error bars represent SD. (n = 3 mice per genotype). C. <i>Gpr116</i> mRNA expression in the published brain-specific vascular and EC mRNA dataset [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137949#pone.0137949.ref046" target="_blank">46</a>]. D. Schematic representation of the area targeted by homologous recombination in the <i>Gpr116</i> locus. Dotted lines indicate the regions of homology in between the Gpr116 locus and the cassette. The dark grey arrow indicates the position of WT primers: both are located in the untranslated region of exon 21, but the area recognized by the forward primer is lost in the mutant allele. The light grey arrow represents the knockout primer, specific for the cassette. Critical Gpr116 domains (SEA, IgG, GAIN and transmembraine, TM) are indicated above the corresponding encoding exons. E. Example of genotyping PCR products on genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. WT primers amplify a 325-bp fragment in the 3´UTR exon 21 of <i>Gpr116</i> gene representing the wild type allele. The 401 bp band is specific for the mutant allele. F. Example of genotyping PCR products using genomic DNA (toe) from <i>Gpr116</i> WT, heterozygous and knockout littermates. LacZ primers amplify a 210 bp fragment in LacZ gene present in the insert replacing exon 4 to 21. G. <i>Gpr116</i> exon 17–18 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). H. <i>Gpr116</i> exon 2–3 mRNA expression assessed by qRT-PCR in <i>Gpr116</i> WT, heterozygous and knockout organs at P4 (n = 3 mice per genotype). I. mRNA detection by RNAscope in brain cortical capillary vessels from <i>Gpr116</i> WT (top row), knockout (middle row) and ROSA^mTmG X Tie2-Cre mice (lower row) at 3 weeks. On the left column, note that only the probe signal (red) and the nuclear staining (blue) are visible. On the right column, an endothelial staining (green) is merged to the probe and the nuclear signal: a CD31 antibody staining is on the two upper rows, while Tie-2 Cre mediated GFP is on the lower row. (n = 1 mouse per genotype).</p