Effect of <i>PDGFRB</i> mutations on PDGF-B signaling.

<p>(A) Western blot for known downstream targets of PDGF-B signaling. PAE cells were stimulated with 40 ng/ml PDGF-BB for the indicated time periods. Autophosphorylation of PDGF-Rβ mutants was investigated with a phosphospecific antibody raised against tyrosine 771, while downstream PDGF-BB signaling was assessed with antibodies directed against phosphorylated activated forms of ERK 1/2, Akt and PLCγ. β-actin was used as a loading control. (B). To quantify ERK 1/2, Akt and PLCγ activations over time, the signals were normalized over β-actin levels and plotted against time. Error bars indicate the standard deviation of three independent experiments. *<i>p</i>˂0,05 when compared to wild-type PDGFRβ (red), with the color of the star indicating the mutant PDGFRβ that is being compared to wild-type PDGFRβ (blue: L658P, yellow: R987W, brown: E1071V).</p

Expression and autophosphorylation of PDGF-Rβ mutants in PAE cells.

<p>PAE cells were stably transfected with vectors expressing the different PFBC mutations found in <i>PDGFRB</i>. (<u>A</u>) After mRNA extraction, the total expression of human <i>PDGFRB</i> was detected with qPCR. Error bars indicate standard deviation between 3 independent experiments. ND: Not detected. (<u>B</u>) Immunoblot demonstrating the protein expression levels of the different <i>PDGFRB</i> mutants. Stable clones of mutant <i>PDGFRB</i> expressing PAE cells were treated with a proteasomal inhibitor (MG-132), lysosomal inhibitor (chloroquine) or vehicle for 4 hours before lysis. Total levels of PDGF-Rβ were visualized with anti-total PDGF-Rβ (28E1) antibody. The graphs indicate relative expression level of the inhibitor-treated conditions <i>vs</i> control condition for three individual experiments. Error bars indicate standard deviation. *<i>p</i><0.05 compared to basal wild type (WT) expression, ^#<i>p</i><0.05 when comparing inhibitor-treated <i>vs</i> control condition for each mutant. (CD) Autophosphorylation of the PDGF-Rβ mutants. PAE cells stably expressing mutant PDGF-Rβ were exposed to 40 ng/ml of exogenous PDGF-BB for 60 minutes after cooling on ice. A wild-type <i>PDGFRB</i>-expressing construct was used as a positive control, while a kinase dead (KD) variant was used as a negative control. Cell lysates were adjusted to yield a comparable amount of PDGF-Rβ signal. (C) Representative western blot demonstrating autophosphorylation of the different mutants on four residues. (D) Quantification of PDGF-Rβ autophosphorylation signal from tyrosine residues 751, 771, 1009 and 1021 using phospho-specific antibodies. Signals were normalized over total levels of PDGF-Rβ protein, and expressed as a percentage of wild-type PDGF-Rβ autophosphorylation. The graph represents the averaged results from the 4 tyrosine residues that were assessed in three independent experiments. *<i>p</i><0,05 when compared to the positive control (WT PDGF-Rβ).</p

Conditioned medium from mutant <i>PDGFB</i>-transfected HEK cells fails to induce membrane ruffles in human brain pericytes.

<p>After overnight serum starvation, human brain pericytes (HBP) were cooled on ice and exposed to cooled conditioned medium from HEK cells (described in Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143407#pone.0143407.g001" target="_blank">1B and 1C</a>) for 15 minutes, then warmed up in at 37°C for 30 min and fixed for phalloidin staining. A low concentration of exogenous PDGF-BB (2 ng/ml) in serum-free pericyte medium (A) and conditioned medium from wild-type <i>PDGFB-</i>transfected HEKs (B) were used as a positive controls. Supernatant from pcDNA-transfected HEKs was used as a negative control (C). The first two conditions induced widespread circular ruffles (arrowheads), which were absent in the negative control. Likewise, HBP treated with conditioned medium from mutant <i>PDGFB</i> transfected HEKs did not show any ruffles: (D) *242Yext*89 mutation, (E) M1? mutation and (F) L9R mutation. Cyan: DAPI. Green: Alexa 488 conjugated phalloidin. Scale bar: 30 μm.</p

Overview of PFBC-related <i>PDGFB</i> and <i>PDGFRB</i> mutations.

<p>(A) In-scale schematic representation of a PDGF-BB dimer with the location of the known PFBC-associated mutations. Cysteine residues involved in interchain disulfide bonds are indicated in orange. The relative position of different mutations is indicated by stars. The predicted protein extension due to the stop codon mutation is indicated by dashed boxes. (B) Location of the main point mutations in <i>PDGFB</i> in the PDGF-BB:PDGF-Rβ complex. Ribbon diagram of two PDGF-Rβ proteins (in blue) in complex with dimeric PDGF-B (in grey). The location of the 3 stop mutations Q145*, Q147* and R149* are indicated in red. The location of the L119P missense mutation is indicated in green. The image was created from PDB 3MJG (Platelet-derived growth factor subunit B) using PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC. <a href="http://pymol.sourceforge.net/faq.html" target="_blank">http://pymol.sourceforge.net/faq.html</a>). (C) Schematic representation of a PDGFRβ dimer with the location of the known PFBC-associated mutations. Ig-like C2-type domains are indicated by oval shapes. The split tyrosine-kinase domain is indicated by dark grey boxes. Tyrosine autophosphorylation sites Y751, Y771, 71009 and Y1021 assessed in the article are indicated in purple. ATP-binding sites are indicated in black, including the K634 residue mutated in our kinase-dead negative control. The relative position of the mutations is indicated by stars.</p

Assessment of vessel pericyte coverage and blood-brain barrier integrity in aged <i>Pdgfrb^{redeye/redeye}</i> and <i>Pdgfb^+/-; Pdgfrb^+/-</i> mice.

<p>(ABC) Assessment of pericyte coverage. A CD13 and CD31 co-immunolabelling was performed on 50 μm-thick parasagittal vibratome sections. (A) Representative 2D projections of ~40 μm z-stacks taken from the dorsal pons of wild-type (left panels) and double heterozygous (right panels) animals; upper panels (red): CD13 immunostaining; lower panels: merged CD31 (green) and CD13 immunostainings. Inserts provide a more detailed indication of the coverage rate. Scale bar: 30 μm. The capillary surface coverage rate in <i>Pdgfb</i>^<i>+/-</i><i>; Pdgfrb</i>^<i>+/-</i> (B) and <i>Pdgfrb</i>^{<i>redeye/redeye</i>} mice (C) was calculated and is plotted as the percentage of the vessel surface enveloped by pericytes. Two pictures from the dorsal pons and two pictures from the cortex were analyzed for each animal. The standard deviation of pericyte coverage of 4 animals per genotype is indicated by the error bars. WT mice and heterozygous <i>Pdgfrb</i>^{<i>redeye/redeye</i>} mice were used as controls. (DE) Blood-brain barrier permeability assessment in <i>Pdgfb</i>^<i>+/-</i><i>; Pdgfrb</i>^<i>+/-</i> (D) and <i>Pdgfrb</i>^{redeye/redeye} (E) mice. Lysine-fixable cadaverine conjugated to Alexa Fluor-555 was injected intravenously into the tail vein (5 mg/ml in saline) 2 hours before sacrifice. Fluorescence was measured in the brain homogenate and arbitrary fluorescence units (AFU) were normalized to the brain weight. Error bars indicate standard deviation of fluorescence level measurement in 4 different animals. (F). Pericyte coverage of vessels in calcification prone regions compared with non-calcification-prone regions. Different brain regions were assessed for pericyte coverage in <i>Pdgfb</i>^ret/ret mice and the result is plotted as the percentage of the vessel surface enveloped by pericytes. For each animal, two pictures from the dorsal pons (calcification-prone) and two pictures from the cortex (non-calcification-prone) were analyzed. The standard deviation of pericyte coverage of 4 animals per genotype is indicated by the error bars. *<i>p</i><0,05 when comparing <i>Pdgfb</i>^ret/ret with <i>Pdgfb</i>^ret/+, #<i>p</i><0,05 when comparing calcification-prone regions with non-calcification-prone regions in <i>Pdgfb</i>^ret/ret mice. (G). Analysis of blood-brain barrier integrity in calcification prone regions compared to non-calcification-prone regions. Alexa Fluor-555 conjugated cadaverine tracer was allowed to circulate for 2 hours prior to sacrifice of the mice. The calcification prone regions of the brain were microdissected, and after homogenizing of the tissue, fluorescence was measured and normalized over the tissue weight (AFU). The standard deviation of 4 animals per genotype is indicated by the error bars. *<i>p</i><0,05 when comparing <i>Pdgfb</i>^ret/ret with <i>Pdgfb</i>^ret/+, #<i>p</i><0,05 when comparing calcification-prone regions with non-calcification-prone regions in <i>Pdgfb</i>^ret/ret mice.</p

Effect of <i>PDGFRB</i> mutations on membrane ruffle formation and wound healing.

<p>(AB) PDGF-BB—induced membrane ruffling of stably transfected PAE cells expressing mutant PDGF-Rβ receptor. (A) Fluorescent labeling of the actin cytoskeleton in PAE cells after 30 minutes of PDGF-BB exposure. Arrowheads indicate peripheral membrane ruffles. Cyan: DAPI, green: Alexa 488-conjugated phalloidin. Error bar: 30 μm. (B) The total amount of ruffles was counted in 20 fields per condition, and normalized over the total amount of cells. *<i>p</i>˂0.05 as compared to wild-type <i>PDGFRB</i>-expressing PAE cells. Empty vector (pcDNA) and KD <i>PDGFRB</i>-transfected cells were used as negative controls. (CD) Wound healing assays of stably transfected PAE cells expressing different mutant PDGFRβ receptors. Confluent monolayers of PAE cells expressing different <i>PDGFRB</i> constructs were scratched using the WoundMaker™ and wound closure was monitored automatically every hour for 13 hours with the IncuCyte Zoom®. (C) Representative images of the wound at 0 and 13 hours of PDGF-BB stimulation. (D) Quantification of the increase in relative wound density within 13 hours. Error bars indicate standard deviation between 6 individual scratches, 2 images per scratch.</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

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

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