Endothelial regeneration in VGs.

<p>Confocal microscopy images of endothelium in IVC and in VGs. Endothelium of IVC in a Tie2-GFP mouse (A). Photomontage of confocal images of a FVB VG grafted into a Tie2-GFP recipient at 14 days (B) and at 28 days (C). The GFP-labeled endothelial cells located at center of a FVB VG at 28 days (C). Migration of GFP-labeled endothelial cells from aorta is evident by sequent time point. Zoomed images show photos in higher magnification. Isolated GFP-positive cells in center of grafts are also visible. Scale bar = 100 µm. Dotted line = Anastomosis between aorta and VG, Triangle head = isolated endothelial cells in middle of VG, Arrow = direction of blood flow.</p

Leukocyte rolling and adhesion in native vessels and vascular grafts.

<p>Intravital microscopic data on leukocyte recruitment. Bar graphs represent (A) leukocyte rolling and adhesion in native vessels in WT and EP^−/−mice, (B) leukocyte adhesion in AGs and VGs in WT and EP^−/− mice and (C) leukocyte rolling in AGs and VGs in WT mice at different time points. Bar graphs represent number of leukocytes that were visible in a 100×100 µm square during 30 seconds. Error bars represent mean±SEM. *p<0.05.</p

IH in vascular grafts.

<p>Morphologic assessment of vascular grafts stained with Hematoxylin-eosin. (A) Upper panel demonstrates VGs in WT mice and in EP^−/− mice at different time point. Bar graphs represent IH area in AGs and VGs from WT and VGs from EP^−/− mice. (B) Lower panel demonstrates VGs in WT mice without or with treatment with combination of function-blockage antibodies against P- and E-selectin or rat IgG₁ λ isotype control in cuff-assisted vein grafting technique. Bar graph represents IH area. Error bars represent mean±SEM. *p<0.05. Scale bar = 100 µm. Arrows indicated IH of VGs.</p

Endothelial structure in vascular grafts.

<p>Scanning electron microscopy images of endothelium in AGs and VGs (magnification 1K to 1.9K, scale bar = 50 µm). Images demonstrate endothelium in native aorta, native IVC (A), VGs and AGs (B) at different time point in WT mice. Bar graph shows endothelial coverage area change in AGs and VGs (C). Arrow = red blood cells, Triangle arrow = leukocytes, ** = denudated endothelium. Error bars represent mean±SEM. *p<0.05.</p

Schip1 Is a Novel Podocyte Foot Process Protein that Mediates Actin Cytoskeleton Rearrangements and Forms a Complex with Nherf2 and Ezrin

<div><p>Background</p><p>Podocyte foot process effacement accompanied by actin cytoskeleton rearrangements is a cardinal feature of many progressive human proteinuric diseases.</p><p>Results</p><p>By microarray profiling of mouse glomerulus, SCHIP1 emerged as one of the most highly enriched transcripts. We detected Schip1 protein in the kidney glomerulus, specifically in podocytes foot processes. Functionally, Schip1 inactivation in zebrafish by morpholino knock-down results in foot process disorganization and podocyte loss leading to proteinuria. In cultured podocytes Schip1 localizes to cortical actin-rich regions of lamellipodia, where it forms a complex with Nherf2 and ezrin, proteins known to participate in actin remodeling stimulated by PDGFβ signaling. Mechanistically, overexpression of Schip1 in vitro causes accumulation of cortical F-actin with dissolution of transversal stress fibers and promotes cell migration in response to PDGF-BB stimulation. Upon actin disassembly by latrunculin A treatment, Schip1 remains associated with the residual F-actin-containing structures, suggesting a functional connection with actin cytoskeleton possibly via its interaction partners. A similar assay with cytochalasin D points to stabilization of cortical actin cytoskeleton in Schip1 overexpressing cells by attenuation of actin depolymerisation.</p><p>Conclusions</p><p>Schip1 is a novel glomerular protein predominantly expressed in podocytes, necessary for the zebrafish pronephros development and function. Schip1 associates with the cortical actin cytoskeleton network and modulates its dynamics in response to PDGF signaling <i>via</i> interaction with the Nherf2/ezrin complex. Its implication in proteinuric diseases remains to be further investigated.</p></div

Schip1 is expressed by glomerular podocytes.

<p><b>(A)</b> RT-PCR shows SCHIP1 expression in both glomerulus and the kidney fraction lacking glomeruli. In the glomerulus, expression is mostly detected in FACS-sorted podocytes. As an internal control, expression levels of GAPDH were measured. Controls for markers of various fractions are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122067#pone.0122067.s003" target="_blank">S3 Fig.</a> (Pod-podocytes, ROG-rest of glomerulus, GLOM-glomerulus, ROK-rest of kidney). <b>(B)</b> Northern blotting on a mouse multiple tissue panel shows the presence of two SCHIP1 mRNA transcripts enriched in the brain, heart, testes and kidney tissues. <b>(C)</b> Northern blotting on mouse glomerulus and ROK tissue confirms stronger SCHIP1 expression in the glomerulus, and presence of two transcripts. <b>(D)</b> By radioactive <i>in situ</i> hybridization on newborn mouse kidney sections SCHIP1 mRNA is localized to developing podocytes of the capillary loop stage glomerulus. <b>(E)</b> By Western blotting, the mouse 55kDa Schip1 protein is detected mostly in the glomerulus. Podocin was used as a positive control for the glomerular fraction, β-actin as a loading control. <b>(F)</b> Immunofluorescence on mouse and human kidney sections shows Schip1 glomerulus signal that partially overlaps with a podocyte foot process marker synaptopodin (Synpo). <b>(G)</b> By immunoelectron microscopy, Schip1 localizes to the glomerular podocyte foot processes (FP) in human kidney sections (GBM-glomerular basement membrane).</p

Additional evidence of Schip1 expression in podocytes.

<p>SCHIP1 is significantly upregulated in microarrays from human glomeruli vs. tubuli comparison <b>(A)</b>, in mouse podocytes vs. non-podocyte cells <b>(B)</b> and in mouse podocytes (visceral epithelial cells) vs. parietal epithelial cells <b>(C).</b></p

Schip1, ezrin and Nherf2 colocalize in the podocyte foot processes of the human kidney glomerulus.

<p><b>(A)</b> Immunofluorescence on human kidney sections shows partial colocalization of Schip/ezrin and Schip/Nherf2 in the glomerulus (arrowheads). <b>(B)</b> By IEM, Schip1 is localized to the podocyte foot processes (FP), often to the apical but also to the basolateral side. Similar localization is seen for ezrin and Nherf2 (arrowheads). <b>(C)</b> Double IEM for Schip1 (10 nm gold particle) and ezrin or Nherf2 (5 nm gold particle) indicates that the proteins colocalize at the same subcellular area in the foot processes (arrowheads, zoom).</p

Schip1 localizes to cell lamellipodia and associates with the cortical actin cytoskeleton.

<p><b>(A)</b> Both endogenous (arrowheads, upper panel) and ectopic (arrowheads, lower panel) Schip1 localize to lamellipodia in cultured human podocytes. <b>(B)</b> To test Schip1 association to actin-rich lamellipodia regions, transiently transfected human podocytes were treated with the standard procedure (fixation and Triton X-100 permeabilization, upper panel), or incubated with saponin prior to fixation and staining for MycSchip1 (lower panel). Peripheral Schip1 expression is partially retained after saponin treatment, indicating association of the protein with detergent-insoluble cytoskeletal/plasma membrane structures. The same was not observed in control cells transfected with Stx8 (syntaxin 8). <b>(C)</b> Schip1 colocalizes with cortical F-actin in the podocyte lamellipodia. Both the Z- and XY-scanning indicate considerable signal overlap between Schip1 and F-actin along the plasma membrane in cells presenting well-developed lamellipodia. <b>(D)</b> Treatment with latrunculin A results in the dissolution of actin fibers in both control and Schip1-transfected podocytes. However, Schip1 signal remains associated with disturbed F-actin positive residues. In contrast, treatment with cytochalasin D results mostly in preservation of the cortical actin in Schip1 transfected podocytes.</p

Schip1 overexpression promotes cortical F-actin accumulation, dissolution of stress fibers and motility in PDGF-BB-stimulated cells by attenuating actin depolymerisation.

<p><b>(A)</b> In control cells PDGF-BB treatment enhances development of lamellipodia. In Schip1-transfected cells PDGF-BB stimulation induces similar changes but also marked actin cytoskeleton rearrangement with cortical actin accumulation and dissolution of the actin stress fibers (box, zoom). Observe the neighboring non-Myc-Schip1 expressing cells, presenting with normal actin cytoskeleton and pronounced stress fibers. <b>(B)</b> Stable Schip1-expressing and control HEK293 cells were stimulated with 10% FCS or PDGF-BB, scratched, and left to migrate for 24 h (the wound healing assay). Schip1 transfected cells exhibit similar migration rate as controls in medium supplemented with 10% FCS, but migrate faster when induced with PDGF-BB (graph). Microscopic images of control and Schip1-expressing cell monolayers 18 and 24 h after wound scratching (left). <b>(C)</b> In vitro actin polymerization (right panel) and depolymerization (left panel) assays with lysates from GFP–Schip1-expressing HEK293 cells and controls show that Schip1-overexpression slows down actin depolymerization in presence of PDGFBB in comparison to cells treated with 10% FCS (p<0.0001). Results are representative of three separate experiments. RFU-relative fluorescence units.</p