Reduced macrophage and T cell infiltration in adipose tissue from CD36 KO mice fed a HFD compared to WT mice.

<p><b>A–C</b>. Representative images of F4/80 and CD3 stained epididymal fat sections from male WT and CD36 KO mice after 16 wks on a HFD. <b>A</b>, <b>B</b> F4/80-positive macrophages (brown). <b>C.</b> CD3-positive cells (red). <b>D</b>. The number of CLS was calculated as the percentage of adipocytes that are found in crown-like structures. For quantitative analysis, cells from 5 random fields, each containing more than 100 cells/field, were examined and counted. Values shown are mean ± SD (n = 3 mice), *, p<0.05. <b>E.</b> Adipose tissue gene expression. RNA was extracted and gene expression was determined by Q-PCR. Data was normalized to 36B4 expression. Values shown are mean ± SD (n = 5). *, p<0.05. Scale bar, 200 µm (<b>A</b>), 100 µm (<b>B</b>, <b>C</b>).</p

CD36 promotes adipocyte inflammatory cytokine and chemokine production in response to LPS.

<p>Adipocytes were differentiated from the SVF in culture wells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036785#s4" target="_blank">Materials and Methods</a>. <b>A</b>. Lipid accumulation in adipocytes was determined by Oil Red O staining. <b>B</b>. Differentiated (Oil Red O-positive) cells were calculated as a percentage of total cells. <b>C</b>, <b>D</b>. Cytokine levels in culture medium was determined following 4 h treatment of cells with LPS (10 ng/mL). Values shown are mean ± SD (n = 4) from a representative experiment, *, p<0.05; **, p<0.001. Scale bar, 100 µm.</p

CD36 KO mice are resistant to high fat diet-induced obesity compared to WT mice.

<p><b>A–C</b>. WT and CD36 KO male mice were maintained on a HFD for 16 wks. Body weight was measured weekly. After 16 wks on HFD, lean body mass and fat body mass were determined by MRI. Plasma was collected after 6 h of fasting. <b>D</b>. Glucose tolerance test (GTT). Mice were given a bolus of D-glucose (2 g/kg body weight) i.p. after 6 h of fasting. <b>E</b>, <b>F</b>. Fasting (6 h) insulin levels were determined by ELISA kits (BD Bioscience) and plasma lipid levels were determined by commercially available kits (WAKO). Values shown are mean ± SD (n = 7 except GTT, n = 5). *, p<0.05, CD36 KO vs. WT.</p

Macrophages and adipocytes display a synergistic and CD36- dependent cytokine response to LPS. A

<p>. Contact co-culture. Adipocytes were differentiated from the SVF of WT and CD36 KO mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036785#s4" target="_blank">Materials and Methods</a>. Peritoneal macrophages isolated from WT and CD36 KO mice were then layered and cultured on top of the differentiated adipocytes and co-cultured for 16 h. LPS (10 ng/mL) was then applied to the co-cultures for 4 h after which medium and cells were collected for cytokine determination. <b>B</b>, <b>C</b>. Non-contact co-culture in transwells. Primary pre-adipocytes were seeded in the bottom chamber and differentiated into mature adipocytes. Primary peritoneal macrophages were then seeded in the transwell inserts and cells were then co-cultured for 16 h. For LPS treatment groups, both adipocytes and macrophages were exposed to LPS (10 ng/mL) for 4 h after which cellular gene expression was measured by Q-PCR (B). Medium was collected and secreted cytokines were measured by ELISA (C). Values shown are mean ± SD (n = 4). *, p<0.05, **, p<0.001.</p

Cell death in CD36 KO adipose tissue is reduced compared to WT mice. A

<p>. Representative images of F4/80 and perilipin stained epididymal fat sections from male WT and CD36 KO mice after 16 wks on a HFD. Viable adipocytes are shown as perilipin-positive (red) and macrophages as F4/80-positive (brown). Cell nuclei were stained with DAPI (blue). Stars (*) indicate the cells devoid of perilipin expression. <b>B</b>. Quantification of dead adipocytes (perilipin-negative cells) expressed as a percentage of total adipocytes. For quantitative analysis, cells from 5 random fields, each containing more than 100 cells/field, were examined and counted. Values shown are mean ± SD (n = 3 mice), *, p<0.05. Scale bar, 100 µm.</p

CD36 induces pro-inflammatory and pro-apoptotic gene expression in adipose tissue.

<p>Epididymal fat from WT and CD36 KO mice after 16 wks on a HFD. RNA was isolated and Q-PCR was performed. <b>A</b>. Expression of genes associated with inflammation. <b>B</b>. Genes associated with cell death. Data was normalized to 36B4 expression. Values shown are mean ± SD (n = 5), *, p<0.05; **, p<0.001.</p

The Neuropilin-1 Inhibitor, ATWLPPR Peptide, Prevents Experimental Diabetes-Induced Retinal Injury by Preserving Vascular Integrity and Decreasing Oxidative Stress

<div><p>Neuropilin-1 (NRP-1) is a transmembrane glycoprotein. As a VEGF co-receptor, NRP1 significantly enhances VEGFR2 signaling and promotes vascular permeability and migration. The purpose of this study was to evaluate the effects of an NRP-1 inhibitor, ATWLPPR peptide, on the early stages of diabetic retinopathy. Eight-week-old male C57BL/6 mice were divided into three groups: a Normal group, a Diabetes (DB) ATWLPPR treatment group and a DB saline group. Electroretinography (ERG), fundus fluorescence angiography (FFA) and leukostasis were examined to evaluate the retinal injury induced by diabetes at the end of the fifth week after STZ injection. Occludin expression and extravasation of albumin were measured to determine the extent of vascular injury. The oxidative stress level and the levels of inflammation-associated proteins were also assayed. The results indicated that treatment with ATWLPPR prevents the abnormal condition of ERG (amplitudes of b-wave decreased and implicit time increased) and vascular injury (occludin degradation and increase in extravasated albumin). These effects were associated with a reduction in the oxidase stress level and the expression of VEGF, GFAP, and ICAM-1. We conclude that ATWLPPR, an NRP-1 inhibitor, may reduce the early retinal damage induced by diabetes by preserving vascular integrity and decreasing the oxidative stress level. Blockade of NRP-1 may be a new therapeutic strategy for the early stages of DR.</p></div

ATWLPPR inhibited the diabetes-induced up-regulation of inflammatory proteins in the retina.

<p>(A) Representative immunohistochemical images for ICAM-1 (a, b, c), GFAP (d, e, f), and VEGF (g, h, i) staining on retinal sections. Arrows, immune-positive. Scale Bar = 50 mm, 200X. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (B) Western blot analysis of the GFAP, VEGF and ICAM-1 expression levels in retinas. D, Diabetic. *<i>P</i> < 0.05, versus the Normal group; #<i>P</i> < 0.05, versus the D+saline group.</p

Diabetes-induced decrease in occludin was inhibited by treatment with ATWLPPR.

<p>*<i>P</i> < 0.05, versus the Normal group; #<i>P</i> < 0.05, versus the D+saline group. D, Diabetic.</p

ERG responses in the Normal, D+saline and D+ATWLPPR groups.

<p>(A) Representative original recordings from the three groups; (B) Quantitative analysis of b-wave amplitudes (mV); (C) Quantitative analysis of b-wave implicit time (ms). *<i>P</i> < 0.05 versus the Normal group; #<i>P</i> < 0.05 versus the D+saline group. D, Diabetic.</p