Crosstalk between FGF2 and HIF-1α.

<p>A. FGF2 and HIF-1α gene silencing using targeted-siRNA. 911 cells were transfected with FGF2 (left) or HIF-1α (right) targeted siRNA (siFGF2 or siHIF-1α, respectively). Protein contents were measured in normoxic (FGF2) and hypoxic (HIF-1α) cells 48 h after transfection. SiC corresponds to a control scrambled siRNA. B. Effect of FGF2 gene silencing on HIF-1α accumulation in 911 cells cultivated in normoxic (N) and hypoxic conditions. C. Effect of HIF-1α gene silencing on FGF2 accumulation in 911 cells cultivated in normoxic (N) and hypoxic conditions.</p

Endogenous FGF2 and HIF-1α expression in ischemic skin extracts.

<p>A. Representative Western blot analyses of FGF2 and HIF-1α at the indicated post-operative time. The molecular weights of FGF2 mouse isoforms are indicated. B. Levels of endogenous FGF2 mRNA determined by RT-qPCR analysis. Results are expressed relative to the level from control mice and represent mean±SE (n = 5 mice per post-operative time). *P<0.05 vs. control. C. Quantification of total FGF2 detected by Western blot analysis after normalization to GAPDH and to endogenous mRNA level. **P<0.001 vs. control.</p

Ischemia induced by a dorsal skin flap model in IRES FGF2-Luc transgenic mice.

<p>A. Representation of FGF2 mRNA and mouse protein isoforms (left panel) and of the bicistronic mRNA expressed by the RFL12 transgenic mice (right panel). This bicistronic cassette expresses, under control of the CMV (Cytomegalovirus) promoter, LucR and LucF reporter genes in a cap- or FGF2 IRES-dependent manner, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003078#pone.0003078-Creancier1" target="_blank">[18]</a>. B. Ischemia induction using a skin flap model modified from Ceradini <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003078#pone.0003078-Ceradini1" target="_blank">[26]</a> and representative Laser Doppler analysis performed 18, 48 and 72 hours after surgery. A U-shaped peninsular skin incision was created on the dorsal surface of 8-week old female RFL12 mice. The two vascular pedicles arising from the lateral thoracic arteries were sectioned. To avoid necrotic tissues, the study of gene expression was performed on the proximal part of the skin flap indicated by the white square. The color scale illustrates blood flow variations from maximal (red) to minimal perfusion (dark blue). C. Quantification of laser Doppler analysis. Ctr corresponds to non-operated mice. Results represent mean±SE on at least 3 mice per post-operative time.</p

High-Fat Diet Induces Periodontitis in Mice through Lipopolysaccharides (LPS) Receptor Signaling: Protective Action of Estrogens

<div><h3>Background</h3><p>A fat-enriched diet favors the development of gram negative bacteria in the intestine which is linked to the occurrence of type 2 diabetes (T2D). Interestingly, some pathogenic gram negative bacteria are commonly associated with the development of periodontitis which, like T2D, is characterized by a chronic low-grade inflammation. Moreover, estrogens have been shown to regulate glucose homeostasis <em>via</em> an LPS receptor dependent immune-modulation. In this study, we evaluated whether diet-induced metabolic disease would favor the development of periodontitis in mice. In addition, the regulatory role of estrogens in this process was assessed.</p> <h3>Methods</h3><p>Four-week-old C57BL6/J WT and CD14 (part of the TLR-4 machinery for LPS-recognition) knock-out female mice were ovariectomised and subcutaneously implanted with pellets releasing either placebo or 17β-estradiol (E2). Mice were then fed with either a normal chow or a high-fat diet for four weeks. The development of diabetes was monitored by an intraperitoneal glucose-tolerance test and plasma insulin concentration while periodontitis was assessed by identification of pathogens, quantification of periodontal soft tissue inflammation and alveolar bone loss.</p> <h3>Results</h3><p>The fat-enriched diet increased the prevalence of periodontal pathogenic microbiota like <em>Fusobacterium nucleatum</em> and <em>Prevotella intermedia</em>, gingival inflammation and alveolar bone loss. E2 treatment prevented this effect and CD14 knock-out mice resisted high-fat diet-induced periodontal defects.</p> <h3>Conclusions/Significance</h3><p>Our data show that mice fed with a diabetogenic diet developed defects and microflora of tooth supporting-tissues typically associated with periodontitis. Moreover, our results suggest a causal link between the activation of the LPS pathway on innate immunity by periodontal microbiota and HFD-induced periodontitis, a pathophysiological mechanism that could be targeted by estrogens.</p> </div

HFD-fed <i>CD14</i>KO mice are protected from periodontal disease compared with WT. A

<p>) The occurrence of periodontal pathogens was analyzed in 8-wk-old mice after 4 weeks of diet: WT OVX+HFD (n = 10), WT OVX+HFD+E2 (n = 10), CD14KO OVX+HFD (n = 12) and CD14KO OVX+HFD+E2 (n = 12). <b>B–G</b>) Hemi-mandible from each group, as reconstructed by the micro-CT. <b>F</b> CEJ (red line: cemento-enamel junction)-ABC (green line: alveolar bone crest) distance to represent alveolar bone loss (yellow line). *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA followed by Tukey test). Results are presented as means ± SEM.</p

HFD induced periodontal disease in WT mice: reversal effect of E2-treatment. A)

<p>The occurrence of periodontal pathogens was analysed in 8-wk-old mice after 4 weeks of diet : WT OVX+NCD (n = 27), WT OVX+NCD+E2 (n = 16), WT OVX+HFD (n = 15) and WT OVX+HFD+E2 (n = 15). <b>B–G</b>) Hemi-mandible from each group, as reconstructed by the micro-CT. <b>F</b>) CEJ (red line: cemento-enamel junction)-ABC (green line: alveolar bone crest) distance to represent alveolar bone loss (yellow line) (n = 8 for each group). *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA followed by Tukey test). Results are presented as means ± SEM.</p

E2 decreases the expression of gingival inflammatory mediators in HFD-fed ovariectomised (OVX) mice.

<p>mRNA synthesis of TNF-α (<b>A</b>), IL-1β (<b>B</b>), PAI-1(<b>C</b>) and IL-6 (<b>D</b>) in gingival tissue. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA followed by Tukey test). Results are presented as means ± SEM.</p

Positive bacterial cultures : intergroup comparisons among ovariectomised mice.

1<p>Fischer exact tests p-values, significant at p<0.05.</p><p>The table shows the qualitative analysis of periodontal microbiota in each group: WT OVX+NCD (n = 27), WT OVX+HFD (n = 25), WT OVX+HFD+E2 (n = 25) and <i>CD14</i>KO OVX+HFD (n = 12).</p>*<p>P<0.05,</p>**<p>P<0.01 (Fischer exact tests).</p

HFD-fed <i>CD14</i>KO mice did not exhibit metabolic disorders compared with WT. A)

<p>Body weight was assessed in 8-wk-old mice after 4 weeks of diet : WT OVX+HFD (n = 10), WT OVX+HFD+E2 (n = 10), CD14KO OVX+HFD (n = 12) and CD14KO OVX+HFD+E2 (n = 12). <b>B</b>) Time course of glycemia (mg/dl) during an IPGTT. The inset represents the glycemic index for each group. <b>C</b>) Fasted plasma insulin concentration (µg/l) after 4 weeks of diet : WT OVX+HFD (n = 6), WT OVX+HFD+E2 (n = 6), CD14KO OVX+HFD (n = 6) and CD14KO OVX+HFD+E2 (n = 6). *P<0.05, **P<0.01 (One-way ANOVA followed by Tukey’s post-test for <b>A</b> and <b>C</b>; and Two-Way ANOVA with Bonferroni’s post-test for <b>B</b>). Results are presented as means ± SEM.</p

<i>CD14</i>KO mice did not display HFD-induced periodontal inflammation.

<p>mRNA expression of TNF-α (<b>A</b>), IL-1β (<b>B</b>), PAI-1(<b>C</b>) and IL-6 (<b>D</b>) in gingival tissue. *P<0.05, **P<0.01,***P<0.001 (one-way ANOVA followed by Tukey test). Results are presented as means ± SEM.</p