Additional file 1 of NaviSE: superenhancer navigator integrating epigenomics signal algebra

Supplementary Information. Manual for installation, use and running examples of NaviSE. (pdf 33792 kb

Amniotic Membrane Modifies the Genetic Program Induced by TGFß, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds

<div><p>Background</p><p>Post-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM) induced a robust epithelialisation in deep traumatic wounds.</p><p>Methods and Findings</p><p>To better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21) and CDK2B (p15). The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border.</p><p>Conclusions</p><p>The effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.</p></div

AM modified the genetic response induced by TGFß in human primary keratinocytes.

<p>Several TGFß inducible genes were measured in human primary keratinocytes in response to TGFß or in response to the combined treatment of TGFß and AM. Isolated RNA from primary keratinocytes stimulated for 24 h with AM and TGFß for the indicated times, or only with TGFß was analysed by qPCR, represented as a ratio to <i>GAPDH</i> and represented as a fold change of the untreated control sample. The asterisks denote statistically significant differences between the treatments according Student’s <i>t</i>-test. *p<0.05, **p<0.005 and ***p<0.001, ****p<0.0001.</p

AM modified the genetic response induced by TGFß in HaCaT cells.

<p>Several TGFß inducible genes were measured in HaCaT cells stimulated with TGFß or TGFß and AM. (A), isolated RNA from HaCaT stimulated with AM, TGFß, or both was analyzed by qPCR, represented as a ratio to <i>GAPDH</i> and represented as fold change of the untreated control sample. (B), isolated RNA from HaCaT stimulated for 24 h with AM and TGFß for the indicated times, or only with TGFß was analysed by qPCR, represented as a ratio to <i>GAPDH</i> and represented as fold change of the untreated control sample. The asterisks denote statistically significant differences between the treatments according Student’s <i>t</i>-test. *p<0.05, **p<0.005 and ***p<0.001, ****p<0.0001.</p

AM attenuated cell cycle proliferation arrest of TGFß on HaCaT cells and induced the expression of c-Jun protein in HaCaT and in human primary keratinocytes.

<p>Treatment of HaCaT cells with AM attenuates TGFß-induced cell cycle arrest in G1. (A), Cell cycle analysis of HaCaT cells in different conditions, treatment with AM, combined with serum starvation (SS) or TGFß is indicated. The histogram shows cells at G0/G1, S or G2/M stage respectively. AM induced the expression of c-Jun in HaCaT and human primary keratinocytes in clear synergy with TGFß. (B), HaCaT cells or, (D), human primary keratinocytes were stimulated with AM, TGFß or both simultaneously for the indicated times. Additionally, (C), HaCaT cells, or, (E), human primary keratinocytes, were stimulated for 24 h with AM and then treated with TGFß for the indicated times, as a control non treated cells were used. Indicated proteins were detected by Western blot. Grb2 or Zo-1 were used as loading controls where indicated. This experiment was performed at least three times. A representative result is shown.</p

In HaCaT cells, AM induced motility and the expression of c-Jun at the migratory front.

<p>Wound healing scratch assay was performed in HaCaT cells in the presence of AM, EGF or combinations of AM with different inhibitors. (A), cells forming a confluent epithelium were wounded and immediately treated as indicated for 21 h. Representative pictures were taken at the beginning of the treatment and 21 h later. (B), treatment of HaCaT cells with AM caused the cells to express c-Jun at the migratory front. Wound healing scratch assay was treated with AM, EGF or combinations of AM with different inhibitors. Cells were wounded and treated for 24 h, afterwards cells were fixed and immunostained for c-Jun. Images of c-Jun fluorescence were converted into pseudo-colour to show the intensity of c-Jun staining. Colour rainbow scale represents fluorescence intensity for c-Jun. Co-staining with phalloidin and Hoechst-33258 was used to show cells structure and nuclei, respectively. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope. This experiment was repeated at least three times. A representative result is shown. (C), HaCaT cells forming a confluent epithelium were treated with Mitomycin C, wounded and immediately treated for 21 h as indicated. Results were compared to non-treated cells. Representative pictures were taken at the beginning of the treatment and 21 h later. Scale Bars 100 μm.</p

Expression of c-Jun at the epidermal leading edge.

<p>Histopathological study of AM induced epithelialisation from a patient´s wound that had been treated with AM. (A), microscopic section from wound border before AM treatment. (B), and (C), microscopic section of the wound border 5 and 15 days after AM treatment, respectively. Insets in Fig A to C show roughly the area where confocal microscopy images have been taken. (D), to (F), microscopic sections were also immune-stained against c-Jun (green) and F-Actin (red). Cell nuclei were revealed by Hoechst-33258 staining. (D), same as in A; (E), same as in (B) and (F), same as in (C). Arrows in (E) and (F) point to the epidermal leading edge. Several patients were analysed in this experiment, single patient data is shown for illustrative purposes. Images were taken by confocal microscopy using a Zeiss 510 LSM confocal microscope.</p

MAP DNA in the blood of IBD patients who were receiving Infliximab® (n = 13).

<p>“Clearance medications” are agents that, in this study, were associated with the absence of MAP DNA in the blood of IBD patient's samples. These were: methotrexate, 6-MP, ciprofloxacin, and Tacrolimus®.</p

Shown is a <i>post hoc</i> analysis of the 62 IBD patients who were receiving “anti-metabolites”, agents recently shown to be potent antiMAP antibiotics.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002537#pone.0002537-Greenstein4" target="_blank">[20] </a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002537#pone.0002537-Shin1" target="_blank">[21]</a> The control group comprises all IBD patients who were NOT receiving the agent identified. The majority were receiving the precursor of 6-MP, azathioprine. No MAP DNA is found when 6-MP, methotrexate and Tacrolimus are used.</p

Shown is a <i>post hoc</i> analysis of the 16 IBD patients who were receiving conventional antibiotics, ciprofloxacin and metronidazole.

<p>There was no MAP DNA detected in the small number of patients taking ciprofloxacin.</p