Reduced PASMCs proliferation rate as a function of BMP-2 concentration when cultured in GM under hypoxia (A).

<p>PASMCs cultured in GM supplemented with 0–80 ng/ml BMP-2. BMP-2 at the concentrations of 40 and 60 ng/ml significantly inhibited PASMC proliferation as compared with GM without BMP-2. Typical pictures of PASMCs cultured in GM only (<b>B</b>), or GM supplemented with BMP-2 at 40 ng/ml (<b>C</b>). (*: vs 0 ng/ml only, p<0.05) <b>(</b>Magnification = 100×).</p

Growth profile of PASMCs cultured in GM supplemented with BMP-2, GW9662 (PPARγ antagonist) or bpV(HOpic) (PTEN inhibitor).

<p>PPARγ antagonist and PTEN inhibitor were added into respective cell culture medium 1 hour before adding BMP-2 (40 ng/ml). (The number of PASMCs after cultured in GM only was considered as 100%). (*: vs GM only) (Lipo = Lipofetamine −2000).</p

RT-PCR primers and annealing temperature.

<p>RT-PCR primers and annealing temperature.</p

Fringe Controls Naïve CD4⁺T Cells Differentiation through Modulating Notch Signaling in Asthmatic Rat Models

<div><p>The ability of Notch signaling to regulate T helper cell development and differentiation has been widely accepted. Fringe, <em>O-fucose-β1,3-N</em>-acetylglucosaminyltransferases modulate Notch receptor expression and promote the Notch signaling pathway through receptor-ligand binding. In this study, we assayed the expression levels of three Fringe homologs in naive CD4⁺T cells in asthmatic rats. We found that Radical Fringe (Rfng) was highly expressed, whereas both Lunatic Fringe (Lfng) and Manic Fringe (Mfng) were expressed at low levels. Down-regulation of Rfng using siRNA, and overexpression of Lfng or Mfng enhanced Th1 subset lineages and diminished Th2 subset lineages. Notch signaling was more activated in asthmatic naïve CD4⁺T cells than in control cells, and Lfng, but not Mfng or Rfng, partly inhibited Notch signaling in asthmatic naïve CD4⁺T lymphocytes. Lfng overexpression resulted in significantly decreased Th2 cytokine production in asthma, which was the same effect as the GSI (γ-secretase inhibitor) treatment alone, but had an increased effect on Th1 cytokines than GSI treatment. Collectively, these data identify the essential role of Fringe modulating naïve CD4⁺T cells differentiation through Notch signaling. Lfng regulated Th2 cells differentiation via a Notch-dependent manner and Th1 cells differentiation via a Notch-independent manner. Fringe could be a therapeutic strategy for the management and prevention of allergic asthma.</p> </div

Typical pictures of EGFP expression from pEGFP lipoplexes transfected PASMC when the ratio between Lipofectamine-2000 and plasmid DNA was at (A) 1∶1, (B) 2∶1, (C) 3∶1 and (D) 4∶1, when 2 µg plasmid DNA was used per 1×10⁵ cells.

<p>(Magnification = 40×).</p

Down-regulation of Rfng and overexpression of Lfng or Mfng decreased their ability to promote Th2 subsets but elevated the ability to promote Th1 subsets.

<p><b>A</b>, Real-time PCR analysis was performed to detect the IL-4, IL-5, IFN-γ, IL-12, T-bet, GATA-3 levels in SiRNA interference CD4⁺T cells. Blank-treated results were taken as 1. Results are from three independent experiments. The data for each group are expressed as means±SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, significant differences between SiRNA-Rfng group and SiRNA-scramble group (NC), mock control group or blank group. <b>B</b>, the IL-4, IL-5, IFN-γ, IL-12, T-bet, GATA-3 mRNA levels of Lfng plasmid group or Mfng plamid group were determined by real-time PCR analysis. *<i>p</i><0.05, **<i>p</i><0.01, significant differences between Lfng (Mfng) plasmid group and pEGFP-N1 group (NC) or blank control group. <b>C and D</b>, the IL-4, IL-5, IFN-γ, IL-12 concentrations in supernatants were determined by ELISA analysis. The data for each group are expressed as means±SEM. *<i>p</i><0.05.</p

CD4⁺T cells stimulating assay by flow cytometry.

<p><b>A and B</b>, Purified naïve CD4⁺T cells were cultured in wells with PBS, anti-CD3 mAb alone (5 µg/ml), anti-CD3 mAb (5 µg/ml), plus anti-CD28 mAb (2 µg/ml), and PHA-M (10 ng/ml) as indicated. After 3 days culturing, the expression of CD69 was assessed by flow cytometry. The percentages represented positive CD69 populations after CD4⁺T stimulation. The histogram of a representative experiment is presented in A (blank line, CD69 staining). CD4⁺T cells stimulated by anti-CD3 Ab plus anti-CD28 Ab elevated CD69 expression by 67.95%, compared with PBS (2.76%), anti-CD3 alone (26.78%) or PHA-M (31.57%), displaying the most efficient T cells proliferation. The summary of 3 independent experiments is presented in B. <b>C</b>, Cell division of CD4⁺T cell subpopulations were measured by CFSE dilution on day 3 by flow cytometric analyses of CD3/CD28 stimulating cells. (blue line, Isotype control; blank, CD69 staining).</p

OVA-sensitized asthmatic rat models were established successfully.

<p>Airway function was detected and BAL fluid and serum were collected and evaluated by ELISA. <b>A</b>, R_L and Cdyn values were obtained in response to increasing concentrations of inhaled MCh, as described in material and methods. Data represented mean±SEM (n = 20 in each group). *<i>p</i><0.05. <b>B</b>, Cellular composition of BAL fluid. <b>C</b>, Cytokine levels in BAL fluid and serum. <b>D</b>, Eotaxin levels in BAL fluid and serum. IgE levels in serum. Total, total cells; Epi, epithelium; Mac, macrophages; Lym, lymphocytes; Neu, neutrophils; Eos, eosinophils. Date represent the mean±SEM (n = 20 in each group). *<i>p</i><0.05, **<i>p</i><0.01 significant differences comparing asthmatic group and control group.</p

Addition of BMP-2 (40 ng/ml) in GM increased PTEN gene expression of PASMCs.

<p>BMP-2 significantly increased PTEN expression at 8 and 24 hours after treatment. (&: vs any other time point, p<0.05; *: vs 0, 1, and 4 hours, p<0.05).</p

HE staining of lung tissue in OVA-sensitized asthmatic group and control group.

<p><b>A</b>, control group; <b>B</b>, asthmatic group (Original magnification×400). Asthmatic group showed bronchial inflammation that exsisted in the forms of epithelial damage, wall thickening of the tunica mucosa bronchiorum, inflammatory cell infiltration into the lower layer of the mucous membrane and the surrounding bronchus, and accrementition and spasm of airway smooth muscle.</p