26 research outputs found
Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation
<div><p>Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF).</p><p>Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore, endogenous and exogenous semaphorin-7A may have opposite effects on the fibroblast phenotype.</p></div
Semaphorin-7A reduces serum-induced ERK and p38 phosphorylation in HLF.
<p><b>HLF derived from non-fibrotic lungs</b> were treated with control-siRNA (Ctrl.) or semaphorin7A-siRNA (sema7A) for 24 h before starvation in BSA for 24 h. A/ Then cells were activated with 1% or 10% FBS for 15 min. ERK phosphorylation (P-ERK) was analyzed by western-blot. The graph shows ratios of phospho-ERK versus total ERK1, and is an average of 3 experiments. * indicates that the increase of phospho-ERK after semaphorin-7A (Sema7A)-siRNA-treatment is statistically different from phospho-ERK after control-siRNA (Ctrl) treatment. B/ Cells were activated with 1% FBS for 30 min and a representative blot of 2 experiments shows phosphorylation of p38 (P-p38). C/ Cells were treated with the indicated siRNA and cultured for 48 h in 1% FBS along with a phospho-ERK inhibitor (U0126) or its inactive analog (U0124) during the last 24 h. Proliferation was determined by adding BrdU in the last 6 h of the culture. One representative experiment of 2 using 2 different HLF lines is shown. Average ±SEM of 4 wells is shown.</p
Semaphorin-7A diminishes HLF proliferation and migration.
<p>HLF derived from non-fibrotic lungs were treated with either control or semaphorin-7A siRNA and cultured in 1% or 10% FBS for 48 h. A/ BrdU was incubated with cells for 6 h. Absorbance was measured using a spectrophotometric plate reader at dual wavelengths of 450–550 nm. Each condition was performed in quadruplicate (4 wells) and graphs show a mean ± SEM of 3 experiments. B/ Cells were resuspended in 0.1% FBS and migration on plastic or plexin-C1 (10 μg/ml) toward medium with 10% FBS was measured after 20 h. Graph shows an average ± SEM of 4 experiments, and * indicates statistical difference between control-siRNA and sema7A-siRNA-treated HLF.</p
Semaphorin-7A inhibits the expression of myofibroblast markers.
<p>Non-fibrotic HLF were treated with control-siRNA (C), sema7A-siRNA (S) or plexin C1-siRNA (P) for 24 h before starvation in BSA for 24 h. Then, the cells were either kept in BSA or activated with TGF-ß (1 ng/ml) for 20 h. Real-time PCR was used to measure the level of expression of the indicated genes. For each gene, the first 2 graphs show the difference between C, S or P treatment, with C fixed at 1. The third graph displays the difference between TGF-ß and BSA after treatment with the control-siRNA. Graphs are an average of 3 experiments. <i>P</i> values from ANOVA analyses are shown. # indicates a statistical difference between TGF-ß and BSA.</p
Semaphorin-7A blocks α-SMA and periostin production in HLF.
<p>HLF derived from non-fibrotic lungs were treated with the indicated siRNAs for 24 h. HLF were subsequently starved in BSA for 24 h before either adding TGF-ß (1 ng/ml) or left in BSA for 20 h. The amounts of α-SMA and periostin in the cell lysates were determined by western-blot. A/ Graph shown is an average of 3 experiments, and * indicates that control (Ctrl) siRNA-treated HLF produce significantly less α-SMA than sema7A-siRNA-treated HLF. B/ Amount of intracellular periostin is representative of 2 experiments.</p
Semaphorin-7A expression is inversely correlated with periostin expression in HLF.
<p>Expression of semaphorin-7A (sema7A), periostin, fibronectin and collagen-1 (COL1A1) were measured by real-time PCR in fibroblasts from non-fibrotic (●, n = 7) and IPF lung (■, n = 6). Semaphorin-7A expression was inversely associated with periostin expression level (<i>r</i>p = -0.608, <i>p</i> = 0.027, n = 13).</p
Safety of and Cellular Response to Segmental Bronchoprovocation in Allergic Asthma
<div><h3>Rationale</h3><p>Despite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration.</p> <h3>Methods</h3><p>We performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject’s response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms.</p> <h3>Results</h3><p>Among subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×10<sup>4</sup> per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens.</p> <h3>Conclusions</h3><p>In mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.</p> </div
The Peripheral Blood Eosinophil Proteome
A system-wide
understanding of biological processes requires a
comprehensive knowledge of the proteins in the biological system.
The eosinophil is a type of granulocytic leukocyte specified early
in hematopoietic differentiation that participates in barrier defense,
innate immunity, and allergic disease. The proteome of the eosinophil
is largely unannotated with under 500 proteins identified. We now
report a map of the nonstimulated peripheral blood eosinophil proteome
assembled using two-dimensional liquid chromatography coupled with
high-resolution mass spectrometry. Our analysis yielded 100,892 unique
peptides mapping to 7,086 protein groups representing 6,813 genes
as well as 4,802 site-specific phosphorylation events. We account
for the contribution of platelets that routinely contaminate purified
eosinophils and report the variability in the eosinophil proteome
among five individuals and proteomic changes accompanying acute activation
of eosinophils by interleukin-5. Our deep coverage and quantitative
analyses fill an important gap in the existing maps of the human proteome
and will enable the strategic use of proteomics to study eosinophils
in human diseases
Comparison of oxygen saturation and heart rate on D0 and 48 hours after SBP-AG (D2).
<p>On D0, oxygen saturation and heart rate were monitored immediately before initiation of bronchoscopy, during BAL, during SBP-AG, immediately after the procedure, and at discharge. On D2, hemodynamic monitoring was done immediately before bronchoscopy, during BAL, immediately after the procedure, and at discharge. Bars represent median with 25 and 75<sup>th</sup> percentiles for subjects challenged with RW (white), HDM (gray), CAT (black). Whisker lines represent 10 and 90<sup>th</sup> percentiles. *p<0.05 compared to value for respective allergen group immediately before procedure. NS indicates there were no significant differences among allergen groups for the indicated time point.</p
The Peripheral Blood Eosinophil Proteome
A system-wide
understanding of biological processes requires a
comprehensive knowledge of the proteins in the biological system.
The eosinophil is a type of granulocytic leukocyte specified early
in hematopoietic differentiation that participates in barrier defense,
innate immunity, and allergic disease. The proteome of the eosinophil
is largely unannotated with under 500 proteins identified. We now
report a map of the nonstimulated peripheral blood eosinophil proteome
assembled using two-dimensional liquid chromatography coupled with
high-resolution mass spectrometry. Our analysis yielded 100,892 unique
peptides mapping to 7,086 protein groups representing 6,813 genes
as well as 4,802 site-specific phosphorylation events. We account
for the contribution of platelets that routinely contaminate purified
eosinophils and report the variability in the eosinophil proteome
among five individuals and proteomic changes accompanying acute activation
of eosinophils by interleukin-5. Our deep coverage and quantitative
analyses fill an important gap in the existing maps of the human proteome
and will enable the strategic use of proteomics to study eosinophils
in human diseases