Cytosolic calcium response to histamine (10^-5M) on day 1 of ITS medium.

<p>Mean ± SEM. N = 6 (Neonate) and 3 (Adult). Comparisons between populations using Mann & Whitney U-test.</p><p>Cytosolic calcium response to histamine (10^-5M) on day 1 of ITS medium.</p

Patients’ characteristics.

<p>*CLE: Congenital lobar emphysema, non-ventilated patient; FEF_25–75: forced expiratory flow between 25% and 75% of forced vital capacity; FEV1: forced expiratory volume in 1 second; PO₂: arterial partial pressure of oxygen; RDS: Respiratory Distress Syndrome; RV: residual volume; %: percentage of predicted values; sGaw: specific airway conductance; TLC: total lung capacity.</p><p>Patients’ characteristics.</p

Effect of mitochondrial metabolism (aerobic glycolysis) on ASMC proliferation.

<p><b>A. Cell count according to the presence of glucose or not (galactose) in the culture medium.</b> Results are means ± SEM. In galactose + 10% FCS medium, proliferation of neonatal ASMC (open circles, n = 6) is still present, unlike in adult ASMC (closed circles, n = 4). *<i>p < 0</i>.<i>05</i> neonate vs. adult at the corresponding time using MANOVA. <b>B. DNA synthesis according to the presence or absence of glucose in the culture medium.</b> Values are means ± SEM. After 24h in a glucose-free (galactose) medium, neonatal ASMC (white bars, n = 5), DNA synthesis is still present, in contrast with adult ASMC (black bars, n = 3). *<i>p</i> < 0.05 using Mann-Whitney U-test.</p

Proliferation of neonatal and adlult cultured human airway smooth muscle cells in the presence of pro-inflammatory stimuli.

<p>Results are means ± SEM. Values were normalized to proliferation in ITS medium. Cells cultured in 100 ng/ml IL-4 (black bars, adults; white bars, neonates)), IL-6, TNFalpha or 10^-4M Histamine, SLIGKV were assayed 24 following synchronization in ITS medium. There were no significant difference between the 2 age groups regarding all experimental conditions (N = 3 to 6 per group, Mann-Whitney U-test).</p

Porin expression according to age groups.

<p><b>A. Immunoblot for porin and actin in neonatal and adult ASMC. B. Expression of porin in human cultured ASMC.</b> A significant difference between the two cell populations after incubation in ITS medium for 1 day was found (neonates, white bars, n = 4 and adults, black bars, n = 3, *<i>p < 0</i>.<i>05</i> neonate vs. adult using Mann-Whitney U-test.</p

Proliferation of neonatal and adult cultured human airway smooth muscle cells in the presence of FCS and PDGF-AA.

<p><b>A. Cell counts according to time.</b> Results are means ± SEM. In glucose + 10% FCS medium, ASMC proliferation was greater in neonatal cells (open symbols, n = 5) vs. adult cells (closed symbols, n = 5). *<i>p</i> < 0.05 neonate vs. adult at the corresponding time using MANOVA. <b>B. DNA synthesis in adult and neonatal cultured human airway smooth muscle cells.</b> Results are means ± SEM. Values were normalized to proliferation in ITS medium. Cells cultured in 10% FCS (white bars, neonates, n = 5; black bars, adults, n = 7)), 15 ng/ml PDGF-AA (neonate, n = 4; adults, n = 10). *<i>p</i> < 0.05 using Mann-Whitney U-test.</p

Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

<div><p>Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation.</p><p>BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH₂ or trypsin for 1 to 3 days.</p><p>Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells.</p><p>In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation.</p></div

Increased PAR-2 expression in asthmatic bronchial smooth muscle cells.

<p>PAR-2 levels were assessed by flow cytometry (A), western blot (B) and quantitative RT-PCR (C). Normalized median fluorescence intensities were calculated by dividing median fluorescence intensity of PAR-2 by that of isotype control (A). Representative blots stained with anti–PAR-2 or anti–β-actin antibodies are shown (B). The RT-PCR expression of PAR-2 was presented as an arbitrary unit and normalized to endogenous references (geometric averaging of three internal control genes; <i>i.e.</i> YWHAZ, HPRT-1, and PO) according to GeNorm (C). Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 7) and control subjects (white bars, n = 7). Results are expressed as mean ± SD. *<i>P</i><0.05 using Mann & Whitney test.</p

Increased PAR-2 dependent calcium response in asthmatic bronchial smooth muscle cells.

<p>Representative intracellular calcium responses following stimulation by 10⁻⁴ M SLIGKV-NH₂ for 30 sec are presented in bronchial smooth muscle cells from asthmatic (black line) or control subjects (grey line) (A). Basal calcium concentration (Basal [Ca²⁺]_i, B), relative calcium response ([Ca²⁺]_i peak, C) and area under the curve (AUC [Ca²⁺]_i, D) were assessed from cell response to 10⁻⁴ M SLIGKV-NH₂. Bronchial smooth muscle cells were obtained from asthmatic (black bars, n = 3) and control subjects (white bars, n = 3). Results are expressed as mean ± SEM from a range of 14 to 41 cells per patient. *<i>P</i><0.05 using Mann & Whitney test.</p

Clinical and functional characteristics of subjects.

<p>Data are mean ± SD. BMI: body mass index. OCS: oral corticosteroid. ICS: inhaled corticosteroid. LABA: long acting beta2 agonist. FEV1: forced expiratory volume in one second. FVC: forced vital capacity. FEF 25–75: forced expiratory flow between 25 and 75% of FVC.</p