BMP-7 inhibits SMADs phosphorilation in mouse lung fibroblasts from chronic asthmatic mice.

<p>Mouse lung fibroblasts were treated with TGF-β1 (5 ng/ml) alone or in combination with BMP-7 (300 ng/ml). After 15 min, samples were analyses for SMAD 2 and 3 or p-p38 expression. Proteins were analyzed by western blot and normalized by their respective total proteins. Data are representative of 5 animals in each of 3 independent experiments. *p<0.05 comparing with the control group; Δ p<0.05 comparing TGF-β1-treated with TGF-β1/BMP-7-treated groups.</p

TGF-β and BMP-7 mRNA expression in lung tissue.

<p>BMP-7 (A) and TGF-β (B) mRNA in whole lung preparations. The TGF-β/BMP-7 ratio was calculated (C). HPRT was used as housekeeping gene. Data are representative of 5 animals in each of 3 independent experiments. *p<0.05 comparing with control group; Δ p<0.05 comparing acute with chronic groups. In the insert, collagen-I (area/um²).</p

Fibrosis markers in the asthmatic lung.

<p>Lung sections were taken from animals with asthma (6 challenges), stained with PAS for mucus (A) or prepared for immunohistochemical and morphometric analysis of α-SMA (B) and collagen type I (C). Data are representative of 5 animals in each of 3 independent experiments. Bars  = 50 mm. *p<0.05 comparing with control group.</p

Intranasal treatment with BMP-7 reduces cellular infiltrate in the airways of chronic asthmatic lungs.

<p>Total cells in BAL were counted in a hemocytometer. After cytocentrifugation cells were stained with H.E. for determination of eosinophil and neutrophil numbers. Data are representative of 5 animals in each of 3 independent experiments. *p<0.05 comparing with the non asthmatic control; Δ p<0.05 comparing asthmatic mice treated or not with BMP-7.</p

Intranasal treatment with BMP-7 reduces collagen-I expression in chronic asthmatic lungs.

<p>Lung slides were stained with Masson's Trichrome (blue) and the morphometric analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095959#s2" target="_blank">methods</a>. Control (A), Chronic asthmatic (B), Chronic asthmatic treated with BMP-7 (300 µg/kg) by intranasal route (C). Morphometric analysis of the stained area (blue)(D). Data are representative of 5 animals in each of 3 independent experiments. Bars  = 50 µm. *p<0.05 comparing with the non asthmatic control; Δ p<0.05 comparing asthmatic mice treated or not with BMP-7.</p

BMP-7 inhibits TGF-β1-induced synthesis of collagen-I by lung fibroblasts.

<p>Mouse lung fibroblasts from control animals (A) were stimulated with TGF-β1 (5 ng/ml) alone or in combination with BMP-7 (30, 100 ou 300 ng/ml) during 18 hours. Mouse lung fibroblasts from asthmatic animals (B) were treated with TGF-β1 (5 ng/ml) alone or in combination with BMP-7 (300 ng/ml). Type I collagen was analyzed by western blot and normalized by β-actin expression. Results are presented as percentage of the control or as arbitrary units. Data are representative of 5 animals in each of 3 independent experiments. *p<0.05 comparing with the control group.</p

Tail-cuff pressures (TCP, mmHg) in Protocol 1 (a) and Protocol 2 (b).

<p>S, Sham-operated (clear circles); Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V (filled circles), untreated Nx; Nx+L (triangles), losartan-treated Nx; Nx+LH (diamonds), Nx treated with losartan+hydrochlorothiazide; Nx+AHHz (squares), Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.</p

Quantitative analysis of percent renal area occupied by collagen I in Protocol 1 and Protocol 2.

<p>S, Sham-operated; Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V, untreated Nx; Nx+L, losartan-treated Nx; Nx+LH, Nx treated with losartan+hydrochlorothiazide; Nx+AHHz, Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.</p

Urinary albumin excretion rates (U_albV, mg/24 h) in Protocol 1 (a) and Protocol 2 (b).

<p>S, Sham-operated (clear circles); Nx_pre, pretreatment Nx (60 or 120 days after renal ablation); Nx+V (filled circles), untreated Nx; Nx+L (triangles), losartan-treated Nx; Nx+LH (diamonds), Nx treated with losartan+hydrochlorothiazide; Nx+AHHz (squares), Nx treated with amlodipine, hydrochlorothiazide, and hydralazine. Results expressed as Mean ± SE. ^a, p<0.05 vs. Sham; ^b, p<0.05 vs. Nx_pre; ^c, p<0.05 vs. Nx+V; ^d, p<0.05 vs. Nx+L and ^e, p<0.05 vs. Nx+LH.</p

Representative microphotographs of renal tissue obtained for Nx_pre (60 days after renal ablation) and for all other groups (150 days after renal ablation) in Protocol 1.

<p>AII, tubulointerstitial cells staining positively for AII; PCNA, proliferating-cell nuclear antigen; NCC, sodium-chloride cotransporter, specific for distal convoluted tubule (DCT). Arrowheads in Figs. 10c, f, i, l, o and r (double staining for PCNA and NCC) indicate examples of PCNA-positive cells in DCT.</p