BMP-7 Does Not Protect against Bleomycin-Induced Lung or Skin Fibrosis

Bone morphogenic protein (BMP)-7 is a member of the BMP family which are structurally and functionally related, and part of the TGFβ super family of growth factors. BMP-7 has been reported to inhibit renal fibrosis and TGFβ1-induced epithelial-mesenchymal transition (EMT), in part through negative interactions with TGFβ1 induced Smad 2/3 activation. We utilized in vivo bleomycin-induced fibrosis models in the skin and lung to determine the potential therapeutic effect of BMP-7. We then determined the effect of BMP-7 on TGFβ1-induced EMT in lung epithelial cells and collagen production by human lung fibroblasts. We show that BMP-7 did not affect bleomycin-induced fibrosis in either the lung or skin in vivo; had no effect on expression of pro-fibrotic genes by human lung fibroblasts, either at rest or following exposure to TGFβ1; and did not modulate TGFβ1 -induced EMT in human lung epithelial cells. Taken together our data indicates that BMP-7 has no anti-fibrotic effect in lung or skin fibrosis either in vivo or in vitro. This suggests that the therapeutic options for BMP-7 may be confined to the renal compartment

Serum Amyloid P Therapeutically Attenuates Murine Bleomycin-Induced Pulmonary Fibrosis via Its Effects on Macrophages

Macrophages promote tissue remodeling but few mechanisms exist to modulate their activity during tissue fibrosis. Serum amyloid P (SAP), a member of the pentraxin family of proteins, signals through Fcγ receptors which are known to affect macrophage activation. We determined that IPF/UIP patients have increased protein levels of several alternatively activated pro-fibrotic (M2) macrophage-associated proteins in the lung and monocytes from these patients show skewing towards an M2 macrophage phenotype. SAP therapeutically inhibits established bleomycin-induced pulmonary fibrosis, when administered systemically or locally to the lungs. The reduction in aberrant collagen deposition was associated with a reduction in M2 macrophages in the lung and increased IP10/CXCL10. These data highlight the role of macrophages in fibrotic lung disease, and demonstrate a therapeutic action of SAP on macrophages which may extend to many fibrotic indications caused by over-exuberant pro-fibrotic macrophage responses

Serum amyloid P ameliorates radiation-induced oral mucositis and fibrosis

Purpose: To evaluate the effect of the anti-fibrotic protein serum amyloid P (SAP) on radiation-induced oral mucositis (OM) and fibrosis in a hamster cheek-pouch model. Experimental Design: Hamsters received a single dose of radiation (40 Gy) to the left everted cheek pouch to induce significant OM. The protective therapeutic potential of SAP was evaluated using varying dosing regimens. The extent of OM was measured using a validated six-point scoring scheme ranging from 0 (normal tissue, no mucositis) to 5 (complete ulceration). Fibrotic remodeling was also visualized histologically and quantified at later time points using collagen gene expression. Results: SAP treatment attenuated the profile of radiation-induced oral mucositis by delaying the time of onset, reducing the peak value, and enhancing the resolution of injury. The peak mucositis score was reduced by approximately 0.5 grade in SAP-treated animals. The number of animal days with a score of ≥ 3 was reduced by 48% in the SAP-treated group, compared with the saline control group (P < 0.01). SAP also inhibited the extent of tissue remodeling and decreased radiation-induced increases in myofibroblast number. Attenuated collagen deposition and gene expression was also observed in the cheek pouches of hamsters treated with SAP at both 16 and 28 days post-radiation. Conclusions: SAP treatment significantly attenuated radiation-induced injury. In particular, SAP attenuated the severity of OM and inhibited pathogenic remodeling. This suggests that SAP may be a useful therapy for the palliation of side effects observed during treatment for head and neck cancer.Medicine, Faculty ofNon UBCAnesthesiology, Pharmacology and Therapeutics, Department ofReviewedFacult

BMP-7 knock-down does not augment the ability of TGFβ₁ to induced EMT in A549 cells.

<p>(A) Representative photomicrographs of A549 cultures (n = 3) transfected with BMP-7 siRNA (10 or 50 ng/ml), or non-silencing scrambled siRNA for 24 hours followed by the presence or absence of rhTGFβ₁ (10 ng/ml) for 72 hours. (B) Western blot analysis of A549 cell lysates indicated increased expression of mesenchymal marker Fibronectin-EDA and loss of epithelial marker E-Cadherin following rhTGFβ₁ treatment which was not enhanced following endogenous knock down of BMP-7. (C) Western blot analysis of BMP-7 showed efficient knock down of the protein following BMP-7 siRNA treatment.</p

BMP-7 does not inhibit TGFβ₁ induced gene expression in fibroblasts.

<p>Primary human lung fibroblasts (n = 3) were serum starved for 24 hours before incubation with rhBMP-7 at 10 or 50 ng/ml for one hour and then stimulated with rhTGFβ₁ for 24 hours. Relative changes in mRNA expression of (A) procollagen-1, (B) αSMA, (C) CTGF and (D) TGFβ₁ were analyzed using real-time RT-PCR. Protein expression of αSMA and phosphoSMAD 1,5,8, was (E) visualized and (F) quantitated to β-tubulin by Western blot analysis. * indicates p<0.05 significant difference in comparison to unstimulated cells.</p

BMP-7 does not inhibit subcutaneous bleomycin-induced skin and lung fibrosis.

<p>Total collagen in the (A) skin or (B) lungs of mice challenged daily with subcutaneous bleomycin and treated daily with either BMP-7 (hatched bars) or vehicle control (filled bars) (mean±S.E.M. of n = 8 mice/group). JE/CCL2 protein levels were measured by luminex in (C) skin and (D) lung homogenates of subcutaneous bleomycin-challenged mice. Each data point represents one animal, with bars representing the mean. For differences in Bleomycin challenged in comparison to naïve control groups * indicates p<0.05, **p<0.01, ***p<0.005 statistical significance.</p

Enhanced M2 monocyte/macrophage phenotype in IPF/UIP.

<p>(A–D) Levels of macrophage-associated mediators measured in lung tissue biopsies taken from IPF/UIP patients (filled bars) or from the normal margins of lung tumors following resection (open bars). M2 macrophage-associated IL13 (A), IL1RA (B) and resistin (C); and M1-associated fractalkine/CX3CL1 (D) from non-fibrotic (<i>n</i> = 11) or IPF/UIP (<i>n</i> = 13) lung tissue were assessed using Luminex technology. *<i>P</i>≤0.05 significance using Mann Whitney analysis.</p

Local and abbreviated SAP treatment significant reduces bleomycin-induced collagen generation and deposition.

<p>Mice were challenged with intratracheal bleomycin (0.03 U) on day 0 and dosed with SAP (6 mg/kg) every other day on days 11–19 intraperitoneally (i.p., filled bar) or intranasally (i.n., down-hatched bar); or dosed with SAP (10 mg/kg, i.p.) on days 11,12,13 only (cross-hatched bar); or dosed with control (P5S, i.n., open bar) every other day on days 11–19. Collagen was quantitated biochemically (A) and visualized histologically (B). Original magnification, ×20. Bars represent the mean ± s.e.m. of a minimum of <i>n</i> = 5 per group. *<i>P</i>≤0.05 significance using Mann Whitney t-test.</p