TGF-beta driven lung fibrosis is macrophage dependent and blocked by Serum amyloid P.

The pleiotropic growth factor TGFß(1) promotes many of the pathogenic mechanisms observed in lung fibrosis and airway remodeling, such as aberrant extracellular matrix deposition due to both fibroblast activation and fibroblast to myofibroblast differentiation. Serum amyloid P (SAP), a member of the pentraxin family of proteins inhibits bleomycin-induced lung fibrosis through an inhibition of pulmonary fibrocyte and pro-fibrotic alternative (M2) macrophage accumulation. It is unknown if SAP has effects downstream of TGFß(1), a major mediator of pulmonary fibrosis. Using the lung specific TGFß(1) transgenic mouse model, we determined that SAP inhibits all of the pathologies driven by TGFß(1) including apoptosis, airway inflammation, pulmonary fibrocyte accumulation and collagen deposition, without affecting levels of TGFß(1). To explore the role of monocyte derived cells in this model we used liposomal clodronate to deplete pulmonary macrophages. This led to pronounced anti-fibrotic effects that were independent of fibrocyte accumulation. Administration of SAP mirrored these effects and reduced both pulmonary M2 macrophages and increased chemokine IP10/CXCL10 expression in a SMAD 3-independent manner. Interestingly, SAP concentrations were reduced in the circulation of IPF patients and correlated with disease severity. Last, SAP directly inhibited M2 macrophage differentiation of monocytes obtained from these patients. These data suggest that the beneficial anti-fibrotic effects of SAP in TGFß(1)-induced lung disease are via modulating monocyte 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.

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 Fcgamma 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.

Generation of bleomycin-induced lung fibrosis is independent of IL-16.

Given that CD4+ cells are found in the lungs of patients with fibrotic lung diseases such as idiopathic pulmonary fibrosis (IPF) we hypothesized that IL-16, a potent chemoattractant for CD4+ cells, may be involved in the pathogenesis of this disease. We found that baseline IL-16 gene expression is greater in fibroblasts isolated from IPF patients compared to non-fibrotic fibroblasts. Furthermore, IL-16 gene expression increased in IPF fibroblasts following stimulation with either of the pro-fibrotic growth factors TGFb1 or PDGF. In contrast, PDGF had no effect on IL-16 gene expression in non-fibrotic lung fibroblasts, whereas TGFb1 down-regulated IL-16 gene expression in non-fibrotic fibroblasts. To gain a better understanding of an association of IL-16 with fibrosis, we used the bleomycin-induced mouse model of fibrosis to examine IL-16 gene expression. Our current study demonstrates that IL-16, and its activator caspase 3, are highly expressed at the mRNA level in the lungs of mice prior to the deposition of collagen following intratracheal bleomycin administration. We then sought to determine the role of IL-16 in the generation of fibrosis in the mouse by using IL-16KO mice. There were no differences observed between IL-16WT and IL-16KO mice (cellular infiltrate, collagen deposition, total lung collagen generation and cytokine expression) following bleomycin instillation. These results indicate that IL-16 is prominently expressed in both murine and human fibrosis however as complete loss of this cytokine did not modulate pulmonary fibrosis, IL-16 is a candidate biomarker for IPF.

BMP-7 fails to attenuate TGF-beta1-induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells.

BACKGROUND: In rodent models of chronic renal disease bone morphogenetic protein-7 (BMP-7) has been shown to halt disease progression and promote recovery. Subsequent studies utilizing immortalized rodent renal cell lines showed that BMP-7 was renoprotective by antagonizing TGF-beta1-stimulated epithelial-to-mesenchymal transition (EMT). The present study sought to determine if BMP-7 prevents TGF-beta1-induced EMT in primary (RPTEC) and immortalized (HK-2) human proximal tubule epithelial cells. METHODS: EMT was determined by quantitative real-time PCR analysis of e-cadherin, vimentin, CTGF and TGF-beta1 transcript expression and immunocytochemical analysis of ZO-1 and alpha-smooth muscle actin (alpha-SMA) protein expression following TGF-beta1 treatment in RPTEC and HK-2 cells. RESULTS: In RPTEC and HK-2 cells, TGF-beta1 significantly reduced e-cadherin expression and significantly increased vimentin, CTGF and TGF-beta1 expression. TGF-beta1 also diminished ZO-1 immunoreactivity and increased alpha-SMA expression in confluent cell monolayers. Co-incubation of TGF-beta1 with an anti-TGF-beta1 neutralizing antibody substantially reduced the cytokine's effects, which indicated EMT in these cells was inhibitable. Co-administration of BMP-7 over a broad concentration range (0.01-100 microg/ml) with TGF-beta1 failed to attenuate EMT in RPTEC or HK-2 cells, as demonstrated by no inhibition of altered e-cadherin, vimentin, CTGF and TGF-beta1 expression and no restoration of ZO-1 immunoreactivity. Furthermore, when BMP-7 was applied to proximal tubule cells alone, it also decreased e-cadherin expression and increased vimentin, CTGF and TGF-beta1 expression. Additionally, BMP-7 failed to induce the mesenchymal-to-epithelial transition (MET) in NRK-49F rat renal fibroblasts. BMP-7 did however prevent TGF-beta1-mediated e-cadherin downregulation in TCMK-1 mouse renal tubular epithelial cells. BMP-7 activity was routinely confirmed by examining BMP-7-induced phosphorylation of SMADs 1/5/8, BMP-7 regulation of BMPR-IA, BMP-7-mediated reduction of IL-6 transcript expression and BMP-7-mediated reduction of secreted IL-6 and IL-8 proteins. CONCLUSIONS: In the present study, despite confirming BMP-7 regulation of receptor expression and induction of downstream signalling events, we were unable to demonstrate BMP-7 inhibition of EMT in either primary or immortalized human proximal tubule cells. Moreover, we were unable to demonstrate BMP-7-stimulated MET in rat renal fibroblasts. A protective effect was however observed at an elevated BMP-7 concentration in mouse renal tubular epithelial cells.

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 TGFbeta super family of growth factors. BMP-7 has been reported to inhibit renal fibrosis and TGFbeta1-induced epithelial-mesenchymal transition (EMT), in part through negative interactions with TGFbeta1 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 TGFbeta1-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 TGFbeta1; and did not modulate TGFbeta1-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.

Virtual screening to successfully identify novel janus kinase 3 inhibitors: a sequential focused screening approach.

In an effort to identify novel Janus kinase 3 inhibitors, a sequential focused screening approach was adopted to search our in-house chemical database. By biologically testing only 79 selected compounds, we successfully identified 19 compounds showing IC 50 < 20 microM, with four of them in the nanomolar range. Particularly, a 3,5-disubstituted pyrazolo[4,3- d]pyrimidine scaffold emerged as a promising candidate for further lead optimization. With the advantages of efficiency and flexibility, this approach may be utilized to identify leads for other therapeutic targets.

Hyper-responsiveness of IPF/UIP fibroblasts: interplay between TGFbeta1, IL-13 and CCL2.

One of the hallmarks of idiopathic pulmonary fibrosis with a usual interstitial pneumonia histological pathology (IPF/UIP) is excess collagen deposition, due to enhanced fibroblast extracellular matrix synthetic activity. Studies using murine models of lung fibrosis have elucidated a pro-fibrotic pathway involving IL-13 driving CCL2, which in turn drives TGFbeta1 in lung fibroblasts. Therefore, we sought to determine whether this pathway exists in the human fibrotic setting by evaluating human IPF/UIP fibroblasts. IPF/UIP fibroblasts have an increased baseline fibrotic phenotype compared to non-fibrotic fibroblasts. Interestingly, non-fibrotic fibroblasts responded in a pro-fibrotic manner to TGFbeta1 but were relatively non-responsive to IL-13 or CCL2, whereas, IPF/UIP cells were hyper-responsive to TGFbeta1, IL-13 and CCL2. Interestingly, TGFbeta1, CCL2 and IL-13 all upregulated TGFbeta receptor and IL-13 receptor expression, suggesting an ability of the mediators to modulate the function of each other. Furthermore, in vivo, neutralization of both JE and MCP5, the two functional orthologs of CCL2, during bleomycin-induced pulmonary fibrosis significantly reduced collagen deposition as well as JE and CCR2 expression. Also in the bleomycin model, CTGF, which is highly induced following TGFbeta stimulation, was attenuated with anti-JE/anti-MCP5 treatment. Overall this study demonstrates an interplay between TGFbeta1, IL-13 and CCL2 in IPF/UIP, where these three mediators feedback on each other, promoting the fibrotic response.

Targeting cytosolic phospholipase A2 by arachidonyl trifluoromethyl ketone prevents chronic inflammation in mice.

Cytosolic phospholipase A(2) (cPLA(2)) plays a pivotal role in inflammation by catalyzing the release of arachidonic acid, a substrate for lipoxygenase and cyclooxygenase enzymes, from membrane phospholipids. In the present study we examined the role of cPLA(2) in inflammatory responses through the use of a specific inhibitor of the enzyme, cPLA(2), arachidonyl trifluoromethyl ketone (AACOCF3). Interestingly, we observed that AACOCF3 is an inhibitor of chronic but not acute inflammatory responses. Specifically, AACOCF3 inhibited phorbol 12-myristate 13-acetate (PMA)-induced chronic ear edema in mice. Additionally, oral treatment of ovalbumin-sensitized/ovalbumin-challenged BALB/c mice with 20 mg/kg AACOCF3 prevented the development of airway hyper-responsiveness in a model of asthma. Furthermore, AACOCF3 decreased cellular recruitment in the airway lumen and airway inflammation after the ovalbumin challenge. Taken together, these results suggest that a potent and specific chemical inhibitor of cPLA(2) may be useful for the treatment of chronic inflammatory diseases including rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma.