Therapeutic targets in lung tissue remodelling and fibrosis.

Structural changes involving tissue remodelling and fibrosis are major features of many pulmonary diseases, including asthma, chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Abnormal deposition of extracellular matrix (ECM) proteins is a key factor in the development of tissue remodelling that results in symptoms and impaired lung function in these diseases. Tissue remodelling in the lungs is complex and differs between compartments. Some pathways are common but tissue remodelling around the airways and in the parenchyma have different morphologies. Hence it is critical to evaluate both common fibrotic pathways and those that are specific to different compartments; thereby expanding the understanding of the pathogenesis of fibrosis and remodelling in the airways and parenchyma in asthma, COPD and IPF with a view to developing therapeutic strategies for each. Here we review the current understanding of remodelling features and underlying mechanisms in these major respiratory diseases. The differences and similarities of remodelling are used to highlight potential common therapeutic targets and strategies. One central pathway in remodelling processes involves transforming growth factor (TGF)-ß induced fibroblast activation and myofibroblast differentiation that increases ECM production. The current treatments and clinical trials targeting remodelling are described, as well as potential future directions. These endeavours are indicative of the renewed effort and optimism for drug discovery targeting tissue remodelling and fibrosis.

Semicarbazide-sensitive amine oxidase inhibition ameliorates albuminuria and glomerulosclerosis but does not improve tubulointerstitial fibrosis in diabetic nephropathy.

Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme with a unique dual function in controlling inflammation as well as reactive oxygen species (ROS) generation. We have demonstrated benefit of SSAO inhibition in acute kidney fibrosis. However the function of SSAO in chronic kidney disease (CKD) and diabetic kidney disease (DKD) is yet to be determined. We aimed to assess the effectiveness of a SSAO inhibitor (SSAOi; PXS-4728A) as an antifibrotic agent using a diabetic model of CKD. Diabetic mice were treated with SSAOi for 24 weeks and outcomes compared with untreated diabetic mice and telmisartan treated animals as a standard of care comparator. Extracellular matrix markers, fibronectin and oxidative stress, were downregulated in diabetic mice treated with SSAOi compared with untreated diabetic mice. Expression of the pan-leukocyte marker CD45 was also supressed by SSAOi. SSAO inhibition in diabetic mice resulted in a significant reduction in glomerulosclerosis and associated albuminuria compared to untreated diabetic mice. However, the effect of SSAO inhibition was less obvious in the tubulointerstitial compartment than in the glomeruli. Therefore, SSAO may be a potential target for diabetic glomerulosclerosis.

Identification and Optimization of Mechanism-Based Fluoroallylamine Inhibitors of Lysyl Oxidase-like 2/3.

Lysyl oxidase-like 2 (LOXL2) is a secreted enzyme that catalyzes the formation of cross-links in extracellular matrix proteins, namely, collagen and elastin, and is indicated in fibrotic diseases. Herein, we report the identification and subsequent optimization of a series of indole-based fluoroallylamine inhibitors of LOXL2. The result of this medicinal chemistry campaign is PXS-5120A (12k), a potent, irreversible inhibitor that is >300-fold selective for LOXL2 over LOX. PXS-5120A also shows potent inhibition of LOXL3, an emerging therapeutic target for lung fibrosis. Key to the development of this compound was the utilization of a compound oxidation assay. PXS-5120A was optimized to show negligible substrate activity in vitro for related amine oxidase family members, leading to metabolic stability. PXS-5120A, in a pro-drug form (PXS-5129A, 12o), displayed anti-fibrotic activity in models of liver and lung fibrosis, thus confirming LOXL2 as an important target in diseases where collagen cross-linking is implicated.

The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-5153A, reduces crosslinks and ameliorates fibrosis.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Lysyl oxidase-like 2 inhibition ameliorates glomerulosclerosis and albuminuria in diabetic nephropathy.

Diabetic nephropathy is characterised by the excessive amount of extracellular matrix in glomeruli and tubulointerstitial space. Lysyl oxidase-like 2 (LOXL2) is elevated in renal fibrosis and known to play key roles in ECM stabilisation by facilitating collagen cross-links, epithelial to mesenchymal transition and myofibroblast activation. Thus, targeting LOXL2 may prove to be a useful strategy to prevent diabetic nephropathy. We explored the renoprotective effect of a selective small molecule LOXL2 inhibitor (PXS-S2B) in a streptozotocin-induced diabetes model. Diabetic mice were treated with PXS-S2B for 24 weeks and outcomes compared with untreated diabetic mice and with telmisartan treated animals as comparator of current standard of care. Diabetic mice had albuminuria, higher glomerulosclerosis scores, upregulation of fibrosis markers and increased renal cortical LOXL2 expression. Treatment with PXS-S2B reduced albuminuria and ameliorated glomerulosclerosis. This was associated with reduced expression of glomerular fibronectin and tubulointerstitial collagen I. The renoprotective effects of both PXS-S2B and telmisartan were more marked in the glomerular compartment than in the tubulointerstitial space. The study reveals that LOXL2 inhibition was beneficial in preserving glomerular structure and function. Thus, LOXL2 may be a potential therapeutic target in diabetic nephropathy.

The mannose-6-phosphate analogue, PXS64, inhibits fibrosis via TGF-ß1 pathway in human lung fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-ß1 to induce increases in fibrotic markers. rhTGF-ß1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-ß3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-ß1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-ß1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis.

Effects of an anti-inflammatory VAP-1/SSAO inhibitor, PXS-4728A, on pulmonary neutrophil migration.

BACKGROUND AND PURPOSE: The persistent influx of neutrophils into the lung and subsequent tissue damage are characteristics of COPD, cystic fibrosis and acute lung inflammation. VAP-1/SSAO is an endothelial bound adhesion molecule with amine oxidase activity that is reported to be involved in neutrophil egress from the microvasculature during inflammation. This study explored the role of VAP-1/SSAO in neutrophilic lung mediated diseases and examined the therapeutic potential of the selective inhibitor PXS-4728A. METHODS: Mice treated with PXS-4728A underwent intra-vital microscopy visualization of the cremaster muscle upon CXCL1/KC stimulation. LPS inflammation, Klebsiella pneumoniae infection, cecal ligation and puncture as well as rhinovirus exacerbated asthma models were also assessed using PXS-4728A. RESULTS: Selective VAP-1/SSAO inhibition by PXS-4728A diminished leukocyte rolling and adherence induced by CXCL1/KC. Inhibition of VAP-1/SSAO also dampened the migration of neutrophils to the lungs in response to LPS, Klebsiella pneumoniae lung infection and CLP induced sepsis; whilst still allowing for normal neutrophil defense function, resulting in increased survival. The functional effects of this inhibition were demonstrated in the RV exacerbated asthma model, with a reduction in cellular infiltrate correlating with a reduction in airways hyperractivity. CONCLUSIONS AND IMPLICATIONS: This study demonstrates that the endothelial cell ligand VAP-1/SSAO contributes to the migration of neutrophils during acute lung inflammation, pulmonary infection and airway hyperractivity. These results highlight the potential of inhibiting of VAP-1/SSAO enzymatic function, by PXS-4728A, as a novel therapeutic approach in lung diseases that are characterized by neutrophilic pattern of inflammation.

Semicarbazide-sensitive amine oxidase (SSAO) inhibition ameliorates kidney fibrosis in a unilateral ureteral obstruction murine model.

Semicarbazide-sensitive amine oxidase (SSAO) is an enzyme known for its dual function in mediating inflammation and reactive oxygen species production. However, the role of SSAO inhibitors in limiting kidney fibrosis is unclear. We aimed to determine the effectiveness of a SSAO inhibitor (SSAOi; PXS4728A) as an antifibrotic agent using a 7-day unilateral ureteric obstruction (UUO) model of acute kidney fibrosis in 6- to 8-wk-old mice. The experimental groups were 1) Sham operated; 2) UUO; 3) UUO+SSAOi (2 mg/kg); 4) UUO+telmisartan, an angiotensin receptor blocker (3 mg/kg); and 5) UUO+SSAOi+telmisartan. Kidney tissue was analyzed for histological evidence of tubulointerstitial fibrosis, nitrotyrosine staining, and mRNA expression of markers associated with fibrosis and inflammation. Kidney SSAO activity was determined by radiometric [(14)C]benzylamine methodology. Our results show that SSAOi effectively suppresses UUO-mediated SSAO activity. Extracellular matrix markers, namely, fibronectin, collagen IV protein, and nitrotyrosine staining, were lower in UUO+SSAOi mice compared with untreated UUO mice. This was consistent with the attenuated mRNA expression of collagen IV and fibronectin. SSAOi effectively inhibited transforming growth factor-ß1 (TGF-ß1) and monocyte chemoattractant protein-1 (MCP-1) expression to a similar extent to that observed with telmisartan. Individually, SSAOi and telmisartan induced a reduction in interstitial leukocyte and macrophage accumulation. However, the combination of SSAOi and telmisartan was more effective at reducing inflammatory cell infiltration. These results demonstrate that SSAO inhibition significantly suppresses profibrotic and proinflammatory cytokine secretion, reduces oxidative stress, and limits inflammatory cell accumulation and extracellular matrix expression in an acute model of renal fibrosis.

PXS-4681A, a potent and selective mechanism-based inhibitor of SSAO/VAP-1 with anti-inflammatory effects in vivo.

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Chlamydia muridarum lung infection in infants alters hematopoietic cells to promote allergic airway disease in mice.

BACKGROUND: Viral and bacterial respiratory tract infections in early-life are linked to the development of allergic airway inflammation and asthma. However, the mechanisms involved are not well understood. We have previously shown that neonatal and infant, but not adult, chlamydial lung infections in mice permanently alter inflammatory phenotype and physiology to increase the severity of allergic airway disease by increasing lung interleukin (IL)-13 expression, mucus hyper-secretion and airway hyper-responsiveness. This occurred through different mechanisms with infection at different ages. Neonatal infection suppressed inflammatory responses but enhanced systemic dendritic cell:T-cell IL-13 release and induced permanent alterations in lung structure (i.e., increased the size of alveoli). Infant infection enhanced inflammatory responses but had no effect on lung structure. Here we investigated the role of hematopoietic cells in these processes using bone marrow chimera studies. METHODOLOGY/PRINCIPAL FINDINGS: Neonatal (<24-hours-old), infant (3-weeks-old) and adult (6-weeks-old) mice were infected with C. muridarum. Nine weeks after infection bone marrow was collected and transferred into recipient age-matched irradiated naïve mice. Allergic airway disease was induced (8 weeks after adoptive transfer) by sensitization and challenge with ovalbumin. Reconstitution of irradiated naïve mice with bone marrow from mice infected as neonates resulted in the suppression of the hallmark features of allergic airway disease including mucus hyper-secretion and airway hyper-responsiveness, which was associated with decreased IL-13 levels in the lung. In stark contrast, reconstitution with bone marrow from mice infected as infants increased the severity of allergic airway disease by increasing T helper type-2 cell cytokine release (IL-5 and IL-13), mucus hyper-secretion, airway hyper-responsiveness and IL-13 levels in the lung. Reconstitution with bone marrow from infected adult mice had no effects. CONCLUSIONS: These results suggest that an infant chlamydial lung infection results in long lasting alterations in hematopoietic cells that increases the severity of allergic airway disease in later-life.

Protection against Nippostrongylus brasiliensis infection in mice is independent of GM-CSF.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine with the capacity to promote inflammation in a wide variety of infectious and inflammatory diseases. These conditions include allergic airway inflammation, which is driven by T-helper 2 (Th2) cells. Because of the importance of Th2 cells in parasite infections, we have investigated the role of GM-CSF in mice infected with the nematode Nippostrongylus brasiliensis. The effect of primary and secondary infection was investigated in mice lacking functional genes for GM-CSF (CSF2 genes) (ΔGM-CSF mice), and in mice lacking the cytokine receptor common ß chain (Δß mice), the latter being unable to signal in response to GM-CSF and interleukin (IL)-5. ΔGM-CSF mice showed no significant defect in parasite immunity, measured by larval numbers in the lungs, worm numbers in the intestine or egg numbers in the faeces, in either primary or secondary infection. By contrast, the Δß mice showed increased parasite burden, with higher numbers of lung larvae after secondary infection and higher numbers of intestinal worms and faecal eggs after both primary and secondary infection. Unexpectedly, there were increased numbers of circulating eosinophils in the ΔGM-CSF mice, associated with significantly reduced larval numbers in the lungs. These results indicate that GM-CSF is redundant in protection against N. brasiliensis infection, and that the increased susceptibility of Δß mice to infection is likely to be attributed to the lack of IL-5 signalling in these mice. The results suggest that clinical use of agents that neutralise GM-CSF may not be associated with increased risk of parasite infection.

Responses of mouse airway epithelial cells and alveolar macrophages to virulent and avirulent strains of influenza A virus.

Influenza A virus (IAV) infection is associated with outcomes ranging from subclinical infection to severe pneumonia. In this study, we compared IAV strains BJx109 (H3N2), HKx31 (H3N2), and PR8 (H1N1), for their ability to elicit innate immune responses from mouse airway cells in vitro and their virulence in mice. The viruses differed markedly in their ability to induce disease in mice (PR8 > HKx31 > BJx109). In particular, PR8 infection was associated with high levels of virus replication and pulmonary inflammation. We next compared the ability of each virus strain to infect and induce inflammatory mediators from mouse airway cells. First, major differences were observed in the ability of viruses to infect and induce chemokines and cytokines from mouse alveolar macrophages (BJx109 > HKx31 > PR8), but not from airway epithelial cells (AEC) in vitro. Second, C-type lectins of the innate immune system in mouse lung fluids blocked the ability of BJx109, but not PR8, to infect mouse macrophages and AEC. The failure of the virulent PR8 virus to elicit responses from airway macrophages, combined with resistance to antiviral proteins in mouse airway fluids, likely contribute to virulence in mice. These findings provide insight into the mechanisms underlying disease severity in the mouse model of influenza infection.

Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43.

The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in 'healthy' microbiota such as Bifidobacterium and Bacteriodes, and a concurrent reduction in SCFAs. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2), and here we show that SCFA-GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43(-/-)) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43(-/-) immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.