Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown cause. Key signaling developmental pathways are aberrantly expressed in IPF. The hedgehog pathway plays a key role during fetal lung development and may be involved in lung fibrogenesis. We determined the expression pattern of several Sonic hedgehog (SHH) pathway members in normal and IPF human lung biopsies and primary fibroblasts. The effect of hedgehog pathway inhibition was assayed by lung fibroblast proliferation and differentiation with and without transforming growth factor (TGF)-β1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-β pathway in human lung fibroblasts. TGF-β1 modulated the expression of key components of the hedgehog pathway independent of Smoothened, the obligatory signal transducer of the pathway. Smoothened was required for TGF-β1-induced myofibroblastic differentiation of control fibroblasts, but differentiation of IPF fibroblasts was partially resistant to Smoothened inhibition. Furthermore, functional hedgehog pathway machinery from the primary cilium, as well as GLI-dependent transcription in the nucleus, was required for the TGF-β1 effects on normal and IPF fibroblasts during myofibroblastic differentiation. These data identify the GLI transcription factors as potential therapeutic targets in lung fibrosis.

Idiopathic pulmonary fibrosis has been associated with the reactivation of developmental pathways, notably the Hedgehog-Glioma-associated oncogene homolog (GLI) pathway. In this study, we determined whether the Hedgehog pathway was activated in bleomycin-induced lung injury in mice, and whether targeting the Hedgehog-Gli pathway could decrease bleomycin-induced lung fibrosis. After intratracheal injection of bleomycin on Day 0, C57Bl6 mice received GDC-0449 (an inhibitor of Smoothened, the transducer of the pathway), or 2,2'-[[Dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N dimethylbenzenamine (GANT61; an inhibitor of GLI transcription factors in the nucleus), from Day 7 to Day 13. At Day 14, whole-lung homogenates were obtained for morphological analysis, assessment of cell apoptosis and proliferation, collagen quantification, and evaluation of profibrotic (transforming growth factor-β, connective tissue growth factor, plasminogen activator inhibitor 1, vascular endothelial growth factor-A) and proinflammatory mediators (IL-1β) expression. We showed that the Hedgehog pathway was activated in bleomycin-induced lung fibrosis on Day 14 after injury, with an increased lung expression of the ligand, Sonic Hedgehog, and with increased messenger RNA expression and nuclear localization of GLI1 and GLI2. Inhibition of Smoothened with GDC-0449 did not influence the development of bleomycin-induced lung fibrosis. By contrast, the inhibition of GLI activity with GANT61 decreased lung fibrosis and lung collagen accumulation, and promoted an antifibrotic and anti-inflammatory environment. Our results identify the hedgehog-Gli pathway as a profibrotic pathway in experimental fibrosis. Inhibition of the Hedgehog-Gli pathway at the level of GLI transcriptional activity could be a therapeutic option in fibrotic lung diseases.

Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT) n polymorphism and single nucleotide polymorphisms located at -1135 and -413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT) n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1. 46-3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT) n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30-3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter

Summary.  Background:  Heme is a redox active macrocyclic compound that is released upon tissue damage or hemorrhages. The extracellular release of large amounts of heme saturates scavenging heme‐binding proteins. Free heme has been proposed to affect coagulation and has been co‐purified with the factor VIII (FVIII)‐von Willebrand factor (VWF) complex. The sites from which heme is released upon injury overlap with the sites to which FVIII is targeted for performing its hemostatic functions. Objectives:  To investigate the interaction of heme with FVIII and the consequence for the procoagulant activity of FVIII in vitro. Methods and results:  Heme bound to several sites on FVIII with high apparent affinity. Heme‐binding inhibited FVIII procoagulant activity in a dose‐dependent manner. FVIII inactivation in the presence of saturating amounts of heme implicated a reduced interaction of FVIII with activated FIX, as shown by ELISA, surface plasmon resonance and fluorescence quenching. Heme‐mediated inactivation of FVIII was prevented by VWF, but not by human serum albumin, a heme‐binding protein known for its protective activity in hemolytic conditions. Conclusions:  Our data identify FVIII as a novel heme‐binding protein. Occupation of high affinity heme‐binding sites on FVIII at low concentrations of free heme did not inactivate FVIII. Conversely, large molar excesses of heme over FVIII, which correspond to conditions of extensive heme release, inhibited FVIII activity in vitro. It remains to be demonstrated whether, under such conditions, heme‐mediated modulation of the activity of FVIII plays some role in the regulation of coagulation.

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38 The occurrence of inhibitory anti-factor VIII (FVIII) antibodies is the major complication of replacement therapy in patients with hemophilia A. Heme oxygenase-1 (HO-1) is a stress inducible enzyme with anti-inflammatory activity. Induction of HO-1 in hemophilic mice reduces the immunogenicity of therapeutic FVIII. Interestingly, polymorphisms in the promoter of the HO-1-encoding gene (HMOX1) modulate the expression of HO-1. We investigated the relationship between polymorphisms in the promoter of HMOX1 in severe hemophilia A patients and the development of FVIII inhibitors. We analyzed 362 patients with severe hemophilia A involving 99 patients with FVIII inhibitors and 263 patients who did not develop inhibitor within the first 150 cumulative exposure days to therapeutic FVIII. Direct sequencing and DNA fragment analysis were used to study the variable (GT)n polymorphism and single nucleotide polymorphisms located at −1135 and −413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies and genotypes, and the development of inhibitors. Our results demonstrate a higher frequency of alleles with large (GT)n repeat (n≥30, L, associated with a lesser expression of HO-1) in inhibitor-positive patients [odds ratio (OR) 2.31; 95% CI 1.46–3.66, p<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% vs 17.1%) [OR 2.21, 95% CI 1.30–3.76, p<0.01]. To our knowledge, this is the first association between HMOX1 promoter polymorphism and development of anti-drug antibodies. Modulating the endogenous anti-inflammatory machinery of hemophilia A patients appears as a plausible therapeutic option for reducing the risk of inhibitor development. Disclosures: No relevant conflicts of interest to declare.

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