Fibroblast growth factor 10 (Fgf10) is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10^LacZ), which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10^Cre-ERT2 knock-in line (Fgf10^iCre) in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10^iCre; Tomato^flox double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10^iCre line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10^iCre line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a poor prognosis. Pirfenidone is the first antifibrotic agent to be approved for IPF-treatment as it is able to slow down disease progression. However, there is no curative treatment other than lung transplantation. Because epigenetic alterations are associated with IPF, histone deacetylase (HDAC)-inhibitors have recently been proven to attenuate fibrotic remodeling in vitro and in vivo. This study compared the effects of pirfenidone with the pan-HDAC-inhibitor panobinostat/LBH589, a FDA-approved drug for the treatment of multiple myeloma, head-to-head on survival, fibrotic activity and proliferation of primary IPF-fibroblasts in vitro. Primary fibroblasts from six IPF-patients were incubated for 24h with vehicle (0.25% DMSO), panobinostat (LBH589, 85 nM) or pirfenidone (2.7 mM), followed by assessment of proliferation and expression analyses for profibrotic and anti-apoptosis genes, as well as for ER stress and apoptosis-markers. In addition, the expression status of all HDAC enzymes was examined. Treatment of IPF-fibroblasts with panobinostat or pirfenidone resulted in a downregulated expression of various extracellular matrix (ECM)-associated genes, as compared to vehicle-treated cells. In agreement, both drugs decreased protein level of phosphorylated (p)-STAT3, a transcription factor mediating profibrotic responses, in treated IPF-fibroblasts. Further, an increase in histone acetylation was observed in response to both treatments, but was much more pronounced and excessive in panobinostat-treated IPF-fibroblasts. Panobinostat, but not pirfenidone, led to a significant suppression of proliferation in IPF-fibroblasts, as indicated by WST1- and BrdU assay and markedly diminished levels of cyclin-D1 and p-histone H3. Furthermore, panobinostat-treatment enhanced α-tubulin-acetylation, decreased the expression of survival-related genes Bcl-XL and BIRC5/survivin, and was associated with induction of ER stress and apoptosis in IPF-fibroblasts. In contrast, pirfenidone-treatment maintained Bcl-XL expression, and was neither associated with ER stress-induction nor any apoptotic signaling. Pirfenidone also led to increased expression of HDAC6 and sirtuin-2, and enhanced α-tubulin-deacetylation. But in line with its ability to increase histone acetylation, pirfenidone reduced the expression of HDAC enzymes HDAC1, -2 and -9. We conclude that, beside other antifibrotic mechanisms, pirfenidone reduces profibrotic signaling also through STAT3 inactivation and weak epigenetic alterations in IPF-fibroblasts, and permits survival of (altered) fibroblasts. The pan-HDAC-inhibitor panobinostat reduces profibrotic phenotypes while inducing cell cycle arrest and apoptosis in IPF-fibroblasts, thus indicating more efficiency than pirfenidone in inactivating IPF-fibroblasts. We therefore believe that HDAC-inhibitors such as panobinostat can present a novel therapeutic strategy for IPF.

Idiopathic pulmonary fibrosis (IPF) features excessive extracellular matrix deposition driven by activated fibroblasts and dysregulated signaling pathways. To assess the anti-fibrotic effects of LTI-03, a caveolin-1 scaffolding domain peptide, ex vivo precision-cut lung slices (PCLS) from patients with IPF were evaluated using bulk RNA sequencing, Ingenuity Pathway Analysis, and immunofluorescence. LTI-03 dose-dependently reduced collagen protein levels, suppressed pro-fibrotic cytokines, inhibited pro-fibrotic pathways, and activated protective mechanisms such as PTEN and PPAR signaling. Modulation was comparable to nintedanib but without the induction of apoptosis or necrosis pathways. These results demonstrate LTI-03’s potential therapeutic efficacy in a highly relevant translational disease model and support LTI-03 as a promising next-generation therapeutic to halt IPF progression and improve patient outcomes.

Rationale LTI-03 is a novel inhaled therapeutic in development for idiopathic pulmonary fibrosis (IPF). LTI-03 has been shown to promote alveolar epithelial cell survival and reduce profibrotic protein expression in experimental models of lung disease. Objectives To evaluate the safety and pharmacokinetics (PK) of LTI-03 and effects on disease biomarkers in patients with IPF. Methods This Phase 1b, randomized, controlled, dose-escalation study randomized 24 participants, 3:1 into 2 sequential dose cohorts, to receive inhaled LTI-03 (5 or 10 mg/day) or placebo for 14 days. The primary endpoint was the incidence of treatment-related adverse events (TEAEs). Exploratory analyses included PK and change from baseline in fibrosis-related and epithelial integrity-related biomarkers in plasma, peripheral blood mononuclear cells (PBMCs), and deep bronchial brushings (DBB). Measurements and Main Results Inhaled LTI-03 was well-tolerated, with no treatment-related AEs leading to treatment discontinuation. All TEAEs were mild or moderate in severity. Cough was the most common TEAE and the only treatment-related TEAE experienced by more than 1 participant. There was no evidence of airway obstruction by symptoms or spirometry. LTI-03 did not induce inflammation (phosphorylated AKT) in PBMCs. In DBB samples, LTI-03 significantly reduced the expression of interleukin-11, chemokine ligand 7, thymic stromal lymphopoietin, and galectin 7; dose-related reductions were also observed for collagen type 1 alpha chain 1 and plasma surfactant protein D. Conclusions Inhaled LTI-03 exhibited a favorable safety and tolerability profile over 14 days. Exploratory biomarker analyses suggest a positive effect on epithelial homeostasis and corresponding antifibrotic effects. Scientific Knowledge on the Subject Current standard of care therapies for IPF do not halt or reverse disease progression, and systemic side effects commonly result in discontinued use. LTI-03 is a caveolin scaffolding domain (CSD) peptide formulated as an excipient-free dry powder for the treatment of IPF. It has demonstrated anti-fibrotic and epithelial protective effects in animal models of fibrosis and in IPF precision cut lung slices via putative replenishment of endogenous Cav-1 signaling, which is lost in fibrotic diseases. What This Study Adds to the Field The results of this study support the conclusion that LTI-03 was well tolerated in patients with IPF at doses of 5 and 10 mg/day. Exploratory biomarker analyses from deep bronchial brushings suggested that positive pharmacodynamic effects were achieved on target cell types. Taken together, these results support the initiation of a Phase 2 efficacy study in patients with IPF. Data sharing statement Rein Therapeutics, Inc. (“Rein”) understands and acknowledges the need to share clinical study data with the research community in an open and transparent manner and has provided de-identified patient data in the manuscript. Rein will not consider further requests pertaining to clinical data outside of what has been accepted and published by the journal. Any queries regarding clinical study data must be submitted in writing to https://info{at}reintx.com. A data supplement for this article is available via the Supplements tab at the top of the online article.

Wnt signaling is important for the formation of different organs during embryonic development. It also plays a key role in lung development, as it is required for branching morphogenesis, proliferation, differentiation and survival of lung progenitors in both the epithelium and mesenchyme. Upregulation of Wnt signaling in the mesenchymal cells is associated with lung pathologies, such as asthma, lung fibrosis, and pulmonary arterial hypertension. After naphthalene injury, an established mouse model of airway epithelial damage, the surviving ciliated airway epithelial cells express Wnt7b. Wnt7b then acts on parabronchial smooth muscle cells (PBSMCs) to induce Fibroblast Growth Factor 10 (Fgf10) expression leading to the expansion of epithelial progenitor cells required for epithelial restoration after injury. We will further investigate the role of Wnt signaling in PBSMCs (using the SMA-Cre-ERT2 mouse driver line, which we recently validated) by gain and loss of function approaches of Wnt signaling during development and in the context of naphthalene injury. Loss of function of Wnt signaling will be performed by using mouse lines that will allow specific deletion of β-catenin in smooth muscle cells. Gain of function will be performed by using mouse line, which allows expression of stable form of β-catenin in smooth muscle cells. We will also use different reporter lines that will allow us to visualize the smooth muscle cells which underwent Cre activation (Tomatoflox/flox mice) and monitor Wnt signaling (Topgal and Axin2LacZ lines).