Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor–β ₁ (TGF-β ₁ ), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-β ₁ transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-β ₁ transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-β ₁ –induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-β ₁ –induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-β ₁ –induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

Nintedanib is an anti-fibrotic medication endowed with a multi-kinase inhibitor profile and approved for the treatment of Idiopathic Pulmonary Fibrosis (IPF). Nintedanib is believed to inhibit mainly Vascular Endothelial Growth Factor (VEGF), Platelet-Derived Growth Factor (PDGF), and Fibroblast Growth Factor (FGF) receptor kinases. The main objective was to identify potential tissue and/or circulating biomarkers to demonstrate Nintedanib’s target engagement and support its in vivo pharmacodynamic activity, consistent with its proposed mechanism(s) of action. In four independent experiments of bleomycin (BLM)-induced lung fibrosis model in rats, animals received Nintedanib (oral, 100 mg/kg/day) from day 7 post-BLM for 3 weeks. As expected, Nintedanib significantly reduced lung weight, the levels of lung fibrotic markers, and fibrotic areas. Moreover, Nintedanib-treated animals expressed lower levels of FGF2 in lung homogenates and higher plasma and lung levels of VEGF (≥3-fold, p < 0.05) compared to control animals. Lung proteomic analysis revealed the inhibition of receptor tyrosine kinases signaling in Nintedanib-treated animals. Circulating and lung levels of Nintedanib confirmed an optimal tissue distribution in the rat, consistent with the data reported for humans. Although VEGF ligand levels are elevated in the lungs of Nintedanib-treated animals, the VEGF signaling pathway remained functionally downregulated, strongly suggesting compensatory VEGF feedback delivery to its receptor blockade by Nintedanib. In summary, based on the present experimental findings in rats and supporting clinical preliminary evidence, increased VEGF levels can be reasonably considered an indicator of target engagement for Nintedanib and potentially for other VEGF modulators.

Background: Extensive studies have suggested epithelial-mesenchymal transition (EMT) and fibroblast-myofibroblast transformation (FMT) as major drivers of fibrosis. Both EMT and FMT are strongly regulated by TGFβ1. Objectives. The aim of this study was to assess a specific transcriptional analysis of a selected panel of genes involved in fibrogenesis by profiling TGFβ-stimulated human lung fibroblasts (HLFs) and human bronchial epithelial cells (HBECs) derived from IPF patients and healthy donors. Moreover, the effects of Nintedanib and the ALK5 inhibitor, SB525334, were evaluated. Methods: Cultured primary HLFs and HBECs were stimulated with TGF-β1 (1.25 and 5 ng/ml, respectively). IPF-derived cells were exposed to Nintedanib (3 µM) and SB525334 (1 µM). RNA was extracted and subsequently reverse transcribed and amplified. RT2 Profiler™ PCR Arrays Human Fibrosis (Qiagen) were used to gene expression analysis. Results: Approximately half of the assessed genes were endowed with a differential expression (≥1.5 fold) between non-stimulated IPF and control HLFs (e.g. GREM1, HGF) and HBECs (e.g. ITGB3, ENG). The above expression pattern was further modified when TGF-β1 was applied to IPF and control cells. Both Nintedanib and SB525334 mainly induced a significant effect on TGFβ1-induced differential expression of genes related to extracellular matrix remodelling (e.g. COL1A1 and COL3A1). Conclusions: Our study revealed a basal differential expression of a selected panel of genes among control and IPF cells. Furthermore, the subsequent validation step with Nintedanib may provide insights on new targets endowed with a potential anti-fibrotic effect.

Idiopathic pulmonary fibrosis (IPF) remains a progressive and fatal disease despite major advances in antifibrotic therapy. Pirfenidone and nintedanib slow lung function decline, and the PDE4B inhibitor nerandomilast and treprostinil have recently shown promise as next-generation treatments. However, current therapies neither halt nor reverse disease progression, and tolerability issues often limit adherence. We compared the antifibrotic activity and plasma levels of nintedanib administered by either oral gavage or dietary supplementation in a rat model of pulmonary fibrosis. Male Sprague-Dawley rats received intratracheal bleomycin (1 U/kg) on days 0 and 4. From day 7 to day 28, animals were treated with nintedanib (100 mg/kg/day) by either oral gavage or medicated chow. Lung weight, fibrosis biomarkers (procollagen-I, metalloproteinase-7 or MMP7, WNT1-inducible signaling pathway protein or WISP-1), epithelial injury marker KL-6, target engagement biomarkers (Fibroblast Growth Factor 2 or FGF-2, Vascular Endothelial Growth Factor or VEGF), plasma drug levels, and histological fibrosis scores were evaluated. Both administration regimens significantly reduced procollagen-I, WISP-1 and KL-6, and histological fibrosis scores. Oral gavage produced approximately fourfold-higher peak plasma concentrations of nintedanib 30 min after dosing, whereas dietary administration resulted in lower, more stable plasma levels with reduced variability while resulting in comparable antifibrotic efficacy. In conclusion, nintedanib retained robust antifibrotic activity when administered via dietary supplementation despite lower, but continuous, active plasma concentrations. Collectively, these findings indicate that optimizing drug delivery and absorption kinetics may achieve sustained therapeutic efficacy while reducing unnecessary exposure to supratherapeutic concentrations, thereby potentially improving tolerability.

Background: A growing body of evidence suggests an altered role of epigenetic regulators like histone deacetylases (HDACs) in IPF. Targeting the epigenetic regulation by HDACs modulation could offer a promising approach to control and revert pulmonary fibrosis. Aims and objectives. Test the efficacy of Panobinostat, an FDA approved pan-HDAC inhibitor for the treatment of cancer, in a therapeutic Bleomycin (BLM) lung fibrosis rat model in comparison to a reference drug for IPF, Nintedanib (NINT). Methods. For pharmacokinetic (PK) and target engagment (TE) experiments, two groups of male naive SD rats received Panobinostat 10 mg/kg (IP) and were sacrified until 24 h and 72h post-dosing respectively. For efficacy experiment, male SD rats received a double intratracheally BLM administration or its vehicle (saline) on day 0 and day 4. According to therapeutic regimen, Panobinostat or NINT were administered from day 7 post-BLM at the respective doses of 1 and 3 mg/kg (IP, three times per week) and 100 mg/kg/day (oral, once daily) for three weeks. Automated histomorphometric analysis and biomarkers levels in lung homogenates were used to evaluate the efficacy of treatment. Results. The PK analysis indicated a high tissue partition after IP administration and a sustained target engagement (H3 acetylation quantification) supporting a less-frequent dosing respect to NINT. Panobinostat significantly reduced lung weight, pro-collagen-I, collagen-I and WISP-1 levels in lung homogenates and showed a trend in reduction of fibrotic area as highlighted by the histological analysis. Conclusions. Systemic administration of a pan-HDAC inhibitor demonstrated an anti-fibrotic effect comparable to that of NINT.

Background: Chronic Obstructive Pulmonary Disease (COPD) is a progressive, incurable condition marked by irreversible airflow limitation and systemic inflammation. Cardiovascular comorbidities, particularly pulmonary hypertension (PH), exacerbate disease severity. While cigarette smoke is a well-known trigger, non-smoking-related inflammatory pathways remain underexplored. This study investigates vascular remodeling in a murine model of inflammation induced by chronic exposure to house dust mite Farinae (HDM). Methods: Female C57BL/6 mice were sensitized with HDM in Freund’s Complete Adjuvant and challenged intranasally with HDM for six weeks. Lung inflammation, mucus hypersecretion, and vascular remodeling were evaluated via BAL, histology, immunofluorescence, echocardiography, gene expression, proteomics, and FlexiVent pulmonary function tests (FlexiVent system). Results: HDM exposure induced a mixed inflammatory response, with elevated neutrophils, monocytes, and lymphocytes in BALF. Mucus hyperproduction (increase in MUC5AC/MUC5B) and impaired lung function (reduced FEV0.1/FVC) were observed. Vascular remodeling was evidenced by increased wall thickness, α-SMA expression, and collagen deposition. Proteomic analysis revealed dysregulation of endothelial markers and protease/antiprotease imbalance. HIF1-α was significantly upregulated in lung tissue and correlated with vascular and epithelial remodeling. Conclusions: Chronic HDM exposure in mice recapitulates key features observed in subsets of COPD and PH, including inflammation-driven airway and vascular remodeling. HIF1-α emerges as a central regulator, linking hypoxia to structural changes. This model offers insights into the effect of non-smoking-related inflammatory pathways on bronchial and vascular remodeling that are potentially relevant for subgroups of COPD patients and highlights HIF1-α as a potential therapeutic target.