B79-20 Hexosamine Biosynthetic Pathway Modulation Reduces Tgf-β-induced Fibrotic Endpoints in Human Precision Cut Lung Slices

Rationale

Idiopathic Pulmonary Fibrosis (IPF) is a progressive and terminal lung disease driven by fibroblast-to-myofibroblast differentiation and excessive extracellular matrix (ECM) deposition, with limited therapies. The Hexosamine Biosynthetic Pathway (HBP), including enzymes glutamine-fructose-6 phosphate transaminase 2 (GFPT2) and phosphoglucomutase 3 (PGM3), contributes to myofibroblast differentiation and ECM macromolecule production. We hypothesize inhibiting this pathway via AMP activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and PGM3 inhibitor FR054 may attenuate fibrosis.

Methods

Precision cut lung slices (PCLS) were derived from three human donor lungs. Lungs were inflated with 5% low-melt agarose and cooled to harden in cold PBS. 10mm core samples were sectioned into 500µm PCLS via vibratome and rested for 24 hours. PCLS were cultured in media under the following conditions: vehicle control, and TGF-β1 (5 ng/ml) ± AICAR (1µM) or FR054 (5 ng/ml). Media was refreshed at 48 hours and harvested at day 5 for RNA or fixed for immunohistochemistry (IHC). In parallel, primary human lung fibroblasts (HLFs) were stimulated with the same conditions. In both experimental models, RT-qPCR quantified fibrotic markers and HBP enzymes, and IHC assessed GFPT2 and fibronectin expression.

Results

Compared with TGF-β1 alone, FR054 + TGF-β1 significantly decreased fibronectin (FN1) and collagen (COL1A1) gene expression, while GFPT2 mRNA increased. Consistent with transcriptional changes, fibronectin staining was reduced in FR054 + TGF-β1 compared to TGF-β1 alone. Interestingly, GFPT2 reduced expression was observed via IHC despite an increase in transcript levels. Similarly, AICAR + TGF-β1 significantly decreased FN1 and COL1A1 gene expression with increased GFPT2, and IHC showed reduced fibronectin and GFPT2 staining compared with TGF-β1 alone. Findings were directionally consistent in HLFs, with AICAR and FR054 reducing TGF-β1-induced FN1 and COL1A1 gene and protein expression.

Conclusion

The data in human PCLS shows both FR054 and AICAR attenuated TGF-β1-induced fibrotic endpoints by gene expression and histology, with FR054 producing a larger and more consistently significant effect. This increased effect may reflect more direct constraint of HBP flux via PGM3 inhibition, limiting ECM/glycosylation-dependent profibrotic programs, whereas AMPK activation via AICAR exerts broader, indirect effects. These findings, along with complementary HLF studies, support further investigation of HBP-targeted modulation as a potential anti-fibrotic strategy in intact human lung tissue.

This abstract is funded by: Department of Defense