Extracellular Matrix Protection Factor-2, a metalloprotease inhibitor and potential lung fibrosis therapeutic, targets Transforming Growth Factor-β storage through the Latent Transforming Growth Factor-β Binding Protein
Location
Philadelphia, PA
Start Date
3-5-2023 1:00 PM
End Date
3-5-2023 4:00 PM
Description
Introduction: Pulmonary fibrosis is a disease that presents as an accumulation of scar tissue by the fibroblasts in the lungs. The scar tissue results from the excessive remodeling of the alveolar air spaces and the breakdown of components of the extracellular matrix (ECM) via elastolysis. Latent transforming growth factor-β binding protein (LTBP-1) is a key regulator of transforming growth factor-β (TGF-β) activation by storing the TGF-β in the ECM, thus maintaining the growth factor in a latent state. In cultured lung fibroblasts, decreased LTBP-1 leads to increased expression of fibrosis-causing TGF-β target genes. Therefore, increased production of LTBP-1 should lead to increased storage of TGF-β in the ECM and reduced fibrotic activity in the disease state.
Methods: In the current study, we measured LTBP-1 production in Human Lung Fibroblasts (HLFB) reared in serum-free media (normal (NHLFB) and COPD Diseased (DHLFB)). Commercially available early passage HLFB cells established from lung tissue, obtained during a biopsy, were cultured through passage 4 and transferred to serum-free media for treatment. Serum-free cultures were treated with a novel MMP-12 inhibitor, Extracellular Matrix Protection Factor-2 (ECPF-2), at 5 ug/ml and 10 ug/ml for 24 hours. Conditioned media (secreted fraction) and cell-associated matrix (incorporated fraction) were collected, and the total LTBP-1 produced was measured by enzyme-linked immunosorbent assay (ELISA).
Results: Normal fibroblasts (NHLFB) produced an average of 65pg/ml (secreted) and 340pg/ml (incorporated) LTBP-1. This was significantly different from the 47pg/ml (secreted) and 237pg/ml (incorporated) LTBP-1 produced by the diseased fibroblasts (DHLFB) (one-way ANOVA p< 0.01). These data are in line with published findings that demonstrate a decrease in incorporated LTBP-1 is associated with increased fibrosis in the lung. Treatment of diseased cultures with 10 ug/ml ECPF-2, an inhibitor of MMP-12 enzyme activity, resulted in an increased incorporated fraction of LTBP-1 compared to diseased serum-free control (p<0.02 by student’s t-test). At the same time, no significant effect was detected in the normal fibroblast cultures.
Discussion: This finding highlights the therapeutic potential of inhibition of MMP-12 that can lead to increased TGF-β storage, thereby slowing the progression of lung fibrosis. An Alumni Grant through the Division of Research at the Philadelphia College of Osteopathic Medicine supported this study.
Embargo Period
5-31-2023
Extracellular Matrix Protection Factor-2, a metalloprotease inhibitor and potential lung fibrosis therapeutic, targets Transforming Growth Factor-β storage through the Latent Transforming Growth Factor-β Binding Protein
Philadelphia, PA
Introduction: Pulmonary fibrosis is a disease that presents as an accumulation of scar tissue by the fibroblasts in the lungs. The scar tissue results from the excessive remodeling of the alveolar air spaces and the breakdown of components of the extracellular matrix (ECM) via elastolysis. Latent transforming growth factor-β binding protein (LTBP-1) is a key regulator of transforming growth factor-β (TGF-β) activation by storing the TGF-β in the ECM, thus maintaining the growth factor in a latent state. In cultured lung fibroblasts, decreased LTBP-1 leads to increased expression of fibrosis-causing TGF-β target genes. Therefore, increased production of LTBP-1 should lead to increased storage of TGF-β in the ECM and reduced fibrotic activity in the disease state.
Methods: In the current study, we measured LTBP-1 production in Human Lung Fibroblasts (HLFB) reared in serum-free media (normal (NHLFB) and COPD Diseased (DHLFB)). Commercially available early passage HLFB cells established from lung tissue, obtained during a biopsy, were cultured through passage 4 and transferred to serum-free media for treatment. Serum-free cultures were treated with a novel MMP-12 inhibitor, Extracellular Matrix Protection Factor-2 (ECPF-2), at 5 ug/ml and 10 ug/ml for 24 hours. Conditioned media (secreted fraction) and cell-associated matrix (incorporated fraction) were collected, and the total LTBP-1 produced was measured by enzyme-linked immunosorbent assay (ELISA).
Results: Normal fibroblasts (NHLFB) produced an average of 65pg/ml (secreted) and 340pg/ml (incorporated) LTBP-1. This was significantly different from the 47pg/ml (secreted) and 237pg/ml (incorporated) LTBP-1 produced by the diseased fibroblasts (DHLFB) (one-way ANOVA p< 0.01). These data are in line with published findings that demonstrate a decrease in incorporated LTBP-1 is associated with increased fibrosis in the lung. Treatment of diseased cultures with 10 ug/ml ECPF-2, an inhibitor of MMP-12 enzyme activity, resulted in an increased incorporated fraction of LTBP-1 compared to diseased serum-free control (p<0.02 by student’s t-test). At the same time, no significant effect was detected in the normal fibroblast cultures.
Discussion: This finding highlights the therapeutic potential of inhibition of MMP-12 that can lead to increased TGF-β storage, thereby slowing the progression of lung fibrosis. An Alumni Grant through the Division of Research at the Philadelphia College of Osteopathic Medicine supported this study.