The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA Activity

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, interstitial lung disease with a poor prognosis. Although specific anti-fibrotic medications are now available, the median survival time following diagnosis remains very low, and new therapies are urgently needed. To uncover novel therapeutic targets, we examined how biochemical properties of the fibrotic lung are different from the healthy lung. Previous work identified lactate as a metabolite that is upregulated in IPF lung tissue. Importantly, inhibition of the enzyme responsible for lactate production prevents fibrosis in vivo. Further studies revealed that fibrotic lesions of the lung experience a significant decline in tissue pH, likely due to the overproduction of lactate. It is not entirely clear how cells in the lung respond to changes in extracellular pH, but a family of proton sensing G-protein coupled receptors has been shown to be activated by reductions in extracellular pH. This work examines the expression profiles of proton sensing GPCRs in non-fibrotic and IPF-derived primary human lung fibroblasts. We identify TDAG8 as a proton sensing GPCR that is upregulated in IPF fibroblasts and that knockdown of TDAG8 dampens myofibroblast differentiation. To our surprise, BTB, a proposed positive allosteric modulator of TDAG8, inhibits myofibroblast differentiation. Our data suggest that BTB does not require TDAG8 to inhibit myofibroblast differentiation, but rather inhibits myofibroblast differentiation through suppression of RhoA mediated signaling. Our work highlights the therapeutic potential of BTB as an anti-fibrotic treatment and expands upon the importance of RhoA-mediated signaling pathways in the context of myofibroblast differentiation. Furthermore, this works also suggests that TDAG8 inhibition may have therapeutic relevance in the treatment of IPF

Prevention and treatment of bleomycin-induced pulmonary fibrosis with the lactate dehydrogenase inhibitor gossypol

<div><p>Pulmonary fibrosis is a chronic and irreversible scarring disease in the lung with poor prognosis. Few therapies are available; therefore it is critical to identify new therapeutic targets. Our lab has previously identified the enzyme lactate dehydrogenase-A (LDHA) as a potential therapeutic target in pulmonary fibrosis. We found increases in LDHA protein and its metabolic product, lactate, in patients with idiopathic pulmonary fibrosis (IPF). Importantly, we described lactate as a novel pro-fibrotic mediator by acidifying the extracellular space, and activating latent transforming growth factor beta (TGF-β1) in a pH-dependent manner. We propose a pro-fibrotic feed-forward loop by which LDHA produces lactate, lactate decreases pH in the extracellular space and activates TGF-β1 which can further perpetuate fibrotic signaling. Our previous work also demonstrates that the LDHA inhibitor gossypol inhibits TGF-β1-induced myofibroblast differentiation and collagen production <i>in vitro</i>. Here, we employed a mouse model of bleomycin-induced pulmonary fibrosis to test whether gossypol inhibits pulmonary fibrosis <i>in vivo</i>. We found that gossypol dose-dependently inhibits bleomycin-induced collagen accumulation and TGF-β1 activation in mouse lungs when treatment is started on the same day as bleomycin administration. Importantly, gossypol was also effective at treating collagen accumulation when delayed 7 days following bleomycin. Our results demonstrate that inhibition of LDHA with the inhibitor gossypol is effective at both preventing and treating bleomycin-induced pulmonary fibrosis, and suggests that LDHA may be a potential therapeutic target for pulmonary fibrosis.</p></div

Gossypol prevents TGF-β1 induced fibronectin expression.

<p>Mice were exposed to bleomycin and treated with gossypol as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197936#pone.0197936.g002" target="_blank">Fig 2</a> and sacrificed on day 21. Fibronectin expression was measured in whole lung homogenates by Western blot (A) which was quantified using densitometry (B). Data are displayed as mean ± SEM. **p<0.01 by ANOVA. n = 8 mice per treatment group. (C-F) Lung tissue sections were stained for fibronectin in red and counterstained with hematoxylin. (C) Saline + vehicle, (D) saline + gossypol 20mg/kg, (E) Bleomycin + vehicle at (F), bleomycin + gossypol 20mg/kg. All images were taken at 20X magnification (G). Scale bars represent 50 μm. The inset in (C) is an isotype negative control stain.</p

Gossypol is an effective anti-fibrotic treatment.

<p>Mice were exposed to 1.5 U/kg bleomycin by OA. (A) Starting at day 7 post-bleomycin, mice were dosed with 20 mg/kg gossypol or vehicle until day of sacrifice at 21 days. (B) Collagen levels in the right lung lobes were measured by hydroxyproline assay. Data are displayed as mean ± SEM. *p≤0.05 by ANOVA. n = 7–10 mice per treatment. (C) Fibrosis scoring was performed on trichrome stained lung tissue sections. (D-F) Collagen fibers were visualized by Trichrome stain in blue. (D) Saline + vehicle, (E) bleomycin + vehicle, (F) saline + gossypol 20mg/kg, (G) bleomycin + gossypol 20mg/kg. Images were taken at 20X objective, scale bar represents 100 μm.</p

LDHA is increased in bleomycin-induced pulmonary fibrosis.

<p>Mice were exposed to saline or bleomycin via oropharyngeal aspiration (OA) and euthanized at indicated time points. (A) Total RNA was isolated from the right lung lobes and qRT-PCR was performed to measure LDHA mRNA. Data are displayed as fold change from saline controls normalized to 18S. *p≤0.05 compared to saline controls by t-test. n = 4–5 per group. (B) LDH activity was measured on lung tissue homogenates in mice administered either saline or bleomycin. ***p<0.001. (C-D) Lung tissue from mice at day 21 post bleomycin was stained for LDHA by immuno-histochemistry in red. Images were taken at 20X magnification, scale bars represent 50 μm. (E-F) Serial section immunohistochemistry was performed for LDH shown in panel E and αSMA shown in panel F. Images were taken at 40X magnification. LDHA expression localized to both macrophages (bold arrows) and areas co-staining for αSMA (open arrows), a marker of myofibroblast differentiation.</p

Gossypol prevents collagen gene expression.

<p>Mice were exposed to bleomycin and treated with gossypol as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197936#pone.0197936.g002" target="_blank">Fig 2</a> and sacrificed on day 7. Collagen 1α1 and collagen 3α1 gene expression were measured by qRT-PCR. Data are displayed as fold changes from saline and vehicle treated controls normalized to 18S. Collagen 1α1 mRNA was measured on day 7 (A) and day 21 (B). Collagen 3α1 mRNA was measured on day 7(C) and day 21 (D). Data are displayed as mean ± SEM. *p≤0.05 by ANOVA. n = 3–9 mice per group.</p

Gossypol treats bleomycin-induced fibronectin expression.

<p>Mice were exposed to bleomycin and treated with gossypol as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197936#pone.0197936.g007" target="_blank">Fig 7</a>. Lung homogenates were probed for fibronectin protein by western blot and quantified using densitometry (A). One representative blot is shown, each lane represents one mouse (B). Data are displayed as mean ± SEM. *p≤0.05 by ANOVA. n = 3–6 mice per group. (C-H) Lung tissue sections were stained for fibronectin in red and counterstained with hematoxylin. (C) Saline + vehicle, (D) saline + gossypol 20mg/kg, Bleomycin + vehicle at 10X magnification (E) and 20X magnification (F), bleomycin + gossypol 20mg/kg at 10X magnification (G) and 20X magnification (H). Scale bars represent 50 μm. Inset in (C) is an isotype negative control stain.</p

Gossypol prevents bleomycin-induced pulmonary fibrosis.

<p>(A) Mice were exposed to 1.5U/kg bleomycin via OA and treated daily with sub-cutaneous injections of the LDHA inhibitor gossypol at 5, 10, or 20 mg/kg until euthanasia at day 21. (B) Collagen levels in the right lung lobes were measured by hydroxyproline assay. Data are displayed as mean ± SEM. *p≤0.05 by ANOVA. n = 9–10 mice per group. (C) Fibrosis scores were performed on trichrome stained sections. Data are displayed as mean ± SEM. *p≤0.05 by ANOVA (D-H) Lung tissue sections were Trichrome stained for collagen fibers in blue. (D) saline + vehicle, (E) saline + gossypol 20mg/kg, (F) bleomycin + vehicle, (G) bleomycin + gossypol 20mg/kg, (H) bleomycin + gossypol 10mg/kg, (I) bleomycin + gossypol 5mg/kg. Images were taken at 20X magnification, scale bar represents 50 μm.</p