Pharmacologic inhibition of lactate production prevents myofibroblast differentiation

Abstract

Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. Transforming growth factor-β (TGF-β) has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-β. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-β-induced myofibroblast differentiation. Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-β and or gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Gossypol inhibited TGF-β-induced expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in a dose-dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3, and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose-dependent manner. Furthermore, gossypol inhibited TGF-β bioactivity in a dose-dependent manner. Concurrent treatment with an LDH siRNA increased the ability of gossypol to inhibit TGF-β-induced myofibroblast differentiation. Gossypol inhibits TGF-β-induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid, and inhibition of TGF-β bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis.

pulmonary fibrosis is a devastating disease that currently has few effective therapies (24). Although the pathogenesis of pulmonary fibrosis is not completely understood, the differentiation of fibroblasts into myofibroblasts is thought to play a central role (11). Myofibroblasts are one of the primary cell types responsible for the generation of extracellular matrix proteins such as collagen (5). Targeting excess myofibroblast differentiation has become an attractive target for therapies for pulmonary fibrosis.

Transforming growth factor-β (TGF-β) is widely considered to be the primary cytokine responsible for the induction of myofibroblast differentiation and has been implicated as being a critical factor in the development of pulmonary fibrosis in vivo (8, 17, 29). Fibroblasts treated with TGF-β differentiate to a myofibroblast phenotype that generates excess extracellular matrix proteins, expresses markers of myocytes, and exhibits enhanced contractile activity (4, 5). These cellular properties are thought to contribute to the progressive restriction and diffusion impairment seen in patients with pulmonary fibrosis. Thus inhibiting the profibrotic properties of TGF-β has become an important strategy for the treatment of pulmonary fibrosis.

TGF-β is produced endogenously as a latent protein that requires activation to have bioactivity. TGF-β can be activated by mechanical stretch, by enzymatic degradation of the latency associated peptide, and by changes in temperature or pH (1, 9, 32, 36, 37). We have recently identified that physiologic alterations in pH resulting from the endogenous production of lactic acid are capable of activating latent TGF-β in cell culture (18). We have also demonstrated that overexpression of lactate dehydrogenase (LDH), the enzyme responsible for the production of lactic acid, induces myofibroblast differentiation in vitro in a pH-dependent and TGF-β-dependent manner (18). Importantly, genetic inhibition of LDH using LDHA siRNA significantly inhibits TGF-β-induced myofibroblast differentiation (18). Therefore, we hypothesized that pharmacologic inhibition of LDH will also inhibit TGF-β-induced myofibroblast differentiation.

Gossypol, a naturally occurring derivative of cotton seed oil, has been identified as an inhibitor of lactate dehydrogenase (3, 10, 13, 14). Gossypol has also been shown to promote cellular apoptosis and interfere with the regulation of the cell cycle and/or cellular proliferation (20, 21, 31, 34). Many of the properties of gossypol appear to be dose and cell type specific. Gossypol also effectively inhibits cell differentiation in several types of malignancy (19, 34, 35) and is being investigated as a potential cancer therapy (25, 27, 30). Because of these properties, we wanted to determine whether gossypol would also be effective at inhibiting TGF-β-induced myofibroblast differentiation and may therefore represent a possible treatment for pulmonary fibrosis. In this article we investigate the ability of gossypol to inhibit TGF-β-induced myofibroblast differentiation and explore the potential mechanisms through which it exerts its effects.

METHODS

Cells and reagents.

Healthy and fibrotic human lung fibroblasts were derived from tissue explants as described previously (2, 7). Informed written consent was obtained from all donors, and the collection protocol was approved by the University of Rochester Institutional Review Board. Cells were cultured in MEM (Gibco/ThermoFisher Scientific, Grand Island, NY) containing 1 g/l glucose and 10% FBS (VWR, Randor, PA). NADH and pyruvate supplements were not added to the cell culture media. TGF-β (R&D Systems, Minneapolis, MN) and gossypol (Cayman, Ann Arbor, MI) were added to the media at the concentrations indicated in the figure legends, and cells were harvested after 24–72 h as indicated in the figure legends.

LDH assay.

Cell lysates were collected as above and subsequently incubated in the presence of the working buffer containing 0.05 M potassium phosphate pH 7.5, 30 mM pyruvic acid, and 0.25 μM β-NADH with and without gossypol at 1, 5, and 10 μM concentrations for 15 min. The LDH activity was assessed using spectrophotometry using the millimolar extinction coefficient of NADH of 6.33 (28).

Alamar blue and trypan blue assays.

Fibroblasts were cultured with and without TGF-β and/or gossypol for 72 h and subsequently incubated with Alamar blue (Invitrogen, Grand Island, NY). Alamar blue reduction was determined by spectrophotometric analysis per the manufacturer's protocol. Similarly, fibroblasts were cultured with and without TGF-β and/or gossypol for 72 h, collected after exposure to 0.01% trypsin, and counted after addition of 1:1 trypan blue.

Lactate assay.

Lactate was measured in the supernatants using a commercially available assay kit (BioVision, Milpitas, CA). Fibroblasts were cultured with and without TGF-β and/or gossypol for 72 h to allow for the induction of myofibroblast differentiation. Supernatants were collected and analyzed for lactate concentrations per the manufacturer's protocol.

Western blot.

Cell lysates were analyzed on SDS-PAGE and examined for expression of α-smooth muscle actin (α-SMA; Sigma-Aldrich, St. Louis, MO), calponin (Dako, Carpinteria, CA), or LDHA (Abcam, Cambridge, MA) by Western blot as previously described (7). GAPDH (Abcam) and β-tubulin (Abcam) were utilized as loading controls.

TGF-β bioactivity assay.

Mv1Lu mink lung epithelial cells (American Type Culture Collection CCl-64) were cultured in 96-well plates as previously described (15). The Mv1Lu cells were cultured for 12 h with conditioned media from fibroblasts treated with TGF-β and/or gossypol. [³H]thymidine (1 μCi/well) was added and the cells were incubated for a further 4 h. Incorporation was measured with a Topcount Luminometer (PerkinElmer, Boston, MA).

Quantitative RT-PCR.

RNA was isolated from primary human lung fibroblast cultures as previously described (7). Quantitative (q)RT-PCR reactions were performed for COL1A and COL3A1 and compared with GAPDH as previously described (7).

Seahorse bioassay.

Extracellular acidification rates of human lung fibroblasts treated with TGF-β and/or gossypol were measured using the Seahorse XF96 system (Seahorse Bioscience, North Billerica, MA) according to the manufacturer's protocol. Fibroblasts were cultured with and without TGF-β for 72 h to allow for the induction of myofibroblast differentiation. The differentiated fibroblasts were then transferred to XF96 cell culture plates and incubated a further 24 h with TGF-β and/or gossypol, followed by determination of the extracellular acidification rate.

siRNA transfection.

LDH5 siRNA (ON-TARGET SMART-pool Thermo Scientific) was transfected using Simporter transfection reagent (Millipore, Temecula, CA). A nonspecific scrambled siRNA was used as a control. Protein knockdown was confirmed by Western blot.

Statistical analyses.

All data are expressed as means ± SD. A Student's unpaired t-test and one-way ANOVA with Tukey posttest comparisons were used to establish statistical significance using Graph Pad Prism software (version 5.04). Results were considered significant if p < 0.05.

RESULTS

Gossypol inhibits TGF-β-induced myofibroblast differentiation in healthy and fibrotic primary human lung fibroblasts in a dose-dependent manner.

Primary human lung fibroblasts were cultured with and without 1 ng/ml TGF-β and/or 1, 5, or 10 μM gossypol for 72 h. Markers of myofibroblast differentiation, including expression of α-SMA and calponin, were determined by Western blot. Gossypol inhibited both α-SMA and calponin expression in a dose-dependent manner (Fig. 1, A–D). There was some variability among normal fibroblast strains. All strains were strongly inhibited by 10 μM gossypol, while the effectiveness of 5 μM gossypol varied with different strains (Fig. 1, E and F). Immunofluorescence staining for α-SMA demonstrated increased expression of actin filaments in fibroblasts treated with TGF-β. This change in morphology was prevented by gossypol (Fig. 1G). We also tested the efficacy of gossypol in primary human lung fibroblasts isolated from donors with pulmonary fibrosis. α-SMA and calponin expression were determined by Western blot. Gossypol also inhibited α-SMA (Fig. 2, A and B) and calponin (Fig. 2, C and D) expression in fibrotic primary human lung fibroblasts in a dose-dependent manner. Among three strains of idiopathic pulmonary fibrosis (IPF) fibroblasts tested, 10 μM gossypol were effective in all three strains, and 5 μM gossypol were significantly effective in two of three strains (Fig. 2, E and F).

Fig. 1.

The LDH inhibitor gossypol inhibits transforming growth factor-β (TGF-β)-induced myofibroblast differentiation. Normal primary human lung fibroblasts were cultured with and without 1 ng/ml TGF-β and/or 1, 5, or 10 μM gossypol for 72 h. Western blot analysis of protein lysates were performed for markers of myofibroblast differentiation, α-smooth muscle actin (α-SMA; A and B) and calponin (C and D). Densitometry for n = 3 replicates per condition and representative Western blot images are shown. E and F: 2 additional strains of normal fibroblasts were treated with TGF-β and gossypol as described. α-SMA expression was determined by Western blot and densitometry. Results shown are means ± SD for n = 3 independent replicates per experiment. *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA compared with cells treated with TGF-β alone. G: immunofluorescence staining for α-SMA was performed on cell cultures treated with and without TGF-β and/or 10 μM gossypol.

Fig. 2.

Gossypol inhibits TGF-β-induced myofibroblast differentiation in fibroblasts obtained from donors with idiopathic pulmonary fibrosis (IPF). IPF fibroblasts were cultured with and without 1 ng/ml TG-β and/or 1, 5, or 10 μM gossypol for 72 h. Western blot analysis of protein lysates was performed for markers of myofibroblast differentiation α-SMA (A and B) and calponin (C and D). E and F: 2 additional strains of normal fibroblasts were treated with TGF-β and gossypol as described. α-SMA expression was determined by Western blot and densitometry. Results shown are means ± SD for n = 3 independent replicates per experiment. **P < 0.01, ***P < 0.001, by ANOVA compared with cells treated with TGF-β alone.

Gossypol inhibits TGF-β-induced extracellular matrix generation.

Primary human lung fibroblasts were cultured with and without 1 ng/ml TGF-β and/or 1, 5, or 10 μM gossypol for 24 h. Col1A1 and Col3A1 mRNA expression was determined by qRT-PCR and fibronectin expression was determined by Western blot. Gossypol inhibited both Col1A1 and Col3A1 mRNA expression (Fig. 3, A and B) and fibronectin expression (Fig. 3, C and D) in a dose-dependent manner.

Fig. 3.

Gossypol inhibits TGF-β-induced extracellular matrix generation. Primary human lung fibroblasts were cultured with and without 1 ng/ml TGF-β and/or 1, 5, or 10 μM Gossypol for 24 h. Col1A1 (A) and Col3A1 (B) mRNA induction were analyzed using quantitative RT-PCR. C and D: Western blot analysis of protein lysates was performed for fibronectin expression. Densitometry for n = 3 replicates and representative Western blot images are shown. Results shown are means ± SD for n = 3 independent replicates per experiment. *P < 0.05, **P < 0.01, ***P < 0.001, by ANOVA compared with cells treated with TGF-β alone.

Gossypol inhibits LDH activity, extracellular acidification, lactic acid production, and TGF-β bioactivity in primary human lung fibroblasts.

We hypothesized that gossypol inhibits endogenous LDH activity, thus reducing lactate production and subsequent lactate-dependent activation of latent TGF-β in the medium. To investigate the mechanism of action of gossypol, we first treated fibroblasts with TGF-β for 72 h, harvested protein lysates, and incubated the lysates with increasing concentrations of gossypol for 1 h. The LDH activity of the lysates was then determined with a standardized LDH kinetic activity assay. Gossypol inhibited LDH activity in protein lysates in a concentration-dependent manner (Fig. 4A). Next, we investigated whether gossypol could prevent the extracellular accumulation of lactate by treated fibroblasts. Lactic acid generation was also inhibited in a concentration-dependent manner (Fig. 4B). To confirm that gossypol inhibits extracellular accumulation of lactate, we measured the rate of extracellular acidification using the commercially available Seahorse bioassay. TGF-β alone significantly increased the extracellular acidification rate in the fibroblasts, expressed as milli-pH units of change per minute (Fig. 4C). Gossypol significantly inhibited TGF-β stimulated extracellular acidification (Fig. 4C). To determine whether gossypol was associated with cellular toxicity, we utilized both the Alamar blue assay and the trypan blue exclusion assay. Gossypol at 10 μM caused a 50% reduction in Alamar blue reduction, which measures mitochondrial activity (Fig. 4D). However, 3 days of continuous treatment with 10 μM gossypol only reduced the percent of cells excluding trypan blue dye by 15% (Fig. 4E). The disproportionate response in the Alamar blue assay may indicate that this highest concentration of gossypol also inhibits other NADH dependent enzymes in the mitochondria (13). Gossypol at 5 μM did not affect either Alamar blue reduction or trypan blue exclusion.

Fig. 4.

Gossypol inhibits LDH activity, extracellular acidification rates, the generation of extracellular lactic acid, and TGF-β bioactivity. A: primary human lung fibroblasts were cultured with and without 1 ng/ml TGF-β for 72 h at which time cells were lysed in a buffer that did not contain SDS. Gossypol was added to the lysates to achieve a final concentration of 1, 5, or 10 μM. LDH activity was assessed using a standardized spectrophotometric assay for the extinction of NADH. Results shown are means ± SD for 2 independent experiments with 3 replicates each. **P < 0.01, ***P < 0.001, by ANOVA compared with TGF-β treated cells; †P < 0.05, compared with untreated cells. B: lung fibroblasts were cultured with and without 1 ng/ml TGF-β and/or 1, 5, or 10 μM gossypol for 72 h. Lactic acid was measured in the supernatants using a commercially available assay. **P < 0.01, by ANOVA compared with TGF-β alone. C: fibroblasts were cultured with and without 1 ng/m, TGF-β and/or 1, 5, or 10 μM gossypol for 72 h and subsequently transferred to a Seahorse bioassay plate for an additional 24 h with fresh TGF-β and gossypol. The Seahorse XF96 analyzer was used to measure extracellular acidification rates (ECAR), expressed as milli-pH units of change per minute. ***P < 0.001, by ANOVA compared with untreated cells; n = 22 replicates each. D: fibroblasts were cultured with and without 1 ng/ml TGF-β and/or 1, 5, or 10 μM gossypol for 72 h. Cells were subsequently incubated with Alamar blue and intracellular reduction to the fluorescent product was assessed after 4 h. Results expressed as percentage of control and means ± SD for n = 4 wells per group. **P < 0.01, ***P < 0.001, by ANOVA. E: fibroblasts were cultured for 72 h as described, trypsinized, and incubated with trypan blue dye. The percentage of cells excluding the dye was determined and normalized to untreated control cells. Results shown are means ± SD for n = 3 wells per group. ***P < 0.001, by ANOVA. F: active TGF-β was determined in conditioned medium from the fibroblast cultures from C using the mink lung epithelial cell bioassay. Results are shown as percent incorporation normalized to control medium. In this assay, active TGF-β suppresses proliferation of the cells. **P < 0.01, by ANOVA compared with TGF-β alone.

Finally, we wished to confirm that reducing extracellular lactate production inhibited the activation of latent TGF-β in the culture medium. Lung fibroblasts were treated for 72 h with 1 ng/ml TGF-β and/or 1, 5, or 10 μM gossypol, and the conditioned medium was assessed in the standardized mink lung epithelial cell bioassay. In this assay, active TGF-β suppresses proliferation of the mink lung cells. Conditioned medium from gossypol-treated fibroblasts had less of a suppressing effect, demonstrating that gossypol-conditioned medium had less active TGF-β and confirming that gossypol inhibited the activation of latent TGF-β in the fibroblast cultures (Fig. 4F).

Genetic knockdown of LDH enhances the ability of gossypol to inhibit TGF-β-induced myofibroblast differentiation.

We previously reported that genetic knockdown of LDH expression inhibited TGF-β stimulated myofibroblast differentiation. Here, we wanted to compare the efficacy of gossypol as a pharmaceutical approach to the genetic approach. Primary lung fibroblasts were transfected with an LDHA siRNA or control (scrambled) siRNA, followed by treatment with TGF-β and gossypol as indicated (Fig. 5). Gossypol alone significantly inhibited TGF-β-stimulated myofibroblast differentiation, with similar efficacy to LDH siRNA alone (Fig. 5, A and C). Interestingly, LDHA siRNA also reduced background levels of α-SMA expression in cells without TGF-β stimulation (Fig. 5B), suggesting that even under unstimulated conditions, there is some basal level of TGF-β activation driven by lactate. The combination of LDH knockdown and gossypol together completely inhibited TGF-β driven myofibroblast differentiation (Fig. 5C).

Fig. 5.

Gossypol is more effective at inhibiting myofibroblast differentiation when there is concurrent genetic inhibition of LDH. Fibroblasts were transfected with either LDHA siRNA or a control siRNA and subsequently cultured with and without 1 ng/ml TGF-β and/or 5 μM gossypol for 72 h. Western blot analysis of protein lysates was performed for α-SMA expression. A: densitometry for n = 3 replicates per group (means ± SD). *P < 0.05, ***P < 0.001, by ANOVA compared with untreated cells. B: representative Western blot of cells treated with gossypol and LDH or scrambled siRNA without TGF-β. C: representative Western blot of cells treated with gossypol and LDH or scrambled siRNA plus 1 ng/ml TGF-β.

DISCUSSION

We hypothesize that TGF-β and LDH act together to create a profibrotic feed-forward loop in pulmonary fibrosis. Increased expression of LDH results in the release of lactic acid into the extracellular matrix and activation of latent TGF-β. TGF-β, in addition to promoting myofibroblast differentiation directly, also upregulates expression of LDH and production of lactic acid. Inhibition of LDH activity should interrupt this cycle by reducing activation of latent TGF-β and consequently downregulating profibrotic effector functions including myofibroblast differentiation and extracellular matrix production. Here, we have demonstrated that gossypol, a pharmacologic inhibitor of lactate dehydrogenase, does indeed inhibit TGF-β-induced myofibroblast differentiation with great efficacy. Gossypol inhibits upregulation of α-SMA and calponin, makers of myofibroblast differentiation, in normal primary human lung fibroblasts (Fig. 1), as well as in fibroblasts obtained from fibrotic lung tissue (Fig. 2). In addition, gossypol also inhibits the generation of extracellular matrix components such as collagen 1, collagen 3, and fibronectin (Fig. 3). We confirmed that the primary mechanism by which gossypol inhibits myofibroblast differentiation is by inhibiting acid-dependent activation of latent TGF-β.

We also demonstrated that gossypol inhibits myofibroblast differentiation with similar efficacy to genetic inhibition of LDH expression (Fig. 5). This is significant because genetic inhibition of gene expression presents significant challenges in a clinical setting. LDH is a promising therapeutic target for a variety of malignancies (22, 25, 27, 30, 33), and systemic inhibition of LDH is clinically feasible and can be well tolerated. Additional anti-LDH pharmacotherapies are in development along with strategies to more precisely target specific organs and cell types. It is interesting to note that gossypol was effective in inhibiting myofibroblast differentiation even in LDHA knockdown cells (Fig. 5C). There are several possible explanations for this effect, the simplest of which is that we did not achieve complete knockdown. However, it should also be remembered that lactate dehydrogenase encompasses five tetrameric isozymes made up of varying ratios of the LDH-M and LDH-H subunit peptides. RNA silencing of the LDHA gene will reduce the expression of the M subunit of LDH causing a reduction in the protein levels of the LDH2-5 isoenzymes (H₃M₁, H₂M₂, H₁M₃, and M₄, respectively); it will not affect expression of LDH1 (H₄). Although LDH5 the isoenzyme (M₄), is reported to be the primary generator of lactate, we cannot rule out a contribution from LDH1 and the LDHB gene. Thus pharmacologic inhibition of LDH activity is likely more promising than knockdown of specific subunits.

Also, it is worth noting that lactic acid will not induce the myofibroblast differentiation without a source of latent TGF-β (such as FBS) (18). This indicates that activation of latent TGF-β is an integral and necessary step in myofibroblast differentiation. As it is well known that the extracellular matrix in human lung tissue contains large stores of latent TGF-β, this suggests that inhibition of LDH activity might be a surprisingly potent clinical strategy.

While genetic inhibition of LDH activity would be difficult to implement clinically, it is worth noting that some individuals have disorders related to LDH deficiency, such as type XI glycogen storage disease (23). It would be very interesting to determine whether individuals with LDH deficiency have a lower rate of pulmonary fibrosis. Due to the sporadic nature of both diseases, this would be a difficult hypothesis to test in humans. However, the question could be addressed through development of novel mouse models, such as conditional knockdown of LDHA.

Gossypol was less effective at inhibiting TGF-β-stimulated myofibroblast differentiation of IPF fibroblasts than normal fibroblasts at the 5 μM concentration but comparably effective at the 10 μM concentration. This is consistent with our previous reports that IPF fibroblasts are comparatively resistant to other antifibrotic treatments (4, 7). IPF fibroblasts express constitutively higher levels of LDHA and higher rates of extracellular acidification (18). We hypothesize that pharmacologic inhibition of LDH activity in vivo may have two separate but related effects: inhibition of extracellular matrix production by existing myofibroblasts, and inhibition of differentiation of fibroblasts and other cells to the myofibroblast phenotype. Even if pharmacologic inhibition of LDH activity is less effective in fibrotic fibroblasts, it may prove effective in slowing disease progression. Studies are also underway to determine whether gossypol has antifibrotic activity in vivo.

Gossypol is reported to target other cellular processes in addition to LDH activity. In cancer cells it can inhibit cell cycle progression and promote apoptosis (6, 12, 19, 21, 31). Although we have focused on inhibition of LDH activity as a pathway to blocking TGF-β activity, these other activities could potentially be important in treating fibrosis. Gossypol caused a small reduction in cell viability measured by the trypan blue assay and a larger reduction in mitochondrial function measured by the Alamar blue assay, but only at the 10 μM concentration. Five micromoles of gossypol was significantly effective at inhibiting α-SMA expression in most cell strains tested (Figs. 1, 2, and 5), as well as inhibiting calponin and collagen (Fig. 3). Overall, we believe our evidence confirms that the effect of gossypol is due to inhibition of extracellular lactate, rather than a nonspecific effect of cell toxicity. Gossypol is also an inhibitor of other NAD-linked enzymes involved in the citric acid cycle including glucelaldehyde-3-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase (13). We suspect that this may be one reason for the difference in cell viability measurements between the Alamar blue and trypan blue methods, and we cannot rule out that inhibition of other enzymes in the citric acid cycle contributes to the reduction in extracellular lactate with gossypol. Inhibition of these additional enzymes would not diminish our findings that gossypol has demonstrable effects on LDH activity and the production of lactic acid (Fig. 4) but could provide additional routes by which gossypol or other LDH inhibitors could attenuate activation of latent TGF-β. We also recognize that gossypol itself is unlikely to be adopted as a human therapeutic. We show here mechanistic proof that pharmacologic inhibition of LDH activity reduces myofibroblast differentiation by blocking an important profibrotic feed-forward loop involving extracellular activation of latent TGF-β by lactate. Our expectation is that second- and third-generation LDH inhibitors currently under development in the cancer field may also be promising antifibrotic therapies. Despite the recent discovery of two new medications (Pirfenidone and Nintedanib), pulmonary fibrosis remains a progressive disease (16, 24, 26). Our data support the role of LDH inhibition in the prevention of TGF-β-induced myofibroblast differentiation and potentially in the treatment of pulmonary fibrosis.

GRANTS

This research was supported in part by National Institutes of Health Grants T32-HL-066988 and P30-ES-01247, the James W. Doran Family Endowment, the C. Jane Davis and C. Robert Davis Fund, the Greg Chandler and Guy F. Solimano Fibrosis Research Fund, and the Pulmonary Fibrosis Foundation. L. A. Wahl was supported in part by NIH Grant TL1TR000096. R. M. Kottmann was supported in part by an Empire Clinical Research Investigator Program Career Development Award, a Parker B. Francis Fellowship Award, and the William G. Stuber Medicine Fellowship Award. The project described was supported by National Center for Research Resources Grant KL2-RR-024136.

DISCLAIMER

The content is solely the responsibility of the authors and does not necessarily represent official views if the National Center for Research Resources or the National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

R.M.K., E.T., T.H.T., and P.J.S. conception and design of research; R.M.K., E.T., J.L.J., L.A.W., A.E., and K.M.O. performed experiments; R.M.K., E.T., J.L.J., L.A.W., A.E., K.M.O., and T.H.T. analyzed data; R.M.K., E.T., J.L.J., L.A.W., T.H.T., R.P.P., and P.J.S. interpreted results of experiments; R.M.K. and T.H.T. prepared figures; R.M.K., T.H.T., and P.J.S. drafted manuscript; R.M.K., J.L.J., T.H.T., R.P.P., and P.J.S. edited and revised manuscript; R.M.K., E.T., J.L.J., L.A.W., A.E., K.M.O., T.H.T., R.P.P., and P.J.S. approved final version of manuscript.