(B-41) Myeloperoxidase regulates extracellular matrix accumulation and fibroblast contractility

Introduction:: Fibrotic diseases are persistent and progressive diseases characterized by deposition of excess extracellular matrix (ECM) which thickens and stiffens the interstitial space. Fibroblasts are recognized to be a major player in propagating fibrotic diseases by depositing ECM and contracting, increasing the local tissue stiffness.^1,2 Recently, we showed fibroblast control of matrix degradation through collagenolytic protease cathepsin k.³ Fibroblasts are regulated by many factors,⁴but increasingly immune-fibroblast crosstalk is being recognized as a contributor to pro-fibrotic regulation.⁵

Fibrotic diseases are characterized by increased recruitment of neutrophils.⁶ Neutrophils can release many factors, one which may play a role in fibrosis progression is myeloperoxidase (MPO). MPO has been implicated in many fibrotic disorders,^7–10 and has been shown to play a role in regulating the ECM through increasing ECM cross-linking and aggregation,¹¹ increasing collagen synthesis in fibroblasts,¹² and inhibiting collagenolytic proteases.¹³

Our hypothesis is myeloperoxidase activity enhances the pro-fibrotic environment through direct effects on fibroblasts. We show here that fibroblast contractility and ECM accumulation are regulated by MPO in vitro while MPO knockout or inhibition prevents bleomycin-induced fibrosis in vivo.


Materials and Methods::
Fibroblast contractility




We performed traction force microscopy as we have previously described.^2,14 On 25 kPa polyacrylamide plates coated with fluorescent beads and bovine collagen type I, we plated 2,500 IMR-90 fibroblasts in each well and allowed them to attach overnight. After attachment, we added MPO (0.2 µg/ml), a nitric oxide donor (SNAP, 100 µM), and H₂O₂ (450 µM) or TGFβ (2 ng/ml) to the cells. We replenished the H₂O₂ daily for two days before performing traction force microscopy on the wells.




ECM accumulation assay




We plated IMR-90 fibroblasts into each well of a 96-well plate. Once confluent, we replaced the media with 0.1% FBS, containing ascorbic acid (20 µg/ml) and TGFβ (2 ng/ml) as we have shown previously.³ On the fourth day after confluence, we added MPO (0.2 µg/ml), H₂O₂ (450 µM) and NaCl (750 mM), replenishing the H₂O₂ on day five. On the sixth day we measured collagenolytic protease activity using fluorescent probes and a plate reader.




In vivo studies




To model fibrosis in vivo, we administered bleomycin (1.5 U/kg) intratracheally, or PBS as a vehicle control, to global MPO-knockout (MPO-ko) or C57BL/6 mice, as we have described previously.¹⁵ For MPO inhibition studies, we administered PF1355 (50 mg/kg) via oral gavage daily for 7 days, beginning on day 7. On day 13, we administered a collagenolytic probe of collagenolytic protease activity and imaged activity in vivo. All animals were harvested 14 days after bleomycin administration, and we performed hydroxyproline to quantify total lung collagen.




Results, Conclusions, and Discussions::
To evaluate MPO’s role in promoting a pro-fibrotic environment, we tested MPO as a regulator of contractility and ECM accumulation. Exogenous MPO addition increased the contractility of fibroblasts by more than 200% over control cells (Fig. 1A). Further, MPO increased contractile forces of fibroblasts slightly higher than the positive control TGFβ. As further evaluation of MPO’s role in promoting fibrosis, we identified exogenous addition of MPO to fibroblasts in vitroincreased ECM accumulation (Fig. 1B, C) and decreased ECM proteolytic activity (Fig. 1D, E). These results show MPO exhibits a direct effect on fibroblasts to promote a pro-fibrotic environment, and we questioned if MPO exhorted a pro-fibrotic role in vivo.




We used a loss-of-function model of pulmonary fibrosis to test global knockout of MPO as a regulator of total collagen deposition in bleomycin-induced fibrosis. Fourteen days after intratracheal injection of bleomycin, we harvested the lungs of MPO-ko and wild-type mice and found a significant reduction in total lung collagen compared to wild-type (WT) mice in a bleo model of fibrosis (Fig. 1F). Similarly, hydroxyproline levels were significantly reduced in C57BL/6 when treated with an oral MPO inhibitor for 7 days. To begin identifying possible mechanisms, we then investigated whether MPO-ko mice had differences in CTSK activity and found that while WT mice had consistently reduced CTSK activity (33% decrease) in the bleo model of fibrosis, bleo treated MPO-ko mice retained baseline CTSK activity (Fig. 1G). These results suggest inhibiting MPO as a target to regulate fibrotic diseases, reduce fibroblast contractility, and increase ECM proteolytic activity.




Acknowledgements (Optional): :

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