Background
Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease associated with high mortality [
1]. Two anti-fibrotic agents, nintedanib and pirfenidone, have been approved for the treatment of IPF and demonstrated to slow the progression of the disease [
2,
3]. However, the diagnosis and management of IPF remain challenging, with no clinically available biomarkers to serve as adjuncts in diagnosis or prediction of prognosis or treatment response.
The pathobiology of IPF involves excess production of extracellular matrix (ECM) and dysregulated matrix remodeling [
4]. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases important in ECM degradation. Expression of MMPs and their physiological inhibitors, tissue inhibitors of MMPs (TIMPs), is tightly regulated in the lung, with notable upregulation during lung development, tissue injury, and host defense [
5].
Several basic and clinical studies have underscored the importance of MMPs and TIMPs in the pathobiology of IPF, as recently reviewed [
6]. In particular, murine models of bleomycin-induced pulmonary fibrosis demonstrated increased expression of MMPs and TIMPs, while mice with genetic deletions in select MMPs had reduced lung fibrosis after bleomycin administration compared with wild type mice [
6,
7]. Patients with IPF showed increased MMP and TIMP expression in the lungs [
8‐
10], including in structural cells (for example, the epithelium) and immune cells (for example, interstitial macrophages) [
8]. Circulating levels of MMPs 1, 3, 7, 8, and 9 have been shown to be elevated in patients with IPF [
9,
11] and higher circulating levels of MMP7 to be associated with more severe disease [
11], a higher risk of disease worsening over a 3-year period [
12], and shorter survival time [
13]. However, there remains a relative paucity of information on the full range of MMPs and TIMPs detectable in the blood of patients with IPF and their utility as biomarkers. We sought to determine expression of MMPs 1, 2, 3, 7, 8, 9, 12, and 13 and TIMPs 1, 2, and 4 in a cohort of well-characterized patients with IPF, to understand if combinations of MMPs and TIMPs could distinguish patients with IPF from controls, and to investigate associations between MMPs/TIMPs and measures of IPF severity.
Discussion
We present the first study to quantify expression of a broad array of circulating MMPs and TIMPs in a multicenter cohort of well-characterized patients with IPF and in controls without known lung disease. This study not only provides insights into how single MMPs/TIMPs relate to IPF status and severity, but also considers the influence of combinations of these proteins. Our results demonstrate that circulating MMPs and TIMPs are generally elevated among patients with IPF, consistent with altered extracellular matrix remodeling. The combination of MMPs 8 and 9 and TIMP1 demonstrated good performance characteristics in differentiating patients with IPF from controls with a similar distribution of age, sex, and smoking status. Moreover, several MMPs, in addition to TIMP4, strongly associated with CPI. The association between MMPs/TIMPs and CPI appeared to be driven mostly by DLCO % predicted, as no MMP/TIMP significantly associated with FVC % predicted.
In our analyses, circulating levels of MMPs 7, 8, 12, and 13 associated with both DL
CO and CPI, while MMP9 associated with CPI only. Though we found MMP2 concentrations to be higher among IPF patients with clinically significant radiographic emphysema, we did not find a significant association between MMP2 and DL
CO. MMP7 had the largest estimated effect size in our disease severity analyses. Prior work has shown that MMP7 is elevated in patients with IPF [
11,
12,
23‐
25] and is negatively correlated with DL
CO [
11,
24]. Previous studies have also suggested that circulating MMP7 concentrations increase as FVC % predicted values decline [
12] and that elevated MMP7 concentrations may identify patients with IPF with a worse prognosis [
13,
23]. However, a recent study found that while MMP7 was elevated in patients with IPF compared with controls, there was no difference in baseline concentrations of MMP7 between patients whose disease progressed or did not progress over a 52-week follow-up period [
25].
In addition to supporting the value of MMP7 as a marker of IPF severity, our work identified other MMPs associated with disease severity measures. Previous studies have shown that circulating levels of MMP8 are increased in patients with IPF [
9,
11], although they did not correlate with disease severity measures, including DL
CO, in a cohort of 74 patients [
9]. Increased circulating levels of MMP3 and MMP9 in patients with IPF have also been reported, although their association with pulmonary function was not investigated [
11]. We found that circulating concentrations of MMPs 12 and 13 were increased in patients with IPF and associated with DL
CO and CPI. Studies in murine models of pulmonary fibrosis have yielded inconsistent results regarding the roles of these MMPs after exposure to bleomycin, radiation, or other insults [
26‐
31]. Few clinical data exist regarding circulating levels of these MMPs, but MMP13 has been shown to be overexpressed in the lungs of patients with IPF [
30]. Among patients with systemic sclerosis, MMP12 concentrations were increased in patients who had interstitial lung disease compared with those who did not, and correlated with the degree of pulmonary restriction [
32]. Together, these observations support further investigation into the potential value of MMPs 12 and 13 to predict clinically relevant outcomes among patients with IPF.
In our IPF cohort, we observed higher levels of TIMP4 to be associated with lower DL
CO and higher (worse) CPI. This finding is of interest, as TIMP4 differs from other TIMPs in that at homeostasis, its expression is restricted to cardiovascular structures [
33]. Moreover, studies in individuals with pulmonary hypertension have indicated that TIMP4 levels correlate with hemodynamic parameters [
34]. It is unknown whether the association we observed between DL
CO or CPI and peripheral blood TIMP4 expression reflects pulmonary vascular remodeling and development of pulmonary hypertension, accumulating lung fibrosis, or the effects of angiogenesis supporting pulmonary hypertension and fibroplasia.
Consistent with our results, a prior study implicated MMP8 as a differentiating protein in a 5-protein classifier (MMP1, MMP7, MMP8, IGFBP-1, TNFRS1A) of IPF status, although the classification model did not select MMP9 and did not consider TIMP1 [
11]. Our results suggest that the combination of MMP8, MMP9 and TIMP1 provides good discriminatory ability in classifying individuals as having or not having IPF, with TIMP1 being the most important distinguishing variable in two of the best performing models. Collectively, these studies support the role of evaluating multiple MMP/TIMP sets to aid in determining the presence and severity of IPF. Additionally, such data may provide useful guidance for future investigations, such as those focused on MMP/TIMPs that discriminate IPF from other fibrosing lung diseases or those examining patients with early interstitial lung abnormalities of uncertain clinical significance.
When our data on circulating MMP or TIMP levels in patients with IPF are considered in the context of data from experimental models of fibrosis, a clearer view emerges of the potential mechanism(s) by which altered MMP/TIMP expression contributes to lung fibrosis. Both MMP3 and MMP7 appear critical to the development of experimental lung fibrosis, with rodents genetically deficient in MMP3 or MMP7 demonstrating a reduction in pulmonary fibrosis after bleomycin challenge [
35‐
37]. MMP7 can promote pulmonary neutrophil recruitment and chemokine dependent angiogenesis, two important processes in the development of lung fibroplasia [
37‐
42]. The pro-fibrotic nature of MMPs 3 and MMP7 is supported by our observation that higher levels of these MMPs are associated with higher CPI, i.e. more severe disease, among patients with IPF. MMP12 has been found to be pro-fibrotic during bleomycin-induced lung fibrosis [
27,
43‐
45], in line with our findings that elevated levels of MMP12 are associated with worse CPI.
In contrast to these pro-fibrotic MMPs, animal models suggest that augmented levels of MMP1 decrease fibroplasia in liver, muscle and heart [
46‐
48], while MMP2 decreased type I collagen production in experimental liver fibrosis [
49]. This could imply that the elevations in circulating MMP1 and MMP2 expression we observed in patients with IPF are a failed attempt to control lung fibrosis. MMP13 cleaves and so reduces the activity of CCL2 and CXCL12 [
29,
50]. This suggests that augmented levels of MMP13 may be anti-fibrotic by decreasing the recruitment of CCR2-expressing “pro-fibrotic” macrophages [
51] and CCR2- and CXCR4-expressing fibrocytes [
52,
53]. TIMP1−/− mice have not been shown to develop reduced lung fibrosis in response to bleomycin [
31,
54]; however, our finding of augmented levels of human TIMP1 suggests an unsuccessful attempt at regulating fibrosis in IPF. Collectively, our human data, in conjunction with data from animal models, insinuate that pro-fibrotic mediators (MMP3, MMP7, and particularly MMP12) overwhelm any anti-fibrotic effects mediated by MMP1, MMP2, MMP13, and TIMP1, leading to increased extracellular matrix deposition and impairments in pulmonary function and gas exchange.
While our study has several strengths, including the multicenter nature of the IPF cohort, we acknowledge that it has inherent limitations. First, although we characterized a broad array of MMPs, we did not include all described MMPs nor TIMP3. Additionally, while our MMP assay provides precise quantification of circulating MMP concentrations, MMP activity and organ specificity cannot be inferred. Second, as pulmonary function data were not available for the majority of subjects prior to the date of enrolment, we cannot ascertain whether patients were experiencing significant disease progression or were relatively stable at the time of sampling. Finally, although this work contributes important new information regarding circulating MMP and TIMP expression in patients with IPF, our study was not designed to understand whether the observed changes are specific to IPF as compared with non-IPF fibrosing lung diseases. As the IPF-PRO Registry has recently expanded to include patients with non-IPF interstitial lung diseases in the ILD-PRO Registry, we anticipate that future studies will address the specificity of candidate biomarkers for IPF as compared with other fibrosing lung diseases.
Acknowledgements
The authors acknowledge the IPF-PRO Registry participants and principal investigators: Wael Asi, Renovatio Clinical, The Woodlands, TX; Albert Baker, Lynchburg Pulmonary Associates, Lynchburg, VA; Scott Beegle, Albany Medical Center, Albany, NY; John A. Belperio, University of California Los Angeles, Los Angeles, CA; Rany Condos, NYU Medical Center, New York, NY; Francis Cordova, Temple University, Philadelphia, PA; Daniel A. Culver, Cleveland Clinic, Cleveland, OH; Tracey Luckhardt (formerly Joao A.M. de Andrade), University of Alabama at Birmingham, Birmingham, AL; Daniel Dilling, Loyola University Health System, Maywood, IL; Kevin R. Flaherty, University of Michigan, Ann Arbor, MI; Marilyn Glassberg, University of Miami, Miami, FL; Mridu Gulati, Yale School of Medicine, New Haven, CT; Kalpalatha Guntupalli, Baylor College of Medicine, Houston, TX; Nishant Gupta, University of Cincinnati Medical Center, Cincinnati, OH; Amy Hajari Case, Piedmont Healthcare, Austell, GA; David Hotchkin, The Oregon Clinic, Portland, OR; Tristan Huie, National Jewish Hospital, Denver, CO; Robert Kaner, Weill Cornell Medical College, New York, NY; Hyun Kim, University of Minnesota, Minneapolis, MN; Maryl Kreider, University of Pennsylvania, Philadelphia, PA; Lisa Lancaster, Vanderbilt University, Nashville, TN; Joseph Lasky, Tulane University, New Orleans, LA; David Lederer, Columbia University Medical Center/New York Presbyterian Hospital, New York, NY; Doug Lee, Wilmington Health and PMG Research, Wilmington, NC; Timothy Liesching, Lahey Clinic, Burlington, MA; Randolph Lipchik, Froedtert & The Medical College of Wisconsin Community Physicians, Milwaukee, WI; Jason Lobo, UNC Chapel Hill, Chapel Hill, NC; Yolanda Mageto, Baylor University Medical Center at Dallas, Dallas, TX; Prema Menon, Vermont Lung Center, Colchester, VT; Lake Morrison, Duke University Medical Center, Durham, NC; Andrew Namen, Wake Forest University, Winston Salem, NC; Justin Oldham, University of California, Davis, Sacramento, CA; Rishi Raj, Stanford University, Stanford, CA; Murali Ramaswamy, PulmonIx LLC, Greensboro, NC; Tonya Russell, Washington University, St. Louis, MO; Paul Sachs, Pulmonary Associates of Stamford, Stamford, CT; Zeenat Safdar, Houston Methodist Lung Center, Houston, TX; Barry Sigal, Salem Chest and Southeastern Clinical Research Center, Winston Salem, NC; Leann Silhan, UT Southwestern Medical Center, Dallas, TX; Mary Strek, University of Chicago, Chicago, IL; Sally Suliman, University of Louisville, Louisville, KY; Jeremy Tabak, South Miami Hospital, South Miami, FL; Rajat Walia, St. Joseph’s Hospital, Phoenix, AZ; Timothy P. Whelan, Medical University of South Carolina, Charleston, SC.
The authors acknowledge the contribution of Katey Durham, formerly of Boehringer Ingelheim Pharmaceuticals, Inc., to this work.
Writing support was provided by Elizabeth Ng and Wendy Morris of FleishmanHillard Fishburn, London, UK, which was contracted and funded by Boehringer Ingelheim Pharmaceuticals, Inc. The authors meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICMJE). Boehringer Ingelheim was given the opportunity to review the manuscript for medical and scientific accuracy as well as intellectual property considerations.
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