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(Chest. 2003;124:1724-1732.)
© 2003 American College of Chest Physicians

All-trans Retinoic Acid Modulates the Balance of Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 in Patients With Emphysema*

Jenny T. Mao, MD; Donald P. Tashkin, MD; Paula N. Belloni, PhD; Irene Baileyhealy, BA; Felicita Baratelli, MD and Michael D. Roth, MD

* From the Division of Pulmonary and Critical Care Medicine (Drs. Mao, Tashkin, Baratelli, and Roth), David Geffen School of Medicine at UCLA, Los Angeles; and Respiratory Diseases (Dr. Belloni and Ms. Baileyhealy), Roche Biosciences, USA, Palo Alto, CA.

Correspondence to: Jenny T. Mao, MD, Division of Pulmonary and Critical Care Medicine, Department of Medicine, CHS 37-131, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1690; e-mail: jmao{at}mednet.ucla.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objective: The balance between proteases and antiproteases plays an essential role in the pathogenesis of emphysema. This study was designed to evaluate the impact of all-trans retinoic acid (ATRA) on the balance of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in patients with emphysema.

Design and setting: As part of a clinical study, ATRA was administered to 20 patients with emphysema for 12 weeks and evaluated for its effects on plasma levels of MMP-9 and TIMP-1. Plasma MMP-9 levels were also measured in a separate cohort of patients with emphysema and matched control subjects to evaluate the relationship of circulating enzyme levels to lung disease. To further investigate the effects of ATRA on protease activity within the lung microenvironment, alveolar macrophages (AM) recovered from the lungs of active smokers with COPD were cultured with ATRA in vitro.

Measurements and results: Administration of ATRA to patients with emphysema produced a 45 ± 14% reduction (mean ± SEM) in plasma MMP-9 by enzyme-linked immunosorbent assay and a similar reduction in MMP-9 enzyme activity, while having little effect on TIMP-1 levels. Baseline MMP-9 levels were higher in patients with emphysema compared to nonsmoking control subjects, suggesting a relationship between plasma levels and the presence of lung disease. In vitro, concentrations of ATRA similar to those achieved in the plasma of study subjects significantly reduced both the production and enzyme activity of MMP-9 by AM. In the same experiments, TIMP-1 levels increased significantly, resulting in a marked reduction in the MMP-9/TIMP-1 molar ratio.

Conclusion: We conclude that ATRA can modulate protease/antiprotease balance in a manner that may impact on disease pathogenesis.

Key Words: all-trans retinoic acid • COPD • human alveolar macrophage • matrix metalloproteinase-9 • tissue inhibitor of metalloproteinase-1


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Emphysema results from chronic lung inflammation and progressive destruction of the extracellular matrix (ECM) of the lung.1 2 3 4 Alterations in protease/antiprotease balance are thought to play an important role in the pathogenesis of this disease.5 6 7 8 In a landmark study by Massaro and Massaro,9 all-trans retinoic acid (ATRA) was demonstrated to reverse anatomic and physiologic injury in an elastase-induced model of emphysema. Similar in vivo results were duplicated by Belloni et al,10 when elastase-exposed animals were treated with either ATRA or 9-cis retinoic acid. In vitro studies suggest that retinoids may interfere with the pathogenesis of COPD by regulating protease/antiprotease balance and promoting matrix regeneration. Retinoids have been shown to modulate production of matrix-degrading metalloproteinase (matrix metalloproteinase [MMP]) and tissue inhibitors of metalloproteinase (TIMP) by endothelial cells and alveolar macrophages (AM)11 12 ; regulate transforming growth factor-ß-induced collagen production13 ; attenuate cytokine-driven degradation of ECM14 ; and suppress collagenase gene expression.12 15 In addition, endogenous retinoids were found to increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants.16 Collectively, these preclinical studies suggest a potential role for ATRA as a disease-modifying treatment for emphysema.

While it is clear that alveolar septal collagen destruction and aberrant collagen repair contribute to pathogenesis of COPD, not much is known about the mechanism(s) associated with collagen turnover. AM have been implicated as a major inflammatory effector cell involved in matrix destruction.7 The presence of macrophages in bronchial biopsy samples correlates with airflow obstruction,17 and AMs are the most prominent cell recovered by BAL.18 They have the capacity to degrade and remodel the ECM, as well as the basement membrane, through the secretion of proteases and antiproteases, including MMPs and TIMPs.7 19 20 21 Activation of AM by inflammatory agents such as lipopolysaccharide and interleukin-1ß stimulates the production of MMP-9, a 92-kd gelatinase with collagenolytic and elastolytic activities. AMs also produce TIMPs, which bind to the active forms of MMPs and inactivate their proteolytic function.19 22 The balance of MMP-9 and TIMP-1, and their production by AM are believed to be an important determinant of the clinical expression of COPD.7 8 17 18 19 20 21

As part of a pilot study23 to evaluate the feasibility of ATRA as a medical therapy for emphysema, 20 patients with moderate-to-advanced emphysema were treated with a 3-month course of oral ATRA. Serial blood samples were collected and examined for changes in plasma MMP-9 and TIMP-1 as potential biomarkers of treatment-related effects. Administration of ATRA was associated with a significant reduction in plasma MMP-9 and a shift in protease/antiprotease balance in the blood. To evaluate whether this modulation of protease/antiprotease balance reflected changes occurring in the lung microenvironment, we also examined the in vitro effects of ATRA on the production of MMP-9 and TIMP-1 by AM recovered from active smokers with COPD. ATRA not only suppressed the release of MMP-9 in this setting, but increased production of TIMP-1. Our findings support the hypothesis that ATRA may be capable of modulating proteolytic activity within the lung microenvironment and favorably impact on the pathogenesis of emphysema.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
ATRA Clinical Study Design
A double-blind, placebo-controlled crossover design was employed in which qualified patients with moderate-to-advanced emphysema were randomized at a 1:1 ratio to receive either 12 weeks of ATRA followed by 12 weeks of a matching placebo, or 12 weeks of placebo followed by 12 weeks of ATRA. The randomization list was generated by a statistician without stratifications.23 Subjects met two of three pulmonary function criteria (FEV1 < 60% predicted, total lung capacity [TLC] > 110% predicted, or diffusing capacity of the lung for carbon monoxide [DLCO] < 60% predicted) and demonstrated visual evidence of emphysema on CT scan. All subjects were former tobacco smokers (>= 6 months abstinence), including two subjects with {alpha}1-antitrypsin deficiency. Written informed consent was obtained in accordance with the University of California, Los Angeles Institutional Review Board, and subjects were continued on standard medical therapy at the direction of their primary physician. Study medications were administered at a dose of 50 mg/m2/d po (10 mg gel caps) in divided doses on 4 consecutive days of every week.

Blood Samples
Serial heparinized plasma samples were obtained from each subject at multiple time points including immediately prior to the first dose (baseline) and then 3 h after administering the first dose of medications on weeks 1, 3, 8, and 12 of each treatment cycle. Samples were stored at - 80°C until analyzed. Samples from all 20 subjects were available for measurement of serum ATRA concentrations, but a complete set of plasma samples was available from only 16 of the 20 participating subjects for measurement of MMP-9 and TIMP-1.

Comparison Between Smokers With Emphysema and Paired Nonsmokers
Twenty-five lifetime nonsmokers (age range, 50 to 70 years) and 18 current tobacco smokers (age range, 55 to 71 years; 25 to 254 pack-years tobacco use) with physiologic evidence of COPD (FEV1 of 56 ± 12% predicted [mean ± SEM]) and visual evidence of emphysema on high-resolution CT scan, were recruited from the Palo Alto Veterans Affairs Hospital for studies into the pathophysiology of emphysema. Plasma samples from all of the nonsmokers and 11 of the tobacco smokers were stored at - 80°C and available for determination of MMP-9 concentrations.

BAL and AM Culture
Bronchoscopy was performed on five current heavy smokers with COPD (> 20 pack-years, FEV1/FVC < 70, age >= 45 years). Written informed consent was obtained. Subjects were prepped with a combination of topical anesthesia (20% benzocaine spray to pharynx plus 2% lidocaine as needed) and conscious sedation using midazolam and meperidine. A fiberoptic videobronchoscope (Pentax; Inglewood, CO) was advanced into the airway and wedged into a subsegment of the right middle lobe. Four 60-mL aliquots of room temperature saline solution were serially lavaged and recovered by manual syringe suction. Recovered fluid was passed through a 100-µm sterile nylon filter (Becton Dickinson; San Jose, CA) to remove mucus and particulates, pooled, and centrifuged at 300g for 8 min at 4°C. Cell pellets were washed twice in phosphate-buffered saline solution (Irvine Scientific; Santa Ana, CA) and resuspended in X-Vivo serum free medium (Biowhittaker; Walkersville, MD) to a concentration of 0.5 x 106/mL. Unstimulated control cells and cells stimulated with lipopolysaccharide (5 µg/mL; Sigma-Aldrich; St. Louis, MO) were cultured with or without ATRA (0.7 µM, Sigma-Aldrich) at 37°C. Following 24 h of incubation, the conditioned supernatants were harvested and stored at - 80°C until analyzed. Stock solutions of ATRA were prepared at a concentration of 10 mM in 100% ethanol and stored at - 80°C.

Measurement of Plasma ATRA Levels
Heparinized plasma samples were collected in foil-covered tubes, processed under yellow light, and stored in amber colored vials at - 80°C. Analyses were carried out by Cedra Corporation (Austin, TX) using a good laboratory practice-validated liquid chromatography/mass spectroscopy assay.

Measurement of MMP-9 and TIMP-1 by Enzyme-Linked Immunosorbent Assay
Concentrations of pro-MMP-9 and TIMP-1 in plasma from ATRA-treated subjects, nonsmoking control subjects, and smokers with emphysema, and in supernatants from AM cultured in vitro, were measured according to the protocol of the manufacturer using commercial enzyme-linked immunosorbent assay (ELISA) kits (Biotrak; Amersham Pharmacia Biotech; Little Chalfont, UK). One hundred microliter samples were analyzed in duplicate with cytokine standards and measured on a microplate reader (Spectra/SLT Lab Instruments; Salzburg, Austria). A standard curve was constructed and sample values determined using automated regression software (WinSeLecT; Tecan United States; Research Triangle Park, NC). The molar ratio of MMP-9/TIMP-1 was calculated using a molecular weight for pro-MMP-9 of 92 kd and a molecular weight for TIMP-1 of 28.5 kd.

Measurement of MMP-9-Specific Enzyme Activity
To confirm that changes in pro-MMP-9 concentrations at the protein level correlate with actual changes in MMP-9-specific proteolytic function, plasma and culture supernatant samples were concurrently evaluated using a MMP-9 bioassay (Biotrak; Amersham Pharmacia Biotech LTD). According to the protocol of the manufacturer, 100 µL of test sample was incubated in 96-well plates in duplicates with a modified pro-urokinase and chromogenic peptide substrate. When incubated at 37°C, the pro-urokinase was cleaved in a specific manner by biologically active MMP-9 contained in the samples. Enzyme activity was then measured by the amount of cleaved indicator peptide detected at a 405-nm wavelength and transformed into relative enzyme concentration by comparison to a standard curve.

Statistical Analysis
The effects of ATRA on plasma MMP-9 and TIMP-1 levels in vivo were determined by comparing baseline levels with those obtained at different time points during treatment using paired t tests. Similarly, the effects of ATRA on the production of MMP-9 and TIMP-1 by cultured AM were determined by paired t tests. MMP-9 plasma values in nonsmokers and tobacco smokers with emphysema were compared by t test and by a Fisher exact test with values > 35 ng/mL characterized as elevated. Changes in MMP-9/TIMP-1 ratios were determined by comparing the percentage change in MMP-9 between baseline (or control) and follow-up (or experimental conditions) to the percentage change in TIMP-1 for each subject (or experiment) by paired t test. Batch analysis was used for each subject/comparison group in order to eliminate interassay variability.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subject Characteristics and Plasma ATRA Levels Following Oral Administration
Patient demographics and baseline studies for the ATRA clinical trial are summarized in Table 1 . Overall, FEV1 averaged 1.24 L (43% predicted), TLC averaged 7.95 L (129% predicted), and mean DLCO was 11.2 mL/min/mm Hg (36% predicted). CT confirmed heterogeneously distributed emphysema of a moderate-to-severe grade in all cases. Only one patient had symptoms consistent with chronic bronchitis in addition to the presence of emphysema. As an estimate of peak ATRA plasma levels, blood was collected at 3 h after oral administration of the first dose of therapy on weeks 1, 3, 8, and 12 of active therapy. Peak ATRA levels for all subjects ranged from 0.22 to 2.27 µM, with an average of 0.77 µM (Fig 1 ).


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  		Table 1. Baseline Demographic and Pulmonary Function Characteristics*

 


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  		Figure 1. Plasma ATRA levels were determined by liquid chromatography/mass spectroscopy on blood samples collected 3 h after a single oral dose of 25 mg/m2 of ATRA. Values represent highest recorded value for each of the 20 subjects with corresponding group mean ± SD.

 
ATRA Modulates the Protein Levels of MMP-9 and TIMP-1 In Vivo
Patients with emphysema were treated with 12 weeks of oral ATRA, and plasma samples were analyzed for relative changes in the concentrations of MMP-9 and TIMP-1 protein as a measure of its effect on protease/antiprotease balance (Fig 2 ). Baseline MMP-9 levels varied considerably between different subjects, but averaged 100.3 ± 35 ng/mL. With treatment, there was a 45 ± 14% reduction in plasma MMP-9 levels 2 weeks after the start of ATRA (p < 0.01). This reduction was maintained throughout the rest of the active treatment period but with a decrease in statistical difference at the 12-week follow-up. TIMP-1 levels, however, remained relatively stable with no significant increases or decreases observed at anytime during treatment. As a result, the molar ratio of MMP-9/TIMP-1 significantly decreased during treatment (p < 0.05). Plasma levels of MMP-9 and TIMP-1 obtained during the placebo phase of therapy showed some variability over time, but these changes were not statistically significant from baseline. In the group of patients who received ATRA first, plasma MMP-9/TIMP-1 ratio measured 2 months after treatment showed an overall trend toward returning to baseline (baseline value, 115 ± 49 ng/mL; 2 months after treatment, 89.9 ± 45 ng/mL), but four subjects still had sustained reduction of their plasma MMP-9/TIMP-1 ratio at this time point (data not shown). No significant differences were seen in response patterns for the two subjects with {alpha}1-antitrypsin deficiency.



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  		Figure 2. Left, A: Oral administration of ATRA decreased plasma MMP-9 levels and the MMP-9/TIMP-1 ratios at all time points during active treatment. Plasma samples were collected at baseline and 3 h after the administration of the first dose of ATRA on weeks (Wk) 1, 3, 8, and 12 of therapy. Concentrations of MMP-9 and TIMP-1 were determined in duplicate for each time point by ELISA assay. Data shown as mean ± SEM (n = 16 subjects); *p < 0.05. The variability in MMP-9 and TIMP-1 levels primarily reflects the variation in baseline levels between different subjects rather than variability in the assay or in the pattern of change over time. Right, B: Actual pattern of changes of MMP-9 and TIMP-1 in one representative subject, over time, in response to ATRA. Results for individual subjects demonstrated limited variability for replicate measurements at each time point. Data shown as mean ± SD.

 
Plasma MMP-9 Levels Are Elevated in Smokers With Emphysema
The high baseline levels for circulating MMP-9 in our patients with emphysema, and their down-regulation in response to ATRA, suggested that plasma MMP-9 might act as a disease-related marker of pulmonary protease activity. To evaluate this further, plasma samples collected from an unrelated cohort of 25 nonsmoking control subjects and 11 patients with emphysema were evaluated for MMP-9 levels (Fig 3 ). The value for one nonsmoker was excluded from analysis as it was five SDs higher than the mean value for the group and did not correlate with the result of a simultaneous serum MMP-9 determination, which was within the normal range. Excluding this result, the presence of emphysema in current smokers was associated with a higher mean MMP-9 concentration (42.3 ± 12.7 ng/mL vs 18.3 ± 2.7 ng/mL; p < 0.05, t test) and a significantly higher incidence of elevated MMP-9 levels than observed in age-matched nonsmoking control subjects (55% vs 8%; p < 0.01, Fisher exact test).



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  		Figure 3. A significant proportion of smokers with emphysema have higher-than-normal plasma MMP-9 levels. Plasma samples from 24 lifelong nonsmokers and 11 active smokers with emphysema were evaluated for MMP-9 levels by ELISA. Both the mean value and the percentage with elevated MMP-9 levels were significantly increased in the patients with emphysema. *p < 0.05.

 
ATRA Modulates the Production of MMP-9 and TIMP-1 Protein by Bronchoalveolar Cells Collected From Smokers With COPD
Bronchoalveolar cells prepared from the BAL fluid of five smoking subjects with COPD consisted of predominately AM (> 90%). When BAL cells from patients with COPD were cultured for 24 h in media containing 0.7 µM ATRA, concentrations of MMP-9 protein in the culture supernatant were reduced by 41 ± 5% compared to untreated cells (p < 0.05; Fig 4 ). In contrast to the effects on MMP-9, treatment with ATRA increased the TIMP-1 levels to 174 ± 21% of control values (p < 0.01). As a consequence, the relative molar ratio of MMP-9/TIMP-1 decreased dramatically by 67 ± 10% (p < 0.05). Stimulation with lipopolysaccharide increased the production of MMP-9 in all cases, an effect that was similarly reduced by the presence of ATRA (data not shown). In contrast to the effects observed with AM from smokers with COPD, exposure of BAL cells collected from nonsmokers to ATRA in vitro increased the release of TIMP-1, but did not alter the production of MMP-9 protein (data not shown).



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  		Figure 4. ATRA suppressed levels of MMP-9 and increased concentrations of TIMP-1 when added to cultures of AM collected from smokers with COPD. AM were collected from the lungs of active smokers with evidence of COPD and cultured in vitro for 24 h with control media, or media containing 0.7 µM ATRA. Supernatants were analyzed for MMP-9 and TIMP-1 by ELISA. Data are shown as mean ± SEM (n = 5 subjects). *p < 0.05.

 
Changes in MMP-9 Protein Levels Reflect Changes in MMP-9 Proteolytic Activity With Both In Vivo and In Vitro Samples
MMP-9 may exist in several forms, each associated with a different level of activity, making it difficult to imply changes in proteolytic function from measured changes in MMP-9 concentration. In order to confirm that therapy with ATRA modified MMP-9 activity, plasma samples from four treated patients were re-evaluated by MMP-9 bioassay. While MMP-9 concentrations by bioassay were lower than those measured by ELISA, they clearly demonstrated reduced MMP-9 activity in response to treatment (Fig 5 , top, A). A similar relationship between ELISA measurements and bioassay results was observed in culture supernatants collected from AM exposed to ATRA in vitro. MMP-9 protein levels were reduced on average by 41 ± 5% in supernatants containing ATRA, while MMP-9 proteolytic activities were reduced on average by 30 ± 5% (Fig 5 , center, B). Further, there was a definite linear correlation (r2 = 0.82, p < 0.01) when values as determined by the two assays were compared (Fig 5 , bottom, C). The same correlation was observed with supernatants collected from the AM of nonsmoking control subjects, where exposure to ATRA failed to alter either protein levels or enzyme activity (data not shown).



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  		Figure 5. ATRA decreased MMP-9 protein levels and proteolytic activity with both in vivo and in vitro samples. Top, A: Plasma samples from four patients treated with ATRA were re-evaluated by MMP-9 bioassay and demonstrated decreased enzyme activity in response to treatment. *p <= 0.05. Center, B: AMs from patients with COPD secreted less MMP-9, as measured by both MMP-9 ELISA (left) and MMP-9 enzyme assay (right), when cultured in vitro for 24 h with 0.7 µM ATRA. MMP-9 protein levels were reduced on average by 41 ± 5% in supernatants containing ATRA, while MMP-9 proteolytic activities were reduced on average by 30 ± 5% (p < 0.01). Bottom, C: A linear correlation was observed when MMP-9 protein values obtained by ELISA were compared to enzyme activity values obtained by bioassay for AM culture supernatants from both COPD and nonsmokers (r2 = 0.82, p < 0.01).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Retinoids are pleiotropic regulatory molecules that mediate their biological activity by binding to specific intracellular retinoic acid receptors.24 25 They are important regulators of epithelial cell proliferation, differentiation, and morphogenesis.26 In vitro studies12 14 26 have demonstrated that retinoids can modulate factors involved in ECM turnover, including the production of MMPs and TIMPs. During lung organogenesis, retinoids are believed to play a critical role in alveolarization.27 28 29 Endogenous retinoids increase perinatal elastin gene expression in rat lung fibroblasts and fetal explants16 and induce expression of surfactant genes.30 In the developing rat lung, expression of retinoic acid receptors and retinoic acid storage granules correlates with alveolar septation.31 Vitamin A deficiency inhibits normal lung development, and the administration of exogenous ATRA enhances alveolarization.28 Collectively, this information suggests that retinoid therapy might counteract the pathogenesis of emphysema by decreasing ongoing destruction, orchestrating matrix regeneration, and promoting alveolar septation and growth.

Because emphysema is a heterogeneous process resulting from years of inflammation and destruction, we anticipated that standard pulmonary function tests might miss early changes in tissue remodeling. Pulmonary function measurements act as global indicators of lung function and are relatively insensitive to regional changes. Their measurement is further complicated by variability in effort, technique, bronchomotor tone, and other factors that change over time.32 33 34 As such, we were not surprised when our short-term pilot treatment with ATRA did not duplicate the striking physiologic results observed when ATRA was administered to rats.23 Although there was a suggestion of improved quality-of-life scores 3 months after completing treatment with ATRA, none of our patients had significant improvement in their pulmonary function as previously described.23

However, working under the hypothesis that ATRA-induced regulation of protease/antiprotease balance might predate the onset of clinical improvements, we examined our patients for changes in plasma MMP-9 and TIMP-1 levels as potential biochemical evidence of lung remodeling. We found that oral administration of ATRA significantly reduced plasma MMP-9 levels in these patients, while the level of TIMP-1 remained the same, resulting in a reduced MMP-9/TIMP-1 ratio. In addition, we demonstrated that pharmacologically achievable levels of ATRA reduce the production of MMP-9, while simultaneously increasing the production of TIMP-1, in BAL cells recovered from active smokers with COPD. These changes in MMP-9 and the MMP-9/TIMP ratio were not observed when patients with emphysema were treated with placebo, further suggesting a specific drug-related effect on metalloproteinase balance.

MMP-9 is secreted in a latent form as pro-MMP-9, which is biologically activated following cleavage of its 10-kd proenzyme peptide. Both pro-MMP-9 and its active form are bound by TIMP-1 in a 1:1 molar ratio, which can retard both activation and proteolytic activity. As a result, levels of pro-MMP-9 measured by ELISA may not reflect biological activity.35 To further define the functional impact of ATRA on proteolytic activity, we also examined our samples using a MMP-9–specific enzyme assay. MMP-9 activity correlated with MMP-9 protein levels in both plasma and conditioned AM culture supernatant, and decreased in response to ATRA in a similar fashion. Our data suggest that ATRA is capable of modulating plasma MMP-9 and shifting the proteolytic balance in emphysema patients in vivo. This is the first evidence that ECM remodeling may occur in human lungs in response to clinical treatment with ATRA.

In considering the response to ATRA therapy, the reduction of MMP-9 and MMP-9/TIMP-1 ratio appeared to wane after 12 weeks of ATRA treatment and did not quite reach statistical significance. A secondary analysis was performed to see if this lack of response correlated with the declining ATRA plasma levels observed at this time point.23 Six subjects had less significant reductions in MMP-9 at week 12. Four of these subjects also had decreases in their plasma ATRA levels. However, of the 10 patients with sustained reductions of MMP-9, 9 patients also had decreased plasma ATRA level after 12 weeks of therapy. We therefore did not observe a direct correlation between drug levels and modulation of MMP-9 following prolonged drug exposure. Similarly, no correlations were obvious between severity of airflow obstruction or diffusion and changes in MMP-9 over time. While the majority of subjects experienced sustained reductions in MMP-9 with extended drug use, the exact cause for waning responses in a minority of subjects remains to be elucidated.

Based on the concept that tissue-derived MMPs contribute to blood levels of these enzymes, we hypothesized that plasma levels might act as a surrogate measure and allow us to follow the pulmonary parenchymal response to therapy. The feasibility of this approach has been evaluated in several conditions including cancer, rheumatoid arthritis, systemic lupus erythematosus, vascular disease, and liver disease.36 37 38 Bosse and associates37 found that serum plasma MMP-9 levels, and the ratio of MMP-9/TIMP-1, correlated with bronchodilator responsiveness in patients with moderate-to-severe asthma. Toward this end, we measured MMP-9 protein levels in a separate cohort of subjects and found that plasma MMP-9 levels were significantly higher in patients with moderate emphysema than in healthy nonsmokers.

The differences in baseline MMP-9 values observed between patients with emphysema at the Palo Alto Veterans Affairs Hospital (Fig 3) and those studied in the ATRA study cohort likely results from the fact that these represent different cohorts of subjects assayed at different institutions. While both groups had emphysema, the cohort from the Veterans Affairs hospital had only mild-to-moderate disease on CT and as measured by FEV1 (average FEV1 of 56%), while the ATRA study cohort had moderate- to-severe lung destruction as determined by these parameters (average FEV1 only 43%). The relationship between plasma MMP-9 levels and the severity of lung disease is not yet known but may account for some of the observed differences. Interassay variability may also play a factor. While all samples at each site were run in batches and the results were highly reproducible (Fig 2 , right, B), it is more difficult to comment on interassay variation when the two groups, assayed at different sites, are compared.

To gain further insight into whether ATRA can modulate ECM turnover in the lung microenvironment, we obtained bronchoalveolar cells from active smokers with airflow obstruction and healthy nonsmokers as control. Incubation of these cells with ATRA, at average levels found within the plasma of our treated subjects, significantly reduced the production of MMP-9 while increasing the release of TIMP-1. These alterations resulted in a marked reduction of the MMP-9/TIMP-1 relative ratio in active heavy smokers with COPD, under both basal and lipopolysaccharide-stimulated conditions. This strongly suggests that plasma MMP changes in our treated patients may reflect similar changes occurring within their lungs, but a direct correlation between plasma and lung MMP-9 levels/enzymatic activity, and their modulation with ATRA in vivo, remains to be proven.

It was also interesting that the response to ATRA in cells recovered from patients with COPD differed slightly from that observed with bronchoalveolar cells obtained from healthy nonsmokers. In nonsmokers with normal lungs, ATRA had no effect on MMP-9 production, but significantly increased the production of TIMP-1. ATRA may therefore be more active at protecting against active destructive influences than in regulating ECM turnover in the normal adult lung. Our findings suggest a more targeted effect of ATRA on the pathogenesis of emphysema than on normal tissue homeostasis.

The concentration of ATRA used for our in vitro studies (0.7 µM) was chosen based on the average peak plasma concentration measured in our treated patients. This level was also within the concentration range found to mediate biological effects by others in vitro.39 Frankenberger et al12 reported that free ATRA, as well as liposomal preparations of ATRA, mediated similar effects on the production of MMP-9 and TIMP-1 in human bronchoalveolar cells recovered from five patients with COPD, five patients with sarcoidosis, and one patient with Churg-Strauss disease. They demonstrated that these alterations were regulated at the level of transcription. In that study, however, free ATRA was used at a concentration of 5 µM, a level far exceeding that achievable in plasma following oral administration. The results presented here not only confirm the capacity for ATRA to regulate MMP-9 and TIMP-1 in vitro, but correlate the changes observed in vitro to those occurring in patients in vivo at a comparable drug concentration.

In summary, results from this pilot study suggest that oral ATRA might restore more favorable protease/antiprotease balance in emphysema patients by reducing the production of MMP-9 and/or increasing the tissue levels of TIMP-1. Although this pilot work did not demonstrate that a 3-month course of ATRA reverses emphysema, the effects on MMP-9 and TIMP-1 are nevertheless interesting. MMP-9 is one of the main enzymes responsible for the macrophage-derived elastase activity in smokers.18 Release of MMP-9 and TIMP-1 is under the influence of several inflammatory mediators, cytokines, and surface molecules.21 40 41 To this end, even if retinoids are only capable of altering the proteolytic forces in the lung microenvironment and prevent the lungs from further deterioration, retinoid therapy may represent a significant new approach to the management of COPD. Clinical studies evaluating higher doses, longer treatments, and alternative retinoids are currently ongoing and should provide more evidence about biological and clinical efficacy.


   Acknowledgements
 
The authors thank John Dermand, Grace Ibrahim, Francine Estrada, and Jessica Bailow for technical assistance. Laboratory space and resources to conduct some of the in vitro studies was provided by Steven M. Dubinett, MD, David Geffen School of Medicine at University of California, Los Angeles.


   Footnotes
 
Abbreviations: AM = alveolar macrophages; ATRA = all-trans retinoic acid; DLCO = diffusing capacity of the lung for carbon monoxide; ECM = extracellular matrix; ELISA = enzyme-linked immunosorbent assay; MMP = matrix metalloproteinase; TIMP = tissue inhibitor of metalloproteinase; TLC = total lung capacity

Studies were supported by a research donation from John and Alice Moore, a Clinical Research Grant from the American Lung Association (JTM #GC022N), and with medications generously provided by Roche Laboratories, Nutley, NJ.

Received for publication January 17, 2003. Accepted for publication May 27, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Thurlbeck, WM (1990) Pathophysiology of chronic obstructive pulmonary disease. Clin Chest Med 11,389-403[ISI][Medline]
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  3. Sethi, JM, Rochester, CL Smoking and chronic obstructive pulmonary disease. Clin Chest Med 2000;21,67-86[CrossRef][ISI][Medline]
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