Author + information
- Received October 10, 2013
- Revision received February 25, 2014
- Accepted March 7, 2014
- Published online August 1, 2014.
- Zeenat Safdar, MD∗∗ (, )
- Emilio Tamez, BA∗,
- Wenyaw Chan, PhD†,
- Basant Arya, MD‡,
- Yimin Ge, MD§,
- Anita Deswal, MD, MPH‡,
- Biykem Bozkurt, MD, PhD‡,
- Adaani Frost, MD∗ and
- Mark Entman, MD‡
- ∗Division of Pulmonary, Critical Care, and Sleep Medicine, Baylor College of Medicine, and Houston Methodist Hospital, Houston, Texas
- †University of Texas School of Public Health, Houston, Texas
- ‡Department of Cardiology, Baylor College of Medicine, Houston, Texas
- §Houston Methodist Hospital, Houston, Texas
- ↵∗Reprint requests and correspondence:
Dr. Zeenat Safdar, Division of Pulmonary, Critical Care, and Sleep Medicine, Baylor College of Medicine, 6620 Main Street, Suite 11B.09, Houston, Texas 77030.
Objectives The goal of this study was to determine if biomarkers of collagen metabolism in PAH identify patients with worse disease and higher risk of death.
Background The relationship between circulating markers of collagen metabolism, degree of disease severity, and outcome in pulmonary arterial hypertension (PAH) is unknown.
Methods Patients with stable idiopathic, anorexigen-associated, and hereditary PAH were prospectively enrolled. Levels of the following collagen biomarkers were measured: N-terminal pro-peptide of type III procollagen (PIIINP), C-terminal telopeptide of collagen type I (CITP), matrix metalloproteinase (MMP)-9, and tissue inhibitor of metalloproteinase (TIMP)-1. Patients were divided into mild, moderate, and severe PAH groups. Data were compared between tertiles of each biomarker. Pearson correlation and Spearman rank coefficient analyses were performed. Data on time to death or transplantation were examined by Kaplan-Meier survival curves.
Results Circulating levels of PIIINP, CITP, MMP-9, and TIMP-1 were higher in the PAH group (n = 68) as compared with age- and sex-matched healthy controls (n = 37) (p < 0.001 for each). PIIINP levels increased with the severity of disease (p = 0.002). PIIINP tertile data indicated that with increasing levels, 6-min walk distance and cardiac index decreased, World Health Organization functional classification worsened, and resting heart rate increased. A significant correlation existed between PIIINP levels and worsening World Health Organization functional classification (rs = 0.320; p < 0.01), and there was a negative correlation between cardiac index and 6-min walk distance (r = −0.304 and r = −0.362, respectively; p < 0.05). PIIINP tertiles showed a trend toward worse outcome in patients with higher tertiles (lung transplant or death) (p = 0.07; log-rank test).
Conclusions Markers of collagen metabolism were associated with worse disease in patients with PAH.
Pulmonary arterial hypertension (PAH) is a terminal disease characterized by pulmonary vascular remodeling resulting in right heart failure and death. Vascular remodeling and fibrosis are among the key pathological features in PAH. One of the main features of vascular remodeling seen in PAH is collagen deposition in the remodeled pulmonary vessels. The best way to quantify collagen deposition in the pulmonary vasculature is by tissue analysis at autopsy or of explanted lungs. Antemortem assessment of collagen in the pulmonary vasculature is not possible with current imaging, and lung biopsy is not considered safe.
Type I and III collagen, the most abundant forms of collagen in the human lungs, provide architectural support for the alveolar walls, vessels, visceral pleura, and tracheobronchial tree and are primarily synthesized and secreted by lung fibroblasts as procollagen precursor molecules with pro-peptides at both ends (1). The N-terminal pro-peptide of type III procollagen (PIIINP) is used as a biological marker of collagen metabolism because it is not completely removed from its procollagen precursor (2). Carboxy-terminal telopeptide of type I collagen (CITP) is a marker of extracellular collagen I degradation. Historically, matrix metalloproteinase (MMP)-9, a gelatinase that degrades most fibrillar collagen, is considered a marker of extracellular matrix breakdown. However, recent data suggest that MMP-9 may play an important role in the inflammatory response and control of angiogenesis (3–6). Tissue inhibitor of metalloproteinase (TIMP)-1 is a ubiquitous inhibitor of all MMPs.
Collagen production and smooth muscle cell proliferation occurs in small pulmonary arteries of patients with severe PAH (7,8). Studies have shown that the transpulmonary gradient of procollagen III occurs in healthy subjects undergoing cardiac catheterization, suggesting that normal human lungs can actively synthesize collagen (9). It has been established that elevated procollagen III levels in the serum mirror changes in bronchoalveolar lavage of patients with sarcoidosis (10), interstitial pulmonary fibrosis (11), Pneumocystis carinii pneumonia (12), acute lung injury (13), and acute respiratory distress syndrome (14,15), indicating that such parenchymal changes are reflected in peripheral blood samples. These studies suggest that ongoing collagen metabolism in the pulmonary vascular bed can be assessed by measuring circulating levels of collagen metabolites. Accordingly, the objectives of this study were to investigate the relationship between circulating markers of collagen metabolism, degree of disease severity, and outcome in a well-characterized PAH cohort.
After obtaining institutional review board approval and written informed consent, consecutive subjects with PAH and age- and sex-matched healthy controls who met inclusion/exclusion criteria were prospectively enrolled in a cross-sectional observational study. Patients with PAH were enrolled from the new and established patient population followed at the Pulmonary Hypertension Clinic at Baylor College of Medicine. The diagnosis of PAH was established by the presence of mean pulmonary artery pressure ≥25 mm Hg and pulmonary capillary wedge pressure ≤15 mm Hg. The mean time between right heart catheterization and study enrollment was 1.3 ± 1.6 years. Inclusion criteria for patients with PAH were age of 18 years or older, ability to provide written informed consent, and stable PAH therapy for 1 month before enrollment. Detailed inclusion/exclusion criteria for the patients with PAH and controls are outlined in the Online Appendix. Patients with PAH were followed for up to 61 months (2.8 ± 1.4 years; range: 1.2 to 5.1 years) after enrollment, and data on transplant-free survival, transplantation, and death were collected. Histologically, the available lungs and heart tissue of 2 patients with PAH who underwent transplantation were examined. Hematoxylin and eosin stain was used to outline the pulmonary vascular remodeling, and trichrome stain was used to document collagen staining. To define the severity of PAH, patients were divided into mild, moderate, and severe PAH groups. The mild PAH group was defined by a 6-min walk distance (6MWD) of >440 m, World Health Organization functional classification (WHO FC) I to II, and right atrial pressure (RAP) ≤10 mm Hg. The severe PAH group was defined by a 6MWD of ≤350 m, WHO FC III to IV, and RAP ≥15 mm Hg. The moderate PAH group fell between the criteria for mild and severe PAH (16).
Biochemical measurements of indices of collagen metabolism
Blood was drawn once from a peripheral vein for biomarker measurements, transferred immediately into a glass tube, and allowed to clot. Serum was separated from the blood cells by centrifugation at 3000 rpm for 10 minutes; supernatant was collected and immediately stored at −80°C until simultaneous analysis. Blood was also collected in ethylenediaminetetraacetic acid collection tubes and centrifuged, and plasma was stored at −20°C for analysis. Enzyme-linked immunosorbent assays were used to determine serum CITP levels, and double antibody radioimmunoassays were used to determine PIIINP levels according to the manufacturer’s specifications. Serum MMP-9 and TIMP-1 levels were measured using 2-site sandwich enzyme-linked immunosorbent assays per the manufacturer’s protocol. A commercially available immunoassay was used for quantitative determination of brain natriuretic peptide (BNP) levels on an ADVIA Centaur analyzer system (Siemens, Erlangen, Germany) according to the manufacturer’s recommendation. Detailed methodology for biomarker measurements and echocardiography is included in the Online Appendix.
All echocardiograms were obtained by registered diagnostic cardiac sonographers and interpreted by a certified cardiologist. The mean time between echocardiography and study enrollment was 2 ± 9 months. Complete echocardiography was performed with 2-dimensional, spectral Doppler, and Doppler tissue imaging modes. Tissue Doppler imaging of the right ventricular (RV) lateral annulus obtained from the apical 4-chamber view was used to calculate the peak systolic excursion of the RV annulus (S′), and calculation of the myocardial performance index of the right ventricle was performed as described by Rudski et al (17). Notch ratio was calculated using pulse wave (doppler) signal in the RV outflow tract (18). Peak pulmonary artery systolic pressure was calculated on the basis of peak tricuspid regurgitation velocity using the following formula: pulmonary artery systolic pressure = 4 (peak velocity of TR)2 + estimated RAP, where TR = tricuspid regurgitation.
Baseline characteristics are presented as mean ± SD for continuous variables and as percentages for categorical variables. Comparisons between the control and PAH groups were performed using Student t test for most continuous variables except for skewed variables, in which Wilcoxon rank sum test was used. Fisher exact test or chi-square test was used for comparing categorical variables. Collagen biomarker levels are presented in box-and-whisker plots, in which boxes represent the 25th and 75th percentiles, the horizontal line is the median, and bars indicate the lowest 5th and highest 95th percentiles. Clinical parameters and patient baseline characteristics were compared by tertiles of each biomarker and by disease severity using chi-square test for categorical variables and analysis of variance for continuous variables. Each biomarker was also compared by disease etiology using analysis of variance.
Further analysis between biomarker levels and hemodynamic and clinical variables was performed using Pearson correlation coefficient analysis, and Spearman rank correlation coefficient analysis was used for categorical variables. For survival analysis, time to death or lung or heart/lung transplantation was estimated using the Kaplan-Meier method, and log-rank analysis used to compare differences between biomarker tertiles and disease severity. All analyses were 2 sided, and significance was indicated by a p value <0.05.
Baseline patient characteristics
A total of 105 subjects were prospectively enrolled in the study: 68 patients with idiopathic, anorexigen-associated, and hereditary PAH and 37 healthy controls (Table 1). The mean duration of disease was 2.6 ± 2.8 years for patients with PAH. As expected, BNP levels, 6MWD, Borg dyspnea score, WHO FC, and maximum heart rate and minimum oxygen saturation (during the walk test) all showed significant differences between the healthy controls and the PAH group (p < 0.001). Most notably, circulating levels of PIIINP, CITP, MMP-9, and TIMP-1 were significantly elevated in the PAH group as compared with the controls (p < 0.001 for each) (Fig. 1).
Biomarker levels according to disease severity
We explored the possibility that, because increasing disease severity is associated with progressive vascular remodeling, circulating levels of collagen deposition markers would be elevated in patients with worse disease. Consistent with this hypothesis, PIIINP levels increased with the severity of disease; they were lowest in patients with mild PAH and highest in patients with severe PAH (p = 0.002) (Fig. 2A). Similarly, a trend towards higher CITP levels was also noted with increasing disease severity (p = 0.07) (Fig. 2B), although this was not evident for MMP-9 and TIMP-1 data (Figs. 2C and 2D). As would be expected for a disease that often ends in heart failure, BNP levels followed a similar pattern, increasing with worsening disease (p = 0.002). Just as with BNP, RAP and RV systolic pressure increased with disease severity (p < 0.01), and a trend toward lower S′ was noted with worsening disease (p = 0.053). In addition, and consistent with prior reports, pericardial effusion occurred more frequently in patients with severe PAH (p = 0.021). In our cohort, patients with severe PAH were less likely to be on anticoagulant therapy (p < 0.001) and calcium channel blocker therapy (p = 0.087). There were no other appreciable differences in the medication profiles of patients on the basis of disease severity. Although age was not different between the groups, patients in the severe PAH group had shorter disease duration than those in the mild or moderate PAH groups (p = 0.001).
Disease severity and biomarker tertiles
Complementary to the preceding findings, higher tertiles of serum PIIINP levels were accompanied by worsening 6MWD (p < 0.001), elevated RAP (p = 0.013), and worsening WHO FC (p = 0.024) (Table 2). Contrary to what would be expected, patients in the higher PIIINP tertiles were younger than those in the lower tertiles (p < 0.001). Similar but less significant results were obtained by dividing data into tertiles on the basis of other biomarkers. For example, resting heart rate was significantly higher in the higher tertile groups of PIIINP, CITP, and TIMP-1 (p < 0.05 for each). The higher CITP tertile group had worse Borg dyspnea scores (p = 0.040). Also, trends toward a higher occurrence rate of pericardial effusion (p = 0.058) and worse WHO FC (p = 0.087) were seen with higher CITP tertiles. Similar to PIIINP tertiles, maximum heart rate from the walk test was increased in the higher tertiles of MMP-9 (p = 0.016).
Correlations with collagen biomarkers
We explored relationships between the collagen biomarkers and the hemodynamic and clinical indicators of PAH (Fig. 3). Of note, a weak but significant correlation existed between PIIINP and worsening WHO FC (rs = 0.320, p < 0.01), while a weak correlation was seen between CITP and Borg dyspnea score (r = −0.264; p = 0.031). In addition, a negative correlation between PIIINP and cardiac index as well as 6MWD (r = −0.304 and r = −0.362, respectively; p < 0.05) was noted. Moreover, PIIINP was negatively correlated with age (r = −0.394; p < 0.01) and duration of disease (r = −0.263; p = 0.031), further reinforcing the results of the severity and tertile analyses. These data suggest that PIIINP could be linked to more aggressive and rapid disease progression as well as severe disease. BNP was shown to have a weak, positive correlation to PIIINP (rs = 0.243; p = 0.055).
Time to death or transplantation
A total of 6 deaths occurred and 7 patients underwent transplantation (5 double lung and 2 heart/double lung transplantations) over the 61-month period of investigation, representing 19% of the sample population. Review of histology slides from transplant recipients showed characteristic lesions of PAH and increased collagen deposition in both the heart (in heart-lung transplant recipients) and lungs, as shown in Figure 4. PIIINP tertiles showed a trend toward worse outcome in patients with higher tertiles (p = 0.07; log-rank test) (Fig. 5), suggesting that increasing levels may be a marker of impending need for lung transplantation or death. However, unlike those for PIIINP, Kaplan-Meier survival curves for CITP, MMP-9, and TIMP-1 tertiles were not significantly different between tertiles (Fig. 5). As expected, the patient group with severe disease had worse outcomes (i.e., lung transplant or death) than the groups with less severe disease (p = 0.0009; log-rank test).
We report novel findings that circulating levels of PIIINP, CITP, MMP-9, and TIMP are elevated in patients with PAH as compared with age- and sex-matched healthy controls. In a small number of patients with connective tissue disease–associated PAH (n = 9) and congenital heart disease–associated PAH (n = 11), levels of PIIINP, CITP, MMP-9, and TIMP-1 were high and not significantly different from those of subjects with idiopathic, hereditary, and anorexigen-associated PAH (data not shown). These results suggest that the elevated levels are markers of disease state rather than markers of the etiology of PAH. In addition, our data suggest that PIIINP levels may be a good indicator of disease severity. Furthermore, circulating markers of new collagen formation, type 1 collagen degradation, elastase (MMP-9) activity, and inhibition of MMP by a ubiquitous inhibitor of MMPs (TIMP-1) may be indicative of active vascular remodeling and reflect clinically relevant, peripherally measurable markers of disease and outcome in PAH.
PAH causes significant morbidity and mortality, which is commonly due to progressive right heart failure and death (19–21). Our results showed that PIIINP levels are elevated in patients with PAH compared with healthy controls, and these levels correlated with markers of disease severity such as worsening WHO FC, cardiac index, and 6MWD. It was interesting that, although PIIINP levels have been shown elsewhere to increase with age (22), PIIINP levels in our study were negatively correlated with age and duration of disease. We hypothesize that this is likely because of a role in increased collagen turnover due to more aggressive vascular remodeling in patients with more severe disease. This suggestion is supported by the result that PIIINP levels were higher in patients with worsening disease and by the fact that PIIINP correlated positively with markers of worsening disease. These findings agree with those from a cohort of systemic hypertensive patients with left ventricular hypertrophy showing elevated levels of PIIINP and TIMP1 studied by Agrinier et al. (23), and the presence of excess serum PIIINP and TIMP-1 was suggested to be an indicator of cardiac fibrosis. In addition, Gonzalez et al. (24) explored the role of collagen metabolism in hypertensive subjects with heart failure, specifically the role of MMPs and TIMP-1. True to their findings, we observed increased levels of both MMP-9 and TIMP-1 in our patients with PAH, suggesting that their vasculature undergoes active and possibly excessive remodeling.
Our patients were normotensive and had higher pulmonary artery pressures, reflective of vascular remodeling in the pulmonary arteries rather than the systemic vascular bed. A significant proportion of patients with PAH were exposed to mineralocorticoid antagonists, which have been reported to influence collagen biomarkers in patients with heart failure (25,26). Consistent with that, our results also showed elevated PIIINP levels but in patients who were not being treated with a mineralocorticoid blocker (p = 0.028). Furthermore, we are investigating the effects of the mineralocorticoid blocker spironolactone on circulating collagen biomarkers in patients with PAH in an ongoing randomized clinical trial (Effects of Spironolactone on Collagen Metabolism in Patients With Pulmonary Arterial Hypertension; NCT01468571). It is possible that the treatment of patients with PAH with spironolactone may result in changes in collagen biomarker levels, and this study will provide further insight.
Because the samples were drawn from peripheral blood, the source of collagen could be the right ventricle, pulmonary vasculature, or left ventricle. However, on the basis of echocardiography performed as a part of standard care for each patient, there was no evidence of hypertensive left ventricular hypertrophy or left ventricular systolic or diastolic dysfunction. Levels of collagen metabolites have been shown to be elevated in other conditions associated with RV remodeling, such as congenital heart disease and RV pressure overload induced by pulmonary artery banding, and in animal models of RV hypertrophy (27–30). Therefore, the degree of adverse RV remodeling in more advanced cases of PAH could explain the increased levels of circulating collagen metabolites. Although the source of collagen remains unclear, the disease target and pathology suggest that it likely originated from the right ventricle and/or the pulmonary vascular bed. These claims, however, require further investigation that is beyond the scope of this study.
Severity of PAH
Consistent with the previously observed association between rapid onset and progression of disease with worse outcome, our data indicated that patients with shorter disease duration have more severe PAH, as established by parameters such as lower walk distance, worse hemodynamic indices (RAP, RV systolic pressure, pericardial effusion), worse WHO FC, and higher BNP levels (16). These findings indicated that patients were correctly assigned to their respective severity group. There was a trend toward lower S′ with increasing disease severity, suggesting worsening RV systolic function as the disease progresses. Elevated BNP levels are considered a poor prognostic indicator (31), a marker of ventricular overload (32,33), and a marker of disease severity in PAH (34). Interestingly, PIIINP levels were positively correlated with BNP levels, suggesting that PIIINP may be eligible for use as a potential indicator of disease severity as well. In addition, there was a trend toward higher CITP levels as the disease worsened, suggesting that circulating collagen I degradation markers may be reflective of ongoing vascular remodeling. Because only a trend was observed, a larger sample size may be needed to further elucidate this finding.
Biomarker tertile data
Published data suggest that PIIINP increases with age (22). Interestingly, we found that our study patients who had higher PIIINP levels were significantly younger. This counterintuitive phenomenon could be due to the potential link between increased levels of the biomarker and rapid disease progression, which we suggest and support in this paper. Accordingly, we found that higher PIIINP tertiles were indeed associated with worse disease, as indicated by low walk distance, higher RAP, and worse WHO FC. The highest tertile group for PIIINP had higher RAP, and this parameter tends to be associated with worse renal function, which may in turn mediate circulating levels of these markers. To investigate this possibility, we evaluated the glomerular filtration rate for each biomarker tertile and found no significant difference between glomerular filtration rate values across the tertiles.
Kaplan-Meier survival analysis showed a trend toward a higher rate of lung transplant or death in patients with increasing PIIINP tertiles, which is entirely congruent with our finding that higher PIIINP levels are associated with disease severity. As expected, there was a significantly higher rate of lung transplant or death in the group with severe PAH. It would be interesting to explore whether it is the pro-collagens themselves or rather the vascular remodeling in patients with worsening disease that leads to increased mortality. Perhaps a longer study with more patients could provide additional data on possible links between collagen biomarkers and outcome in PAH.
More advanced forms of PAH may lead to reduction in left ventricular mass as a result of impaired filling. It may be the case that a biological response to decreased filling/stretch may also involve activation of collagen synthesis/turnover. However, there are data linking left ventricular remodeling and left ventricular hypertrophy in left heart failure with increased levels of collagen metabolites in the peripheral circulation (27). In fact, there are reports stating that decreased left ventricular mass may result in decreased collagen levels in the left ventricle (35). Further studies will be needed to explore this possibility. Additional considerations are listed in the Online Appendix.
This study indicates that levels of circulating collagen biomarkers are elevated in patients with idiopathic, hereditary, and anorexigen-associated PAH. These findings suggest that the state of active vascular remodeling in the pulmonary vascular bed or right ventricle may be represented by circulating collagen biomarkers. We propose that circulating collagen biomarkers provide clinically relevant, peripherally measurable indices of disease severity and outcome and that elevated PIIINP levels may prove to be a useful marker for the identification of patients with severe disease and poor outcome. However, because this is one of the first studies to investigate these relationships, further research is necessary.
The authors thank Dorellyn Lee for help in sample analysis, Praveen Konasagar for sample collection, and Janice Brister for editorial support.
This study was supported in part by the National Heart, Lung, and Blood Institutehttp://dx.doi.org/10.13039/100000050 (grant K23 HL093214 to Dr. Safdar). Dr. Safdar has received support (advisory board, speakers' bureau, and consulting) from United Therapeutics, Gilead Sciences, Actelion Pharmaceuticals, and Bayer Pharmaceuticals. Dr. Frost has received clinical research support from Gilead Scienceshttp://dx.doi.org/10.13039/100005564, Pfizer Inc., Actelion Pharmaceuticalshttp://dx.doi.org/10.13039/100005646, United Therapeutics, Eli Lilly and Companyhttp://dx.doi.org/10.13039/100004312, Bayerhttp://dx.doi.org/10.13039/100004326, Novartishttp://dx.doi.org/10.13039/100004336, InterMune, GlaxoSmithKlinehttp://dx.doi.org/10.13039/100004330, Ikariahttp://dx.doi.org/10.13039/100006970, and Aires Pharmaceuticals; has served as an advisory board member/consultant for Bayer, Actelion Pharmaceuticals, Gilead Sciences, and United Therapeutics; has served as a steering committee member for study, grant awards, or registry for REVEAL (funded by Actelion Pharmaceuticals); has served as a member of the ENTELLIGENCE grant award committee (funded by Actelion Pharmaceuticals); has served as a member of the endpoint adjudication committee for the beraprost study (funded by United Therapeutics); has served as a steering committee member for AMBITION (funded by Gilead Sciences, GlaxoSmithKline, and Eli Lilly and Company); and has served as a speaker for Actelion Pharmaceuticals, Gilead Sciences, Bayer, and Pfizer Inc. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- 6-min walk distance
- brain natriuretic peptide
- carboxy-terminal telopeptide of type I collagen
- matrix metalloproteinase
- pulmonary arterial hypertension
- N-terminal pro-peptide of type III procollagen
- right atrial pressure
- right ventricular
- tissue inhibitor of metalloproteinase
- WHO FC
- World Health Organization functional classification
- Received October 10, 2013.
- Revision received February 25, 2014.
- Accepted March 7, 2014.
- American College of Cardiology Foundation
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