REJUVENATION RESEARCH Volume 17, Number 1, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/rej.2013.1478
Role of Vasoactive Intestinal Peptide in Chronic Obstructive Pulmonary Disease with Pulmonary Hypertension Donato Lacedonia,1 Giuseppe Valerio,2 Grazia Pia Palladino,1 Giovanna Elisiana Carpagnano, 1 Michele Correale, 3 Matteo Di Biase, 3 and Maria Pia Foschino Barbaro1
Abstract
Background: The aim of this study was to define the involvement of some biomarkers in patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH), with particular attention to subgroups with a PH that is ‘‘out of proportion’’ (OP). Materials and Methods: Patients with COPD without PH, with PH and marked airways obstruction, and with PH and mild airways obstruction were compared. Assays for human interleukin-6 (IL-6), leukotriene B4 (LTB4), vasoactive intestinal peptide (VIP), and endothelin-1 (ET-1) were performed on the blood samples taken during right heart catheterization (RHC) in a pulmonary artery. Results: In all, 83 patients were enrolled and divided into three groups: 37 simple COPD (mean pulmonary artery pressure [mPAP] < 25 mmHg) and 46 COPD with PH (mPAP ‡ 25 mmHg). Among the latter, those who had a mPAP ‡ 35 mmHg and forced expiratory volume in 1 sec [FEV1] ‡ 50% were classified as OP (7 patients). Patients with PH were older and had a body mass index (BMI) higher than the other groups; moreover, they had lower FEV1 and carbon monoxide diffusion (DLCO) values. A lower level of partial pressure of oxygen in arterial blood (PaO2) was observed in the group of OP patients. The levels of ET-1, IL-6, and LTB4 were similar in each group; VIP was higher in the OP patients than in simple COPD and was related to PAP. Conclusions: In the patients with COPD and PH and in particular in the group of OP PH, VIP is significantly increased, probably to correct the imbalance between vasoconstrictor and vasodilatator mediators
severe PH (systolic mean pulmonary arterial pressure [mPAP] > 45 mmHg) that cannot be related solely with COPD severity.4 This subgroup of patients defined as ‘‘out of proportion’’ (OP)5 represents a challenge for the physician, because it is not clear if this condition is due to the association of two diseases, namely pulmonary arterial hypertension (PAH) and COPD, or if it is a particular phenotype of COPD in which inflammation6 and/or vascular remodeling7 may promote the development of PH. Different cytokines, interleukin-6 (IL-6) in particular, were investigated to support the theory according to which pulmonary inflammation is also involved in vascular remodeling and thus promotes the development of PH.8 On the other hand, the imbalance between vasoconstrictor/ vasodilatation mediators such as endothelin-1 (ET-1) and vasoactive intestinal peptide (VIP), caused by endothelial dysfunction, could also have an important role in this process.9 However, whereas evidence confirms VIP
Introduction
A
ccording to the latest international guidelines, pulmonary hypertension (PH), which is secondary to chronic obstructive pulmonary diseases (COPD), is placed in the third group of the World Health Organization (WHO) classification. Furthermore, along with other lung diseases, it represents the second most frequent cause of PH after leftheart diseases.1 PH is thus a frequent co-morbidity, or maybe a complication, of COPD with a strong impact on the prognosis and outcome (under exacerbations) of these patients.2 Usually the prevalence of PH in COPD increases with the progression of disease so that mild-to-moderate PH is more probable in patients who have a forced expiratory volume in 1 sec (FEV1) less than 50%.3 However, a minority of these patients develop a PH in the first stages of the disease when FEV1 is over 50% (I–II stage Global Initiative for Chronic Obstructive Lung Disease [GOLD]) or have a
1 Institute of Respiratory Diseases and 3Department of Medical and Surgical Sciences, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy. 2 San Pietro Vernotico’s Hospital, San Pietro Vernotico, Italy.
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involvement in PAH, no data are available about its role in the development of PH in COPD. That being said, the aim of this study was to define the levels and the role of some biomarkers involved in inflammation, oxidative stress, and vascular tone in COPD with and without PH. Materials and Methods
Patients were enrolled for 2 years in the Hospital of San Pietro Vernotico (Brindisi, Italy). All consecutive patients with COPD who had low levels of oxygen (partial pressure of oxygen in arterial blood [PaO2] < 75 mmHg) and/or high levels of dyspnea (Medical Research Council [MRC] scale > 2) were evaluated by echocardiographic study. The exclusion criteria were: PAP values < 40 mmHg, history of left-cardiac disease (ejection fraction < 50%, early wave/ atrial wave [E/A] ratio < 1), aortic or mitral valve pathologies, oncology diseases, COPD exacerbation in the last 3 months, and previous treatment with long-term oxygen therapy. All subjects were former smokers for at least 1 year. All subjects gave written informed consent, and the study was approved by Ethics committee of ‘‘Ospedali Riuniti of Foggia’s University.’’ Right-heart catheterization
Right-heart catheterization (RHC; Swan-Ganz catheter, Baxter, CA) was performed to evaluate pulmonary pressures according to standard criteria: Right-atrial pressure (RAP), pulmonary artery pressure (systolic, diastolic, mean), and pulmonary artery wedge pressure (PWP) were measured at the end of expiration. Cardiac output (Q¢) and cardiac index (CI) were calculated by the Fick method (Q¢ = V¢O2/C (a-v¢)O2), measuring oxygen consumption (V¢O2) by a polarographic analyzer (Cortex, FRG) and O2 content by simultaneous arterial and mixed venous blood sampling and analysis (Omni, Chiba, Switzerland). Indexed pulmonary vascular resistance (iPVR) was calculated as follows: iPVR = [(mPAP - Pw)/CI] · 79.9. Reversibility of pulmonary artery pressure was assessed by measuring the drop of mPAP following inhalation of air containing nitric oxide (NO), in accordance with standard procedures. Pulmonary function testing
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performed with a specific commercial enzyme-linked immunosorbent assay (ELISA) (for LTB4, IL-6, Cayman Chemical Company, Ann Arbor MI; for VIP, ET-1, Cusabio Biotech Co., LTD) by following the manufacturers’ protocols. All ELISAs were tested by the internal quality management systems of Thermo Scientific and Cusabio Company. All samples were assayed in duplicate, and the reproducibility of repeated measurements was assessed by the Bland and Altman method, which shows a good reproducibility of test. Statistical analysis
Descriptive statistics (i.e., means, standard deviations, percentages) were applied to summarize the continuous and categorical variables. Overall differences among the three groups of patients (COPD, PH, OP) were tested by the oneway analysis of variance (ANOVA) and the Tukey post hoc analysis with the Spjotvol–Stoline test for unequal samples, which was performed to assess the differences between each group; the Kruskal–Wallis test was used for non-parametric variables. The relationship between the two continuous variables was determined by measuring the Pearson or Spearman correlation coefficient, according to the normal distribution of variables. Statistical Software (Statistica v. 8.0, StatSoft, Inc. 2007) was used to analyze the data. A p value £ 0.05 was considered to be significant. Results
A total of 112 patients were potentially suitable for the study; 29 patients were excluded from the study (20 refused RHC and 9 showed a PWP > 15 mmHg without clear leftheart disease on the echocardiographic study). As a result, 83 patients were enrolled. According to the RHC data, the patients were divided into three groups: (1) Simple COPD, when mPAP was less then 25 mmHg (37 patients); (2) COPD with PH, if mPAP ‡ 25 mmHg (46 pz); and (3) of the latter those who had a mPAP ‡ 35 mmHg and FEV1 ‡ 50% were classified as OP (7 pz) (Fig. 1). Patients with PH were older and had a BMI higher than those with COPD without PH; moreover, they had a worse functional status at spirometry with lower FEV1 and carbon
Baseline and post-bronchodilatator (Salbutamol 400 lg) spirometry and single-breath CO diffusion (after 10-sec breathhold) were performed in the pulmonary function laboratory of hospital by using a spirometer (Sensormedics, USA). The equipment was calibrated daily using a 3-L syringe. In accordance with GOLD guidelines, the subjects were defined as COPD when FEV1/forced vital capacity (FVC) was £ 70%. Blood sample
During RHC, 10 mL of blood was collected with the catheter when inside the pulmonary artery to obtain the concentration of biomarkers in the pulmonary circulation. After sampling, the blood was centrifuged, and the serum obtained was then stored at - 80C. Laboratory test
The assays for IL-6, leukotriene B4 (LTB4), VIP, and ET1 in the serum of all subjects enrolled in the study were
FIG. 1. Study design. RHC, right heart catherization; PWP, pulmonary artery wedge pressure; COPD, chronic obstructive pulmonary disease; PH, pulmonary hypertension; OP, out of proportion.
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Table 1. General Characteristics, Functional Status, Right Heart Catheterization Parameters, and Biomarker Levels of the Study Population COPD Mean
SD
No. (%) 37 (45%) Male/female 34/3 Age (years) 64.35 8.25 BMI (kg/m2) 24.81 3.8 FVC% 53.54 16.61 FEV1% 44.11 18.51 FEV1/FVC 55.15 9.57 DLCO% 46.19 20.69 DLCO/Va 89.46 37.47 PaO2 (mmHg) 65.32 8.75 PaCO2 (mmHg) 40.68 6.05 SaO2% 92.73 3.53 pH 7.42 0.04 6MWT (m) 341.39 135.81 PAPs (mmHg) 35.27 7.99 PAP mean (mmHg) 19.05 4.34 PWP (mmHg) 6.35 2.03 PVR (dyn*sec/cm5) 237.92 92.65 Q¢ (L/m) 5.31 1.47 VIP (pg/mL)a 42.64 3.6–153.9 ET-1 (pg/mL) a 9.9 4.05–29.68 IL-6 (pg/mL)a 10.4 8.79–22.34 7871.6 7310.3–8269.6 LTB4 (pg/mL)a
PH Mean
OP SD
39(47%) 34/5 69.9 7.1 27.43 4.04 45.74 15.79 33.69 12.07 45.24 7.89 31.33 17.44 73.15 43.17 60.3 10.2 48.28 11.54 90.35 4.41 7.37 0.06 315.69 138.2 54.97 12.28 31.82 5.7 8.89 3.09 338.17 120.74 5.49 1.85 63.48 10.2–325.2 10.38 4.5–47.7 10.04 8.79–15.75 7727.4 5081.1–8320.7
Mean
ANOVA SD
7 (8%) ’7/0 67.85 14.28 28.75 5.79 67.28 12.51 67.71 16.01 58.67 8.96 40 34.24 58.30 37.96 49.42 5.15 36.57 9.31 83.85 4.56 7.43 0.03 285.71 102.93 80.14 19.96 46 8.4 8.71 2.62 433.28 122.33 4.85 1.77 131.8 42.6–338.68 7.505 6.07–26.85 10.49 9.15–14.32 7847.4 7679.9–7969.3
p
< 0.05 < 0.01 < 0.01 < 0.001 < 0.001 < 0.01 < 0.05 < 0.001 < 0.001 < 0.001 < 0.001 0.52 < 0.001 < 0.001 < 0.001 < 0.001 0.64 < 0.05 0.61 0.23 0.10
Post hoc analysis
OP = COPD < PH OP = PH > COPD OP > PH = COPD PH < COPD < OP PH < (PH = OP) PH < COPD = OP OP < PH < COPD OP < COPD = PH PH > COPD = OP OP < PH < COPD OP = PH < COPD OP > PH > COPD OP > PH > COPD PH > COPD = OP (OP = PH) > COPD PH = OP > COPD
Two groups together in brackets in post hoc analysis indicates that both are significantly different from one another. a Results shown as median with range. COPD, chronic obstructive pulmonary disease; PH, pulmonary hypertension; OP, out of proportion; ANOVA, analysis of variance; SD, standard deviation; BMI, body mass index; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 sec; DLCO, carbon monoxide diffusing capacity; PaO2, partial pressure of oxygen in arterial blood; SaO2, oxygen saturation; 6MWT, Six-Minute Walk Test; PAP, pulmonary arterial pressure; PWP, pulmonary artery wedge pressure; PVR, pulmonary vascular resistance; Q¢, cardiac output; VIP, vasoactive intestinal protein; ET-1, endothelin-1; IL-6, interleukin-6; LTB4, leukotriene B4.
monoxide diffusing capacity (DLCO) values. Despite these differences, a lower level of PaO2 was observed in the group of patients with PH-OP and no differences were measured on the Six-Minute Walk Test (6MWT) among the three groups (Table 1). The levels of ET-1, IL-6, and LTB4 were found to be similar in each group (see Table 1 for details); VIP was higher in the patients with PH and OP than in simple COPD, even if only OP patients had significant differences when compared to simple COPD (Fig. 2). The involvement of VIP was assessed by the relationship between VIP and mPAP. The role of inflammatory factors by the relationship between VIP and IL-6 or LTB4. The analysis of correlations (Table 2) showed that there is an inverse correlation between mPAP and FEV1, but only if patients with OP were not included in the analysis (Fig. 3). This means that there is a proportional relationship between respiratory disease evolution and increasing PH. In the PHOP patients, pulmonary pressure increase is partially independent from the worsening respiratory scenario. There was a strongly negative correlation between PaO2 and mPAP; furthermore, there was a weak positive correlation between mPAP with BMI and VIP (Fig. 4). Discussion
For the first time, our data have allowed us to assess a significant increase of VIP in the patients affected by COPD
and PH that higher in OP patients and significantly related to mPAP. VIP seems not to be related to the degree of pulmonary inflammation or oxidant burden as assessed by IL-6 and LTB4. Moreover, the data confirm that when carefully evaluated, PH is frequently present in patients with COPD,
FIG. 2. Differences between vasoactive intestinal peptide (VIP) levels in each group of patients. ‘‘out-of-proportion’’ (OP) patients had a higher level of VIP than chronic obstructive pulmonary disease (COPD) patients without pulmonary hypertension (PH), although no other differences were found among the other groups. NS, not significant.
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Table 2. Correlation (Expressed as R) Between Main Functional Parameters and Biomarkers
PaO2 FEV1% DLCO% 6MWT BMI VIP ET-1 IL-6 LTB4
mPAP
PaO2
FEV1
- 0.44* - 0.38xa - 0.28# - 0.23# 0.22x 0.26x 0.00 - 0.01 0.01
- 0.06 - 0.06 0.14 - 0.07 0.09 - 0.03 - 0.22 0.03
0.47* 0.25x 0.15 0.15 - 0.28x 0.01 0.02
Bold indicates correlations that were statistically significant. a PAPm and FEV1 are correlated when patients with OP were excluded from the analysis (see Fig. 3). *p < 0.001. #p < 0.01. xp < 0.05. mPAP, mean pulmonary arterial pressure; PaO2, partial pressure of oxygen in arterial blood; FEV1, forced expiratory volume in 1 sec; DLCO, carbon dioxide diffusing capacity; 6MWT, SixMinute Walk Test; BMI, body mass index; VIP, vasoactive intestinal protein; ET-1, endothelin-1; IL-6, interleukin-6; LTB4, leukotriene B4.
and there is a subgroup of patients (8% in our population) who have a PH that cannot be strictly related with disease severity. These patients, defined as OP, are characterized by low levels of DLCO/VA and diurnal PaO2. The main limitations of the study lie in the absence of a normal control group or of healthy smoker subjects, the lack of patients with PAH, and in the choice of blood sampling during RHC, which could have influenced the results. The definition of the OP subgroup delineates patients affected by an increase of pulmonary pressure exceeding the value expected for the severity of airway obstruction. As previously demonstrated,4,5 even if these patients had relatively good FEV1 and FVC values, they had a reduction of gas exchange at the alveolar–capillary membrane level, as is shown by a worsening in DLCO and a lower level of PaO2 in comparison with the other groups of patients. The high standard deviation of ET-1 and VIP levels demonstrates that the level of these proteins is very variable; what is more, many factors can influence their production and metabolism in the pulmonary vessels bed, such as the sampling during a stressful RHC and the different extents of vascular rarefaction due to emphysema, not appropriately quantified by computed tomography or vascular scintigraphy study. VIP is a 28-amino-acid pleiotropic neuropeptide with several features—potent anti-inflammatory and immunomodulatory effects; high bronchodilator, anti-proliferative, and strong vasodilatory effects through direct action on vessels; variation of plasma volume; and modulation of vascular constriction mediated by ET-1. VIP-expressing nerve fibers are present in the tracheobronchial smooth muscle layer, sub-mucosal glands, and the walls of pulmonary and bronchial arteries and veins. Some aspects of its functions are still debated, in particular regarding whether the vasodilatation response is mediated by the presence of endothelium or not. Studies have shown that the relaxation of the arteries mediated by VIP is endothelium-independent,10,11 but other studies contradict this
hypothesis.12,13 Current opinion has it that the vessels’ response to VIP is different in the presence or absence of endothelium, because different pathways are activated.14 Indeed, when endothelium is removed, activation of K + channels in smooth muscle cells can be mediated by VIP through an increase in cyclic adenosine monophosphate (cAMP).15 On the other hand, in the presence of endothelium, VIP increases the production of NO and in this way contributes to vasodilatation.16 Such evidence suggests that in both cases VIP can relax the vascular tone, even if in the presence of endothelium its action is stronger. The development of PH in PAH is claimed to be dependent upon different pathways, such as the lack or polymorphism of genes, endothelial dysfunction, and vessel inflammation. Knockout mice lacking the VIP gene or humans presenting polymorphism of sequences encoding VIP are affected by PAH,17–19 as are patients with an underexpression of constitutive secretion of NO.20 Thus, a diminution of VIP was expected; however, the measurement showed an unexpected and interesting increase. Our data tally fairly well with the reported increased immunoreactivity for VIP and receptors21 in the epithelium and glands of the airways together with a decreased expression in the smooth muscle layer in patients affected by COPD. The increase of VIP found in this study was also measured under experimental hypoxia,22,23 which could mean that in the PH associated with COPD there is a different physiopathological mechanism compared to the PAH. Our study in humans tallies fairly well with the outcomes observed in mice, indicating that the most probable pathway is endothelial dysfunction24,25 with the imbalance between increased levels of vasoconstrictors, proliferative and pro-coagulant mediators (such as ET-1, thromboxanes, etc.), and diminished levels of vasodilators, antiproliferative, and anti-coagulant substances (such as NO, prostacyclin, VIP, and adrenomedullin, etc.). Within vasoconstrictors, previous studies found that in different respiratory diseases and in the case of COPD, ET-1 is increased in the endothelium of the pulmonary vessels,26 in pulmonary circulation during advanced GOLD levels, and during reacutization. One of the causes that can explain why ET-1 is not increased in the current study could be that our patients were studied at least 3 months after the last exacerbation and had better functional levels than previous studies. Indeed, recent studies show that even in exhaled breath condensate ET-1 is higher in patients with COPD and PH than in ones who do not have a PH.27,28 These reports, together with our results, suggest that local and not systemic production of ET-1 could be involved in the pathogenesis of PH in COPD. Therefore, in accordance with these studies, we can speculate that the increase of VIP, which we found in COPD with PH and in particular in the OP group, is a reaction against vasoconstriction. The pulmonary vessels’ rarefaction with reduction of surface-active endothelium leads to diminished secretion of mediators and under-expression of NO, the main endogenous vasodilator, whereas VIP is increased to compensate for the vessels’ vasoconstriction, although its action is not efficient enough. The supposed drivers leading to VIP secretion could be inflammation, oxidative stress, hypoxia, correlative networks within autocrine peptides, and development of PH.
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FIG. 3. Correlation between forced expiratory volume in 1 sec (FEV1) and mean pulmonary arterial pressure (mPAP) (R = - 0,38, p < 0.05). Correlation analysis referred only to patients without ‘‘out-of-proportion’’ (OP) status (A). (See the Discussion section for explanation.) Concerning inflammation and oxidant load, in our study no difference of IL-6 was found in patients with or without PH, such as in OP patients. Moreover, the level of LTB4, a marker of oxidative stress, was the same in three groups. These results, in contrast with previous ones,29 seem to suggest that inflammation and oxidative stress are not involved in the pathogenesis of PH during stable phases of COPD; however, the measurement of only two markers does not give us sufficient information about the level of inflammation. Moreover, in COPD, inflammation and oxidative stress are influenced by different factors, and the concentration of IL-6 and LTB4 measured does not reflect either the ‘‘history’’ or the evolution of diseases but shows only an ‘‘instant photo’’ in which many confounding factors can be present at the same time (bacteria and virus colonization, co-morbidities, different phenotypes, drugs, etc.). In this light, other molecular or alternative study designs are
necessary to ascertain the importance of the inflammatory process in development of PH in COPD. In conclusion, our opinion is that VIP is not directly involved in the pathogenesis of PH in COPD, but, on the contrary, its production is increased so as to try and compensate for the vasoconstriction and pulmonary pressure elevation. However, this mechanism does not seem to be efficient. This hypothesis should be confirmed by further studies. That said, our study appears to confirm that the mechanisms that lead to PH in some COPD patients are complex and need to be better understood. Furthermore, even if to date there has not been any specific treatment for PH when it is associated with COPD, it could be useful to identify this subgroup of patients to improve their follow-up, clinical approach, and, at some future date, a course of treatment.
FIG. 4. Correlation between vasoactive intestinal peptide (VIP) and mean pulmonary arterial pressure (mPAP) (R = 0.32, p < 0.05). () ‘‘Out-of-proportion’’ (OP) patients.
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Author Disclosure Statement
All authors declare that they have no conflict of interest in the subject matter or materials discussed in the manuscript. The study has not received any financial support. No offlabel investigation has been performed in the present study. D.L. designed the study, analyzed the data, and wrote the manuscript; G.V., determined study concept and performed RHC; G.P.P., performed biological investigation; G.E.C., collected and analyzed the data; M.C., analysed the data and performed the echocardiography; M.D.B., interpreted the results and made a critical review; M.P.F.B., supervised the study, interpreted the results, and provided the final approval of the version submitted. References
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Address correspondence to: Donato Lacedonia Department of Medical and Surgical Sciences Institute of Respiratory Diseases University of Foggia viale degli Aviatori 71100 – Foggia Italy E-mail: [email protected] Received: August 5, 2013 Accepted: November 11, 2013
Role of Vasoactive Intestinal Peptide in Chronic Obstructive Pulmonary Disease with Pulmonary Hypertension Donato Lacedonia,1 Giuseppe Valerio,2 Grazia Pia Palladino,1 Giovanna Elisiana Carpagnano, 1 Michele Correale, 3 Matteo Di Biase, 3 and Maria Pia Foschino Barbaro1
Abstract
Background: The aim of this study was to define the involvement of some biomarkers in patients with chronic obstructive pulmonary disease (COPD) and pulmonary hypertension (PH), with particular attention to subgroups with a PH that is ‘‘out of proportion’’ (OP). Materials and Methods: Patients with COPD without PH, with PH and marked airways obstruction, and with PH and mild airways obstruction were compared. Assays for human interleukin-6 (IL-6), leukotriene B4 (LTB4), vasoactive intestinal peptide (VIP), and endothelin-1 (ET-1) were performed on the blood samples taken during right heart catheterization (RHC) in a pulmonary artery. Results: In all, 83 patients were enrolled and divided into three groups: 37 simple COPD (mean pulmonary artery pressure [mPAP] < 25 mmHg) and 46 COPD with PH (mPAP ‡ 25 mmHg). Among the latter, those who had a mPAP ‡ 35 mmHg and forced expiratory volume in 1 sec [FEV1] ‡ 50% were classified as OP (7 patients). Patients with PH were older and had a body mass index (BMI) higher than the other groups; moreover, they had lower FEV1 and carbon monoxide diffusion (DLCO) values. A lower level of partial pressure of oxygen in arterial blood (PaO2) was observed in the group of OP patients. The levels of ET-1, IL-6, and LTB4 were similar in each group; VIP was higher in the OP patients than in simple COPD and was related to PAP. Conclusions: In the patients with COPD and PH and in particular in the group of OP PH, VIP is significantly increased, probably to correct the imbalance between vasoconstrictor and vasodilatator mediators
severe PH (systolic mean pulmonary arterial pressure [mPAP] > 45 mmHg) that cannot be related solely with COPD severity.4 This subgroup of patients defined as ‘‘out of proportion’’ (OP)5 represents a challenge for the physician, because it is not clear if this condition is due to the association of two diseases, namely pulmonary arterial hypertension (PAH) and COPD, or if it is a particular phenotype of COPD in which inflammation6 and/or vascular remodeling7 may promote the development of PH. Different cytokines, interleukin-6 (IL-6) in particular, were investigated to support the theory according to which pulmonary inflammation is also involved in vascular remodeling and thus promotes the development of PH.8 On the other hand, the imbalance between vasoconstrictor/ vasodilatation mediators such as endothelin-1 (ET-1) and vasoactive intestinal peptide (VIP), caused by endothelial dysfunction, could also have an important role in this process.9 However, whereas evidence confirms VIP
Introduction
A
ccording to the latest international guidelines, pulmonary hypertension (PH), which is secondary to chronic obstructive pulmonary diseases (COPD), is placed in the third group of the World Health Organization (WHO) classification. Furthermore, along with other lung diseases, it represents the second most frequent cause of PH after leftheart diseases.1 PH is thus a frequent co-morbidity, or maybe a complication, of COPD with a strong impact on the prognosis and outcome (under exacerbations) of these patients.2 Usually the prevalence of PH in COPD increases with the progression of disease so that mild-to-moderate PH is more probable in patients who have a forced expiratory volume in 1 sec (FEV1) less than 50%.3 However, a minority of these patients develop a PH in the first stages of the disease when FEV1 is over 50% (I–II stage Global Initiative for Chronic Obstructive Lung Disease [GOLD]) or have a
1 Institute of Respiratory Diseases and 3Department of Medical and Surgical Sciences, Department of Medical and Surgical Sciences, University of Foggia, Foggia, Italy. 2 San Pietro Vernotico’s Hospital, San Pietro Vernotico, Italy.
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involvement in PAH, no data are available about its role in the development of PH in COPD. That being said, the aim of this study was to define the levels and the role of some biomarkers involved in inflammation, oxidative stress, and vascular tone in COPD with and without PH. Materials and Methods
Patients were enrolled for 2 years in the Hospital of San Pietro Vernotico (Brindisi, Italy). All consecutive patients with COPD who had low levels of oxygen (partial pressure of oxygen in arterial blood [PaO2] < 75 mmHg) and/or high levels of dyspnea (Medical Research Council [MRC] scale > 2) were evaluated by echocardiographic study. The exclusion criteria were: PAP values < 40 mmHg, history of left-cardiac disease (ejection fraction < 50%, early wave/ atrial wave [E/A] ratio < 1), aortic or mitral valve pathologies, oncology diseases, COPD exacerbation in the last 3 months, and previous treatment with long-term oxygen therapy. All subjects were former smokers for at least 1 year. All subjects gave written informed consent, and the study was approved by Ethics committee of ‘‘Ospedali Riuniti of Foggia’s University.’’ Right-heart catheterization
Right-heart catheterization (RHC; Swan-Ganz catheter, Baxter, CA) was performed to evaluate pulmonary pressures according to standard criteria: Right-atrial pressure (RAP), pulmonary artery pressure (systolic, diastolic, mean), and pulmonary artery wedge pressure (PWP) were measured at the end of expiration. Cardiac output (Q¢) and cardiac index (CI) were calculated by the Fick method (Q¢ = V¢O2/C (a-v¢)O2), measuring oxygen consumption (V¢O2) by a polarographic analyzer (Cortex, FRG) and O2 content by simultaneous arterial and mixed venous blood sampling and analysis (Omni, Chiba, Switzerland). Indexed pulmonary vascular resistance (iPVR) was calculated as follows: iPVR = [(mPAP - Pw)/CI] · 79.9. Reversibility of pulmonary artery pressure was assessed by measuring the drop of mPAP following inhalation of air containing nitric oxide (NO), in accordance with standard procedures. Pulmonary function testing
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performed with a specific commercial enzyme-linked immunosorbent assay (ELISA) (for LTB4, IL-6, Cayman Chemical Company, Ann Arbor MI; for VIP, ET-1, Cusabio Biotech Co., LTD) by following the manufacturers’ protocols. All ELISAs were tested by the internal quality management systems of Thermo Scientific and Cusabio Company. All samples were assayed in duplicate, and the reproducibility of repeated measurements was assessed by the Bland and Altman method, which shows a good reproducibility of test. Statistical analysis
Descriptive statistics (i.e., means, standard deviations, percentages) were applied to summarize the continuous and categorical variables. Overall differences among the three groups of patients (COPD, PH, OP) were tested by the oneway analysis of variance (ANOVA) and the Tukey post hoc analysis with the Spjotvol–Stoline test for unequal samples, which was performed to assess the differences between each group; the Kruskal–Wallis test was used for non-parametric variables. The relationship between the two continuous variables was determined by measuring the Pearson or Spearman correlation coefficient, according to the normal distribution of variables. Statistical Software (Statistica v. 8.0, StatSoft, Inc. 2007) was used to analyze the data. A p value £ 0.05 was considered to be significant. Results
A total of 112 patients were potentially suitable for the study; 29 patients were excluded from the study (20 refused RHC and 9 showed a PWP > 15 mmHg without clear leftheart disease on the echocardiographic study). As a result, 83 patients were enrolled. According to the RHC data, the patients were divided into three groups: (1) Simple COPD, when mPAP was less then 25 mmHg (37 patients); (2) COPD with PH, if mPAP ‡ 25 mmHg (46 pz); and (3) of the latter those who had a mPAP ‡ 35 mmHg and FEV1 ‡ 50% were classified as OP (7 pz) (Fig. 1). Patients with PH were older and had a BMI higher than those with COPD without PH; moreover, they had a worse functional status at spirometry with lower FEV1 and carbon
Baseline and post-bronchodilatator (Salbutamol 400 lg) spirometry and single-breath CO diffusion (after 10-sec breathhold) were performed in the pulmonary function laboratory of hospital by using a spirometer (Sensormedics, USA). The equipment was calibrated daily using a 3-L syringe. In accordance with GOLD guidelines, the subjects were defined as COPD when FEV1/forced vital capacity (FVC) was £ 70%. Blood sample
During RHC, 10 mL of blood was collected with the catheter when inside the pulmonary artery to obtain the concentration of biomarkers in the pulmonary circulation. After sampling, the blood was centrifuged, and the serum obtained was then stored at - 80C. Laboratory test
The assays for IL-6, leukotriene B4 (LTB4), VIP, and ET1 in the serum of all subjects enrolled in the study were
FIG. 1. Study design. RHC, right heart catherization; PWP, pulmonary artery wedge pressure; COPD, chronic obstructive pulmonary disease; PH, pulmonary hypertension; OP, out of proportion.
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Table 1. General Characteristics, Functional Status, Right Heart Catheterization Parameters, and Biomarker Levels of the Study Population COPD Mean
SD
No. (%) 37 (45%) Male/female 34/3 Age (years) 64.35 8.25 BMI (kg/m2) 24.81 3.8 FVC% 53.54 16.61 FEV1% 44.11 18.51 FEV1/FVC 55.15 9.57 DLCO% 46.19 20.69 DLCO/Va 89.46 37.47 PaO2 (mmHg) 65.32 8.75 PaCO2 (mmHg) 40.68 6.05 SaO2% 92.73 3.53 pH 7.42 0.04 6MWT (m) 341.39 135.81 PAPs (mmHg) 35.27 7.99 PAP mean (mmHg) 19.05 4.34 PWP (mmHg) 6.35 2.03 PVR (dyn*sec/cm5) 237.92 92.65 Q¢ (L/m) 5.31 1.47 VIP (pg/mL)a 42.64 3.6–153.9 ET-1 (pg/mL) a 9.9 4.05–29.68 IL-6 (pg/mL)a 10.4 8.79–22.34 7871.6 7310.3–8269.6 LTB4 (pg/mL)a
PH Mean
OP SD
39(47%) 34/5 69.9 7.1 27.43 4.04 45.74 15.79 33.69 12.07 45.24 7.89 31.33 17.44 73.15 43.17 60.3 10.2 48.28 11.54 90.35 4.41 7.37 0.06 315.69 138.2 54.97 12.28 31.82 5.7 8.89 3.09 338.17 120.74 5.49 1.85 63.48 10.2–325.2 10.38 4.5–47.7 10.04 8.79–15.75 7727.4 5081.1–8320.7
Mean
ANOVA SD
7 (8%) ’7/0 67.85 14.28 28.75 5.79 67.28 12.51 67.71 16.01 58.67 8.96 40 34.24 58.30 37.96 49.42 5.15 36.57 9.31 83.85 4.56 7.43 0.03 285.71 102.93 80.14 19.96 46 8.4 8.71 2.62 433.28 122.33 4.85 1.77 131.8 42.6–338.68 7.505 6.07–26.85 10.49 9.15–14.32 7847.4 7679.9–7969.3
p
< 0.05 < 0.01 < 0.01 < 0.001 < 0.001 < 0.01 < 0.05 < 0.001 < 0.001 < 0.001 < 0.001 0.52 < 0.001 < 0.001 < 0.001 < 0.001 0.64 < 0.05 0.61 0.23 0.10
Post hoc analysis
OP = COPD < PH OP = PH > COPD OP > PH = COPD PH < COPD < OP PH < (PH = OP) PH < COPD = OP OP < PH < COPD OP < COPD = PH PH > COPD = OP OP < PH < COPD OP = PH < COPD OP > PH > COPD OP > PH > COPD PH > COPD = OP (OP = PH) > COPD PH = OP > COPD
Two groups together in brackets in post hoc analysis indicates that both are significantly different from one another. a Results shown as median with range. COPD, chronic obstructive pulmonary disease; PH, pulmonary hypertension; OP, out of proportion; ANOVA, analysis of variance; SD, standard deviation; BMI, body mass index; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 sec; DLCO, carbon monoxide diffusing capacity; PaO2, partial pressure of oxygen in arterial blood; SaO2, oxygen saturation; 6MWT, Six-Minute Walk Test; PAP, pulmonary arterial pressure; PWP, pulmonary artery wedge pressure; PVR, pulmonary vascular resistance; Q¢, cardiac output; VIP, vasoactive intestinal protein; ET-1, endothelin-1; IL-6, interleukin-6; LTB4, leukotriene B4.
monoxide diffusing capacity (DLCO) values. Despite these differences, a lower level of PaO2 was observed in the group of patients with PH-OP and no differences were measured on the Six-Minute Walk Test (6MWT) among the three groups (Table 1). The levels of ET-1, IL-6, and LTB4 were found to be similar in each group (see Table 1 for details); VIP was higher in the patients with PH and OP than in simple COPD, even if only OP patients had significant differences when compared to simple COPD (Fig. 2). The involvement of VIP was assessed by the relationship between VIP and mPAP. The role of inflammatory factors by the relationship between VIP and IL-6 or LTB4. The analysis of correlations (Table 2) showed that there is an inverse correlation between mPAP and FEV1, but only if patients with OP were not included in the analysis (Fig. 3). This means that there is a proportional relationship between respiratory disease evolution and increasing PH. In the PHOP patients, pulmonary pressure increase is partially independent from the worsening respiratory scenario. There was a strongly negative correlation between PaO2 and mPAP; furthermore, there was a weak positive correlation between mPAP with BMI and VIP (Fig. 4). Discussion
For the first time, our data have allowed us to assess a significant increase of VIP in the patients affected by COPD
and PH that higher in OP patients and significantly related to mPAP. VIP seems not to be related to the degree of pulmonary inflammation or oxidant burden as assessed by IL-6 and LTB4. Moreover, the data confirm that when carefully evaluated, PH is frequently present in patients with COPD,
FIG. 2. Differences between vasoactive intestinal peptide (VIP) levels in each group of patients. ‘‘out-of-proportion’’ (OP) patients had a higher level of VIP than chronic obstructive pulmonary disease (COPD) patients without pulmonary hypertension (PH), although no other differences were found among the other groups. NS, not significant.
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Table 2. Correlation (Expressed as R) Between Main Functional Parameters and Biomarkers
PaO2 FEV1% DLCO% 6MWT BMI VIP ET-1 IL-6 LTB4
mPAP
PaO2
FEV1
- 0.44* - 0.38xa - 0.28# - 0.23# 0.22x 0.26x 0.00 - 0.01 0.01
- 0.06 - 0.06 0.14 - 0.07 0.09 - 0.03 - 0.22 0.03
0.47* 0.25x 0.15 0.15 - 0.28x 0.01 0.02
Bold indicates correlations that were statistically significant. a PAPm and FEV1 are correlated when patients with OP were excluded from the analysis (see Fig. 3). *p < 0.001. #p < 0.01. xp < 0.05. mPAP, mean pulmonary arterial pressure; PaO2, partial pressure of oxygen in arterial blood; FEV1, forced expiratory volume in 1 sec; DLCO, carbon dioxide diffusing capacity; 6MWT, SixMinute Walk Test; BMI, body mass index; VIP, vasoactive intestinal protein; ET-1, endothelin-1; IL-6, interleukin-6; LTB4, leukotriene B4.
and there is a subgroup of patients (8% in our population) who have a PH that cannot be strictly related with disease severity. These patients, defined as OP, are characterized by low levels of DLCO/VA and diurnal PaO2. The main limitations of the study lie in the absence of a normal control group or of healthy smoker subjects, the lack of patients with PAH, and in the choice of blood sampling during RHC, which could have influenced the results. The definition of the OP subgroup delineates patients affected by an increase of pulmonary pressure exceeding the value expected for the severity of airway obstruction. As previously demonstrated,4,5 even if these patients had relatively good FEV1 and FVC values, they had a reduction of gas exchange at the alveolar–capillary membrane level, as is shown by a worsening in DLCO and a lower level of PaO2 in comparison with the other groups of patients. The high standard deviation of ET-1 and VIP levels demonstrates that the level of these proteins is very variable; what is more, many factors can influence their production and metabolism in the pulmonary vessels bed, such as the sampling during a stressful RHC and the different extents of vascular rarefaction due to emphysema, not appropriately quantified by computed tomography or vascular scintigraphy study. VIP is a 28-amino-acid pleiotropic neuropeptide with several features—potent anti-inflammatory and immunomodulatory effects; high bronchodilator, anti-proliferative, and strong vasodilatory effects through direct action on vessels; variation of plasma volume; and modulation of vascular constriction mediated by ET-1. VIP-expressing nerve fibers are present in the tracheobronchial smooth muscle layer, sub-mucosal glands, and the walls of pulmonary and bronchial arteries and veins. Some aspects of its functions are still debated, in particular regarding whether the vasodilatation response is mediated by the presence of endothelium or not. Studies have shown that the relaxation of the arteries mediated by VIP is endothelium-independent,10,11 but other studies contradict this
hypothesis.12,13 Current opinion has it that the vessels’ response to VIP is different in the presence or absence of endothelium, because different pathways are activated.14 Indeed, when endothelium is removed, activation of K + channels in smooth muscle cells can be mediated by VIP through an increase in cyclic adenosine monophosphate (cAMP).15 On the other hand, in the presence of endothelium, VIP increases the production of NO and in this way contributes to vasodilatation.16 Such evidence suggests that in both cases VIP can relax the vascular tone, even if in the presence of endothelium its action is stronger. The development of PH in PAH is claimed to be dependent upon different pathways, such as the lack or polymorphism of genes, endothelial dysfunction, and vessel inflammation. Knockout mice lacking the VIP gene or humans presenting polymorphism of sequences encoding VIP are affected by PAH,17–19 as are patients with an underexpression of constitutive secretion of NO.20 Thus, a diminution of VIP was expected; however, the measurement showed an unexpected and interesting increase. Our data tally fairly well with the reported increased immunoreactivity for VIP and receptors21 in the epithelium and glands of the airways together with a decreased expression in the smooth muscle layer in patients affected by COPD. The increase of VIP found in this study was also measured under experimental hypoxia,22,23 which could mean that in the PH associated with COPD there is a different physiopathological mechanism compared to the PAH. Our study in humans tallies fairly well with the outcomes observed in mice, indicating that the most probable pathway is endothelial dysfunction24,25 with the imbalance between increased levels of vasoconstrictors, proliferative and pro-coagulant mediators (such as ET-1, thromboxanes, etc.), and diminished levels of vasodilators, antiproliferative, and anti-coagulant substances (such as NO, prostacyclin, VIP, and adrenomedullin, etc.). Within vasoconstrictors, previous studies found that in different respiratory diseases and in the case of COPD, ET-1 is increased in the endothelium of the pulmonary vessels,26 in pulmonary circulation during advanced GOLD levels, and during reacutization. One of the causes that can explain why ET-1 is not increased in the current study could be that our patients were studied at least 3 months after the last exacerbation and had better functional levels than previous studies. Indeed, recent studies show that even in exhaled breath condensate ET-1 is higher in patients with COPD and PH than in ones who do not have a PH.27,28 These reports, together with our results, suggest that local and not systemic production of ET-1 could be involved in the pathogenesis of PH in COPD. Therefore, in accordance with these studies, we can speculate that the increase of VIP, which we found in COPD with PH and in particular in the OP group, is a reaction against vasoconstriction. The pulmonary vessels’ rarefaction with reduction of surface-active endothelium leads to diminished secretion of mediators and under-expression of NO, the main endogenous vasodilator, whereas VIP is increased to compensate for the vessels’ vasoconstriction, although its action is not efficient enough. The supposed drivers leading to VIP secretion could be inflammation, oxidative stress, hypoxia, correlative networks within autocrine peptides, and development of PH.
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FIG. 3. Correlation between forced expiratory volume in 1 sec (FEV1) and mean pulmonary arterial pressure (mPAP) (R = - 0,38, p < 0.05). Correlation analysis referred only to patients without ‘‘out-of-proportion’’ (OP) status (A). (See the Discussion section for explanation.) Concerning inflammation and oxidant load, in our study no difference of IL-6 was found in patients with or without PH, such as in OP patients. Moreover, the level of LTB4, a marker of oxidative stress, was the same in three groups. These results, in contrast with previous ones,29 seem to suggest that inflammation and oxidative stress are not involved in the pathogenesis of PH during stable phases of COPD; however, the measurement of only two markers does not give us sufficient information about the level of inflammation. Moreover, in COPD, inflammation and oxidative stress are influenced by different factors, and the concentration of IL-6 and LTB4 measured does not reflect either the ‘‘history’’ or the evolution of diseases but shows only an ‘‘instant photo’’ in which many confounding factors can be present at the same time (bacteria and virus colonization, co-morbidities, different phenotypes, drugs, etc.). In this light, other molecular or alternative study designs are
necessary to ascertain the importance of the inflammatory process in development of PH in COPD. In conclusion, our opinion is that VIP is not directly involved in the pathogenesis of PH in COPD, but, on the contrary, its production is increased so as to try and compensate for the vasoconstriction and pulmonary pressure elevation. However, this mechanism does not seem to be efficient. This hypothesis should be confirmed by further studies. That said, our study appears to confirm that the mechanisms that lead to PH in some COPD patients are complex and need to be better understood. Furthermore, even if to date there has not been any specific treatment for PH when it is associated with COPD, it could be useful to identify this subgroup of patients to improve their follow-up, clinical approach, and, at some future date, a course of treatment.
FIG. 4. Correlation between vasoactive intestinal peptide (VIP) and mean pulmonary arterial pressure (mPAP) (R = 0.32, p < 0.05). () ‘‘Out-of-proportion’’ (OP) patients.
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Author Disclosure Statement
All authors declare that they have no conflict of interest in the subject matter or materials discussed in the manuscript. The study has not received any financial support. No offlabel investigation has been performed in the present study. D.L. designed the study, analyzed the data, and wrote the manuscript; G.V., determined study concept and performed RHC; G.P.P., performed biological investigation; G.E.C., collected and analyzed the data; M.C., analysed the data and performed the echocardiography; M.D.B., interpreted the results and made a critical review; M.P.F.B., supervised the study, interpreted the results, and provided the final approval of the version submitted. References
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Address correspondence to: Donato Lacedonia Department of Medical and Surgical Sciences Institute of Respiratory Diseases University of Foggia viale degli Aviatori 71100 – Foggia Italy E-mail: [email protected] Received: August 5, 2013 Accepted: November 11, 2013
