Ghrelin is an endogenous vasodilatory peptide also stimulating the release of growth hormone and has been demonstrated to attenuate the development of pulmonary arterial hypertension (PAH) in a monocrotaline-treated animal model of PAH ⁸. A previous study demonstrated that ghrelin improved endothelial dysfunction and increased endothelial NOS expression through a growth hormone (GH)-independent mechanism ⁹. Moreover, ghrelin improved cardiac function and cardiopulmonary-associated cachexia ¹⁰. Thus, it can be suggested that ghrelin may become a potential therapeutic agent for PH in the near future ¹¹.

Pulmonary and systemic inflammation might contribute to weight loss in patients with COPD ¹². Inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) are contributed to the development of weight loss in COPD patients. Several studies have demonstrated that plasma ghrelin levels are increased and positively correlated with TNF-α but negatively correlated with body mass index (BMI) in cachectic patients with chronic hearth failure ¹³ and cancer ¹⁴. A causal link between inflammatory status, weight maintenance and PH in COPD remains unclear.

We hypothesized that increased or decreased ghrelin levels may play a role on development of PH in patients with COPD. In addition changed ghrelin levels would be associated with evidence of an inflammatory response and weight loss in these patients. To test this we investigated the serum ghrelin levels and systemic inflammation by measuring serum levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in underweight and normal weight COPD patients with and without PH.

Pulmonary Function Testing
Pulmonary function parameters (forced expiratory volume in 1 second [FEV₁; and forced vital capacity [FVC] were measured using a spirometer (Ultima CPX 790705-205; Medgraphics Corporation, St. Paul, MN, USA). The values were interpreted according to the predicted values of the European Respiratory Society ¹⁵. Pulmonary function values were expressed as a percentage of the predicted values.

Arterial Blood Gases
Arterial blood gases were measured at rest with a blood gas analyzer (Rapid lab 348; Biobak, Chiron, Bayer Diagnostic, UK).

Fat-free mass
Fat-free mass (FFM) was measured by single-frequency (50 kHz) bioelectrical impedance analysis (Tanita TBF 300A Body Composition Analyzer, Tanita Corporation, Tokyo, Japan).

Measurement of serum IL-6, TNF-α and ghrelin levels
In all patients, venous blood samples from the antecubital vein were collected between 7:00 and 8:00 A.M. after overnight fasting and the patients seated and resting comfortably. The blood was centrifuged immediately at 4°C and stored at -800C. Serum TNF-α and IL-6 levels were measured using appropriate commercial kits (Biosource, Biosource International Inc., Camarillo, CA, USA and Orgenium Laboratories, Helsinki, Finland, respectively) by enzyme-linked immunosorbent assay (ELISA) method ⁵. Plasma acylated and non-acylated ghrelin levels were measured with two commercially available ELISA kits (the Active Ghrelin ELISA kit and the Desacyl-Ghrelin ELISA kit, respectively), according to the manufacturer's protocol (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan). The Active Ghrelin ELISA Kit was used to measure n-octanoyl ghrelin. The Desacyl-ghrelin ELISA Kit was used for the measurement of desacyl-ghrelin. The minimal detection limits for acylated and desacyl ghrelin in this assay system were 2.5 and 12.5 fmol/ml, respectively. The intra-and inter-assay coefficients of variation were 6.5% and 9.8% for acylated ghrelin and 3.7% and 8.1% for desacyl ghrelin, respectively.

Echocardiography
Systolic pulmonary arterial pressure (P_pas) in patients with COPD were assessed by Doppler echocardiography (Acuson Sequa 512 device with a 3.5 MHz transducer; Acuson Corporation, Mountain View, CA, USA) by a single expert cardiologist who was blinded to the results of the biochemical analysis. While patients were in semi-supine position, continuous wave Doppler recordings of maximal velocity were obtained from apical, parasternal long-short axis, and subcostal transducer positions. Tricuspid regur

gitant flow identified by color flow Doppler techniques and the maximum jet velocity was measured by continuous-wave Doppler recording. Regurgitation flow obtained from the apical 4 cavity images by continuous-wave Doppler were inserted into the Bernoulli equation and tricuspid regurgitation was calculated automatically by device ^{16, 17}.

ΔPRV-RA [the pressure gradient between the right atrium and right ventricle]=4[VTR]^² [tricuspid regurgitant flow rate] and right atrial pressure (appraisal of right atrial pressure was taken up to 10 mmHg) was added to this value to calculate the P_pas. Systolic Ppa was calculated according to this equation and PH was defined as P_pas ≥30 mmHg ^18-20.

Statistical Analysis
All statistical analyses were performed using the SPSS 12.0 program. The results were expressed as the mean±standard deviation (SD). Significance level was accepted as p<0.05. Kruskal-Wallis, Mann-Whitney U and chi-square (for sex distribution) tests were used to compare the data.

Comparisons of the COPD patients with and without PH
There was no significant difference between COPD patients with or without PH in terms of age, BMI, and FFM (p>0.05). The FEV₁, FEV₁/FVC, and partial pressure of oxygen in arterial blood (PaO₂) values were significantly lower and the duration of disease and smoking history were significantly higher in COPD patients with PH compared to those without PH (Table 1).

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Table 1: Demographic data and other parameters in COPD patients with or without pulmonary hypertension and control subjects.

Serum active ghrelin and TNF-α levels were significantly lower in COPD patients with PH than without PH patients. A trend toward higher IL-6 levels in COPD patients with PH compared to those without PH was noted; however, it did not reach statistically significance level (Table 1).

Comparisons of COPD patients with or without PH and control subjects
Serum IL-6 and TNF-α levels were significantly higher in COPD patients with or without PH group compared to control subjects. Serum active ghrelin levels were significantly lower in COPD patients with or without PH group than control subjects. In particular, the serum ghrelin levels were lower in patients with PH than those without PH and control subjects (Table 1).

PH and cachectic status in patients with COPD

Underweight patients
There were no statistically significant differences between groups Ia and Ib in terms of age, duration of disease, smoking history, BMI, and FFM (p>0.05). Serum active ghrelin levels were significantly lower and IL-6 levels were significantly higher in group Ia than group Ib. Serum TNF-α levels were lower in group Ia compared with group Ib, but were not statistically significant (Table 2).

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Table 2: Demographic data and other parameters in underweight COPD patients with and without pulmonary hypertension.

Normal weight patients
No differences existed between group Ic and group Id in terms of age, BMI, FFM, and smoking history (p>0.05) (Table 3). TNF-α and IL-6 levels were significantly lower in group Ic compared to group Id; however, there was no statistically significant differences in serum active ghrelin levels between the two groups.

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Table 3: Demographic data and other parameters in normal weight COPD patients with and without pulmonary hypertension.

There was no statistically significant difference in mean pulmonary artery pressures between groups Ia and Ic (59.93±16.14 and 56.46±12.33, respectively; p>0.05).

Wiley and Davenport ²³ have reported that ghrelin is a potent vasodilator in isolated human arteries. Nagaya et al. ²⁴ have demonstrated that intravenous administration of ghrelin decreases systemic vascular resistance in rats, at least in part through GH-independent mechanisms. Several studies have demonstrated that ghrelin improves endothelial dysfunction and increases endothelial nitric oxide secretion through a GH-independent mechanism and ghrelin has a potent vasodilatory effect ^{9, 23-25}. Thus, at least one ghrelin signaling pathway appears to be involved in the regulation of vascular tone ²⁶. Previous experimental studies demonstrated that ghrelin was produced in the heart, lungs, and stomach of animals and the exogenous administration of ghrelin decreased the development of PH, right ventricular hypertrophy, and vascular remodeling in pulmonary arteries in animals ^{27, 28}. Ghrelin has important direct cardiovascular effects that ghrelin administration reduced cardiac afterload and increased cardiac output in both normal subjects and patients with dilated cardiomyopathy ^{10, 29}.

On the basis of the beneficial effects of ghrelin on the pulmonary vasculature and heart, we hypothesized that ghrelin might be beneficial in COPD patients with PH by improving cardiac and/or pulmonary vascular structure and function.

In the present study, it was demonstrated that serum active ghrelin levels significantly decreased in COPD patients with or without PH compared to control subjects. In particular, the decrease in serum active ghrelin levels was more prominent in COPD patients with PH. Thus, the serum active ghrelin level was decreased in patients with COPD and this decrease became more pronounced when COPD patients had PH. To our knowledge, there are no clinical studies evaluating the effects of ghrelin on the development of PH in COPD patients. For this reason, our results cannot be compared with other data. Surprisingly, the ability of systemic ghrelin in the regulation of its local production was apparently lost in PH. One experimental study demonstrated that endogenous production of ghrelin was almost abolished in normal rats treated with ghrelin. Furthermore, in PH the pulmonary expression of ghrelin was preserved and right ventricle myocardial expression was increased in monocrotalin-treated animals, and exogenous administration of ghrelin attenuated in PH and pulmonary vascular remodeling ²⁷. Similarly, another experimental study demonstrated that exogenous ghrelin attenuated the progression of PH, which was induced by chronic hypoxia in rats ²⁸. At the pulmonary level, ghrelin has a determinant role in fetal lung development ^30, and the adult human lung is a major source of ghrelin mRNA gene expression ³¹. GH secretagogue receptors were detected in the adult lung parenchyma and pulmonary artery wall ^{32, 33}. According to all these findings and our results, decreased ghrelin levels may contribute to the development of PH.

Ghrelin induces a positive energy balance and weight gain by decreasing fat utility and stimulating food intake ^{34, 35}. When the serum active ghrelin levels were evaluated in both underweight and normal weight COPD patients with and without PH, it was demonstrated that the serum active ghrelin levels were lower in underweight COPD patients with PH than underweight COPD patients without PH. In contrast to these findings, there was no significant difference in serum active ghrelin levels between normal weight COPD patients with and without PH. These observations may implicate that ghrelin has a potential role in the development of PH, especially in underweight COPD patients. The potential role of inflammation in the pathogenesis of vessel disease was not well-studied until the past few years. Humbert et al ^36, reported that elevated serum concentrations of IL-1 and IL-6, but not TNF- α, in patients with idiopathic pulmonary arterial hypertension (IPAH). Hoeper and Welte ⁷ explored that none of these cytokines was elevated in patients with IPAH. It has been recently shown that systemic inflammation in COPD appears to increase the risk for developing PH in COPD. Pro-inflammatory cytokines, such as IL-6, was correlated with the level of pulmonary artery pressure ⁶ and patients with significant pulmonary hypertension had higher levels of TNF-α ⁵. Inflammation, most likely involving IL-6, may contribute substantially to PH complicating COPD ³⁷ Similarly, we also determined that serum IL-6 levels were significantly higher in underweight COPD patients with PH than those without PH; however, this relationship was not observed for TNF-α. In contrast to this finding, TNF-α and IL-6 levels were significantly lower in normal weight COPD patients with PH compared to normal weight COPD patients without PH. We believe that systemic inflammation may be at least partially responsible for pulmonary hypertension especially underweight COPD patients. Pro-inflammatory cytokines, such as TNF-α, was negatively correlated with plasma ghrelin in COPD ²². Decreased ghrelin levels in patients with COPD may contribute to alterations in metabolic status during inflammatory stress and may cause cachexia ²². Cachexia impacts both respiratory musculature and peripheral skeletal muscle function. Generalized muscle weakness is caused by deconditioning, malnutrition, electrolyte disturbances, cardiac failure, and systemic inflammation ³⁸. Malnutrition has a tremendous impact on respiratory functions. It affects respiratory muscle performance, lung structure, defense mechanisms, and control of ventilation and predisposes to respiratory failure ³⁹. In addition, a decline in FFM in COPD is associated with worse lung function ⁴⁰. It has been suggested that low body weight may be associated with decreased respiratory muscle function in COPD; however, the precise mechanism is not known 42 demonstrated that repeated administration of ghrelin improves body composition, muscle wasting, and functional capacity. The present study results and previous data suggested that decreased ghrelin levels due to systemic inflammation and/or other reasons, such as right ventricular hypertrophy in cachectic COPD patients, contributed to the decreased respiratory function ^{22, 43}. Ghrelin may act synergistically with severe airflow limitation effects and this may predispose to PH in patients with COPD especially underweight patients. However, in patients with severe PH frequently report loss of appetite and weight ⁴⁴.

Ghrelin divided into two main groups according to octanoyl side chain is present. Octanoylated ghrelins have an endocrine activity (“active ghrelin”), but nonoctanoylated ghrelins do not have this activity (“inactive ghrelin”) ⁴⁵. In the human stomach, the ratio of octanoylated to decanoylated ghrelin was found to be roughly 3:1. Because acylation of ghrelin is essential for its activity, the enzyme that catalyzes this modification step should be an important regulator of ghrelin biosynthesis. Plasma concentrations of active ghrelin constitute about 20% of total ghrelin levels and total ghrelin may reflect the level of the active form in plasma ^{22, 46}. We measured in both total and active ghrelin levels. Our results showed that there was no significantly different between groups in total ghrelin levels despite active ghrelin levels changed. For this reason, measure of active ghrelin may be rely on active rather than total for the evaluation of ghrelin effects.

In conclusion, this was a pilot study which should generate additional ideas for future study. It was considered that ghrelin may be contributing factor to the development of PH other than the severity of COPD, hypoxemia, and systemic inflammation in underweight COPD patients but decreased ghrelin levels also might be actually reflect BMI that patients with more severe COPD have lost body weight. In addition, the loss of ghrelin as an endogenous pulmonary vasodilator leads to worsening PAP. Decreased ghrelin levels may be an important medical issue in cachectic COPD patients with PH. The role of ghrelin in weight loss and pathogenesis of PH in patients with COPD, and its potential beneficial effects on the treatment of COPD patients with PH should be investigated with further studies.

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