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Endocrinology, doi:10.1210/en.2007-0833
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Endocrinology Vol. 149, No. 1 237-244
Copyright © 2008 by The Endocrine Society

Exogenous Ghrelin Attenuates the Progression of Chronic Hypoxia-Induced Pulmonary Hypertension in Conscious Rats

Daryl O. Schwenke, Takeshi Tokudome, Mikiyasu Shirai, Hiroshi Hosoda, Takeshi Horio, Ichiro Kishimoto and Kenji Kangawa

Department of Biochemistry (D.O.S., T.T., H.H., I.K., K.K.), and Division of Hypertension and Nephrology (T.H.), Department of Medicine, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan; Faculty of Health Sciences (M.S.), Hiroshima International University, Hiroshima 730-0016, Japan; and Department of Physiology (D.O.S.), School of Medical Sciences, University of Otago, Dunedin 9016, New Zealand

Address all correspondence and requests for reprints to: Ichiro Kishimoto, Department of Biochemistry, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. E-mail: kishimot{at}ri.ncvc.go.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic exposure to hypoxia, a common adverse consequence of most pulmonary disorders, can lead to a sustained increase in pulmonary arterial pressure (PAP), right ventricular hypertrophy, and is, therefore, closely associated with heart failure and increased mortality. Ghrelin, originally identified as an endogenous GH secretagogue, has recently been shown to possess potent vasodilator properties, likely involving modulation of the vascular endothelium and its associated vasoactive peptides. In this study we hypothesized that ghrelin would impede the pathogenesis of pulmonary arterial hypertension during chronic hypoxia (CH). PAP was continuously measured using radiotelemetry, in conscious male Sprague Dawley rats, in normoxia and during 2-wk CH (10% O2). During this hypoxic period, rats received a daily sc injection of either saline or ghrelin (150 µg/kg). Subsequently, heart and lung samples were collected for morphological, histological, and molecular analyses. CH significantly elevated PAP in saline-treated rats, increased wall thickness of peripheral pulmonary arteries, and, consequently, induced right ventricular hypertrophy. In these rats, CH also led to the overexpression of endothelial nitric oxide synthase mRNA and protein, as well as endothelin-1 mRNA within the lung. Exogenous ghrelin administration attenuated the CH-induced overexpression of endothelial nitric oxide synthase mRNA and protein, as well as endothelin-1 mRNA. Consequently, ghrelin significantly attenuated the development of pulmonary arterial hypertension, pulmonary vascular remodeling, and right ventricular hypertrophy. These results demonstrate the therapeutic benefits of ghrelin for impeding the pathogenesis of pulmonary hypertension and right ventricular hypertrophy, particularly in subjects prone to CH (e.g. pulmonary disorders).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ALVEOLAR HYPOXIA IS a common adverse consequence of most chronic obstructive pulmonary disorders and, when sustained, can ultimately lead to the irreversible remodeling of the pulmonary vasculature (1, 2). The consequential sustained increase in pulmonary arterial pressure (PAP) increases the workload of the heart, and is, therefore, closely associated with congestive heart failure and increased morbidity and mortality. Although the exact mechanisms responsible for the pathogenesis of pulmonary arterial hypertension (PAH) are yet to be fully elucidated, numerous studies have shown that disruption of the pathways for several vascular vasoactive substances, such as endothelin-1 (ET-1) (3, 4, 5) and nitric oxide (NO) (6, 7, 8, 9), contribute to the pathogenesis of PAH.

Ghrelin is a GH-releasing peptide, originally isolated from the stomach (10), and has been identified as an endogenous ligand for the GH secretagogue receptor (GHS-R). Ghrelin has improved cardiac function and cardiopulmonary associated cachexia, partly attributable to the anabolic properties of the GH-IGF-I axis (11, 12, 13, 14).

Animal and human experimental studies have also shown that ghrelin has direct vasodilatory properties that are GH independent (15, 16). Wiley and Davenport (17) attributed the vasodilatory properties of ghrelin to the potent antagonism of ET-1, a powerful vasoconstrictor. These observations may implicate ghrelin as a potential therapeutic treatment for PAH, especially because several reports have shown that ET-1 antagonism significantly attenuates PAH, right ventricular hypertrophy, and, thus, improves survival prognosis (18, 19). Recently, the research team of Leite-Moreira (20, 21, 22, 23, 24, 25) has demonstrated the direct actions of ghrelin in treating monocrotaline-induced cardiopulmonary disease. Monocrotaline is a pyrrolizidine alkaloid that causes pathological inflammation and remodeling of the pulmonary vasculature. Henriques-Coelho et al. (20) showed that exogenous ghrelin can attenuate monocrotaline-induced PAH.

However, the etiology for monocrotaline-induced PAH, including responses to potential therapeutic treatments, differs considerably to that for chronic hypoxia (CH)-induced PAH (26, 27, 28). Moreover, CH is a common adverse complication for most pulmonary disorders that compromises the physiological status of the whole individual, whereas monocrotaline is commonly used in experimental settings for inducing selective vascular damage of the pulmonary circulation.

Therefore, in this study we aimed to monitor PAP during the progression of pulmonary hypertension and determine the physiological benefits of ghrelin during the pathogenesis of PAH during CH in the conscious rat. In addition, we aimed to ascertain whether ghrelin could prevent changes in pulmonary vascular reactivity, which are normally associated with the vascular structural changes after CH. Pulmonary vascular reactivity was assessed by the magnitude of acute hypoxic pulmonary vasoconstriction (HPV) response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experiments were conducted on 14 male Sprague Dawley rats [6 wk old; body weight (BW) ~180–220 g]. Rats were divided into normoxic rats (N-rats) (treated with ghrelin; n = 4) and two groups of chronic hypoxic rats: saline-treated (CH-rats) (n = 5) and ghrelin-treated groups (n = 5). All rats were on a 12-h light, 12-h dark cycle at 25 ± 1 C, and provided with food and water ad libitum. All experiments were approved by the Animal Ethics Committee of the National Cardiovascular Center Research Institute and conducted in accordance with the guidelines of the Physiological Society of Japan.

After surgical implantation of a telemetric transmitter, all rats were housed in standard normoxic conditions for 1 wk (recovery period). Subsequently, two groups of CH-rats were continuously housed in a hypoxic chamber (10 ± 0.1% O2) for 2 wk, except for a 10-min interval each day when the chamber was cleaned (N-rats remained in normoxic conditions for 1 wk). The hypoxic gas mixture was prepared from N2 (gas cylinders) and compressed air, and was continuously delivered to the hypoxic chamber at a flow rate of approximately 8 liter/min.

Anesthesia and surgical preparation
All surgical procedures were performed using standard aseptic techniques. Rats were anesthetized with pentobarbital sodium (50 mg/kg, ip). Supplementary doses of anesthetic (~15 mg/kg/h ip) were administered if a limb withdrawal reflex was elicited in response to pinching of a hind footpad. Immediately before the first incision, all animals received an injection (im) of Temgesic analgesia (0.05 mg/kg, Buprenorphine; Reckitt & Colman Ltd., UK). Throughout the surgery, body temperature was maintained at 38 C using a rectal thermistor coupled with a thermostatically controlled heating pad.

Pulmonary artery catheterization and pressure measurement
PAP was measured in the conscious rat using a telemetry system (Data Sciences, St. Paul, MN) as described previously (29). The system consisted of an implantable transmitter (model TA11PA-C20) for continuously monitoring PAP, a receiver (RPC-1), and an ambient-pressure monitor (C11PR; Data Sciences). The pressure signal from the transmitter was calibrated in reference to atmospheric pressure.

With the rat in a supine position on the operating table, the trachea was intubated, and the lungs were ventilated with a rodent ventilator (SN-480-7; Shinano, Tokyo, Japan). A right thoracotomy was made between the second and third rib, and the conus of the right ventricle (RV) was exposed. The abdomen was opened with a midline abdominal laparotomy, and an Angiocath needle (21 gauge; BD Medical-Medical Surgical Systems, Sandy, UT) was used as a trocar to pass the gel-filled sensing catheter of a telemetric transmitter from the peritoneal cavity into the thorax. The body of the transmitter was placed into the abdominal cavity and sutured to the abdominal wall. A 23-gauge needle was used to pierce the ventricle wall, and then the tip of the transmitter catheter (~0.5-mm, thin-walled thermoplastic membrane) was inserted anteriorly into the RV and advanced into the pulmonary artery. The catheter was fixed in position with 7.0 Prolene suture (Ethicon, Inc., Johnson & Johnson, Somerville, NJ) to the epicardium. Subsequently, the thoracotomy, laparotomy, and all skin incisions were closed with sutures.

Immediately after surgery, all animals received 30 mg/kg (im) ampicillin (Viccillin antibiotic; Meiji Seika Kaisha, Ltd., Tokyo Japan) and remained on the heating blanket during recovery from anesthesia. Rats had 7 d to recover from surgery before data were collected. During this period the welfare of the rat was monitored by recording BW, food and water consumption, observing general appearance and behavior, and cleaning and dressing wounds if required.

Measurement of arterial blood pressure
Systolic and diastolic blood pressures were recorded in conscious animals using an automatic sphygmomanometer tail-cuff pressure transducer (BP-98A; Softron Co. Ltd., Tokyo, Japan). Each value recorded was derived from five consecutive measurements (within ~2 min), which were then averaged to give one value representative of each experimental condition.

Experimental protocol
Training.
All rats were subjected to the acute hypoxic protocol in the conscious state 1 wk after surgery. Each rat was first trained to sit restfully within a Perspex chamber (~2.5 liter; custom made by the author D.O.S.) to avoid stress or anxiety influencing the results. The airtight Perspex chamber was incorporated in a unidirectional flow circuit into which either normoxia or hypoxia was delivered. Gases flowing into the chamber were first humidified, and the chamber was heated (28.5–29.8 C) to ensure that rats had adequate blood flow to the tail, an essential requisite for measuring ambulatory blood pressure (ABP) via the tail-cuff method (30). Chamber temperature was continuously measured (to one decimal place) with a Fluke thermocouple thermometer (model 52II; Fluke, Everett, WA). The acute hypoxic test gas (i.e. 8% O2) was created using gas rotameters (O2 and N2). Training started 4 d after surgery and was achieved by exposing each rat to air for 60 min (d 1 and 2 training) or the acute hypoxic protocol (d 3 training). By the fourth day (i.e. 1 wk after surgery), rats were considered trained, and cardiovascular data were collected from conscious rats during normoxia and in response to acute hypoxia.

Acute hypoxic protocol.
Cardiovascular responses to acute hypoxia (10 min 8% O2) were tested in all rats 1 wk after surgery (first acute test; Fig. 1Go) and then again 1) after 2 wk of CH (10% O2; CH-groups only); and 2) after 1 wk of daily ghrelin injections in normoxia (N-group). On the day of an experiment, each rat was initially placed inside the Perspex chamber and allowed 30 min to become accustomed, after which data collection began. Rats were first subjected to normoxia for 5 min before being exposed to acute hypoxia (8% O2 in N2) for 10 min.


Figure 1
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  		FIG. 1. A schematic illustration of the 3-wk experimental protocol with which rats were subjected. Rats were exposed to the acute hypoxic test (8% O2 for 10 min) after 7-d normoxia (N), and then again after 2-wk CH (10% O2).

 
Drug administration.
After the acute hypoxic protocol, rats received a sc injection of either saline (n = 5) or ghrelin (150 µg/kg in 0.3 ml; n = 5), and were then placed into the hypoxic chamber (CH-rats) or returned to their normal cage (N-rats). CH-rats continued to receive daily injections of either saline or ghrelin during the 2-wk CH (CH-rats). N-rats received daily ghrelin injections for 7 consecutive days, under standard normoxic conditions (N-rats). Ghrelin was obtained from the Peptide Institute Inc. (Osaka, Japan), and administered dissolved in saline as the vehicle.

All experiments were performed in a random order, so that each cage within the hypoxic chamber often housed both saline-treated and ghrelin-treated rats.

Data acquisition and analysis.
The telemetric PAP signal was continuously sampled at 200 Hz with an eight-channel MacLab/8s interface hardware system (AD Instruments Pty. Ltd., Japan Inc.), and recorded on a Macintosh Power Book G4 using Chart (version 5.0.1; AD Instruments). Heart rate (HR) was derived from the pulmonary arterial systolic peaks. A 30-sec block of data was analyzed –5 min and immediately before (time "0") acute hypoxic exposure, and then after 1, 2, 4, 6, 8, and 10 min exposure to 8% O2. Normoxic baseline data for individual rats in each group were averaged from values acquired at –5 min and zero time. ABP was periodically measured (approximately once per minute) during normoxia and after the eighth minute of acute hypoxia. The mean ABP (MABP) values during normoxia and hypoxia were entered into spreadsheets for further statistical analysis.

Morphometric analysis
After hemodynamic data had been collected, each rat was euthanized, and the heart and lungs were excised for mRNA and morphometric analyses. An additional four rats were used (8 wk old; BW ~240–290 g) to obtain morphometric and mRNA expression data of normoxic, untreated rats. The atria were removed, and the RV wall was separated from the left ventricle (LV) and septum (Sep). Tissues were blotted, and weighed and normalized to 100-g BW. Right and left ventricular weights were expressed as the ratio of the RV to the LV + Sep weight (WRV/WLV + Sep; Fulton’s ratio).

Cross-sections of the middle lobe of the right lung were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Sections 2-µm thick were stained with hematoxylin and eosin for examination by light microscopy. The wall-to-lumen ratio of the pulmonary arteries was measured in 20 muscular arteries, ranging in size from 25–100 µm in external diameter, as previously reported (31).

Northern blot analysis
Northern blot analyses were performed as described previously (32). Total RNA (15 mg/lane) was extracted from whole left lung tissue using TRIZOL (Invitrogen Corp., Carlsbad, CA) reagent, then denatured with formaldehyde and formamide, and electrophoresed on a 1% agarose gel containing formaldehyde. RNA in the gel was then transferred to a nylon membrane and fixed by UV irradiation. Hybridization of the membrane was performed using 32P-labeled cDNA probes for rat endothelial nitric oxide synthase (eNOS), ET-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs according to methods previously reported (33). The band intensity was estimated using a radioimage analyzer (BAS-5000; Fujifilm Corp., Tokyo, Japan). Each mRNA value was corrected for GAPDH mRNA and expressed as fold increase over normoxia control.

Immunohistochemistry
The following antibodies were purchased as indicated: rabbit polyclonal NOS3 (C-20) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and horseradish peroxidase-coupled antirabbit IgG antibodies from Dako Corp. (Carpinteria, CA). After perfusion fixation with 4% paraformaldehyde and embedding in paraffin, sections (2 µm thick) were deparaffinized and stained with rabbit antirat NOS3 antibody, followed by horseradish peroxidase-coupled antirabbit IgG antibodies, respectively. Immunostaining was completed by 5 min incubation with 3,3' diaminobenzidine tetrahydrochloride/hydrogen peroxide, which results in a brown precipitate at the antigen site. The sections were then lightly counterstained with hematoxylin to visualize nuclei.

Blood glucose/free fatty acid
A blood sample was taken from each rat immediately before euthanasia. Plasma samples were subsequently sent to Tokyo Special Reference Laboratories, Inc. (SRL, Inc., Tokyo, Japan) for analyses of plasma glucose and free fatty acid levels.

Statistical analysis
All statistical analyses were conducted using StatView (version 5.01; SAS Institute Inc., Cary, NC). All results are presented as means ± SEM. Two-way ANOVA (repeated measures) was used to test significance for: 1) temporal changes in hemodynamic variables in response to 10 min acute hypoxia, and 2) alterations in the hypoxic response by ghrelin or CH. One-way ANOVA (factorial) was used to test for differences in baseline values for normoxia compared with CH. Where statistical significance was reached, post hoc analyses were performed using the Dunnet’s test for multiple comparisons. A P value equal to or less than 0.05 was predetermined as the level of significance for all statistical analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BW/glucose/free fatty acids
In general, rats experienced weight loss within 1–2 d after surgery but had fully regained BW by 1 wk after surgery (204 ± 6 g; 7 wk old). By the end of 2-wk CH (9 wk old), saline-treated and ghrelin treated rats had: 1) an equal increase in weight (246 ± 10 and 248 ± 10 g, respectively); 2) similar plasma glucose (198 ± 4 and 172 ± 12 mg/dl, respectively); and 3) similar free fatty acid levels (66 ± 3 and 59 ± 3 mEQ/liter, respectively).

In N-rats, daily injections of ghrelin for 1 wk increased weight from 208 ± 3 g (7 wk old) to 253 ± 5 g (8 wk old). Ghrelin did not significantly alter plasma glucose (219 ± 6 mg/dl) or free fatty acid levels (38 ± 14 mEQ/liter), compared with that of untreated rats.

Hemodynamic analysis
Baseline pre-chronic hypoxic hemodynamic data are presented in Table 1Go. Two weeks of CH induced pulmonary hypertension in saline-treated rats, evident by a significant 110 ± 15% increase in mean PAP (MPAP) above normoxic values (P < 0.01). CH did not modify HR, but it did induce a significant 26 ± 8% increase in systemic MABP. The daily administration of ghrelin (150 µg/kg·d) during CH significantly attenuated the development of pulmonary hypertension (48 ± 10% increase in MPAP) and the magnitude of systemic hypertension (17 ± 9% increase in MABP).


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  		TABLE 1. Cardiovascular variables of ''pre-chronic hypoxic'' (Pre-CH) rats (n = 10)

 
Morphometric analysis
The sustained increase in MPAP during CH resulted in right ventricular hypertrophy in saline-treated rats (RV/LV + Sep ratio of 0.68, cf. 0.31 in N-rats; Table 2Go). CH also induced significant medial thickening of the small pulmonary arteries (25–100 µm), resulting in a decrease in vessel caliber (Fig. 2AGo) and an increase in the wall thickness to lumen diameter ratio (Fig. 2BGo). Ghrelin treatment reduced not only the magnitude of pulmonary hypertension, but also the degree of right ventricular hypertrophy (RV/LV + Sep ratio of 0.52; Table 2Go) and medial thickening of small pulmonary arteries in response to CH (Fig. 2Go).


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  		TABLE 2. Heart weight from three groups of rats: 1) normoxia (n = 4); 2) CH and saline for 2 wk (n = 5); and 3) CH and ghrelin (n = 5)

 

Figure 2
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  		FIG. 2. A, Histology photomicrographs of peripheral pulmonary arteries from normoxic rats (Norm; n = 4) and chronic hypoxic rats treated with either saline (CH; n = 5) or ghrelin (CH + Ghr; 150 µg/kg·d; n = 5). For clarity purposes, a photomicrograph displaying a high degree of medial thickening after CH has been presented. Magnification, x200. B, Quantitative analysis of wall to lumen ratio in pulmonary arteries. Data are presented as mean ± SEM *, Significantly different from normoxia (P < 0.05).{dagger}, Significant difference between chronic hypoxic rats treated with saline vs. ghrelin (P < 0.05).

 
mRNA expression analysis
The mRNA expression of eNOS and ET-1 in the left lung of normoxic and chronic hypoxic rats was assessed by Northern blot analyses (Fig. 3Go). The expression of both eNOS and ET-1 mRNAs was significantly elevated in chronic hypoxic rats (saline-treated) compared with normoxic control rats (Fig. 3BGo). However, ghrelin treatment prevented the CH-induced overexpression of eNOS and ET-1 mRNAs.


Figure 3
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  		FIG. 3. A, Representative images from Northern blot analysis of eNOS, ET-1, and GAPDH mRNA from lung tissue of normoxic rats (Norm; n = 4) and chronic hypoxic rats treated with either saline (CH; n = 5) or ghrelin (CH + Ghr; 150 µg/kg·d; n = 5). B, Quantitative analysis of the gene expression for eNOS and ET-1. For each individual sample, the mRNA value was corrected by using GAPDH mRNA. The expression levels in normoxia were arbitrarily assigned a value of 1.0. *, Significantly different from normoxia (P < 0.05).{dagger}, Significant difference between chronic hypoxic rats treated with saline vs. ghrelin (P < 0.05).

 
Immunohistochemistry of eNOS
Immunostaining was performed to assess the expression of eNOS protein in the left lung of normoxic and chronic hypoxic rats. As illustrated in Fig. 4Go, positive staining (brick-red) increased after 2-wk CH. However, the concomitant administration of ghrelin tended to attenuate the magnitude of positive staining after CH (Fig. 4Go).


Figure 4
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  		FIG. 4. Representative images from immunohistochemistry of eNOS from lung tissue of normoxic rats and chronic hypoxic rats treated with either saline (CH) or ghrelin (CH + Ghr; 150 µg/kg·d). Ghrelin administration attenuated CH-induced overexpression of eNOS protein.

 
Hemodynamic responses to acute hypoxia
Normoxia (before CH).
Rats were exposed to acute hypoxia (8% O2 for 10 min) before being placed into the chronic hypoxic chamber and before saline or ghrelin treatment. In normoxic, pretreated rats, acute hypoxia provoked an increase in MPAP significant by the second minute, reaching a plateau by the fourth minute, and remaining above baseline values by the tenth minute ({Delta}MPAP 7.2 ± 0.6 mm Hg) (Fig. 5Go). Acute hypoxia caused mild systemic hypotension ({Delta}MABP 9 ± 2 mm Hg; P < 0.05) and a significant increase in HR ({Delta}HR 120 ± 15 beats/min).


Figure 5
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  		FIG. 5. MPAP and HR responses (transient) and MABP response (steady state) to acute hypoxia (8% O2 for 10 min) of rats housed in normoxia (Norm; n = 10) and then again after 2-wk CH (10% O2). During CH, rats received daily injections of either saline (n = 5) or before ghrelin (150 µg/kg·d; n = 5). *, Significantly different from pre-acute hypoxia values (P < 0.05). {dagger}, Significant difference between chronic hypoxic rats treated with saline vs. ghrelin (P < 0.05). b, Beats.

 
CH.
The acute HPV response was significantly accentuated after 2-wk CH. The response was similar for saline-treated rats and ghrelin-treated rats ({Delta}MPAP 10.2 ± 1.1 and 12.4 ± 2.4 mm Hg, respectively), although the response curve was shifted downwards in ghrelin-treated rats (Fig. 5Go). The HR response to acute hypoxia was not modified by either CH or ghrelin (Fig. 5BGo). However, the systemic hypotensive response to acute hypoxia was accentuated after CH in saline-treated rats ({Delta}MABP 24 ± 7 mm Hg), but not in ghrelin-treated rats (Fig. 5Go).

Responses to ghrelin of N-rats
Daily ghrelin administration under standard normoxic conditions did not alter hemodynamic variables (Table 3Go), or the acute response to hypoxia (data not presented) or pulmonary expression of ET-1 and eNOS mRNA. Heart weight/100-g body mass was slightly reduced compared with that of control rats (significant only for LV + Sep), although the distribution of weight between the left and right sides of the heart was not altered (RV/LV + Sep ratio of 0.32 ± 0.01; Table 4Go).


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  		TABLE 3. Cardiovascular variables of ''Normoxic'' rats (n = 4) before and after 1-wk ghrelin administration

 

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  		TABLE 4. Heart weight of two groups of N-rats: 1) control rats (n = 4; see Table 2Go); and 2) ghrelin-treated rats (n = 4)

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The primary findings of this study demonstrate that exogenous ghrelin was able to attenuate the magnitude of PAH, pulmonary vascular remodeling, right ventricular hypertrophy, and the overexpression of ET-1 and eNOS, which are collectively adverse pathological responses to CH.

Ever since its discovery in 1999 (10), ghrelin has been implicated as an important modulator of numerous physiological functions, such as orexigenic regulation, inhibition of cell apoptosis, the release of GH, central modulation of sympathetic tone, and direct hemodynamic control [reviewed by Nagaya et al. (34)]. Nagaya et al.34 also demonstrated that ghrelin was effective in improving left ventricular dysfunction in humans with end-stage chronic heart failure (11, 12, 13, 14) as well as a rat model with chronic heart failure (13, 16). The actions of ghrelin have largely been linked to the release of GH after activation of GHS-Rs within the anterior pituitary. However, GHS-Rs are also located within the myocardium and vessels (35), indicating a direct cardioprotective and vasodilatory effect of ghrelin.

The mechanisms responsible for the pathogenesis of PAH during CH are yet to be fully elucidated, although disruption of ET-1 and eNOS vasoactive pathways have been implicated. In this study we observed that CH provoked the overexpression of ET-1 within the lung, which is consistent with previous reports (36, 37). It is this increase in ET-1 that has been attributed, at least in part, to the pathogenesis of PAH (36, 38, 39). When the overexpression of ET-1 is prevented during CH, as was reported for ghrelin-treated rats in our study, the magnitude of PAH was significantly attenuated. Indeed, conventional ET-1 antagonists (e.g. bosentan) have significantly attenuated the development of PAH (18, 19, 37).

Wiley and Davenport (17) reported that ghrelin was a potent physiological antagonist of ET-1. Moreover, the chronic administration of exogenous ghrelin leads to the down-regulation of ET-1 mRNA expression (21, 40). These observations suggest that ghrelin is inherently beneficial for pulmonary hypertensive subjects. Henriques-Coelho et al. (20) reported an increase in ghrelin mRNA within the RV, but not the lungs, of pulmonary hypertensive rats (monocrotaline treatment). They speculated that the enhanced expression of ghrelin within the RV may possibly be a compensatory response to increased afterload, acting locally within the heart and hypertensive lung.

In this study we reported that ghrelin significantly attenuated CH-induced right ventricular hypertrophy. This was most likely an indirect effect from attenuating the magnitude of PAH (i.e. reducing RV afterload). Ghrelin also has negative inotropic (contractility) and lusitropic (relaxation) attributes (22, 23) that may aid in attenuating the development of right ventricular hypertrophy.

Impairment of the endothelial NO pathway has also been implicated as a significant contributing factor in the pathophysiology of PAH (7, 41, 42). NO is a potent vasodilator and an inhibitor of vascular remodeling (9, 43, 44). In this study CH induced an overexpression of eNOS mRNA within the rat lung, which is consistent with previous reports (45, 46, 47). Despite the elevated levels of eNOS, reports suggest that NO bioavailability is reduced in CH (48) and, furthermore, that NO-dependent vasodilation is blunted (42, 49, 50).

Impairment of the NO-dependent vasodilation during CH may be caused by an increase in ET-1, as reported in this study, because the overexpression of ET-1 has directly inhibited NO-mediated dilatation (51, 52). Therefore, these reports may imply that ghrelin could indirectly increase NO bioavailability, although further studies are required to verify this assumption. Another factor that is likely to impair NO-dependent vasodilation may be an increase in reactive oxygen species during CH, which subsequently reduces NO availability (53, 54).

It has been suggested that the elevated level of eNOS after CH may be due to the hypoxic stimulus itself, independent of changes in flow/wall shear stress (55, 56). However, in our study we reported that ghrelin prevented the overexpression of eNOS, even though ghrelin-treated rats were subjected to the same hypoxic stimulus as saline-treated rats. These results may suggest that the increase in eNOS is a compensatory response to the increase in pressure/shear stress, rather than a response to the hypoxic stimulus itself. Therefore, the attenuated overexpression of eNOS in ghrelin-treated rats may be solely due to the reduced magnitude of PAH. However, we cannot exclude the possibility that ghrelin directly attenuated the overexpression of eNOS in CH. This scenario seems unlikely because previous studies have shown that ghrelin directly improves endothelial dysfunction, up-regulates eNOS expression (57), and increases NO bioactivity (58, 59).

Acute HPV
CH has altered acute HPV due to structural changes and endothelial dysfunction within the pulmonary vasculature (62). According to the literature, CH can potentially attenuate (63, 64, 65, 66), enhance, or have no effect on acute HPV (67, 68, 69). In this study CH accentuated the acute HPV response in saline-treated rats, which is consistent with our previous report (29). Interestingly, although ghrelin was able to attenuate CH-induced PAH, it was not able to prevent accentuation of the acute HPV. Essentially, the acute HPV response in ghrelin-treated rats was shifted downwards and was similar to saline-treated rats.

Despite decades of research concerning the HPV, the exact mechanism(s) that govern acute HPV are yet to be elucidated fully, although modulating influences from the endothelium (e.g. NO and ET-1), vasculature smooth muscle, and sympathetic nervous system are critical for full expression of acute HPV (see Ref. 70). In this study ghrelin attenuated pulmonary vascular remodeling and endothelial dysfunction during CH, so we expected that ghrelin would also prevent alterations in acute HPV. We are unsure as to the reasons why ghrelin did not prevent alterations in the HPV, although it may be possible that the O2-sensing mechanisms that initiate acute HPV are modified by CH, and in this study, such alterations could not be prevented by ghrelin.

Controversy currently exists as to whether the O2 sensor is located within the vascular endothelium or arterial smooth muscle cells. Consequently, some authors implicate endothelial dysfunction as the primary cause for alterations in acute HPV (39), whereas others implicate impairment of voltage-dependant K+ channels or redox sensors within smooth muscle cells (60). In this study it may be possible that ghrelin, which attenuated dysfunction of the eNOS and ET-1 pathways, was not able to prevent changes of the O2-sensor components within smooth muscle cells that facilitate HPV. This is an important area that warrants future research to provide further insight into acute HPV.

Limitations of this study
A significant limitation of this study is that we had limited data regarding protein levels for eNOS and ET-1. Indeed, we "predicted" ET-1 protein content based on mRNA expression alone. In this study we semiquantitatively assessed eNOS protein content, showing that changes in eNOS mRNA after CH (with or without ghrelin) were mirrored by similar changes in eNOS protein expression. Although we did not measure ET-1 protein content, previous studies have shown that ghrelin causes a concomitant reduction in both the expression of ET-1 mRNA and plasma levels of ET-1 protein (40).

Ghrelin, an orexigenic compound, can potentially increase appetite and food consumption, whereas hypoxia reduces food intake and attenuates weight gain. Therefore, we expected a greater weight gain for ghrelin-treated rats compared with saline-treated rats. Yet, weight gain was similar for all rats. The reason for this "lack" of difference is uncertain, although the fact that plasma glucose levels and free fatty acids were similar for all chronic hypoxic rats may indicate that the dose of ghrelin used in our study was not sufficient to modify metabolic status.

Baseline MABP in our study (~85 mm Hg) was slightly lower than that typical for a conscious rat (~90–110 mm Hg). Although the accuracy of the tail-cuff method has previously been validated in rats (30), Bunag (61) did warn that even with a properly validated tail-cuff method, errors could still be inadvertently incorporated into an experimental design. Consequently, it may be possible that MABP was underestimated in conscious rats of this study.

In summary, the results of this study indicate that ghrelin may be an effective prophylactic therapy for attenuating the adverse responses to CH, such as pulmonary vascular remodeling, PAH, and right ventricular hypertrophy. The mechanisms by which ghrelin improves cardiopulmonary prognosis are likely to involve attenuating the overexpression of ET-1 and possibly preventing impairment of the NO-mediated vasodilation. However, ghrelin was not able to prevent alterations of pulmonary vascular reactivity (i.e. HPV) after CH. This study highlights the therapeutic use of ghrelin for impeding the pathogenesis of pulmonary hypertension, particularly in subjects prone to CH (e.g. pulmonary disorders).


   Footnotes
 
This study was supported by the "Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation" of Japan.

Disclosure Summary: The authors have nothing to disclose.

First Published Online October 4, 2007

Abbreviations: ABP, Arterial blood pressure; BW, body weight; CH, chronic hypoxia; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GHS-R, GH secretagogue receptor; HPV, hypoxic pulmonary vasoconstriction; HR, heart rate; N-rat, normoxic rat; LV, left ventricle; MABP, mean arterial blood pressure; MPAP, mean pulmonary arterial pressure; NO, nitric oxide; PAH, pulmonary arterial hypertension; PAP, pulmonary arterial pressure; RV, right ventricle; Sep, septum.

Received June 25, 2007.

Accepted for publication September 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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