INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Pulmonary hypertension is a manifestation of a wide variety of cardiac and pulmonary diseases and a consequence of profound structural alterations in the pulmonary vasculature, commonly called pulmonary vascular remodeling (1). This process is known to involve medial smooth-muscle cell hypertrophy and hyperplasia, fibroblast proliferation, and matrix protein synthesis (2). The mechanism governing these architectural changes is unknown, although angiotensin II (AII), among other mediators, may be involved (5). In the systemic circulation, AII formed by the action of angiotensin converting enzyme (ACE) on AI has been shown to be an important mediator of vascular smooth-muscle cell growth (6). It seems likely that AII also contributes to the development of pulmonary hypertension via its vasoconstrictor action or via effects on vascular smooth-muscle cell migration and growth (7). Moreover, increasing evidence that ACE plays an important role in systemic vascular pathology led us to question whether ACE participates in the structural remodeling associated with pulmonary hypertension. Indeed, ACE is present in very high concentrations in the lungs, and its activity is further increased by chronic hypoxia (8).

The ACE gene has been considered a candidate gene for contributing to the development of hypertension and cardiovascular diseases. The ACE gene contains a polymorphism based on the presence (insertion [I]) or absence (deletion [D]) within an intron of a 287-bp nonsense DNA domain, resulting in three genotypes (DD and II homozygotes, and DI heterozygotes) (9). The ACE DD genotype is associated with increased circulating and cellular concentrations of ACE (10, 11), and the observed codominant association between D-I polymorphism and ACE activity could be consistent with the reported increase in cardiovascular risk associated with the DD genotype (12).

Recent studies have shown that the renin-angiotensin system is not only a hormonal system of the circulation, but also a tissue system, widespread in cardiovascular organs, that has been implicated in vascular remodeling accompanying various cardiovascular diseases (13). However, substantive evidence of a role for locally increased ACE activity in pulmonary hypertension resulting from chronic obstructive pulmonary disease (COPD) is lacking. We hypothesized that though pulmonary hypertension may develop at some point in most patients with COPD, a genetic predisposition to pulmonary vascular remodeling may exist. In the present study we investigated whether D-I polymorphism in the ACE gene is a genetic factor for the development of pulmonary hypertension in patients with COPD, using exercise challenge during breathing of room air or oxygen.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Nineteen patients with COPD (all males; mean age: 67.6 yr) from the respiratory outpatient clinic of our institution were randomly enrolled. They had not been recruited as part of another study. They had a history of former smoking and severe irreversible airflow limitation. All patients satisfied the American Thoracic Society criteria for COPD (14) and high-resolution computed tomographic scanning (HRCT) was performed on all patients, with the degree of emphysema quantified as previously described by our colleagues (HRCT emmphysema score in COPD subjects > 8%) (15). Patients with evidence of coronary artery disease, valvular heart disease, systemic hypertension, or primary myocardial disease were excluded from the study. None of the study subjects had radiologic or clinical evidence of pulmonary congestion or right heart failure. Concomitant left ventricular dysfunction was excluded in all patients by echocardiography performed prior to the study. All patients with COPD were clinically stable, and none had a history of respiratory infection for at least the 4-wk period preceding the study. All patients gave their written informed consent for participation in the study, which was approved by the Ethics Committee of Osaka City University, Japan.

Genomic DNA was extracted from peripheral blood leukocytes by standard methods. The ACE genotypes of the subjects were determined by polymerase chain reaction (PCR), using the primers and methods described by Rigat and colleagues (9). Under some conditions, the ACE D allele amplifies more effectively than the longer I allele, resulting in mistyping of the ID as the DD genotype (16). Therefore, all DD genotypes were reconfirmed. Briefly, the sense primer used in the PCR was replaced with an insertion-specific primer that leads to selective amplification of the I allele and absence of the D allele in mistyped ID genotypes. No mistyping was identified.

On the first day of the study, the subjects underwent a progressive incremental exercise test while sitting on an ergometer (EM840; Siemens, Germany), starting at 0 W for 3 min and adding 10 W every minute until a symptom-limited maximum was reached. Expired gas analysis was performed during the exercise test with a Respiromonitor RM-300 (Minoto Medical Science, Osaka, Japan) to continuously measure oxygen consumption (O₂), carbon dioxide production (CO₂), and minute ventilation (E). Heart rate (HR) was continuously monitored with standard electrocardiographic equipment. The purpose of the incremental exercise test was to determine maximal exercise capacity.

On the day following the test, all subjects underwent right heart catheterization. All cardiopulmonary medications were withheld for at least 12 h before the study. A balloon-tipped pulmonary arterial catheter was inserted percutaneously into an internal jugular vein and advanced to the pulmonary artery for measurement of pulmonary arterial pressure (Ppa) and pulmonary artery wedge pressure (Ppaw). In addition, a plastic catheter was placed percutaneously into the brachial artery to monitor systemic arterial pressure (Psa) and to sample systemic arterial blood. HR and heart rhythm were monitored continuously. Intravascular pressures were measured with a transducer (UK901; Baxter, Tokyo, Japan) located at the level of the anterior fourth intercostal space, with the patient sitting upright, and were recorded on photographic paper. Pressures were averaged over three respiratory cycles. Mean pressures were obtained by electronic integration. Cardiac output (CO) was determined by the thermodilution method, using a Fukuda Denshi CO computer. Arterial blood gas tensions were measured with a blood gas analyzer (Model IL1312; Instrumentation Laboratory).

Resting hemodynamic and blood gas data were obtained about 20 min after the patient had been seated comfortably on the ergometer. Each patient then performed a constant-load exercise test for 5 min while on the ergometer at a workload corresponding to 60% of the previously determined maximal workload. Hemodynamic and blood gas measurements were made during the final minute of constant-load exercise.

After exercise with breathing of room air, 100% oxygen was given to the patient for 60 min via nasal cannula at a rate of 3 L/min. All of the protocols described earlier were repeated while the patient breathed oxygen.

Statistical Analysis

All values are presented as mean ± SD. When multiple comparisons were made between groups, significant intergroup variability was first established with the Kruskal-Wallis test. The Mann-Whitney U test was then used for intergroup comparisons. The significance level was set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Pulmonary function data for all 19 study subjects with COPD are shown in Table 1. Baseline spirometry revealed obstructive defects in all of the subjects. Because all of the subjects were Japanese, population stratification was unlikely. The seven patients with the II genotype, six with the ID genotype, and six with DD genotype constituted well-matched subgroups with respect to age, blood gas data at rest or after exercise, baseline lung function, HRCT emphysema score, results of incremental exercise testing, and hemodynamic data at rest. The at rest in all patients averaged 23.0 mm Hg. Twelve of the 19 patients had resting pulmonary hypertension ( not less than 20 mm Hg). The mean at rest did not differ significantly in the three subgroups during breathing of room air (II: 22.3 ± 3.6 mm Hg; ID: 22.2 ± 4.4 mm Hg; DD: 24.5 ± 4.2 mm Hg) or oxygen (II: 20.7 ± 3.6 mm Hg; ID: 20.0 ± 5.2 mm Hg; DD: 21.8 ± 3.9 mm Hg). After constant-load exercise testing, the in all patients increased markedly. The after exercise challenge in patients with the DD genotype (55.7 ± 4.9 mm Hg) was significantly higher than that in patients with the II genotype (42.6 ± 7.1 mm Hg, p = 0.008) (Figure 1). The Rpv after exercise in patients with the DD genotype (8.9 ± 0.3 mm Hg/L/min/m²) was also significantly higher than in patients with the ID genotype (8.0 ± 0.3 mm Hg/L/min/m²) or the II genotype (7.1 ± 0.3 mm Hg/L/min/m²) (Table 2). During breathing of oxygen, the in patients with the DD genotype (52.3 ± 3.1 mm Hg) was also higher than that in patients with the ID genotype (40.5 ± 5.9 mm Hg, p = 0.0049) or the II genotype (37.7 ± 5.9 mm Hg, p = 0.0027) (Figure 2). In addition, the Rpv in patients with the DD genotype (8.7 ± 0.3 mm Hg/L/min/m²) was higher than that in patients with the ID genotype (7.6 ± 0.4 mm Hg/L/min/m²) or the II genotype (6.9 ± 0.4 mm Hg/L/min/m²).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Ppa before and after exercise in patients with the ACE II, ID and DD genotypes during breathing of room air.

View larger version (22K): [in this window] [in a new window]   Figure 2.   Ppa before and after exercise in patients with the ACE II, ID and DD genotypes during breathing of oxygen.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study we found that the significant increase in Ppa and Rpv evoked by exercise challenge is associated with the ACE DD genotype in patients with COPD. The 12 subjects with baseline  > 20 mm Hg were evenly distributed (II: n = 5; ID: n = 3; DD: n = 4). Thus, greater severity of disease, as reflected by a higher baseline Ppa and Rpv, did not occur in patients with the DD genotype, and could not have caused greater increases in Ppa and Rpv with exercise. In normal subjects, there is only a minimal increase in Ppa during exercise, due to recruitment of collateral vessels and distension of pulmonary vessels themselves (17). In contrast, in our patients, increased markedly with exercise despite a slightly elevated Ppa at rest. Thus, pulmonary hypertension became particularly pronounced in patients with COPD during exercise, indicating that the increase in pulmonary blood flow during exercise resulted in a steep increase in Ppa. In this study, the patients with the DD genotype did not have an increased baseline Ppa or Rpv. For that reason, we thought that our patients had the ability to accommodate pulmonary blood flow at rest, but that they had lost the ability to accommodate increased pulmonary blood flow through distension of pulmonary vessels during exercise. This loss is due principally to pulmonary vascular remodeling.

Pulmonary vascular remodeling is known to occur as a consequence of persistent pulmonary vasoconstriction in response to chronic hypoxia, in addition to following pulmonary vascular destruction secondary to emphysematous change (18). In the present study, the three subgroups of COPD patients did not differ significantly with regard to their blood gas data, diffusing capacity of carbon monoxide, HRCT emphysema score, or exercise capacity on incremental exercise testing, suggesting that the patients had the same degree of emphysematous change and pulmonary vascular destruction. Moreover, it is well known that one of the most important determinants of Ppa and Rpv is hypoxia. The three subgroups in the study were similar in their degree of exercise-induced hypoxia. Therefore, these findings suggested that the D-I polymorphism in the ACE gene may be associated with pulmonary hypertension evoked by exercise challenge in patients with COPD.

Considerable controversy has recently surrounded the role of the D-I polymorphism in pulmonary hypertension. For example, Abraham and coworkers found that the DD genotype was associated with right-ventricular hypertrophy in patients with primary pulmonary hypertension (19). In contrast, van Suylen and associates found less right-ventricular hypertrophy in COPD patients who had the DD genotype (20). However, a limitation of their study was that evidence of right-ventricular hypertrophy was based on electrocardiographic criteria, and this technique is rather insensitive compared with hemodynamic and echocardiographic measurements.

Chronic hypoxia is a well-characterized experimental model of pulmonary hypertension, which is caused by vasoconstriction and pulmonary vascular remodeling (21). In recent studies, messenger RNA for ACE and ACE antigen expression were found to be increased locally in the walls of small pulmonary arteries in rat hypoxic pulmonary hypertension (22). Our findings in the present study suggested that patients with the DD genotype might have a greater increase in tissue ACE activity than those with the II and ID genotypes, and that increased production of AII causes proliferation and hypertrophy of vascular smooth-muscle cells in small pulmonary arteries. Moreover, to enable testing with a decreased degree of acute hypoxic pulmonary vasoconstriction, we performed another exercise challenge in which our subjects breathed oxygen. Induction of pulmonary hypertension by exercise challenge with breathing of oxygen was also greater in patients with the DD genotype than in those with the II or ID genotype.

We have also examined other possible ACE phenotypes. However, the D-I genotypes examined in the present study did not affect the attenuation of the increase in Ppa following exercise with breathing of oxygen, or the magnitude of the increase in following exercise, or the magnitude of the increase in exercise-induced systemic hypertension. On the other hand, several additional possible explanations exist for the observed association between ACE polymorphism genotypes and pulmonary hypertension with exercise in COPD patients. Population stratification based on ethnicity or other factors could have contributed to the differences in across the genotypes examined in our study. However, all of the subjects in this study were Japanese, and population stratification is therefore less likely to have occurred. In addition, it is also possible that the deletion polymorphism is associated with because this polymorphism is in linkage disequilibrium with another causative variant in or near the ACE gene. Further research will be required to clarify this issue.

In conclusion, the number of patients in this study was very small for a genetic association study, and our results should be examined in larger studies. However, since invasive physical monitoring such as that used in this study is difficult to perform on large numbers of subjects, further studies will probably be required to use noninvasive methods, including echocardiographic measurements. Identification of the D-I polymorphisms in the ACE gene may be valuable in the management of COPD, since there could be a genotype-based variation in response to ACE inhibitors that wouid make genotyping for these polymorphisms a clinically useful exercise. The effects of ACE genotypes on the response to therapy with ACE inhibitors or AII receptor antagonists should be studied.