INTRODUCTION

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Chronic alveolar hypoxia is associated with the development of pulmonary hypertension and right ventricular hypertrophy. Both hypoxia-induced vasoconstriction and structural pulmonary vascular changes contribute to an increased pulmonary artery pressure. Hypoxia-induced structural pulmonary vascular changes include increased medial thickness of small muscular pulmonary arteries and peripheral extension of smooth muscle cells into normally nonmuscular arterioles, commonly called pulmonary vascular remodeling (1, 2). These structural changes are a result of vascular smooth muscle cell proliferation, hypertrophy, and deposition of extracellular matrix components (3). In the systemic circulation angiotensin II (AII) has been shown to be a mediator of vascular smooth muscle cell growth and/or proliferation (7) and may therefore also be regarded as a potential modulator of the structural pulmonary vascular changes associated with pulmonary hypertension. During the development of hypoxic pulmonary hypertension, local angiotensin-converting enzyme (ACE) expression is increased in the walls of small pulmonary arteries (10). Several studies have investigated the effects of ACE inhibitors on pulmonary artery pressure and pulmonary artery structure under normoxic and chronic hypoxic conditions. Under normoxic conditions ACE inhibition has no effect on rat pulmonary artery pressure and pulmonary artery structure (11, 12). Most studies report that in chronic hypoxic rats, ACE inhibitors prevent or attenuate the development of pulmonary artery medial thickening and/or muscularization of arterioles (12). The literature is, however, less clear about the effects of ACE inhibitors on pulmonary artery pressure. In chronic hypoxic rats, cilazapril completely prevents medial thickening of pulmonary arteries without a significant effect on pulmonary artery pressure and right ventricular weight (15). Data from our group show that captopril prevented the increase in medial cross-sectional area of small (30-100 µm) muscular pulmonary arteries in chronic hypoxic rats and reduced the percentage of muscularized arterioles (16). This effect occurred, as in the study with cilazapril, without a decrease in pulmonary artery pressure or right ventricular hypertrophy. Others, however, did observe a preventive effect of ACE inhibition on the increase in pulmonary artery pressure during hypoxia (12).

Tissue ACE-deficient mice have been generated by introducing a modified ACE allele into a mouse line (17). Mice homozygous for this mutant allele (-/-) have only 34% of the ACE activity in plasma compared with wild-type mice, and ACE activity in lung tissue is undetectable. They also have a reduced systemic blood pressure, thickening of small renal arteries and arterioles, and a urine-concentrating defect. There are no data available on the pulmonary vasculature. On the basis of our data on rats (16) we hypothesized that under normoxic conditions tissue ACE-deficient mice have a normal mean right ventricular pressure and no structural pulmonary vascular changes. Under chronic hypoxic conditions, however, they should exhibit less remodeling of arterioles compared with wild-type mice, yet mean right ventricular pressure increases to a comparable extent as in hypoxic wild-type mice.

	METHODS

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Experimental Animals

Mice had a targeted disruption of the ACE gene (homozygous mutant, -/-) on a C57BL/6J genetic background. Wild-type (+/+) animals were littermates. They were a kind gift of K. E. Bernstein (Atlanta, GA). Male and female mice at an age of 6-10 wk, weighing 18 to 30 g, were used for the experiment. The generation of these mice has been described by Esther and coworkers (17). Heterozygous male and female were bred and at the age of 21 d offspring was weaned and genotyped by polymerase chain reaction (PCR) analysis (18). Animals were provided food (RMH-TM; Hope Farms, Woerden, The Netherlands) and water ad libitum and were maintained on a 12-h light/dark cycle. The experiments were performed according to institutional guidelines for the use and care of animals and approved by the local ethics committee. All mice were killed 4 wk after the start of the experiment.

Chronic Hypoxia

On Day 1 mice were placed into a normobaric chamber for 4 wk. The chamber was maintained at 10% O₂, which was accomplished by controlling the inflow rates of room air and nitrogen. The O₂ concentration was checked daily, using an O₂ analyzer (AVL 993; AVL List, Graz, Austria). Each day the chamber was opened for approximately 1 min to replenish food and water.

Experimental Groups

A total of 82 mice was investigated. Wild-type (+/+) and tissue ACE-deficient (-/-) mice were studied under normoxic (-/-, n = 19; +/+, n = 24) and chronic hypoxic (-/-, n = 19; +/+, n = 20) conditions. Groups were matched for age and sex.

Hemodynamic Studies

At the end of the experiment the mice were anesthetized (pentobarbital [110 mg/kg], administered intraperitoneally; Sanofi Sante, Maassluis, The Netherlands) and placed on a heated table. The trachea was intubated, and the lungs were ventilated with room air at 80 strokes/min and 1.5-ml tidal volume. A parasternal thoracotomy from the xiphoid to the pretracheal region was performed. A needle (o.d. 0.6 mm) connected to a silastic catheter was placed into the right ventricular cavity by direct puncture of the right ventricle. The catheter was connected to a pressure monitor. After stabilization for 10 min, the right ventricular systolic and diastolic pressures were recorded during a 10-min period, using a miniature pressure transducer (CP-01; Century Technology, Inglewood, CA) and a computerized data-acquisition system (HDAS; Instruments Services Department, Maastricht University, Maastricht, The Netherlands). Mean right ventricular pressure (MRVP) was calculated by digital integration.

Hematocrit

After induction of anesthesia with pentobarbital, blood samples were obtained by orbital puncture. The hematocrit was determined with a microcapillary centrifuge.

Assessment of Right Ventricular Hypertrophy

The heart was dissected free and both atria, the aorta, and the pulmonary trunk were removed. The right ventricle was separated from the left ventricle and the ventricular septum. Right ventricular hypertrophy was expressed as the ratio of the weight of the right ventricular wall (RV) and that of the left ventricular wall and ventricular septum (LV + S).

Assessment of Muscularization of Arterioles

After the hemodynamic studies 10% phosphate-buffered formalin (pH 7.4) was infused into the lung via a trachea tube, at a pressure of 20 cm H₂O. Subsequently the heart and lungs were excised and fixed for 24 h in 10% phosphate-buffered formalin (pH 7.4). Both lungs were paraffin embedded, and 4-µm sections, parallel to the hilus, were stained with a monoclonal antibody against -smooth muscle actin (diluted 1:500; Dako, Carpinteria, CA) using diaminobenzidine as the chromogen and hematoxylin as the counterstain (19).

At ×400 magnification 50 alveolar wall and alveolar duct vessels were located, and the proportion of muscularized and partially muscularized vessels was calculated and recorded as a percentage of the total alveolar wall and alveolar duct vessels. A completely muscularized arteriole was defined as an arteriole with -smooth muscle actin-positive cells forming a subendothelial layer of more than 50% of its circumference. A partially muscularized arteriole was defined as an arteriole where -smooth muscle actin-positive cells formed a subendothelial layer of less than 50% of its circumference. If -smooth muscle actin staining was absent, the vessel was termed nonmuscular (Figure 1). This analysis was performed by a single observer (R.J.v.S.), who was unaware of the experimental conditions.

View larger version (81K): [in this window] [in a new window]   Figure 1.   Immunoperoxidase staining for -smooth muscle actin of completely muscularized (A), partially muscularized (B), and nonmuscular (C ), arteriole. Arrows indicate -smooth muscle actin-positive smooth muscle cells (brown). All bars: 20 µm.

Statistical Analysis

All results were expressed as means ± SD. Differences between groups were determined by a nonparametric Mann-Whitney U test (20). Statistical significance was defined as p < 0.05.

	RESULTS

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Effects of Chronic Hypoxia in Tissue ACE-deficient Mice

Chronic hypoxic conditions induced an increase in mean right ventricular pressure (normoxia -/- = 7.7 ± 1.9 vs. hypoxia -/- = 11.7 ± 1.7 mm Hg, p < 0.05) and right ventricular hypertrophy [RV/(LV + S): normoxia -/- = 0.29 ± 0.05 vs. hypoxia -/- = 0.49 ± 0.1, p < 0.05] and an increase in hematocrit (normoxia -/- = 38 ± 3 vs. hypoxia -/- = 60 ± 6%, p < 0.05) (Table 1) and pulmonary vascular remodeling, especially the muscularization of arterioles (Figure 2).

View larger version (21K): [in this window] [in a new window]   Figure 2.   The percentage of nonmuscular (NM), partially muscularized (PM), and completely muscularized (M) arterioles in wild-type (+/+) and tissue ACE-deficient (-/-) normoxic and hypoxic mice. All values are presented as means ± SD. #p < 0.05 compared with genotype-matched normoxic mice. p < 0.05 compared with wild-type mice with the same exposure.

Absence of Tissue ACE: Effect on Hemodynamic and Structural Pulmonary Vascular Parameters under Normoxic and Hypoxic Conditions

To determine whether the absence of tissue ACE has an effect on the parameters being measured, normoxic and chronic hypoxic tissue ACE-deficient mice were compared to normoxic and hypoxic wild-type littermates.

Normoxia. Under normoxic conditions right ventricular weight and the ratio of right ventricular weight to body weight were comparable in wild-type and tissue ACE-deficient mice (Table 1). The LV + S/BW ratio and the mean weight of the LV + S were higher in normoxic wild-type mice compared with tissue ACE-deficient mice. The higher ratio of RV and LV + S in tissue ACE-deficient mice is therefore likely due to their lower LV + S weight. The hematocrit in normoxic wild-type mice was slightly higher than the hematocrit in tissue ACE-deficient mice. The percentages of nonmuscular, partially muscularized, and completely muscularized arterioles were not significantly different between wild-type and tissue ACE-deficient mice. Also, the MRVP in normoxic tissue ACE-deficient mice was not different from the MRVP in normoxic wild-type mice.

Chronic hypoxia. The right ventricular weights of wild-type mice were not significantly different compared with tissue ACE-deficient mice, whereas the weight of LV + S was lower in tissue ACE-deficient mice (Table 1). The higher RV/LV + S ratio in tissue ACE-deficient mice is therefore mainly due to their lower LV + S weight.

The rise in hematocrit was less in tissue ACE-deficient mice compared with wild-type littermates. The percentage of completely muscularized arterioles was lower in chronic hypoxic tissue ACE-deficient mice compared with chronic hypoxic wild-type mice whereas the percentage of partially muscularized arterioles was higher (Figure 2). Thus, hypoxia-induced pulmonary vascular remodeling was attenuated in tissue ACE-deficient mice. There was no difference in MRVP between wild-type and tissue ACE-deficient hypoxic mice.

Sex and Genotype

No differences in MRVP, cardiac weights, and extent of muscularization of pulmonary arterioles were observed between hypoxic male and female mice, either in wild-type or in tissue ACE-deficient mice (Table 2).

	DISCUSSION

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The aim of this study was to assess the effects of chronic hypoxia on hemodynamic and structural pulmonary vascular changes in mice lacking tissue-bound ACE. Under chronic hypoxic conditions tissue ACE-deficient mice showed an increase in MRVP and right ventricular hypertrophy. These effects were comparable to those found in chronic hypoxic wild-type mice. Hypoxia also induced structural pulmonary vascular changes in tissue ACE-deficient mice; however, the extent of these structural vascular changes was smaller than those in wild-type mice. The data suggest that, in mice, local pulmonary production of AII by ACE plays an important role in pulmonary vascular remodeling secondary to chronic hypoxia. Experiments performed in hypoxic rats corroborate with these findings. Morrell and coworkers have demonstrated that ACE expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension (10). Moreover, several studies have shown that ACE inhibitors can at least partially prevent the development of hypoxia-induced structural pulmonary vascular changes (11, 13, 15), probably via inhibition of pulmonary vascular smooth muscle cell proliferation and/or growth (12). This effect is angiotensin type 1 receptor mediated (21) and apparently pressure independent (16). Although we showed that pulmonary vascular remodeling was attenuated in hypoxic tissue ACE-deficient mice, a majority (77%) of the arterioles in these animals was partially or completely muscularized. These residual structural pulmonary vascular changes could be a result of AII formation by plasma ACE, which is reduced but not absent in these mice (17). On the other hand, alternative pathways of AII formation as, for instance, by chymase (22), or compensatory mechanisms may compensate for the loss of tissue ACE in these mice. However, in two separate studies, the present study and a pharmacologic study (with an ACE inhibitor in rats) (16), we found similar effects of chronic hypoxia on right ventricular hypertrophy, right ventricular pressure, and pulmonary vascular remodeling. We therefore think that alternative AII-forming pathways or compensatory mechanisms do not play an important role in chronic hypoxic tissue ACE-deficient mice.

Another interesting result of the present study is that the mean right ventricular pressure was not different in wild-type and tissue ACE-deficient hypoxic mice despite a significant reduction of completely muscularized arterioles in tissue ACE-deficient hypoxic mice. This result is in accordance with the results from a previous study from our group, in which we demonstrated that the ACE inhibitor captopril also attenuated the increase in the percentage of completely muscularized arterioles, without any effect on the pulmonary artery pressure and right ventricular hypertrophy in chronic hypoxic rats (16). A possible explanation for the dissociation between structural changes of arterioles and right ventricular pressure is that other structural changes, such as medial thickening of muscular pulmonary arteries (external diameter between 30 and 200 µm), contribute to an increase in MRVP. In rats, we have shown that chronic hypoxia only slightly increased the media cross-sectional area without a change in the lumen area (16), suggesting that neither changes in media cross-sectional area nor changes in lumen area are significant contributors to hypoxia-induced pulmonary hypertension. In the mouse lung the number of these muscular pulmonary arteries suitable for morphometrical analysis is too low to allow reliable measurements with respect to total vessel area, medial area, and lumen area. An alternative explanation for the dissociation between arteriolar structure and right ventricular pressure could be the fact that, despite a significant reduction of the percentage of completely muscularized arterioles in hypoxic tissue ACE-deficient mice, the percentage of partially muscularized arterioles was significantly increased compared with hypoxic wild-type mice. Constriction of these partially muscularized arterioles, even under normoxic conditions, may obscure potential effects of a reduced percentage of completely muscularized arterioles on the MRVP.

Hematocrit levels were increased in hypoxic wild-type mice as compared with hypoxic tissue ACE-deficient mice. It is, however, unlikely that a difference in the hematocrit between wild-type and tissue ACE-deficient hypoxic mice obscures potential effects of a reduced percentage of completely muscular arterioles on the MRVP. Steudel and coworkers have shown that in hypoxic mice, even a high hematocrit (65-75%) does not impose sufficient additional work load on the right ventricle to cause hypertrophy (23).

In the systemic circulation there are some contradictory data with respect to sex specificity of the ACE gene and the presence of hypertension. In white individuals, O'Donnell and coworkers observed an association with the I/D polymorphism of the ACE gene and hypertension in men but not in women (24), whereas data from Sugiyama and coworkers did not support the existence of a sex-specific association between the ACE I/D polymorphism and essential hypertension (25). With respect to the pulmonary circulation, few data exist about a sex-specific association between polymorphisms of the ACE gene and pulmonary hypertension. Data from our group show that only in male patients with chronic alveolar hypoxia (chronic obstructive pulmonary disease) the DD variant of the ACE gene is negatively associated with right ventricular hypertrophy (26). In the present study we did not observe a male/female difference with respect to MRVP, hematocrit, cardiac weights, and structural pulmonary vascular changes in hypoxic mice, neither in wild-type nor in tissue ACE-deficient animals.

In summary, this study demonstrates that chronic hypoxia induces an increase in MRVP, right ventricular hypertrophy, and pulmonary vascular remodeling in these mice despite an absence of tissue-bound ACE. However, pulmonary vascular remodeling is attenuated in hypoxic tissue ACE-deficient mice compared with hypoxic wild-type littermates. This implies that local production of AII by ACE in pulmonary arterioles plays an important role in the pathogenesis of pulmonary vascular remodeling secondary to chronic hypoxia, but not in the development of hypoxia-induced increase in mean right ventricular pressure.