INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Pulmonary hypertension is a common complication of cardiopulmonary diseases. Regardless of the etiology, all patients with chronic pulmonary hypertension have structural pulmonary vascular changes, right ventricular hypertrophy and, in the long term, right ventricular failure (1). In patients with congenital heart disease, however, preoperative hemodynamic findings and postoperative clinical outcome do not always correlate with structural pulmonary artery changes (4). This discrepancy between hemodynamics and pulmonary artery structure is also true for animal studies. For example, the angiotensin-converting enzyme (ACE) inhibitor cilazapril completely prevented the thickening of the media of pulmonary arteries in chronic hypoxic rats but did not decrease the pulmonary artery pressure (PAP) or right ventricular weight (9). The apparent discrepancies between changes in pulmonary vascular structure and hemodynamic function may be explained by the fact that vasoconstriction, rather than structural narrowing of pulmonary arteries, plays a pivotal role in the rise of PAP. An alternative explanation is that previous studies have focused on the wrong parameters of structural pulmonary vascular changes. In most studies medial wall thickness is expressed as a percentage of the arterial external diameter (10). This parameter, however, does not adequately reflect changes in medial cross-sectional area and lumen area since the external diameter of arteries may also change during the complex process of vascular remodeling (13). Recently, Mulvany and coworkers introduced an adaptation of terminology to allow a precise description of the structural changes that can occur in the vasculature (13). The media to lumen ratio and the lumen area, rather than medial wall thickness, are essential parameters in this analysis. In contrast to the systemic circulation, description of structural vascular changes according to the concept of Mulvany (14) has not yet been employed in the pulmonary circulation. We therefore determined the lumen area, the medial cross-sectional area, and the ratio of media to lumen area of muscular pulmonary arteries with an external diameter between 30-100 and 101-200 µm in two classic rat models of pulmonary hypertension, the rat hypoxia and monocrotaline models (11, 20, 21). To optimize structural analyses, the pulmonary vessels were fixed under standard intravascular pressure and maximal vasodilatation. Right ventricular weight and PAP were measured to correlate structural pulmonary vascular changes with hemodynamic function. Moreover, we investigated the effects of the ACE inhibitor captopril on pulmonary vascular remodeling and pulmonary hemodynamics, because angiotensin II (AII) is believed to be involved in hypoxia and MCT-induced pulmonary hypertension. In both the systemic and pulmonary circulation AII acts as a vasoconstrictor and can induce structural arterial changes like smooth muscle hypertrophy and proliferation as well as matrix protein synthesis (22). In animal models of pulmonary hypertension, several ACE inhibitors are known to prevent or attenuate the development of pulmonary hypertension and medial thickening of pulmonary arteries (9, 27). The consequences of this intervention for the lumen area of pulmonary arteries, however, have not been described.

Thus we hypothesized that both chronic hypoxia and monocrotaline would increase the medial cross-sectional area and decrease the lumen area of muscular pulmonary arteries. Second, we hypothesized that changes in the media to lumen ratio and lumen area of muscular pulmonary arteries would correlate with changes in PAP. Finally we hypothesized that captopril would prevent or attenuate the reduction of the lumen area of the pulmonary arteries, right ventricular hypertrophy, and the increase in PAP in these two rat models.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Morphological Studies

Forty young male Wistar rats (Iffa Credo, Someren, The Netherlands) were used. Body weight was 145-200 g at the start of the experiments. The rats were randomly assigned to one of four treatment groups. Food (RMH-TM; Hope Farms, Woerden, The Netherlands) and water were freely available to all groups of animals. The experiments were performed according to institutional guidelines for the use and care of animals. All rats were killed 4 wk after the start of the experiment.

Group 1: n = 8, Monocrotaline (MCT)

At Day 1 these rats received a single subcutaneous injection of monocrotaline (300 µl, 60 mg/kg in 0.2 N HCl; Sigma, St. Louis, MO).

Group 2: n = 8, Hypoxia (H)

At Day 1 these rats were placed into a normobaric chamber for 4 wk. Hypoxic exposure was accomplished by ventilation with room air (2.5 L/min) and N₂ (2.5 L/min) resulting in a constant O₂ concentration of 10%. The O₂ concentration was checked every day using an O₂ analyzer (AVL 993; AVL LIST GmbH, Graz, Austria). Each day the chamber was opened for approximately 2 min to replenish food and water.

Group 3: n = 8, Monocrotaline and Captopril (MCTcap)

At Day 1 these rats received a single subcutaneous injection of monocrotaline (300 µl, 60 mg/kg in 0.2 N HCl). The ACE inhibitor captopril (0.5 mg/kg/h in 0.9% NaCl; Sigma) was given for 4 wk via osmotic minipumps (Alzet Model 2002; Alza Corp., Palo Alto, CA) implanted subcutaneously between the shoulder blades under ether anesthesia. Before implantation the minipumps were placed in 0.9% NaCl at 37° C for 4 h. After 14 d the minipumps were replaced and a new pump filled with captopril was inserted at the same site.

Group 4: n = 8, Hypoxia and Captopril (Hcap)

These rats were kept under hypoxic conditions for 4 wk as described for Group 2. During this period the rats received captopril in the same way as described for Group 3.

Group 5: n = 8, Control Rats (C)

These rats were kept under normoxic conditions (room air) without intervention.

Preparation of Lung Tissue for Morphological Measurements

After induction of anesthesia with sodium pentobarbital (60 mg/kg intraperitoneally), the right jugular vein was cannulated and the tip of the Silastic cannula was positioned into the right atrium. After opening the abdominal wall and the thoracic cavity a clamp was placed on the inferior caval vein. The abdominal aorta was cut open to allow maximal perfusion of the lung vessels. A solution containing phosphate-buffered saline (PBS, pH 7.4) and nitroprusside (1 mg/ml; Sigma) was perfused at a pressure of 18 mm Hg for 10 min through the pulmonary vessels via the cannula in the right atrium. Subsequently, a solution of 10% phosphate-buffered formalin (pH 7.4) and nitroprusside (1 mg/ ml) was infused for 10 min for perfusion fixation under maximal vasodilatation. At the same time 10% phosphate-buffered formalin (pH 7.4) was administered into the lungs via a trachea tube, at a pressure of 20 cm H₂O. Subsequently, heart and lungs were removed and fixed overnight in 10% phosphate-buffered formalin (pH 7.4). Both left and right lung were processed for light microscopy. The lungs were paraffin embedded and 4-µm sections were stained with Lawson's stain (Klinipath, Zevenaar, The Netherlands).

Assessment of Right Ventricular Hypertrophy

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

Hematocrit

The hematocrit was determined using a microcapillary centrifuge for 5 min. After induction of anesthesia with pentobarbital blood samples were drawn from the orbita.

Light Microscopic Analysis of Pulmonary Arteries

Remodeling of pulmonary arteries. Slides were analyzed by light microscopy by one observer (R.J.v.S.) who was unaware of the treatment group. To assess the type of remodeling of muscular pulmonary arteries, microscopic images were analyzed using a computerized morphometric system (Quantimet 570; Leica, Cambridge, UK). Total vessel area was defined as the area within the lamina elastica externa, and lumen area was defined as the area within the lamina elastica interna. Medial area is the area between the lamina elastica interna and the lamina elastica externa. Arteries were categorized depending on the external diameter. Category I included arteries with an external diameter between 30 and 100 µm, and category II included arteries with an external diameter between 101 and 200 µm. Within each category 10 arteries were measured per animal. The average of 10 values obtained was used for calculations.

Assessment of muscularization of arterioles. Extension of smooth muscle cells into usually nonmuscular arterioles of the alveolar wall and alveolar duct was assessed as follows. At ×400 magnification 100 alveolar wall and alveolar duct vessels were counted and noted as muscular, partially muscular, or nonmuscular. These alveolar wall and alveolar duct vessels included small venules which could not be distinguished from normal pulmonary arterioles. A completely muscular arteriole was defined as an arteriole with a double elastic lamina for more than half of its circumference. An arteriole was categorized as partially muscular if the double elastic lamina was less than half of its circumference. A normal nonmuscular arteriole had a single elastic lamina. Intraobserver variation was 10%.

Hemodynamic Studies

For hemodynamic measurements, separate groups of male Wistar rats (mean initial body weight 178 g) were used and treated in an identical way as the rats used for structural pulmonary vascular analysis (MCT n = 6, MCTcap n = 7, H n = 7, Hcap n = 7, C n = 6).

PAP was measured using the technique described by Rabinovitch and coworkers (33). In brief, the animals were anesthetized (60 mg/kg pentobarbital, intraperitoneally) and placed on a heated table (37° C). The trachea was intubated and the animals were respirated with room air at 60 strokes/min and 2.5 ml tidal volume. The right jugular vein was isolated and a stainless steel introducer and catheter were passed via a small transverse cut. The catheter was connected to a pressure monitor and was advanced into the right ventricle and pulmonary trunk under guidance of the pressure tracing. With the catheter in place, the introducer was carefully retracted. Following stabilization during 15 min, PAP was recorded during a 15-min period using a miniature pressure transducer (CP-01); Century Technology, Inglewood, CA) and a computerized data-acquisition system (MDAS; Instruments Services Dept., UM, Maastricht, The Netherlands). Mean pulmonary artery pressure was calculated by digital integration.

Statistical Analysis

Data are expressed as means ± SD. Statistical significance was defined at p < 0.05. Intergroup differences were evaluated with a nonparametric Mann-Whitney U test with a Bonferroni correction for multiple group comparison (34).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Morphological Studies

Body and cardiac weights. Rats in the control group gained 124 ± 24 g during 4 wk. Rats exposed to hypoxia or treated with MCT only gained half the weight compared with the control group. In the MCT group, captopril had no significant influence on the body weight change (Table 1). In the hypoxic group captopril treatment diminished the gain in body weight further. The MCT and hypoxic groups exhibited significant right ventricular hypertrophy, defined as an increase of the ratio of right ventricular weight and the sum of the weight of the left ventricular free wall and interventricular septum (RV/LV + S ratio) (MCT = 0.69 ± 0.16, H = 0.51 ± 0.09, C = 0.22 ± 0.03, both p < 0.001) compared with the control group. In both groups captopril treatment had no significant effect on the RV/LV + S ratio. Hematocrit values significantly increased in the hypoxic rats (H = 72 ± 6, C = 47 ± 2, p < 0.001), whereas the hematocrit significantly decreased in the MCT-treated rats (MCT = 42 ± 4, C = 47 ± 2, p < 0.05). Captopril treatment had no effect on the hematocrit (Table 1).

Vessel Dimensions

Category I arteries (external diameter 30-100 µm). MCT significantly increased the media to lumen ration (MCT = 46 ± 9, C = 24 ± 4, p < 0.001) and the medial cross-sectional area (MCT = 1,435 ± 344, C = 961 ± 387, p < 0.05) of category I pulmonary arteries. The lumen area showed a trend to decrease although this does not reach significant levels (p = 0.2) Table 2 and Figure 1A and B). Hypoxia also increased the media to lumen ratio (H = 38 ± 13, C = 24 ± 4, p = 0.02). The medial cross-sectional area showed a trend to increase (H = 1,326 ± 410, C = 961 ± 387, p = 0.07) whereas the lumen area was not changed. In MCT-treated rats captopril did not affect the media to lumen ratio nor the lumen area. In the hypoxic rats captopril prevented an increase in the media to lumen ratio (Hcap = 28 ± 17, H = 38 ± 13, p < 0.05) and medial cross-sectional area (Hcap = 944 ± 115, H = 1,326 ± 410, p < 0.05). Captopril had no effect on the lumen area.

View larger version (11K): [in this window] [in a new window]   View larger version (11K): [in this window] [in a new window]   Figure 1.   Media to lumen ration (MA/LA) of category I pulmonary arteries in relation to (A) the ratio of weight of the right ventricular wall and that of the free left ventricular wall and ventricular septum (RV/LV + S) and (B) pulmonary artery pressure (PAP) in control rats (C), monocrotaline-treated rats (MCT), monocrotaline and captopril treated rats (MCTcap), chronic hypoxic rats (H), and chronic hypoxic and captopril treated rats (Hcap). All values are presented as means. For standard deviations see Tables 1 and 2. *p < 0.05 versus control. p < 0.05 versus hypoxia.

Category II arteries (external diameter 101-200 µm). MCT significantly increased the media to lumen ratio (MCT = 38 ± 10, C = 21 ± 6, p < 0.05) and medial cross-sectional area (MCT = 5,112 ± 1,502, C = 3,826 ± 1,545, p < 0.05) of category II pulmonary arteries whereas the lumen area decreased compared with control rats (MCTcap = 13,514 ± 1,555, C = 17,463 ± 3,601, p < 0.05). Hypoxia did not affect the media to lumen ratio or the lumen area, whereas the medial cross-sectional area increased to some extent (H = 4,807 ± 1,167, C = 3,826 ± 1,545, p = 0.07). Captopril treatment had no effect on the media to lumen ratio, medial cross-sectional area, and lumen area neither in MCT-treated rats nor in hypoxic rats (Table 2).

Arterioles. Both MCT and hypoxia increased the percentage of partly muscularized and muscularized arterioles and decreased the percentage of nonmuscularized arterioles (Figure 2). In the MCT-treated rats captopril had no effect on muscularization of arterioles, whereas in hypoxic rats it reduced the percentage of muscularized arterioles and significantly increased the percentage of partly muscularized and nonmuscularized arterioles.

View larger version (23K): [in this window] [in a new window]   Figure 2.   The percentage of nonmuscular (NM), partially muscular (PM), and muscular (M) arterioles in control rats (C), monocrotaline-treated rats (MCT), monocrotaline and captopril treated rats (MCTcap), chronic hypoxic rats (H), and chronic hypoxic and captopril treated rats (Hcap). All values are presented as mean ± SD. *p < 0.05 versus control. p < 0.05 versus hypoxia.

Hemodynamic Studies

MCT and chronic hypoxia induced a significant increase in PAP (MCT = 42.4 ± 10 mm Hg, H = 42.7 ± 5.6 mm Hg, C = 15.5 ± 1.5 mm Hg, p < 0.005, Table 1). In both groups captopril did not affect PAP. Captopril treatment had no significant effect on the PAP in control rats (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This is one of the first morphometrical studies of muscular pulmonary arteries, analyzed under standard intravascular pressure and maximal vasodilatation, in which changes of pulmonary artery structure and changes in pulmonary artery pressure are compared.

Although the increases in PAP and right ventricular hypertrophy were comparable in the rat hypoxia and MCT model, structural analysis of muscular pulmonary arteries reveals major differences between the two models which may be due to the way we analyzed the pulmonary vascular structure. In general, structural pulmonary vascular changes in animal models of chronic pulmonary hypertension include thickening of the media of muscular pulmonary arteries and muscularization of normally nonmuscular arterioles. Some studies have shown that chronic hypoxia and MCT induce different patterns of pulmonary vascular remodeling. Muscularization of arterioles is seen in both models, however, medial hypertrophy of muscular pulmonary arteries is more extensive in monocrotaline-treated rats (35, 36). Only a few studies report the consequences of these structural vascular changes for the lumen diameter (11, 20, 37). These investigators stated that thickening of the wall of pulmonary arteries contributed to a decrease in the lumen diameter. Changes in lumen diameter, however, were based on arteriograms and were investigated without maximal vasodilatation. Thus vasoconstriction rather than structural vascular changes may have been responsible for the reported reduction of the lumen diameter. To exclude the possible effects of vasoconstriction on medial cross-sectional area and lumen area, we performed morphometrical analysis of pulmonary arteries after maximal vasodilatation and fixed the vessels in situ at standard intravascular pressures. By this way we are assured that an increase in medial cross-sectional area indeed reflects a structural increase of the medial cross-sectional area rather than active pulmonary vasoconstriction. Moreover, in those conditions a reduction of the lumen area reflects a structural reduction rather than a functional reduction of the lumen area as a consequence of pulmonary vasoconstriction.

Under these conditions MCT increased the medial cross-sectional area and media to lumen ratio of pulmonary arteries with an external diameter between 30-100 µm and 101-200 µm. The consequences for the lumen area, however, were different in these two categories of pulmonary arteries. As expected, an increase in medial cross-sectional area was associated with a reduction of the lumen area of pulmonary arteries with an external diameter between 101-200 µm. The lumen area of pulmonary arteries with an external diameter between 30-100 µm, however, did not change. This indicates that the type of pulmonary vascular remodeling in MCT-induced pulmonary hypertension differs in different segments of the pulmonary arterial tree.

In contrast to MCT, hypoxia only slightly increased the medial area and did not alter the lumen area of pulmonary arteries with an external diameter between 30-200 µm. Several studies have shown a significant increase in media thickness of muscular pulmonary arteries in chronic hypoxic rats whereas we only showed a trend to an increase of the medial cross-sectional area (20, 38). We assume that, in those studies in which pulmonary arteries were studied without fixation under standard intravascular pressure and without maximal vasodilatation, a significant part of the media thickening can be attributed to active pulmonary vasoconstriction rather than a structural increase in media thickness. Previous studies have shown that the development of an increase in the PAP and media thickening of pulmonary arteries differs in the hypoxia and MCT model (11, 20). Hypoxia induces a significant increase in the PAP and media thickness after 3 d, whereas in MCT-treated rats significant changes in the PAP and media thickness were observed after 21 d. In these studies the level of PAP correlated with the degree of arterial wall thickness. The present study shows differences of pulmonary artery remodeling, not only between the two models but also in different segments of the pulmonary artery tree. This suggests that different mechanisms other than pressure contribute to pulmonary artery remodeling.

These structural pulmonary vascular changes, such as an increase in medial cross-sectional area and a reduction of the lumen area in MCT-treated rats, correlate well with an increased PAP and right ventricular hypertrophy. Although chronic hypoxia also increased the PAP and induced right ventricular hypertrophy, the medial area only slightly increased in absence of changes of the lumen area. As stated earlier, vasoconstriction rather than structural vascular changes of muscular pulmonary arteries may contribute to hypoxia-induced pulmonary hypertension. Structural vascular changes of arterioles rather than small-sized muscular pulmonary arteries could provide an alternative explanation for hypoxia-induced pulmonary hypertension as hypoxia increased the number of muscularized and partially muscularized arterioles. Hypoxia-induced polycythemia, however, could be a confounding factor, because an increase in blood viscosity contributes to an increase in pulmonary vascular resistance (39).

Previous studies have demonstrated that ACE inhibitors can at least partially prevent the development of MCT and hypoxia-induced pulmonary hypertension and structural pulmonary vascular changes (9, 27). In the present study the ACE inhibitor captopril did not prevent the MCT-induced increase in medial mass of muscular pulmonary arteries, had no effect on the lumen area, and did not reduce the percentage of muscularized arterioles. Moreover, captopril did not prevent MCT-induced increase in PAP and right ventricular hypertrophy. We therefore believe that angiotensin II (AII) is an unimportant modulator of structural pulmonary vascular changes in this model. This hypothesis is supported by a previous report of Cassis who showed that the specific nonpeptide AII type 1 receptor antagonist, Losartan, could not prevent MCT-induced PAP and structural pulmonary vascular changes (10).

In contrast to the lack of effect in MCT-treated rats, captopril prevented the increase in medial cross-sectional area of pulmonary arteries in chronic hypoxic rats and reduced the percentage of muscularized arterioles. This effect was, however, without a decrease in PAP or right ventricular hypertrophy. From this observation we conclude that the effect of captopril on the pulmonary vascular structure is a direct one and not due to an effect on pulmonary hemodynamics.

There may be several explanations for the inability of captopril to reduce PAP and right ventricular hypertrophy despite its potential to prevent an increase in medial cross-sectional area and reduce the percentage of muscularized arterioles. As previously stated, one possibility is that the increased blood viscosity due to polycythemia is responsible for the increased PAP and right ventricular hypertrophy, as captopril had no effect on the increased hematocrit in chronic hypoxic rats. Another possibility is that, despite a significant reduction of the percentage of muscular arterioles in captopril-treated hypoxic rats, the percentage of muscularized and partially muscularized arterioles is still significantly increased compared with control rats (Figure 2). The vascular tone of these muscularized and partially muscularized arterioles may, even under normoxic conditions, still be increased and therefore contribute to a sustained increased PAP (40).

One of the limitations of this study is that we did not analyze the lumen area of arterioles because it is technically impossible to perform on these small vessels the type of morphometrical analysis that was performed on muscular arteries. To optimize morphometrical analysis of pulmonary artery dimensions we compared pulmonary arteries with approximately the same external diameter. Within each experimental group we therefore analyzed two categories of pulmonary arteries (30- 100 and 101-200 µm), with comparable median external diameters between the groups.

In conclusion, this study demonstrates that the type of pulmonary artery remodeling in chronic hypoxic and MCT-treated rats is heterogeneous, not only between the groups but also in different segments of the pulmonary arterial tree. Moreover, these data suggest that AII plays a role in hypoxia but not in MCT-induced pulmonary artery remodeling. Finally, we showed that morphometric analysis of structural changes of muscular pulmonary arteries, in the rat hypoxia and MCT models of pulmonary hypertension, under optimal conditions only reveals a limited correlation between changes in pulmonary artery structure and changes in pulmonary artery pressure. Other factors like vasoconstriction or structural vascular changes at the level of arterioles may play a more important role in the increase in pulmonary artery pressure.