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Am. J. Respir. Crit. Care Med., Volume 158, Number 3, September 1998, 939-950

Angiotensin-converting Enzyme Inhibition Delays Pulmonary Vascular Neointimal Formation

KAZUSHIGE OKADA, MATTHEW L. BERNSTEIN, WEI ZHANG, DANIEL P. SCHUSTER, and MITCHELL D. BOTNEY

Respiratory and Critical Care Division, Washington University Medical Center, St. Louis, Missouri

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary pulmonary hypertension (PPH) is a disease characterized pathologically by pulmonary artery medial hypertrophy, adventitial thickening, and neointimal proliferation. Increasing recognition of the importance of remodeling to the pathogenesis of PPH suggests new therapeutic possibilities, but it will be necessary to (1) identify essential mediators of remodeling, and (2) demonstrate that inhibiting those mediators suppresses remodeling before new antiremodeling therapies can be considered feasible. The effect of angiotensin-converting enzyme (ACE) inhibition on pulmonary vascular remodeling was studied in a newly developed rat model in which neointimal lesions develop between 3 and 5 wk after monocrotaline injury is coupled with increased pulmonary artery blood flow after contralateral pneumonectomy. Neointimal formation was significantly suppressed at 5 wk by ACE inhibition whether it was started 10 d before or 3 wk after remodeling was initiated, although medial hypertrophy and adventitial thickening still developed. By 11 wk, the extent of neointimal formation in rats treated with ACE inhibition was similar to rats without ACE inhibition at 5 wk. Pulmonary artery pressures and right ventricular weights correlated with the extent of neointimal formation. Northern blot analysis and in situ hybridization demonstrated marked suppression of lung tropoelastin and type I procollagen gene expression in the presence of ACE inhibition. An angiotensin II type I receptor antagonist partially, but not completely, replicated the effects of ACE inhibition. These data suggest that the tissue angiotensin system may be a target for therapeutic efforts to suppress the vascular remodeling that is characteristic of primary pulmonary hypertension.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Primary pulmonary hypertension (PPH) is a disease characterized by the progressive increase in pulmonary artery impedance, which ultimately produces right ventricular failure and death. At the time most patients with PPH present to clinical attention, pathologic lesions include pulmonary artery medial hypertrophy, neointimal proliferation, and adventitial thickening (1). These pathologic lesions contribute to the high vascular impedance found in PPH. Thus, our understanding of the pathophysiology of PPH has undergone radical changes in the last three decades. Initially considered a problem of vasoconstriction (5, 6), vascular remodeling is now considered an important component in the pathophysiology of PPH (7, 8).

Although the contribution of vascular remodeling to the pathophysiology of PPH is becoming increasingly recognized, successful therapies aimed at interfering with this process have not yet emerged. An antiremodeling approach appears feasible for several reasons. First, several studies have demonstrated hypertensive pulmonary arteries from patients with PPH are actively remodeling (9). Interference with remodeling should slow, halt, or even reverse, the progression of disease. Second, factors that likely promote remodeling such as transforming growth factor-beta (TGF-beta ), angiotensin-converting enzyme (ACE), as well as possibly others, are present at sites of active remodeling (11). Third, inhibiting the synthesis and deposition of extracellular matrix proteins with antifibrotic agents such as cis-hydroxyproline or beta -aminoproprionitrile, prevented remodeling in a rat chronic hypoxia model of pulmonary hypertension (16, 17). Elevated pulmonary artery pressures, and the severity of medial hypertrophy, also could be reduced in established hypoxia-induced pulmonary vascular remodeling by inhibitors of collagen deposition (18). Unfortunately, these agents cause severe side effects that limit their clinical usefulness. Thus, identification of drugs with satisfactory safety profiles that interfere with pulmonary vascular remodeling would represent a significant advance in the therapy of PPH.

We used a recently developed animal model, in which the pattern of vascular remodeling resembles the neointimal lesions seen in PPH, to address the problem of determining whether pulmonary vascular remodeling could be halted or slowed. Neointimal lesions develop in this animal model, accompanied by severe pulmonary hypertension and right ventricular hypertrophy when monocrotaline injury is coupled with increased pulmonary artery flow or pressure (19, 20). Like remodeling pulmonary arteries in PPH, extracellular matrix genes are expressed within the developing neointima in both preacinar (19) and intra-acinar pulmonary arteries (unpublished observations).

ACE inhibition was chosen to demonstrate the feasibility of an antiremodeling therapeutic approach to PPH for several reasons. First, ACE expression is increased in both the endothelium and the neointima of hypertensive pulmonary arteries compared with normal arteries (14), and focal expression of ACE protein increases in rat intra-acinar pulmonary arteries during chronic hypoxia (21). Second, ACE inhibitors and angiotensin II receptor antagonists attenuate the development of pulmonary hypertension in animal models characterized by medial hypertrophy rather than neointimal formation (22), although whether remodeling per se was inhibited was not examined. Third, ACE inhibition suppresses carotid neointima formation after balloon endarterectomy (26). Final, ACE inhibitors have minimal side effects in patients.

The purpose of this study was to determine whether inhibiting ACE activity would suppress pulmonary vascular remodeling in an animal model with neointimal lesions that resemble the neointimal lesions of PPH.

    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Model and Study Design

Pathogen-free 12-wk-old male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 380 to 400 g were used for this experiment. Large animals were chosen to minimize the influence of remodeling associated with growth and development, and because the inflammatory response to monocrotaline diminishes with age (29, 30).

Monocrotaline (MCT) (Sigma Chemical, St. Louis, MO) was prepared as described (31, 32). Rats were injected subcutaneously in the right hindlimb with MCT (60 mg/kg) or vehicle. Left pneumonectomy was performed 1 wk later. Rats were anesthetized by a subcutaneous injection of ketamine chloride (0.1 mg) and atropine sulphate (0.1 mg), placed in the supine position, and tracheally intubated with a 14G catheter (Baxter, Deerfield, IL). Anesthesia was maintained with halothane inhalation (0.5%). Ventilation was maintained with a Harvard ventilator (tidal volume, 3.0 ml; respiratory rate, 60/min; PEEP, 1.0 cm H2O) (Harvard Apparatus Co., South Natick, MA). Animals were killed 28 d after surgery (5 wk after MCT).

Animals receiving continuous quinapril (generously provided by S. Haleen, Parke-Davis, Ann Arbor) began quinapril (30 mg/kg/d supplied daily in the drinking water) 10 d before MCT injection. Animals receiving delayed quinapril began drug 2 wk before harvest (3 wk after MCT and 2 wk after pneumonectomy). Once begun, quinapril was continued until harvest. Control animals received water alone.

Losartan (40 mg/kg/d supplied daily in the drinking water) was started 10 d before MT injection and continued until harvest.

All animals received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Science and published by the National Institute of Health (NIH Publication No. 86-23, revised 1985).

Hemodynamic Studies and Tissue Preparation

Rats were anesthetized by an intraperitoneal injection of ketamine HCl (0.1 mg) and atropine sulfate (0.1 mg) and placed in the supine position, and tracheal intubation was performed with a 14 G catheter (Angiocath). Ventilation was maintained with a Harvard ventilator (tidal volume, 3.0 ml; respiratory rate, 60/min; PEEP, 1.0 cm H2O). After a midline thoracotomy a 24G catheter was inserted into the main pulmonary artery under direct visualization and pulmonary arterial pressure (Ppa) was recorded. Systemic arterial pressure (Psa) was determined in the descending aorta. After hemodynamic measurements, the right lung was flushed with 0.9% saline under 20 cm H2O pressure and the heart-lung block was excised. The right upper lobe was fixed in neutral-buffered formalin for histologic studies, whereas the rest of the right lung was frozen immediately in liquid nitrogen. The ratio of right ventricular/left ventricular plus septal weight (RV/ LV + S) for each animal was determined.

Angiotensin-converting Enzyme Assay

ACE activity was determined in extracts from rat lungs. Briefly, lungs were homogenized with a Brinkman Polytron PT for 30 s at 4° C in 12 volumes of ACE assay buffer (50 mM HEPES HCl and 300 mM NaCl at pH 8.3). The homogenate was filtered through Whatman no. 2 paper and centrifuged at 5,000 g for 30 min at 4° C. ACE activity was determined using the method of Cushman and Cheung (33) and normalized to the total protein concentration of the supernatant, which was determined by Bradford assay (BioRad Laboratories, Richmond, CA).

Immunohistochemistry

Immunohistochemistry was performed with a polyclonal rabbit antihuman ACE antibody as previously described (14).

Northern Blot Analysis

Total lung RNA was extracted from the right lower lobe by the Chomczynski and Sacchi method (34). Total RNA was electrophoresed through a 1% agarose-formaldehyde gel followed by capillary transfer onto Hybond nylon membrane. Steady-state levels of tropoelastin and type I procollagen mRNA were determined with a rat tropoelastin cDNA probe (kindly provided by Dr. R. Pierce) and a 1.8 kB bovine alpha 1(I) procollagen cDNA (HF677; kindly provided by Dr. Jeanne Myers).

In Situ Hybridization

The right upper lobe was perfused via the airway with 10% neutral-buffered formalin at a pressure of 20 cm H2O, fixed overnight at room temperature, and subsequently dehydrated in sequential 30, 50, and 70% ethanol washes. Tissues were embedded in paraffin. In situ hybridization was performed as previously described (9, 11). Rat tropoelastin and bovine alpha 1(I) procollagen cDNAs were used to prepare [35S]- labeled antisense cRNA probes as described (9). The [35S]-labeled sense cRNA probes served as negative controls. Hybridization solution containing 2.5 × 105 cpm of [35S]-labeled cRNA probe was added to the processed sections, and slides where incubated overnight at 55° C. After hybridization, slides were washed extensively under stringent conditions. To decrease background, slides were incubated with 20 mg/ml RNase A to remove unhybridized probe. Washed slides were then processed for autoradiography and developed after 3 wk.

Image Analysis

Precapillary intra-acinar pulmonary arteries located at alveolar septal junctions were randomly selected to determine the effect of quinapril on remodeling. Pulmonary arteries were judged as either free of neointima or containing neointima within the vascular lumen. If neointima was present, vessels were subcategorized as either containing neointima within < 50% or > 50% of the vascular lumen. Results of 25 randomly selected intra-acinar pulmonary arteries on two separate tissue sections from each rat were summed, combined with totals from other rats in that treatment group, and expressed as average ± standard deviation.

Statistical Analysis

Data are presented as mean ± SD. In general, statistical significance was determined by analysis of variance (with corrections for repeated measures when appropriate). Post hoc testing (including Tukey's Studentized range test) was limited to predetermined pairs.

For the data presented in Figures 3 and 9D, the issue was whether the overall distribution of vessel pathology was different between the experimental groups. To evaluate this, we gave each normal vessel that was examined an arbitrary score of zero, each vessel that was < 50% occluded by neointima an arbitrary score of 1, and each vessel that was > 50% occluded by neointima an arbitrary score of 2. An "occlusion index" was then computed for each animal by multiplying the proportion of vessels at each grade with the score for that grade. Mean values for the "occlusion index" were computed for each group and analyzed for differences using a standard General Linear Model technique.


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  		Figure 3.   ACE inhibition suppresses pulmonary artery neointimal formation. Animals were treated with monocrotaline plus pneumonectomy (M + P), monocrotaline plus pneumonectomy plus continuous quinapril (M + P + CQ), monocrotaline plus pneumonectomy plus delayed quinapril (M + P + DQ), or were left untreated (C) as described in METHODS. Neointimal lesions were judged as absent (closed columns), filling less than 50% of the vascular lumen (shaded columns), or filling more than 50% of the vascular lumen (open columns) in 50 randomly chosen intra-acinar pulmonary arteries from each animal in each treatment group. A histogram of the distribution of lesions is shown in panel A, and the actual data are shown in panel B.


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  		Figure 9.   Type I angiotensin II receptor blockade with losartan significantly inhibits pulmonary hypertension (panel A) in animals subjected to monocrotaline plus pneumonectomy (MCT + P) but not monocrotaline alone (MCT). There was no significant difference in <OVL>Ppa</OVL> between control and monocrotaline-only animals, but <OVL>Ppa</OVL> was significantly increased in monocrotaline plus pneumonectomy compared with control animals. Significant right ventricular hypertrophy developed in animals treated with monocrotaline plus pneumonectomy but not monocrotaline alone (panel B). There was a trend toward a significant decrease in right ventricular hypertrophy with losartan. Animals were treated with losartan (40 mg/ kg/d) (dashed columns) or vehicle (dotted columns) starting 10 d before monocrotaline was given. *p < 0.05 compared with control or groups treated with monocrotaline only; +p < 0.05 compared with control, the group treated with monocrotaline only, or the group treated with monocrotaline plus pneumonectomy plus losartan.The pattern of vascular remodeling was significantly different in animals treated with losartan (panels C and D). Animals were treated with monocrotaline plus pneumonectomy (MCT + P) (n = 6), monocrotaline plus pneumonectomy plus continuous losartan (MCT + P + L) (n = 6), or were left untreated (C) (n = 4). Neointimal lesions were judged as absent (closed columns), filling less than 50% of the vascular lumen (shaded columns), or filling more than 50% of the vascular lumen (open columns) in 50 randomly chosen intra-acinar pulmonary arteries from each animal in each treatment group.

Because all vessels in the control group were normal, an occlusion index was not computed for this group per se. Rather, we evaluated whether the index for each treatment group was significantly different from 0 (the value of a normal vessel) as well as whether the mean values for each treatment group were significantly different from each other.

We accepted p < 0.05 as indicating statistical significance. The Statistical Analysis System (SAS) was used for all data manipulations and statistical calculations.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Preliminary experiments were performed to determine whether pulmonary ACE activity could be inhibited and whether neointimal lesions contain cells immunoreactive for ACE. To determine whether pulmonary ACE activity could be inhibited, rats were administered quinapril (30 mg/kg/d, supplied fresh daily in the drinking water) for 10 d. Pulmonary ACE activity in two animals receiving quinapril was decreased by more than 95% compared with that in two control animals (Figure 1). Maximal inhibition of pulmonary ACE activity was demonstrated by the absence of any further decrease in ACE activity when additional quinapril was added in the in vitro ACE assay. Immunohistochemistry, with a rabbit antihuman ACE antibody, demonstrated cells within pulmonary artery neointimal lesions that were immunoreactive for ACE (Figure 2). Immunoreactivity was greatest in medial and adventitial cells compared with neointimal cells. Control studies with normal rabbit serum were negative.


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  		Figure 1.   Lung ACE activity is completely inhibited by 10 d of orally administered quinapril (stipled columns) (30 mg/kg/d) compared with control lung ACE activity. Maximal inhibition is demonstrated by the absence of further inhibition when additional quinapril was added to the ACE activity assay in vitro (dashed columns).


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  		Figure 2.   Immunohistochemistry, using a rabbit antihuman ACE polyclonal antibody, demonstrates ACE is present in cells located within the pulmonary artery neointimal lesions (a). Immunoreactivity is also present in non-neointimal cells. Control studies with normal rabbit serum were negative (b). Original magnification: ×1,000.

Quinapril was administered to animals receiving monocrotaline plus pneumonectomy to determine whether ACE inhibition suppressed pulmonary vascular neointimal formation. The extent of neointimal development was determined in 50 randomly chosen precapillary pulmonary arteries, located at alveolar septal junctions, from each rat in each group. Neointima developed in 98 ± 1% of acinar arteries from rats receiving monocrotaline plus pneumonectomy alone (mean occlusion index, 1.67 ± 0.08) (Figures 3 and 4C). In contrast, only 24 ± 4% acinar vessels developed neointimal lesions in animals treated continuously with quinapril in addition to monocrotaline plus pneumonectomy (M + P + CQ) (mean occlusion index, 0.27 ± 0.09) (Figure 3). In those vessels that did form neointimal lesions, the extent of vascular occlusion was decreased. Medial hypertrophy was the typical pattern of remodeling in the remaining vessels not developing neointima (Figure 4D), similar to the medial hypertrophy seen with monocrotaline injury alone (Figure 4B). A typical pattern of remodeling was not seen in animals beginning treatment with quinapril 3 wk after remodeling began (M + P + DQ); 76 ± 4% of acinar arteries developed neointimal lesions, leaving 24 ± 11% of arteries without neointima (mean occlusion index, 1.20 ± 0.26) (Figure 3). The mean occlusion index of each treatment group was significantly different from 0, and the mean values for each treatment group were significantly different from each other. Adventitial thickening was seen in all remodeling vessels and appeared similar regardless of treatment. No structural abnormalities were seen in the pulmonary arteries from control animals (Figure 3).


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  		Figure 4.   ACE inhibition suppresses development of pulmonary neointimal formation after monocrotaline injury and left pneumonectomy. Animals were treated with monocrotaline plus pneumonectomy (M + P), monocrotaline plus pneumonectomy plus continuous quinapril (M + P + CQ), monocrotaline plus pneumonectomy plus delayed quinapril (M + P + DQ), or were left untreated (C) as described in METHODS. Tissues were then stained with a Virhoff-van Gieson elastin stain. Neointimal lesions occluding more than 50% of the vascular lumen were the predominant abnormality in the intra-acinar arteries of animals treated with monocrotaline plus pneumonectomy (panel C ). In contrast, medial hypertrophy was the typical pattern of remodeling in animals treated with monocrotaline plus pneumonectomy plus continuous ACE inhibition (panel D). These structural changes resemble the medial hypertrophy that develops with monocrotaline only (panel B). Adventitial thickening was seen in all remodeling vessels. No structural abnormalities were seen in intra-acinar pulmonary arteries from control animals (panel A).

Mean pulmonary artery pressures (Ppa) and the ratio of right ventricular and left ventricular plus septal weights (RV/LV + S) increased significantly in animals with a neointimal pattern of remodeling when compared with control animals (Figure 5). In contrast, <OVL>Ppa</OVL> and RV/LV + S were significantly lower in animals in which ACE activity was inhibited beginning 10 d before remodeling was initiated (M + P + CQ). Likewise, <OVL>Ppa</OVL> and RV/ LV + S were significantly lower in animals which ACE activity was inhibited beginning 3 wk after remodeling was initiated, although the effect was less dramatic (M + P + DQ).


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  		Figure 5.   ACE inhibition prevents development of pulmonary hypertension and right ventricular hypertrophy. Mean pulmonary artery pressure (<OVL>Ppa</OVL>) and the ratio of right ventricular weight to left ventricular plus septal weight (RV/LV + S), indirect indices of pulmonary vascular remodeling, were measured in the animals from each treatment group described in Figures 2 and 3. <OVL>Ppa</OVL> and RV/LV + S were significantly decreased in animals continuously treated with quinapril (MP + CQ) compared with animals treated with monocrotaline plus pneumonectomy only (MP). <OVL>Ppa</OVL> and RV/LV + S were also decreased in animals receiving delayed quinapril treatment (MP + DQ), suggesting that ACE inhibition suppresses preexisting, active remodeling. *p < 0.05 compared with control values; Dagger p < 0.05 compared with monocrotaline plus pneumonectomy.

To determine whether delayed ACE inhibition slowed the progression of remodeling, halted, or even reversed the development of remodeling, a longer time-course study with quinapril was performed (Figure 6). Quinapril was started 3 wk after neointimal remodeling was initiated, and pulmonary artery pressures and right ventricular hypertrophy were determined at 5, 7, 9, and 11 wk. Increases in pulmonary artery pressure and right ventricular weight were delayed by ACE inhibition but eventually reached levels comparable to those in animals treated with monocrotaline plus pneumonectomy but no quinapril. Histologic examination showed progressively more extensive and severe neointimal lesion formation with time, correlating with the increases in pulmonary hypertension and right ventricular hypertrophy (data not shown).


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  		Figure 6.   Delayed ACE inhibition delays development of pulmonary hypertension and right ventricular hypertrophy 3 wk after remodeling is initiated. Quinapril (30 mg/kg/d) was started 3 wk after monocrotaline injection (2 wk after pneumonectomy). <OVL>Ppa</OVL> and RV/LV + S were measured in animals from each treatment group at weeks 0, 3, 5, 7, 9, and 11 (n = 6 for each group at each time point). <OVL>Ppa</OVL> and RV/LV + S are significantly lower in animals receiving delayed quinapril (open circles) at 7 wk compared with animals receiving vehicle only (closed circles). No significant difference is seen between animals receiving delayed quinapril at 11 wk and those receiving vehicle only at 7 wk. *p < 0.05 compared with control values. +p < 0.05 compared with the 7-wk data in the group treated with quinapril.

Monocrotaline alone initiates a non-neointimal pattern of vascular remodeling characterized by moderate pulmonary hypertension and right ventricular hypertrophy (31, 32). That intra-acinar pulmonary arteries in quinapril-treated monocrotaline plus pneumonectomy animals develop medial hypertrophy, rather than neointima, prompted us to reexamine whether the development of medial hypertrophy after monocrotaline injury would be suppressed by ACE inhibition. RV/LV + S and <OVL>Ppa</OVL> increased moderately in animals receiving monocrotaline when compared with control animals, but they remained at control levels in animals receiving both monocrotaline and continuous quinapril (data not shown). Right ventricular hypertrophy and pulmonary artery hypertension were also significantly suppressed when quinapril was administered on a delayed-dose schedule.

The effects of ACE inhibition on matrix gene expression are poorly understood. Because pulmonary vascular remodeling is characterized in part by active matrix gene expression (9, 10, 32), we used Northern blot analysis and in situ hybridization to determine whether pulmonary vascular matrix gene expression was decreased by ACE inhibition. However, quinapril inhibits the formation of neointimal lesions in the monocrotaline plus pneumonectomy model that are the predominant site of matrix gene expression. We thought it would be difficult to distinguish in the monocrotaline plus pneumonectomy model between a "direct" effect of ACE on matrix gene expression versus an "indirect" effect of ACE on matrix gene expression because neointimal lesions did not develop. Therefore, we chose to study the effects of ACE inhibition on matrix expression in the monocrotaline-only model. Northern blot analysis demonstrated increased lung tropoelastin and type I procollagen gene expression after monocrotaline-injury when compared with control tissues (Figure 7). Tropoelastin and type I procollagen gene expression, both basal and monocrotaline-induced, were decreased by quinapril. Thus, changes in steady-state levels of tropoelastin and type I procollagen mRNA paralleled changes in <OVL>Ppa</OVL> and RV/LV + S and corroborate the use of tropoelastin and type I procollagen gene expression as markers of vascular injury in this model.


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  		Figure 7.   Monocrotaline-induced tropoelastin and type I procollagen gene expression is suppressed by quinapril. Quinapril (30 mg/kg/d) was orally administered for 10 d prior to subcutaneous monocrotaline injection (60 mg/kg) and continued until lungs were harvested 26 d later. Northern blot analysis using total lung RNA (10 µg/lane) demonstrates that inhibiting ACE activity suppresses monocrotaline-induced type I collagen (top) and tropoelastin (middle) gene expression (panel A). Control (lanes 1 to 4), quinapril only (lanes 5 to 8), monocrotaline (lanes 9 to 11) and monocrotaline plus quinapril (lanes 12 to 15). One animal receiving monocrotaline only died before harvest. Equivalent loading is demonstrated by ethidium bromide staining (bottom). The histogram (panel B) demonstrates relative densitometries, with control = 100. Tropoelastin (dotted columns), type I procollagen (dashed column). *p < 0.05 compared with control values. +p < 0.05 compared with monocrotaline alone.

In situ hybridization was used to determine the location of tropoelastin and type I procollagen expressing cells after MCT injury in the absence or presence of ACE inhibition. Minimal tropoelastin gene expression was seen in the pulmonary arteries and capillary bed from control lungs (Figures 8A and 8B), consistent with the normal low level of expression of these genes in healthy adult lung. There was a remarkable increase in tropoelastin gene expression after MCT injury in pulmonary arteries, capillaries, and bronchial adventitia (Figures 8C and 8D) when compared with control tissues. This increase was nearly totally ablated by quinapril, with minimal tropoelastin gene expression remaining in large elastic pulmonary arteries (Figures 8E and 8F). Minimal procollagen gene expression was seen in the pulmonary arteries and capillary bed from control lungs and in lungs from animals treated with quinapril, although procollagen gene expression was significantly elevated in remodeling pulmonary arteries (data not shown). Studies performed with sense probes were negative.


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  		Figure 8.   Monocrotaline-induced tropoelastin gene expression is suppressed by quinapril. In situ hybridization with [35S]-labeled antisense tropoelastin cRNA probes was performed on lung tissue harvested from control rats, rats treated with monocrotaline, or rats treated with monocrotaline plus quinapril (the same rat lungs used for Northern blot analysis). Representative figures demonstrate minimal signal in control lungs (panels A and B) but significant tropoelastin gene expression in the pulmonary arteries, capillaries, and bronchial adventitia of animals receiving monocrotaline (panels C and D). In contrast, induction of lung tropoelastin gene expression by monocrotaline is remarkably suppressed by ACE inhibition (panels E and F  ). In situ hybridization with [35S]-labeled sense tropoelastin cRNA probes was negative. Original magnification: ×100.

The above studies strongly support a role for ACE in the pathogenesis of pulmonary vascular remodeling in this model. However, because ACE converts angiotensin I to the potent vasoconstrictor angiotensin II and inactivates the vasodilator bradykinin, it was unclear whether the neointimal formation was mediated by increased angiotensin II, decreased bradykinin, or some other mechanism. Therefore, losartan, a type I angiotensin II receptor antagonist (generously donated by Merck), was studied in both the monocrotaline plus pneumonectomy and monocrotaline models. Losartan had no effect in the monocrotaline model, similar to the results of Cassis and coworkers (35), but it significantly inhibited pulmonary artery pressures in the monocrotaline plus pneumonectomy model (Figure 9). There was a trend toward a significant decrease in right ventricular hypertrophy with losartan, probably not reaching significance because of the few number of animals in this study. Losartan also significantly affected the development of neointima in vessels affected by monocrotaline plus pneumonectomy, similar to that seen after delayed (not continuous) quinapril (vessel occlusion index of 1.57 ± 0.30 after monocrotaline plus pneumonectomy alone versus 1.18 ± 0.39 after monocrotaline plus pneumonectomy plus losartan, p < 0.05). The relatively weaker effect of losartan compared with that of quinapril suggests the angiotensin II type 1 receptor has a role, albeit minor, in neointimal formation in this model.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

As vascular remodeling becomes increasingly recognized as an important component in the pathogenesis of PPH it will be necessary to (1) identify essential mediators of remodeling, and (2) develop therapeutic strategies to interrupt that process. Recent development of an animal model of pulmonary vascular remodeling with neointimal lesions that resemble the neointimal lesions seen with PPH (20) should facilitate these goals. An essential role for ACE in the pathogenesis of PPH is strongly suggested by the presence of increased ACE immunoreactivity at sites of increased matrix gene expression in hypertensive human pulmonary arteries (14) and by this study, in which an ACE inhibitor suppressed matrix gene expression and delayed development of pulmonary vascular lesions. Further supporting a role for ACE in pulmonary vascular remodeling are the observations that ACE protein and mRNA expression are focally increased in intra-acinar rat pulmonary arteries developing medial hypertrophy during chronic hypoxia (21), whereas ACE inhibition is associated with reduced numbers of 5'-bromo-deoxyuridine-labeled SMCs (25) and decreased medial hypertrophy (24) in hypoxic rats when compared with normoxic control rats. A role for ACE in pulmonary vascular remodeling is consistent with studies demonstrating a role for ACE in systemic vascular remodeling, suggesting similarities between the two vascular beds in their response to injury. Furthermore, the early development of medial hypertrophy followed eventually by neointima in quinapril-treated monocrotaline plus pneumonectomy animals is consistent with our hypothesis that medial hypertrophy and neointimal formation are unique patterns of remodeling that proceed sequentially (19, 20).

Although these studies strongly support a role for ACE in the pathogenesis of PPH, it is less clear what the role of angiotensin II per se is in pulmonary vascular remodeling. Angiotensin II stimulates cell proliferation, extracellular matrix protein synthesis, and SMC migration (36, 37). Angiotensin II also induces TGF-beta gene expression and protein synthesis in vitro (38) and in vivo (39), whereas antibodies against TGF-beta ablate angiotensin-induced matrix gene expression (40). Losartan, a type I angiotensin II receptor antagonist, inhibited the development of medial hypertrophy during chronic hypoxia (24) but failed to suppress MCT-induced medial hypertrophy or pulmonary hypertension (35). The discrepancies in the effect of losartan between the hypoxic and monocrotaline models of pulmonary vascular remodeling may simply reflect idiosyncracies of the models since inflammation is a feature of monocrotaline injury, whereas hypoxia induces expression of a unique group of genes. The extent of inhibition with losartan was not equivalent to continuous quinapril in this study (compare Figures 3 and 4 with Figure 9), implying angiotensin II may not be the most important mediator of remodeling. ACE inhibitors not only inhibit the formation of angiotensin II but allow kinins (such as bradykinin) to accumulate in tissues by inhibiting their degradation (41). Bradykinin, an oligopeptide with potent biologic activities that can be locally synthesized in vascular tissue (42), is a potential candidate for this additional ACE-dependent factor since bradykinin levels increase during ACE inhibition (41). Whether tissue kinin levels change in remodeling human pulmonary arteries or whether changes in tissue kinin levels would modulate either smooth muscle proliferation and migration, or extracellular matrix production is unknown. Rat lung kininogen mRNA and protein levels progressively rise after monocrotaline injection, and immunohistochemistry shows increased kininogen immunoreactivity in endothelium of remodeling pulmonary arteries (43). A possible role for kinins in systemic vascular remodeling is indirectly suggested by the observation that, although ACE inhibition markedly reduces carotid neointimal formation after balloon endarterectomy (26), the effect of ACE inhibition is significantly blunted when given together with a bradykinin receptor inhibitor (27). On the other hand, bradykinin has been reported to stimulate cultured lung fibroblast collagen protein synthesis (44, 45). Gene expression and other studies to determine the mechanism by which bradykinin induces matrix (other than prostaglandin inhibition) have not been reported. Thus, whether bradykinin mediates the effect of ACE inhibition on pulmonary vascular remodeling is unclear.

Another interpretation of our data (but one that does not exclude a role for bradykinin) is that the proremodeling effect of angiotensin II acting at the type 1 receptor is somewhat balanced by angiotensin II acting at the type 2 receptor. Recent studies suggest the type 2 receptor antagonizes the growth effects of the type 1 receptor in vascular SMC (46) and endothelial cells (47). Overexpressing the type 2 receptor in injured rat carotid arteries attenuated neointimal formation after balloon endarterectomy (46). The type 2 receptor may mediate this antigrowth effect by inducing apoptosis (48). Losartan interferes with remodeling by allowing unimpeded type 2 receptor activity, which subsequently interferes with neointimal formation. The availability of selective angiotensin type 2 and bradykinin receptor antagonists will allow us in future studies to directly determine the relative roles of the type 2 receptor and bradykinin in pulmonary vascular neointimal formation in vivo.

That intra-acinar pulmonary arteries in quinapril-treated monocrotaline plus pneumonectomy animals develop medial hypertrophy, rather than neointima, prompted us to reexamine whether the development of medial hypertrophy after monocrotaline injury would be suppressed by ACE inhibition. This study has confirmed previous studies showing that ACE inhibition significantly suppresses medial hypertrophy after monocrotaline alone (22, 23). In addition, this study extends those earlier observations by showing that reinitiation of pulmonary vascular matrix gene expression after injury is inhibited by ACE. Northern blot analysis demonstrated decreases in both baseline and injury-induced tropoelastin and type I procollagen gene expression, whereas in situ hybridization confirmed that the differences in matrix gene expression were mostly localized to vascular structures. In contrast, however, a neointimal pattern of remodeling was not induced after infusion of angiotensin II in chronically hypoxic animals (49). Moreover, it appeared as if angiotensin II attenuated the extent of hypoxia-induced remodeling, presumably via an indomethacin-dependent pathway. The reasons for this difference are unclear, but two possibilities are (1) chronic hypoxia and monocrotaline are different models with distinct types of injury and patterns of gene expression, and (2) pharmacologic doses of exogenous angiotensin II were infused rather than blocking production of endogenous angiotensin II as in this study. Our results, however, are consistent with several studies in the systemic literature showing ACE inhibitors block neointimal formation in systemic arteries (26, 27), presumably by inhibiting cell proliferation. Whether ACE inhibitors suppress neointimal formation by inhibiting matrix production is largely unexplored but a possible additional mechanism by which ACE inhibitors act to delay neointimal formation.

Right ventricular hypertrophy and hypertrophy of the arterial medial layer are commonly used indices of pulmonary vascular remodeling. Both end points are useful since they reflect a physiologically relevant response to pulmonary vascular injury. Nevertheless, these gross morphologic changes are not primary biologic responses but cumulative and indirect reflections of more basic responses to injury such as matrix synthesis and degradation, smooth muscle replication, and migration. Defining these primary responses to vascular injury will be necessary to understand vascular remodeling. Several observations support the use of matrix gene expression as a primary response to injury. First, matrix gene expression is a pathologically relevant marker of remodeling since it occurs in remodeling human pulmonary arteries (9, 10). Tropoelastin and type I procollagen gene expression is not observed in noninjured pulmonary arteries once expression associated with growth and development ceases. Second, reinduction of matrix gene expression occurs quickly after injury, within 7 d after monocrotaline injury, and prior to the development of neointimal formation, medial hypertrophy, or right ventricular hypertrophy (32). Third, right ventricular hypertrophy progressively increases as matrix gene expression increases (32). However, extracellular matrix gene expression is only one of several primary responses to vascular injury and may be regulated independently of other responses such as smooth muscle cell replication. That quinapril inhibits both SMC replication (25) and matrix gene expression suggests a central role for ACE in pulmonary vascular remodeling.

The participation of ACE in pulmonary vascular remodeling suggests the possible participation of several other mediators of vascular remodeling since basic fibroblast growth factor (50) and endothelin (51) can induce ACE expression, whereas angiotensin II induces PDGF (52, 53), plasminogen activator inhibitor-1 (54), and IGF-1 (55) gene expression. Angiotensin II also induces TGF-beta gene expression and protein synthesis in vitro (38, 53) and in vivo (39), whereas antibodies against TGF-beta ablate angiotensin-induced matrix gene expression in vitro (40). Previous studies have shown that TGF-beta is closely associated with matrix gene expression in hypertensive pulmonary arteries from patients with PPH (12, 13) and increases after monocrotaline injury (32). On the other hand, bradykinin increases both nitric oxide and prostacyclin, which may inhibit cell proliferation and matrix production. As clinically useful inhibitors of these putative mediators of pulmonary vascular remodeling are developed, future experiments will be required to determine whether these inhibitors, alone or in combination, suppress pulmonary vascular remodeling.

Given the inhibitory effects of continuous ACE inhibition on rat pulmonary artery neointimal formation, as well as the dramatic effects of ACE inhibition on rat systemic artery neointimal hyperplasia, the lack of a therapeutic benefit in patients with neointimal lesions of either the systemic or the pulmonary artery has been disappointing. Explanations for the discrepancy between results in experimental animals and patients have been ascribed to inadequate dosing, species differences, or differences between inhibiting established complex lesions in patients versus newly developing lesions in experimental animals. Our results demonstrate that pulmonary artery remodeling proceeds at a slower pace in the presence of ACE inhibition but that eventually neointimal lesions develop that are comparable to those seen in animals that did not receive ACE inhibitor. This suggests that species differences or lesion complexity are not responsible for therapeutic failure, but other possible explanations remain. First, pulmonary vascular ACE activity may be insufficiently inhibited. Focal ACE activity could persist at sites of remodeling despite the results of a total-lung ACE assay indicating maximal ACE inhibition. Unfortunately, no assay is available to determine whether focal ACE activity is completely inhibited. Second, resistance to ACE inhibitors could develop as neointimal SMCs proliferate. SMC diversity is being increasingly recognized in systemic (56) and pulmonary arteries (57). For example, heparin inhibits SMC growth and migration in vivo (58) and decreases the elastin and collagen content of neointima (61). The putative endogenous equivalents of heparin are heparan sulfate proteoglycans in the basement membrane of blood vessels (62) since they also inhibit SMC growth and migration (63- 66). Recent studies have demonstrated both heparin-sensitive and heparin-insensitive subpopulations of SMC within arteries (67), and selective growth of heparin-insensitive SMC clones may explain eventual neointimal formation after endarterectomy (71). Whether similar phenotypic diversity exists for ACE inhibition is unknown, but it could explain eventual development of neointima in our model. Third, there may be redundancy in mediators of remodeling. Although our study suggests ACE-dependent mediators participate in pulmonary vascular neointimal formation, non-ACE-dependent mediators might also participate. These would not be inhibited by quinapril and with time neointimal lesions would develop. Incomplete suppression of matrix gene expression by quinapril after monocrotaline injury alone is consistent with eventual neointimal formation and compatible with any of the reasons listed above.

The utility of this new animal model is that the pattern of pulmonary vascular remodeling more closely resembles the pathology of PPH than do other animal models. That quinapril delays the pace of remodeling suggests that the therapeutic role of ACE inhibitors in PPH could be readdressed. Chronic ACE inhibition in a few small, nonrandomized, uncontrolled studies significantly reduced elevated pulmonary artery pressures in patients with pulmonary hypertension secondary to collagen vascular disease (72) or congenital heart disease (73). Chronic ACE inhibition also either reduced pulmonary artery pressures in a heterogeneous group of patients with both primary and secondary pulmonary hypertension (74) or was associated with a lack of hemodynamic deterioration in patients with primary pulmonary hypertension (75). However, ACE inhibitors have not been considered beneficial. Perhaps by understanding how ACE inhibition delays but does not completely suppress remodeling, pharmacologic manipulations could be achieved to develop a more useful therapeutic agent. For example, if focal pulmonary vascular ACE activity is insufficiently inhibited, inhibitors with higher tissue affinities might be the answer. If ACE inhibition acts as a selective pressure against ACE-sensitive SMC, but ACE-resistant SMC can still proliferate, understanding mechanisms of resistance might provide new insights into the biology of SMC and neointimal hyperplasia as well as suggest possible new therapeutic avenues. Finally, if neointimal lesions develop because of redundant mediators, a "cocktail" of antiremodeling agents, including new endothelin inhibitors, calcium-channel blockers (76, 77), and prostacyclin (78) may prove more beneficial to patients with PPH than any single agent.

    Footnotes

Correspondence and requests for reprints should be addressed to Mitchell D. Botney, M.D., Respiratory and Critical Care Division, Jewish Hospital of St. Louis, 216 S. Kingshighway Blvd., St. Louis, MO 63110. E-mail: mbotney{at}imgate.wustl.edu

(Received in original form October 1, 1997 and in revised form April 10, 1998).

Dr. Botney is the recipient of a Career Investigator Award from the American Lung Association.

Acknowledgments: The writers gratefully acknowledge T. Toley, J. Roby, and N. Persons for their excellent technical assistance.

Supported by grants HL-02425 and HL-29594 from the National Institutes of Health and by the Alan A. and Edith L. Wolff Charitable Trust.

    References
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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