INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Primary pulmonary hypertension (PPH) is a devastating and incurable disease that leads to relentless elevations in pulmonary arterial pressures and death due to circulatory failure (1, 2). The pathology of PPH is characterized by abnormal expansions of endothelial cells, medial hypertrophy, and adventitial thickening of pulmonary arteries (3). Each appears to play a role in the development and progression of pulmonary hypertension (PH). (3). In spite of research that suggests an important role for endothelial proliferation and neointimal formation in the development and progression of PH, there are no therapeutic strategies designed to suppress neointimal formation being used to treat pulmonary hypertension (4). The current medical management of PPH is directed at anticoagulation and vasodilatation rather than the prevention of endothelial proliferation and neointimal formation (2). Prostacyclin may have beneficial effects on vascular remodeling, because some patients who do not demonstrate a vasodilator response to prostacyclin appear to benefit from its use (2, 5, 7).

Monocrotaline (MCT) is a toxin derived from plants of the Crotalaria species (8). When MCT is injected into rats it causes pulmonary arterial endothelial cell injury, pulmonary artery medial hypertrophy, and PH (8, 9). The average pulmonary arterial pressure () rises to approximately 32 mm Hg, 4 wk after monocrotaline administration (10, 11). The combination of monocrotaline administration with pneumonectomy results in higher (mean , 45 mm Hg) in addition to pulmonary arterial neointimal formation (12). The development of pulmonary vascular neointimal formation in this rat model of pulmonary hypertension is thought to be due to increases in shear stresses (the entire cardiac output flows to the right lung) because neointimal formation does not occur after monocrotaline toxin alone (12). Disturbed laminar shear stresses on cultured endothelial cells have been shown to activate transcription factors associated with inflammation and cell proliferation, including nuclear factor B (NF-B), early growth response protein 1 (Egr-1), and activator protein 1 (AP-1, composed of Fos and Jun proteins) (13).

Rapamycin is a macrolide immunosuppressant that is currently being used as a novel therapy for chronic allograft rejection (14). Rapamycin exerts antiproliferative effects on lymphoid and nonlymphoid cells by inhibiting growth factor receptor-mediated signaling at the level of protein translation. The target of rapamycin (TOR) is large protein kinase of the phosphoinositide 3-kinase family (15). Rapamycin binding to TOR interferes with downstream activation of ribosomal p70 S6 kinase, and with phosphorylation and activation of translation initiation factor 4E-binding protein 1 (4E-BP1) (15). Rapamycin has been shown to prevent vascular endothelial growth factor (VEGF)-mediated and serum-stimulated proliferation of endothelial cells in vitro (18, 19). In addition, rapamycin reduced the arterial proliferative response, intimal thickening, and vascular smooth muscle proliferation after angioplasty in pigs (20, 21). We hypothesized that rapamycin might prevent pulmonary vascular neointimal formation associated with shear stress and thereby attenuate the development of pulmonary hypertension. This study investigates the effects of 40-O-(2-hydroxyethyl)-rapamycin (RAD), an orally active derivative of rapamycin (22), on monocrotaline-induced PH in the pneumonectomized rat.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Study Sample

Twenty pathogen-free, 13-wk-old, male Sprague-Dawley rats (body weight, 350-400 g) were studied.

Left Pneumonectomy

On Day 0, rats were anesthesized with atropine sulfate (50 µg, intramuscular), ketamine hydrochloride (10 mg, intramuscular), and xylazine (3 mg, subcutaneous). After oral endotracheal intubation (with a 14-gauge catheter; Baxter, Deerfield, IL), anesthesia was maintained with halothane inhalation (0.5%) and rats were ventilated with a Harvard rodent ventilator (tidal volume, 3.0 ml; respiratory rate, 60/min; positive end-expiratory pressure [PEEP], 1 cm H₂O) (Type 683; Harvard Apparatus, South Natick, MA). Left pneumonectomy was performed via left thoracotomy, using aseptic technique.

Monocrotaline Administration

On Day 7, rats were injected subcutaneously in the right hindlimb with monocrotaline (MCT, 60 mg/kg; Sigma, St. Louis, MO) (dissolved in distilled water, adjusted to pH 7.40 with 0.5 N HCl).

Treatment Groups

Rats were randomized to receive 40-O-(2-hydroxyethyl)-rapamycin (RAD; gift of Novartis Pharma, Basel, Switzerland), or vehicle by daily gavage. Five groups were studied: Group PMR_5-35 received RAD (2% solution, 2.5 mg/kg/d) from Day 5 to Day 35 (n = 5), Group PMR_5-14 (early treatment group) received RAD (2.5 mg/kg/d) from Day 5 to Day 14 (n = 5), and Group PMR_15-35 (late treatment group) received RAD (2.5 mg/kg/d) from Day 15 to Day 35 (n = 5). Group PMV (vehicle group) received a vehicle (placebo) solution from Day 5 to Day 35 (n = 5). An additional five rats were studied as a control (N) group. These rats did not undergo pneumonectomy, nor did they receive monocrotaline or RAD.

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 Institutes of Health (NIH Publication No. 86-23, revised 1985). The study was approved by the Stanford Panel on Laboratory Animal Care and was conducted in compliance with Stanford University regulations.

Hemodynamic Studies and Tissue Preparation

On Day 35, rats were anesthesized by intramuscular injections of atropine sulfate (50 µg, intramuscular) and ketamine hydrochloride (10 mg, intramuscular) and placed in the supine position. Anesthesia was maintained with inhalation halothane (0.5%, by hood). A carotid arterial catheter (PE-50, 0.58-mm i.d.) was placed after cutdown. A pulmonary artery catheter (PV 1, 0.28-mm i.d.) was inserted into the right internal jugular vein through an introducer. The pulmonary artery catheter was passed (under pressure wave guidance) through the right ventricle into the pulmonary artery. Right ventricular systolic pressure measurements were also obtained by percutaneous needle (27 gauge) puncture of the right ventricle. Mean arterial blood pressure, pulmonary artery blood pressure, and right ventricular systolic blood pressure were recorded. After exsanguination, the right lung, right ventricle, left ventricle plus septum, liver, spleen, kidney, testis, and thymus were collected for histology. Tissues were fixed in 10% neutral buffered formalin. The lungs were axially sectioned, processed, and embedded in paraffin wax. Five-micron sections were prepared and stained with elastin-van Gieson (EVG). The severity of neointimal formation was scored according to the criteria of Okada and coworkers (12). Briefly, the absence of neointimal formation equals 0; the presence of neointimal proliferation causing less than 50% lumenal narrowing equals 1; lumenal narrowing greater than 50% equals 2. To facilitate comparisons across groups of rats, organ weights are presented per kilogram of body weight.

Statistical Analysis

Data are presented as means ± standard deviation. First, the data from normal rats were compared with data for Group PMV (the disease model), using the Student t test (statistical significance was indicated by p < 0.05). Next, Groups PMV, PMR_5-35, PMR_5-14, and PMR_15-35 were analyzed by two-way analysis of variance (ANOVA) and multiple comparisons in order to determine the effects of early and late therapy. A value of p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Twenty-five rats were studied. Five rats were used as a control group. Twenty rats received monocrotaline (60 mg/kg, subcutaneous) 7 d after left pneumonectomy. One rat in Group PMV and one rat in Group PMR_15-35 died from mediastinitis (secondary to gavage tube trauma) on postoperative Days 14 and 21, respectively, and were excluded from the analysis. The remaining 18 rats underwent hemodynamic measurements and were killed on postoperative Day 35. The four treatment groups did not differ in terms of body mass.

Hemodynamic Parameters (Day 35)

Pulmonary arterial pressure. Rats that received RAD had lower mean than rats that received vehicle (Figure 1A). Rats that received RAD from Day 5 to Day 35 (n = 5) had the lowest (25 ± 3 mm Hg). Rats that received RAD from Day 5 to Day 14 (n = 5) had lower (29 ± 4 mm Hg) than those treated from Day 15 to Day 35 (n = 4) (38 ± 5 mm Hg). Of the four treatment groups, vehicle-treated rats (n = 4) had the highest (41 ± 3 mm Hg). Two-way ANOVA and multiple comparison revealed that the main effect of therapy occurred between Days 5 and 14 (Group PMR_5-14) (p < 0.001).

View larger version (21K): [in this window] [in a new window]   Figure 1.   RAD prevents the development of pulmonary arterial hypertension. Measurements of (A) mean pulmonary artery pressures (mPAP, Ppa) and (B) right ventricular systolic pressures (RVSP, Prv,s); in normal rats (n = 5), Group PMV (pneumonectomized rats that received monocrotaline [60 mg/kg] and vehicle treatment) (n = 4), Group PMR5-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 5 to Day 35) (n = 5), Group PMR5-14 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 5 to Day 14) (n = 5), and Group PMR15-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 15 to Day 35) (n = 4). ## p < 0.01 by Student t test; **p < 0.01 by ANOVA.

Right ventricular systolic pressure. Rats that received RAD had lower right ventricular systolic pressure (Prv,s) than rats that received vehicle (Figure 1B). Rats that received RAD from Day 5 to Day 35 had a Prv,s (32 ± 7 mm Hg). Rats that received RAD from Day 5 to Day 14 had lower Prv,s (30 ± 5 mm Hg) than those treated from Day 15 to Day 35 (42 ± 7 mm Hg). Of the four treatment groups, vehicle-treated rats had the highest Prv,s (45 ± 2 mm Hg). Two-way ANOVA and multiple comparison revealed that the main effect of therapy occurred between Days 5 and 14 (Group PMR_5-14) (p < 0.01).

Mean arterial blood pressure measurements were not significantly different between the four groups. Note that values for Group N (a group of five normal, healthy rats) were included as a reference control. Group N demonstrated systemic blood pressures (126 ± 9 mm Hg), pulmonary arterial blood pressures (17 ± 1 mm Hg), and right ventricular systolic blood pressures (25 ± 2 mm Hg) that were typical of healthy adult rats (23, 24) (Figure 1). Measurements of blood hematocrit were not significantly different among the four groups.

Organ Weights

Right ventricular hypertrophy. The development of chronic pulmonary arterial hypertension results in a compensatory hypertrophy of the right ventricle (increased ratio of [right ventricle]/[left ventricle and septum] [RV/LV&S]). Rats that received RAD had lower (RV/LV&S) than did rats that received vehicle (Group PMV = 0.55 ± 0.05) (Figure 2). The mean ratios for the RAD-treated animals were as follows: Group PMR_5-35, 0.42 ± 0.06; Group PMR_5-14, 0.43 ± 0.07; and Group PMR_15-35, 0.45 ± 0.08. Two-way ANOVA and multiple comparison revealed that significant effects of therapy occurred between Days 5 and 14 (Group PMR_5-14) (p < 0.01) and between Days 15 and 35 (Group PMR_15-35) (p = 0.038).

View larger version (22K): [in this window] [in a new window]   Figure 2.   RAD prevents the development of right ventricular hypertrophy in pneumonectomized, monocrotaline-treated rats. Measures of (right ventricular mass)/(left ventricle and septal mass) (RV/LV&S) in normal rats (n = 5), Group PMV (pneumonectomized rats that received monocrotaline [60 mg/kg] and vehicle treatment) (n = 4), Group PMR5-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 5 to Day 35) (n = 5), Group PMR5-14 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 5 to Day 14) (n = 5), and Group PMR15-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 15 to Day 35) (n = 4). ## p < 0.01 by Student t test; *p < 0.05 by ANOVA.

Other organs. Rats that received RAD had lower thymus weights than rats that received vehicle (Figure 3A). Two-way ANOVA and multiple comparison revealed that the main effect of therapy occurred between Days 15 and 35 (p = 0.012). Rats in Group PMR_5-35 had lower spleen weights than rats in Group PMV (Figure 3B). Two-way ANOVA and multiple comparison revealed there was a significant interaction between early and late treatments (p = 0.04). Liver, kidney, and testis weights were not significantly different among treatment groups.

View larger version (22K): [in this window] [in a new window]   Figure 3.   The effects of RAD therapy on organ weights. Thymus (A) and spleen (B) organ weight (g)/body weight (kg) in normal rats (n = 5); Group PMV (pneumonectomized rats that received monocrotaline [60 mg/kg] and vehicle treatment) (n = 4), Group PMR5-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 5 to Day 35) (n = 5), Group PMR5-14 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 5 to Day 14) (n = 5), and Group PMR15-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD [2.5 mg/kg/d] from Day 15 to Day 35) (n = 4). NS = p > 0.05 by Student t test; *p < 0.05, **p < 0.01 by ANOVA.

Histopathology

A quantitative analysis of lumenal obstruction on 50 consecutive small pulmonary arteries from each rat in Groups PMV and PMR was performed (Figure 4). The distribution of the vascular lesions, and an average vascular occlusion score (between 0 and 2), are presented (Figure 4). Two-way ANOVA and multiple comparison revealed that significant effects of therapy occurred between Days 5 and 14 (Group PMR_5-14) (p < 0.01).

View larger version (150K): [in this window] [in a new window]   View larger version (31K): [in this window] [in a new window]   Figure 4.   RAD suppresses pulmonary artery neointimal formation. The "vascular occlusion score" is the average score for 25 intraacinar pulmonary arteries. Vessels were assessed for neointimal formation, and scored: Grade 0 for no evidence of neointimal formation, Grade 1 for less than 50% lumenal occlusion, Grade 2 for more than 50% lumenal occlusion. (A) Normal rat intraacinar artery without evidence of neointimal proliferation (Grade 0). (B) Grade 2 neointimal lesion (> 50% lumenal occlusion) in pneumonectimized rats, 4 wk after receiving monocrotaline (60 mg/kg, subcutaneous). (C) Grade 1 lesion (< 50% lumenal occlusion) in Group PMR5-14 (early treatment group). (D) Predominance of Grade 2 lesion in Group PMR15-35 (late treatment group). All samples prepared with elastin-van Geison (EVG). Original magnification ×600. (E) Grades of vascular occlusion in right lung. Groups included N (normal), PMV (pneumonectomized rats that received monocrotaline [60 mg/kg] and vehicle treatment), RAD5-35 (pneumonectomized rats that received monocrotaline [60 mg/kg] and RAD treatment [0.25 mg/kg/d] from Day 5 to Day 35) (n = 5), Group PMR5-14 (early treatment group, receiving RAD [2.5 mg/kg/d] from Day 5 to Day 14) (n = 5), and Group PMR15-35 (late treatment group, receiving RAD [2.5 mg/kg/d] from Day 15 to Day 35) (n = 5). ## p < 0.01 by Student t test; **p < 0.01 by ANOVA.

In summary, these data demonstrate that RAD therapy, when administered at the same time as monocrotaline, results in significantly lower pulmonary arterial pressures, less right ventricular hypertrophy, and a lower vascular occlusion score in this rat model. When initiated more than 1 wk after monocrotaline administration (Group PMR_15-35), RAD treatment decreases right ventricular hypertrophy.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this investigation, we demonstrate that treatment with an oral derivative of rapamycin (RAD) attenuates the development of pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular neointimal formation in pneumonectomized rats that receive monocrotaline. Moreover, we demonstrate that early therapy with RAD (simultaneous with the administration of monocrotaline) appears more effective at preventing vascular remodeling and PH than late therapy (commencing 1 wk after monocrotaline administration). Our data suggest that RAD therapy is most effective when initiated before monocrotaline administration. Late therapy (beginning 1 wk after monocrotaline) does not significantly attenuate the development of pulmonary arterial neointimal formation and pulmonary hypertension but does attenuate the development of right ventricular hypertrophy. Our laboratory has previously demonstrated that right ventricular hypertrophy (RVH) is present as early as Day 21 (RV/LV&S = 0.4) in this disease model (25). Therefore, the effect of late RAD treatment probably results from direct effects on heart muscle proliferation rather than from changes in pulmonary vascular resistance.

The mechanisms that result in neointimal formation in this disease model are still unknown. In rats, monocrotaline administration leads to vascular smooth muscle cell hypertrophy and an increase in medial wall thickness (9, 26). It is believed that an increase in vascular shear stress (left pneumonectomy results in a relative increase in blood flow to the remaining lung) is necessary to produce neointimal formation, because the lesion does not occur after monocrotaline administration alone (12). This pathologic neointimal formation leads to lumenal occlusion and marked increases in pulmonary vascular resistance. Okada and coworkers (27) demonstrated that the angiotensin-converting enzyme inhibitor, quinapril, effectively delayed the development of PH and RVH in a rat model that involved monocrotaline injection followed by pneumonectomy. Five weeks after monocrotaline administration, pneumonectomized rats showed = 45 ± 5 mm Hg, RV/LV&S = 0.6 ± 0.1, and vascular occlusion score = 1.67 ± 0.08 (27). These values closely match our results in Group PMV, measured 4 wk after monocrotaline administration. Early treatment with quinapril (30 mg/kg/d, commencing 10 d before monocrotaline) resulted in = 21 ± 3 mm Hg, RV/LV&S = 0.39 ± 0.05, and vascular occlusion score = 0.27 ± 0.09 (27). Delayed treatment with quinapril (initiated 3 wk after MCT) resulted in = 28 ± 3 mm Hg, RV/LV&S = 0.48 ± 0.1, and vascular occlusion score = 1.20 ± 0.26 (27). The current data indicate that RAD is as effective as quinapril in this disease model when therapy is commenced before monocrotaline administration. However, RAD is probably less effective than quinapril when treatment is started after monocrotaline administration.

There is no evidence to suggest that immunosuppression has a beneficial effect on the development of monocrotaline-induced PH. Several investigations have suggested that T lymphocytes probably do not contribute to the development of monocrotaline-induced PH. First, monocrotaline-induced pulmonary hypertension has been reported to be more severe, rather than less severe, in athymic rats, compared with euthymic littermates, suggesting that T lymphocytes are not required to develop the disease (28). Second, neither the adoptive transfer of lymphocytes, nor anti-lymphocyte serum, has a significant effect on the development or progression of monocrotaline-induced pulmonary hypertension (29). In addition, therapy with the immunosuppressant cyclosporin does not protect against the development of monocrotaline-induced PH (29). Unlike cyclosporin, rapamycin inhibits growth factor receptor-mediated proliferation in both hematopoietic and nonhematopoietic cells (including endothelial cells) (18, 19). There are no data on the impact of the immune system on the development and progression of neointimal lesions in pneumonectomized rats that receive monocrotaline. In other disease models it appears that rapamycin and RAD have effects on vascular remodeling that are independent of immunosuppression (30, 31). Chronic graft vascular disease in rat cardiac allografts is generally unresponsive to immunosuppression, but it is reversed by rapamycin (14). Rapamycin also inhibits the arterial proliferative response after balloon angioplasty in pigs, a process that is also generally unresponsive to immunosuppression (20, 21). On the basis of the above, we believe that the efficacy of RAD in this disease model is likely due to its direct antiangiogenic effects, rather than to immunosuppressive effects.

Current therapies for PPH include vasodilators (such as calcium channel blockers and prostacyclin) and anticoagulation (2). Currently, there are no data on the use of nonvasoactive antiproliferative agents to treat PPH. Rapamycin and RAD have not been used as therapy for PH in patients. In this study, RAD helps to prevent (but does not appear to reverse) vascular neointimal formation in this model of pulmonary hypertension. Most cases of human pulmonary hypertension are advanced by the time of diagnosis. Therefore, rather than curing pulmonary hypertension, RAD might help to prevent further disease progression. This study, and our study of the effect of triptolide in this disease model (25), demonstrate the usefulness of antiangiogenic compounds in the development and progression of pulmonary hypertension, at least in rats. Such strategies might represent a new approach to the management of pulmonary hypertension in humans.