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This paper reports the effect of triptolide (a diterpenoid triep- oxide) on the development of monocrotaline (MCT)-induced pul- monary hypertension in ...
Triptolide Attenuates Pulmonary Arterial Hypertension and Neointimal Formation in Rats JOHN L. FAUL, TOSHIHIKO NISHIMURA, GERALD J. BERRY, GAIL V. BENSON, RONALD G. PEARL, and PETER N. KAO The Division of Pulmonary and Critical Care Medicine, and the Departments of Pathology and Anesthesiology, Stanford University Medical Center, Stanford, California

This paper reports the effect of triptolide (a diterpenoid triepoxide) on the development of monocrotaline (MCT)-induced pulmonary hypertension in pneumonectomized rats. Male Sprague– Dawley rats were injected with MCT (60 mg/kg) on Day 7 after left pneumonectomy. Rats received therapy from Day 5 to 35 with triptolide (0.25 mg/kg intraperitoneally, every other day, n ⫽ 10), or vehicle (0.1 ml of ethanol/cremophor intraperitoneally, every other day, n ⫽ 10). By Day 35, triptolide-treated rats demonstrated lower mean pulmonary arterial pressure (mPAP) than vehicle-treated rats (mPAP 21 ⫾ 3 versus 42 ⫾ 5 mm Hg, p ⬍ 0.001). Triptolide-treated rats also had significantly less right ventricular hypertrophy (RVH) and pulmonary arterial neointimal formation. In a rescue experiment, rats initiated therapy on Day 21. At Day 35, vehicle-treated rats (n ⫽ 4) had higher mPAP (40 ⫾ 9 mm Hg), greater RVH, and more severe pulmonary arterial neointimal formation than rats that received triptolide (0.25 mg/kg every other day, n ⫽ 7, mPAP 30 ⫾ 4 mm Hg) and rats that received triptolide (0.2 mg/kg daily, n ⫽ 7, mPAP 25 ⫾ 5 mm Hg, p ⬍ 0.01). In pneumonectomized rats that receive MCT, triptolide attenuates the development of pulmonary hypertension and RVH, and promotes regression of pulmonary arterial neointimal formation.

Primary pulmonary hypertension (PPH) is an incurable disease that causes progressive elevation of pulmonary arterial blood pressure and life-threatening right heart failure (1). Continuous intravenous prostacyclin, calcium channel blockers, and lung and heart/lung transplantation provide the only hopes for prolonged survival (2). The etiology of PPH is unknown, but there is evidence that endothelial cell proliferation is important in its development and progression (3–5). Abnormal proliferations of endothelial cells that occlude the vascular lumen have been described in the onion-skin and plexiform vascular lesions of humans with PPH (5). A recently developed animal model produces certain pathologic changes that resemble those seen in humans with PPH (6). In this model, monocrotaline (MCT) is administered to pneumonectomized rats. Three to five weeks later, pulmonary arterial hypertension and pulmonary vascular neointimal formation are evident. The severity of pulmonary hypertension (PH) (mean pulmonary arterial pressure [mPAP] ⫽ 45 mm Hg) in this model is greater than that seen 4 wk after monocrotaline administration alone (mPAP ⫽ 32 mm Hg) (6, 7). Changes in vascular shear stress are essential to the development of neointimal lesions in the pneumonectomy/mono-

crotaline model of PH, because pulmonary arterial neointimal formation does not occur after monocrotaline alone (6). Tripterygium wilfordii Hook F. (TWHF) is an herb that is used in traditional Chinese medicine for rheumatoid arthritis and other autoimmune diseases (8, 9). Diterpene lactone epoxide compounds extracted from TWHF have immunosuppressive, antifertility, and antiinflammatory properties (10). One of the principal active agents from TWHF, a highly oxygenated diterpenoid triepoxide, is called triptolide (11). Extracts of TWHF, and pure triptolide, exhibit antitumor effects in vitro and in vivo (11–16). Extracts of TWHF have been shown to demonstrate antiangiogenic effects, including the inhibition of both the vascular endothelial cell growth and neovascularization of tumors (17), and the attenuation of graft coronary artery neointimal formation in rats (18). We have characterized some of the intracellular actions of triptolide in vitro (19, 20). Triptolide potently inhibits T-cell activation at the level of cytokine gene transcription, through mechanisms that differ from cyclosporin A and FK506. Triptolide inhibits transcriptional activation of NF-␬B in numerous cell types, including lymphocytes, epithelial cells, and fibroblasts (16, 19, 20). The antiproliferative effects of triptolide involve enhanced apoptosis (15, 16, 21) and the inhibition of expression of genes involved in cell cycle progression and survival (20). Because it potently inhibits gene transcription in a variety of cell types, and because it demonstrates beneficial effects in coronary arterial neointimal formation in rats, we hypothesized that triptolide would attenuate the development of pulmonary arterial neointimal formation and pulmonary hypertension in pneumonectomized rats that received monocrotaline. This study describes the effect of triptolide on the development of PH, when it is given prior to (and 2 wk after) monocrotaline administration.

METHODS Animal Model and Study Design

(Received in original form February 7, 2000 and in revised form June 16, 2000)

Twelve-week-old, male, pathogen-free, Sprague–Dawley rats (weight: 350–400 g) underwent left pneumonectomy. Rats were anesthesized with atropine sulfate (0.1 mg) intermuscularly, ketamine hydrochloride (0.1 mg) intermuscularly, and xylazine (0.15 mg) subcutaneously. They were placed in a supine position and underwent oral endotracheal intubation with a 14-gauge catheter (Baxter, Deerfield, IL). Anesthesia was maintained with halothane inhalation (0.5%) and rats were ventilated using a Harvard (Type 683, Harvard Apparatus, MA) rodent ventilator (tidal volume 3.0 ml, respiratory rate 60/min, positive end expiratory pressure [PEEP] 1 cm H2O). Seven days after pneumonectomy rats were injected subcutaneously in the right hind limb with monocrotaline (60 mg/kg) (Sigma).

Supported by a gift from the Donald E. and Delia B. Baxter Foundation and NIH Grants AI39624 and HL62588 to P.N.K.

Continuous Triptolide Therapy

Correspondence and requests for reprints should be addressed to Peter N. Kao, M.D., Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, CA 94305-5236. E-mail: [email protected] Am J Respir Crit Care Med Vol 162. pp 2252–2258, 2000 Internet address: www.atsjournals.org

Ten rats (PMT5–35qod group) received triptolide (PG490; a generous gift of Pharmagenesis, Palo Alto, CA) (0.25 mg/kg, intraperitoneally, every other day, dissolved in ethanol/cremophor [Sigma]) commencing on Day 5. Ten rats received ethanol/cremophor vehicle (PMV5–35qod group) commencing on Day 5. Identical volumes of solution (approxi-

Faul, Nishimura, Berry, et al.: Effect of Triptolide on Pulmonary Hypertension in Rats mately 0.1 ml per rat per injection) were administered every other day by intraperitoneal injection.

Delayed Triptolide Therapy Seven rats (PMT5–35qod group) received alternate day dosing of triptolide (PG490; a generous gift of Pharmagenesis, Palo Alto, CA) (0.25 mg/kg, intraperitoneally, every other day, dissolved in ethanol/cremo phor [Sigma]) commencing on Day 21. Seven rats (PMT21–35qd group) received daily doses of triptolide (PG490; Pharmagenesis) (0.2 mg/kg, intraperitoneally, daily, dissolved in ethanol/cremophor [Sigma]) commencing on Day 21. Four rats received ethanol/cremophor vehicle every other day (PMV21–35qod group) commencing on Day 21. Identical volumes of solution (approximately 0.1 ml per rat per injection) were administered by intraperitoneal injection. An additional three rats were sacrificed on Day 21 (14 d post-MCT) to provide a reference point for disease progression in this model. 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 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). The study was approved by the Stanford Administrative Panel on Laboratory Animal Care, and was conducted in compliance with Stanford University regulations.

Hemodynamic Studies and Tissue Preparation On Day 35, 28 d postmonocrotaline administration, rats were anesthetized by intramuscular injections of ketamine HCl (0.1 mg) and atropine sulfate (0.1 mg). An additional six rats were studied as a normal (control) group. These six rats underwent neither pneumonectomy nor monocrotaline therapy, nor did they receive triptolide. Anesthesia was maintained with inhalation halothane (0.5%, by hood). An arterial catheter was placed in the left carotid artery by cutdown. An arterial blood gas sample was obtained. The right internal jugular vein was dissected and a pulmonary artery catheter (PV 1, 0.28 mm internal diameter) was inserted through an introducer under pressure waveform monitoring. Mean arterial blood pressure, right ventricular systolic blood pressure, and pulmonary arterial blood pressure were recorded. Following tracheostomy, rats were ventilated for 6 min (tidal volume 0.3 ml, rate 60 breaths/min, PEEP 1 cm H2O) on 99% oxygen, 0.5% halothane. A sternotomy was performed and the chest was opened. Repeat measures of right ventricular systolic pressure and pulmonary artery pressure were obtained by direct needle puncture (27 gauge needle connected to a pressure transducer) of the right ventricle and right pulmonary artery, respectively. Rats were sacrificed by exsanguination via the carotid arterial catheter. The right lung, right ventricle, left ventricle and septum, liver, spleen, kidney, testis, and thymus were weighed and collected for histology in formalin-buffered saline. In contrast to previous studies (6, 22), we measured pulmonary arterial pressure (PAP) and right ventricular systolic pressure (RVSP) by two separate techniques: a “closed chest” technique: pulmonary artery catheterization and percutaneous right ventricular needle puncture; and an “open chest” technique: right ventricular and pulmonary arterial needle puncture under direct vision after sternotomy and blunt dissection. Both sets of measurements were closely matched (intraclass correlation coefficient ⫽ 0.95, data not shown), indicating that open and closed chest techniques are equally accurate for the measurement of PAP and RSVP. The use of closed chest techniques may allow serial measurements of pulmonary artery pressures in future studies. Organs were fixed in formalin, then paraffin embedded and sectioned. After EVG (elastin) staining, lung sections were examined histologically for evidence of pulmonary vascular disease. The severity of pulmonary vascular neointimal formation was assessed in 50 panacinar arteries from each animal. The severity of neointimal formation was scored by a blinded investigator (G.J.B.). The absence of neointimal formation or luminal occlusion equals Grade 0; the presence of neointimal formation causing less than 50% luminal occlusion equals Grade 1; the presence of neointimal formation causing greater than 50% luminal occlusion equals Grade 2. An average score for 50 vessels (bounded by 0 and 2) was calculated for each animal.

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Statistical Analysis Organ weights are presented as grams per kilogram body weight. Data are presented as mean and standard deviation. Between-group differences (between vehicle-treated and triptolide-treated groups) in the study of continuous therapy were determined using Student’s t test, with significance indicated by p ⬍ 0.05. Significant between-group differences in the study of delayed triptolide therapy were analyzed using ANOVA, with statistical significance indicated by p ⬍ 0.05. Values for normal rats are supplied for comparison.

RESULTS Continuous Triptolide Therapy

This experiment investigated whether triptolide treatment would attenuate the development of pulmonary arterial hypertension. Twenty-six rats were studied, including six normal rats that served as a reference control. Ten pneumonectomized rats received monocrotaline (60 mg/kg) on Day 7 and sham treatment with vehicle commencing on Day 5: Group PMV5–35qod. Ten pneumonectomized rats received monocrotaline (60 mg/kg, subcutaneously) and triptolide (0.25 mg/ kg, intraperitoneally, every other day), commencing on Day 5: Group PMT5–35qod. Three deaths occurred in Group PMV5–35qod, on postoperative Days 29, 30, and 33, respectively, and three deaths occurred in Group PMT5–35qod, on postoperative Days 32, 33, and 33, respectively. Rats that died in Group PMV5–35qod had evidence of right ventricular hypertrophy (their average right ventricle/left ventricle and septum [RV/(LV and S)] was 0.58 ⫾ 0.1), and neointimal proliferation on histopathology (their mean vascular occlusion score was 1.89 ⫾ 0.03). In contrast, the rats that died in Group PMT5–35qod had less right ventricular hypertrophy (RVH) (their average RV/[LV and S] was 0.42 ⫾ 0.01) and less pulmonary vascular disease (their mean vascular occlusion score was 0.98 ⫾ 0.17). The seven surviving rats in Groups PMV5–35qod and PMT5–35qod underwent measurements of pulmonary physiology. The organ weights and histopathology from all 10 rats in Groups PMV5–35qod and PMT5–35qod were analyzed together in an intention to treat analysis. Delayed Triptolide Therapy

This experiment investigated whether triptolide, administered after monocrotoline, would ameliorate the development of pulmonary arterial hypertension, right ventricular hypertrophy, and neointimal proliferation in pneumonectomized rats. Triptolide therapy was commenced on postoperative Day 21, 14 d after monocrotaline administration. Pulmonary artery pressures, right ventricular hypertrophy, and scores of neointimal proliferation were measured in three pneumonectomized, monocrotaline-treated rats on Day 21 (to study the relative effects of delayed therapy). Four rats received vehicle every other day, commencing on Day 21: Group PMV21–35qod. Seven rats received triptolide treatment every other day (0.25 mg/kg, intraperitoneally): Group PMT21–35qod. Seven rats received triptolide daily (0.2 mg/kg, intraperitoneally): Group PMT21–35qd. There were no early deaths in any of the delayed therapy groups. There were no significant between-group differences in activity or body mass. Physiology

Continuous triptolide therapy. Physiologic data were obtained on Day 35 from the seven surviving members in Groups PMV5–35qod and PMT5–35qod (Figures 1A and 1B). Note that values for Group N (a group of six normal, healthy rats) are included as a reference control. Group N demonstrated systemic blood pressures (mean arterial pressure [MAP] ⫽ 100 ⫾

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5 mm Hg), pulmonary arterial pressures (mPAP ⫽ 18 ⫾ 2 mm Hg), and right ventricular systolic blood pressures (RVSP ⫽ 20 ⫾ 2 mm Hg) that were typical of healthy adult rats (23, 24). There was no significant difference in MAP between normal rats and rats in Group PMV5–35qod or PMT5–35qod (data not shown), indicating that triptolide produces no vasodilator effects on the systemic circulation. Group PMV5–35qod demonstrated severe pulmonary arterial hypertension (PAP ⫽ 42 ⫾ 5 mm Hg, Figure 1A) and right ventricular systolic hypertension (RVSP ⫽ 47 ⫾ 6 mm Hg, Figure 1B). In contrast, Group PMT5–35qod demonstrated lower pulmonary artery pressures (mPAP ⫽ 21 ⫾ 3 mm Hg, Figure 1A) and right ventricular systolic pressures (RVSP ⫽ 29 ⫾ 5 mm Hg, Figure 1B) (p ⬍ 0.001). The hematocrit in Group PMV5–35qod (52 ⫾ 2 g/dl) was significantly higher than that in Group PMT5–35qod (44 ⫾ 2 g/ dl) (p ⬍ 0.01). Delayed triptolide therapy. Rats sacrificed on Day 21 did not demonstrate pulmonary arterial hypertension (mPAP ⫽ 18 ⫾ 3 mm Hg, Figure 1C). By Day 35, rats in Group PMV21–35qod demonstrated pulmonary hypertension (mPAP ⫽ 40 ⫾ 9 mm Hg, Figure 1C) and right ventricular systolic hypertension (RVSP ⫽ 46 ⫾ 11, Figure 1D). The similarity between these results and the results of the study of continuous therapy (Group PMV5–35qod, Figures 1A and 1B) demonstrates that the development of PH in this disease model is very reproducible. Compared to the vehicle group, rats in Group PMT21–35qod had lower pulmonary artery pressures (mPAP ⫽ 30 ⫾ 4 mm Hg, Figure 1C) and right ventricular systolic pressures (RVSP ⫽ 39 ⫾ 3, Figure 1D). Rats in Group PMT21–35qd (triptolide 0.2 mg/kg/d) had even lower pulmonary pressures (mPAP ⫽ 25 ⫾ 5 mm Hg, Figure 1C) (p ⬍ 0.01) and right ventricular pressures (RVSP ⫽ 32 ⫾ 8 mm Hg, Figure 1D) (p ⬍ 0.01), suggest-

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ing a dose-dependent effect on the progression of pulmonary hypertension in this model. Organ weights. By Day 35, rats in Groups PMV5–35qod and PMT5–35qod had an average weight loss of 50 g compared with normal rats (data not shown). This weight loss is probably due to the stress of surgical pneumonectomy combined with monocrotaline injection. To facilitate comparisons across groups of rats, organ weights are presented per kilogram of body weight (Figure 2). The RV/(LV and S) ratio is 0.30 in normal rats (Figure 2A). Right ventricular hypertrophy is a characteristic feature of chronic pulmonary arterial hypertension. An RV/(LV and S) ratio greater than 0.5 indicates right ventricular hypertrophy (6, 23, 24). In the study of continuous triptolide therapy, the RV/(LV and S) in Group PMV5–35qod was 0.69 ⫾ 0.18 (Figure 2A). In contrast, the RV/(LV and S) ratio in Group PMT5–35qod was 0.39 ⫾ 0.1, consistent with the absence of RVH (p ⬍ 0.001). In the study of delayed triptolide therapy, the RV/(LV and S) ratio was 0.40 ⫾ 0.05 at Day 21 (Figure 2B), but was 0.79 ⫾ 0.18 in Group PMV21–35qod (Figure 2B), suggesting that RVH develops between Day 21 and Day 35 in this model. Rats in Group PMT21–35qod showed an RV/(LV and S) ratio of 0.45 ⫾ 0.06, and rats in Group PMT21–35qd showed an RV/(LV and S) ratio of 0.4 ⫾ 0.07 (Figure 2B) (p ⬍ 0.001). There was no significant between-group difference in liver, kidney, or spleen weights (data not shown). The weights of the thymus and testis were significantly decreased in Group PMT5–35qod compared with Group PMV5–35qod (p ⬍ 0.01; Figures 3A and 3B). Delayed triptolide therapy resulted in significantly lower weights of thymus and testis in Groups PMT21–35qod and PMT21–35qd compared with Group PMV21–35qd (p ⬍ 0.01; Figures 3C and 3D). Previous reports have demonstrated that

Figure 1. Triptolide prevents the development and progression of pulmonary arterial hypertension and right ventricular systolic hypertension in pneumonectomized, monocrotaline-treated rats. (A) Mean pulmonary arterial pressures (mPAP) in Group PMV5–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 5 to Day 35 [n ⫽ 7]), Group PMT5–35qod (monocrotalinetreated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 5 to Day 35 [n ⫽ 7]), and normal rats (n ⫽ 6). Bars are means ⫾ standard deviations. Group PMT5–35qod had significantly lower mPAP than PMV5–35qod (**p ⬍ 0.001). (B) Right ventricular systolic pressures (RSVP) in Group PMV5–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 5 to Day 35 [n ⫽ 7]), Group PMT5–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 5 to Day 35 [n ⫽ 7]), and normal rats (n ⫽ 6). Bars are means ⫾ standard deviations. Group PMT5–35qod had significantly lower RSVP than Group PMV5–35qod (**p ⬍ 0.001). (C) Mean pulmonary arterial pressures (mPAP) in Group PMV21–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 21 to Day 35 [n ⫽ 7], Group PMT21–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 21 to Day 35 [n ⫽ 7], Group PMT21–35qd (monocrotaline-treated, pneumonectomized rats that received triptolide [0.2 mg/kg, daily] from Day 21 to Day 35 [n ⫽ 7]), and Day 21 rats (monocrotaline-treated, pneumonectomized rats that were sacrificed on Day 21 [n ⫽ 3]). Bars are means ⫾ standard deviations. Group PMT21–35qod and PMT21–35qd had significantly lower PAP than PMV21–35qod (**p ⬍ 0.01). (D) Right ventricular systolic pressures (RSVP) in Group PMV21–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qd (monocrotaline-treated, pneumonectomized rats that received triptolide [0.2 mg/ kg, daily] from Day 21 to Day 35 [n ⫽ 7]), and Day 21 rats (monocrotaline-treated, pneumonectomized rats that were sacrificed on Day 21 [n ⫽ 3]). Bars are means ⫾ standard deviations. Groups PMT21–35qod and PMT21–35qd had significantly lower RSVP than PMV21–35qod (**p ⬍ 0.01).

Faul, Nishimura, Berry, et al.: Effect of Triptolide on Pulmonary Hypertension in Rats

Figure 2. Triptolide prevents the development and progression of right ventricular hypertrophy in pneumonectomized, monocrotaline-treated rats. (A) Right ventricle left ventricle and septum (RV/LV and S) in Group PMV5–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 5 to Day 35 [n ⫽ 10]), Group PMT5–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 5 to Day 35 [n ⫽ 10]), and normal rats (n ⫽ 6). Bars are means ⫾ standard deviations. Group PMT5–35qod had significantly lower RV/LVand S than PMV5–35qod (**p ⬍ 0.001). (B) RV/LV and S in Group PMV21–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qod (monocrotaline-treated pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qd (monocrotaline-treated, pneumonectomized rats that received triptolide [0.2 mg/kg, daily] from Day 21 to Day 35 [n ⫽ 7]), and Day 21 rats (monocrotalinetreated, pneumonectomized rats that were sacrificed on Day 21 [n ⫽ 3]). Bars are means ⫾ standard deviations. Group PMT21–35qod and PMT21–35qd had significantly lower RV/LV and S than PMV21–35qod (**p ⬍ 0.001).

triptolide has inhibitory effects on T-cell proliferation (19, 21) and spermatogenesis (25, 26). Histopathology

Representative morphologies of small pulmonary arteries in normal rats, in pneumonectomized and monocrotaline-treated rats (PM), and in Groups PMV5–35qod and PMT5–35qod are

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shown (Figures 4A–4D). A quantitative analysis of luminal obstruction on 50 consecutive small pulmonary arteries from each rat was performed, and a vascular occlusion score (VOS, between 0 and 2) was calculated for each animal (Figure 4E). The combination of pneumonectomy and monocrotaline results in severe changes of neointimal proliferation and vascular occlusion (Figure 4B versus 4A). The VOS of 1.92 in Group PMV5–35qod is similar to an average VOS of 1.91 for our laboratory’s historical data on pneumonectomized rats that receive monocrotaline. This suggests that the admininstration of ethanol/cremophore vehicle does not alter the histopathology of this disease model. Compared with Group PMV5–35qod, Group PMT5–35qod had a lower VOS (1.92 ⫾ 0.11 versus 1.27 ⫾ 0.61) (p ⬍ 0.001) (Figure 4E). Rats that were sacrificed on postoperative Day 21 (14 d after monocrotaline injection) showed a high degree of occlusion of small pulmonary arteries (score 1.92 ⫾ 0.001; Figure 4F). By Day 35, the vascular occlusion score was even higher in vehicle-treated animals (score 1.96 ⫾ 0.02), and the lumen of many small pulmonary arteries appeared completely obliterated. Rats that received triptolide between Day 21 and Day 35 (Group PMT21–35qod) showed less vascular occlusion (score 1.49 ⫾ 0.3) than both Group PMV21–35qd and Group PMT21–35qd (score 1.84 ⫾ 0.12; Figure 4F) (p ⬍ 0.01). Taken together, these results indicate that both continuous and delayed treatment with triptolide significantly attenuate the development of pulmonary arterial hypertension, right ventricular hypertrophy, and pulmonary arterial occlusion in this model. Furthermore, triptolide therapy can lead to a reversal of pulmonary neointimal formation.

DISCUSSION This investigation demonstrates that triptolide attenuates the development and progression of monocrotaline-induced pul-

Figure 3. The effect of triptolide on thymus and testis weights in rats. (A) Thymus weight (grams per kilogram body weight) in Group PMV5–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 5 to Day 35 [n ⫽ 10], Group PMT5–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/ kg, every other day] from Day 5 to Day 35 [n ⫽ 10]), and normal rats (n ⫽ 6). Bars are means ⫾ standard deviations. Group PMT5–35qod had significantly lower thymus weight than PMV5–35qod (**p ⬍ 0.001). (B) Testis weight (grams per kilogram body weight) in Group PMV5–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 5 to Day 35 [n ⫽ 10]), Group PMT5–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 5 to Day 35 [n ⫽ 10]), and normal rats (n ⫽ 6). Bars are means ⫾ standard deviations. Group PMT5–35qod had significantly lower testis weight than PMV5–35qod (**p ⬍ 0.001). (C) Thymus weight (grams per kilogram body weight) in Group PMV21–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qod that (monocrotaline-treated pneumonectomized rats received triptolide [0.25 mg/kg, every other day] from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qd (monocrotaline-treated, pneumonectomized rats that received triptolide [0.2 mg/kg, daily] from Day 21 to Day 35 [n ⫽ 7]), and Day 21 rats (monocrotaline-treated, pneumonectomized rats that were sacrificed on Day 21 [n ⫽ 3]). Bars are means ⫾ standard deviations. Group PMT21–35qod and PMT21–35qd had significantly lower thymus weight than PMV21–35qod (**p ⬍ 0.001). (D) Testis weight (grams per kilogram body weight) in Group PMV21–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qod (monocrotaline-treated pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qd (monocrotaline-treated, pneumonectomized rats that received triptolide [0.2 mg/kg, daily] from Day 21 to Day 35 [n ⫽ 7]), and Day 21 rats (monocrotaline-treated, pneumonectomized rats that were sacrificed on Day 21 [n ⫽ 3]). Bars are means ⫾ standard deviations. Group PMT21– 35qod and PMT21–35qd had significantly lower testis weight than PMV21–35qod (**p ⬍ 0.001).

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monary hypertension, right ventricular hypertrophy, and pulmonary arterial neointimal formation in pneumonectomized rats. Moreover, triptolide ameliorates the development of pulmonary hypertension and pulmonary arterial neointimal formation, even when administered 2 wk after monocrotaline. In this study, pulmonary arterial neointimal proliferation and vascular occlusion occur in the setting of pulmonary arterial hemodynamic stress (increased blood flow to the right

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lung) combined with a toxic injury to the rat lung by monocrotaline pyrrole (a toxic metabolite of monocrotaline) (6, 27). The severity of pulmonary arterial hypertension, the degree of right ventricular hypertrophy, and the histological evidence of neointimal proliferation in the vehicle-treated groups of this study are similar to those previously reported by other workers who developed this model (6, 22). This study supports previous findings that the combination of pneumonectomy and

Figure 4. Triptolide prevents the development and progression of pulmonary arterial neointimal formation in pneumonectomized, monocrotalinetreated rats. (A) Normal rat arterioles do not have evidence of neointimal formation (Grade 0). (B) A predominance of Grade 2 neointimal lesions (⬎ 50% occlusion) is seen in pneumonectomized rats that receive monocrotaline without therapy. (C) A similar predominance of Grade 2 neointimal lesions (⬎ 50% occlusion) is seen in pneumonectomized rats that receive monocrotaline and vehicle (sham) therapy (Group PMV5–35qod). (D) A predominance of Grade 1 neointimal lesions (⬍ 50% occlusion) is seen in pneumonectomized rats that receive monocrotaline and triptolide therapy (Group PMT5–35qod). (E) The “vascular oclusion score” is the average score for 50 intraacinar pulmonary arteries. Vascular occlusion scores (VOS) in Group PMV5–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 5 to Day 35 [n ⫽ 10]), Group PMT5–35qod (monocrotaline-treated, pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 5 to Day 35 [n ⫽ 10]), and normal rats (n ⫽ 6). Bars are means ⫾ standard deviations. Group PMT5–35qod had significantly lower VOS than PMV5–35qod (**p ⬍ 0.001). (F) Vascular occlusion scores (VOS) in Group PMV21–35qod (monocrotaline-treated, pneumonectomized rats that received vehicle from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qod (monocrotaline-treated pneumonectomized rats that received triptolide [0.25 mg/kg, every other day] from Day 21 to Day 35 [n ⫽ 7]), Group PMT21–35qd (monocrotaline-treated, pneumonectomized rats that received triptolide [0.2 mg/kg, daily] from Day 21 to Day 35 [n ⫽ 7]), and Day 21 rats (monocrotaline-treated, pneumonectomized rats that were sacrificed on Day 21 [n ⫽ 3]). Bars are means ⫾ standard deviations. Group PMT21–35qod and PMT21–35qd had significantly lower VOS than PMV21–35qod (**p ⬍ 0.001). All photomicrographs (original magnification: ⫻600), elastin Virhoff van Gieron (EVG) stain.

Faul, Nishimura, Berry, et al.: Effect of Triptolide on Pulmonary Hypertension in Rats

MCT administration in rats appears to produce pulmonary arterial hypertension that more closely resembles the physiology and pathology of human PPH than administration of MCT alone (6, 7, 22, 28, 29). Administration of MCT (without pneumonectomy) leads to medial hypertrophy and a rise in PAP (approximately 32 mm Hg) and RVH (RV/LV and S ⫽ 0.5) after 4 wk. In this study, and in other studies from our laboratory, pneumonectomized rats that received monocrotaline and vehicle treatment not only consistently demonstrate neointimal formation, but also higher PAP (42 ⫾ 5 mm Hg) and higher RV/LV and S (0.69 ⫾ 0.18). Previously, treatment with the angiotensin-converting enzyme inhibitor, quinapril, was shown to attenuate the development of pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial neointimal formation in a model of PH that combined pneumonectomy with monocrotaline (60 mg/kg) administration. (22). In our study we used a different strategy. We treated rats with triptolide to investigate whether a potent transcriptional inhibitor with antiinflammatory and antiproliferative effects (19, 20) might inhibit the development of pulmonary arterial neointimal formation. Unlike quinapril, triptolide does not cause hypotension or vasodilatation. Nevertheless our data demonstrate that triptolide is effective at attenuating pulmonary hypertension in this disease model. Moreover, the current data from the study of delayed therapy suggest that delayed triptolide is effective even when therapy is started 2 wk after monocrotaline administration and after pulmonary neointimal formation has developed. The current findings are consistent with previous data that triptolide inhibits neointimal formation in coronary arteries of heterotopically transplanted hearts in rats (18). These protective effects were not observed using cyclosporin or other immunosuppressant therapies, suggesting that inhibition of coronary neointimal formation occurs independently of T-cell immunosuppression (18). In spite of triptolide’s beneficial effects on the development of pulmonary hypertension in this model, there are several unresolved issues with its use. First, there were six early deaths (before Day 35) in the study of continuous triptolide therapy. The three early deaths in the vehicle group probably resulted from severe pulmonary hypertension, because each rat demonstrated right ventricular hypertrophy and pulmonary vascular disease on histopathology (RV/LV and S ⫽ 0.58 ⫾ 0.1, vascular occlusion score ⫽ 1.89 ⫾ 0.03). In contrast, the three early deaths in the triptolide-treated group had less RVH and lower vascular occlusion scores (RV/LV and S ⫽ 0.42 ⫾ 0.01, vascular occlusion score ⫽ 0.98 ⫾ 0.17). The deaths in the triptolide-treated group, therefore, may have been due to factors other than severe pulmonary hypertension. We cannot exclude death due to triptolide toxicity, even though we employed a relatively low dose on an alternate-day dosing regimen. In the study of delayed triptolide therapy, we employed a daily dose of triptolide (0.2 mg/kg/d) without morbidity or early mortality. In humans, the main toxicities of TWHF are gastrointestinal disturbance and infertility (25), and one case of death due to a septic shock-like syndrome has been reported following an overdose of TWHF (30). Although TWHF is widely used in China as an herbal remedy, triptolide’s potential for toxicity will require further investigation. Triptolide significantly reduced testis and thymus weights in both the continuous and delayed treatment studies. In rats, low doses of triptolide (0.1 mg/kg/d) cause reversible inhibition of spermatogenesis (25). This is the first report that triptolide treatment significantly reduces thymus weight. This reduction in thymus weight is consistent with our laboratory’s previous findings that triptolide not only inhibits cytokine

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gene transcription, including IL-2 (19), but also enhances lymphocyte apoptosis (21). Although the triptolide-treated rats were probably immunosuppressed, there was no evidence of opportunistic infection. The role of the immune system, if any, in the development of PH in this disease model (monocrotaline combined with pneumonoectomy) is unclear. Immunosuppression does not appear to have beneficial effects of the development of monocrotaline-induced pulmonary hypertension. Indeed, greater degrees of RVH and vascular smooth muscle hypertrophy are seen in nude athymic rats following MCT compared with euthymic rats (31). Triptolide’s attenuation of monocrotaline-induced pulmonary hypertension in this study is comparable to the effect of RAD (an oral derivative of rapamycin). RAD and triptolide have similar beneficial effects on the development of PH in this disease model, when therapy is commenced before monocrotaline. The data in this study indicate that PAP, RVSP, and RV/ (LV and S) increase dramatically between Day 21 and Day 35 (see Figures 1C, 1D, and 2) (22). In contrast to RAD, however, delayed triptolide therapy (commencing 2 wk after monocrotaline) not only reverses pulmonary arterial neointimal formation but also effectively attenuates the progression of pulmonary hypertension and right ventricular hypertrophy. At Day 21 (14 d after monocrotaline injection), severe pulmonary arterial vascular occlusion is present, although the mPAP at rest is not significantly elevated above that in normal rats. The modest increase in RVH may be a consequence of exercise-induced pulmonary hypertension. By Day 35 there was even greater vascular occlusion, significant pulmonary hypertension, and RVH. We observed numerous pulmonary arteries with complete luminal obliteration by neointimal formation. Delayed triptolide therapy from Day 21 to Day 35 produced regression in pulmonary vascular occlusion, and decreased the development of pulmonary arterial hypertension and RVH. Triptolide, therefore, has the potential to produce regression of endothelial proliferation and neointimal formation, even following toxic injury with monocrotaline. The mechanisms by which triptolide produces regression of neointimal formation may include antiproliferative and antiinflammatory effects or enhancement of apoptosis in pulmonary arterial endothelial cells. In PPH, death generally occurs due to right heart failure. Even a partial attenuation of endothelial proliferation and neointimal formation might lead to clinically significant reductions in pulmonary vascular resistance. Although triptolide appears to show promise in this model, its precise mode of action is unknown. Further study of antiproliferative strategies for the management of pulmonary hypertension in humans is warranted. Acknowledgment : The authors thank members of the Kao and Pearl laboratories for helpful discussions.

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