Pulmonary hypertension (PH) is one of the causes of death in systemic lupus erythematosus, mixed connective tissue disease, and progressive systemic sclerosis. Severe symptomatic PH is a rare manifestation of lung involvement in patients with these diseases, but mild subclinical cases are probably not uncommon. Most patients whose cases have been reported were treated with various vasodilator agents, anticoagulants, systemic corticosteroids, or cytotoxic agents, but the overall prognosis of PH is poor. Its pathogenesis indicates that hypercoagulability and vasospasm of pulmonary vessels play important roles in the development of PH (1-3).
Prostacyclin is a potent inhibitor of platelet aggregation in vitro and in vivo in various animal species and has an antithrombotic effect in various species. Prostacyclin is less inactivated as it passes through the pulmonary circulation (4) and is the major metabolite generated during infusion of arachidonic acid in dog lung (5). Gryglewski and colleagues (6) concluded that prostacyclin is continuously generated and released from cat lung in vivo. Therefore, prostacyclin probably plays an important role in the modulation of vasomotor tone in the lung (7). Clinical data demonstrate that prostacyclin is effective in the treatment of primary and secondary PH of various origins (8,9). However, its susceptibility to metabolic degradation in vivo has limited its therapeutic application.
Beraprost (sodium ((±)-1R*, 2R*, 3aS*, 8bS*)-2, 3, 3a, 8b-tetrahydro-2-hydroxyl-1-[(E)-(3S*)-3-hydroxy-4-methyl-1-octen-6-ynyl]-1H-cyclopenta [b] benzofuran-5-butyrate) is a chemically stable prostacyclin analogue which can be administered orally (p.o.) and has a pharmacological profile similar to that of prostacyclin, i.e., potent platelet aggregation inhibitory effect in vitro and in vivo in various animal species (10,11), antithrombotic effect in the hamster cheek pouch (12), and vasodilating effects in dogs (13).
Administration of monocrotaline (MCT) to rats causes endothelial injury and medial hypertrophy of pulmonary vessels with microthrombus formation and cellular infiltration into the interstitium, resulting in PH. Because of the similarity of the pathology of MCT-induced rat PH to that of primary PH in humans, this animal model may provide an insight into possible treatment for human PH. In the present study, we investigated whether beraprost sodium inhibits MCT-induced PH in rats.
The study was performed under the control of the Animal Research Committee in accordance with The Guidelines on Animal Experiments at Fukushima Medical College, Japanese Government Animal Protection and Management Law (No. 105) and Japanese Government Notification of Feeding and Safekeeping of Animals (No. 6).
Induction of PH by MCT in rats
Six-week-old male Sprague-Dawley rats (Funabashi Farm, Sendai, Japan) weighing 150-200 g were injected subcutaneously (s.c.) with 40 mg/kg MCT (Wako Pure Chemical Industries, Tokyo, Japan) prepared as described previously (14). They were housed in cages of an animal isolator and subjected to a light/dark cycle (12/12 h). They were given food (CE-2; Clea Japan, Tokyo, Japan) and tap water ad libitum.
Assessment of PH
The extent of right ventricular hypertrophy reflecting the degree of PH (15) was assessed by the method described by Fulton and associates (16). Atria were trimmed off, and the right ventricular free wall (RV) was carefully separated from the left ventricle plus septum (LV + S). The ratio of weight of RV to that of LV + S [(RV/(LV + S)] was then used as an index of the extent of right ventricular hypertrophy.
Administration of beraprost sodium
Beraprost sodium at a dose of 30 μg/kg/day was administered orally. Administration of beraprost sodium was initiated on the same day as MCT injection, and the administration period was 1, 2, or 4 weeks. Four weeks after the start of the experiment, all rats were killed and the degrees of PH were determined. The numbers of normal rats, MCT-injected rats, and rats treated with beraprost sodium for 1, 2, or 4 weeks were 6, 7, 7, 8, and 7, respectively.
Administration of beraprost sodium at a dose of 10, 30, or 100 μg/kg/day was initiated 1 week after MCT injection. Three weeks after the start of the experiment, rats were killed and the degrees of PH were determined. The numbers of normal control rats, MCT-injected rats, and rats treated with beraprost sodium at a dose of 10, 30, or 100 μg/kg/day were 6, 9, 9, 9, and 9, respectively.
To compare the effect of beraprost sodium administration and that of prostaglandin E1 (PGE1) administration on the extent of PH, administration of beraprost sodium at a dose of 30 μg/kg/day p.o. or PGE1 (Sigma Chemical, St. Louis, MO, U.S.A.) at a dose of 200 μg/kg/day s.c. was initiated 1 week after MCT injection, and the administration was continued until the animals were killed (3 weeks after the start of the experiment) and the extents of PH were determined. Alveolar macrophages were collected and cytokines produced by alveolar macrophages were determined as described herein. The numbers of normal control rats, MCT-injected rats, rats treated with beraprost sodium at a dose of 30 μg/kg/day, and rats treated with PGE1 at a dose of 200 μg/kg/day were 6, 9, 9, and 9, respectively.
Determination of cytokines produced by alveolar macrophages
Rats were anesthetized with 50 mg/kg pentobarbital sodium, and the trachea was cannulated with a 22-gauge vinyl catheter (Surflow needle, Termo, Tokyo, Japan). Both lungs were lavaged twice with 3-ml aliquots of sterilized saline and the total of 5- to 5.5-ml vol was recovered. Alveolar macrophages in bronchoalveolar lavage fluids were washed three times with RPMI-1640 containing 10% fetal calf serum (FCS). Alveolar macrophages (1 × 104) in RPMI-1640 containing 10% FCS were incubated for 3 days at 37 °C. The supernatant of the culture medium was collected by centrifugation, and the amounts of cytokines were determined as described herein. There were no significant differences among experimental groups in terms of the amount of lavage fluid recovered.
Interleukin 1 (IL-1) Thymocytes of a C3H/HeJ mouse, 1.5 × 106/200 μl/well, were cultured for 2 days with 0.1 ml of 1:2 to 1:1,024 dilutions of the supernatant of the alveolar macrophages, followed by a pulse of 0.5 μCi of [3H]thymidine/well and incubation for 12 h. Thymocytes were harvested, and their proliferative activity was determined based on the extent of [3H]thymidine incorporation measured with a liquid scintillation system (Aloka, Tokyo, Japan). A standard amount of human IL-1 β (Otsuka, Tokyo, Japan) added to the untreated thymocytes was used to estimate the amount of IL-1 (17).
Interleukin-6 (IL-6) IL-6 activity was measured with the IL-6-dependent murine hybridoma MH 60. BSF 2 cell clone, a gift from Dr. T. Hirano, Osaka University. The growth of MH 60. BSF 2 cells was confirmed to be dependent on the presence of IL-6 (18). MH60.BSF 2 cells, 1 × 105/200 μl/well, were cultured for 2 days with 0.1 ml 1:2 to 1:2,048 dilutions of the supernatant already described, followed by a pulse of [3H]thymidine during the final 6 h of incubation. The incorporated [3H]thymidine was counted with a liquid scintillation counter. A standard amount of human IL-6, supplied by Dr. Hirano, was added to the untreated MH 60. BSF 2 cells and used to estimate the amount of IL-6.
Tumor necrosis factor (TNF)
L 929 cells (derived from an ATCC cell line), which are mouse fibrosarcoma cells sensitive to TNF, were used to determine the amount of TNF (19); 100-μl aliquots of 4 × 104/ml L 929 cells were added to culture plates (Nunclon, Koskilde, Denmark) and cultured for 2 days to achieve confluence. L 929 cells were then cultured with 1:1 to 1:4 dilutions of the supernatant for 18 h. The culture medium was discarded, and cells were stained with 100 μl 0.2% crystal violet. After 15 min, cells were lysed by adding 100 μl 0.5% sodium dodecyl sulfate and the lysate was read at 600 nm with an enzyme-linked immunosorbent assay reader (Bio-Rad Laboratories, CA, U.S.A.). A standard amount of human TNF added to the untreated L 929 cells was used to estimate the amount of TNF (Dainippon Pharmaceutical, Tokyo, Japan).
Drugs and statistical analysis
Beraprost sodium prepared at the chemical laboratory of the Basic Research Laboratories (Toray Industries) was dissolved in distilled water to prepare a series of test solutions. The data are mean ± SE of six to nine experiments. Statistical analyses were made with Student's t test.
The effect of beraprost sodium administration at a dose of 30 μg/kg/day initiated on the same day as MCT injection is shown in Fig. 1. The ratio of RV/(LV + S) in normal rats was 0.271 ± 0.0095, and the ratio of RV/(LV + S) increased significantly to 0.584 ± 0.025 in MCT-injected rats (p < 0.01). The rats treated with MCT and beraprost sodium for 1 week showed a lower RV/(LV + S) (i.e., 0.508 ± 0.026) than the MCT-injected rats who received no beraprost sodium; however, this difference was not statistically significant. The rats treated with beraprost sodium for 2 or 4 weeks showed significantly lower RV/(LV + S) (i.e., 0.467 ± 0.020 and 0.353 ± 0.015, respectively) than the MCT-injected rats who received no beraprost sodium (p < 0.01).
The effects of beraprost sodium administration initiated 1 week after MCT injection are shown in Fig. 2. The ratio of RV/(LV + S) in normal rats was 0.280 ± 0.014, and the ratio of RV/(LV + S) increased significantly to 0.501 ± 0.029 in MCT-injected rats (p < 0.01). The administration of beraprost sodium at a dose of 10, 30, or 100 μg/kg/day decreased the ratio of RV/(LV + S) to 0.444 ± 0.025 (NS), 0.361 ± 0.026 (p < 0.01), or 0.390 ± 0.033, (p < 0.05), respectively, as compared with that of the MCT-injected rats who received no beraprost sodium.
A comparison between the effect of beraprost sodium administration and that of PGE1 administration on development of PH in MCT-injected rats is shown in Fig. 3. The ratio of RV/(LV + S) in normal rats was 0.284 ± 0.0007, and that in MCT-injected rats increased significantly to 0.526 ± 0.038 (p < 0.01). Rats administered PGE1 at a dose of 200 μg/kg/day s.c. had lower RV/(LV + S) (0.435 ± 0.023) than those that received only MCT injection; however, this difference was not statistically significant. Rats that received beraprost sodium at a dose of 30 μg/kg/day s.c. showed a significantly lower RV/(LV + S) (0.398 ± 0.014) than those that received only MCT injection (p < 0.01).
The amounts of cytokines produced by alveolar macrophages are shown in Fig. 4. The amount of IL-1 in MCT-injected rats, 4.667 U, was significantly higher than that in normal rats, 0.553 U (p < 0.01). The amount of IL-1 in MCT-injected rats decreased significantly to 0.847 U in rats that received beraprost sodium (p < 0.01) and to 2.400 U in rats that received PGE1 (p < 0.05).
The amount of IL-6 in MCT-injected rats, 42.74 × 10-4 U, was significantly higher than that in normal rats, 18.36 × 10-4 U (p < 0.01). The amount of IL-6 in MCT-injected rats decreased to 27.01 × 10-4 U in rats that received beraprost sodium (p < 0.05) and to 33.26 × 10-4 U in rats that received PGE1 (NS).
The amount of TNF in MCT-injected rats, 5.444 U, was significantly higher than that in normal rats, 2.800 U (p < 0.01). The amounts of TNF in MCT-injected rats decreased significantly to 3.933 U in rats that received beraprost sodium (p < 0.01) and to 4.300 U in rats that received PGE1 (p < 0.01). There was no significant difference between rats that received beraprost sodium and those that received PGE1 in terms of amounts of IL-1, IL-6, and TNF produced.
In the present study, beraprost sodium, a stable analogue of prostacyclin, significantly inhibited the development of PH induced by MCT injection. Endothelial injury was demonstrated in MCT-treated rats as an early event followed by gradual development of PH (20). Damage to endothelial cells (EC) leads to decreased production of prostacyclin by EC and inhibits the production of tissue plasminogen activator, resulting in thrombosis. In this regard, prostacyclin, which is produced by EC, shows antiplatelet aggregation effects and vasodilative effects. Moreover, it inhibits damage to EC (21) and the cytoprotective effect of beraprost sodium against oxygen radical-induced damage to EC has been well documented (22).
The amounts of cytokines produced by alveolar macrophages were significantly increased in MCT-injected rats, as we previously described (23), and beraprost sodium administration suppressed the production of IL-1, IL-6, and TNF by alveolar macrophages.
IL-1 is one of the mediators that potentiate coagulation by promoting the expression of platelet-activating factor inhibitor or plasminogen-activator inhibitor (24,25). Gillespie and associates demonstrated that IL-1 induced pathogenic changes similar to those observed in MCT-induced PH in rats (26).
IL-6 induces platelet proliferation in bone marrow (27) and stimulates fibrinogen production by hepatocytes. We induced PH in rats by chronic injection of IL-6 (28).
TNF is known to increase EC permeability directly (29) and to enhance polymorphonuclear leukocyte adhesion to EC (30). As a result of these reactions, pulmonary vascular changes occur, and PH develops as demonstrated in sheep by Amari and co-workers (31).
Beraprost sodium decreased the production of IL-6 from J 744, derived from mouse macrophage, in a dose-dependent manner (manuscript submitted for publication). These findings and those obtained in the present study indicate that beraprost sodium inhibited the development of PH in MCT-injected rats not only through its effects of vasodilation and antiplatelet aggregation in pulmonary circulation but also through its antiinflammatory effects.
PGE1 administration initiated 1 week after MCT injection did not inhibit development of PH, although beraprost sodium administration initiated 1 week after MCT injection did. In another experiment PGE1 administration at a dose of 200 μg/kg/day initiated on the same day as MCT injection suppressed the development of PH significantly (manuscript submitted for publication). These results suggest that PGE1 suppresses only an early event in the development of PH, but that beraprost sodium suppresses an early event and subsequent events. Beraprost sodium suppressed cytokine production by alveolar macrophages to a greater extent than did PGE1 suggesting pivotal roles of cytokines in the development of PH in MCT-injected rats. These findings observed in MCT-induced rat PH, which mimics human PH to some degree, will be helpful in development of a treatment strategy for human PH.
Acknowledgment: This work was supported by a grant from the foundation on Rheumatism Research Committee, Ministry of Health and Welfare, Japan and a grant from the Mixed Connective Tissue Disease Research Committee of the Ministry of Health and Welfare, Japan.
We thank Kyoko Onuma for excellent technical assistance in measuring cytokines.
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