Chung-Hsin Ts'ao

Monocrotaline-Induced Pulmonary Fibrosis in Rats: Amelioration by Captopril and Penicillamine

Abstract The purpose of this study was to determine whether Captopril (an angiotensin converting enzyme inhibitor) or D-penicillamine (an inhibitor of collagen crosslinking) can ameliorate pulmonary fibrosis induced by the plant alkaloid monocrotaline. Rats were randomly assigned to one of six treatment groups: (1) control; (2) Captopril, 60 mg/kg/day, p.o.; (3) D- penicillamine, 30 mg/kg/day, p.o.; (4) monocrotaline, 2.4 mg/kg/day, p.o.; (5) monocrotaline plus Captopril, as above; (6) monocrotaline plus penicillamine, as above; and were killed after 6 weeks of continuous drug administration. Monocrotaline-treated rats exhibited several anatomic correlates of pulmonary hypertension, including cardiomegaly, right heart enlargement, and muscularization of the pulmonary arteries and arterioles. These monocrotaline reactions were accompanied by decreased lung activities of angiotensin converting enzyme (ACE) and plasminogen activator (PLA), indicative of endothelial dysfunction; and by increased lung hydroxyproline concentration, indicative of interstitial fibrosis. The presence of interstitial fibrosis was confirmed by electron microscopy. When given concomitantly with monocrotaline, both Captopril and penicillamine partially prevented the cardiomegaly, right heart enlargement, and vascular muscularization. Both agents also diminished the decreased lung PLA activity and increased hydroxyproline concentration observed in monocrotaline-treated animals. Neither modifying agent influenced the monocrotaline-induced decrease in lung ACE activity. Compared with control rats, the rats receiving Captopril alone exhibited decreased heart weight and increased serum ACE activity, and animals receiving penicillamine alone did not differ significantly from control animals for any of the endpoints studied. These data demonstrate that Captopril and penicillamine ameliorate monocrotaline-induced pulmonary fibrosis in rats. Penicillamine, known to inhibit radiation-induced lung injury, thus is shown to be effective in a second model of pulmonary fibrosis. Perhaps more importantly, the hydroxyproline data demonstrate that the ACE inhibitor Captropril exhibits antifibrotic activity in monocrotaline-treated rat lung.

Monocrotaline-induced cardiopulmonary damage in rats: amelioration by the angiotensin-converting enzyme inhibitor CL242817

Abstract Pulmonary injury induced by the plant alkaloid monocrotaline is partially prevented by the angiotensin-converting enzyme (ACE) inhibitor captopril. CL242817 [(S-[R∗, S∗])-1-([3-acetylthio]-3-benzoyl-2-methyl-propionyl)-L-proline] is a new orally active ACE inhibitor under evaluation as an antihypertensive agent. To determine whether CL242817 also can modify mono-crotaline-induced pulmonary injury, male rats were divided into four groups: control; CL242817 (60 mg/kg/day, po); monocrotaline (2.4 mg/kg/day, po); or monocrotaline plus CL242817, and were sacrificed after 6 weeks of continuous treatment. Rats receiving monocrotaline alone exhibited occlusive medial thickening of the pulmonary arteries, cardiomegaly, and right ventricular hypertrophy. Electron micrographs of monocrotaline-treated lung revealed degeneration of both endothelial and Type I epithelial cells, as well as marked interstitial hypercellularity and fibrosis. Hydroxyproline (collagen) content of monocrotaline-treated lung also increased significantly, confirming the fibrosis observed in the electron micrographs. These structural changes were accompanied by decreased lung ACE and plasminogen activator (PLA) activities, indicative of pulmonary endothelial dysfunction. Concomitant CL242817 treatment ameliorated all anatomic manifestations of monocrotaline injury, particularly the right ventricular hypertrophy, pulmonary arterial occlusion, epithelial degeneration, and interstitial fibrosis. CL242817 also significantly prevented the monocrotaline-induced increase in lung hydroxyproline content. In contrast, concomitant CL242817 did not significantly influence the suppressed lung ACE and PLA activities in monocrotaline-treated rats. CL242817 alone produced retarded weight gain, decreased heart weight relative to body weight, decreased lung hydroxyproline content and ACE activity, and increased serum ACE activity and plasma All concentration. Thus CL242817 resembles captopril, both in its ability to ameliorate monocrotaline-induced pulmonary injury in rats, and in many of its side effects.

Effect of an Aortic Proteoglycan on Platelet Aggregation and Thrombin Time: Plasma Requirement and Active Moieties

Summary A highly purified bovine aortic proteoglycan (PG) prolongs thrombin-in-duced clotting and inhibits thrombin-in-duced platelet aggregation, but has no appreciable effect on platelet aggregation induced by ADP, epinephrine, or collagen. Inhibition of thrombin-induced aggregation requires the presence of plasma factors. Glycosaminoglycans (GAG) isolated from the PG contained all the antithrombic activity. Hydrolysis of GAG by treating the PG with chondroitinase abolishes the activity. The antithrombic action of the PG was compared with that of a commercial heparin. Twice as much protamine sulfate was required to neutralize the antithrombic effect of the PG as to neutralize that of heparin. The location of the PG in the arterial wall and its antithrombic activity suggest it plays a role in regulating the response of the circulating blood to vascular injury. We thank Miss Cassandra Smith and Miss Rose Lo for excellent technical assistance. Miss Ellen Rohde typed the manuscript.

FUNCTIONAL RESPONSES OF THE PULMONARY ENDOTHELIUM TO THORACIC IRRADIATION IN RATS: DIFFERENTIAL MODIFICATION BY D-PENICILLAMINE

Male rats were sacrificed 2 or 6 months after a range of single doses of gamma rays (0-30 Gy) to the right hemithorax. Half of each dose group consumed control feed continuously after irradiation, and half consumed feed containing the collagen antagonist D-penicillamine (10 mg/rat/day). Four markers of pulmonary endothelial function were monitored: angiotensin converting enzyme (ACE) activity, plasminogen activator (PLA) activity, and prostacyclin (PGI2) and thromboxane (TXA2) production. Bronchoalveolar lavage (BAL) fluid also was obtained from the right lung, and was analyzed for macrophage number, and PGI2 and TXA2 concentration. Right lung ACE and PLA activities decreased linearly with increasing dose at both 2 and 6 months postirradiation, and penicillamine had no significant effect on either response. In contrast, PGI2 and TXA2 production by the right lung increased linearly with increasing radiation dose at both autopsy times. Penicillamine significantly ameliorated the increase in PGI2 production at 2 months, and the increase in TXA2 production at both 2 and 6 months postirradiation. Penicillamine dose-reduction factors (DRF) for PGI2 and TXA2 production were 1.3-1.4, and the response curve slope ratios were 1.7-2.5 (p less than 0.05). Penicillamine also ameliorated the dose-dependent increase in TXA2 concentration in the BAL fluid at 2 months. These data indicate that the four markers of radiation-induced pulmonary endothelial dysfunction do not respond identically to penicillamine dose-modification. Of the four markers, TXA2 production exhibits the most significant and widespread penicillamine sparing. TXA2 is a potent vasoconstrictor, promoter of platelet aggregation, and mediator of inflammation, and partial prevention of the radiation-induced hyperproduction of this eicosanoid may account in part for penicillamines therapeutic action in this model.

Monocrotaline-Induced Cardiopulmonary Injury in Rats: Modification by Thiol and Nonthiol Ace Inhibitors

The pyrrolizidine alkaloid monocrotaline produces pulmonary hypertension by an as yet unknown mechanism (1). The possibility that inappropriate angiotensin converting enzyme (ACE) activity may contribute to monocrotaline-induced lung injury led to this study of ACE inhibitors as modifying agents in monocrotaline-treated rats.

Stimulation of Radiation-Impaired Plasminogen Activator Release by Phorbol Ester in Aortic Endothelial Cells

Abstract Ionizing radiation has been reported to affect the fibrinolytic activity of exposed tissue. With cultured bovine aortic endothelial cells, radiation suppresses the release of plasminogen activator to the conditioned media, with a concomitant increase in intracellular plasminogen activator. Thus study was undertaken to determine whether radiation-impaired plasminogen activator release can be modified by phorbol ester. We exposed cultured bovine aortic endothelial cells to a sterilizing dose of 10 Gy of γ-rays and found the treatment led to cell injury, as evidenced by an increased release of prelabeled chromium, and to a reduction of plasminogen activator in the conditioned media with elevated intracellular plasminogen activator in irradiated cells. Phorbol ester enhanced plasminogen activator activity in both sham-irradiated and irradiated endothelial cells. It was interesting to note that the increased plasminogen activator in phorbol ester-stimulated sham-irradiated cells was largely retained inside the cell, while it was released to the conditioned media in irradiated cells. Apparently, altered plasminogen activator activity of radiation-sterilized endothelial cells can be modified by exogenous stimuli.

Potentiation of Platelet Aggregation by Heat-Precipitated Plasma Proteins

Summary Aggregation of human PRP by low concentrations of ADP, epinephrine or collagen, and of human WPS by collagen, was markedly potentiated if the PRP or WPS was premixed with small amounts of heated (56°, 30 min) normal plasma, thrombin-clotted plasma, hemophilic or von Willebrand plasma. Heated plasma samples by themselves did not induce platelet aggregation. The platelet aggregation potentiating activity of these samples resides with the heat-precipitated proteins. Neither serum nor a saline solution of fibrinogen was effective. However, the serum solution of fibrinogen was effective after heating. The inhibitory effect of aspirin was completely counteracted by small quantities of heated plasma in the reaction mixture. The effect of heat-precipitated plasma proteins appears to be on the platelet release mechanism, making it highly sensitive to minute amounts of aggregants.

Evaluation of a thrombin-sensitive fluorogenic substrate for heparin concentration in plasma

Heparin levels in 76 heparinized patient plasma samples were determined with a thrombin-sensitive fluorogenic substrate. Results were compared with heparin values derived from an activated partial thromboplastin time test. The correlation of heparin concentrations obtained by these two methods was poor (r = 0.56). The amidolytic assay gave higher heparin values than the clotting method. The discrepancy is attributed to factors inherent to the methodology of these assay systems.

Combined effect of prostaglandins and an aortic proteoglycan on platelet aggregation and plasma clotting

The individual and combined effects of PGD2, PGI2 and an aortic proteoglycan on human platelet aggregation and plasma clotting were studied. PGI2 was at least 10 times more potent than PGD2 in inhibiting platelet aggregation. Small doses of prostaglandins inhibited ADP- and thrombin-induced aggregation, but only prolonged aggregation time without affecting the extent of arachidonic acid (AA)-induced aggregation. Small doses of prostaglandins did not affect thrombin-induced clotting of PRP. Large doses of prostaglandins abolished platelet aggregation and prolonged the onset of thrombin-induced clotting. The aortic proteoglycan (APG) had no appreciable effect on ADP- or AA-induced aggregation. Small doses of APG abolished thrombin-induced clotting, while large doses of APG suppressed both clotting and aggregation induced by thrombin. PGI2 and PGD2 showed additive inhibition of platelet aggregation regardless how the aggregation was induced. APG and prostaglandins showed additive inhibition of only thrombin-induced aggregation. APG, but not any of the prostaglandins, prolonged clotting time of PPP. This prolongation was not potentiated by PGI2 or PGD2.

Annexin I concentration and prostacyclin production in rat lung and alveolar macrophages following irradiation

The purpose of this study was to gather additional evidence in irradiated rat lung on the relationship between annexin I and prostaglandin synthesis. The right hemithorax of the animal was exposed to a single dose of 0 or 30 Gy of X-rays, and the animals were killed 3 months postirradiation. Levels of annexin I and synthesis of prostacyclin (PGI2) were determined in lungs, in cell-free bronchoalveolar lavage (BAL) fluid, and in macrophages lavaged from those lungs. In addition, protein concentration, lactate dehydrogenase (LDH) activity and macrophage count in BAL fluid obtained from irradiated lung were compared with that from sham-irradiated (0 Gy) lung. Levels of annexin I, the putative inhibitor of phospholipase A2, in lung and cell-free BAL fluid were decreased in samples from irradiated animals. By contrast, the level of annexin I in macrophages lavaged from irradiated lung was higher than that in macrophages from sham-irradiated lung. The irradiated lung produced nearly 3.5 times more prostacyclin than did the control lung. However, prostacyclin synthesis by macrophages lavaged from irradiated lung was no different than that of macrophages from sham-irradiated lung. Protein, LDH and macrophage number in BAL fluid from irradiated lungs were significantly higher than in corresponding specimens from sham-irradiated lungs. These data demonstrate that reduced levels of annexin I, as well as increased protein concentration, LDH activity and macrophage numbers in irradiated rat lung are reflected in BAL fluid. Therefore, information obtained from BAL fluid, but not from BAL macrophages, reflects lung status, and may serve as a minimally invasive index of radiation pneumonitis in this model. In irradiated lung, increased PGI2 synthesis coupled with a decreased annexin I level are consistent with the hypothesis of an inhibitory role of annexin I in prostaglandin metabolism. However, this hypothesis is not supported by findings in BAL macrophages, where increased annexin I concentration is not accompanied by a decrease in PGI2 production. In view of the latter findings, and a previous study from our laboratory demonstrating that phospholipase activity in irradiated rat lung is in fact decreased, despite the reduction in annexin I concentration and the hyperproduction of prostanoids, it would seem unlikely that annexin I negatively modulates prostaglandin synthesis via inhibition of phospholipase in this model.