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Defibrotide decreases cholesterol amount in hypercholesterolemic rabbit aorta, with no modification of plasma or lipoprotein cholesterol
Life Sciences, Vol. 44, pp. Printed in the U.S.A.
Pergamon
789-797
Press
DEFIBROTIDE DECREASES CHOLESTEROL AMOUNT IN HYPERCHOLESTEROLEMIC RABBIT AORTA, WITH NO MODIFICATION OF PLASMA OR LIPOPROTEIN CHOLESTEROL Rodolfo Pescador, Roberto Porta, Angelo Conz*, Marisa Mantovani and Giuseppe Prino Crinos Biological Research Laboratories, Piazza XX Settembre 2, 22079 Villa Guardia (Comol, Italy and Istituto di Ricerche Biomediche Antoine Marxer*, Strada per Castellamonte, 10010 Colleretto Giacosa (Torino), Italy. (Received
in final
form
January
25,
1989)
Summary Defibrotide, (D) an antithrombotic agent, when administered i.v. to cholesterol-fed rabbits decreased cholesterol in the aorta without changing total plasma cholesterol, triglyceride or phospholipid, nor the cholesterol, triglyceride, phospholipid and protein of plasma lipoproteins. Platelet aggregation was decreased in rabbits treated with D. There were fewer vascular lesions in the hearts and kidneys of animals treated with D than in animals fed cholesterol treated the and with placebo. These data suggest that antithrombotic activity of D and its ability to reduce platelet sensitivity could help to reduce the amount of cholesterol in the cardiovascular system in atherosclerosis-prone situations. Defibrotide (D) is single-strand polydeoxyribonucleotides extracted from mammalian lungs by controlled depolymerization (1). The antithrombotic activity of D has been demonstrated in a variety of experimental models: venous thrombosis induced in the rabbit and in the dog by a collagen-coated nylon thread (1,2), arterial thrombosis induced in the rat and in the dog by electrical stimulation (1,31, venular thrombosis in the hamster cheek pouch caused by iontophoretic application of ADP (11, venous thrombosis induced in the rat by stasis (21. The antithrombotic activity of D has also been demonstrated in a typical local thrombonecrotic hemorrhagic lesion caused in the rabbit by endotoxin (Shwartzman reaction) (4). Hypercholesterolemia is a relevant situation associated with intravascular platelet activation (5,6) and a thrombosis tendency as a result of damage to endothelium (7,81. Platelets are involved both in atherosclerosis and in thrombosis in a chicken and egg vicious circle (91. In the present study we have investigated the effect of chronic i.v. D treatment on a model of hypercholesterolemia in the rabbit and present data suggesting that Defibrotide might reduce the severity of atherosclerosis in this model.
0024-3205189 Copyright
$3.00 + .Oo
(c) 1989 Pergamon
Press
plc
790
Defibrotide Decreases Aorta Cholesterol
Vol. 44, No. 12, 1989
Materials and Methods Male, white New Zealand rabbits (Allevamento de1 Verbano, Arona, Italy) weighing 2.70 + 0.028 Kg at the beginning of the experiment were used throughout. The-animals (45 rabbits) were divided randomly into 2 groups: A and [B + Cl (30 rabbits). Group A was fed a standard pellet diet ;;:,;;;l$fi1 Type MSK, Rieper, Bolzano,Italy) while group [B + C] was fed the same diet ~1;s 0.5% cholesterol for 2 weeks (the diet containing cholesterol was prepared by the manufacturer, mixing cholesterol with other diet components before pelleting). After this time, after overnight fasting, blood was collected with heparin as anticoagulant, centrifuged at 3500 g for 15' at 4" C and plasma lipids were determined. The group [B + Cl was further divided into 2 groups B and C, with mean cholesterol plasma levels not statistically different. Treatment was begun at this point (group A, placebo (sterile saline), i.v., once a day; group B, placebo (sterile saline), i.v., once a day, group C, D 128 mg/kg, i.v., once a day) and was carried on until the end of the experiment (1.5 months later). The diet containing cholesterol was fed for one month. Thereafter, groups B and C were again fed with the standard pellet diet, because the plasma cholesterol levels in human lipidemias are not so high as those of rabbits fed a cholesterol diet (Type I, 324 + 57 mg/dl; Type II, 354 + 4; Type III, 441 + 54; Type IV, 251 + 4; Type V, 373 + 19) (10). The rabbits were weighed regularly until the endof the experiment. Plasma cholesterol, triglyceride and phospholipid were determined at the beginning of the experiment, after 2 weeks and after 1 month of cholesterol feeding and at the end of the experiment. The blood was collected after an overnight fasting with heparin as anticoagulant and centrifuged at 3500 g for 15' at 4" C to obtain plasma. Cholesterol, triglyceride and phospholipid were determined with the Boehringer Biochemia Kits no 125512, 240052 and 691844. Plasma lipoproteins were separated as previously described (11) after one month of cholesterol feeding, i.e., when plasma lipids were at the peak. Cholesterol, triglyceride, phospholipid and protein of lipoproteins were assayed as previously described (11). Platelet aggregation was carried out with platelet rich plasma. The platelets were tested with various ADP concentrations to find the 50% effective concentration (EC50), that is, the concentration of ADP able to induce half of the maximum aggregatory response. 10 ADP concentrations were used, ranging from 0.0976 !JM to 3.23 PM. The aortas were removed, cleaned (adventitia and related adipose tissue were completely dissected off the vessel), opened longitudinally and grossly examined for atherosclerotic lesions. Only tiny lesions were observed in cholesterol fed animals. Moderate or severe lesions were absent. For that reasons an estimation based on a subjective evaluation grading aortas by a score was ruled out because the subjective score appeared to be a too undiscriminating tool to assess the degree of atherosclerosis. Then the aorta cholesterol concentration was used as the primary objective expression of the extent of atherosclerotic involvement. The aortas were carefully weighed, cut into small pieces and homogenized in isopropyl alcohol in a Potter homogenizer with a glass pestle. The homogenate was centrifuged at 3500 g for 15' at 15" C. The supernatant was dried under vacuum, the residue saponified with alcoholic KOH, diluted with water and applied to an Extrelut column (Merck
Vol.
44, No. 12, 1989
Defibrotide
Decreases
Aorta
Cholesterol
791
no 11737). The sample was then eluted with petroleum ether and dried under vacuum. The residue was dissolved in a mixture of ethyl alcohol and acetone (1 : 1) containing cholesterol acetate as internal standard. The samples were analyzed by a Carlo Erba gas chromatograph (model 2457 - T) equipped with a column packed with 2% SE 30 on 80 - 100 mesh Gas Chrom P (Q) Silanized. Rabbits were anesthetized with 30 mg/Kg pentobarbital sodium, i.v., and killed by exsanguination. Hearts of all the animals were sliced horizontally from the apex to the base. A cross-sectional slice including both right and left ventricle was fixed in 10% neutral (pH 7.0) buffered formalin, embedded in paraffin, cut 4 pm thick and stained with hematoxylin and eosin (HE). A cross-sectional slice of kidneys was processed as described in the instance of hearts. Data were analyzed by ANOVA.
Results There were no differences in body weight gains between the 3 groups of animals (Fig. 1). Table I lists the plasma levels of total cholesterol, phospholipid and triglyceride, evaluated at different times during the experiment. The cholesterol levels of Groups B and C were not significantly different after 2 weeks of cholesterol feeding, after 1 month and at the end of the experiment. The lipids and protein of plasma lipoproteins of groups B and C were not significantly different after one month of cholesterol feeding (Table II). The weights of kidneys and hearts were not affected by cholesterol feeding and/or treatment (Table III). Cholesterol feeding increased liver weights but no difference was observed between group B and C (Table III). Cholesterol feeding increased aorta cholesterol 7 fold. D treatment reduced aorta cholesterol by 48.6% (P <0.05) (Table IV). Cholesterol feeding decreased the EC50 for platelet aggregation of ADP by 32% (PC 0.05 versus control group A); the treatment with D brought back the EC50 toward the control value (N.S. versus group A) (Table V). In the hearts of rabbits, cholesterol feeding caused vascular lesions of, severity from slight to moderate, consisting of thickening of the intima and oedema, in 9 of 15 animals (60%) (Group B). Treatment with D (Group C) reduced the number of affected animals: 3 of 15 (20%). Just one animal (7%) of group A showed a focal thickening of the vascular intima, with no oedema. It must be stressed that the increase of vascular lesions, in group B, as compared to groups A and C, seemed to intensify slight inflammatory and/or degenerative phenomena in the hearts of rabbits. Thickening and oedema of the intima was found in renal arteries of group B (54%) and in group C (14%) animals. Figure 2 shows, for example, 1 heart histological sample from each of groups A, B and C. Sample A was from the worst of the control animals (group A). Figure 3 shows,for example,1 kidney histological sample from each of groups A, B and C.
Discussion Data summarized in Figure 1 and Table III
demonstrated
that
treatment
with
792
Defibrotide Decreases Aorta Cholesterol
Vol. 44, No. 12, 1989
:I:. 0
12
3
5 4 Weeks
6
7
6
FIG. 1 Time-course of Body Weight Gains of rabbits at various times during the experiment. o animals fed the Standard Diet and treated with Placebo, i.v., (group A); a,animals fed the Cholesterol Diet and treated with Placebo, i.v., (group B); q , animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day (group C). the drug was not debilitating ruling out the possibility of fewer lesions. The treatment with D decreased the amount of cholesterol in the aortic wall of rabbits fed with cholesterol by 48.6% , with no modification of total plasma cholesterol or cholesterol of plasma lipoproteins. The treatment with D also decreased the frequency of heart and kidney vascular lesions in rabbits fed with cholesterol as compared to rabbits treated with placebo and fed with cholesterol. The focal lesions shown in figure 3 (panel B and C) were considered intima modifications rather than organized thrombi. In fact in some areas the endothelium present on the surface of these lesions appears to lie directly on the internal elastic lamina (12). A modified response-to-injury hypothesis of atherosclerosis suggests that one pathway, demonstrated in hypercholesterolemia, involves monocyte and platelet interactions which may stimulate fibrous plaque formation (13). Platelet activity in patients with familial hypercholesterolemia is elevated (14) and pigs homozygous for von Willebrand disease (impaired platelet function) are resistant to the initiation and progression of atherosclerotic disease (15). Cholesterol accumulates in vascular smooth muscle cells incorporated into platelet rich plasma clots (16) while the sterol does not accumulate when smooth muscle cells are incorporated into poor platelet plasma clots (16). Treatment with D shifted the EC for platelet aggregation of ADP in cholesterol-fed rabbits toward normal $!lues. It must be stressed that platelet aggregation was evaluated 24 h after the last administration of D. D
Defibrotide Decreases Aorta Cholesterol
Vol. 44, No. 12, 1989
793
TABLE I Plasma Lipids of rabbits at various Times during the experiment. A,animals fed the Standard Diet and treated with Placebo i.v.; B,animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day.
A
BASAL VALUES B
AFTER 2 WEEKS OF CHOLESTEROL FEEDING A B c-
C
CHOLESTEROL (mg/dl) 48.172 2.36
47.61+ 5.22-A
47.20+ 4.33-A
37.12+ 2.42-
661.91+ 60.85-
577.28+ 75.91-o
43.12+ 7.14-
42.96+ 5.80-e
TRIGLYCERIDE (mg/dl) 47.69+ 2.28-
49.73+ 2.72-A
53.42+ 9.39-A
49.88+ 4.13PHOSPHOLIPID (mg/dl)
82.12+ 3.18-
87.34+ 5.74-A
83.48+ 4.97-A
72.31+ 2.89-
185.20+ 31.50-
182.94+ 26.215
AT THE END OF EXPERIMENT A B C
AFTER 1 MONTH OF CHOLESTEROL FEEDING A B C CHOLESTEROL (mg/dl) 43.00+ 6.60_
965.30+ 184.59-
987.162 128.65 .
31.82+ 1.77~-
213.14+ 46.27-
180.00+ 41.11-0
82.722 36.30
48.82~ 11.62 l
134.69+ 22.92-
117.22+ 19.64-o
TRIGLYCERIDE (mg/dlI 35.54+ 2.59-
71.85+ 27.53-
54.99+ 11.96-e
35.03+ 2.94PHOSPHOLIPID (mg/dll
64.85+ 4.16-
321.47~ 58.87
317.06+ 30.58-e
A N.S. vs. group A
58.40+ 3.44-
l N.S. vs. group B
was shown to have a deaggregating activity on blood-superfused Achilles Tendon (1) and the ability to inhibit leukocyte participation to the thrombotic process (171. Studies by other investigators have suggested that hyperlipemia may increase thrombogenicity as a result of damage to the endothelium (7,8). The damage to the endothelium could, in turn, be the substrate for the
6.55+ 2.31-
6.48+ 1.22-
23.612 1.82
0.20+ 0.04-
IDL
LDL
HDL
d> 1.21
987.16+ 128.65-o 35.54+ 2.59-
5.13+ 0.28-
1.14+ 0.64;
0.76+ 0.43-
14.73+ 0.72-
40.46+ 1.49-0
4.05+ 0.32-
4.44+ 0.82-
39.49t 2.28-
155.22+ 19.58%
153.03t 39.91-
7.68+ 1.92-
71.36+ 14.49-o
536.24+ 84.663
516.81+ 106.08_
A
71.85+ 27.53-
6.36+ 1.54-
16.84+ 5.23-
5.27+ 3.06-
12.76+ 8.18_
31.73t 14.66-
B
TRIGLYCERIDE
55.51+ 12.54-
C
B
TOTAL PLAS- 40.85+ 965.30+ 184.59MA 5.04l N.S. vs. group B.
2.692 0.61
VLDL
A
CHOLESTEROL C
54.99t 11.96-a
7.48+ 1.75-a
15.01+ 3.42%
4.28+ 1.26-o
7.74+ 1.82-e
21.84+ 5.78-e
L I P I D S (mg/dl)
321.47t 58.87-
15.69+ 1.66_
11.61+ 0.56-
64.85+ 4.16-
36.88+ 3.06-
25.67+ 5.26-
58.05+ 17.83-
138.72+ 27.92-
B
45.53+ 3.19-
4.45+ 0.84-
3.96+ 1.37-
2.39t 0.58-
A
PHOSPHOLIPID
317.06+ 30.58-o
15.56+ 1.19.
36.29+ 2.41-iD
29.60+ 6.06%
54.16+ 7.37%
137.64+ 20.57-e
C
78.2+ 10.0-
77.9+ 9.8-m
34.9+ 7.1-o
54.9+ 7.5.
87.72 12.2.
C
5794.8t 6460.0+ 6878.2+ 497.7- 105.4- 353.5-e
4721.5+ 5542.5+ 5619.4+ 60.7- 171.0- 199.6-o
80.1+ 8.1-
29.6+ 6.8-
59.2+ 17.5-
4.6+ 1.26.6+ 0.9-
90.42 18.1
B
2.6+ 0.6-
A
P R 0 T E I N (mg/dll
Composition of Plasma Lipoproteins of rabbits after One Month of Cholesterol Feeding and after Two Weeks of treatment with Placebo or Oefibrotide. A, animals fed the Standard Oiet and treated with Placebo, i.v.; B, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day.
TABLE II
Vol. 44, No. 12, 1989
Defibrotide Decreases Aorta Cholesterol
795
TABLE III
Weights of Kidneys, Livers and Hearts of rabbits at the end of experiment. A, animals fed the Standard Diet and treated with Placebo, i.v.; B, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day. Group A B C A N.S. vs. group A
Liver(g)
Kidneys(g) 17.3 + 0.4 18.0 7 0.9 A 17.9 7 0.6 A IJ P <
88.0 + 3.0 122.7 7 9.5 0 131.2 + - 7.0 q
Heart(g) 8.0 + 0.2 7.8 7 0.2 A 8.2 7 - 0.3 A
0.01 vs. group A
TABLE IV
Cholesterol Levels in rabbit Aortas. A, animals fed the Standard Diet and treated with Placebo, i.v.; B, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day. Group
Cholesterol (mg/g wet tissue)
A B C
1.42 + 0.05 10.00 + 2.10 5.14 z 0.82 (-48.6%) 0
l P< 0.05 vs. group B
TABLE V
Platelet Aggregation: 50% effective ADP concentration (EC50). A, rabbits fed the Standard Diet and treated with Placebo, i.v.; 8, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day. Group A B C A N.S. vs. group A.
AD!' EC50 (FM) 0.273 + 0.021 0.187 T 0.015 (-32%) 0 0.248 z 0.031 (- 9%)A 0 P < 0.05 vs. group A.
FIG.
2
(the worst animal in the control group). B, Kidney Artery of Cholesterol-fed rabbit treated with Placebo, showing thickening of intima and oedema. C, Kidney Artery of Cholesterol-fed rabbit treated with 128 mg/Kg Defibrotide, i.v. once a day, showing moderate intimal thickening and oedema. H.E. x 250.
A Kidney Artery of Control rabbit (fed the standard diet and treated with placebo), with slight intimal thickening
A Coronary Artery of Control rabbit (fed the standard diet and treated with placebo), with slight intimal thickening (the worst animal in the control group). B, Coronary Artery of Cholesterol-fed rabbit treated with Placebo, showing thickening of intima and oedema. C, Coronary Artery of Cholesterol-fed rabbit i.v. once a day, showing moderate intimal thickening and oedema. H.E. x 250.
Vol.
44, No.
12, 1989
Defibrotide
Decreases
Aorta
Cholesterol
797
lead to eventually thrombi which could mural formation of focal atherosclerosis (18,191. Whatever the situation, the ability of D to decrease platelet reactivity and its antithrombotic activities could be helpful in decreasing the amount of cholesterol in the cardiovascular system in atherosclerosis-prone situations.
Acknowledgements We are indebted for technical assistance to Miss Tiziana Romano, Mr. Giovanni Terzaghi and Mr. Giorgio Meroni.
References 1. R. NIADA, M. MANTOVANI, G. PRINO, R. PESCADOR, F. BERTI, C. OMINI and G.C. FOLCO, Thromb. Res. 23 233-246 (19811. 2. R. NIADA, R. TETTAMAEI, P. ALBERICO and M. MANTOVANI, Societa Italiana di Biologia Sperimentale, Societa Italiana di Fisiologia, Societa Italiana di Nutrizione Umana. Riunione Congiunta (Napoli, Italia, Settembre 19861. Riassunto n. 50, pag. 89. 3. 0. N. ULUTIN, H. TUNALI, M.S. UGUR, S. AYTIS, T. ERBENGI and S. BALKUV-ULUTIN, Haemostasis 16 (suppl 11 9-12 (1986). R. PESCADOR, R. PORTA, F. BERTI, G.C. 4. R. NIADA, M. MANTOVANI, G.PRINO, FOLCO, C. OMINI and T. VIGANO’, Pharmacol. Res. Commun., -14 949-957 (1982). 5. K.K. WU, M.L. ARMSTRONG, J.C. HOAK and M.B. MEGAN, Thromb. Res. -7 917-924 (1975). 6. R.D. MACA and J.C. HOAK, Lab. Invest. 30 589-595 (1974). 7. M.L. ARMSTRONG, R.E. PETERSON, J.C. HOE, M.B. MEGAN, F.H. CHENG and W.R. CLARKE, Atherosclerosis 36 89-100 (1980). and B. CARLANDER, Scand.J.Clin.Lab.Invest. -39 8. T. HENRIKSEN, S.A. EVEN= 361-368 (1979). 9. S. MOORE, Atherosclerosis vol. 454 p. 146, The New York Academy of Sciences, New York, New York (1985). 10. E.J. SCHAEFER and R.I. LEVY, N. Engl. J. Med. -312 1300-1310 (1985). 11. R. PESCADOR, Life Sciences 32 625-633 (1983). 12. C. GOPINATH, D.E. PRENTICE and D.J. LEWIS, Atlas of Experimental Toxicological Pathology, p. 18-20, MTP PRESS LIMITED, LANCASTER/BOSTON/THE HAGuEIDORDRECHT (19871. 13. R. ROSS, N. Engl. J. Med. -314 488-500 (1986). 14. M. AVIRAM and J.G. BROOK, Thromb. Res. 26 101-109 (1982). 15. W. FUSTER, E.J. BOWIE, J.C. LEWIS, D.N.FASS, C.A. OWEN and A.L. BROWN, J. Clin. Invest. 61 722-730 (1978). 16. H.S. KRUTH, Lab. Invest. 53 634-638 (1985). 17. G. FUMAGALLI, E. ANGELACCX, N. LOMBARD0 and F. CLEMENTI, Haemostasis -17 361-370 (1987). 18. S.K. PENG, C.B. TAYLOR, J.C. HILL and R.J. MORIN, Atherosclerosis -54 121-133 (1985). 19. Editorial, Lancet I791-793 (1985).
Pergamon
789-797
Press
DEFIBROTIDE DECREASES CHOLESTEROL AMOUNT IN HYPERCHOLESTEROLEMIC RABBIT AORTA, WITH NO MODIFICATION OF PLASMA OR LIPOPROTEIN CHOLESTEROL Rodolfo Pescador, Roberto Porta, Angelo Conz*, Marisa Mantovani and Giuseppe Prino Crinos Biological Research Laboratories, Piazza XX Settembre 2, 22079 Villa Guardia (Comol, Italy and Istituto di Ricerche Biomediche Antoine Marxer*, Strada per Castellamonte, 10010 Colleretto Giacosa (Torino), Italy. (Received
in final
form
January
25,
1989)
Summary Defibrotide, (D) an antithrombotic agent, when administered i.v. to cholesterol-fed rabbits decreased cholesterol in the aorta without changing total plasma cholesterol, triglyceride or phospholipid, nor the cholesterol, triglyceride, phospholipid and protein of plasma lipoproteins. Platelet aggregation was decreased in rabbits treated with D. There were fewer vascular lesions in the hearts and kidneys of animals treated with D than in animals fed cholesterol treated the and with placebo. These data suggest that antithrombotic activity of D and its ability to reduce platelet sensitivity could help to reduce the amount of cholesterol in the cardiovascular system in atherosclerosis-prone situations. Defibrotide (D) is single-strand polydeoxyribonucleotides extracted from mammalian lungs by controlled depolymerization (1). The antithrombotic activity of D has been demonstrated in a variety of experimental models: venous thrombosis induced in the rabbit and in the dog by a collagen-coated nylon thread (1,2), arterial thrombosis induced in the rat and in the dog by electrical stimulation (1,31, venular thrombosis in the hamster cheek pouch caused by iontophoretic application of ADP (11, venous thrombosis induced in the rat by stasis (21. The antithrombotic activity of D has also been demonstrated in a typical local thrombonecrotic hemorrhagic lesion caused in the rabbit by endotoxin (Shwartzman reaction) (4). Hypercholesterolemia is a relevant situation associated with intravascular platelet activation (5,6) and a thrombosis tendency as a result of damage to endothelium (7,81. Platelets are involved both in atherosclerosis and in thrombosis in a chicken and egg vicious circle (91. In the present study we have investigated the effect of chronic i.v. D treatment on a model of hypercholesterolemia in the rabbit and present data suggesting that Defibrotide might reduce the severity of atherosclerosis in this model.
0024-3205189 Copyright
$3.00 + .Oo
(c) 1989 Pergamon
Press
plc
790
Defibrotide Decreases Aorta Cholesterol
Vol. 44, No. 12, 1989
Materials and Methods Male, white New Zealand rabbits (Allevamento de1 Verbano, Arona, Italy) weighing 2.70 + 0.028 Kg at the beginning of the experiment were used throughout. The-animals (45 rabbits) were divided randomly into 2 groups: A and [B + Cl (30 rabbits). Group A was fed a standard pellet diet ;;:,;;;l$fi1 Type MSK, Rieper, Bolzano,Italy) while group [B + C] was fed the same diet ~1;s 0.5% cholesterol for 2 weeks (the diet containing cholesterol was prepared by the manufacturer, mixing cholesterol with other diet components before pelleting). After this time, after overnight fasting, blood was collected with heparin as anticoagulant, centrifuged at 3500 g for 15' at 4" C and plasma lipids were determined. The group [B + Cl was further divided into 2 groups B and C, with mean cholesterol plasma levels not statistically different. Treatment was begun at this point (group A, placebo (sterile saline), i.v., once a day; group B, placebo (sterile saline), i.v., once a day, group C, D 128 mg/kg, i.v., once a day) and was carried on until the end of the experiment (1.5 months later). The diet containing cholesterol was fed for one month. Thereafter, groups B and C were again fed with the standard pellet diet, because the plasma cholesterol levels in human lipidemias are not so high as those of rabbits fed a cholesterol diet (Type I, 324 + 57 mg/dl; Type II, 354 + 4; Type III, 441 + 54; Type IV, 251 + 4; Type V, 373 + 19) (10). The rabbits were weighed regularly until the endof the experiment. Plasma cholesterol, triglyceride and phospholipid were determined at the beginning of the experiment, after 2 weeks and after 1 month of cholesterol feeding and at the end of the experiment. The blood was collected after an overnight fasting with heparin as anticoagulant and centrifuged at 3500 g for 15' at 4" C to obtain plasma. Cholesterol, triglyceride and phospholipid were determined with the Boehringer Biochemia Kits no 125512, 240052 and 691844. Plasma lipoproteins were separated as previously described (11) after one month of cholesterol feeding, i.e., when plasma lipids were at the peak. Cholesterol, triglyceride, phospholipid and protein of lipoproteins were assayed as previously described (11). Platelet aggregation was carried out with platelet rich plasma. The platelets were tested with various ADP concentrations to find the 50% effective concentration (EC50), that is, the concentration of ADP able to induce half of the maximum aggregatory response. 10 ADP concentrations were used, ranging from 0.0976 !JM to 3.23 PM. The aortas were removed, cleaned (adventitia and related adipose tissue were completely dissected off the vessel), opened longitudinally and grossly examined for atherosclerotic lesions. Only tiny lesions were observed in cholesterol fed animals. Moderate or severe lesions were absent. For that reasons an estimation based on a subjective evaluation grading aortas by a score was ruled out because the subjective score appeared to be a too undiscriminating tool to assess the degree of atherosclerosis. Then the aorta cholesterol concentration was used as the primary objective expression of the extent of atherosclerotic involvement. The aortas were carefully weighed, cut into small pieces and homogenized in isopropyl alcohol in a Potter homogenizer with a glass pestle. The homogenate was centrifuged at 3500 g for 15' at 15" C. The supernatant was dried under vacuum, the residue saponified with alcoholic KOH, diluted with water and applied to an Extrelut column (Merck
Vol.
44, No. 12, 1989
Defibrotide
Decreases
Aorta
Cholesterol
791
no 11737). The sample was then eluted with petroleum ether and dried under vacuum. The residue was dissolved in a mixture of ethyl alcohol and acetone (1 : 1) containing cholesterol acetate as internal standard. The samples were analyzed by a Carlo Erba gas chromatograph (model 2457 - T) equipped with a column packed with 2% SE 30 on 80 - 100 mesh Gas Chrom P (Q) Silanized. Rabbits were anesthetized with 30 mg/Kg pentobarbital sodium, i.v., and killed by exsanguination. Hearts of all the animals were sliced horizontally from the apex to the base. A cross-sectional slice including both right and left ventricle was fixed in 10% neutral (pH 7.0) buffered formalin, embedded in paraffin, cut 4 pm thick and stained with hematoxylin and eosin (HE). A cross-sectional slice of kidneys was processed as described in the instance of hearts. Data were analyzed by ANOVA.
Results There were no differences in body weight gains between the 3 groups of animals (Fig. 1). Table I lists the plasma levels of total cholesterol, phospholipid and triglyceride, evaluated at different times during the experiment. The cholesterol levels of Groups B and C were not significantly different after 2 weeks of cholesterol feeding, after 1 month and at the end of the experiment. The lipids and protein of plasma lipoproteins of groups B and C were not significantly different after one month of cholesterol feeding (Table II). The weights of kidneys and hearts were not affected by cholesterol feeding and/or treatment (Table III). Cholesterol feeding increased liver weights but no difference was observed between group B and C (Table III). Cholesterol feeding increased aorta cholesterol 7 fold. D treatment reduced aorta cholesterol by 48.6% (P <0.05) (Table IV). Cholesterol feeding decreased the EC50 for platelet aggregation of ADP by 32% (PC 0.05 versus control group A); the treatment with D brought back the EC50 toward the control value (N.S. versus group A) (Table V). In the hearts of rabbits, cholesterol feeding caused vascular lesions of, severity from slight to moderate, consisting of thickening of the intima and oedema, in 9 of 15 animals (60%) (Group B). Treatment with D (Group C) reduced the number of affected animals: 3 of 15 (20%). Just one animal (7%) of group A showed a focal thickening of the vascular intima, with no oedema. It must be stressed that the increase of vascular lesions, in group B, as compared to groups A and C, seemed to intensify slight inflammatory and/or degenerative phenomena in the hearts of rabbits. Thickening and oedema of the intima was found in renal arteries of group B (54%) and in group C (14%) animals. Figure 2 shows, for example, 1 heart histological sample from each of groups A, B and C. Sample A was from the worst of the control animals (group A). Figure 3 shows,for example,1 kidney histological sample from each of groups A, B and C.
Discussion Data summarized in Figure 1 and Table III
demonstrated
that
treatment
with
792
Defibrotide Decreases Aorta Cholesterol
Vol. 44, No. 12, 1989
:I:. 0
12
3
5 4 Weeks
6
7
6
FIG. 1 Time-course of Body Weight Gains of rabbits at various times during the experiment. o animals fed the Standard Diet and treated with Placebo, i.v., (group A); a,animals fed the Cholesterol Diet and treated with Placebo, i.v., (group B); q , animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day (group C). the drug was not debilitating ruling out the possibility of fewer lesions. The treatment with D decreased the amount of cholesterol in the aortic wall of rabbits fed with cholesterol by 48.6% , with no modification of total plasma cholesterol or cholesterol of plasma lipoproteins. The treatment with D also decreased the frequency of heart and kidney vascular lesions in rabbits fed with cholesterol as compared to rabbits treated with placebo and fed with cholesterol. The focal lesions shown in figure 3 (panel B and C) were considered intima modifications rather than organized thrombi. In fact in some areas the endothelium present on the surface of these lesions appears to lie directly on the internal elastic lamina (12). A modified response-to-injury hypothesis of atherosclerosis suggests that one pathway, demonstrated in hypercholesterolemia, involves monocyte and platelet interactions which may stimulate fibrous plaque formation (13). Platelet activity in patients with familial hypercholesterolemia is elevated (14) and pigs homozygous for von Willebrand disease (impaired platelet function) are resistant to the initiation and progression of atherosclerotic disease (15). Cholesterol accumulates in vascular smooth muscle cells incorporated into platelet rich plasma clots (16) while the sterol does not accumulate when smooth muscle cells are incorporated into poor platelet plasma clots (16). Treatment with D shifted the EC for platelet aggregation of ADP in cholesterol-fed rabbits toward normal $!lues. It must be stressed that platelet aggregation was evaluated 24 h after the last administration of D. D
Defibrotide Decreases Aorta Cholesterol
Vol. 44, No. 12, 1989
793
TABLE I Plasma Lipids of rabbits at various Times during the experiment. A,animals fed the Standard Diet and treated with Placebo i.v.; B,animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day.
A
BASAL VALUES B
AFTER 2 WEEKS OF CHOLESTEROL FEEDING A B c-
C
CHOLESTEROL (mg/dl) 48.172 2.36
47.61+ 5.22-A
47.20+ 4.33-A
37.12+ 2.42-
661.91+ 60.85-
577.28+ 75.91-o
43.12+ 7.14-
42.96+ 5.80-e
TRIGLYCERIDE (mg/dl) 47.69+ 2.28-
49.73+ 2.72-A
53.42+ 9.39-A
49.88+ 4.13PHOSPHOLIPID (mg/dl)
82.12+ 3.18-
87.34+ 5.74-A
83.48+ 4.97-A
72.31+ 2.89-
185.20+ 31.50-
182.94+ 26.215
AT THE END OF EXPERIMENT A B C
AFTER 1 MONTH OF CHOLESTEROL FEEDING A B C CHOLESTEROL (mg/dl) 43.00+ 6.60_
965.30+ 184.59-
987.162 128.65 .
31.82+ 1.77~-
213.14+ 46.27-
180.00+ 41.11-0
82.722 36.30
48.82~ 11.62 l
134.69+ 22.92-
117.22+ 19.64-o
TRIGLYCERIDE (mg/dlI 35.54+ 2.59-
71.85+ 27.53-
54.99+ 11.96-e
35.03+ 2.94PHOSPHOLIPID (mg/dll
64.85+ 4.16-
321.47~ 58.87
317.06+ 30.58-e
A N.S. vs. group A
58.40+ 3.44-
l N.S. vs. group B
was shown to have a deaggregating activity on blood-superfused Achilles Tendon (1) and the ability to inhibit leukocyte participation to the thrombotic process (171. Studies by other investigators have suggested that hyperlipemia may increase thrombogenicity as a result of damage to the endothelium (7,8). The damage to the endothelium could, in turn, be the substrate for the
6.55+ 2.31-
6.48+ 1.22-
23.612 1.82
0.20+ 0.04-
IDL
LDL
HDL
d> 1.21
987.16+ 128.65-o 35.54+ 2.59-
5.13+ 0.28-
1.14+ 0.64;
0.76+ 0.43-
14.73+ 0.72-
40.46+ 1.49-0
4.05+ 0.32-
4.44+ 0.82-
39.49t 2.28-
155.22+ 19.58%
153.03t 39.91-
7.68+ 1.92-
71.36+ 14.49-o
536.24+ 84.663
516.81+ 106.08_
A
71.85+ 27.53-
6.36+ 1.54-
16.84+ 5.23-
5.27+ 3.06-
12.76+ 8.18_
31.73t 14.66-
B
TRIGLYCERIDE
55.51+ 12.54-
C
B
TOTAL PLAS- 40.85+ 965.30+ 184.59MA 5.04l N.S. vs. group B.
2.692 0.61
VLDL
A
CHOLESTEROL C
54.99t 11.96-a
7.48+ 1.75-a
15.01+ 3.42%
4.28+ 1.26-o
7.74+ 1.82-e
21.84+ 5.78-e
L I P I D S (mg/dl)
321.47t 58.87-
15.69+ 1.66_
11.61+ 0.56-
64.85+ 4.16-
36.88+ 3.06-
25.67+ 5.26-
58.05+ 17.83-
138.72+ 27.92-
B
45.53+ 3.19-
4.45+ 0.84-
3.96+ 1.37-
2.39t 0.58-
A
PHOSPHOLIPID
317.06+ 30.58-o
15.56+ 1.19.
36.29+ 2.41-iD
29.60+ 6.06%
54.16+ 7.37%
137.64+ 20.57-e
C
78.2+ 10.0-
77.9+ 9.8-m
34.9+ 7.1-o
54.9+ 7.5.
87.72 12.2.
C
5794.8t 6460.0+ 6878.2+ 497.7- 105.4- 353.5-e
4721.5+ 5542.5+ 5619.4+ 60.7- 171.0- 199.6-o
80.1+ 8.1-
29.6+ 6.8-
59.2+ 17.5-
4.6+ 1.26.6+ 0.9-
90.42 18.1
B
2.6+ 0.6-
A
P R 0 T E I N (mg/dll
Composition of Plasma Lipoproteins of rabbits after One Month of Cholesterol Feeding and after Two Weeks of treatment with Placebo or Oefibrotide. A, animals fed the Standard Oiet and treated with Placebo, i.v.; B, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day.
TABLE II
Vol. 44, No. 12, 1989
Defibrotide Decreases Aorta Cholesterol
795
TABLE III
Weights of Kidneys, Livers and Hearts of rabbits at the end of experiment. A, animals fed the Standard Diet and treated with Placebo, i.v.; B, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day. Group A B C A N.S. vs. group A
Liver(g)
Kidneys(g) 17.3 + 0.4 18.0 7 0.9 A 17.9 7 0.6 A IJ P <
88.0 + 3.0 122.7 7 9.5 0 131.2 + - 7.0 q
Heart(g) 8.0 + 0.2 7.8 7 0.2 A 8.2 7 - 0.3 A
0.01 vs. group A
TABLE IV
Cholesterol Levels in rabbit Aortas. A, animals fed the Standard Diet and treated with Placebo, i.v.; B, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day. Group
Cholesterol (mg/g wet tissue)
A B C
1.42 + 0.05 10.00 + 2.10 5.14 z 0.82 (-48.6%) 0
l P< 0.05 vs. group B
TABLE V
Platelet Aggregation: 50% effective ADP concentration (EC50). A, rabbits fed the Standard Diet and treated with Placebo, i.v.; 8, animals fed the Cholesterol Diet and treated with Placebo, i.v.; C, animals fed the Cholesterol Diet and treated with 128 mg/Kg Defibrotide, i.v. once a day. Group A B C A N.S. vs. group A.
AD!' EC50 (FM) 0.273 + 0.021 0.187 T 0.015 (-32%) 0 0.248 z 0.031 (- 9%)A 0 P < 0.05 vs. group A.
FIG.
2
(the worst animal in the control group). B, Kidney Artery of Cholesterol-fed rabbit treated with Placebo, showing thickening of intima and oedema. C, Kidney Artery of Cholesterol-fed rabbit treated with 128 mg/Kg Defibrotide, i.v. once a day, showing moderate intimal thickening and oedema. H.E. x 250.
A Kidney Artery of Control rabbit (fed the standard diet and treated with placebo), with slight intimal thickening
A Coronary Artery of Control rabbit (fed the standard diet and treated with placebo), with slight intimal thickening (the worst animal in the control group). B, Coronary Artery of Cholesterol-fed rabbit treated with Placebo, showing thickening of intima and oedema. C, Coronary Artery of Cholesterol-fed rabbit i.v. once a day, showing moderate intimal thickening and oedema. H.E. x 250.
Vol.
44, No.
12, 1989
Defibrotide
Decreases
Aorta
Cholesterol
797
lead to eventually thrombi which could mural formation of focal atherosclerosis (18,191. Whatever the situation, the ability of D to decrease platelet reactivity and its antithrombotic activities could be helpful in decreasing the amount of cholesterol in the cardiovascular system in atherosclerosis-prone situations.
Acknowledgements We are indebted for technical assistance to Miss Tiziana Romano, Mr. Giovanni Terzaghi and Mr. Giorgio Meroni.
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