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Effect of bacterial lipopolysaccharide on serum lipids and on the development of aortic atherosclerosis in rabbits
Atherosclerosis, 59 (1986) 307-312 Elsevier Scientific Publishers Ireland,
307 Ltd.
ATH 03745
Effect of Bacterial Lipopolysaccharide on Serum Lipids and on the Development of Aortic Atherosclerosis in Rabbits Yrjii Kerttula
‘**,Martti Vaara ‘, Liisa PyhG I, Hannu Jussi K. Huttunen’
Sariola 3, Ella Kostiainen
’ and
’National Public Health Institute, 2 Aurora Hospital and’ Department of Pathology, Umversity of Helsinki, Helsinki (Finland) (Received 6 March, 1985) (Revised, received 30 September, 1985) (Accepted 18 October, 1985)
Summary The effect of repeated intravenous administration of bacterial lipopolysaccharide (LPS) on serum lipids and on aortic atherosclerosis was studied in rabbits on basal diet and on hypercholesterolemic diets containing 0.15-1.0% cholesterol. LPS (10 or 100 ng/kg body weight) was administered 3 times per week for 3 or 6 weeks. No difference was observed in serum lipid levels or in aortic atherosclerosis between LPSand saline-treated animals. These observations do not support the hypothesis that LPS has an effect on the progression of atherosclerosis.
Key words:
Atherosclerosis
- Bacterial lipopolysaccharide
Introduction Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) is a potent inflammatory agent with numerous biological effects such as fever, complement activation, blood coagulation abnormalities and changes in the plasma concentration of lipoproteins and acute phase proteins. The effects of LPS on serum lipoprotein metabolism include inhibition of lipoprotein lipase activity [l], increase in serum triglyceride (TG) and cholesterol levels [2-51, decrease in serum high density lipoCorrespondence should be addressed to: Yjij Aurora Hospital, Nordenskibldinkatu 20, SF-00250 Finland.
0021-9150/86/$03.50
0 1986 Elsevier Scientific
Kerttula, Helsinki,
Publishers
Ireland,
- Serum lipids
protein (HDL) cholesterol level [6,7] and impaired secretion of apolipoprotein E by murine macrophages [8]. Under experimental conditions LPS has been shown to damage vascular endothelial cells and to strip them off from the vessel wall [9], although denution has not been demonstrated in all experiments [lo]. Vascular endothelial lesion is thought to be an initiating factor in the development of arteriosclerotic disease [ll]. The biological effects of LPS and the fact that small continuous leakage of LPS takes place from the intestinal microflora into blood even under physiological circumstances [12-151 prompted us to study the role of LPS in the development of atherosclerosis. In this paper we have examined the effect of repeated adminisLtd.
308
tration of LPS (designed to simulate ‘physiological’ exposure to LPS) on serum lipids and on the development of atheromatous lesions in the aorta of rabbits. Materials and Methods Experimental animals Male albino rabbits of 2.3-3.6 kg body weight (b.wt.) were used. They were fed on commercial rabbit pellets (OTK, Hgmeenlinna, Finland) containing 2.8% fat and various concentrations of cholesterol. Water was given ad libitum. The rabbits were housed in individual cages. The marginal vein of the ear was used both for obtaining blood samples and for intravenous injections. Preparation and characterization of LPS LPS was extracted from the smooth Salmonella typhimurium LT2 strain SL 696 [16] by the hot phenol-water method [17]. The contaminating RNA was removed by treatment with RNase (Boehringer Mannheim, Mannheim, F.R.G.) The LPS preparation was washed twice using ultracentrifugation (100 000 X g, 3 h), electrodialyzed [18] and converted into its sodium salt. This preparation was found to give the pyrogenic response in rabbits at a dose of 1 ng/kg b.wt. Two rabbits given 0.1 pg/kg b.wt. remained healthy, 2 others given 1 pg/kg b.wt. became hypotonic and the 2 given 10 pg/kg b.wt. died. On the basis of these observations a dose of 10 or 100 ng/kg b.wt. of LPS was chosen for the experiments. Tissue samples and evaluation of the degree of atheromatosis The rabbits were killed using pentobarbital 100 mg/kg i.v. The heart and the aorta were immediately removed, the aorta was opened longitudinally and stained with Sudan IV [19]. Sudanpositive atheromatous plaques were recorded by counting 550-650 points in every case. The number of plaques was counted without knowing to which group the sample belonged. The correlation between the absolute number of Sudan-positive plaques and their relative share of all the points counted was highly significant (r = 0.98; P < 0.001) Therefore, the absolute number of the Sudan-positive plaques was used in calculations.
Chemical methods An enzymatic assay (CHOD-PAP, Nyegaard Ab, Oslo, Norway), was used for cholesterol determination with an OLLI-C (Kone Oy, Espoo, Finland) discrete analyzer [20]. For HDL cholesterol determination the VLD and LD lipoproteins were precipitated with dextran sulphate-magnesium chloride reagent [21]. Triglycerides were analyzed with an enzymatic assay from Boehringer, Mannheim, F.R.G. [22]. Statistical methods The statistical significance of difference between means was tested with t-test (or t-test for paired samples) [23]. Correlation coefficients were calculated with manual electronic calculator (Texas Instruments TI-51-111). Results Effect of repeated LPS injections on serum lipids The effect of repeated LPS injections on serum lipids was studied under basal conditions and during a diet with high content of cholesterol. Thirty rabbits were randomly allocated into 6 groups. Three groups (A, B and C) were maintained on normal pellets. Three groups (D, E and F) were fed with pellets containing 1% cholesterol. Ten days after commencement of the diets the rabbits were given an intravenous injection of saline (0.9%) (Groups A and D), 10 ng LPS/kg body weight (Groups B and E) or 100 ng LPS/kg body weight (Groups C and F), and the injections were repeated 3 times a week for 6 weeks. Serum lipids were determined immediately before the beginning of the injections (i.e. 10 days after the onset of the diet) and 6 weeks later. The concentrations of serum lipids and lipoproteins in various experimental groups are shown in Table 1. Serum lipids remained essentially similar in animals fed on normal pellets. No difference was observed in serum cholesterol, HDL cholesterol or triglycerides between control animals (Group A) and animals receiving intravenous injections of LPS (Groups B and C). On the other hand, a significant increase was observed in serum cholesterol in animals fed with pellets containing 1% cholesterol. However, no difference was observed in any of the lipid parameters between the
1
No No No 1.0% 1.0% 1.0%
A (5) B (5) C (5) D (5) E (5) F (4)
A-F
LPS
ON
SERUM
LIPIDS
1.15 2.29 1.75 5.07 4.60 9.92
I f * f f + f
1.15 3.44 1.49 2.70 4.04 4.86
Cholesterol
0.75+ 1.00* 0.96* 51.5 f 39.1 + 46.6 5
II
Serum lipid concentration
0.29 0.09 0.22 27.0 * 13.9 ** 15.1 **
(mmol/l)
(I) and at the end (II) of the experiment
OF
IN
FED
0.42 + 0.30 0.40*0.10 0.44*0.13 0.51+0.16 0.46 + 0.20 0.57+0.14
I
HDL cholesterol
0.45 0.55 0.50 0.99 0.86 0.55
II + 0.24 kO.12 f 0.09 f 0.57 f 0.24 +0.15
DIET
A DIET
of observations
OR
1.0%
Ten days after the
0.81 f 0.26 0.68 f 0.36 0.69kO.24 4.51 k 6.01 2.03 k 0.40 *** 2.21& 1.23
II
in parentheses.
CONTAINING
with 1% cholesterol.
1.64*0.51 1.46 k 0.73 1.38_+1.01 1.55 * 0.59 1.23 f 0.30 1.95 * 0.77
I
Triglycerides
SD. Number
A NORMAL
are given. Values are mean?
RABBITS
at random into 6 groups (5 rabbits in each). Groups D, E, and F were fed on pellets supplemented feeding, LPS or saline injections (3 times a week) were given for 6 weeks. the initial level, P c 0.02. the initial level. P < 0.01. the initial level, P -c 0.05.
10 100
10 100 _
(ng/kg b.wt.)
Dose of LPS
at the beginning
ADMINISTRATION
a Thirty rabbits were divided beginning of the cholesterol * Significant difference from ** Significant difference from *** Significant difference from
Cholesterol in food
Group
Serum lipids in the groups
EFFECT OF REPEATED CHOLESTEROL =
TABLE
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311
tween 5 and 13 mmol/l were allocated into 2 groups (A and B), and the groups were maintained on the same diet for further 3 weeks. During these 3 weeks rabbits in Group B received i.v. injections of LPS, 100 ng/kg body weight, and rabbits in Group A injections of 0.9% saline, 3 times a week. No difference was observed in feed intake, body weight or serum lipid concentrations between the 2 groups during the study period (Table 2). After 3 weeks of LPS or saline administration the animals were killed and the number of aortic atheroma plaques was counted. No difference was observed between the LPS- and saline-treated animals (Table 2). Discussion According to current theories, the initial step in the formation of the atherosclerotic plaque results from injury that damages the histologic and functional ingegrity of the vascular endothelium [11,24]. A number of causative factors have been identified in endothelial damage. Hemodynamic shearing at bends and bifurcations of the arterial system, low density lipoprotein in the blood stream, immune complex and other factors may play a role. Once the intact arterial endothelium is disrupted, plasma components, especially low density lipoproteins, are deposited in the denuded area, followed by migration of the smooth muscle cells into the subintimal space. At the same time circulating platelets adhere, aggregate to seal off the injury and release proaggregatory prostaglandins with further platelet clumping, thrombosis and vasoconstriction [25]. Single injection of large amounts of bacterial lipopolysaccharide has been shown to influence processes involved in the formation of atherosclerotic plaque [9]. On the other hand, little is known of the effects of repeated administration of small amounts of LPS. Such effects, if any, would be more relevant to the atherosclerotic process characteristic for man for several reasons. Thus, there is a continuous ‘physiological’ leakage of LPS from the gastrointestinal tract into blood, although most of the leaked LPS is normally removed by liver macrophages before entering the systemic circulation [12-151. Transient bacteremia from the gastrointestinal tract has been assumed
to occur during daily bowel movements [26] and is well documented in connection with sigmoideoscopy without biopsy [27] and barium enema
WI. Theoretically, repeated leakage of small amounts of LPS into the systemic circulation could promote atherosclerosis either by modifying plasma lipoprotein metabolism or by inducing damage to the vascular endothelium. No changes were seen in this study in serum lipids or lipoproteins in rabbits after 3-6 weeks of repeated administration of small amounts (lo-100 ng/kg body weight) of bacterial LPS. The results were similar both under basal conditions and during diets containing 0.15 or 1.0% cholesterol. Lack of an effect of LPS on serum lipid was unexpected as an increase in serum triglycerides [2-41 and cholesterol [2,3,5] and a decrease in HDL cholesterol [6,7] has been described in several animal species after a single intravenous injection of LPS. The conflicting results can be explained in several ways. First, tolerance has been shown to develop during repeated administration of LPS [3,5]. Second, the dose of LPS used in most of the previous studies has been relatively high as shown by high mortality among the experimental animals. In contrast, the doses used in the present study were smaller, and are presumably closer to the amounts of LPS leaking to the circulation under ‘physiological’ conditions. Theoretically, continuous leakage of small amounts of LPS into the circulation could enhance atherosclerosis also by inducing endothelial damage or by enhancing platelet aggregation, thrombus formation or smooth cell proliferation in the damaged endothelium. To test this hypothesis we studied the effects of repeated administration of LPS on rabbits fed on diets containing moderate or high amounts of cholesterol. No difference was observed in the degree of aortic atherosclerosis between control animals and those treated with LPS, despite highly significant differences in the plaque formation amoung groups receiving various amounts of cholesterol. It should be emphasized, however, that the cholesterol content of the aortic wall was not determined. In conclusion, our results do not support the hypothesis that leakage of bacterial lipopolysaccharide from the gastrointestinal tract into blood
312
is an important factor in the development of arteriosclerotic disease. Our results should, however, be interpreted with caution. Differences may exist in biological response between different LPS preparations and between various animal species. Thus, the denuding effect of LPS on endothelial cells is not invariable [lo]. Furthermore, the dose and the mode of administration of LPS used in this study are not necessarily comparable to the continuous or transient release of LPS from the intestine into blood. References 1 Kawakami, M. and Cerami, A., Studies of endotoxin-induced decrease in lipoprotein lipase activity, J. Exp. Med., 154 (1981) 631. 2 Hirsch, R.L., McKay, D.G., Travers, R.I. and Skraly, R.K., Hyperlipidemia, fatty liver and bromsulfophthalein retention in rabbits injected intravenously with bacterial endotoxins, J. Lipid Res., 5 (1964) 563. 3 Foldvari, P. and Kertai, P., Changes in the serum lipid fractions of cholesterol-fed and control rabbits induced by Salmonella typhi endotoxin, J. Atheroscler. Res., 7 (1976) 714. 4 Fiser, R.H., Denniston, J.C. and Beisel, W.R., Endotoxemia in the rhesus monkey Alterations in host lipid and carbohydrate metabolism, Pediat. Res., 8 (1974) 13. 5 Lequire, V.S., Hutcherson, J.D., Hamilton, R.L. and Gray, M.E., The effects of bacterial endotoxin on lipid metabolism, Part 1 (The responses of the serum lipids of rabbits to single and repeated injections of Shear’s polysaccharide), J. Exp. Med., 110 (1959) 293. in 6 Sakaguchi, S., Metabolic disorders of serum lipoproteins endotoxin-poisoned mice - The role of high density lipoprotein (HDL) and triglyceride-rich lipoproteins, Microbial. Immunol., 26 (1982) 1017. 7 Kerttula, Y., Vaara, M. and Pyh;ila, L., Effect of bacterial lipopolysaccharide on serum high density lipoprotein cholesterol in rabbits, Atherosclerosis, 52 (1984) 123. 8 Werb, Z. and Chin, J.R., Endotoxin suppresses expression of apoprotein E by mouse macrophages in vivo and in culture, J. Biol. Chem., 258 (1983) 10642. 9 Morrison, D.C. and Ulevitch, R.J., The effects of bacteriil endotoxins on host mediation systems, Amer. J. Path., 93 (1978) 527. 10 Reidy, M.A. and Schwartz, S.M., Endothelial injury and regeneration, Part 4 (Endotoxin: a nondenuding injury to aortic endothelium), Lab. Invest., 48 (1983) 25. 11 Ross, R. and Glomset, J.A., The pathogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369, 420. 12 Jacob, A.I., Goldberg, P.K., Blood, N., Degenshein, G.A.
13
14
15
16
17
18
19
20
21
22
23 24 25
26 27
28
and Kzinin, P.J., Endotoxin and bacteria in portal blood, Gastroenterology, 72 (1977) 1268. Ravin, H.A., Rowley, D., Jenkins, C. and Fine, J., On the absorption of bacterial endotoxin from the gastro-intestinal tract of the normal and shocked animal, J. Exp. Med., 112 (1960) 783. Wilkinson, S.P., Arroyo, V., Gazzard, B.G., Moodie, H. and Williams, R., Relation of renal impairment and haemorrhagic diathesis to endotoxaemia in fulminant hepatic failure, Lancet, i (1974) 521. Camaro, D.S., Cariana, Jr., J.A. Schwartz, K.A., Montes, M. and Nolan, J.P., o-Galactosamine liver injury - Absorption of endotoxin and protective effect of small bowel and colon resection in rabbits, Proc. Sot. Exp. Biol., 172 (1983) 255. Wilkinson, R.G., Gemski, Jr., P. and Stocker, B.A.D., Non-smooth mutants of Salmonella fyphimurium differentiation by phage sensitivity and genetic mapping, J. Gen. Microbial., 70 (1972) 527. Westphal, 0. and Jann, K., Bacterial lipopolysaccharides Extraction with phenol-water and further applications of the procedure, Meth. Carbohydr. Chem., 5 (1965) 63. Galanos, C., Luderitz, 0. and Estphal, O., A new method for the extraction of R-lipopolysaccharide, Europ. J. Biothem., 9 (1969) 245. Granston, W.L., Mitchell, J.R.A., Russell, R.W. and Schwarz, C.J., The assessment of aortic disease, J. Atheroscler. Res., 4 (1964) 29. Roschlau, P., Bernt, E. and Gruber, W., Enzymatische Bestimmung des Gesamt-Cholesterins im Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403. Kostner, G.M., Enzymatic determination of cholesterol in high-density lipoprotein fractions prepared by polyanion precipitation, Clin. Chem., 22 (1976) 695. Wahlefeld, A.W., Triglycerides - Determination after enzymatic hydrolysis. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1974, pp. 1831-1835. Swinscow, T.D.V., Statistics at Square One, 3rd edition, The Macmillan Press, London, Basingstoke, 1978. Ross, R. and Harker, L., Hyperlipidemia and atherosclerosis, Science, 193 (1976) 1094. Weksler, B.B. and Goldstein, J.M., Prostaglandins - Interactions with platelets and polymorphonuclear leucocytes in hemostasis and inflammation, Amer. J. Med., 68 (1980) 419. Weinstein, L., Chemoprophylaxis for bacterial endocarditis, N. Engl. J. Med., 292 (1975) 427. LeFrock, J.L., Ellis, C.A., Turchik, J.B. and Weinstein, L., Transient bacteremia associated with sigmoideoscopy, N. Engl. J. Med., 289 (1973) 467. LeFrock, J.L., Ellis, C.A., Klainer, AS. and Weinstein, L., Transient bacteremia associated with barium enema, Arch. Intern. Med., 135 (1975) 835.
307 Ltd.
ATH 03745
Effect of Bacterial Lipopolysaccharide on Serum Lipids and on the Development of Aortic Atherosclerosis in Rabbits Yrjii Kerttula
‘**,Martti Vaara ‘, Liisa PyhG I, Hannu Jussi K. Huttunen’
Sariola 3, Ella Kostiainen
’ and
’National Public Health Institute, 2 Aurora Hospital and’ Department of Pathology, Umversity of Helsinki, Helsinki (Finland) (Received 6 March, 1985) (Revised, received 30 September, 1985) (Accepted 18 October, 1985)
Summary The effect of repeated intravenous administration of bacterial lipopolysaccharide (LPS) on serum lipids and on aortic atherosclerosis was studied in rabbits on basal diet and on hypercholesterolemic diets containing 0.15-1.0% cholesterol. LPS (10 or 100 ng/kg body weight) was administered 3 times per week for 3 or 6 weeks. No difference was observed in serum lipid levels or in aortic atherosclerosis between LPSand saline-treated animals. These observations do not support the hypothesis that LPS has an effect on the progression of atherosclerosis.
Key words:
Atherosclerosis
- Bacterial lipopolysaccharide
Introduction Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) is a potent inflammatory agent with numerous biological effects such as fever, complement activation, blood coagulation abnormalities and changes in the plasma concentration of lipoproteins and acute phase proteins. The effects of LPS on serum lipoprotein metabolism include inhibition of lipoprotein lipase activity [l], increase in serum triglyceride (TG) and cholesterol levels [2-51, decrease in serum high density lipoCorrespondence should be addressed to: Yjij Aurora Hospital, Nordenskibldinkatu 20, SF-00250 Finland.
0021-9150/86/$03.50
0 1986 Elsevier Scientific
Kerttula, Helsinki,
Publishers
Ireland,
- Serum lipids
protein (HDL) cholesterol level [6,7] and impaired secretion of apolipoprotein E by murine macrophages [8]. Under experimental conditions LPS has been shown to damage vascular endothelial cells and to strip them off from the vessel wall [9], although denution has not been demonstrated in all experiments [lo]. Vascular endothelial lesion is thought to be an initiating factor in the development of arteriosclerotic disease [ll]. The biological effects of LPS and the fact that small continuous leakage of LPS takes place from the intestinal microflora into blood even under physiological circumstances [12-151 prompted us to study the role of LPS in the development of atherosclerosis. In this paper we have examined the effect of repeated adminisLtd.
308
tration of LPS (designed to simulate ‘physiological’ exposure to LPS) on serum lipids and on the development of atheromatous lesions in the aorta of rabbits. Materials and Methods Experimental animals Male albino rabbits of 2.3-3.6 kg body weight (b.wt.) were used. They were fed on commercial rabbit pellets (OTK, Hgmeenlinna, Finland) containing 2.8% fat and various concentrations of cholesterol. Water was given ad libitum. The rabbits were housed in individual cages. The marginal vein of the ear was used both for obtaining blood samples and for intravenous injections. Preparation and characterization of LPS LPS was extracted from the smooth Salmonella typhimurium LT2 strain SL 696 [16] by the hot phenol-water method [17]. The contaminating RNA was removed by treatment with RNase (Boehringer Mannheim, Mannheim, F.R.G.) The LPS preparation was washed twice using ultracentrifugation (100 000 X g, 3 h), electrodialyzed [18] and converted into its sodium salt. This preparation was found to give the pyrogenic response in rabbits at a dose of 1 ng/kg b.wt. Two rabbits given 0.1 pg/kg b.wt. remained healthy, 2 others given 1 pg/kg b.wt. became hypotonic and the 2 given 10 pg/kg b.wt. died. On the basis of these observations a dose of 10 or 100 ng/kg b.wt. of LPS was chosen for the experiments. Tissue samples and evaluation of the degree of atheromatosis The rabbits were killed using pentobarbital 100 mg/kg i.v. The heart and the aorta were immediately removed, the aorta was opened longitudinally and stained with Sudan IV [19]. Sudanpositive atheromatous plaques were recorded by counting 550-650 points in every case. The number of plaques was counted without knowing to which group the sample belonged. The correlation between the absolute number of Sudan-positive plaques and their relative share of all the points counted was highly significant (r = 0.98; P < 0.001) Therefore, the absolute number of the Sudan-positive plaques was used in calculations.
Chemical methods An enzymatic assay (CHOD-PAP, Nyegaard Ab, Oslo, Norway), was used for cholesterol determination with an OLLI-C (Kone Oy, Espoo, Finland) discrete analyzer [20]. For HDL cholesterol determination the VLD and LD lipoproteins were precipitated with dextran sulphate-magnesium chloride reagent [21]. Triglycerides were analyzed with an enzymatic assay from Boehringer, Mannheim, F.R.G. [22]. Statistical methods The statistical significance of difference between means was tested with t-test (or t-test for paired samples) [23]. Correlation coefficients were calculated with manual electronic calculator (Texas Instruments TI-51-111). Results Effect of repeated LPS injections on serum lipids The effect of repeated LPS injections on serum lipids was studied under basal conditions and during a diet with high content of cholesterol. Thirty rabbits were randomly allocated into 6 groups. Three groups (A, B and C) were maintained on normal pellets. Three groups (D, E and F) were fed with pellets containing 1% cholesterol. Ten days after commencement of the diets the rabbits were given an intravenous injection of saline (0.9%) (Groups A and D), 10 ng LPS/kg body weight (Groups B and E) or 100 ng LPS/kg body weight (Groups C and F), and the injections were repeated 3 times a week for 6 weeks. Serum lipids were determined immediately before the beginning of the injections (i.e. 10 days after the onset of the diet) and 6 weeks later. The concentrations of serum lipids and lipoproteins in various experimental groups are shown in Table 1. Serum lipids remained essentially similar in animals fed on normal pellets. No difference was observed in serum cholesterol, HDL cholesterol or triglycerides between control animals (Group A) and animals receiving intravenous injections of LPS (Groups B and C). On the other hand, a significant increase was observed in serum cholesterol in animals fed with pellets containing 1% cholesterol. However, no difference was observed in any of the lipid parameters between the
1
No No No 1.0% 1.0% 1.0%
A (5) B (5) C (5) D (5) E (5) F (4)
A-F
LPS
ON
SERUM
LIPIDS
1.15 2.29 1.75 5.07 4.60 9.92
I f * f f + f
1.15 3.44 1.49 2.70 4.04 4.86
Cholesterol
0.75+ 1.00* 0.96* 51.5 f 39.1 + 46.6 5
II
Serum lipid concentration
0.29 0.09 0.22 27.0 * 13.9 ** 15.1 **
(mmol/l)
(I) and at the end (II) of the experiment
OF
IN
FED
0.42 + 0.30 0.40*0.10 0.44*0.13 0.51+0.16 0.46 + 0.20 0.57+0.14
I
HDL cholesterol
0.45 0.55 0.50 0.99 0.86 0.55
II + 0.24 kO.12 f 0.09 f 0.57 f 0.24 +0.15
DIET
A DIET
of observations
OR
1.0%
Ten days after the
0.81 f 0.26 0.68 f 0.36 0.69kO.24 4.51 k 6.01 2.03 k 0.40 *** 2.21& 1.23
II
in parentheses.
CONTAINING
with 1% cholesterol.
1.64*0.51 1.46 k 0.73 1.38_+1.01 1.55 * 0.59 1.23 f 0.30 1.95 * 0.77
I
Triglycerides
SD. Number
A NORMAL
are given. Values are mean?
RABBITS
at random into 6 groups (5 rabbits in each). Groups D, E, and F were fed on pellets supplemented feeding, LPS or saline injections (3 times a week) were given for 6 weeks. the initial level, P c 0.02. the initial level. P < 0.01. the initial level, P -c 0.05.
10 100
10 100 _
(ng/kg b.wt.)
Dose of LPS
at the beginning
ADMINISTRATION
a Thirty rabbits were divided beginning of the cholesterol * Significant difference from ** Significant difference from *** Significant difference from
Cholesterol in food
Group
Serum lipids in the groups
EFFECT OF REPEATED CHOLESTEROL =
TABLE
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tween 5 and 13 mmol/l were allocated into 2 groups (A and B), and the groups were maintained on the same diet for further 3 weeks. During these 3 weeks rabbits in Group B received i.v. injections of LPS, 100 ng/kg body weight, and rabbits in Group A injections of 0.9% saline, 3 times a week. No difference was observed in feed intake, body weight or serum lipid concentrations between the 2 groups during the study period (Table 2). After 3 weeks of LPS or saline administration the animals were killed and the number of aortic atheroma plaques was counted. No difference was observed between the LPS- and saline-treated animals (Table 2). Discussion According to current theories, the initial step in the formation of the atherosclerotic plaque results from injury that damages the histologic and functional ingegrity of the vascular endothelium [11,24]. A number of causative factors have been identified in endothelial damage. Hemodynamic shearing at bends and bifurcations of the arterial system, low density lipoprotein in the blood stream, immune complex and other factors may play a role. Once the intact arterial endothelium is disrupted, plasma components, especially low density lipoproteins, are deposited in the denuded area, followed by migration of the smooth muscle cells into the subintimal space. At the same time circulating platelets adhere, aggregate to seal off the injury and release proaggregatory prostaglandins with further platelet clumping, thrombosis and vasoconstriction [25]. Single injection of large amounts of bacterial lipopolysaccharide has been shown to influence processes involved in the formation of atherosclerotic plaque [9]. On the other hand, little is known of the effects of repeated administration of small amounts of LPS. Such effects, if any, would be more relevant to the atherosclerotic process characteristic for man for several reasons. Thus, there is a continuous ‘physiological’ leakage of LPS from the gastrointestinal tract into blood, although most of the leaked LPS is normally removed by liver macrophages before entering the systemic circulation [12-151. Transient bacteremia from the gastrointestinal tract has been assumed
to occur during daily bowel movements [26] and is well documented in connection with sigmoideoscopy without biopsy [27] and barium enema
WI. Theoretically, repeated leakage of small amounts of LPS into the systemic circulation could promote atherosclerosis either by modifying plasma lipoprotein metabolism or by inducing damage to the vascular endothelium. No changes were seen in this study in serum lipids or lipoproteins in rabbits after 3-6 weeks of repeated administration of small amounts (lo-100 ng/kg body weight) of bacterial LPS. The results were similar both under basal conditions and during diets containing 0.15 or 1.0% cholesterol. Lack of an effect of LPS on serum lipid was unexpected as an increase in serum triglycerides [2-41 and cholesterol [2,3,5] and a decrease in HDL cholesterol [6,7] has been described in several animal species after a single intravenous injection of LPS. The conflicting results can be explained in several ways. First, tolerance has been shown to develop during repeated administration of LPS [3,5]. Second, the dose of LPS used in most of the previous studies has been relatively high as shown by high mortality among the experimental animals. In contrast, the doses used in the present study were smaller, and are presumably closer to the amounts of LPS leaking to the circulation under ‘physiological’ conditions. Theoretically, continuous leakage of small amounts of LPS into the circulation could enhance atherosclerosis also by inducing endothelial damage or by enhancing platelet aggregation, thrombus formation or smooth cell proliferation in the damaged endothelium. To test this hypothesis we studied the effects of repeated administration of LPS on rabbits fed on diets containing moderate or high amounts of cholesterol. No difference was observed in the degree of aortic atherosclerosis between control animals and those treated with LPS, despite highly significant differences in the plaque formation amoung groups receiving various amounts of cholesterol. It should be emphasized, however, that the cholesterol content of the aortic wall was not determined. In conclusion, our results do not support the hypothesis that leakage of bacterial lipopolysaccharide from the gastrointestinal tract into blood
312
is an important factor in the development of arteriosclerotic disease. Our results should, however, be interpreted with caution. Differences may exist in biological response between different LPS preparations and between various animal species. Thus, the denuding effect of LPS on endothelial cells is not invariable [lo]. Furthermore, the dose and the mode of administration of LPS used in this study are not necessarily comparable to the continuous or transient release of LPS from the intestine into blood. References 1 Kawakami, M. and Cerami, A., Studies of endotoxin-induced decrease in lipoprotein lipase activity, J. Exp. Med., 154 (1981) 631. 2 Hirsch, R.L., McKay, D.G., Travers, R.I. and Skraly, R.K., Hyperlipidemia, fatty liver and bromsulfophthalein retention in rabbits injected intravenously with bacterial endotoxins, J. Lipid Res., 5 (1964) 563. 3 Foldvari, P. and Kertai, P., Changes in the serum lipid fractions of cholesterol-fed and control rabbits induced by Salmonella typhi endotoxin, J. Atheroscler. Res., 7 (1976) 714. 4 Fiser, R.H., Denniston, J.C. and Beisel, W.R., Endotoxemia in the rhesus monkey Alterations in host lipid and carbohydrate metabolism, Pediat. Res., 8 (1974) 13. 5 Lequire, V.S., Hutcherson, J.D., Hamilton, R.L. and Gray, M.E., The effects of bacterial endotoxin on lipid metabolism, Part 1 (The responses of the serum lipids of rabbits to single and repeated injections of Shear’s polysaccharide), J. Exp. Med., 110 (1959) 293. in 6 Sakaguchi, S., Metabolic disorders of serum lipoproteins endotoxin-poisoned mice - The role of high density lipoprotein (HDL) and triglyceride-rich lipoproteins, Microbial. Immunol., 26 (1982) 1017. 7 Kerttula, Y., Vaara, M. and Pyh;ila, L., Effect of bacterial lipopolysaccharide on serum high density lipoprotein cholesterol in rabbits, Atherosclerosis, 52 (1984) 123. 8 Werb, Z. and Chin, J.R., Endotoxin suppresses expression of apoprotein E by mouse macrophages in vivo and in culture, J. Biol. Chem., 258 (1983) 10642. 9 Morrison, D.C. and Ulevitch, R.J., The effects of bacteriil endotoxins on host mediation systems, Amer. J. Path., 93 (1978) 527. 10 Reidy, M.A. and Schwartz, S.M., Endothelial injury and regeneration, Part 4 (Endotoxin: a nondenuding injury to aortic endothelium), Lab. Invest., 48 (1983) 25. 11 Ross, R. and Glomset, J.A., The pathogenesis of atherosclerosis, N. Engl. J. Med., 295 (1976) 369, 420. 12 Jacob, A.I., Goldberg, P.K., Blood, N., Degenshein, G.A.
13
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23 24 25
26 27
28
and Kzinin, P.J., Endotoxin and bacteria in portal blood, Gastroenterology, 72 (1977) 1268. Ravin, H.A., Rowley, D., Jenkins, C. and Fine, J., On the absorption of bacterial endotoxin from the gastro-intestinal tract of the normal and shocked animal, J. Exp. Med., 112 (1960) 783. Wilkinson, S.P., Arroyo, V., Gazzard, B.G., Moodie, H. and Williams, R., Relation of renal impairment and haemorrhagic diathesis to endotoxaemia in fulminant hepatic failure, Lancet, i (1974) 521. Camaro, D.S., Cariana, Jr., J.A. Schwartz, K.A., Montes, M. and Nolan, J.P., o-Galactosamine liver injury - Absorption of endotoxin and protective effect of small bowel and colon resection in rabbits, Proc. Sot. Exp. Biol., 172 (1983) 255. Wilkinson, R.G., Gemski, Jr., P. and Stocker, B.A.D., Non-smooth mutants of Salmonella fyphimurium differentiation by phage sensitivity and genetic mapping, J. Gen. Microbial., 70 (1972) 527. Westphal, 0. and Jann, K., Bacterial lipopolysaccharides Extraction with phenol-water and further applications of the procedure, Meth. Carbohydr. Chem., 5 (1965) 63. Galanos, C., Luderitz, 0. and Estphal, O., A new method for the extraction of R-lipopolysaccharide, Europ. J. Biothem., 9 (1969) 245. Granston, W.L., Mitchell, J.R.A., Russell, R.W. and Schwarz, C.J., The assessment of aortic disease, J. Atheroscler. Res., 4 (1964) 29. Roschlau, P., Bernt, E. and Gruber, W., Enzymatische Bestimmung des Gesamt-Cholesterins im Serum, Z. Klin. Chem. Klin. Biochem., 12 (1974) 403. Kostner, G.M., Enzymatic determination of cholesterol in high-density lipoprotein fractions prepared by polyanion precipitation, Clin. Chem., 22 (1976) 695. Wahlefeld, A.W., Triglycerides - Determination after enzymatic hydrolysis. In: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1974, pp. 1831-1835. Swinscow, T.D.V., Statistics at Square One, 3rd edition, The Macmillan Press, London, Basingstoke, 1978. Ross, R. and Harker, L., Hyperlipidemia and atherosclerosis, Science, 193 (1976) 1094. Weksler, B.B. and Goldstein, J.M., Prostaglandins - Interactions with platelets and polymorphonuclear leucocytes in hemostasis and inflammation, Amer. J. Med., 68 (1980) 419. Weinstein, L., Chemoprophylaxis for bacterial endocarditis, N. Engl. J. Med., 292 (1975) 427. LeFrock, J.L., Ellis, C.A., Turchik, J.B. and Weinstein, L., Transient bacteremia associated with sigmoideoscopy, N. Engl. J. Med., 289 (1973) 467. LeFrock, J.L., Ellis, C.A., Klainer, AS. and Weinstein, L., Transient bacteremia associated with barium enema, Arch. Intern. Med., 135 (1975) 835.