Oxygen free radicals and hypercholesterolemic atherosclerosis: Effect of vitamin E

Oxygen free radicals hypercholesterolemic of vitamin E

and atherosclerosis:

Effect

We investigated the effects of a high-cholesterol diet in the presence and absence of vitamin E on the lipid peroxidation product malondialdehyde of blood and aortic tissue, the oxygen-free-radical-producing activity of polymorphonuclear leukocytes (PMNs) (PMN chemiluminescence), and the blood lipid profile in rabbits. The animals were divided into four groups each of which comprised 10 rabbits. Rabbits in group I received a regular rabbit chow diet; those in group II received vitamin E; those in group Ill received high cholesterol + vitamin E; and those in group IV received a high-cholesterol diet. Blood concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), malondialdehyde, and PMN chemiluminescence were measured. The aorta of each rabbit was removed at the end of the protocol for assessment of atherosclerotic changes (gross and microscopic) and malondialdehyde. Serum triglycerides, total cholesterol, HDL-C, LDL-C, and VLDL-C increased while HDL/LDL ratio decreased in groups Ill and IV but remained unchanged in group I. There was an increase in the HDL-C component and HDLlLDL ratio and a decrease in the LDL-C component and triglycerides in group II. Blood and aortic tissue malondialdehyde increased in group IV but decreased in groups II and Ill. PMN chemiluminescence increased in groups Ill and IV. Atherosclerotic changes were marked in group IV as compared with those in group Ill. However, histologic changes in the aortas were similar in groups Ill and IV. The increased levels of blood and aortic tissue malondialdehyde and PMN chemiluminescence, which were associated with development of atherosclerosis, suggest a role of oxygen free radicals in the pathogenesis of hypercholesterolemia-induced atherosclerosis. The protection afforded by vitamin E, which was associated with a decrease in blood and aortic tissue malondialdehyde concentration in spite of hypercholesterolemia, supports the hypothesis that oxygen free radicals are involved in the development of hypercholesterolemic atherosclerosis. (AM HEART J 1993;125:959.)

Kailash

Prasad, MD, PhD, and Jawahar Kalra, MD, PhD

Saskatoon, Saskatchewan, Canada

Hypercholesterolemia is a major risk factor for atherosclerosis and related occlusive vascular disease.lm6 According to “response-to-injury” theory of the genesis of atherosclerosis, endothelial cell injury is the basic mechanism for initiation and maintenance of atherosclerosiseg and is followed by development of From the Departments of Physiology and Pathology, College of Medicine, University of Saskatchewan and Royal University Hospital, Saskatoon, Saskatchewan, Canada Supported by a grant from Heart and Stroke Foundation of Saskatchewan, Saskatoon, Canada. Received for publication June 3, 1992; accepted Oct. 19, 1992. Reprint requests: K. Prasad, MD, PhD, Department of Physiology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N OWO Canada Copyright cc’1993 by Mosby-Year Book, Inc. 0002.8703/93/$1.00

+

4/l/43806

fatty streaks, fibrous plaques, and “complicated lesions”.7-10 Hypercholesterolemia has been reported to cause endothelial injury,6p lip l2 but the mechanism by which this happens is not clear. Endothelial and smooth muscle cells, neutrophils, monocytes, and platelets may be the source of oxygen free radicals (OFRs) in hypercholesterolemia. Hypercholesterolemia could increase levels of OFRs in various ways. Hypercholesterolemia increases cholesterol content of platelets, polymorphonuclear leukocytes (PMNs) and endothelial cells.13-15 Cholesterol enhances platelet function, and cholesterol-rich platelets release substances, which include thrombin, histamine, and adenosine diphosphate (ADP).16, l7 Histamine and ADP activate phospholipase A2,18 which acts on phospholipids to release arachidonic acid.ig Increase

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Journal

in phospholipase As activity may also arise from increases in intracellular Ca++, ig which accompany 2o Activation of phospholipase hypercholesterolemia. A2 would result in increased release of arachidonic acid with increased synthesis of prostaglandins and leukotrienes in various cells. Increased production of thromboxane As and prostacyclin in the aorta in experimental atherosclerosis has been reported.21 The intermediate steps (conversion of prostaglandin Gz to prostaglandin Hz) in biosynthesis of prostaglandins from arachidonic acid produce OFRS.“-~~ Activated leukocytes also produce OFRs and hypochlorous acid.25-27 During hypercholesterolemia, leukocytes may be activated by various agents including leukotriene B4 (LTBJ),~~ platelet-activating factor (PAF),2g and activated complement components (Csa, c&).30 LTB4 production would be expected to increase because of increased arachidonic acid release by phospholipase A2 activity as stated earlier. PAF synthesis and release are dependent on thrombin and intracellular Cam’+, 31 which are increased during hy2oActivation of complement compercholesterolemia. ponents (C3, Cs) by cholesterol is well known.32-35 OFRs (superoxide anion [O,], hydrogen peroxide [HsOz], hydroxyl radicals [oOH]) have been implicated in tissue injury and various cardiac disorders.36-4” OFRs have been suggested to exert their cytotoxic effects by causing peroxidation of membrane phospholipids, which can result in an elevation in membrane fluidity, which increases permeability, and loss of membrane integrity.43s 44 Lipid peroxidation results in the formation of malondialdehyde (MDA), a lipid peroxidation product. An increase in blbcd and aortic tissue MDA would suggest involve-

ment of OFRs in the tissue damage. We shown that blood MDA is increased in terolemic atherosclerosis.45 According to the evidence presented possible that hypercholesterolemia may

have already hypercholes-

above, it is increase pro-

duction of OFRs and lead to endothelial cell injury,

which sets the stage for atherosclerosis.

If OFRs are

involved in the genesis and maintenance of hyper-

cholesterolemic

atherosclerosis,

the development

of

atherosclerosis should be associated with a rise in blood and aortic tissue MDA. Also, vitamin E, an an-

tioxidant,46-4g would be expected to prevent atherosclerosis and the increase in blood and aortic tissue MDA that is associated with hypercholesterolemia. We have studied the effects of a high-cholesterol diet on the genesis of atherosclerosis and on blood lipid profile, blood and aortic tissue MDA, and OFF&producing activity of PMNs (PMN chemiluminescence) in rabbits. We have also determined the effects of vi-

959

Table I. Experimental diet regimen Group

Diet

I II III

Control (lab rabbit chow, 150 gm/day) Vitamin E (0.04 gm/kg/day) Cholesterol (0.5 gm/kg/day) plus vitamin E (0.04 gm/kg/day) Cholesterol (0.5 gm/kg/day)

IV

tamin E on the development of hypercholesterolemia-induced atherosclerosis (gross and microscopic) and on the changes in blood and aortic tissue

MDA. METHODS Six- to eight-week-old female New Zealand white rabbits, which weighedbetween 1.8 and 2 kg, were used.After a 2-weekperiod of adaptation, the rabbits weredivided into four groups of 10 as shown in Table I. Rabbits in group I (control) were fed laboratory rabbit chow pellets (150gml day). The other groupsreceived cholesterolor vitamin E or cholesterolplus vitamin E in addition to the sameamount of rabbit chow pellets. Water wasgiven ad libitum. Food consumption was determined every day throughout the experimental period. The rabbits on various experimental diets were killed at the end of 4 months. Blood samplesfor measurement

of triglycerides,

total cholesterol,

low-den-

sity lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol (HDL-C), very low-density lipoprotein cholesterol (VLDL-C), blood MDA, OFR-producing activity of PMNs were collected before (0) and after 1,2,3, and 4 months

on the experimental

diets. Food was withdrawn

from rabbits for 18 hours before blood sampleswere taken. Aortas were removed for the assessment of atherosclerotic plaquesand measurementof tissueMDA. The rabbits were weighed before and after 1, 2, 3, and 4 months on the experimental

diets.

Serum triglycerides and cholesterol. Serum triglycerides were measuredby enzymatic hydrolysis of triglycerides with subsequent enzymatic determination of liberated glycerol by calorimetry. 5o Measurement of serum total cholesterol was made by a kinetic enzymatic method.51 Se-

rum HDL-C and LDL-C were measuredby the methodsof Lopes-Virella et al.% and Assmann,‘” respectively. The values for VLDL-C were calculated by subtracting of HDL-C and LDL-C from total cholesterol.

the sum

MDA (thiobarbituric acid-reactive substances). Blood for MDA measurementwas collected in tubes that contained ethylenediaminetetraacetic acid. The MDA levels in blood and aortic tissue were estimated as thiobarbituric

acid (TBA)-reactive substancesby a method similar to that described by Prasad et a1.s6 and by Yagi.54 In brief, blood (0.2 ml) was added to normal saline solution (2 ml) in a centrifuge tube and shaken gently. After centrifugation at 3000 rpm for 10 minutes, 0.5 ml of the supernatant was

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Prasad and Kalra

transferred to another centrifuge tube that contained 4.0 ml of 0.083 N sulfuric acid. Half a milliliter of 10cr phosphotungstic acid was then added to this tube and mixed, After this mixture was allowed to stand at room temperature for 5 minutes, the tube was centrifuged at 3000 rpm for 10 minutes, the sediment was suspended in 2 ml of 0.083 N sulfuric acid and 0.3 ml of lO’(, phosphotungstic acid, and the mixture was centrifuged at 3000 rpm for 10 minutes. The sediment was suspended in 4 ml of distilled water, and 1.0 ml of TBA reagent (mixture of equal volumes of 0.67 ‘, thiobarbituric acid aqueous solution and glacial acetic acid) was added. The reaction mixture was heated for 60 minutes at 95” C in a glycol bath. After the mixture had cooled, 5.0 ml of n-butanol was added, the mixture was centrifuged at 3000 rpm for 15 minutes, and the n-butanol layer was used for fluorometric measurement. at 553 nm with excitation at 515 nm. Tetraethoxypropane was used as a standard, and the results were expressed as nanomoles of MDA equivalents. It is noteworthy that the TBA reagent method measures substances other than MDA. Hence, the substances measured by this method are called “TBA-reactive substances.” However, we are using the term MDA, which is synonymous with TBA-reactive substances. The aortas between the origin and bifurcation into iliac arteries were removed, cleaned of gross adventitial tissue, and immersed into cold Hank’s balanced salt solution (HBSS) for the measurement of MDA in the aortic tissue. The aort.ic tissue was added to 10 volumes (Wt/vol) of HBSS, and homogenized with a Polytron homogenizer (PT-10, Brinkman Instruments, Rexdale, Ontario, Canada) at a setting of 5 for two periods of 10 seconds each at 0’ C to 4’ C. Homogenate (0.1 ml) was used for determination of MDA as described above. MDA data were expressed in nanomoles per milligram of protein. PMN counts and chemiluminescence measurements. Samples of mixed venous blood were collected in tubes that contained ethylenediaminetetraacetic acid for PMN counts and chemiluminescence studies. Total white blood cells (WBCs) and PMN counts were made with the Technicon H6000 system (Technicon Diagnostics, Tarrytown, N.Y. I. OFR-producing activity of PMNs was measured with PMN chemiluminescence. Luminol-dependent chemiluminescence provides a highly sensitive and continuous method for monitoring the rate of production of OFRs by PMNs.‘” The method for measurement of chemiluminescence was similar to that described earlier by US.‘)~,x In brief, 0.05 ml (approximately 2.5 X 1e5 PMNs) of blood was added to a counting vial that contained HBSS at pH 7.4 and luminol at a final concentration of lo-’ mol/L. The final volume of the mixture was 0.5 ml. Samples were placed in a luminometer for 10 minutes at 37” C, and phagocytosis was initiated by addition of 0.1 ml (10 mg/ml) of opsonized zymosan prepared as described previously.Z” The chemililminescence was monitored for 60 minutes in the presence and in the absence of 0.1 ml superoxide dismutase (SOD) (1 mg/ml; 3050 U/mg protein). With the use of a LKBWallac 1251 luminometer (Wallac OY, Turku, Finland) the counts were made for 4 seconds every 4 minutes for a period of 60 minutes. The area under the curve was in-

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1993

Heart Journal

tegrated to give total chemiluminescence. The diff’erence in area in the presence and absence of SOD is designated as SOD-inhibitable chemiluminescence. The unit t’cu chemiluminescence is millivolts X minutes per IO” PMNs. The results are expressed in absolute values (mv min/l#’ PMN). Gross and histologic appearance of aortic wall, The aorta between its origin and bifurcation into the iliac arteries was dissected, opened longitudinally, and prepared for accurate detection and estimation of lipid deposits in the intima by the method of Holman et a1.‘7i The aortic strips were immersed in 10’ <,buffered formalin solution for 24 hours and then rinsed brieHy in 70’, alcohol. The t,issue was then immersed in Herxheimer’s solution that contained Sudan IV (5 gm), ethyl alcohol 70’, (500 ml). and acetone (500 ml) at room temperature for 15 minutes. The tissue was transferred to 80’, alcohol for 20 minutes and washed in running water for 1 hour. The surface areas 01‘ atheromatous lesions were measured from a photograph and expressed as a percentage of’ total aortic intimal surface area. A small portion of the plaques and of adjacent normal aortic area from groups III and IV and from comparable areas of the aortas of groups I and II were cut across and embedded in paraffin. Paraffin sections of 4 Frn thickness of aortas were cut and stained with the standard hematoxylin-eosin stain, hematoxylin-oil red 0 for lipids, toluidine blue for acid mucopolysaccharides, and Verhoeff’s---van Gieson stain for elastic fibers by the method described by Drury and Wallington”s fhr morphologic assessment of atherosclerotic lesions. Statistical analysis. All values are expressed as means _t SEM. Statistical analysis was performed with the use of repeated measures of analysis of variance (BMDP Statistical Software, University of California, Berkeley, Calif.). Analysis for significant differences between individual pairs of means was made by the method of least significant diff’erence.“” A p value of less than 0.05 was considered significant. RESULTS Body weight.

The changes

in body weight

with time

in all groups of rabbits are summarized in Table II. There was a progressive increase in the body weight of the rabbits in all groups. However, there were no significant differences in body weight among the four groups. Triglycerides. The serum triglyceride levels in groups I (control), II (vitamin E), III (cholesterol plus vitamin E), and IV (cholesterol) before they were placed on experimental diets were 0.68 -t 0.10 mmol/L, 0.85 i 0.16 mmol/L, 1.03 i 0.15 mmol/L, and 0.71 + 0.07 mmol/L @EM), respectively. The prediet serum triglyceride levels in group III were significantly higher than those in group I. The changes in the serum triglyceride levels of all groups during

the 4 months

of observation

are given

in Fig.

Volume 125, Number 4 American Heart Journal

Prasad

[O Control

v Vit. E

q

and

K&a

961

T S.E.

Chol + Vit. E o Chol

YY

T

Obc abc

Fig. 1. Sequential changesin the serumtriglyceride concentrations of the four experimental groups.The

results are expressedasmean + SEM. Vit. E, Vitamin E; Chol, cholesterol. *p < 0.05, Comparisonof the valuesat different times with respectto “0” month in the respectivegroups.ap < 0.05,group I versusgroups II, III, and IV. b p < 0.05, group II versus groups III and IV. c p < 0.05, group III versus group IV.

Table

II. Changesin the weight of the rabbits on various experimental diets Body Time Groups

0

I (Control) II (Vitamin El III (Choiesterol+ vitamin E IV (Cholesterol) Results are expressed *p < 0.05, comparison

weight

period

(kg) (months)

2

I

3

4

2.407 2.675 2.595

+ 0.149 k 0.170 * 0.073

3.286 3.175 3.035

+ 0.161* + 0.182* k 0.123*

3.764 k 0.174* 3.680 i 0.139* 3.425 t 0.135*

3.907 k 0.172* 3.780 k o.zoo* 3.445 + 0.126*

4.050 3.920 3.675

t 0.161* ri: 0.165* + 0.159*

2.393

? 0.050

2.931

+ 0.165*

3.344

3.387

3.487

i. 0.206*

as mean f SEM. of the values at different

+ 0.172*

times with respect to “0” in the respective

1. Serum triglyceride levels remained unchanged in group I, increased in groups III and IV, and decreased in group II. The values in groups III and IV were greater than those in groups I and II, and those in group III were greater than those in group IV. Serum cholesterol and lipoprotein. The changes in the serum total cholesterol in the four groups are summarized in Fig. 2. Total serum cholesterol before exposure to experimental diets in groups I, II, III, and IV were 1.84 k 0.11 mmol/L, 1.97 f 0.27 mmol/L, 1.73 -t 0.14mmol/L,and1.63 f O.l5mmol/L (SEM), respectively. Serum cholesterol levels in groups I and II remained unchanged during the 4-month period,

+ 0.252*

groups

but there were progressive increases in groups III and IV. The increases were of similar magnitude up to 2 months in both these groups but were greater in group III than in group IV thereafter. The levels in groups III and IV were higher than those in groups I and II. The changes in HDL-C are summarized in Fig. 3. The prediet serum HDL-C levels in groups I, II, III, and IV were 0.94 + 0.07 mmol/L, 0.76 -t 0.07 mmol/L, 0.69 f 0.05 mmol/L, and 0.78 f 0.06 mmol/L @EM), respectively. Serum HDL-C levels remained unchanged in groups I and II but increased progressively up to 3 months in group III and up to 2 months in group IV and returned to prediet levels

962

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American

0 Control

v Vit. E D Chol + Vit. E o Chol

April 1993 Heart Journal

T S.E

80 -

80 -

1

0

.

2

3

4

(Eiih) Fig. 2. Sequential changesin the total serumcholesterolconcentrations of the four experimental groups. The results are expressedas mean t SEM. Vit. E, Vitamin E; Chol, cholesterol. *p < 0.05, comparisonof the values at different times with respect.to “0” in the respective groups.ap < 0.05, group I versusgroups II, III and IV. b p < 0.05, group II versus groups III and IV. c p < 0.05, group III versusgroup IV.

Control

v i/it. E o Chat + Vit. E o Chol

T S.E.

l ab

I

I

1

k

1

3

4

Tine

(month) Fig. 3. Sequential changesin the serumHDL-C concentrations of the four experimental groups.The resultsare expressedasmean k SEM. Vit. E, Vitamin E; Chol, cholesterol. *p < 0.05,comparisonof the valuesat different times with respect to “0” in the respective groups. ap < 0.05, group I versusgroupsII, III, and IV. b p < 0.05, group II versus groups III and IV.

tbereafter. There were no significant differences between group III and group IV. The changes in the serum LDL-C levels are shown in Fig 4. The prediet levels of LDL-C in groups I, II. III, and IV were

0.47 + 0.07 mmol/L, 0.56 _t 0.15 mmol/L, 0.41 t_ 0.06 mmol/L, and 0.45 + 0.10 mmol/L (SEMI, respectively. The values of LDL-C in groups I and II did not change throughout the period of observation

Volume 125, Number 4 American Heart Journal

Prasad and Kalra

0 Control

v Vit. E a Chol + Vit. E o Chol

60 -

963

T S.E.

+ab

l ab 40 -

0

1

2

3

4

(2%) Fig. 4. Sequential changesin the serumLDL-C concentrations of the four experimental groups. The results are expressedasmean i SEM. Vit. E, Vitamin E; Chol, cholesterol. *p < 0.05,comparisonof the valuesat clifferent times with respect to “0” in the respective groups.ap < 0.05, group I versusgroupsII, III, and IV. b p < 0.05, group II versus groups III and IV.

10 Control

v Vit. E o Chol + Vit. E o Chol

T S.E.l

6 *ab

Fig. 5. Sequential changesin the serumVLDL-C concentrations in the four experimental groups.The results are expressedasmean + SEM. Vit. E, Vitamin E; Chol, cholesterol.*p < 0.05,comparisonof the val-

uesat different times with respectto “0” in the respective groups.ap < 0.05, group I versusgroups II, III, and IV. b p < 0.05, group II versus groups III and IV.

but increased progressively and to a similar extent in groups III and IV. The changes in serum VLDL-C levels are summarized in Fig. 5. The prediet levels of LVDL-C

in groups I, II, III and, IV were 0.43 f 0.11

mmol/L, 0.65 k 0.17 mmol/L, 0.63 ? 0.10 mmol/L, 0.40 + 0.06 mmol/L (SEM), respectively. There were no changes in groups I and II. However, the levels increased progressively in groups III and IV, but there

964

Prasad and Kalra

o CONTROL

: v

American

‘AT. E ; q CHOL + WT. E ; 0 CHOL ;T S.E.

*a

l

o CONTROL

: v

April 1993 Heart Journal

VlT. E : q CHOL + WT. E : 0 CHOL :T

S.E.

ob

00 =

:

6-

d i

‘-

ab

P 2-

l ab ,I.

, 0

l ab l ab

l ab

. 1

2

Time

*ab l ab 3

rab 0 *ab I

l ab

*ab

4

(month)

Fig. 6. Sequential changesin the HDL-C component as percentage of total cholesterol (upper panel) and HDL/ LDL ratio flower panel) of four experimental groups. The results are expressedas mean k SEM. Vit. E, Vitamin E; Chol, cholesterol. *p < 0.05, comparison of the values at

different timeswith respectto “0” in the respectivegroups. a p < 0.05, group I versus groups II, III, and IV. b p < 0.05,

group II versus groups III and IV.

Time

(month)

Fig. 7. Sequential changesin the LDL-C component (upper panel) and VLDL-C component (lower panel) as per-

cent of total cholesterol in the four experimental groups. The results are expressedasmean -+ SEM. Vit. E, Vitamin E; Chol, cholesterol. *p < 0.05,comparisonof the values at different times with respect to “0” in the respective groups. ap < 0.05, group I versus groups II, III and IV. bp < 0.05, group II versusgroupsIII and IV. cp < 0.05, group III ver-

susgroup IV. were no significant differences between these groups. The changes in the various lipoprotein cholesterol components as a percentage of total cholesterol are summarized in Figs. 6 and 7. Prediet levels of HDL-C were51.53 k 3.33%,44.73 i- 5.99%,41.09 k 3.18%, and 48.54 + 2.49% (SEM) of total cholesterol in groups I, II, III, and IV, respectively. These values were not significantly different from each other. The HDL-C component of total cholesterol decreased in groups III and IV, increased in group II, and remained unchanged in group I. Prediet levels of LDL-C were 25.44 i 3.48%, 24.98 i 4.65%,22.91 f 2.64%,and25.57 -t 5.08% (SEM) of total cholesterol in groups I, II, III, and IV, respectively. The LDL-C component of total cholesterol increased significantly in groups III and IV, decreased in group II, and remained practically un-

changed in group I. Prediet levels of VLDL-C were 23.03 k 5.18%, 30.29 f 4.60%, 36.00 + 4.36%, and 25.89 k 4.05% (SEM) of total cholesterol in groups I, II, III, and IV, respectively. These values were different in the four groups. The VLDL-C component decreased in groups III and IV, whereas it remained practically unchanged in groups I and II. The prediet values of the HDL/LDL ratio were 2.27 i 0.43, 3.32 k 1.08, 2.04 + 0.031, and 2.63 k 0.76 in groups I, II, III, and IV, respectively. The HDL/LDL ratio decreased in groups III and IV, increased in group II, and remained practically unchanged in group I. Macroscopic and microscopic evaluation of fatty steak formation. The endothelial surface of the aortic walls

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Fig. 8. Intimal surface of the aortas from different groups of rabbits showing Sudan IV stainable lipid deposits. I, Control diet; II, vitamin E; Ill, cholesterol plus vitamin E; and IV, cholesterol. Note marked atherosclerotic bright-red lipid deposit changes in group IV and minimal changes in group III.

was examined for atherosclerotic plaques. Representative photographs of the endothelial surface of the aorta from four experimental groups are shown in Fig. 8, and the results are summarized in Fig. 9. Fatty streaks were not observed in groups I and II. A significant number of visible atherosclerotic plaques were noted in group IV. For the aortas that were obtained from group III, the atherosclerotic plaques were significantly smaller than those in group IV. The atherosclerotic plaques were distributed all over the aorta in cholesterol-fed rabbits (group IV). However, they were present mainly in the proximal segment of the aortas in rabbits that were fed cholesterol plus vitamin E. Histologic sections of aortas of the four groups stained with hematoxylin and eosin, hematoxylinoil red 0, Verhoeff s-van Gieson, and toluidine blue are shown in Figs. 10 to 13. The intima of the aortas from normal and vitamin-E-treated rabbits was composed of a thin usually mechanically disrupted unicellular layer of endothelial cells that covered the intact internal elastic lamina. The media contained a laminated layer of longitudinal elastic fiber. Toluidine blue staining showed the normal amount of acid mucopolysaccharides in the media. There were no differences in the histologic sections of the aortas from the normal rabbits and those from vitamin-Etreated rabbits. Histologic section through the atherosclerotic

plaques of the aortas from rabbits on a high cholesterol diet showed thickening of the intima, which consisted of foam cells that contained oil red 0 stainable lipids (Fig. 11). The internal elastic lamina and the elastic fibers in the subintimal media were intact and arranged in a normal fashion (Fig. 12). Toluidine blue staining showed no abnormal accumulation of acid mucopolysaccharides in the media; however, there was deposition of some acid mucopolysaccharides in the atherosclerotic plaques (Fig. 13). The histologic changes in the aortas of the rabbits on a combined high-cholesterol and vitamin E diet were similar to those in rabbits on a high-cholesterol diet without vitamin E. Aortic tissue MDA. MDA contents of aortic tissue in the four experimental groups are summarized in Fig. 9. MDA content in control aortic tissue was 0.46 t 0.04 nmol/mg protein @EM). The content decreased significantly in groups II and III, whereas it was significantly higher in group IV than in all other groups. Vitamin E prevented the increase in MDA in high-cholesterol-fed rabbits (group III). Blood MDA. The sequential changes in the blood levels of MDA in the four groups are given in Table III. Blood MDA increased in group IV and decreased in groups II and III at 1 month and remained low throughout the period of observation. There were no changes in MDA of group I throughout the period of observ&ion. The decreases in the values of MDA

April

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Prasad and Kalra

1.5

100

I

American

1993

Heart Journal

abc T

r

Fig. 9. Effects of four different types of diets on the development of atherosclerotic plaques in aorta (lower panel) and aortic tissueMDA (upper panel). The results are expressedas mean ? SEM. Vit. E, Vitamin E; Chol, cholesterol.*p < 0.05, group I versusgroupsII, III, and IV. bp < 0.05, group II versus groups III and IV. “p < 0.05, group III versusgroup IV. Note that group I and II in the lower panel show somevalues for atherosclerotic plaques. This is just to showthe location of groups I and II. There were no atherosclerotic plaquesin these two groups.

were significant throughout the period of observation in group II, whereas they were significant only after 1 month in group III as compared with group I. The increases were significant throughout in group IV when compared with group I. The values of MDA were greater in group III when compared with group II. The MDA levels were higher in group IV than in group III. These results indicated that vitamin E decreased the MDA levels of blood in groups that were fed a control diet and in groups that were fed highcholesterol diets. Chemiluminescence activity of PMNs. The results of

Fig. 10. Microscopic section of aortas of control (I), cholesterol plus vitamin E (Ill), and cholesterol (IV) groups stained with hematoxylin and eosinshowinginternal elastic lamina, elastic fibers, and atherosclerotic plaques.IE, Internal elastic lamina; M. media; A, atherosclerotic plaques. (Original magnificat.ion X175.1

the serial measurements of SOD-inhibitable

chemilu-

minescence of PMNs in the four groups are summarized in Fig. 14. SOD-inhibitable chemiluminescent activity of PMNs increased in groups III and IV at 1 month and remained elevated for the whole period of

observation.

The values in groups III and IV were

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967

Fig. 11. Histologic sectionsof the aortasfrom three groups of rabbits as above stained with hematoxylin-oil red 0. Note the brig’ht-red-stained lipids in the atherosclerotic plaquesin groups III and IV. Abbreviations as in Fig. 10. (Original magnification X175.)

Fig. 12. Histologic sectionsof aortas from three groupsof rabbits stained with Verhoeffs-van Gieson stain. Note the black-stained elastic fibers. Abbreviations asin Fig. 10. (Original magnification X175.)

higher than those in groups I and II after 1 month.

and that vitamin PMNs.

There were no significant differences in the values of chemiluminescent activity between group I and group II. The initial values in the four groups were similar. These results suggest that OFR-producing activity of PMNs is increased in hypercholesterolemic rabbits

E does not affect this activity

of

DISCUSSION

The dose of cholesterol used in this study was similar to that used by other investigators.60-62 In our

966

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I

I

Fig. 13. Histologic section of aortas from three groupsof rabbits stained with toluidine blue for acid mucopolysaccharides. Note the purple-stained acid mucopolysaccharides. Abbreviations asin Fig. 10. (Original magnification x175.)

study the levels of serum total cholesterol, LDL-C, VLDL-C, HDL-C increased in rabbits that received a high-cholesterol diet. The increase in total cholesterol was mainly due to an increase in the LDL-C component. The HDL-C and VLDL-C components of total cholesterol were lower in the cholesterol-fed

April 1993 Heart Journal

rabbits t,han those in control rabbits. Also, the values of the HDL/LDL ratio decreased significantly in the cholesterol-fed rabbits. Similar changes in serum concentrations of triglycerides,45, 63 total cholestero1,45, 63,64 LDL-C,45,63> 6.5VLDL-C,““, 6% 65 and HDLC4”, 63.65have been reported. However, no change in serum triglyceride levels has also been reported.64j 65 The variations in the serum triglyceride levels could not be due to the duration of the cholesterol diet. A rise in serum triglycerides has been observed after 2 weeks.6” The duration of cholesterol diet in the studies of Wojcicki et a1.64and Sugano et al.s5varied between 10 weeks and 3 months. The HDL-C level in the present study increased for up to 2 months, after which it returned to the prediet level. In general, the levels of serum triglycerides and total cholesterol were higher in rabbits that were kept on a high-cholesterol diet plus vitamin E than in those on a high-cholesterol diet only. The HDL-C, LDL-C, VLDL-C, and HDL/LDL ratios were similar in the above two groups. Also, the changes in HDL-C, LDL-C, and VLDL-C components in terms of percentage of total cholesterol were similar in these two groups. Higher levels of LDL-C in rabbits that were fed cholesterol plus vitamin E than in those that were fed cholesterol alone have also been reported.“” However, these authors observed that the rises in the serum cholesterol and triglyceride concentrations were significantly lower in the group that consumed cholesterol plus vitamin E as compared with the group that consumed cholesterol alone. The serum concentrations of total cholesterol and triglycerides remained unchanged with vitamin E treatment in patients with hyperproteinemias.66 Lack of effect of tocopherol on plasma lipid and lipoproteins in patients with hypertriglyceridemia has also been reported.67 These differences may have been due to differences in the dosesused by various investigators. Viswanathan et a1.63used 1100 U/kg of dl-cu-tocopherol administered orally. We used 40 mg/kg administered orally, which translates into 140 U/kg. Other investigators@. ” used low doses(300 to 600 mg daily administered orally in human beings). However, an oral dose of 40 mg of vitamin E daily was effective in amelioration of Adriamycin-induced cardiotoxicity in rabbits. 68The dose we used is greater than that used by Milei et al.“i” and would be expected to be an effective dose for antioxidant activity. Vitamin E in control rabbits produced a decrease in serum triglyceride concentrations but no significant changes in concentrations of serum total cholesterol, HDL-C, LDL-C, and VLDL-C. It produced an increase in the HDL-C component and a decrease in the LDL-C and VLDL-C components of total cho-

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Prasad and Kalra

! 0 CONTROL;

0 VIT. E; o CHOL; I S.E,

1 o CHOL + VIT. E;

1

969

2

4

3

TIME (Month) Fig. 14. Sequential changesin the PMN chemiluminescence of the four experimental groups.The results

are expressedas mean + SEM. *p < 0.05, comparisonof the values at different times with respect to “0” in the respective groups. Vit. E, Vitamin E; Chol, cholesterol.ap < 0.05, group I versusgroups II, III, and IV. b p < 0.05, group II versus groups III and IV. Table

III. Changesin the blood MDA of the four groups of rabbits Blood

MDA

Time 0

Groups I II III IV

0.95 1.00 1.05 1.10

+ * k i

0.90 0.55 0.60 1.30

Tk 0.05 + 0.05*7 f O.O5*t f o.ll*f$§

(months) 2

1 0.15 0.01 0.10 0.12

(mmolfL)

0.80 0.55 0.65 1.55

* L f +-

0.90 0.35 0.75 1.55

0.10 0.10*t O.lO*$ o.lz*t$§

Results are expressed as mean + SEM. *p < 0.05, comparison of the values at different times with respect to “0” in the respective tp < 0.05, group I versus groups II, III, and IV. $p < 0.05, group I1 versus groups III and IV. §p < 0.05, group 1r1 versus group IV.

lesterol. The HDL/LDL ratio increased significantly in group II. Both decreases and increases in the concentration of serum lipids have been reported with vitamin E administration in control rabbits and human beings.63,6gViswanathan et a1.63have reported an increase in serum triglycerides, a decrease in LDL-C, and no change in total serum cholesterol in control rabbits that were treated with vitamin E. Komaratat et a1.6gobserved a decrease in total cholesterol, LDL-C, and VLDL-C and an increase in HDL-C with increasing dosesof vitamin E in control rabbits. Various investigators66s67,6g-71have indi-

3 k c + +

0.15 o.o5*t 0.05*$ o.os*t$§

4 0.70 0.35 0.55 1.75

k It k ?

0.10 o.o5*t 0.05*1 0.18*t$§

groups.

cated that vitamin E does not alter plasma lipid concentration in normal subjects. These differences in results could be due to the differences in the dosesof vitamin E or the method of measurement of the lipoproteins. There were no changes in the lipid profile of the rabbits on the control diet in our study. Blood MDA concentration increased in group IV, decreased in groups II and III, and remained unchanged in group I in our study. Vitamin E prevented the increase in MDA levels in group III. Szczeklik et a1.66observed no change in plasma MDA levels in vitamin-E-treated rabbits on a normal or a high-cho-

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American

lesterol diet. They did, however, find an increase in plasma MDA levels in rabbits that consumed a highcholesterol diet. We have reported an increase in the blood MDA concentration with a high-cholesterol diet.4” This discrepancy in the results with vitamin E cannot be attributed to the dose of vitamin E used, because it was similar to that in our study. Aortic tissue MDA concentrations increased in group IV and decreased in groups II and III as compared with group I in the present study. Vitamin E not only prevented the rise in aortic tissue MDA in cholesterolfed rabbits but also reduced the MDA content of aortic tissue of rabbits on the control diet. No data are available on the effects of vitamin E on the aortic tissue MDA level of rabbits that were fed a high-cholesterol or a control diet. Vitamin E was reported to reduce the plasma MDA level in stressed susceptible pigs.72 The water-soluble vitamin E analog Trolox C has been shown to be a powerful scavenger of hydroxyl radicals. 73 Vitamin E added to the culture medium that contained fibroblasts decreased the lipid peroxidationY4 Luminal-dependent chemiluminescence provides a highly sensitive and continuous method for monitoring the rate of production of OFRs by PMNs.“~ Whole blood was used to measure PMN chemiluminescent activity in this study. However, the chemiluminescence of blood was expressed in terms of the blood PMN count. Whole blood has been used by various investigators for chemiluminescence studies.* The increase in OFR-producing activity of PMNs in rabbits on a high-cholesterol diet may be due to increased levels of LTB4,“s PAF 2g and activated complement (C&, CS,).~O It is also possible that receptors for opsonized zymosan on the PMN are increased in hypercholesterolemia. Vitamin E did not affect the OFR-producing activity of PMNs from hypercholesterolemic rabbits. This might be because vitamin E is a lipid-soluble chain-breaking antioxidant that interrupts free radical peroxidative chain reactions.47, 4g.76 If vitamin E limits lipid peroxidation through the chain-breaking mechanism, then one would not expect a change in the OFR-producing activity of PMNs to be caused by vitamin E. However, a water-soluble vitamin E analog, Trolox C, has been shown to be a powerful scavenger of hydroxyl radicals.73+ 76,77Vitamin E is expected to be present in the blood in group 111 and hence should have a chemiluminescent activity lower than that in group IV. We, however, found no difference in the chemiluminescent activity of PMNs from these two groups. In this study we measured only SOD-in-

*References

26, 40, 42, 56, and

75

April 1993 Heart Journal

hibitable chemiluminescence, which represents superoxide anion and not l OH. This may be a reason for the absence of a difference in the PMN chemiluminescence between the two groups in spite of the l OH-scavenging property of vitamin E. In this study the severity of atheromatous lesions in the aorta was associated with hypercholesterolemia in rabbits that were fed a high-cholesterol diet. Similar results have been obtained by other investigators.4”. ‘I* 63-65Ath erogenic diet in the present study produced intimal thickening, which consisted of foam cells that contained lipids. Similar findings have been reported by other investigators in rabbits with atherogenic diets.78, ig Hollander et a1.7R reported subintimal proliferation of collagen fibers, accumulation of acid mucopolysaccharides, breakage of internal elastic lamina, and disorganization of elastic lamellae in addition to intimal thickening. Typical fibrous plaques with subcellular lipoproteinaceous debris observed in the spontaneous atherosclerosis in WHHL rabbitssO were not observed in our study. Our study did not show any such changes in the aortas. The difference in findings could be due to the severity of the atherosclerosis. Our study indicated that vitamin E reduced the extent of plaque formation but did not significantly change the histologic appearance of the lesion. Hypercholesterolemic atherosclerosis was associated with an increase in the OFR-producing activity of PMNs and blood and aortic tissue MDA levels. Increased levels of blood and aortic tissue MDA suggest an increased level of OFRs. Increased OFRs may be due to increased OFR-producing activity of PMNs in this study. However, reactive oxygen metabolites could also be generated by endothelial cells and other bloodbourne or vessel-wall cells.81 Possibly, an inadequate cellular antioxidant defense system may also lead to increased levels of OFRs. OFRs are known to produce endothelial cell injury. 82 Endothelial cell dysfunction may include alterations in regulatory mechanisms such as receptor binding, protein synthesis, and secretion of growth factors.“” A cascade of events, including release of growth-promoting factors and formation of foam cells, will contribute further to development of fibrous plaque, the advanced atherosclerotic lesions. Free radicals and lipid peroxidation may play an important role in the pathogenesis of atherosclerosis. Endothelial cell injury represents a critical initiating event in the pathogenesis of atherosclerosis.‘~” In this study the severity of atherosclerotic lesions in the aorta in high-cholesterol-fed rabbits was effectively prevented by vitamin E. The protective effect of vitamin E could not possibly be attributed to lowering of blood cholesterol levels, since they did

Volume 125, Number 4 American Heart Journal

not decrease in rabbits on a high-cholesterol diet. Total cholesterol, HDL-C, LDL-C, and VLDL-C concentrations were similar in vitamin-E-treated groups and untreated groups on a high-cholesterol diet. The protective effect of vitamin E was associated with a decrease in the blood and aortic tissue MDA in spite of hypercholesterolemia. It, however, did not reduce the OFR-producing activity of PMNs. Vitamin E could have protected against OFRinduced endothelial injury through various mechanisms. Vitamin E is a potent chain-breaking antioxidant, and its primary role is to prevent lipid peroxidative damage in the tissue.47l 4g,I6 Vitamin E traps the chain-propagating peroxyl radicals and thereby reduces the length of the autoxidation chains.76 The effectiveness of vitamin E in prevention of hypercholesterolemic atherosclerosis could also be due to prevention of LDL oxidation. Oxidatively modified LDL has been implicated in atherogenesis.84-s7 This is supported by the fact that probucoi, an antioxidant, which has been shown to attenuate atherogenesis .in rabbits on a high-cholesterol diet,s8 inhibits oxidation of LDL.8g However, it was found that the susceptibility of LDL to oxidation does not correlate well with vitamin E content of the LDL preparations.87 This has been interpreted as the presence of other factor(s) that override it in determining overall susceptibility of LDL to oxidative modification. The results of the present studies suggest that hypercholesterolemia increases the level of OFRs, which would produce endothelial damage and set the stage for development and maintenance of atherosclerosis. In conclusion, these results suggest that hypercholesterolemia increases the level of lipid peroxidation product (MDA) in blood and aorta, and OFR-producing activity of PMNs concomitant with the development of atherosclerosis. Vitamin E prevented the development of atherosclerosis in rabbits on a high-cholesterol diet without lowering the blood cholesterol level and without affecting cholesterolinduced augmentation of OFR-producing activity of PMNs. The protection was associated with a decrease in the blood and aortic tissue MDA levels because vitamin E is a chain-breaking antioxidant. These results suggest a role of OFRs in the genesis and maintenance of hypercholesterolemia-induced atherosclerosis. We thank Dr. John Nyssen for assistance with the histologic studies and Mr. D. Duncan, Ms. Susanne Marcotte, and Mr. P. K. Chattopadhyay for technical assistance. REFERENCES

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