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Glycosaminoglycans and atherosclerosis in animal aortas
Journal of Atherosclerosis Research Elsevier Publishing C o m p a n y , A m s t e r d a m - Printed in The N e t h e r l a n d s
GLYCOSAMINOGLYCANS AND ATHEROSCLEROSIS IN ANIMAL AORTAS
R. N. M U L L I N G E R AND G. M A N L E Y
Nuf~eld Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford (Great Britain) (Received l l t h September, 1968)
Key words:
Glycosaminoglycans Aorta
- Animal
-
Acid
mucopolysaccharides
-
Atherosclerosis
-
disease
Experimental atherosclerosis was first produced in rabbits by feeding them animal protein 1. It was later found to be the cholesterol in the dietary preparations which caused the disease 1. Some species such as the rat and hamster are resistant to experimental atherosclerosis; other species such as the chicken pigeon and pig develop it as readily as the rabbit. More recently spontaneous atherosclerosis has been studied in ageing animals. This resembles more closely the human form of the disease. Pigeons and chickens in captivity are particularly susceptible~. Histological studies have shown greater metachromasia in the aortas of animals susceptible to atherosclerosis than in those resistant to the disease 3. This implies that susceptible aortas contain more glycosaminoglycans. In this study chemical methods have been used to examine the glycosaminoglycan content of 9 animals who differ in their susceptibility to the disease. MATERIALS AND METHODS
The ascending and thoracic aortas were removed from freshly killed adult animals (cat, chicken, guinea-pig, hamster, mouse, pig, pigeon, rabbit, and rat). A segment was taken from each aorta, fixed in mercuric-formol for 24 h and embedded in paraffin for histological studies. Transverse sections (5 #) were stained with Azur A (G. T. Gurr) in 30 % ethanol 4. The remaining aorta was stripped of its adventitial layer, diced, and dried i n v a c u o over silica gel. Single batches of pooled dried aortas (50-100 rag) were digested with twice recrystallized papain5 in phosphate buffer (5-10 ml) and dialysed against distilled water6. Samples from each digest were dried i n v a c u o and taken up in water (50/zl). The glycosaminoglycans in the concentrate were separated by electrophoresis on cellulose acetate and measured by Alcian blue staining and reflection densitometry6. Control samples were treated with DNAase (B.D.H. Dn-c) in 0.02 M magnesium chloride and analysed similarly. The glycosaminoglycans in the remaining aortic digest were purified by cetyl J. Atheroscler: Res., 1969, 9:108~111
SHORT
109
COMMUNICATIONS
pyridinium chloride and alcohol precipitation 7. The alcohol precipitate was finally taken up in water (5 ml) and dialysed (48 h, 4~ The uronic acid content of the purified glycosaminoglycan samples was determined b y a modified carbazole method s. RESULTS
The histochemical results agreed with previous studies 3 with the exception of the pig and rabbit; in this study aortic metachromasia was found to be greater in the pig than in the rabbit. Analysis of the purified samples showed that susceptible species had the largest uronic acid content on a dry weight basis (Table 1), suggesting a higher glycosaminoglycan content. The pigeon and chicken had the highest uronic acid content. TABLE 1 THE
URONIC
ACID CONTENT
OF PURIFIED
GLYCOSAMINOGLYCAN
PREPARATIONS
(HA), H E P A R A N
TIONS OF HYALURONIC ACID DETERMINED BY ELECTROPHORESIS
Species
Cat Chicken Guinea-pig Hamster Mouse Pig Pigeon Rabbit Rat
SULPHATE (HS), AND CHONDROITIN AND ALCIAN BLUE STAINING
AND THE SULPHATE
Uronic acid content (l~g/mg dry weight)
Proportion of glycosaminoglycans HA
HS
CSA
2.47 5.32 2.37 1.76 1.07 4.02 7.60 3.18 1.54
12.0 13.6 7.0 6.5 7.2 10.9 12.8 15.6 12.8
30.7 11:2 10.2 32.5 27.8 17.6 1.0 23.4 30.0
57.3 75.2 82.8 61.0 65.0 71.5 86.2 61.0 57.2
PROPOR(CSA),
Electrophoresis of the aortic digests yielded 3 Alcian blue-positive bands in each case corresponding in mobility with chondroitin sulphates, heparan sulphates, and hyaluronic acid. Treatment with DNAase had no effect on the reflection densitometer tracings. The tracings showed a particularly low level of heparan sulphate relative to chondroitin sulphate and hyaluronic acid in several of the species susceptible to atherosclerosis (Fig. 1; Table 1). The susceptible species in most cases also showed a higher propertion of chondroitin sulphate than the resistant species. DISCUSSION
In a previous study it was shown that the human internal iliac artery, which commonly develops atheroma, contains a lower proportion of heparan sulphate and a higher proportion of chondroitin sulphate than the external iliac 9. In this study a low proportion of heparan sulphate and high proportion of chondroitin sulphate has been found in m a n y of the susceptible species. The particularly low proportion of heparan J. Atheroscler. Res., 1969, 9:108-111
110
SHORT COMMUNICATIONS (a)
(b)
(c)
I, -
HA HS
CSA
+
-
HA HS
CSA
+
-
HA HS
CSA+
Fig. 1. Reflection densitometer tracings of electrophoretically separated glycosaminoglycans showing the proportions of hyaluronic acid (HA), heparan sulphates (HS), and chondroitin sulphates (CSA). The atherosclerosis-susceptible species, chicken (a) and pig (b), are compared with the resistant species rat (c). sulphate relative to chondroitin sulphate and hyaturonic acid, found in some susceptible animals (chicken, guinea-pig, pig, pigeon), is of interest as this glycosaminoglycan pattern is found in embryonic arterial tissue6. It also occurs in the intimal layer of the atheromatous plaque7,10: it is thought to be characteristic of newly synthesized connective tissue. This pattern is most noticeable in the chicken and pigeon, the two animals most prone to develop spontaneous atherosclerosis. The high concentration of glycosaminoglycans found in the arterial wall of susceptible species may encourage the development of atherosclerosis. Glycosaminoglycans because of their high charge and molecular weight are able to inhibit the diffusion of other moleculesll, 1~. The high glycosaminoglycan content of the arterial walls in susceptible species may impede the passage of metabolites into and out of the medial regions of the artery wall. Cholesterol deposition occurs in regions of the rabbit aorta that have developed increased metachromasia in response to cauterization 13. This deposition may be explained by the precipitation of the cholesterol-containing fl-lipoprotein fraction of serum by sulphated glycosaminoglycans in the presence of calcium, as has been demonstrated in vitro 14. Although there are several reasons why a different glycosaminoglycan content might make animals prone to atherosclerosis, the relation between the two factors has not yet been established. It is equally possible that the difference in glycosaminoglycan content and susceptibility to atherosclerosis have a common cause. ACKNOWLEDGEMENTS
The authors thank Dr. J. E. French, Mr. J. Lever and Mr. R. G. Pertwee for help in obtaining material and Mr. J. R. P. O'Brien for facilities and advice. One of us (R.N.M.) is in receipt of an M.R.C. training award. J. Atherosder. Res., 1969, 9:108-111
SHORT COMMUNICATIONS
111
REFERENCES 1 ANITSCHKOW, N. N., A h i s t o r y of e x p e r i m e n t a t i o n on arterial atherosclerosis in a n i m a l s . I n ' H. T. BLUMENTHAL (Ed.), Cowdry's Arteriosclerosis, T h o m a s , Springfield, Ill., 1967, p. 21. 2 MALINOW, M. R. AND B. LEMA, S p o n t a n e o u s c o r o n a r y atherosclerosis in birds, Circulation, 1962, 26: 661. MANClNI, M., O. B. R o s s I , P. ORIENTE AND A. CALl, Possible r e l a t i o n s h i p s b e t w e e n aortic acid m u c o p o l y s a c c h a r i d e s a n d species s u s c e p t i b i l i t y to e x p e r i m e n t a l atherosclerosis, Nature (Lond.), 1965, 207: 1206. 4 KRAMER, H. AND G. M. WINDRUM, T h e m e t a c h r o m a t i c s t a i n i n g reaction, J. Histochem. Cytochem., 19S5, 3: 227. 5 KIMMEL, J. R. AND E. L. S m r H , Crystalline p a p a i n . P r e p a r a t i o n , specificity a n d a c t i v a t i o n , J . biol. Chem., 1954, 207: 515. e MANL~7, G., C h a n g e s in v a s c u l a r m u c o p o l y s a c c h a r i d e s w i t h age a n d blood pressure, Brit. J. exp. Path., 1965, 46: 125. 7 MANL~V, G. AND R. N. MULLINGER, M u c o p o l y s a c c h a r i d e s of atherosclerotie p l a q u e s a n d platelets, Brit. J. exp. Path., 1967, 48: 529. s BITTER, T. AND H. M. MUIR, A modified uronie acid carbazole reaction, Anal. Biochem., 1962, 4: 330. 9 MULLINGER, R. N. AND G. MANLEV, G l y c o s a m i n o g l y c a n s a n d a t h e r o m a in t h e iliac arteries, J . Atheroscler. Res., 1967, 7: 401. 10 KLVNSTRA, F. B., C. J. F. BOTTCHER, J. A. VAN MELSEN AND E. J. vAN DER LAAN, D i s t r i b u tion a n d c o m p o s i t i o n of acid m u c o p o l y s a c c h arides in n o r m a l a n d atherosclerotic h u m a n a o r t a s , J. Atheroscler. Res., 1967, 7: 301. 11 LAURENT, T. C. AND A. PIETRUSKIEWICZ, T h e effect of h y a l u r o n i c acid on t h e s e d i m e n t a t i o n rate of o t h e r s u b s t a n c e s , Biochim. biophys. Acta, 1961, 49: 258. 12 LAURENT, T. C. AND n . PERSSON, T h e i n t e r a c t i o n b e t w e e n p o l y s a c c h a r i d e s a n d o t h e r m a c r o molecules, P a r t 7 (The effect of v a r i o u s p o l y m e r s on t h e s e d i m e n t a t i o n r a t e s of s e r u m a l b u m i n a n d a-crystallin), Biochim. biophys. Acta, 1964, 83: 141. is FABER, M., S u l p h a t e c o n t a i n i n g p o l y u r o n i d e s a n d t h e deposition of cholesterol, Arch. Path., 1949, 48: 342. 14 AMENTA, J. S. AND L. L. WATERS, P r e c i p i t a t i o n of fl-lipoproteins b y aortic m u c o p o l y s a c c h a r i d e s in t h e p r e s e n c e of Ca ++, Yale J. biol. Med., 1960, 33: 112.
J. Atheroscler. Res., 1969, 9 : 1 0 8 - 1 1 1
GLYCOSAMINOGLYCANS AND ATHEROSCLEROSIS IN ANIMAL AORTAS
R. N. M U L L I N G E R AND G. M A N L E Y
Nuf~eld Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford (Great Britain) (Received l l t h September, 1968)
Key words:
Glycosaminoglycans Aorta
- Animal
-
Acid
mucopolysaccharides
-
Atherosclerosis
-
disease
Experimental atherosclerosis was first produced in rabbits by feeding them animal protein 1. It was later found to be the cholesterol in the dietary preparations which caused the disease 1. Some species such as the rat and hamster are resistant to experimental atherosclerosis; other species such as the chicken pigeon and pig develop it as readily as the rabbit. More recently spontaneous atherosclerosis has been studied in ageing animals. This resembles more closely the human form of the disease. Pigeons and chickens in captivity are particularly susceptible~. Histological studies have shown greater metachromasia in the aortas of animals susceptible to atherosclerosis than in those resistant to the disease 3. This implies that susceptible aortas contain more glycosaminoglycans. In this study chemical methods have been used to examine the glycosaminoglycan content of 9 animals who differ in their susceptibility to the disease. MATERIALS AND METHODS
The ascending and thoracic aortas were removed from freshly killed adult animals (cat, chicken, guinea-pig, hamster, mouse, pig, pigeon, rabbit, and rat). A segment was taken from each aorta, fixed in mercuric-formol for 24 h and embedded in paraffin for histological studies. Transverse sections (5 #) were stained with Azur A (G. T. Gurr) in 30 % ethanol 4. The remaining aorta was stripped of its adventitial layer, diced, and dried i n v a c u o over silica gel. Single batches of pooled dried aortas (50-100 rag) were digested with twice recrystallized papain5 in phosphate buffer (5-10 ml) and dialysed against distilled water6. Samples from each digest were dried i n v a c u o and taken up in water (50/zl). The glycosaminoglycans in the concentrate were separated by electrophoresis on cellulose acetate and measured by Alcian blue staining and reflection densitometry6. Control samples were treated with DNAase (B.D.H. Dn-c) in 0.02 M magnesium chloride and analysed similarly. The glycosaminoglycans in the remaining aortic digest were purified by cetyl J. Atheroscler: Res., 1969, 9:108~111
SHORT
109
COMMUNICATIONS
pyridinium chloride and alcohol precipitation 7. The alcohol precipitate was finally taken up in water (5 ml) and dialysed (48 h, 4~ The uronic acid content of the purified glycosaminoglycan samples was determined b y a modified carbazole method s. RESULTS
The histochemical results agreed with previous studies 3 with the exception of the pig and rabbit; in this study aortic metachromasia was found to be greater in the pig than in the rabbit. Analysis of the purified samples showed that susceptible species had the largest uronic acid content on a dry weight basis (Table 1), suggesting a higher glycosaminoglycan content. The pigeon and chicken had the highest uronic acid content. TABLE 1 THE
URONIC
ACID CONTENT
OF PURIFIED
GLYCOSAMINOGLYCAN
PREPARATIONS
(HA), H E P A R A N
TIONS OF HYALURONIC ACID DETERMINED BY ELECTROPHORESIS
Species
Cat Chicken Guinea-pig Hamster Mouse Pig Pigeon Rabbit Rat
SULPHATE (HS), AND CHONDROITIN AND ALCIAN BLUE STAINING
AND THE SULPHATE
Uronic acid content (l~g/mg dry weight)
Proportion of glycosaminoglycans HA
HS
CSA
2.47 5.32 2.37 1.76 1.07 4.02 7.60 3.18 1.54
12.0 13.6 7.0 6.5 7.2 10.9 12.8 15.6 12.8
30.7 11:2 10.2 32.5 27.8 17.6 1.0 23.4 30.0
57.3 75.2 82.8 61.0 65.0 71.5 86.2 61.0 57.2
PROPOR(CSA),
Electrophoresis of the aortic digests yielded 3 Alcian blue-positive bands in each case corresponding in mobility with chondroitin sulphates, heparan sulphates, and hyaluronic acid. Treatment with DNAase had no effect on the reflection densitometer tracings. The tracings showed a particularly low level of heparan sulphate relative to chondroitin sulphate and hyaluronic acid in several of the species susceptible to atherosclerosis (Fig. 1; Table 1). The susceptible species in most cases also showed a higher propertion of chondroitin sulphate than the resistant species. DISCUSSION
In a previous study it was shown that the human internal iliac artery, which commonly develops atheroma, contains a lower proportion of heparan sulphate and a higher proportion of chondroitin sulphate than the external iliac 9. In this study a low proportion of heparan sulphate and high proportion of chondroitin sulphate has been found in m a n y of the susceptible species. The particularly low proportion of heparan J. Atheroscler. Res., 1969, 9:108-111
110
SHORT COMMUNICATIONS (a)
(b)
(c)
I, -
HA HS
CSA
+
-
HA HS
CSA
+
-
HA HS
CSA+
Fig. 1. Reflection densitometer tracings of electrophoretically separated glycosaminoglycans showing the proportions of hyaluronic acid (HA), heparan sulphates (HS), and chondroitin sulphates (CSA). The atherosclerosis-susceptible species, chicken (a) and pig (b), are compared with the resistant species rat (c). sulphate relative to chondroitin sulphate and hyaturonic acid, found in some susceptible animals (chicken, guinea-pig, pig, pigeon), is of interest as this glycosaminoglycan pattern is found in embryonic arterial tissue6. It also occurs in the intimal layer of the atheromatous plaque7,10: it is thought to be characteristic of newly synthesized connective tissue. This pattern is most noticeable in the chicken and pigeon, the two animals most prone to develop spontaneous atherosclerosis. The high concentration of glycosaminoglycans found in the arterial wall of susceptible species may encourage the development of atherosclerosis. Glycosaminoglycans because of their high charge and molecular weight are able to inhibit the diffusion of other moleculesll, 1~. The high glycosaminoglycan content of the arterial walls in susceptible species may impede the passage of metabolites into and out of the medial regions of the artery wall. Cholesterol deposition occurs in regions of the rabbit aorta that have developed increased metachromasia in response to cauterization 13. This deposition may be explained by the precipitation of the cholesterol-containing fl-lipoprotein fraction of serum by sulphated glycosaminoglycans in the presence of calcium, as has been demonstrated in vitro 14. Although there are several reasons why a different glycosaminoglycan content might make animals prone to atherosclerosis, the relation between the two factors has not yet been established. It is equally possible that the difference in glycosaminoglycan content and susceptibility to atherosclerosis have a common cause. ACKNOWLEDGEMENTS
The authors thank Dr. J. E. French, Mr. J. Lever and Mr. R. G. Pertwee for help in obtaining material and Mr. J. R. P. O'Brien for facilities and advice. One of us (R.N.M.) is in receipt of an M.R.C. training award. J. Atherosder. Res., 1969, 9:108-111
SHORT COMMUNICATIONS
111
REFERENCES 1 ANITSCHKOW, N. N., A h i s t o r y of e x p e r i m e n t a t i o n on arterial atherosclerosis in a n i m a l s . I n ' H. T. BLUMENTHAL (Ed.), Cowdry's Arteriosclerosis, T h o m a s , Springfield, Ill., 1967, p. 21. 2 MALINOW, M. R. AND B. LEMA, S p o n t a n e o u s c o r o n a r y atherosclerosis in birds, Circulation, 1962, 26: 661. MANClNI, M., O. B. R o s s I , P. ORIENTE AND A. CALl, Possible r e l a t i o n s h i p s b e t w e e n aortic acid m u c o p o l y s a c c h a r i d e s a n d species s u s c e p t i b i l i t y to e x p e r i m e n t a l atherosclerosis, Nature (Lond.), 1965, 207: 1206. 4 KRAMER, H. AND G. M. WINDRUM, T h e m e t a c h r o m a t i c s t a i n i n g reaction, J. Histochem. Cytochem., 19S5, 3: 227. 5 KIMMEL, J. R. AND E. L. S m r H , Crystalline p a p a i n . P r e p a r a t i o n , specificity a n d a c t i v a t i o n , J . biol. Chem., 1954, 207: 515. e MANL~7, G., C h a n g e s in v a s c u l a r m u c o p o l y s a c c h a r i d e s w i t h age a n d blood pressure, Brit. J. exp. Path., 1965, 46: 125. 7 MANL~V, G. AND R. N. MULLINGER, M u c o p o l y s a c c h a r i d e s of atherosclerotie p l a q u e s a n d platelets, Brit. J. exp. Path., 1967, 48: 529. s BITTER, T. AND H. M. MUIR, A modified uronie acid carbazole reaction, Anal. Biochem., 1962, 4: 330. 9 MULLINGER, R. N. AND G. MANLEV, G l y c o s a m i n o g l y c a n s a n d a t h e r o m a in t h e iliac arteries, J . Atheroscler. Res., 1967, 7: 401. 10 KLVNSTRA, F. B., C. J. F. BOTTCHER, J. A. VAN MELSEN AND E. J. vAN DER LAAN, D i s t r i b u tion a n d c o m p o s i t i o n of acid m u c o p o l y s a c c h arides in n o r m a l a n d atherosclerotic h u m a n a o r t a s , J. Atheroscler. Res., 1967, 7: 301. 11 LAURENT, T. C. AND A. PIETRUSKIEWICZ, T h e effect of h y a l u r o n i c acid on t h e s e d i m e n t a t i o n rate of o t h e r s u b s t a n c e s , Biochim. biophys. Acta, 1961, 49: 258. 12 LAURENT, T. C. AND n . PERSSON, T h e i n t e r a c t i o n b e t w e e n p o l y s a c c h a r i d e s a n d o t h e r m a c r o molecules, P a r t 7 (The effect of v a r i o u s p o l y m e r s on t h e s e d i m e n t a t i o n r a t e s of s e r u m a l b u m i n a n d a-crystallin), Biochim. biophys. Acta, 1964, 83: 141. is FABER, M., S u l p h a t e c o n t a i n i n g p o l y u r o n i d e s a n d t h e deposition of cholesterol, Arch. Path., 1949, 48: 342. 14 AMENTA, J. S. AND L. L. WATERS, P r e c i p i t a t i o n of fl-lipoproteins b y aortic m u c o p o l y s a c c h a r i d e s in t h e p r e s e n c e of Ca ++, Yale J. biol. Med., 1960, 33: 112.
J. Atheroscler. Res., 1969, 9 : 1 0 8 - 1 1 1