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Changes in the content of human aortic glycoproteins and acid mucopolysaccharides in atherosclerosis
Atherosclerosis, 18 (1973) 83-91 0 Elsevier Scientific Publishing Company,
83 Amsterdam
- Printed in The Netherlands
CHANGES IN THE CONTENT OF HUMAN AORTIC GLYCOPROTEINS AND ACID MUCOPOLYSACCHARIDES IN ATHEROSCLEROSIS
PREMANAND C. READ
V. WAGH*,
BRUCE
I. ROBERTS,
HAROLD
.J. WHITE
AND
RAYMOND
Connective Tissue Laboratory, Veterans Administration Hospital, and Departments of Biochemistry, Pathology and Surgery, University of Arkansas Medical Center, Little Rock, Ark. 72201 (U.S.A.)
(Received September 7th, 1972)
SUMMARY
The concentration of glycoproteins (GP) and acid mucopolysaccharides (AMPS) in apparently normal and atherosclerotic regions of human aortae taken at autopsy was determined both in terms of intimal surface area and dry, defatted, ash-free residue weight (DDAF). Variability due to age was excluded by choosing samples within the same age group. Total GP concentration increased significantly in atherosclerotic tissue as evidenced by increase in total neutral sugars, hexosamine and sialic acid both in terms of surface area and DDAF. The total uranic acid content was not altered. However, the ratio of sulfated:non-sulfated AMPS was significantly higher in atherosclerotic regions indicating that sulfated AMPS concentration is increased in atherosclerotic plaques at the expense of hyaluronic acid. These observations suggest that increased GP-concentration may have a functional role in the formation ofatheroscleroticplaque.
Key words:
Acid mucopolysaccharides - Aorta - Atherosclerosis - Glycoproteins
INTRODUCTION
From a great number of histochemical and chemical studies, it has been evident that the ground substance of the human aortic wall contains glycoproteins (GP) and acid mucopolysaccharides (AMPS) as its integral components. A great deal of research interest is presently centered upon the relationship of the concentration of these macro-
This study was supported by the Veterans Administration * To whom to address correspondence.
Research Funds.
84
P. V. WAGH,
B. I. ROBERTS,
H. J. WHITE,
R. C. READ
molecules in the aortic wall with atherosclerosis and aging, with a view to understanding the etiology of these processes. There has been a good deal of confusion, however, due to conflicting reports. This subject is extensively reviewedr. The present investigation was undertaken with a view to studying the quantitative changes in the carbohydrate composition of uninvolved and sclerotic regions of the aorta. Since changes in the carbohydrate-containing macromolecules in the aortic wall are associated with both advancing age and atherosclerosis and that the wet weight of the aorta is increased due to the thickening of the blood vessel, we have attempted to evaluate the data in terms of lipid and ash-free tissue dry weight as well as in terms of intimal surface area of aortae from individuals within the same age group. MATERIALS
AND METHODS
Samples
Thoracic aortae were obtained at necropsy from 41 veterans 70 f 2 (mean f standard error) years of age. Cadavers had been held at 4°C between 10-I 5 hours after death. The tissues were stored at -20 “C in a freezer in thick plastic air-tight bags until use. Each aorta was thawed at room temperature and the adventitia was carefully removed with the aid of forceps. The adventitia-free tissue was briefly rinsed in tap water to wash off adherent blood. The cleaned tissue was blotted on paper towels. From each aorta, two discs of constant area were punched out using a l-cm diameter (0.785 cm2 area) stainless steel punch and the wet weight was recorded. One of these represented the apparently “uninvolved” region which did not exhibit abnormal fatty infiltration and the other represented the “fibrosclerotic plaque” region which could be visually identified as a distinct plaque. Throughout this paper, these specimens will be referred to as “normal” and “atherosclerotic” regions, respectively. Ulcerated areas and those with obvious calcification were deliberately excluded from the study. Distribution of specimens used for chemical analyses
Out of the 41 aortae used in this study, 20 were used for analyses of the moisture content. From these 20 dried specimens, 8 were used for the determination of ash content and 9 for the estimation of collagen. Total neutral lipids were determined from six separate aortae. Another 15 were used for the study of carbohydrate composition and AMPS analyses. Moisture, ash and lipid determination
Samples were dried at 70°C for 40 hours in a vacuum oven connected to a Drierite trap. The difference between the initial wet weight and the weight of the dehydrated tissue was expressed as the moisture content. For the estimation of total lipid, onehalf of each sample was minced and ground in 4 ml of chloroform-methanol mixture (2: 1, v/v). After centrifugation, the clear supernatant was decanted off. The sediment was reextracted with 4 ml of the above lipid extractant and the super-
GLYCOPROTEINS IN ATHEROSCLEROSIS
85
natant again decanted off. The sediment was washed with acetone and after decanting off the acetone, 2 ml of anhydrous ether was added to the residue. The contents were held at 37 “C for 30 min and the ether was removed by decantation. The residue was washed with acetone and dehydrated as in the moisture analyses. Throughout this procedure, care was taken to recover the residue quantitatively. The other half of the sample was used for moisture analyses. The total lipid content was determined as the difference between the weight of the dehydrated tissue and the weight of the dehydrated and delipidated material. Ash was determined gravimetrically after ashing the dry samples at 400 “C for 24 hours in a muffle furnace. Collagen determination Dried tissues were hydrolyzed in 6N HCl at 130 “C for 4 hours. The hydroxyproline content in the hydrolysate was determined by the procedure of Woessner2. The percentage of collagen was expressed by multiplying the percent hydroxyproline values by a factor of 6.94 (ref. 3). Carbohydrates and AMPS determination For the determination of carbohydrates and AMPS, the samples were hydrolyzed with papain. In order to avoid interference of lipid in proteolytic digestion, the samples were treated as described by Mier and Wood4. Each sample was washed with 2 ml of acetone and then extracted with 5 ml of ether-acetone mixture (3 : 1, v/v) at 37 “C for one hour. After decanting off the solvent, the tissue was washed twice with acetone and dried at 70 “C for 16 hours. Although this delipidating procedure was not exhaustive, the samples were quite suitable for proteolytic digestion. The incubation mixture contained the partially delipidated and dried tissue, 0.94 ml of sodium acetate buffer (0.2 M containing 2 mg cysteine HCl/ml, final pH 7.0) and papain (2 x crystallized, 2.0 mg in 0.06 ml, Worthington Biochemical Corporation). The incubation was carried out at 65 “C for 72 hours at which time the digestion of the tissue was apparently complete as seen by total disintegration of the tissue. During incubation, the reaction tubes were occasionally agitated to ensure thorough mixing. The incubation mixture was cooled in an ice-bath and 0.2 ml ice-cold trichloroacetic acid (TCA, 50% w/v) was added. The contents were mixed and centrifuged at 3000 r.p.m. for 10 min. The supernatant and the precipitate were separated. The precipitate was extracted with 0.5 ml of distilled water and the extract was centrifuged as above. The supernatant (washing) was pooled with the initial TCAsupernatant. The TCA precipitate was dried in a vacuum oven at 70 “C and stored for analyses. Aliquots from TCA-soluble fraction were analyzed for constituent carbohydrate composition. Hexose was determined by phenol-sulfuric acids, using galactose as the standard. Hexosamine was analyzed by the method of Gatt and Berman6, after hydrolysis in 2N HCI for 16 hours at 100 “C. Glucosamine hydrochloride was used as the standard and the values were reported as free base. Hexuronic acid was quantitated by the carbazole method of Bitter and Muir7 employing glucuronolactate
86
P. V. WAGH, B. I. ROBERTS, H. J. WHITE, R. C. READ
as the standard. Sialic acid was determined by the procedure of Aminoff* after hydrolysis with 0.1 N HCI for one hour at 80 “C. The amount of each carbohydrate component in the total volume of TCAsoluble fraction was divided by a factor of 0.785 and was expressed as the total amount of that component present in one cm2 area of the tissue. The ratio of sulfated:non-sulfated AMPS was estimated as follows: To 0.8 ml of TCA-soluble fraction, 0.4 ml of distilled water was added and the solution was dialyzed against running tap water for 16 hours. The dialyzed material was transferred to a centrifuge tube to which 0.4 ml of cetylpyridinium chloride (CPC, 5 % w/v in 0.15 A4 NaCl) and 0.4 ml celite (IO’% w/v) were added. The contents were mixed and incubated at 37°C for one hour and centrifuged. The supernatant was discarded. The precipitate was washed with 1.0 ml (0.03 M NaCl containing 0.1% w/v CPC), centrifuged and supernatant again discarded. The resulting precipitate was successively extracted with two 0.5 ml portions of each of the following reagents: 0.40 M NaCl, 1.2 M NaCl and 2.1 M NaCl (each containing 0.1% CPC, w/v). The extracts corresponding to each individual reagent were pooled and analyzed for uranic acid. Since in our fractionation procedure, we observed that there was an extensive carry-over of uranic acid from 1.2 M to 2.1 A4 NaCl fraction (due to imcomplete extraction with 1.2 M NaCl), we pooled these fractions. The AMPS occurring in the 0.4 M NaCl fraction was called non-sulfated AMPS (hyaluronic acid) and that in the pooled 1.2 M NaCl fraction was called the sulfated AMPS (chondroitin sulfates, heparitin sulfate and heparin). RESULTS
Table 1 gives the percentage composition of moisture, lipid and ash in normal TABLE 1 MOISTURE,
LIPID
AND
ASH CONTENT
OF NORMAL
AND
ATHEROSCLEROTIC
AORTAE
The components are expressed as % of the wet weight of the tissue. The values for lipid were calculated as: % (lipid plus moisture) - % (moisture). These determinations were made on the same samples using one-half portion for lipid plus moisture and the other half for moisture analyses. The dry, defatted, ash-free residue (DDAF) is given as the percentage by difference. Component
Age (years) mean i- S.E.M.
N
Normal mean f S.E.M.
Atherosclerotic mean & S. E.M.
Paired t
Moisture Lipid Ash Dry, defatted ash-free residue (DDAF)
70.4 f 2.5 72.0 f 1.5 69.7 zt 3.6
20 6 8
73.77 f 0.31 5.46 f 1.15 0.68 f 0.06
73.63 + 0.45 10.82 * 2.02 0.79 & 0.08
0.22NS 9.92& 1.35NS
-
-
20.09
14.76
-
= P < 0.01. NS = not significant.
87
GLYCOPROTEINS IN ATHEROSCLEROSIS
and atherosclerotic regions of the 41 aortae investigated. The moisture and the ash content did not differ. The amount of lipid in atherosclerotic region was twice as much as the quantity present in the apparently normal tissue. In order to determine the concentrations of collagen and carbohydrate constituents it was therefore desirable TABLE 2 COLLAGEN
AND
TOTAL
CARBOHYDRATE
CONTENT
OF NORMAL
AND ATHEROSCLEROTIC
AORTAE
The components which are expressed as % of the dry, defatted, ash-free residue (DDAF) were calculated by multiplying the wet weight of normal and atherosclerotic tissue by 0.2009 and 0.1476, respectively. These factors were derived from data given in Table 1. Total carbohydrate is expressed as the sum of neutral sugars, hexosamine, hexuronic acid and sialic acid. The residue weight is given by difference. Component
Age (yrs) mean f S.E.M.
N
Normal mean + S.E.M.
Atherosclerotic mean + S.E.M.
Paired t
Collagen Total carbohydrate Residue
73.4 f 3.9 70.3 & 2.8 -
9 15
24.8 & 2.3 5.54 * 0.30 69.7
35.4 zt 6.8 6.37 f 0.27 58.2
4.98& 4.61a
a P < 0.01. TABLE 3 CONCENTRATION
OF COMPONENT
SUGARS
OF NORMAL
AND ATHEROSCLEROTIC
AORTAE
The concentration of components are expressed as weight per unit area and weight per unit dry, defatted, ash-free residue (DDAF). The weight of DDAF was calculated by multiplying the wet weight of normal and atherosclerotic tissue by 0.2009 and 0.1476, respectively. These factors were derived from data given in Table 1. All values represent averages from 15 samples of aortae obtained from individuals of 70.3 =t 2.8 (mean & S.E.M.) years of age. Component
Normal mean + S.E.M.
Atherosclerotic mean * S.E.M.
Paired t
Wet weight (mg/cm2) DDAF (mg/cm2) Total carbohydrate (mg/cmz) Hexose (pg/cm2) Hexosamine (pg/cm2) Uranic acid (pg/cm2) Sialic acid @g/cm2) Total carbohydrate (pg/mg DDAF) Hexose (pg/mg DDAF) Hexosamine (pg/mg DDAF) Uranic acid (pg/mg DDAF) Sialic acid (pg/mg DDAF)
195.7 f 9.2 39.3 i 1.9 2.10 i 0.04 870 f 32 651 f 16 297 + 12 285 f 14 55.4 i 3.0 23.1 * 1.6 17.0 f 0.8 7.9 & 0.6 7.5 * 0.4
250.4 36.9 2.30 978 698 308 317 63.7 27.1 19.4 8.5 8.8
8.96a 2.30b 4.15” 3.30” 2.70c I.OlNS 2.19” 4.61” 4.24” 3.25=
&P < 0.01. b P < 0.05. c P > 0.02 NS = Not significant.
5 11.2 I-t 1.7 f 0.06 5 37 + 23 * 15 & 20 f 2.7 f 1.4 i 0.9 zk 0.4 * 0.5
1.80NS 3.87a
88
P. V. WAGH,
B. I. ROBERTS,
H. J. WHITE,
R. C. READ
to express all values in terms of dry, defatted, ash-free residue weight (DDAF) of the tissue. The calculated DDAF values of 20.09 % and 14.76 % were obtained for normal and atherosclerotic tissues, respectively, and were used throughout to express the quantities of various parameters in terms of DDAF. Table 2 shows the values for collagen and total carbohydrate expressed as % of DDAF. It is apparent that both collagen as well as the total carbohydrate content were significantly higher in atherosclerotic regions. The residue weight which was calculated by difference is lower in the atherosclerotic tissue. This residue should mostly consist of elastin and other non-collagenous proteins. The quantities of individual monosaccharides in the carbohydrate component are shown in terms of unit intimal surface area and per unit DDAF (Table 3). The net weight per unit area of the atherosclerotic tissue was significantly higher than the normal. This was expected since the increase in weight may be attributed to deposition of lipid and other materials which infiltrate the vascular wall in the plaque formation. The calculated DDAF per unit area was significantly lower in the atherosclerotic region. Hexose, hexosamine and sialic acid content increased in the atherosclerotic tissue. These increases were statistically significant indicating that there is a quantitative increase in GP-concentration in atherosclerotic plaques. It is interesting to note that total uranic acid content was not altered indicating that the total AMPS remain unchanged in atherosclerotic tissue. The ratio of sulfated:non-sulfated AMPS was significantly higher in atherosclerotic tissue (Table 4) indicating a substantial increase in sulfated AMPS in atherosclerosis and a decrease in hyaluronic acid. The recoveries of AMPS after fractionation were found to be 74 % of the initial concentration. We were unable to detect measurable carbohydrate constituents in the TCA precipitates indicating that virtually all the glycoproteins and AMPS remained in the TCA supernatants.
TABLE 4 RATIOS
OF SULFATED:NON-SULFATED
ACID
MLJCOPOLYSACCHARIDES
IN NORMAL
AND
ATHEROSCLEROTIC
AORTAE
The definition for sulfated and non-sulfated acid mucopolysaccharides is Methods. Percent recovery is expressed as the total uranic acid recovered in precipitation as related to the initial amount of the uranic acid in the original averages from 15 samples of aortae, obtained from individuals of 70.3 5 years of age. Normal mean f
Ratio sulfated: non-sulfated (AMPS) Recovery (%)
a P < 0.05.
S.E.M.
6.33 Ilt 0.44 73.8 IIZ3.3
given in Materials and all fractions after CPCtissue. Values represent 2.8 (mean k S.E.M.)
Atherosclerotic mean f S.E.M.
Paired t
8.00 k 0.61 72.9 f 2.9
2.31” 0.24
GLYCOPROTEINS
IN ATHEROSCLEROSIS
89
DISCUSSION
In order to provide meaningful comparison between normal and atherosclerotic tissues, it is apparent that parameters expressed in terms of tissue wet weights or dry weights would be erroneous since in non-calcific atherosclerotic lesions the amount of lipid is increased two-fold as compared to the normal. Also since atherosclerotic changes are accompanied with increasing age, it seemed necessary to test the comparisons by eliminating the age variable by choosing samples within the same age group. Several conflicting reports on changes in aortic GP and AMPS can be related to the units of various parameters in which they are expressed or to the variability in sampling of the tissue. For instance, in most cases the composition of the tissue has been expressed in terms of wet weight, dry weight or defatted dry weight without regard to the contribution due to minerals. An increase, a decrease or no change in any parameter should be demonstrated best if the results are calculated on the basis of DDAF. This is best exemplified in a recent report of Anastassiades et uZ.~,wherein the analytical results were done on the basis of nitrogen content of the tissue which is a rough estimate of DDAF. If it is assumed that the thickening of the aortic wall is a result of deposition of certain components on or within the intima, then composition expressed in terms of intimal surface area should be a useful measure in comparisons between normal and atherosclerotic tissue. Manley and Mullinger 10 have reported data on carbohydrate composition of unaffected and atherosclerotic human aorta of one individual in term of tissue dry weight and intimal surface area. From our results, it is clear that the total carbohydrate and collagen content in atherosclerotic region is higher than in normal in terms of DDAF and surface area. Although we have not measured elastin and other non-collagenous components in the residual fraction, it is apparent that there should be a corresponding decrease of this component in atherosclerotic tissue. The increase of hexose, hexosamine, and sialic acid both in terms of DDAF and surface area in atherosclerotic tissue indicates a definite increased GP-concentration. Sialic acid is a good marker to support this statement since except for glycolipids which are removed by organic solvents, sialic acid is an integral part of sialoproteins. The observation that GP-concentration increases in atherosclerotic conditions is consonant with that of Anastassiades et ~1.9. Our data on sialic acid are in conflict with those of Murata and Kirkll, Manley and Mullingerls and of Kotoku et al.12, who have reported a decrease in sialic acid in atherosclerotic tissue. The discrepancy in the data of Manley and Mullingerla and those of ours seems to be in the ratio of dry weight/cm2 area of atherosclerotic:normal tissues which by calculations are 2.5 and 1.3, respectively. We have observed that only in highly-infiltrated regions of aorta undergoing necrosis and calcification the ratio was higher than 2. As such we have avoided such samples in our study. Our results indicate that the total AMPS content in normal and atherosclerotic tissue is not changed. This finding is in accordance with the observation of Anastassiades et cd9 who have expressed the results in terms of nitrogen or DNA content of the
P. V. WAGH,
90
B. I. ROBERTS,
H. J. WHITE,
R. C. READ
tissue. It is, however, in disagreement with the observation of Bdttcher and Klynstrara and Nakamura et al.14 who have reported a decrease in AMPS content. The higher ratio of sulfated :non-sulfated AMPS implies that in atherosclerosis the concentration of sulfated AMPS is increased at the expense of hyaluronic acid which is the only known non-sulfated AMPS in the aorta. Presently the role of glycoproteins in atherosclerosis is not known. However, some report+-17 have indicated that certain glycoproteins in serum and various organs are inhibitors of plasma lipoprotein lipase. Indeed, in our laboratory we have observed that a highly-purified glycoprotein isolated from the intimal region of porcine aorta18 acts as an inhibitor of lipoprotein lipase from post-heparin dog plasmalg. As a hypothesis, we suggest that increased concentration of glycoproteins in atherosclerosis may be related to the inhibition of the enzyme lipoprotein lipase which has an important role in the lipid transport. ACKNOWLEDGEMENTS
This study was carried out with the technical assistance of Mrs. Marian Epperson and Mrs. Victoria Drake. We wish to thank Dr. Dennis Robinette for computer analyses and to Mrs. Diane Butler for secretarial assistance.
REFERENCES 1 BERENSON,G. S., RADHAKRISHNAMURTHY, B., DALFERES,E. R., AND SRINIVASAN,S. R., Carbohydrate macromolecules and atherosclerosis, Human Path., 2 (1971) 57. 2 WOESSNER,JR. J. F., The determination of hydroxyproline in tissue and protein samples containing small proportions of this aminoacid, Arch. Biochem. Biophys., 93 (1961) 440. 3 JACKSON,D. S., AND CLEARY, E. G., The determination of collagen and elastin, In: D. GLICK (Ed.), Methods ofBiochemical Analysis, Vol. IS, Interscience, New York, N.Y., 1967, Ch. 15, p. 31. 4 MIER, P. D., AND WOOD, M., A simplified technique for the analysis of tissue acid mucopolysaccharides, Clin. Chim. Acta, 24 (1969) 105. 5 DUBOIS,M., GILLES,K. A., HAMILTON,J. K., REBERS,P. A., AND SMITH, F., Calorimetric method for determination of sugars and related substances, Anal. Chem., 28 (1956) 350. 6 GATT, R., AND BERMAN,E. R., A rapid procedure for the estimation of aminosugars on a micro scale, Anal. Biochem., 15 (1966) 167. 7 BITTER, T., AND MUIR, H. M., A modified uranic acid carbazole reaction, Anal. Biochem., 4
(1962) 330. 8 AMINOFF, D., Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids, Biochem. J., 81 (1961) 384. T., ANASTASSIADIS,P. A., AND DENSTEDT,0. F., Changes in connective tissue 9 ANASTASSIADES, of the atherosclerotic intima and media of the aorta, Biochim. Biophys. Acta, 261 (1972) 418. 10 MANLEY, G., AND MULLINGER, R. N., Mucopolysaccharides of atherosclerotic plaques and platelets, Brit. J. Exp. Path., 58 (1967) 529. 11 MURATA, K., AND KIRK, J. E., Sialic acid content of human arterial and venous tissue, J. Athero-
scler. Rex, 2 (1962) 452, 12 KOTOKLJ,T., WATANABE, K., OYAMA, K., TOKITA, K., OHBA, T., composition
of glycoproteins
AND NAKAMURA, T., Sugar isolated from normal and sclerotic human aortas, Atherosclerosis,
14 (1971) 411. 13 BBT~CHER,C. J. F., AND KLYNSTRA, F. B., Content of acid mucopolysaccharides aorta, J. Atheroscler. Res., 2 (1962) 263.
in the human
91
GLYCOPROTEINS IN ATHEROSCLEROSIS
14NAKAMURA, T., TOKITA, K., TATENO, S., KOTOKU, T., ANDOHBA,T., Human aortic acid mucopolysaccharides and glycoproteins. Changesduringageingand in atherosclerosis, J. Atheroscler. Res., 8 (1968)891. 15 KLEIN, E., AND LEVER, W. F., Inhibition of lipemia-clearing activity by serum of patients with hyperlipemia, Proc. Sot. Exp. Biol. Med., 15 (1957) 565. 16 HOLLETT,C. R., An inhibitor of the lipolytic reaction from post-heparin plasma of normal dogs, Biochim. Biophys. Acta, 98 (1965) 53. 17 ISHII, M., Relationship between a glycoprotein sclerosis, Jap. Heart J., 12 (1971) 22. 18 WAGH, P. V., AND ROBERTS,B. I., Purification intimal region of porcine aorta, Biochemistry, 19 WAGH, P. V., AND ROBERTS,B. I., Inhibition intimal glycoprotein. In: 28th Southwestern
Baton Rouge, La., December 1972, p. 45.
acting as lipoprotein
lipase inhibitor and arterio-
and characterization of a glycoprotein from the 11 (1972) 4222. of plasma lipoprotein lipase by a highly purified Regional Meeting, American Chemical Society,
83 Amsterdam
- Printed in The Netherlands
CHANGES IN THE CONTENT OF HUMAN AORTIC GLYCOPROTEINS AND ACID MUCOPOLYSACCHARIDES IN ATHEROSCLEROSIS
PREMANAND C. READ
V. WAGH*,
BRUCE
I. ROBERTS,
HAROLD
.J. WHITE
AND
RAYMOND
Connective Tissue Laboratory, Veterans Administration Hospital, and Departments of Biochemistry, Pathology and Surgery, University of Arkansas Medical Center, Little Rock, Ark. 72201 (U.S.A.)
(Received September 7th, 1972)
SUMMARY
The concentration of glycoproteins (GP) and acid mucopolysaccharides (AMPS) in apparently normal and atherosclerotic regions of human aortae taken at autopsy was determined both in terms of intimal surface area and dry, defatted, ash-free residue weight (DDAF). Variability due to age was excluded by choosing samples within the same age group. Total GP concentration increased significantly in atherosclerotic tissue as evidenced by increase in total neutral sugars, hexosamine and sialic acid both in terms of surface area and DDAF. The total uranic acid content was not altered. However, the ratio of sulfated:non-sulfated AMPS was significantly higher in atherosclerotic regions indicating that sulfated AMPS concentration is increased in atherosclerotic plaques at the expense of hyaluronic acid. These observations suggest that increased GP-concentration may have a functional role in the formation ofatheroscleroticplaque.
Key words:
Acid mucopolysaccharides - Aorta - Atherosclerosis - Glycoproteins
INTRODUCTION
From a great number of histochemical and chemical studies, it has been evident that the ground substance of the human aortic wall contains glycoproteins (GP) and acid mucopolysaccharides (AMPS) as its integral components. A great deal of research interest is presently centered upon the relationship of the concentration of these macro-
This study was supported by the Veterans Administration * To whom to address correspondence.
Research Funds.
84
P. V. WAGH,
B. I. ROBERTS,
H. J. WHITE,
R. C. READ
molecules in the aortic wall with atherosclerosis and aging, with a view to understanding the etiology of these processes. There has been a good deal of confusion, however, due to conflicting reports. This subject is extensively reviewedr. The present investigation was undertaken with a view to studying the quantitative changes in the carbohydrate composition of uninvolved and sclerotic regions of the aorta. Since changes in the carbohydrate-containing macromolecules in the aortic wall are associated with both advancing age and atherosclerosis and that the wet weight of the aorta is increased due to the thickening of the blood vessel, we have attempted to evaluate the data in terms of lipid and ash-free tissue dry weight as well as in terms of intimal surface area of aortae from individuals within the same age group. MATERIALS
AND METHODS
Samples
Thoracic aortae were obtained at necropsy from 41 veterans 70 f 2 (mean f standard error) years of age. Cadavers had been held at 4°C between 10-I 5 hours after death. The tissues were stored at -20 “C in a freezer in thick plastic air-tight bags until use. Each aorta was thawed at room temperature and the adventitia was carefully removed with the aid of forceps. The adventitia-free tissue was briefly rinsed in tap water to wash off adherent blood. The cleaned tissue was blotted on paper towels. From each aorta, two discs of constant area were punched out using a l-cm diameter (0.785 cm2 area) stainless steel punch and the wet weight was recorded. One of these represented the apparently “uninvolved” region which did not exhibit abnormal fatty infiltration and the other represented the “fibrosclerotic plaque” region which could be visually identified as a distinct plaque. Throughout this paper, these specimens will be referred to as “normal” and “atherosclerotic” regions, respectively. Ulcerated areas and those with obvious calcification were deliberately excluded from the study. Distribution of specimens used for chemical analyses
Out of the 41 aortae used in this study, 20 were used for analyses of the moisture content. From these 20 dried specimens, 8 were used for the determination of ash content and 9 for the estimation of collagen. Total neutral lipids were determined from six separate aortae. Another 15 were used for the study of carbohydrate composition and AMPS analyses. Moisture, ash and lipid determination
Samples were dried at 70°C for 40 hours in a vacuum oven connected to a Drierite trap. The difference between the initial wet weight and the weight of the dehydrated tissue was expressed as the moisture content. For the estimation of total lipid, onehalf of each sample was minced and ground in 4 ml of chloroform-methanol mixture (2: 1, v/v). After centrifugation, the clear supernatant was decanted off. The sediment was reextracted with 4 ml of the above lipid extractant and the super-
GLYCOPROTEINS IN ATHEROSCLEROSIS
85
natant again decanted off. The sediment was washed with acetone and after decanting off the acetone, 2 ml of anhydrous ether was added to the residue. The contents were held at 37 “C for 30 min and the ether was removed by decantation. The residue was washed with acetone and dehydrated as in the moisture analyses. Throughout this procedure, care was taken to recover the residue quantitatively. The other half of the sample was used for moisture analyses. The total lipid content was determined as the difference between the weight of the dehydrated tissue and the weight of the dehydrated and delipidated material. Ash was determined gravimetrically after ashing the dry samples at 400 “C for 24 hours in a muffle furnace. Collagen determination Dried tissues were hydrolyzed in 6N HCl at 130 “C for 4 hours. The hydroxyproline content in the hydrolysate was determined by the procedure of Woessner2. The percentage of collagen was expressed by multiplying the percent hydroxyproline values by a factor of 6.94 (ref. 3). Carbohydrates and AMPS determination For the determination of carbohydrates and AMPS, the samples were hydrolyzed with papain. In order to avoid interference of lipid in proteolytic digestion, the samples were treated as described by Mier and Wood4. Each sample was washed with 2 ml of acetone and then extracted with 5 ml of ether-acetone mixture (3 : 1, v/v) at 37 “C for one hour. After decanting off the solvent, the tissue was washed twice with acetone and dried at 70 “C for 16 hours. Although this delipidating procedure was not exhaustive, the samples were quite suitable for proteolytic digestion. The incubation mixture contained the partially delipidated and dried tissue, 0.94 ml of sodium acetate buffer (0.2 M containing 2 mg cysteine HCl/ml, final pH 7.0) and papain (2 x crystallized, 2.0 mg in 0.06 ml, Worthington Biochemical Corporation). The incubation was carried out at 65 “C for 72 hours at which time the digestion of the tissue was apparently complete as seen by total disintegration of the tissue. During incubation, the reaction tubes were occasionally agitated to ensure thorough mixing. The incubation mixture was cooled in an ice-bath and 0.2 ml ice-cold trichloroacetic acid (TCA, 50% w/v) was added. The contents were mixed and centrifuged at 3000 r.p.m. for 10 min. The supernatant and the precipitate were separated. The precipitate was extracted with 0.5 ml of distilled water and the extract was centrifuged as above. The supernatant (washing) was pooled with the initial TCAsupernatant. The TCA precipitate was dried in a vacuum oven at 70 “C and stored for analyses. Aliquots from TCA-soluble fraction were analyzed for constituent carbohydrate composition. Hexose was determined by phenol-sulfuric acids, using galactose as the standard. Hexosamine was analyzed by the method of Gatt and Berman6, after hydrolysis in 2N HCI for 16 hours at 100 “C. Glucosamine hydrochloride was used as the standard and the values were reported as free base. Hexuronic acid was quantitated by the carbazole method of Bitter and Muir7 employing glucuronolactate
86
P. V. WAGH, B. I. ROBERTS, H. J. WHITE, R. C. READ
as the standard. Sialic acid was determined by the procedure of Aminoff* after hydrolysis with 0.1 N HCI for one hour at 80 “C. The amount of each carbohydrate component in the total volume of TCAsoluble fraction was divided by a factor of 0.785 and was expressed as the total amount of that component present in one cm2 area of the tissue. The ratio of sulfated:non-sulfated AMPS was estimated as follows: To 0.8 ml of TCA-soluble fraction, 0.4 ml of distilled water was added and the solution was dialyzed against running tap water for 16 hours. The dialyzed material was transferred to a centrifuge tube to which 0.4 ml of cetylpyridinium chloride (CPC, 5 % w/v in 0.15 A4 NaCl) and 0.4 ml celite (IO’% w/v) were added. The contents were mixed and incubated at 37°C for one hour and centrifuged. The supernatant was discarded. The precipitate was washed with 1.0 ml (0.03 M NaCl containing 0.1% w/v CPC), centrifuged and supernatant again discarded. The resulting precipitate was successively extracted with two 0.5 ml portions of each of the following reagents: 0.40 M NaCl, 1.2 M NaCl and 2.1 M NaCl (each containing 0.1% CPC, w/v). The extracts corresponding to each individual reagent were pooled and analyzed for uranic acid. Since in our fractionation procedure, we observed that there was an extensive carry-over of uranic acid from 1.2 M to 2.1 A4 NaCl fraction (due to imcomplete extraction with 1.2 M NaCl), we pooled these fractions. The AMPS occurring in the 0.4 M NaCl fraction was called non-sulfated AMPS (hyaluronic acid) and that in the pooled 1.2 M NaCl fraction was called the sulfated AMPS (chondroitin sulfates, heparitin sulfate and heparin). RESULTS
Table 1 gives the percentage composition of moisture, lipid and ash in normal TABLE 1 MOISTURE,
LIPID
AND
ASH CONTENT
OF NORMAL
AND
ATHEROSCLEROTIC
AORTAE
The components are expressed as % of the wet weight of the tissue. The values for lipid were calculated as: % (lipid plus moisture) - % (moisture). These determinations were made on the same samples using one-half portion for lipid plus moisture and the other half for moisture analyses. The dry, defatted, ash-free residue (DDAF) is given as the percentage by difference. Component
Age (years) mean i- S.E.M.
N
Normal mean f S.E.M.
Atherosclerotic mean & S. E.M.
Paired t
Moisture Lipid Ash Dry, defatted ash-free residue (DDAF)
70.4 f 2.5 72.0 f 1.5 69.7 zt 3.6
20 6 8
73.77 f 0.31 5.46 f 1.15 0.68 f 0.06
73.63 + 0.45 10.82 * 2.02 0.79 & 0.08
0.22NS 9.92& 1.35NS
-
-
20.09
14.76
-
= P < 0.01. NS = not significant.
87
GLYCOPROTEINS IN ATHEROSCLEROSIS
and atherosclerotic regions of the 41 aortae investigated. The moisture and the ash content did not differ. The amount of lipid in atherosclerotic region was twice as much as the quantity present in the apparently normal tissue. In order to determine the concentrations of collagen and carbohydrate constituents it was therefore desirable TABLE 2 COLLAGEN
AND
TOTAL
CARBOHYDRATE
CONTENT
OF NORMAL
AND ATHEROSCLEROTIC
AORTAE
The components which are expressed as % of the dry, defatted, ash-free residue (DDAF) were calculated by multiplying the wet weight of normal and atherosclerotic tissue by 0.2009 and 0.1476, respectively. These factors were derived from data given in Table 1. Total carbohydrate is expressed as the sum of neutral sugars, hexosamine, hexuronic acid and sialic acid. The residue weight is given by difference. Component
Age (yrs) mean f S.E.M.
N
Normal mean + S.E.M.
Atherosclerotic mean + S.E.M.
Paired t
Collagen Total carbohydrate Residue
73.4 f 3.9 70.3 & 2.8 -
9 15
24.8 & 2.3 5.54 * 0.30 69.7
35.4 zt 6.8 6.37 f 0.27 58.2
4.98& 4.61a
a P < 0.01. TABLE 3 CONCENTRATION
OF COMPONENT
SUGARS
OF NORMAL
AND ATHEROSCLEROTIC
AORTAE
The concentration of components are expressed as weight per unit area and weight per unit dry, defatted, ash-free residue (DDAF). The weight of DDAF was calculated by multiplying the wet weight of normal and atherosclerotic tissue by 0.2009 and 0.1476, respectively. These factors were derived from data given in Table 1. All values represent averages from 15 samples of aortae obtained from individuals of 70.3 =t 2.8 (mean & S.E.M.) years of age. Component
Normal mean + S.E.M.
Atherosclerotic mean * S.E.M.
Paired t
Wet weight (mg/cm2) DDAF (mg/cm2) Total carbohydrate (mg/cmz) Hexose (pg/cm2) Hexosamine (pg/cm2) Uranic acid (pg/cm2) Sialic acid @g/cm2) Total carbohydrate (pg/mg DDAF) Hexose (pg/mg DDAF) Hexosamine (pg/mg DDAF) Uranic acid (pg/mg DDAF) Sialic acid (pg/mg DDAF)
195.7 f 9.2 39.3 i 1.9 2.10 i 0.04 870 f 32 651 f 16 297 + 12 285 f 14 55.4 i 3.0 23.1 * 1.6 17.0 f 0.8 7.9 & 0.6 7.5 * 0.4
250.4 36.9 2.30 978 698 308 317 63.7 27.1 19.4 8.5 8.8
8.96a 2.30b 4.15” 3.30” 2.70c I.OlNS 2.19” 4.61” 4.24” 3.25=
&P < 0.01. b P < 0.05. c P > 0.02 NS = Not significant.
5 11.2 I-t 1.7 f 0.06 5 37 + 23 * 15 & 20 f 2.7 f 1.4 i 0.9 zk 0.4 * 0.5
1.80NS 3.87a
88
P. V. WAGH,
B. I. ROBERTS,
H. J. WHITE,
R. C. READ
to express all values in terms of dry, defatted, ash-free residue weight (DDAF) of the tissue. The calculated DDAF values of 20.09 % and 14.76 % were obtained for normal and atherosclerotic tissues, respectively, and were used throughout to express the quantities of various parameters in terms of DDAF. Table 2 shows the values for collagen and total carbohydrate expressed as % of DDAF. It is apparent that both collagen as well as the total carbohydrate content were significantly higher in atherosclerotic regions. The residue weight which was calculated by difference is lower in the atherosclerotic tissue. This residue should mostly consist of elastin and other non-collagenous proteins. The quantities of individual monosaccharides in the carbohydrate component are shown in terms of unit intimal surface area and per unit DDAF (Table 3). The net weight per unit area of the atherosclerotic tissue was significantly higher than the normal. This was expected since the increase in weight may be attributed to deposition of lipid and other materials which infiltrate the vascular wall in the plaque formation. The calculated DDAF per unit area was significantly lower in the atherosclerotic region. Hexose, hexosamine and sialic acid content increased in the atherosclerotic tissue. These increases were statistically significant indicating that there is a quantitative increase in GP-concentration in atherosclerotic plaques. It is interesting to note that total uranic acid content was not altered indicating that the total AMPS remain unchanged in atherosclerotic tissue. The ratio of sulfated:non-sulfated AMPS was significantly higher in atherosclerotic tissue (Table 4) indicating a substantial increase in sulfated AMPS in atherosclerosis and a decrease in hyaluronic acid. The recoveries of AMPS after fractionation were found to be 74 % of the initial concentration. We were unable to detect measurable carbohydrate constituents in the TCA precipitates indicating that virtually all the glycoproteins and AMPS remained in the TCA supernatants.
TABLE 4 RATIOS
OF SULFATED:NON-SULFATED
ACID
MLJCOPOLYSACCHARIDES
IN NORMAL
AND
ATHEROSCLEROTIC
AORTAE
The definition for sulfated and non-sulfated acid mucopolysaccharides is Methods. Percent recovery is expressed as the total uranic acid recovered in precipitation as related to the initial amount of the uranic acid in the original averages from 15 samples of aortae, obtained from individuals of 70.3 5 years of age. Normal mean f
Ratio sulfated: non-sulfated (AMPS) Recovery (%)
a P < 0.05.
S.E.M.
6.33 Ilt 0.44 73.8 IIZ3.3
given in Materials and all fractions after CPCtissue. Values represent 2.8 (mean k S.E.M.)
Atherosclerotic mean f S.E.M.
Paired t
8.00 k 0.61 72.9 f 2.9
2.31” 0.24
GLYCOPROTEINS
IN ATHEROSCLEROSIS
89
DISCUSSION
In order to provide meaningful comparison between normal and atherosclerotic tissues, it is apparent that parameters expressed in terms of tissue wet weights or dry weights would be erroneous since in non-calcific atherosclerotic lesions the amount of lipid is increased two-fold as compared to the normal. Also since atherosclerotic changes are accompanied with increasing age, it seemed necessary to test the comparisons by eliminating the age variable by choosing samples within the same age group. Several conflicting reports on changes in aortic GP and AMPS can be related to the units of various parameters in which they are expressed or to the variability in sampling of the tissue. For instance, in most cases the composition of the tissue has been expressed in terms of wet weight, dry weight or defatted dry weight without regard to the contribution due to minerals. An increase, a decrease or no change in any parameter should be demonstrated best if the results are calculated on the basis of DDAF. This is best exemplified in a recent report of Anastassiades et uZ.~,wherein the analytical results were done on the basis of nitrogen content of the tissue which is a rough estimate of DDAF. If it is assumed that the thickening of the aortic wall is a result of deposition of certain components on or within the intima, then composition expressed in terms of intimal surface area should be a useful measure in comparisons between normal and atherosclerotic tissue. Manley and Mullinger 10 have reported data on carbohydrate composition of unaffected and atherosclerotic human aorta of one individual in term of tissue dry weight and intimal surface area. From our results, it is clear that the total carbohydrate and collagen content in atherosclerotic region is higher than in normal in terms of DDAF and surface area. Although we have not measured elastin and other non-collagenous components in the residual fraction, it is apparent that there should be a corresponding decrease of this component in atherosclerotic tissue. The increase of hexose, hexosamine, and sialic acid both in terms of DDAF and surface area in atherosclerotic tissue indicates a definite increased GP-concentration. Sialic acid is a good marker to support this statement since except for glycolipids which are removed by organic solvents, sialic acid is an integral part of sialoproteins. The observation that GP-concentration increases in atherosclerotic conditions is consonant with that of Anastassiades et ~1.9. Our data on sialic acid are in conflict with those of Murata and Kirkll, Manley and Mullingerls and of Kotoku et al.12, who have reported a decrease in sialic acid in atherosclerotic tissue. The discrepancy in the data of Manley and Mullingerla and those of ours seems to be in the ratio of dry weight/cm2 area of atherosclerotic:normal tissues which by calculations are 2.5 and 1.3, respectively. We have observed that only in highly-infiltrated regions of aorta undergoing necrosis and calcification the ratio was higher than 2. As such we have avoided such samples in our study. Our results indicate that the total AMPS content in normal and atherosclerotic tissue is not changed. This finding is in accordance with the observation of Anastassiades et cd9 who have expressed the results in terms of nitrogen or DNA content of the
P. V. WAGH,
90
B. I. ROBERTS,
H. J. WHITE,
R. C. READ
tissue. It is, however, in disagreement with the observation of Bdttcher and Klynstrara and Nakamura et al.14 who have reported a decrease in AMPS content. The higher ratio of sulfated :non-sulfated AMPS implies that in atherosclerosis the concentration of sulfated AMPS is increased at the expense of hyaluronic acid which is the only known non-sulfated AMPS in the aorta. Presently the role of glycoproteins in atherosclerosis is not known. However, some report+-17 have indicated that certain glycoproteins in serum and various organs are inhibitors of plasma lipoprotein lipase. Indeed, in our laboratory we have observed that a highly-purified glycoprotein isolated from the intimal region of porcine aorta18 acts as an inhibitor of lipoprotein lipase from post-heparin dog plasmalg. As a hypothesis, we suggest that increased concentration of glycoproteins in atherosclerosis may be related to the inhibition of the enzyme lipoprotein lipase which has an important role in the lipid transport. ACKNOWLEDGEMENTS
This study was carried out with the technical assistance of Mrs. Marian Epperson and Mrs. Victoria Drake. We wish to thank Dr. Dennis Robinette for computer analyses and to Mrs. Diane Butler for secretarial assistance.
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(1962) 330. 8 AMINOFF, D., Methods for the quantitative estimation of N-acetylneuraminic acid and their application to hydrolysates of sialomucoids, Biochem. J., 81 (1961) 384. T., ANASTASSIADIS,P. A., AND DENSTEDT,0. F., Changes in connective tissue 9 ANASTASSIADES, of the atherosclerotic intima and media of the aorta, Biochim. Biophys. Acta, 261 (1972) 418. 10 MANLEY, G., AND MULLINGER, R. N., Mucopolysaccharides of atherosclerotic plaques and platelets, Brit. J. Exp. Path., 58 (1967) 529. 11 MURATA, K., AND KIRK, J. E., Sialic acid content of human arterial and venous tissue, J. Athero-
scler. Rex, 2 (1962) 452, 12 KOTOKLJ,T., WATANABE, K., OYAMA, K., TOKITA, K., OHBA, T., composition
of glycoproteins
AND NAKAMURA, T., Sugar isolated from normal and sclerotic human aortas, Atherosclerosis,
14 (1971) 411. 13 BBT~CHER,C. J. F., AND KLYNSTRA, F. B., Content of acid mucopolysaccharides aorta, J. Atheroscler. Res., 2 (1962) 263.
in the human
91
GLYCOPROTEINS IN ATHEROSCLEROSIS
14NAKAMURA, T., TOKITA, K., TATENO, S., KOTOKU, T., ANDOHBA,T., Human aortic acid mucopolysaccharides and glycoproteins. Changesduringageingand in atherosclerosis, J. Atheroscler. Res., 8 (1968)891. 15 KLEIN, E., AND LEVER, W. F., Inhibition of lipemia-clearing activity by serum of patients with hyperlipemia, Proc. Sot. Exp. Biol. Med., 15 (1957) 565. 16 HOLLETT,C. R., An inhibitor of the lipolytic reaction from post-heparin plasma of normal dogs, Biochim. Biophys. Acta, 98 (1965) 53. 17 ISHII, M., Relationship between a glycoprotein sclerosis, Jap. Heart J., 12 (1971) 22. 18 WAGH, P. V., AND ROBERTS,B. I., Purification intimal region of porcine aorta, Biochemistry, 19 WAGH, P. V., AND ROBERTS,B. I., Inhibition intimal glycoprotein. In: 28th Southwestern
Baton Rouge, La., December 1972, p. 45.
acting as lipoprotein
lipase inhibitor and arterio-
and characterization of a glycoprotein from the 11 (1972) 4222. of plasma lipoprotein lipase by a highly purified Regional Meeting, American Chemical Society,