259
Atherosclerosis, 24 (1976) 259-266 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SEASONAL VARIATIONS IN THE SUSCEPTIBILITY WALL TO ATHEROSCLEROSIS
OF THE AORTIC
BIOCHEMICAL STUDIES OF GLYCOSAMINOGLYCANS OF RABBIT ATHEROSCLEROSIS
AND COLLAGEN
P. HELIN and I. LORENZEN Hvidovre Gentofte
Hospital, Hospital,
Medical Department, Medical Department
(Received Oktober 4,1975,
Division of Rheumatology C. Copenhagen (Denmark)
and Immunology
and
accepted January 7,1976)
Summary The aortic content of glycosaminoglycans and collagen as well as the uptake of [ lz51] albumin were studied in 53 male albino rabbits during hair-shedding and outside the period of hair-shedding to elucidate the previously reported resistance to experimental arteriosclerosis during the shedding period [l].The concentration of hyaluronic acid was highest during hair-shedding, decreasing towards the non-shedding period. The content of dermatan sulphate, chondroitin4, 6-sulphate and hydroxyproline was lowest during shedding and highest outside the shedding period. Accordingly, the incorporation of [ 35S] sulphate in chondroitin4,6_sulphate and the dermatan plus heparan sulphate fraction was increased outside shedding, consistent with a stimulated synthesis. The concentration of hyaluronic acid was negatively correlated to the uptake of [‘*‘I] albumin, and the dermatan sulphate content was positively correlated to the content of hydroxyproline. The higher concentration of hyaluronic acid during the period of shedding may improve the elastic propeities as well as the ability of the aortic wall to absorbe the haemodynamic strain involved in the vascular injury of this type of experimental arteriosclerosis [2]. The decrease in the concentration of hyaluronic acid simultaneously with an increase in the aortic content of collagen as well as of chondroitin-4, 6-sulphate and dermatan sulphate may imply a greater stiffness of the aorta resulting in a higher susceptibility to injury. The relationship between hyaluronic acid and [ 1251]albumin is consistent with an importance of hyaluronic acid to the susceptibility of the arterial wall to deposition of macromolecules such as the lipids. Our observaCorrespondence to: Ib Lorenzen. M.D., Hvidovre Hospital, Medical Department, Division of Rbeumatology and Immunology, DK-2650 Hvidovre. Copenhagen, Denmark.
260
tions represent an example of endogenous conditioned variations in the aortic content of glycosaminoglycans and hydroxyproline accompanied by a variation in the susceptibility to experimental arteriosclerosis. Key words:
Aorta - Arteriosclerosis - Collagen - Glycosaminoglycans -Hair-shedding Hyaluronic acid - Susceptibility to arteriosclerosis, seasonal variations cular injury
Vas-
Introduction In a previous study we have demonstrated a seasonal variation in the susceptibility of the aortic wall in rabbits to experimental arteriosclerosis, caused by exposure to severe systemic hypoxia [l] . The susceptibility was lowest during the season when the animals were shedding and highest outside this period. One of the pathogenetic mechanisms of the vascular injury in our model seems to be a short-lasting increase in the arterial blood pressure followed by a severe dilatation of the aorta [2]. We therefore advanced the hypothesis, that the variations in the susceptibility of the aortic wall to injury might be conditioned by changes in the physicochemical properties of the aortic wall. The mechanical properties of the aortic wall depend upon the intercellular macromolecules of the connective tissue, including glycosaminoglycans, collagen and elastin. Therefore changes in these macromolecules in relation to the period of hair-shedding might condition variations in the mechanical properties of the aorta. Studies on skin of mice and rabbits during hair growth have demonstrated that changes do take place in the glycosaminoglycans [3,4] . The purpose of the present study was an analysis of the glycosaminoglycans and collagen in the aorta of normal rabbits in relation to the periods of hairshedding and non-shedding corresponding to the periods of lowest and highest susceptibility to vascular injury. The hair-shedding of rabbits usually starts in September and reaches its maximum in November. During December the hairshedding decreases and at the end of January the shedding is completed. Material and methods The study included a total of 53 male albino rabbits of the Danish country strain. The rabbits were of the same breed. Four groups of animals were compared. One group was killed in November during shedding, one group in the beginning of January and one group at the end of this month corresponding to the end of the shedding period. These two groups were included in order to evaluate the transitory period between shedding and non-shedding. Finally, one group was killed outside the shedding period in the month of April. The age of the animals at death was 5 months and the body weights about 3 kg, similar in all 4 groups. The animals were fed a standard laboratory diet containing 12% protein, 54% carbohydrate and 4% fat, only traces of cholesterol. The room temperature in the stable was 20°C. The daily light period for the shedding aninials was 6 h in contrast to more than 12 h for the animals outside the shedding period.
261
Two days before death, the animals received i.v. injection of 0.4 ml of a 2% solution of 1251-labelled human serum albumin with a radioactivity of about 20 PCi and 1 mCi of [35S]sulphate (carrier-free [35S]sulphate diluted to a suitable volume with a 10 mg/lOO ml solution sodium sulphate). The animals were killed by i.v. injection of 300 mg pentobarbital. The aorta was isolated and the adventitia was removed. Only the intima plus the media were used for the biochemical analyses. The tissue was homogenized in water, lyophilized and dried to constant weight. The samples were then stored at room temperature in vacuum of 10m5 torr in the presence of phosphor pentoxide. When the animals from the least group had been killed the tissue samples from all 4 groups were analysed. Samples were selected at random from the 4 groups for each bath of analysis. The water content was determined from the difference between wet weight and dry weight. The aortic content of collagen plus elastin was estimated by analysis of hydroxyproline using the method of Prockop and Udenfriend [ 51 as modified by Jasin and Ziff [6]. The glycosaminoglycans were determined by the method devised by Thunell [7] as described previously [8]. Samples of the defatted tissue were digested with papain at 67°C for 8 h. The glycosaminoglycans were precipitated with cetylpyridinium chloride, dissolved in n-propanol and reprecipitated as sodium salts with absolute alcohol. The precipitate was digested with testicular hyaluronidase whereafter the depolymerized glycosaminoglycans were separated from the non-depolymerized glycosaminoglycans by means of a cetylpyridinium chloride saturated cellulose column. In each fraction glucosamine and galactosamine were separated by a Dowex 50(H’) microcolumn [9]. The content of glucosamine and galactosamine was determined by analysis of the hexosamine content by the method of Elson and Morgan [lo]. The [35S] sulphate radioactivity was determined on the testicular hyaluronidase digestable and the nondigestable fractions by a Beckman LS-250 liquid scintillation counter. The samples were prepared as described by Mahin and Lofberg [ll].A 20% solution of BBS-Biosolv Beckman @was used. The [125I] albumin radioactivity in aorta and serum was measured in a well-type scintillation counter. Results None of the animals in the 4 groups showed macroscope arteriosclerosis of the aorta. The body weights of the animals in the 4 groups did not differ (Table 1). The dry weights of the aortae were significantly lower in the shedding animals compared to the non-shedding rabbits. The water content of aorta showed no systematic changes (Table 1). The concentrations as well as the total amounts of hydroxyproline and hexosamine in aorta were highest outside the shedding period (Table 2). The concentration of hyaluronic acid was highest during the shedding period. The concentration then decreased towards the nonshedding period (Table 3). The changes in the total amounts of hyaluronic acid were not statistically significant. The total amount of chondroitin-4, 6-sulphate was lowest during shedding. Similarly, the content of dermatan sulphate was also lowest during this period increasing to the highest value in the non-shedding period (April). By contrast, the content of heparan sulphate did not
262 TABLE 1 BODY WEIGHT, DRY WEIGHT (DW) AND WATER CONTENT OF AORTA OF MALE ALBINO RABBITS DURING AND OUTSIDE THE SHEDDING PERIOD FROM NOVEMBER TO APRIL. Number of animals is given in parentheses. Other values are means f standard deviation of the mean. P-values were derived by the Mann-Whitney test.
Body weight (kg) Dry weight of aorta (mg) Water content (% of DW)
I (November)
II (Beginning of Januarv)
III (End of January)
(April)
(15)
(12)
(14)
(12)
2.81 +_0.04 97.9 + 5.4 208.1 + 4.9
2.82 k 0.06 118.1 t 5.0 226.9 f 4.6
2.80 f 0.03 110.3 f 3.4 209.9 + 4.4
2.82 f 0.04 119.5 f 5.4 225.4 + 3.7
IV
P-value (I-IV)
n.s. co.01 co.02
change. The uptake of sulphate in vivo into chondroitin4, 6-sulphate and in the dermatan sulphate/heparan sulphate fraction was highest outside the shedding period (Table 4). The uptake of 1251-labelled albumin in the aorta was lowest in the aorta during shedding (Table 4). Regression analysis of the relationship between hyaluronic acid and the [‘*‘I] albumin radioactivity of the aorta showed a negative correlation between the 2 variables (regression coefficient % 0.006, P < 0.01). There was a positive correlation between the concentrations of dermatan sulphate and hydroxyproline (regression coefficient 8.54, P < 0.02) as well as between the total amounts of these two variables (regression coefficient 25.77, P< 0.001). A comparison of the results from the 2 groups of January, the transitory month, shows that the water content, the total content of heparan sulphate and the total amount of chondroitin-4, 6sulphate of the aorta were highest in the animals killed in the beginning of January.
TABLE 2 THE CONTENT OF HYDROXYPROLINE AND HEXOSAMINE IN AORTA OF MALE ALBINO RABBITS DURING AND OUTSIDE THE SHEDDING PERIOD FROM NOVEMBER TO APRIL Number of animals is given in parentheses. Other numbers are means f standard deviation of the mean. P-values were derived by the Mann-Whitney test.
Hydroxyproline Concentration (wg/mg dry tissue) Total amount (fig) Hexosnmine Concentration (pglmg dry tissue) Total amount (fig)
I (November)
II (Beginning of January)
III (End of January)
(April)
(15)
(12)
(14)
(12)
21.0 f 2035
f 94
5.9 + 576
0.7
0.1
+ 16
23.3 k 2745
+ 103
6.3 + 740
0.5
f
0.1 28
22.6 f 2498
0.4
f 90
6.4 f 698
IV
0.2
f 26
P-value (I-IV)
25.2 + 3021
r 208
6.6 f 795
0.7
+
0.2 32
co.01 co.01
Atherosclerosis, 24 (1976) 259-266 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
SEASONAL VARIATIONS IN THE SUSCEPTIBILITY WALL TO ATHEROSCLEROSIS
OF THE AORTIC
BIOCHEMICAL STUDIES OF GLYCOSAMINOGLYCANS OF RABBIT ATHEROSCLEROSIS
AND COLLAGEN
P. HELIN and I. LORENZEN Hvidovre Gentofte
Hospital, Hospital,
Medical Department, Medical Department
(Received Oktober 4,1975,
Division of Rheumatology C. Copenhagen (Denmark)
and Immunology
and
accepted January 7,1976)
Summary The aortic content of glycosaminoglycans and collagen as well as the uptake of [ lz51] albumin were studied in 53 male albino rabbits during hair-shedding and outside the period of hair-shedding to elucidate the previously reported resistance to experimental arteriosclerosis during the shedding period [l].The concentration of hyaluronic acid was highest during hair-shedding, decreasing towards the non-shedding period. The content of dermatan sulphate, chondroitin4, 6-sulphate and hydroxyproline was lowest during shedding and highest outside the shedding period. Accordingly, the incorporation of [ 35S] sulphate in chondroitin4,6_sulphate and the dermatan plus heparan sulphate fraction was increased outside shedding, consistent with a stimulated synthesis. The concentration of hyaluronic acid was negatively correlated to the uptake of [‘*‘I] albumin, and the dermatan sulphate content was positively correlated to the content of hydroxyproline. The higher concentration of hyaluronic acid during the period of shedding may improve the elastic propeities as well as the ability of the aortic wall to absorbe the haemodynamic strain involved in the vascular injury of this type of experimental arteriosclerosis [2]. The decrease in the concentration of hyaluronic acid simultaneously with an increase in the aortic content of collagen as well as of chondroitin-4, 6-sulphate and dermatan sulphate may imply a greater stiffness of the aorta resulting in a higher susceptibility to injury. The relationship between hyaluronic acid and [ 1251]albumin is consistent with an importance of hyaluronic acid to the susceptibility of the arterial wall to deposition of macromolecules such as the lipids. Our observaCorrespondence to: Ib Lorenzen. M.D., Hvidovre Hospital, Medical Department, Division of Rbeumatology and Immunology, DK-2650 Hvidovre. Copenhagen, Denmark.
260
tions represent an example of endogenous conditioned variations in the aortic content of glycosaminoglycans and hydroxyproline accompanied by a variation in the susceptibility to experimental arteriosclerosis. Key words:
Aorta - Arteriosclerosis - Collagen - Glycosaminoglycans -Hair-shedding Hyaluronic acid - Susceptibility to arteriosclerosis, seasonal variations cular injury
Vas-
Introduction In a previous study we have demonstrated a seasonal variation in the susceptibility of the aortic wall in rabbits to experimental arteriosclerosis, caused by exposure to severe systemic hypoxia [l] . The susceptibility was lowest during the season when the animals were shedding and highest outside this period. One of the pathogenetic mechanisms of the vascular injury in our model seems to be a short-lasting increase in the arterial blood pressure followed by a severe dilatation of the aorta [2]. We therefore advanced the hypothesis, that the variations in the susceptibility of the aortic wall to injury might be conditioned by changes in the physicochemical properties of the aortic wall. The mechanical properties of the aortic wall depend upon the intercellular macromolecules of the connective tissue, including glycosaminoglycans, collagen and elastin. Therefore changes in these macromolecules in relation to the period of hair-shedding might condition variations in the mechanical properties of the aorta. Studies on skin of mice and rabbits during hair growth have demonstrated that changes do take place in the glycosaminoglycans [3,4] . The purpose of the present study was an analysis of the glycosaminoglycans and collagen in the aorta of normal rabbits in relation to the periods of hairshedding and non-shedding corresponding to the periods of lowest and highest susceptibility to vascular injury. The hair-shedding of rabbits usually starts in September and reaches its maximum in November. During December the hairshedding decreases and at the end of January the shedding is completed. Material and methods The study included a total of 53 male albino rabbits of the Danish country strain. The rabbits were of the same breed. Four groups of animals were compared. One group was killed in November during shedding, one group in the beginning of January and one group at the end of this month corresponding to the end of the shedding period. These two groups were included in order to evaluate the transitory period between shedding and non-shedding. Finally, one group was killed outside the shedding period in the month of April. The age of the animals at death was 5 months and the body weights about 3 kg, similar in all 4 groups. The animals were fed a standard laboratory diet containing 12% protein, 54% carbohydrate and 4% fat, only traces of cholesterol. The room temperature in the stable was 20°C. The daily light period for the shedding aninials was 6 h in contrast to more than 12 h for the animals outside the shedding period.
261
Two days before death, the animals received i.v. injection of 0.4 ml of a 2% solution of 1251-labelled human serum albumin with a radioactivity of about 20 PCi and 1 mCi of [35S]sulphate (carrier-free [35S]sulphate diluted to a suitable volume with a 10 mg/lOO ml solution sodium sulphate). The animals were killed by i.v. injection of 300 mg pentobarbital. The aorta was isolated and the adventitia was removed. Only the intima plus the media were used for the biochemical analyses. The tissue was homogenized in water, lyophilized and dried to constant weight. The samples were then stored at room temperature in vacuum of 10m5 torr in the presence of phosphor pentoxide. When the animals from the least group had been killed the tissue samples from all 4 groups were analysed. Samples were selected at random from the 4 groups for each bath of analysis. The water content was determined from the difference between wet weight and dry weight. The aortic content of collagen plus elastin was estimated by analysis of hydroxyproline using the method of Prockop and Udenfriend [ 51 as modified by Jasin and Ziff [6]. The glycosaminoglycans were determined by the method devised by Thunell [7] as described previously [8]. Samples of the defatted tissue were digested with papain at 67°C for 8 h. The glycosaminoglycans were precipitated with cetylpyridinium chloride, dissolved in n-propanol and reprecipitated as sodium salts with absolute alcohol. The precipitate was digested with testicular hyaluronidase whereafter the depolymerized glycosaminoglycans were separated from the non-depolymerized glycosaminoglycans by means of a cetylpyridinium chloride saturated cellulose column. In each fraction glucosamine and galactosamine were separated by a Dowex 50(H’) microcolumn [9]. The content of glucosamine and galactosamine was determined by analysis of the hexosamine content by the method of Elson and Morgan [lo]. The [35S] sulphate radioactivity was determined on the testicular hyaluronidase digestable and the nondigestable fractions by a Beckman LS-250 liquid scintillation counter. The samples were prepared as described by Mahin and Lofberg [ll].A 20% solution of BBS-Biosolv Beckman @was used. The [125I] albumin radioactivity in aorta and serum was measured in a well-type scintillation counter. Results None of the animals in the 4 groups showed macroscope arteriosclerosis of the aorta. The body weights of the animals in the 4 groups did not differ (Table 1). The dry weights of the aortae were significantly lower in the shedding animals compared to the non-shedding rabbits. The water content of aorta showed no systematic changes (Table 1). The concentrations as well as the total amounts of hydroxyproline and hexosamine in aorta were highest outside the shedding period (Table 2). The concentration of hyaluronic acid was highest during the shedding period. The concentration then decreased towards the nonshedding period (Table 3). The changes in the total amounts of hyaluronic acid were not statistically significant. The total amount of chondroitin-4, 6-sulphate was lowest during shedding. Similarly, the content of dermatan sulphate was also lowest during this period increasing to the highest value in the non-shedding period (April). By contrast, the content of heparan sulphate did not
262 TABLE 1 BODY WEIGHT, DRY WEIGHT (DW) AND WATER CONTENT OF AORTA OF MALE ALBINO RABBITS DURING AND OUTSIDE THE SHEDDING PERIOD FROM NOVEMBER TO APRIL. Number of animals is given in parentheses. Other values are means f standard deviation of the mean. P-values were derived by the Mann-Whitney test.
Body weight (kg) Dry weight of aorta (mg) Water content (% of DW)
I (November)
II (Beginning of Januarv)
III (End of January)
(April)
(15)
(12)
(14)
(12)
2.81 +_0.04 97.9 + 5.4 208.1 + 4.9
2.82 k 0.06 118.1 t 5.0 226.9 f 4.6
2.80 f 0.03 110.3 f 3.4 209.9 + 4.4
2.82 f 0.04 119.5 f 5.4 225.4 + 3.7
IV
P-value (I-IV)
n.s. co.01 co.02
change. The uptake of sulphate in vivo into chondroitin4, 6-sulphate and in the dermatan sulphate/heparan sulphate fraction was highest outside the shedding period (Table 4). The uptake of 1251-labelled albumin in the aorta was lowest in the aorta during shedding (Table 4). Regression analysis of the relationship between hyaluronic acid and the [‘*‘I] albumin radioactivity of the aorta showed a negative correlation between the 2 variables (regression coefficient % 0.006, P < 0.01). There was a positive correlation between the concentrations of dermatan sulphate and hydroxyproline (regression coefficient 8.54, P < 0.02) as well as between the total amounts of these two variables (regression coefficient 25.77, P< 0.001). A comparison of the results from the 2 groups of January, the transitory month, shows that the water content, the total content of heparan sulphate and the total amount of chondroitin-4, 6sulphate of the aorta were highest in the animals killed in the beginning of January.
TABLE 2 THE CONTENT OF HYDROXYPROLINE AND HEXOSAMINE IN AORTA OF MALE ALBINO RABBITS DURING AND OUTSIDE THE SHEDDING PERIOD FROM NOVEMBER TO APRIL Number of animals is given in parentheses. Other numbers are means f standard deviation of the mean. P-values were derived by the Mann-Whitney test.
Hydroxyproline Concentration (wg/mg dry tissue) Total amount (fig) Hexosnmine Concentration (pglmg dry tissue) Total amount (fig)
I (November)
II (Beginning of January)
III (End of January)
(April)
(15)
(12)
(14)
(12)
21.0 f 2035
f 94
5.9 + 576
0.7
0.1
+ 16
23.3 k 2745
+ 103
6.3 + 740
0.5
f
0.1 28
22.6 f 2498
0.4
f 90
6.4 f 698
IV
0.2
f 26
P-value (I-IV)
25.2 + 3021
r 208
6.6 f 795
0.7
+
0.2 32
co.01 co.01
