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Histological and biochemical studies on the ground substance of the aortas of lathyritic rats
Journal of Atherosclerosis Research
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
H I S T O L O G I C A L AND B I O C H E M I C A L S T U D I E S ON T H E G R O U N D SUBSTANCE OF T H E AORTAS OF L A T H Y R I T I C RATS
R. ALPER*,
J. T. PRIOR
AND
W.
I~. RUEGAMER
Departments of Biochemistry and Pathology, State University of New York, Upstate Medical Center, Syracuse, N.Y. (U.S.A.)
(Received February 13th, 1968)
SUMMARY Aortas from normal and lathyritic rats were examined histologically and chemically. The administration of fl-aminopropionitrile (BAPN) to young animals often resulted in the production of medial lesions of the aortic arch; such lesions did not develop in animals administered semicarbazide (SC). Microscopic examination of the lesions revealed a marked disorganization of the elastic components of the arterial wall, a high degree of metachromasia with toluidine blue and a proliferation of smooth muscle cells. Increased metachromasia and a hypertrophy of the smooth muscle cells were also seen in sections of the thoracic and abdominal aorta in which there was no obvious structural damage in response to the administration of either BAPN or SC. Analyses of the acid mucopolysaccharide (AMPS) composition of aortas from young lathyritic animals revealed a marked increase in the chondroitin sulfate A plus C fraction, but essentially no change in the other AMPS components of the arterial wall. The changes were not observed in adult lathyritic animals. The aortic AMPS changes in young lathyritic rats persist for a long time after the lathyrogenic agent (BAPN) is withdrawn from the diet. The present observations are consistent with the concepts that (a) it is the rapidly growing animal that is particularly susceptible to the effects of lathyrogenic agents on the aorta; (b) smooth muscle is involved in the repair of arterial injury; (c) smooth muscle cells biosynthesize AMPS; and (d) the changes represent a nonspecific response of the aorta to injury.
* Present address: Department of Biochemistry, School of Medicine, State University of New York at Buffalo, Buffalo, N.Y. 14214, U.S.A. J. Atheroscler. Res., 1968, 8:787-801
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R. ALPER, J. T. PRIOR, W. R. RUEGAMER
INTRODUCTION
Experimental lathyrism is a connective tissue disease induced b y the action of certain compounds (lathyrogens), the most commonly used of which are /5-aminopropionitrile (BAPN), aminoacetonitrile (AAN) and semicarbazide. The condition is characterized b y the development of various skeletal abnormalities and b y a predisposition to the development of abdominal wall hernias and aortic aneurysms 1. The most characteristic biochemical defect in lathyrism is the dramatic increase in the proportion of salt-soluble collagen that is extractable from connective tissue2, 3. This has been attributed to a defect in the production of covalent intra- and intermolecular cross linkages within the collagen molecule. A similar defect in the structure of elastin apparently involves an inhibition of desmosine and isodesmosine formation 4. These amino acids are believed to participate in the formation of cross linkages in the elastin molecule. Such defects decrease the tensile strength and elasticity of these proteins. Thus, the lathyritic symptoms that have been described might be expected to be predominant in those tissues which are chronically subjected to strain, e.g. the aorta and the epiphyses of the bones. Although there has been considerable interest in the effects of experimental lathyrism on the acid mucopolysaccharide (AMPS) composition of connective tissue, m a n y of the studies have been rather inconclusive. BERNTSEN5,6 presented evidence t h a t the AMPS content of the epiphysial plate cartilage was decreased in the lathyritic animal and that the uptake of labeled sulfate into the epiphysial growth zones was depressed. LEVENE et al. 6 were unable to demonstrate any alterations either qualitatively or quantitatively in the AMPS content of chick embryo cartilage. PEDRINIMILLE AND PEDRINIs demonstrated a decreased synthesis of hexosamine in lathyritic epiphysial cartilage but KARNOVSKY AND KARNOVSKY9 found no alteration in the hexosamine content of the same tissue. However, chemical, histological and radioautographic studies have shown that there is a marked increase in the sulfated AMPS content of the lathyritic aorta 1~ BICKLEY13 has cited this as being the only consistent alteration in the composition of the ground substance that has been observed in any tissue. GRANT e~ al. 14 found a significant elevation in the hexosamine content of lathyritic aorta and also demonstrated the absence of similar effects in bone, cartilage, and tendon. Thus, the effect of lathyrism on the aortic connective tissue differs from that seen in other types of connective tissue. BURMAN 15, in a review on aortic medial degeneration, pointed to the possibility that the accumulation of interlaminar polysaccharide m a y be a non-specific response of the aorta to a variety of adverse conditions. If this response were an intrinsic property of the aorta, it would not be observed in other connective tissues. This would imply: (1) that there are cellular components of aorta which are absent in other tissues and which are responsible for the increased levels of AMPS seen under various conditions or (2) that the cellular elements in aorta which are involved in the deposition of ground substance behave differently from those in other connective tissues. I t was the purpose of this investigation to further characterize the effects of J. Atheroscler. Res., 1968, 8:787-801
STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITIC RATS
789
experimental lathyrism on the AMPS content of aorta and to attempt to correlate the biochemical events with the histological alterations. The evidence presented in this paper would support the concept that the increased AMPS concentrations found in the lathyritic aorta are part of a non-specific response to injury and that smooth muscle may have an important role in this response. MATERIALS AND METHODS
In the majority of these experiments, weanling (40-60 g body weight) male or female Sprague-Dawley strain albino rats were used. The animals were fed ad libitum a purified basal diet (BD) consisting of 60 % sucrose and 30 % casein plus vitamins and mineralsl6,17 and were housed in a constant temperature room (23~ with controlled lighting (5 a.m.-6 p.m.). Lathyrism was induced by feeding the basal diet containing 0 . 1 % (w/w) of either fl-aminopropionitrile hydrochloride (BAPN) or semicarbazide hydrochloride (SC) for a period of at least 4 weeks. At appropriate time intervals, the animals were sacrificed and the aortas were quickly excised. Adherent blood, fat, and adventitia were removed and the tissues were used directly or were placed in a freezer at --20~ to await analysis. Samples for histological examination were fixed in neutral formalin.
Histological The formalin-fixed aortic samples were imbedded in paraffin, sectioned and stained with (a) hematoxylin-eosin, (b) Weigert-van Gieson's stain, (c) 0.5 % toluidine blue and (d) Masson's trichrome stain. Analytical The aortas from at least 5 animals were pooled. These were finely minced with scissors and then extracted with chloroform-methanol (2 : 1, v/v) for at least 4 h in a Soxhlet apparatus. The defatted tissue was dried to a constant weight and at least 100 mg of dry material was added to 5 ml of 0.1 M phosphate buffer (pH 6.5) which contained 2 mg of papain (2 • recrystallized, Sigma Chemical Co.), 0.01 M ethylenediaminetetra-acetic acid (disodium salt), and 0.005 M L-cysteine. The mixture was incubated at 50 ~ for 24 h. The pH was then readjusted to 6.5 with 0.1 N NaOH, 1 ml of newly prepared papain solution was added and the mixture was re-incubated for another 24 h. The papain digest was centrifuged at 600 • g for 30 rain, the supernatant was decanted and the insoluble residue was washed twice with distilled water. The combined washings and supernate were passed directly onto an anion exchange column (Bio-Rad AG l-X2, 200-400 mesh, C1 form, 1 cm • 15 cm). After the column had been washed with 25 ml of 0.1 M NaC1, the acid mucopolysaccharides (AMPS) were eluted by a modification of the procedure of SCHILLERet al. is. This involved the stepwise application of 50 ml each of 0.5 M, 0.8 M, 1.25 M, and 2.5 M NaC1. The eluate was collected in 5-ml fractions and each fraction was analyzed for hexuronic acid by the j . Atheroscler. Res., 1968, 8 : 7 8 7 - 8 0 1
790
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
carbazole method of DISCHE19 using I~-glucuronolactone as a standard. The results were expressed as #g of glucoronolactone found per 100 mg of dry, defatted tissue for each step of the elution system. In order to evaluate the quantitative and qualitative aspects of this procedure, experiments were performed in which known amounts of standard AMPS* were added to weighed tissue samples prior to the papain digestion. After hydrolysis with the enzyme, the samples were chromatographed as described and the recoveries of the internal standards were determined. A modification of the procedure of SCHMIDT AND DMOCHOWSK120was used for the determination of the relative amounts of chondroitin sulfates A/C and B. The chondroitin sulfate-containing fractions from at least three columns were combined and dialyzed overnight against distilled water. The dialysate was evaporated to dryness on a rotary evaporator and then reconstituted in 5 ml of citrate-phosphate buffer, p H 5.0.2-ml aliquots were treated with 600 I.U. of testicular hyaluronidase in 0.1 M NaC1 for 24 h at 37~ The mixture was then applied to a Sephadex G-25 column (1 cm • 35 cm) and eluted in 1 M NaC1. 3-ml fractions were collected and each was analyzed for hexuronic acid b y the carbazole procedure 19. RESULTS
Virtually all of the weanling animals that were fed lathyrogens developed a marked scoliosis of the spine and thoracic cage after 4 weeks. By the end of this period approximately one-third of the animals receiving BAPN had died of ruptured aneurysms of the ascending or thoracic aorta, whereas all of the animals on semicarbazide survived. Histological Marked lesions in the aortic arch were observed in about half of the survivors of the animals receiving BAPN for 4 weeks or longer. A typical lesion is shown in Fig. 1. There was a visible thickening of the wall of the arch and the internal surface of the lesion was very irregular. The lumen of the arch was much wider than in control animals, which suggests that the lesion was produced in response to the development of an aneurysm. Microscopic examination of these lesions revealed some marked alterations in the tunica media. The lamellar structure of the tissue was severely disrupted and, in m a n y areas of the media, it had entirely disappeared (Fig. 2). There was a profusion within the ground substance of short fibers which stained positively for elastic tissue. Toluidine blue staining revealed a marked metachromasia indicating the presence of large amounts of acid mucopolysaccharides (AMPS). There was a random prolifera-
* Hyaluronic acid and chondroitin sulfate preparations were obtained from Sigma Chemical Co. (St. Louis). Heparitin sulfate was a gift of Dr. J. A. Cifonelli. Chondroitin sulfate 13 was a gift of Dr. Karl Meyer. J. Atheroscler. Res., 1968, 8:787-801
STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITIC RATS
791
Fig. 1. Production of an aortic arch lesion in a rat fed BAPN for 5 weeks. Left: Aorta from a control animal. Right: Aorta from a lathyritic animal. There is a marked thickening of the aorta within the arch. Note the highly irregular intimal surface of the lesion.
tion of s m o o t h muscle t h r o u g h o u t t h e lesion (Fig. 3), giving t h e overall impression t h a t a r e p a i r process h a d been i n a u g u r a t e d in response to t h e d e v e l o p m e n t of t h e a n e u r y s m . I n two animals, dissecting a n e u r y s m s d e v e l o p e d in t h e aortic arch followed b y t h e f o r m a t i o n of a new channel w i t h t h e r e s u l t a n t p r o d u c t i o n of a " d o u b l e b a r r e l e d " a o r t a (Fig. 4). T h e t h i c k e n i n g of t h e wall of the a o r t a was v e r y p r o m i n e n t in t h e v i c i n i t y of t h e dissection as was t h e proliferation of s m o o t h muscle a n d t h e disr u p t i o n of t h e l a m e l l a r s t r u c t u r e of t h e media. A n i m a l s m a i n t a i n e d on the semicarb a z i d e regimen seldom d e v e l o p e d these lesions. Microscopic e x a m i n a t i o n of t h o r a c i c a n d a b d o m i n a l sections of a o r t a s from b o t h n o r m a l a n d l a t h y r i t i c a n i m a l s r e v e a l e d a definite t h i c k e n i n g of t h e a r t e r i a l wall along its entire l e n g t h in t h e a o r t a s of t h e l a t h y r i t i c animals. These sections d i d n o t d i s p l a y t h e lesions which h a v e been described above. F u r t h e r m o r e , this t h i c k e n i n g was o b s e r v e d in a n i m a l s fed s e m i c a r b a z i d e as well as B A P N . T h e l a m e l l a r s t r u c t u r e of J. Ath*roscler. Res., 1968, 8:787-801
792
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
Fig. 2. Histological e x a m i n a t i o n of a typical aortic arch lesion produced b y the a d m i n i s t r a t i o n of B A P N for 5 weeks. The effects on the elastic c o m p o n e n t s of the aorta. Left: Control aorta; note the c o n t i n u o u s lamellar s t r u c t u r e of the artery. Right: A o r t a of treated animal at s a m e magnification. Section t a k e n t h r o u g h an arch lesion; note the e x t r e m e thickening of the wall of the artery, the m a r k e d disorganization of the elastic lamellae and the profusion of s h o r t elastic fibers. V a n Gieson, • 170.
Fig. 3. Histological e x a m i n a t i o n of a typical aortic arch lesion produced b y t h e a d m i n i s t r a t i o n of B A P N for 5 weeks. The effects on the c o n t e n t of s m o o t h muscle, Left: Control aorta. Right: A o r t a of t r e a t e d animal at same magnification. Section t a k e n t h r o u g h the lesion. Note t h e m a r k e d proliferation and disorientation of the s m o o t h muscle. Masson's Trichrome, • 170,
J. Atheroscle~,. Res., 1968, 8:787-801
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793
Fig. 4. The d e v e l o p m e n t of a " d o u b l e barreled" a o r t a in an a n i m a l receiving B A P N for eight weeks. The n e w channel is on the left. H e m a t o x y l i n - e o s i n , X 45.
the artery remained intact but there was an increase in the distance between lamellae. The arteries displayed a marked interlamellar metachromasia (Fig. 5), which gave the impression that there were increased amounts of ground substance within the arterial wall. Further histological examination of these sections revealed some unusual alterations in the smooth muscle of the aorta (Fig. 6). There appeared to be a greater number of smooth muscle cells within the lathyritic aorta, m a n y of the cells being very large. These cells were highly elongated, their long axes being perpendicular to the elastic lamellae. The increased metachromasia and the alterations in the smooth muscle were most prominent in the innermost third of the media.
Analytical Since glycoproteins as well as acid mucopolysaccharides contain hexosamines, it was decided that more meaningful information could be obtained b y performing a chromatographic separation of the AMPS rather than b y relying upon a determination of total hexosamine concentration for an estimate of the AMPS content of aorta, as J. Atheroscler. Res., 1968, 8 : 7 8 7 - 8 0 1
794
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
Fig. 5. M e t a c h r o m a s i a in a section of the thoracic a o r t a of an animal receiving semicarbazide for 5 weeks. Left: ControI aorta. Right: Lathyritic a o r t a a t s a m e magnification. Note the intense m e t a c h r o m a s i a and the increased interlamellar distance. Toluidine blue, • 280.
Fig. 6. Alterations in t h e s m o o t h muscle w i t h i n the thoracic a o r t a of an animal receiving semicarbazide for 5 weeks. Left: Control aorta. Right: Lathyritic a o r t a at the same magnification. Note the h y p e r t r o p h y of the s m o o t h muscle cells and the increased n u m b e r of these cells within the widened interlamellar space. Masson's Trichrome, • 640.
J. Atheroscler. Res., 1968, 8:787-801
STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITIC RATS
795
have m a n y investigators. The ion exchange chromatographic procedure of SCHILLER et al.i8 provided an excellent method for the separation and quantitation of hyaluronic acid, heparitin sulfate, the chondroitin sulfates and heparin. Hyaluronic acid was eluted with 0.5 M NaC1, heparitin sulfate with 1.25 M NaC1, the chondroitin sulfates with 1.5 M NaC1 and heparin with 2.5 M NaC1. The procedure was found to be quantitative in that over 90 % of the standard AMPS applied to the column could be recovered in the appropriate fractions. Preliminary experiments demonstrated that heparin was not a constituent of rat aorta and since considerable "tailing" of the chondroitin sulfate fraction was obtained when it was eluted with 1.5 M NaC1, we decided to elute the chondroitin sulfate fraction in 2.5 M NaC1. This greatly reduced the "tailing", thus providing more reproducible results. The procedure employed had an additional advantage over other procedures in that very little protein remaining in the papain digests of aorta was retained b y the column. The small amount that was adsorbed to the resin was eluted with 0.5 M NaC1. This eliminated the necessity for the removal of protein from the papain digest and for a preliminary precipitation of the AMPS. Recoveries of internal standards were consistently in excess of 85 % and the modified procedure thus provided a convenient and rapid method for the quantitative analysis of the various aortic AMPS. A typical chromatogram of the AMPS in normal rat aorta is presented in Fig. 7. The major AMPS fraction, eluted in 1.2 M NaC1, seems to be heparitin sulfate. The second major fraction was eluted in 2.5 M NaC1 and probably is a mixture of the various chondroitin sulfates. A smaller fraction was eluted in 0.8 M NaCI. No a t t e m p t has been made to characterize this fraction but DALFERES et al. 21 reported that a similar fraction contained a heparitin sulfate-like material of low sulfate content. Little if any AMPS was eluted in 0.5 M NaC1 indicating that hyaluronic acid is not an import a n t component of rat aorta. The AMPS of normal rat aorta consist of an average of 45-50 % heparitin sulfate, 35-40 % chondroitin sulfate and about 15 % of the material eluted in 0.8 M NaC1 (in terms of the percent of the total glucuronolactone equivalent eluted from the 0.SM
O.8M
NaCI
NoEl
1.25M
NoCI
r
r
25M
NoC|
r
6C
-~. 4C 3C o s
20 ~o
t
o
4
8
12
i , i i J 16 20 24 28 Fraction No,
32
36
40
Fig. 7. C h r o m a t o g r a m of t h e AMPS f r o m a p a p a i n digest of n o r m a l r a t aortas on Bio-Rad AG 1 X2.
J. Atheroscler. Res., 1968, 8:787-801
796
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
:['ABLE 1 I'.'FFECTS OF LATHYRISM
ON
AMPS
OF RAT AORTA:
WEANLING
ANIMALS
Weanling animals were maintained for 30 days on a basal diet (BD) containing 0.1% ]3APN or 0.1% semicarbazide (SC). Aortas from 6 rats were pooled and the aortic acid mucopolysaccharides were analyzed as described in the text. Results are expressed as/,g of glucuronolactone per 100 mg of dry, defatted tissue. Regimen
iV[ean body weight (g)
0.8 M NaCl
0 . 2 5 M NaC1
2.5MNaCl
Total
BD BD 0.1 % 0.1 % 0.1% 0.1 % 0.1% 0.1 %
165 165 147 147 147 162 162 162
44 85 64 55 36 39 61 61
130 123 157 115 141 138 166 124
94 109 179 162 197 185 220 216
268 287 400 332 374 362 447 401
BAPN BAPN BAPN SC SC SC
TABLE 2 EFFECTS
OF LATHYRISM
ON
AMPS
OF RAT AORTA: ADULT
ANIMALS
Adult (400-600 g) rats were maintained on a basal diet (BD) containing 0.1% BAPN for 30 or 60 days. Aortas from 6 animals within each group were combined and analyses were performed as described in the text. Results are expressed as #g of glucuronolactone per 100 mg of dry, defatted tissue. Regimen
Mean body weight (g)
0.8 M NaCl
1.25 M NaCl
2.5 M NaCl
Total
BD BAPN, 30 d. BAPN, 60 d.
530 495 510
48
121
63
46 42
149 136
63 62
232 258 240
ion exchange column). Analyses of aortas from weanling a n i m a l s t r e a t e d with l a t h y r o g e n s for 30 days revealed a m a r k e d elevation in the c h o n d r o i t i n sulfate c o n t e n t to a b o u t twice t h a t found in control a n i m a l s (Table 1). T h e other AMPS fractions were relatively unaffected. Thus, in the l a t h y r i t i c animal, the condroitin sulfates c o n s t i t u t ed a b o u t half of the total AMPS f o u n d in the aortas of a n i m a l s fed either B A P N or semicarbazide. However, when B A P N was a d m i n i s t e r e d to adult a n i m a l s (400-600 g b o d y weight), the AMPS profile was unaffected even after c o n t i n u e d feeding of the l a t h y r o g e n i c diet for as long as 2 m o n t h s (Table 2). The chondroitin sulfate concentrations i n the aortas of older a n i m a l s were o n l y a b o u t 70 % of those f o u n d in y o u n g a n i m a l s (Tables 1 a n d 2), whereas the other AMPS fractions appeared to be relatively unaffected b y age. I n order to gain some insight as to which of the various c h o n d r o i t i n sulfates m i g h t be elevated in the l a t h y r i t i c aorta, a d v a n t a g e was t a k e n of the fact t h a t c h o n d r o i t i n sulfates A/C can be depolymerized b y testicular h y a l u r o n i d a s e whereas, J. Atheroscler. Res., 1968, 8:787--801
STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITIC RATS
Void volume
797
Salt volume
70
~ 6o I ~5o ::k E
o40
\
0 30 _o No 2o
-6
f 12
18
,~!"x,..~ , , 24 30 34 42 Eluate (ml)
Fig. 8. Gel filtration on S e p h a d e x G 25 of t h e p r o d u c t s of t h e t r e a t m e n t of t h e c h o n d r o i t i n s u l f a t e f r a c t i o n s of l a t h y r i t i c r a t a o r t a s w i t h t e s t i c u l a r h y a l u r o n i d a s e . - - . . . . , before h y a l u r o n i d a s e t r e a t m e n t ; - - - x - - - x - - -, a f t e r h y a l u r o n i d a s e t r e a t m e n t .
chondroitin treatment
sulfate B (dermatan of the chondroitin
hyaluronidase, demonstrated the enzyme
s u l f a t e ) is u n a f f e c t e d
sulfate containing
f o l l o w e d b y g e l f i l t r a t i o n of t h e p r o d u c t s that the chondroitin
by the enzyme.
fractions from lathyritic
Subsequent aortas with
of the enzymatic
sulfates were depolymerized
( F i g . 8). I t w a s c o n c l u d e d t h a t t h e i n c r e a s e d c h o n d r o i t i n
lathyritic aorta was for the most part due to elevated amounts
digestion,
to a large extent
by
sulfate content of
of chondroitin
sulfates
A/C.
TABLE 3 REGRESSION
OF THE EFFECTS
OF LATHYRISM
ON AORTIC
AMPS
W e a n l i n g m a l e or female r a t s (6 p e r group) were m a i n t a i n e d on a b a s a l diet (BD) c o n t a i n i n g 0 . 1 % B A P N for 4, 8, or 11 weeks. O t h e r g r o u p s received B A P N for 4 weeks after w h i c h t h e l a t h y r o g e n w a s r e m o v e d f r o m t h e diet a n d a n i m a l s (6 per group) were sacrificed 4 a n d 8 weeks later. T h e a o r t a s f r o m t h e a n i m a l s in e a c h g r o u p were pooled a n d tile A M P S were a n a l y z e d as described in t h e t e x t . R e s u l t s are e x p r e s s e d as # g of g l u c u r o n o l a c t o n e per 100 m g dry, d e f a t t e d tissue.
Group
Sex
M B D , 4 weeks F BD, 4 weeks M BAPN 4 weeks F B A P N 4 weeks M BAPN 8 weeks F B A P N 8 weeks B A P N 12 weeks M B A P N 12 weeks F 4 week r e c o v e r y M F 8 week r e c o v e r y M F F
Mean body weight (g)
0.8 M NaCI
1.25 M NaCl
2.5 M NaC1
Total
175 155 160 135 220 185 265 220 245 200 285 235 235
34 39 66 55 81 88 120 97 59 65 63 42 44
101 119 116 126 114 125 122 107 99 97 89 111 124
81 95 338 277 216 205 205 195 205 188 198 176 178
216 253 520 458 411 418 447 399 363 350 350 329 346
j . Atheroscler. Res., 1968, 8 : 7 8 7 - 8 0 1
798
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
All a t t e m p t was made to determine the reversibility of the alterations induced in aortic tissue b y the feeding of BAPN. Male and female weanling rats were fed a diet containing 0 . 1 % BAPN for 4 weeks. The lathyrogen was withdrawn from the diet and animals were sacrificed 4 and 8 weeks later. The results of the AMPS analyses of aortas taken from these animals are shown in Table 3. Although the levels of chondroitin sulfate decreased with time in the aortas of animals taken off the lathyrogenic diet so did the corresponding levels in the animals maintained on the lathyrogenic diet, over the entire experimental period, and the decrease was not such as to reduce the levels of chondroitin sulfate towards normal. There also appeared to be steady increase in the concentration of AMPS found in the 0.8 M NaC1 fraction of aortas taken from animals maintained on BAPN throughout the experiment. Eight weeks after the removal of BAPN from the diet; however, the levels of the 0.8 M NaC1 fraction were comparable to those observed in control animals. Little histological distinction could be made between animals from which BAPN had been withdrawn and those which had been maintained on the lathyrogen for the duration of the experiment. The metachromasia with toluidine blue persisted as did the increased amounts of smooth muscle. I t would appear that unlike the lathyritic skeletal deformities which are reported to be completely and rapidly reversible22, 23, the alterations induced in the aorta are long-lasting. This regression experiment failed to demonstrate a n y sex differences in either the development or regression of lathyrism as far as the aorta is involved. DISCUSSION The present study confirms both histologically and chemically that there is an increased concentration of acid mucopolysaccharides in the aorta of the lathyritic rat. Furthermore, this elevation is due mostly to a fairly rapid increase in the chondroitin sulfate fraction, particularly chondroitin sulfates A/C after the start of the lathyrogenic regime. Some investigators have postulated that the increase in AMPS is the result of reduced collagen and/or elastin concentrations brought about by an increased degradation of the proteins or by some block in their neogenesisll,14, 24. T h a t is, if the amount of protein per unit weight of aorta were reduced, then the AMPS would appear to be elevated. However, GRANT et al. 14 were unable to demonstrate any decrease in the amounts of collagen and elastin in the aortas of lathyritic rats and the present data also do not support this view. If decreased protein levels were responsible for the increased AMPS, then every AMPS component should have been equally affected. Therefore, it is more probable that the increase in AMPS is the result of an increased biosynthesis of chondroitin sulfate. HAM25 observed that the tunica media of the rat aorta contains only a single type of cell and she suggested t h a t this cell must function as smooth muscle and at the same time synthesize collagen, elastin and components of the ground substance of the media. As a corollary to these observations, we propose that the effects of lathyrism on aortic AMPS are mediated through changes in the activity of smooth muscle. The J. Atheroscler. Res., 1968, 8:787-801
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present histological findings support this concept because the only cellular alteration observed was a proliferation of smooth muscle particularly in areas showing structural damage. However, it is unlikely that lathyrogens stimulate smooth muscle directly since aortic AMPS in the adult animals were unaffected by the administration of BAPN (Table 2). Rather, it is suggested that the elevated concentration of aortic AMPS is the result of an increased proliferation of smooth muscle and that the increase in smooth muscle activity is part of a repair mechanism that responds to the development of structural weakness and damage in the wall of the aorta. The young, rapidly growing animal receiving lathyrogens is especially susceptible to aortic damage since the collagen and elastin which are being rapidly synthesized in the aorta are defective. On the other hand, the adult animal has already deposited sufficient quantities of normally structured proteins to maintain the tensile strength and elasticity of the aorta and consequently, little aortic damage or smooth muscle response would be expected during the 4-8 week administration of a lathyrogen. HAM25 also made the observation t h a t the fine structure of the smooth muscle in aorta differs from that of the smooth muscle in other tissues in that it contains fewer myofilaments and more cytoplasmic organelles. These differences as well as the absence of smooth muscle from such tissues as bone, cartilage and tendon could explain why lathyrogens produce no increase in AMPS in tissues other than the aorta. There is some evidence in the literature which supports the above hypothesis. LINKER et al. 26 showed t h a t copper deficiency leads to increased levels of chondroitin sulfate in the aortas of young swine. I t is known that young copper-deficient animals are predisposed towards the development of aortic aneurysms and there is good evidence that this is related to an inhibition of desmosine formation which, in turn, leads to a weakening of aortic elastinlT. Thus, in the absence of lathyrogens, structural aortic weakness as seen in copper deficiency can lead to an accumulation of chondroitin sulfate. LORENZEN also found that high levels of epinephrine and thyroxine produced aortic lesions in rabbits 28-3~ This was accompanied by an increase in the hexosamine content of the aorta and in the uptake of labeled sulfate into the artery. He presented evidence for fibroblast proliferation in the lesions, although the fibroblasts m a y actually have been smooth muscle cells since hematoxylin-eosin staining cannot differentiate between the two. LORENZEN concluded that these changes were the result of a non-specific response to injury. Also implicit in the present hypothesis is the suggestion that smooth muscle is able to synthesize chondroitin sulfate. In support of this concept is the observation by CRANE~1 that mesenteric smooth muscle in animals made hypertensive had an increased ability to utilize inorganic sulfate. HAM32 alSO presented some histological evidence which suggests that smooth muscle has a high level of synthetic activity during the early stages of lathyrism. She observed increased numbers of mitochondria and RNA granules in aortic smooth muscle cells following 3-4 weeks of lathyrogen administration. After 7 or 8 weeks, mitochondria, Golgi elements and components of endoplasmic reticulum were dilated or even absent, indicating cellular degeneration. In the present study, BAPN administration after the first 4 weeks resulted in a steady decline in the chondroitin sulfate fraction and an increase J. Athevoscler. Res., 1968, 8:787-801
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R. ALPER, j . T. PRIOR, W. R. RUEGAMER
in t h e 0.8 M NaCI fraction (Table 3). T h i s suggests a d e r a n g e m e n t in m u c o p o l y s a c c h a ride m e t a b o l i s m t h a t could be r e l a t e d to t h e cellular d e g e n e r a t i o n r e p o r t e d b y HAM. T h e o b s e r v a t i o n t h a t B A P N was m o r e effective t h a n s e m i c a r b a z i d e in p r o d u c i n g a o r t i c d a m a g e is in a g r e e m e n t w i t h t h e o b s e r v a t i o n s of LALICH a n d o t h e r s 33-~5. H o w e v e r , in t h e p r e s e n t s t u d y , i t was f o u n d t h a t s e m i c a r b a z i d e was as effective as B A P N in p r o d u c i n g A M P S a l t e r a t i o n s (Table 1) a n d was also able to p r o d u c e a l t e r a tions in s m o o t h muscle (Fig. 6). T h e a b i l i t y of B A P N to produce angiorrhexis m a y be reflection of its a c t i o n u p o n some s y s t e m which is r e l a t i v e l y unaffected b y s e m i c a r b a zide. Since s e m i c a r b a z i d e is an otherwise effective l a t h y r o g e n , it is possible t h a t t h e difference in a c t i v i t y b e t w e e n t h e two l a t h y r o g e n s m a y be r e l a t e d to a g r e a t e r a b i l i t y of B A P N to p r o d u c e defects in elastin. Since b o t h l a t h y r o g e n s affect t h e struct u r e of collagen, s m o o t h muscle p r o l i f e r a t i o n m a y be closely r e l a t e d to t h e p r o d u c t i o n of collagen defects which l e a d to a decreased tensile s t r e n g t h of t h e aorta. T h e fact t h a t b o t h B A P N a n d s e m i c a r b a z i d e p r o d u c e t h e s a m e increase in t h e A M P S as well as similar changes in t h e c o n t e n t a n d c o n f o r m a t i o n of s m o o t h muscle w o u l d suggest t h a t these effects are n o t p r i m a r y factors in t h e p r o d u c t i o n of aortic d a m a g e . ACKNOWLEDGEMENTS
W e are especially g r a t e f u l to Dr. W. W . W e s t e r f e l d a n d to Dr. R. J a c o b s for t h e i r helpful a d v i c e in the course of this s t u d y . T h i s s t u d y was a i d e d in p a r t b y a g r a n t from t h e U.S. P u b l i c H e a l t h Service, R e s e a r c h G r a n t No. AM-06292 from t h e I n s t i t u t e of A r t h r i t i s a n d Metabolic Diseases, a n d b y a g r a n t from t h e H e a r t Association of U p s t a t e New York, Inc. R. A. is a P r e d o c t o r a l Trainee of t h e U.S. P u b l i c H e a l t h Service, G r a n t No. 5-TIGM-301-05, from the I n s t i t u t e of t h e A c a d e m y of Medical Sciences.
REFERENCES x SELYE, H., Lathyrism, Rev. Can. Biol., 1957, 16: 1. 2 LEVENE, C. I. AND J. GRoss, Alterations in the state of molecular aggregation of collagen induced in chick embryos by beta-aminopropionitrile (Lathyrus factor), J. exp. Med., 1959, 110: 771. MARTIN, G. R., J. GROSS, H. A. PIEZ AND M. S. LEwis, Intramolecular crosslinking of collagen in lathyritic rats, Biochim. biophys. Acta, 1961, 53: 599. 40'DELL, B. L., D. F. ELSDEN, J. THOMAS, S. M. PARTRIDGE, R. I-I. SMITH AND R. PALMER, Inhibition of the biosynthesis of the cross-links in elastin by a lathyrogen, Nature (Lond.), 1966, 209: 401. 5 BERNTSEN, E., Epiphysial growth zones in acute lathyrism uptake and distribution of 85SO4 in cartilage and bone, Proc. Soc. exp. Biol. ( N . Y . ) , 1967, 124: 731. 6 BERNTSEN, E., Epiphysial growth zones in acute lathyrism: determination of water hydroxyproline and mucopolysaccharide, Proc. Soc. exp. Biol. ( N . Y . ) , 1966, 123: 588. 7 LEVENE, C. I., J. KRANZLERAND S. FRANCO-BRoWDER,Acid mucopolysaccharides of chickembryo cartilage in osteolathyrism, Biochem. J., 1966, 101: 435. 8 PEDRINI-3{ILLE, A. AND V. PEDRINI, Mucopolysaccharide defect in lathyrism, Proc. Soc. exp. Biol. ( N . Y . ) , 1965, 119: 322. 9 KARNOVSKY,M. J. AND M. L. KARNOVSKY,Metabolic effects of lathyrogenic agents on cartilage in vivo and in vitro, J. exp. Med., 1961, 113: 381. 18 PYORALA,K., S. PUNSAR,T. SEPPALAAND K. KARLSSON,Mucopolysaccharides of the aorta and epiphysial cartilage in lathyritic growing rat fetuses, A cta path. microbiol, scand., 1957, 41: 497. J. Atheroscler. Res., 1968, 8:787-801
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11 MENZIES, D. W. AND K. W. MILLS, The aortic and skeletal lesions of lathyrism in rats on a diet of sweet pea, ]. Path. Bact., 1957, 23: 223. 12 WIRTSCHAFTER, Z. T., Acid mucopolysaccharides in the histopathogenesis of aortic aneurysms in the lathyrus-fed rat, Arch. Path., 1957, 64: 577. is BICELEY, H., Experimental lathyrism: apparent antithesis of ageing in connective tissue, ] . Amer. geriat. Sot., 1964, 12: 717. 14 GRANT, R. A., M. HATHORN AND T. GILLMAN, Aortic, serum, connective tissue and osseous chemistry in lathyritic rats, Nature (Loud.), 1960, 186: 164. is BURMAN,S. O., Medial degeneration and its relation to dissecting aneurysms, Int. Abstr. Surg., 1960, 110: I. 16 RICHERT, D. A., J. SCHENKMANAND W. W. WESTERFELD, An antithyrotoxic assay based upon the response of rat liver a-glycerophosphate dehydrogenase, J. Nutr., 1964, 83: 322. 17 RUEGAMER, W. R., W. W. WESTERFELD AND D. A. I~ICHERT, a-Glycerophosphate dehydrogenase response to thyroxine in thyroidectomized, thiouracil-fed and temperature-adapted rats, Endocrinology, 1964, 75: 908. is SCHILLER, S., G. A. SLOVER AND A. DORFMAN, A method for the separation of acid mucopolysaccharide: its application to the isolation of heparin from the skin of rats, J. biol. Chem., 1961, 236: 983. 19 DlSCHE, Z., A new specific color reaction of hexuronic acids, J. biol. Chem., 1947, 167: 189. 20 SCI~MIDT,M. AND M. DM0C~OWSKI, The determination of isomeric chondroitin sulfates, Biochim. biophys. Acta, 1964, 83: 137. 21 DALFERES, E. R., JR., B. I~ADAKRISHNAI~URTHYAND G. S. BERENSON, Acid mucopolysaccharides of amyloid tissue, Arch. biochem., 1967, 118: 284. 22 RAMAMURTI,P. AND H. ]~. TAYLOR, Histochemical studies of the evaluation and regression of skeletal deformaties due to fl-aminopropionitrile, Lab. Invest., 1958, 7:115. 23 MARWAH,A. S., W. DASLER AND I. MEYER, Reversibility of lathyric damage to the periodontal structures, J. Periodont., 1963, 34: 1421. 24 CHURCHILL, D. W., S. GELFANT, J. J. LALICH AND D. M. ANGEVINE, Alterations in the polysaccharides and elastic fibers in the aortas of rats fed toxic lathyrus factor, Lab. Invest., 1955, 4: 1. ~5 HAM, K . N . , The fine structure of normal rat aorta, Aust. J. exp. biol. reed. Sci., 1962, 40: 341. 26 LINKER, A., W. F. COULSON AND W. H. CARNY-S, Cardiovascular studies on copper deficient swine, P a r t 6 (The mucopolysaccharide composition of aorta and cartilage), J. biol. Chem., 1964, 239: 1690. 27 MILLER, E. J., G. R. MARTIN, C. E. MECCA AND K. A. PIEZ, The biosynthesis of elastin crosslinks. The effects of copper deficiency and a lathyrogen, J. biol. Chem., 1965, 240: 3623. 2s LORE~ZE~, I., Vascular connective tissue changes induced by catecholamines and thyroid hormone. In: G. ASBOE-HANS~N (Ed.), Hormones and Connective Tissue, Williams and Wilkins, Baltimore, Md., 1966, p. 136. 29 LORENZEN, I., Alterations in acid mucopolysaccharide and r of rabbit aorta related to age of epinephrine-thyroxine induced arteriosclerotic lesions, Acta entoG., (Kbh). 19~2, 3~: 615. s~ LORENZEN,I., Vascular connective tissue under the influence of thyroxine, Part 1 (Epinephrinethyroxine induced alterations in rabbit aorta), Acta endocr. (Kb~).) 1961, 35: 197. sl CRANE, W. A. J., Sulfate utilization and mucopolysaccharide synthesis by m~eat~ric ~rt~ri~s of hypertensive rats, J. Path. Bact., 1962, 84: 113. s~ HAM, K., The fine structure of rat aorta in experimental lathyrism, Aust. J. exp. biol. meal. Sci., 1962, 40: 353. s 3 LAUCH, J. J., Effect of different lathyrogens upon tissue responses in rats. Proc. Soc. exp. Biol. ( N . Y . ) , 1966, 123: 214. s4 D^SLER, W. AND R. V. MILLXSER, OsteolathyTogenic action of mercaptoethylamiue and of cystamine, Proc. Soc. exp. Biol. ( N . Y . ) , 1958, 98: 759. s 5 MILLmEE, R. V. AND W. DASLBR, Osteolathyrism. Histopathological lesions produced by semicarbazide and acetone semicarbazone, Arch. Path., 1959, 67: 427.
j . Atheroscler. Res., 1968, 8:787-801
Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
H I S T O L O G I C A L AND B I O C H E M I C A L S T U D I E S ON T H E G R O U N D SUBSTANCE OF T H E AORTAS OF L A T H Y R I T I C RATS
R. ALPER*,
J. T. PRIOR
AND
W.
I~. RUEGAMER
Departments of Biochemistry and Pathology, State University of New York, Upstate Medical Center, Syracuse, N.Y. (U.S.A.)
(Received February 13th, 1968)
SUMMARY Aortas from normal and lathyritic rats were examined histologically and chemically. The administration of fl-aminopropionitrile (BAPN) to young animals often resulted in the production of medial lesions of the aortic arch; such lesions did not develop in animals administered semicarbazide (SC). Microscopic examination of the lesions revealed a marked disorganization of the elastic components of the arterial wall, a high degree of metachromasia with toluidine blue and a proliferation of smooth muscle cells. Increased metachromasia and a hypertrophy of the smooth muscle cells were also seen in sections of the thoracic and abdominal aorta in which there was no obvious structural damage in response to the administration of either BAPN or SC. Analyses of the acid mucopolysaccharide (AMPS) composition of aortas from young lathyritic animals revealed a marked increase in the chondroitin sulfate A plus C fraction, but essentially no change in the other AMPS components of the arterial wall. The changes were not observed in adult lathyritic animals. The aortic AMPS changes in young lathyritic rats persist for a long time after the lathyrogenic agent (BAPN) is withdrawn from the diet. The present observations are consistent with the concepts that (a) it is the rapidly growing animal that is particularly susceptible to the effects of lathyrogenic agents on the aorta; (b) smooth muscle is involved in the repair of arterial injury; (c) smooth muscle cells biosynthesize AMPS; and (d) the changes represent a nonspecific response of the aorta to injury.
* Present address: Department of Biochemistry, School of Medicine, State University of New York at Buffalo, Buffalo, N.Y. 14214, U.S.A. J. Atheroscler. Res., 1968, 8:787-801
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INTRODUCTION
Experimental lathyrism is a connective tissue disease induced b y the action of certain compounds (lathyrogens), the most commonly used of which are /5-aminopropionitrile (BAPN), aminoacetonitrile (AAN) and semicarbazide. The condition is characterized b y the development of various skeletal abnormalities and b y a predisposition to the development of abdominal wall hernias and aortic aneurysms 1. The most characteristic biochemical defect in lathyrism is the dramatic increase in the proportion of salt-soluble collagen that is extractable from connective tissue2, 3. This has been attributed to a defect in the production of covalent intra- and intermolecular cross linkages within the collagen molecule. A similar defect in the structure of elastin apparently involves an inhibition of desmosine and isodesmosine formation 4. These amino acids are believed to participate in the formation of cross linkages in the elastin molecule. Such defects decrease the tensile strength and elasticity of these proteins. Thus, the lathyritic symptoms that have been described might be expected to be predominant in those tissues which are chronically subjected to strain, e.g. the aorta and the epiphyses of the bones. Although there has been considerable interest in the effects of experimental lathyrism on the acid mucopolysaccharide (AMPS) composition of connective tissue, m a n y of the studies have been rather inconclusive. BERNTSEN5,6 presented evidence t h a t the AMPS content of the epiphysial plate cartilage was decreased in the lathyritic animal and that the uptake of labeled sulfate into the epiphysial growth zones was depressed. LEVENE et al. 6 were unable to demonstrate any alterations either qualitatively or quantitatively in the AMPS content of chick embryo cartilage. PEDRINIMILLE AND PEDRINIs demonstrated a decreased synthesis of hexosamine in lathyritic epiphysial cartilage but KARNOVSKY AND KARNOVSKY9 found no alteration in the hexosamine content of the same tissue. However, chemical, histological and radioautographic studies have shown that there is a marked increase in the sulfated AMPS content of the lathyritic aorta 1~ BICKLEY13 has cited this as being the only consistent alteration in the composition of the ground substance that has been observed in any tissue. GRANT e~ al. 14 found a significant elevation in the hexosamine content of lathyritic aorta and also demonstrated the absence of similar effects in bone, cartilage, and tendon. Thus, the effect of lathyrism on the aortic connective tissue differs from that seen in other types of connective tissue. BURMAN 15, in a review on aortic medial degeneration, pointed to the possibility that the accumulation of interlaminar polysaccharide m a y be a non-specific response of the aorta to a variety of adverse conditions. If this response were an intrinsic property of the aorta, it would not be observed in other connective tissues. This would imply: (1) that there are cellular components of aorta which are absent in other tissues and which are responsible for the increased levels of AMPS seen under various conditions or (2) that the cellular elements in aorta which are involved in the deposition of ground substance behave differently from those in other connective tissues. I t was the purpose of this investigation to further characterize the effects of J. Atheroscler. Res., 1968, 8:787-801
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experimental lathyrism on the AMPS content of aorta and to attempt to correlate the biochemical events with the histological alterations. The evidence presented in this paper would support the concept that the increased AMPS concentrations found in the lathyritic aorta are part of a non-specific response to injury and that smooth muscle may have an important role in this response. MATERIALS AND METHODS
In the majority of these experiments, weanling (40-60 g body weight) male or female Sprague-Dawley strain albino rats were used. The animals were fed ad libitum a purified basal diet (BD) consisting of 60 % sucrose and 30 % casein plus vitamins and mineralsl6,17 and were housed in a constant temperature room (23~ with controlled lighting (5 a.m.-6 p.m.). Lathyrism was induced by feeding the basal diet containing 0 . 1 % (w/w) of either fl-aminopropionitrile hydrochloride (BAPN) or semicarbazide hydrochloride (SC) for a period of at least 4 weeks. At appropriate time intervals, the animals were sacrificed and the aortas were quickly excised. Adherent blood, fat, and adventitia were removed and the tissues were used directly or were placed in a freezer at --20~ to await analysis. Samples for histological examination were fixed in neutral formalin.
Histological The formalin-fixed aortic samples were imbedded in paraffin, sectioned and stained with (a) hematoxylin-eosin, (b) Weigert-van Gieson's stain, (c) 0.5 % toluidine blue and (d) Masson's trichrome stain. Analytical The aortas from at least 5 animals were pooled. These were finely minced with scissors and then extracted with chloroform-methanol (2 : 1, v/v) for at least 4 h in a Soxhlet apparatus. The defatted tissue was dried to a constant weight and at least 100 mg of dry material was added to 5 ml of 0.1 M phosphate buffer (pH 6.5) which contained 2 mg of papain (2 • recrystallized, Sigma Chemical Co.), 0.01 M ethylenediaminetetra-acetic acid (disodium salt), and 0.005 M L-cysteine. The mixture was incubated at 50 ~ for 24 h. The pH was then readjusted to 6.5 with 0.1 N NaOH, 1 ml of newly prepared papain solution was added and the mixture was re-incubated for another 24 h. The papain digest was centrifuged at 600 • g for 30 rain, the supernatant was decanted and the insoluble residue was washed twice with distilled water. The combined washings and supernate were passed directly onto an anion exchange column (Bio-Rad AG l-X2, 200-400 mesh, C1 form, 1 cm • 15 cm). After the column had been washed with 25 ml of 0.1 M NaC1, the acid mucopolysaccharides (AMPS) were eluted by a modification of the procedure of SCHILLERet al. is. This involved the stepwise application of 50 ml each of 0.5 M, 0.8 M, 1.25 M, and 2.5 M NaC1. The eluate was collected in 5-ml fractions and each fraction was analyzed for hexuronic acid by the j . Atheroscler. Res., 1968, 8 : 7 8 7 - 8 0 1
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carbazole method of DISCHE19 using I~-glucuronolactone as a standard. The results were expressed as #g of glucoronolactone found per 100 mg of dry, defatted tissue for each step of the elution system. In order to evaluate the quantitative and qualitative aspects of this procedure, experiments were performed in which known amounts of standard AMPS* were added to weighed tissue samples prior to the papain digestion. After hydrolysis with the enzyme, the samples were chromatographed as described and the recoveries of the internal standards were determined. A modification of the procedure of SCHMIDT AND DMOCHOWSK120was used for the determination of the relative amounts of chondroitin sulfates A/C and B. The chondroitin sulfate-containing fractions from at least three columns were combined and dialyzed overnight against distilled water. The dialysate was evaporated to dryness on a rotary evaporator and then reconstituted in 5 ml of citrate-phosphate buffer, p H 5.0.2-ml aliquots were treated with 600 I.U. of testicular hyaluronidase in 0.1 M NaC1 for 24 h at 37~ The mixture was then applied to a Sephadex G-25 column (1 cm • 35 cm) and eluted in 1 M NaC1. 3-ml fractions were collected and each was analyzed for hexuronic acid b y the carbazole procedure 19. RESULTS
Virtually all of the weanling animals that were fed lathyrogens developed a marked scoliosis of the spine and thoracic cage after 4 weeks. By the end of this period approximately one-third of the animals receiving BAPN had died of ruptured aneurysms of the ascending or thoracic aorta, whereas all of the animals on semicarbazide survived. Histological Marked lesions in the aortic arch were observed in about half of the survivors of the animals receiving BAPN for 4 weeks or longer. A typical lesion is shown in Fig. 1. There was a visible thickening of the wall of the arch and the internal surface of the lesion was very irregular. The lumen of the arch was much wider than in control animals, which suggests that the lesion was produced in response to the development of an aneurysm. Microscopic examination of these lesions revealed some marked alterations in the tunica media. The lamellar structure of the tissue was severely disrupted and, in m a n y areas of the media, it had entirely disappeared (Fig. 2). There was a profusion within the ground substance of short fibers which stained positively for elastic tissue. Toluidine blue staining revealed a marked metachromasia indicating the presence of large amounts of acid mucopolysaccharides (AMPS). There was a random prolifera-
* Hyaluronic acid and chondroitin sulfate preparations were obtained from Sigma Chemical Co. (St. Louis). Heparitin sulfate was a gift of Dr. J. A. Cifonelli. Chondroitin sulfate 13 was a gift of Dr. Karl Meyer. J. Atheroscler. Res., 1968, 8:787-801
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Fig. 1. Production of an aortic arch lesion in a rat fed BAPN for 5 weeks. Left: Aorta from a control animal. Right: Aorta from a lathyritic animal. There is a marked thickening of the aorta within the arch. Note the highly irregular intimal surface of the lesion.
tion of s m o o t h muscle t h r o u g h o u t t h e lesion (Fig. 3), giving t h e overall impression t h a t a r e p a i r process h a d been i n a u g u r a t e d in response to t h e d e v e l o p m e n t of t h e a n e u r y s m . I n two animals, dissecting a n e u r y s m s d e v e l o p e d in t h e aortic arch followed b y t h e f o r m a t i o n of a new channel w i t h t h e r e s u l t a n t p r o d u c t i o n of a " d o u b l e b a r r e l e d " a o r t a (Fig. 4). T h e t h i c k e n i n g of t h e wall of the a o r t a was v e r y p r o m i n e n t in t h e v i c i n i t y of t h e dissection as was t h e proliferation of s m o o t h muscle a n d t h e disr u p t i o n of t h e l a m e l l a r s t r u c t u r e of t h e media. A n i m a l s m a i n t a i n e d on the semicarb a z i d e regimen seldom d e v e l o p e d these lesions. Microscopic e x a m i n a t i o n of t h o r a c i c a n d a b d o m i n a l sections of a o r t a s from b o t h n o r m a l a n d l a t h y r i t i c a n i m a l s r e v e a l e d a definite t h i c k e n i n g of t h e a r t e r i a l wall along its entire l e n g t h in t h e a o r t a s of t h e l a t h y r i t i c animals. These sections d i d n o t d i s p l a y t h e lesions which h a v e been described above. F u r t h e r m o r e , this t h i c k e n i n g was o b s e r v e d in a n i m a l s fed s e m i c a r b a z i d e as well as B A P N . T h e l a m e l l a r s t r u c t u r e of J. Ath*roscler. Res., 1968, 8:787-801
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Fig. 2. Histological e x a m i n a t i o n of a typical aortic arch lesion produced b y the a d m i n i s t r a t i o n of B A P N for 5 weeks. The effects on the elastic c o m p o n e n t s of the aorta. Left: Control aorta; note the c o n t i n u o u s lamellar s t r u c t u r e of the artery. Right: A o r t a of treated animal at s a m e magnification. Section t a k e n t h r o u g h an arch lesion; note the e x t r e m e thickening of the wall of the artery, the m a r k e d disorganization of the elastic lamellae and the profusion of s h o r t elastic fibers. V a n Gieson, • 170.
Fig. 3. Histological e x a m i n a t i o n of a typical aortic arch lesion produced b y t h e a d m i n i s t r a t i o n of B A P N for 5 weeks. The effects on the c o n t e n t of s m o o t h muscle, Left: Control aorta. Right: A o r t a of t r e a t e d animal at same magnification. Section t a k e n t h r o u g h the lesion. Note t h e m a r k e d proliferation and disorientation of the s m o o t h muscle. Masson's Trichrome, • 170,
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Fig. 4. The d e v e l o p m e n t of a " d o u b l e barreled" a o r t a in an a n i m a l receiving B A P N for eight weeks. The n e w channel is on the left. H e m a t o x y l i n - e o s i n , X 45.
the artery remained intact but there was an increase in the distance between lamellae. The arteries displayed a marked interlamellar metachromasia (Fig. 5), which gave the impression that there were increased amounts of ground substance within the arterial wall. Further histological examination of these sections revealed some unusual alterations in the smooth muscle of the aorta (Fig. 6). There appeared to be a greater number of smooth muscle cells within the lathyritic aorta, m a n y of the cells being very large. These cells were highly elongated, their long axes being perpendicular to the elastic lamellae. The increased metachromasia and the alterations in the smooth muscle were most prominent in the innermost third of the media.
Analytical Since glycoproteins as well as acid mucopolysaccharides contain hexosamines, it was decided that more meaningful information could be obtained b y performing a chromatographic separation of the AMPS rather than b y relying upon a determination of total hexosamine concentration for an estimate of the AMPS content of aorta, as J. Atheroscler. Res., 1968, 8 : 7 8 7 - 8 0 1
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Fig. 5. M e t a c h r o m a s i a in a section of the thoracic a o r t a of an animal receiving semicarbazide for 5 weeks. Left: ControI aorta. Right: Lathyritic a o r t a a t s a m e magnification. Note the intense m e t a c h r o m a s i a and the increased interlamellar distance. Toluidine blue, • 280.
Fig. 6. Alterations in t h e s m o o t h muscle w i t h i n the thoracic a o r t a of an animal receiving semicarbazide for 5 weeks. Left: Control aorta. Right: Lathyritic a o r t a at the same magnification. Note the h y p e r t r o p h y of the s m o o t h muscle cells and the increased n u m b e r of these cells within the widened interlamellar space. Masson's Trichrome, • 640.
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have m a n y investigators. The ion exchange chromatographic procedure of SCHILLER et al.i8 provided an excellent method for the separation and quantitation of hyaluronic acid, heparitin sulfate, the chondroitin sulfates and heparin. Hyaluronic acid was eluted with 0.5 M NaC1, heparitin sulfate with 1.25 M NaC1, the chondroitin sulfates with 1.5 M NaC1 and heparin with 2.5 M NaC1. The procedure was found to be quantitative in that over 90 % of the standard AMPS applied to the column could be recovered in the appropriate fractions. Preliminary experiments demonstrated that heparin was not a constituent of rat aorta and since considerable "tailing" of the chondroitin sulfate fraction was obtained when it was eluted with 1.5 M NaC1, we decided to elute the chondroitin sulfate fraction in 2.5 M NaC1. This greatly reduced the "tailing", thus providing more reproducible results. The procedure employed had an additional advantage over other procedures in that very little protein remaining in the papain digests of aorta was retained b y the column. The small amount that was adsorbed to the resin was eluted with 0.5 M NaC1. This eliminated the necessity for the removal of protein from the papain digest and for a preliminary precipitation of the AMPS. Recoveries of internal standards were consistently in excess of 85 % and the modified procedure thus provided a convenient and rapid method for the quantitative analysis of the various aortic AMPS. A typical chromatogram of the AMPS in normal rat aorta is presented in Fig. 7. The major AMPS fraction, eluted in 1.2 M NaC1, seems to be heparitin sulfate. The second major fraction was eluted in 2.5 M NaC1 and probably is a mixture of the various chondroitin sulfates. A smaller fraction was eluted in 0.8 M NaCI. No a t t e m p t has been made to characterize this fraction but DALFERES et al. 21 reported that a similar fraction contained a heparitin sulfate-like material of low sulfate content. Little if any AMPS was eluted in 0.5 M NaC1 indicating that hyaluronic acid is not an import a n t component of rat aorta. The AMPS of normal rat aorta consist of an average of 45-50 % heparitin sulfate, 35-40 % chondroitin sulfate and about 15 % of the material eluted in 0.8 M NaC1 (in terms of the percent of the total glucuronolactone equivalent eluted from the 0.SM
O.8M
NaCI
NoEl
1.25M
NoCI
r
r
25M
NoC|
r
6C
-~. 4C 3C o s
20 ~o
t
o
4
8
12
i , i i J 16 20 24 28 Fraction No,
32
36
40
Fig. 7. C h r o m a t o g r a m of t h e AMPS f r o m a p a p a i n digest of n o r m a l r a t aortas on Bio-Rad AG 1 X2.
J. Atheroscler. Res., 1968, 8:787-801
796
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
:['ABLE 1 I'.'FFECTS OF LATHYRISM
ON
AMPS
OF RAT AORTA:
WEANLING
ANIMALS
Weanling animals were maintained for 30 days on a basal diet (BD) containing 0.1% ]3APN or 0.1% semicarbazide (SC). Aortas from 6 rats were pooled and the aortic acid mucopolysaccharides were analyzed as described in the text. Results are expressed as/,g of glucuronolactone per 100 mg of dry, defatted tissue. Regimen
iV[ean body weight (g)
0.8 M NaCl
0 . 2 5 M NaC1
2.5MNaCl
Total
BD BD 0.1 % 0.1 % 0.1% 0.1 % 0.1% 0.1 %
165 165 147 147 147 162 162 162
44 85 64 55 36 39 61 61
130 123 157 115 141 138 166 124
94 109 179 162 197 185 220 216
268 287 400 332 374 362 447 401
BAPN BAPN BAPN SC SC SC
TABLE 2 EFFECTS
OF LATHYRISM
ON
AMPS
OF RAT AORTA: ADULT
ANIMALS
Adult (400-600 g) rats were maintained on a basal diet (BD) containing 0.1% BAPN for 30 or 60 days. Aortas from 6 animals within each group were combined and analyses were performed as described in the text. Results are expressed as #g of glucuronolactone per 100 mg of dry, defatted tissue. Regimen
Mean body weight (g)
0.8 M NaCl
1.25 M NaCl
2.5 M NaCl
Total
BD BAPN, 30 d. BAPN, 60 d.
530 495 510
48
121
63
46 42
149 136
63 62
232 258 240
ion exchange column). Analyses of aortas from weanling a n i m a l s t r e a t e d with l a t h y r o g e n s for 30 days revealed a m a r k e d elevation in the c h o n d r o i t i n sulfate c o n t e n t to a b o u t twice t h a t found in control a n i m a l s (Table 1). T h e other AMPS fractions were relatively unaffected. Thus, in the l a t h y r i t i c animal, the condroitin sulfates c o n s t i t u t ed a b o u t half of the total AMPS f o u n d in the aortas of a n i m a l s fed either B A P N or semicarbazide. However, when B A P N was a d m i n i s t e r e d to adult a n i m a l s (400-600 g b o d y weight), the AMPS profile was unaffected even after c o n t i n u e d feeding of the l a t h y r o g e n i c diet for as long as 2 m o n t h s (Table 2). The chondroitin sulfate concentrations i n the aortas of older a n i m a l s were o n l y a b o u t 70 % of those f o u n d in y o u n g a n i m a l s (Tables 1 a n d 2), whereas the other AMPS fractions appeared to be relatively unaffected b y age. I n order to gain some insight as to which of the various c h o n d r o i t i n sulfates m i g h t be elevated in the l a t h y r i t i c aorta, a d v a n t a g e was t a k e n of the fact t h a t c h o n d r o i t i n sulfates A/C can be depolymerized b y testicular h y a l u r o n i d a s e whereas, J. Atheroscler. Res., 1968, 8:787--801
STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITIC RATS
Void volume
797
Salt volume
70
~ 6o I ~5o ::k E
o40
\
0 30 _o No 2o
-6
f 12
18
,~!"x,..~ , , 24 30 34 42 Eluate (ml)
Fig. 8. Gel filtration on S e p h a d e x G 25 of t h e p r o d u c t s of t h e t r e a t m e n t of t h e c h o n d r o i t i n s u l f a t e f r a c t i o n s of l a t h y r i t i c r a t a o r t a s w i t h t e s t i c u l a r h y a l u r o n i d a s e . - - . . . . , before h y a l u r o n i d a s e t r e a t m e n t ; - - - x - - - x - - -, a f t e r h y a l u r o n i d a s e t r e a t m e n t .
chondroitin treatment
sulfate B (dermatan of the chondroitin
hyaluronidase, demonstrated the enzyme
s u l f a t e ) is u n a f f e c t e d
sulfate containing
f o l l o w e d b y g e l f i l t r a t i o n of t h e p r o d u c t s that the chondroitin
by the enzyme.
fractions from lathyritic
Subsequent aortas with
of the enzymatic
sulfates were depolymerized
( F i g . 8). I t w a s c o n c l u d e d t h a t t h e i n c r e a s e d c h o n d r o i t i n
lathyritic aorta was for the most part due to elevated amounts
digestion,
to a large extent
by
sulfate content of
of chondroitin
sulfates
A/C.
TABLE 3 REGRESSION
OF THE EFFECTS
OF LATHYRISM
ON AORTIC
AMPS
W e a n l i n g m a l e or female r a t s (6 p e r group) were m a i n t a i n e d on a b a s a l diet (BD) c o n t a i n i n g 0 . 1 % B A P N for 4, 8, or 11 weeks. O t h e r g r o u p s received B A P N for 4 weeks after w h i c h t h e l a t h y r o g e n w a s r e m o v e d f r o m t h e diet a n d a n i m a l s (6 per group) were sacrificed 4 a n d 8 weeks later. T h e a o r t a s f r o m t h e a n i m a l s in e a c h g r o u p were pooled a n d tile A M P S were a n a l y z e d as described in t h e t e x t . R e s u l t s are e x p r e s s e d as # g of g l u c u r o n o l a c t o n e per 100 m g dry, d e f a t t e d tissue.
Group
Sex
M B D , 4 weeks F BD, 4 weeks M BAPN 4 weeks F B A P N 4 weeks M BAPN 8 weeks F B A P N 8 weeks B A P N 12 weeks M B A P N 12 weeks F 4 week r e c o v e r y M F 8 week r e c o v e r y M F F
Mean body weight (g)
0.8 M NaCI
1.25 M NaCl
2.5 M NaC1
Total
175 155 160 135 220 185 265 220 245 200 285 235 235
34 39 66 55 81 88 120 97 59 65 63 42 44
101 119 116 126 114 125 122 107 99 97 89 111 124
81 95 338 277 216 205 205 195 205 188 198 176 178
216 253 520 458 411 418 447 399 363 350 350 329 346
j . Atheroscler. Res., 1968, 8 : 7 8 7 - 8 0 1
798
R. ALPER, J. T. PRIOR, W. R. RUEGAMER
All a t t e m p t was made to determine the reversibility of the alterations induced in aortic tissue b y the feeding of BAPN. Male and female weanling rats were fed a diet containing 0 . 1 % BAPN for 4 weeks. The lathyrogen was withdrawn from the diet and animals were sacrificed 4 and 8 weeks later. The results of the AMPS analyses of aortas taken from these animals are shown in Table 3. Although the levels of chondroitin sulfate decreased with time in the aortas of animals taken off the lathyrogenic diet so did the corresponding levels in the animals maintained on the lathyrogenic diet, over the entire experimental period, and the decrease was not such as to reduce the levels of chondroitin sulfate towards normal. There also appeared to be steady increase in the concentration of AMPS found in the 0.8 M NaC1 fraction of aortas taken from animals maintained on BAPN throughout the experiment. Eight weeks after the removal of BAPN from the diet; however, the levels of the 0.8 M NaC1 fraction were comparable to those observed in control animals. Little histological distinction could be made between animals from which BAPN had been withdrawn and those which had been maintained on the lathyrogen for the duration of the experiment. The metachromasia with toluidine blue persisted as did the increased amounts of smooth muscle. I t would appear that unlike the lathyritic skeletal deformities which are reported to be completely and rapidly reversible22, 23, the alterations induced in the aorta are long-lasting. This regression experiment failed to demonstrate a n y sex differences in either the development or regression of lathyrism as far as the aorta is involved. DISCUSSION The present study confirms both histologically and chemically that there is an increased concentration of acid mucopolysaccharides in the aorta of the lathyritic rat. Furthermore, this elevation is due mostly to a fairly rapid increase in the chondroitin sulfate fraction, particularly chondroitin sulfates A/C after the start of the lathyrogenic regime. Some investigators have postulated that the increase in AMPS is the result of reduced collagen and/or elastin concentrations brought about by an increased degradation of the proteins or by some block in their neogenesisll,14, 24. T h a t is, if the amount of protein per unit weight of aorta were reduced, then the AMPS would appear to be elevated. However, GRANT et al. 14 were unable to demonstrate any decrease in the amounts of collagen and elastin in the aortas of lathyritic rats and the present data also do not support this view. If decreased protein levels were responsible for the increased AMPS, then every AMPS component should have been equally affected. Therefore, it is more probable that the increase in AMPS is the result of an increased biosynthesis of chondroitin sulfate. HAM25 observed that the tunica media of the rat aorta contains only a single type of cell and she suggested t h a t this cell must function as smooth muscle and at the same time synthesize collagen, elastin and components of the ground substance of the media. As a corollary to these observations, we propose that the effects of lathyrism on aortic AMPS are mediated through changes in the activity of smooth muscle. The J. Atheroscler. Res., 1968, 8:787-801
STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITICRATS
799
present histological findings support this concept because the only cellular alteration observed was a proliferation of smooth muscle particularly in areas showing structural damage. However, it is unlikely that lathyrogens stimulate smooth muscle directly since aortic AMPS in the adult animals were unaffected by the administration of BAPN (Table 2). Rather, it is suggested that the elevated concentration of aortic AMPS is the result of an increased proliferation of smooth muscle and that the increase in smooth muscle activity is part of a repair mechanism that responds to the development of structural weakness and damage in the wall of the aorta. The young, rapidly growing animal receiving lathyrogens is especially susceptible to aortic damage since the collagen and elastin which are being rapidly synthesized in the aorta are defective. On the other hand, the adult animal has already deposited sufficient quantities of normally structured proteins to maintain the tensile strength and elasticity of the aorta and consequently, little aortic damage or smooth muscle response would be expected during the 4-8 week administration of a lathyrogen. HAM25 also made the observation t h a t the fine structure of the smooth muscle in aorta differs from that of the smooth muscle in other tissues in that it contains fewer myofilaments and more cytoplasmic organelles. These differences as well as the absence of smooth muscle from such tissues as bone, cartilage and tendon could explain why lathyrogens produce no increase in AMPS in tissues other than the aorta. There is some evidence in the literature which supports the above hypothesis. LINKER et al. 26 showed t h a t copper deficiency leads to increased levels of chondroitin sulfate in the aortas of young swine. I t is known that young copper-deficient animals are predisposed towards the development of aortic aneurysms and there is good evidence that this is related to an inhibition of desmosine formation which, in turn, leads to a weakening of aortic elastinlT. Thus, in the absence of lathyrogens, structural aortic weakness as seen in copper deficiency can lead to an accumulation of chondroitin sulfate. LORENZEN also found that high levels of epinephrine and thyroxine produced aortic lesions in rabbits 28-3~ This was accompanied by an increase in the hexosamine content of the aorta and in the uptake of labeled sulfate into the artery. He presented evidence for fibroblast proliferation in the lesions, although the fibroblasts m a y actually have been smooth muscle cells since hematoxylin-eosin staining cannot differentiate between the two. LORENZEN concluded that these changes were the result of a non-specific response to injury. Also implicit in the present hypothesis is the suggestion that smooth muscle is able to synthesize chondroitin sulfate. In support of this concept is the observation by CRANE~1 that mesenteric smooth muscle in animals made hypertensive had an increased ability to utilize inorganic sulfate. HAM32 alSO presented some histological evidence which suggests that smooth muscle has a high level of synthetic activity during the early stages of lathyrism. She observed increased numbers of mitochondria and RNA granules in aortic smooth muscle cells following 3-4 weeks of lathyrogen administration. After 7 or 8 weeks, mitochondria, Golgi elements and components of endoplasmic reticulum were dilated or even absent, indicating cellular degeneration. In the present study, BAPN administration after the first 4 weeks resulted in a steady decline in the chondroitin sulfate fraction and an increase J. Athevoscler. Res., 1968, 8:787-801
800
R. ALPER, j . T. PRIOR, W. R. RUEGAMER
in t h e 0.8 M NaCI fraction (Table 3). T h i s suggests a d e r a n g e m e n t in m u c o p o l y s a c c h a ride m e t a b o l i s m t h a t could be r e l a t e d to t h e cellular d e g e n e r a t i o n r e p o r t e d b y HAM. T h e o b s e r v a t i o n t h a t B A P N was m o r e effective t h a n s e m i c a r b a z i d e in p r o d u c i n g a o r t i c d a m a g e is in a g r e e m e n t w i t h t h e o b s e r v a t i o n s of LALICH a n d o t h e r s 33-~5. H o w e v e r , in t h e p r e s e n t s t u d y , i t was f o u n d t h a t s e m i c a r b a z i d e was as effective as B A P N in p r o d u c i n g A M P S a l t e r a t i o n s (Table 1) a n d was also able to p r o d u c e a l t e r a tions in s m o o t h muscle (Fig. 6). T h e a b i l i t y of B A P N to produce angiorrhexis m a y be reflection of its a c t i o n u p o n some s y s t e m which is r e l a t i v e l y unaffected b y s e m i c a r b a zide. Since s e m i c a r b a z i d e is an otherwise effective l a t h y r o g e n , it is possible t h a t t h e difference in a c t i v i t y b e t w e e n t h e two l a t h y r o g e n s m a y be r e l a t e d to a g r e a t e r a b i l i t y of B A P N to p r o d u c e defects in elastin. Since b o t h l a t h y r o g e n s affect t h e struct u r e of collagen, s m o o t h muscle p r o l i f e r a t i o n m a y be closely r e l a t e d to t h e p r o d u c t i o n of collagen defects which l e a d to a decreased tensile s t r e n g t h of t h e aorta. T h e fact t h a t b o t h B A P N a n d s e m i c a r b a z i d e p r o d u c e t h e s a m e increase in t h e A M P S as well as similar changes in t h e c o n t e n t a n d c o n f o r m a t i o n of s m o o t h muscle w o u l d suggest t h a t these effects are n o t p r i m a r y factors in t h e p r o d u c t i o n of aortic d a m a g e . ACKNOWLEDGEMENTS
W e are especially g r a t e f u l to Dr. W. W . W e s t e r f e l d a n d to Dr. R. J a c o b s for t h e i r helpful a d v i c e in the course of this s t u d y . T h i s s t u d y was a i d e d in p a r t b y a g r a n t from t h e U.S. P u b l i c H e a l t h Service, R e s e a r c h G r a n t No. AM-06292 from t h e I n s t i t u t e of A r t h r i t i s a n d Metabolic Diseases, a n d b y a g r a n t from t h e H e a r t Association of U p s t a t e New York, Inc. R. A. is a P r e d o c t o r a l Trainee of t h e U.S. P u b l i c H e a l t h Service, G r a n t No. 5-TIGM-301-05, from the I n s t i t u t e of t h e A c a d e m y of Medical Sciences.
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STUDIES ON GROUND SUBSTANCE OF AORTAS OF LATHYRITIC RATS
801
11 MENZIES, D. W. AND K. W. MILLS, The aortic and skeletal lesions of lathyrism in rats on a diet of sweet pea, ]. Path. Bact., 1957, 23: 223. 12 WIRTSCHAFTER, Z. T., Acid mucopolysaccharides in the histopathogenesis of aortic aneurysms in the lathyrus-fed rat, Arch. Path., 1957, 64: 577. is BICELEY, H., Experimental lathyrism: apparent antithesis of ageing in connective tissue, ] . Amer. geriat. Sot., 1964, 12: 717. 14 GRANT, R. A., M. HATHORN AND T. GILLMAN, Aortic, serum, connective tissue and osseous chemistry in lathyritic rats, Nature (Loud.), 1960, 186: 164. is BURMAN,S. O., Medial degeneration and its relation to dissecting aneurysms, Int. Abstr. Surg., 1960, 110: I. 16 RICHERT, D. A., J. SCHENKMANAND W. W. WESTERFELD, An antithyrotoxic assay based upon the response of rat liver a-glycerophosphate dehydrogenase, J. Nutr., 1964, 83: 322. 17 RUEGAMER, W. R., W. W. WESTERFELD AND D. A. I~ICHERT, a-Glycerophosphate dehydrogenase response to thyroxine in thyroidectomized, thiouracil-fed and temperature-adapted rats, Endocrinology, 1964, 75: 908. is SCHILLER, S., G. A. SLOVER AND A. DORFMAN, A method for the separation of acid mucopolysaccharide: its application to the isolation of heparin from the skin of rats, J. biol. Chem., 1961, 236: 983. 19 DlSCHE, Z., A new specific color reaction of hexuronic acids, J. biol. Chem., 1947, 167: 189. 20 SCI~MIDT,M. AND M. DM0C~OWSKI, The determination of isomeric chondroitin sulfates, Biochim. biophys. Acta, 1964, 83: 137. 21 DALFERES, E. R., JR., B. I~ADAKRISHNAI~URTHYAND G. S. BERENSON, Acid mucopolysaccharides of amyloid tissue, Arch. biochem., 1967, 118: 284. 22 RAMAMURTI,P. AND H. ]~. TAYLOR, Histochemical studies of the evaluation and regression of skeletal deformaties due to fl-aminopropionitrile, Lab. Invest., 1958, 7:115. 23 MARWAH,A. S., W. DASLER AND I. MEYER, Reversibility of lathyric damage to the periodontal structures, J. Periodont., 1963, 34: 1421. 24 CHURCHILL, D. W., S. GELFANT, J. J. LALICH AND D. M. ANGEVINE, Alterations in the polysaccharides and elastic fibers in the aortas of rats fed toxic lathyrus factor, Lab. Invest., 1955, 4: 1. ~5 HAM, K . N . , The fine structure of normal rat aorta, Aust. J. exp. biol. reed. Sci., 1962, 40: 341. 26 LINKER, A., W. F. COULSON AND W. H. CARNY-S, Cardiovascular studies on copper deficient swine, P a r t 6 (The mucopolysaccharide composition of aorta and cartilage), J. biol. Chem., 1964, 239: 1690. 27 MILLER, E. J., G. R. MARTIN, C. E. MECCA AND K. A. PIEZ, The biosynthesis of elastin crosslinks. The effects of copper deficiency and a lathyrogen, J. biol. Chem., 1965, 240: 3623. 2s LORE~ZE~, I., Vascular connective tissue changes induced by catecholamines and thyroid hormone. In: G. ASBOE-HANS~N (Ed.), Hormones and Connective Tissue, Williams and Wilkins, Baltimore, Md., 1966, p. 136. 29 LORENZEN, I., Alterations in acid mucopolysaccharide and r of rabbit aorta related to age of epinephrine-thyroxine induced arteriosclerotic lesions, Acta entoG., (Kbh). 19~2, 3~: 615. s~ LORENZEN,I., Vascular connective tissue under the influence of thyroxine, Part 1 (Epinephrinethyroxine induced alterations in rabbit aorta), Acta endocr. (Kb~).) 1961, 35: 197. sl CRANE, W. A. J., Sulfate utilization and mucopolysaccharide synthesis by m~eat~ric ~rt~ri~s of hypertensive rats, J. Path. Bact., 1962, 84: 113. s~ HAM, K., The fine structure of rat aorta in experimental lathyrism, Aust. J. exp. biol. meal. Sci., 1962, 40: 353. s 3 LAUCH, J. J., Effect of different lathyrogens upon tissue responses in rats. Proc. Soc. exp. Biol. ( N . Y . ) , 1966, 123: 214. s4 D^SLER, W. AND R. V. MILLXSER, OsteolathyTogenic action of mercaptoethylamiue and of cystamine, Proc. Soc. exp. Biol. ( N . Y . ) , 1958, 98: 759. s 5 MILLmEE, R. V. AND W. DASLBR, Osteolathyrism. Histopathological lesions produced by semicarbazide and acetone semicarbazone, Arch. Path., 1959, 67: 427.
j . Atheroscler. Res., 1968, 8:787-801