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Elastin degradation in the aorta of Watanabe hereditary hyperlipidemic rabbits
ELSEVIER SCIENCE IRELAND
Mechanisms of Ageing and Development 74 (1994) 117-120
m nm daammt
Elastin degradation in the aorta of Watanabe hereditary hyperlipidemic rabbits Kathie M. Dalessandri a, Pamela Eisele b, Toniel S. Wong c, Jackie Parker b, Robert B. Rucker *a'c aDepartment of Surgery (112), Palo Alto VA Hospital, Palo Alto, CA 94304, USA bAnimal Resources Service, CDepartrnent of Nutrition, University of California, Davis, CA 95616, USA (Received 20 September 1993; revision received 10 January 1994; accepted 27 January 1994)
Abstract The Watanabe hereditary hyperlipidemic (WHHL) rabbit is an animal model that resembles humans with familial hyperlipidemia. In the thoracic aortae, there is also morphologic evidence of marked destruction of medial lamellar elastin fibers. Herein is provided the chemical evidence of elastolytic degradation. The levels of lysyl oxidase (L.Ox.), soluble elastin (SE) and insoluble elastin (IE) were estimated in thoracic aortae samples from New Zealand white (NZW) and WHHL rabbits at 6 months of age or 5 WHHL rabbits at 2.5 years of age. Enzyme-linked immunosorption assays (ELISA) were used in the L.Ox. and SE measurements. IE was measured following alkali extraction of aortae. There was a decrease in IE in thoracic aortae from Watanabe rabbits compared to NZW controls at 6 months of age (P < 0.1), and a further loss of IE in aortae from 2.5-year-old WHHL rabbits relative to the values at 6 months (P < 0.05). Average values for IE were: 130 mg/g for 6-month-old NZW, 100 mg/g for 6-month-old WHHL, and 60 mg/g for 2.5-year-old WHHL rabbits. Moreover, SE was only observed in aorta extracts from the older WHHL rabbits, a sign of elastolytic damage. There was also a five- to sixfold decrease in L.Ox. in the older vs. younger rabbits. Key words: Elastin; Lysyl oxidase; Elastolysis; Watanabe rabbit
1. Introduction We have r e p o r t e d that the focal erosion o f elastin fibers in the aortic m e d i a is a striking feature o f the vascular p a t h o l o g y observed in the W a t a n a b e hereditary * Corresponding author, Department of Nutrition, University of California, Davis, CA 95616, USA. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0047-6374(94)01442-0
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K. M. Dalessandri et al. / Mech. Ageing Dev. 74 (1994) 117-120
hyperlipidemic rabbit (WHHL) [1]. Although the mechanisms for the erosion of elastic fibers is not clear [2-5], the process probably involves poor control of elastolytic proteinases and/or modification of elastin in elastic fibers sufficient to allow increased susceptibility to elastolysis [4-8]. In that there are only limited biochemical data on the extent to which elastic fiber erosion and elastin degradation occur in aged animals, we have measured elastin in aorta in 6-month- or 2.5-year-old W H H L rabbits. We have also used enzyme-linked immunosorption assays to measure soluble elastin and lysyl oxidase to assess: (1) whether elastolysis is of sufficient magnitude to result in measurable levels of solubilized elastin-derived peptides or a decrease in insoluble elastin; and (2) if lysyl oxidase is sufficient in amount to sustain normal elastin crosslinking in aorta from older W H H L rabbits. 2. Materials and methods
Rabbits (WHHL) or New Zealand White (NZW) were fed Purina rabbit chow ad libitum. Rabbits (6 months or 2.5 years) were euthanized with an overdose of pentobarbital according to NIH guidelines. The NZW rabbits were obtained commercially; W H H L rabbits were obtained from an NIH breeding project. The levels of lysyl oxidase (L.Ox.), soluble elastin (SE), and insoluble elastin (IE) were estimated in thoracic aortic samples from five 6-month old NZW and in five of the 6-monthold homozygous W H H L rabbits. These estimates were compared to those from five homozygous W H H L rabbits at 2.5 years of age. Enzyme-linked immunosorption assays (ELISA) were used in the L.Ox. and SE measurements as described by Romero-Chapman et al. [9] and Tinker et al. [10]. For the SE and L.Ox. values, tissues were first extracted into 4 M guanidinium isothionate; a strong denaturant to facilitate complete extraction of these proteins. The extract was next dialyzed against 0.5 M urea and an aliquot was taken for ELISA assays, which employed chicken-derived elastin or lysyl oxidase polyclonal antibodies. A rabbit-derived, anti-chicken horseradish peroxidase conjugate was used for eventual detection (see Refs. 9 and 10 and Refs. cited therein). It was also possible to assess the molecular weight range of the principle soluble elastin peptides in aorta extracts. First, ethanol was added 'to the dialyzed samples containing 0.5 M urea to 60% (v/v, 4°C). Elastin peptides are soluble in ethanol [10]. Thus, following: (1) centrifugation to remove precipitated protein; (2) concentration under vacuum; (3) reconstitution to approximately 6 M urea; and (4) sodium dodecyl sulfate-polyacrylamide gel electrophoresis [10], it was possible to utilize the antielastin antibodies in Western blots to size elastin peptides [10]. Next, the residue obtained after guanidinium extraction was subjected to 0.1 M NaOH extraction (90°C for 45 min) to obtain IE [10]. Sections of the distal thoracic aorta (1 cm sections) were also fixed in formalin in preparation for morphologic examination. Aortic cross sections were stained with hematoxylin-eosin and examined as described previously [11. 3. Results
There was a decrease in IE in aorta from W H H L rabbits compared to NZW controls at 6 months. Insoluble elastin levels in aortae of W H H L rabbits were further
K.M. Dalessandri et al./ Mech. Ageing Dev. 74 (1994) 117-120
119
Table 1 Values for elastin and lysyl oxidase per gram of fresh aortae in 6-month NZW and 6-month or 2.5-year WHHL Rabbits Rabbit
6-month NZW 1. 2. 3. 4. 5. Mean 4- S.D. 6-month WHHL 1. 2. 3. 4. 5. 6. Mean 4- S.D. 2.5-year WHHL I. 2. 3. 4. 5. Mean 4- S.D.
Insoluble elastin (mg)
Soluble elastin (#g)
Lysyl oxidase (/zg)
170 80 140 -130 130 4- 30
Not Detected (ND) ND ND ND ND ND
430 -480 340 330 395 4- 60
150 20 60 90 120 70 85 4- 32**
ND ND ND ND ND ND ND
290 390 200 120 --250 4- 70
1.4 3.0 3.0 1.0 1.8 2.0 4- 0.8*
30 50 -20 130 57 4- 40*
20 80 80 80 40 60 4- 20*
*P < 0.05. **P < 0.1.
reduced at 2.5 years of age (Table 1). In guanidine thiocyanate extracts, soluble elastin was detected only in the older W H H L rabbits, i.e. 1.4-3.0/zg/g of aorta. The immunoreactive material ranged in size from 20 to 50 kDa and as material that was excluded by 10% polyacrylamide gel (see Discussion). Lysyl oxidase was easily detected in all samples. However, the levels of lysyl oxidase were reduced in the older W H H L rabbits; four- to fivefold compared to younger rabbits (Table 1). Upon histopathologic examination, the aorta of 6-month-old W H H L rabbits showed signs of mild atherosclerosis and degeneration of the media and medial lamellar elastin units. The NZW rabbits had intact arterial media with no signs of atherosclerosis. In contrast, degradation of the media was obvious in all sections of aorta from 2.5-year-old W H H L rabbits. These findings were similar or identical to those reported previously by us and others [1-3]. 4. Discussion
In major arteries, elastin normally does not undergo measurable turnover [11]. Consequently, the relative decrease in insoluble elastin in aorta from 6-month-old
120
K.M. Dalessandri et a l . / Mech. Ageing Dev. 74 (1994) 117-120
W H H L rabbits (vs. NZW rabbits) and the further reduction of elastin in older W H H L rabbits was taken as evidence for elastrolysis. Moreover, soluble elastin peptides were only observed in arteries in which medial degeneration and loss of insoluble elastin were obvious. The size range of such peptides was 20-55 kDa, i.e. considerably smaller in size than that of the elastin precursor, tropoelastin (mol. wt., 72 000, Refs. 10 and 11). With regard to lysyl oxidase, an enzyme involved in elastin crosslinking, several hundred micrograms/gram of tissue were detected by ELISA. Assuming that a significant quantity, i.e. one-third or more, was enzymatically functional, then clearly sufficient lysyl oxidase was present in the aorta of 6-month-old rabbits for the efficient crosslinking of any newly synthesized elastin (cf. Ref. 11 for the basis of this assumption). However, whether the five- to sixfold reduction in lysyl oxidase in extracts of older W H H L rabbits is of sufficient magnitude to seriously compromise crosslinking remains to be determined. Nevertheless, it can be speculated that the decrease should lessen the potential for the repair of elastin fibers. Moveover, since lysyl oxidase is also suspected to be a structural component of the elastin fiber matrix [11], the relative decrease with aging deserves further attention as it is related to the maintenance of elastic fiber integrity. In summary, the genetic defects that lead to hyperlipidemia and vascular tissue degeneration in the aged W H H L rabbit have as components, loss of elastin and decreased levels of lysyi oxidase. The changes in elastin and lysyl oxidase in aged WHHL rabbits are easily detected and are consistent with morphologic findings 11-31. 5. References 1
K.M. Dalessandri, H. Bogren, S. Bjorkerud et al., Watanabe hyperlipidemic rabbit as a model of aortic degeneration of the medial lamellar elastin unit. J. Invest. Surg., 5 (1992) 19-23. 2 E. Esper, E.K. Chan and H. Buchwald, Natural history of atherosclerosis and hyperlipidemia in heteroygous WHHL (WHHL-Hh) rabbits. J. Lab. Clin. Meal, 121 (1993) 97-102. 3 E. Esper, W.J. Runge, R. Gunther and H. Buchwald, Natural history of atherosclerosis and hyperlipidemia in heterozygous WHHL (WHHL-Hh) rabbits. 1I. Morphologic evaluation of spontaneously occurring aortic and coronary lesions. J. Lab. Clin. Med., 121 (1993) 103-110. 4 D.K. Stoller, C.T. Campos, C.B. Grorud, V. Michalek and H. Buckwald, Reduction ofatherosclerosis with nonsteroidal anti-inflammatory drugs. J. Surg. Res., 54 (1993) 7-11. 5 D. Steinberg, S. Parthasarthy and T. Carew, In vivo inhibition of foam cell development by probucol in Watanabe rabbits. Am. J. Cardiol., 62 (1988) 6B-12B. 6 J.M. Reilly, C.M. Brophy and D. Tilson, Characterization of an elastase from aneurysmal aorta which degrades intact aortic elastin. Ann. Vasc. Surg., 6 (1992) 495-502. 7 J.S. Campa, R.M. Greenhalgh and J.T. Powell, Elastin degradation in abdominal aortic aneurysms. Alherosclerosis, 65 (1987) 13-21. 8 J.R. Cohen, I. Darfato, L. Ratner and D. Tilson, Alpha 1 antitrypsin phenotypes in patients with abdominal aortic aneurysms. J. Surg. Res., 49 (1990) 319-321. 9 N. Romero-Chapman, K. Reiser, D. Hyde and R. Rucker, Purification properties and influence of dietary copper on accumulation of lysyl oxidase in rat skin. Biochem. J., 275 (1991) 657. 10 D. Tinker, N. Romero-Chapman, K. Reiser, D. Hyde and R. Rucker, Elastin metabolism during recovery from impaired cross-link information. Arch. Biochem. Biophys., 278 (1990) 326. 11 K. Reiser, R.J. McCormick and R.B. Rucker, Enymatic and nonenymatic cross-linking of collagen and elastin. FASEB J., 6 (1992) 2439-2449.
Mechanisms of Ageing and Development 74 (1994) 117-120
m nm daammt
Elastin degradation in the aorta of Watanabe hereditary hyperlipidemic rabbits Kathie M. Dalessandri a, Pamela Eisele b, Toniel S. Wong c, Jackie Parker b, Robert B. Rucker *a'c aDepartment of Surgery (112), Palo Alto VA Hospital, Palo Alto, CA 94304, USA bAnimal Resources Service, CDepartrnent of Nutrition, University of California, Davis, CA 95616, USA (Received 20 September 1993; revision received 10 January 1994; accepted 27 January 1994)
Abstract The Watanabe hereditary hyperlipidemic (WHHL) rabbit is an animal model that resembles humans with familial hyperlipidemia. In the thoracic aortae, there is also morphologic evidence of marked destruction of medial lamellar elastin fibers. Herein is provided the chemical evidence of elastolytic degradation. The levels of lysyl oxidase (L.Ox.), soluble elastin (SE) and insoluble elastin (IE) were estimated in thoracic aortae samples from New Zealand white (NZW) and WHHL rabbits at 6 months of age or 5 WHHL rabbits at 2.5 years of age. Enzyme-linked immunosorption assays (ELISA) were used in the L.Ox. and SE measurements. IE was measured following alkali extraction of aortae. There was a decrease in IE in thoracic aortae from Watanabe rabbits compared to NZW controls at 6 months of age (P < 0.1), and a further loss of IE in aortae from 2.5-year-old WHHL rabbits relative to the values at 6 months (P < 0.05). Average values for IE were: 130 mg/g for 6-month-old NZW, 100 mg/g for 6-month-old WHHL, and 60 mg/g for 2.5-year-old WHHL rabbits. Moreover, SE was only observed in aorta extracts from the older WHHL rabbits, a sign of elastolytic damage. There was also a five- to sixfold decrease in L.Ox. in the older vs. younger rabbits. Key words: Elastin; Lysyl oxidase; Elastolysis; Watanabe rabbit
1. Introduction We have r e p o r t e d that the focal erosion o f elastin fibers in the aortic m e d i a is a striking feature o f the vascular p a t h o l o g y observed in the W a t a n a b e hereditary * Corresponding author, Department of Nutrition, University of California, Davis, CA 95616, USA. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0047-6374(94)01442-0
118
K. M. Dalessandri et al. / Mech. Ageing Dev. 74 (1994) 117-120
hyperlipidemic rabbit (WHHL) [1]. Although the mechanisms for the erosion of elastic fibers is not clear [2-5], the process probably involves poor control of elastolytic proteinases and/or modification of elastin in elastic fibers sufficient to allow increased susceptibility to elastolysis [4-8]. In that there are only limited biochemical data on the extent to which elastic fiber erosion and elastin degradation occur in aged animals, we have measured elastin in aorta in 6-month- or 2.5-year-old W H H L rabbits. We have also used enzyme-linked immunosorption assays to measure soluble elastin and lysyl oxidase to assess: (1) whether elastolysis is of sufficient magnitude to result in measurable levels of solubilized elastin-derived peptides or a decrease in insoluble elastin; and (2) if lysyl oxidase is sufficient in amount to sustain normal elastin crosslinking in aorta from older W H H L rabbits. 2. Materials and methods
Rabbits (WHHL) or New Zealand White (NZW) were fed Purina rabbit chow ad libitum. Rabbits (6 months or 2.5 years) were euthanized with an overdose of pentobarbital according to NIH guidelines. The NZW rabbits were obtained commercially; W H H L rabbits were obtained from an NIH breeding project. The levels of lysyl oxidase (L.Ox.), soluble elastin (SE), and insoluble elastin (IE) were estimated in thoracic aortic samples from five 6-month old NZW and in five of the 6-monthold homozygous W H H L rabbits. These estimates were compared to those from five homozygous W H H L rabbits at 2.5 years of age. Enzyme-linked immunosorption assays (ELISA) were used in the L.Ox. and SE measurements as described by Romero-Chapman et al. [9] and Tinker et al. [10]. For the SE and L.Ox. values, tissues were first extracted into 4 M guanidinium isothionate; a strong denaturant to facilitate complete extraction of these proteins. The extract was next dialyzed against 0.5 M urea and an aliquot was taken for ELISA assays, which employed chicken-derived elastin or lysyl oxidase polyclonal antibodies. A rabbit-derived, anti-chicken horseradish peroxidase conjugate was used for eventual detection (see Refs. 9 and 10 and Refs. cited therein). It was also possible to assess the molecular weight range of the principle soluble elastin peptides in aorta extracts. First, ethanol was added 'to the dialyzed samples containing 0.5 M urea to 60% (v/v, 4°C). Elastin peptides are soluble in ethanol [10]. Thus, following: (1) centrifugation to remove precipitated protein; (2) concentration under vacuum; (3) reconstitution to approximately 6 M urea; and (4) sodium dodecyl sulfate-polyacrylamide gel electrophoresis [10], it was possible to utilize the antielastin antibodies in Western blots to size elastin peptides [10]. Next, the residue obtained after guanidinium extraction was subjected to 0.1 M NaOH extraction (90°C for 45 min) to obtain IE [10]. Sections of the distal thoracic aorta (1 cm sections) were also fixed in formalin in preparation for morphologic examination. Aortic cross sections were stained with hematoxylin-eosin and examined as described previously [11. 3. Results
There was a decrease in IE in aorta from W H H L rabbits compared to NZW controls at 6 months. Insoluble elastin levels in aortae of W H H L rabbits were further
K.M. Dalessandri et al./ Mech. Ageing Dev. 74 (1994) 117-120
119
Table 1 Values for elastin and lysyl oxidase per gram of fresh aortae in 6-month NZW and 6-month or 2.5-year WHHL Rabbits Rabbit
6-month NZW 1. 2. 3. 4. 5. Mean 4- S.D. 6-month WHHL 1. 2. 3. 4. 5. 6. Mean 4- S.D. 2.5-year WHHL I. 2. 3. 4. 5. Mean 4- S.D.
Insoluble elastin (mg)
Soluble elastin (#g)
Lysyl oxidase (/zg)
170 80 140 -130 130 4- 30
Not Detected (ND) ND ND ND ND ND
430 -480 340 330 395 4- 60
150 20 60 90 120 70 85 4- 32**
ND ND ND ND ND ND ND
290 390 200 120 --250 4- 70
1.4 3.0 3.0 1.0 1.8 2.0 4- 0.8*
30 50 -20 130 57 4- 40*
20 80 80 80 40 60 4- 20*
*P < 0.05. **P < 0.1.
reduced at 2.5 years of age (Table 1). In guanidine thiocyanate extracts, soluble elastin was detected only in the older W H H L rabbits, i.e. 1.4-3.0/zg/g of aorta. The immunoreactive material ranged in size from 20 to 50 kDa and as material that was excluded by 10% polyacrylamide gel (see Discussion). Lysyl oxidase was easily detected in all samples. However, the levels of lysyl oxidase were reduced in the older W H H L rabbits; four- to fivefold compared to younger rabbits (Table 1). Upon histopathologic examination, the aorta of 6-month-old W H H L rabbits showed signs of mild atherosclerosis and degeneration of the media and medial lamellar elastin units. The NZW rabbits had intact arterial media with no signs of atherosclerosis. In contrast, degradation of the media was obvious in all sections of aorta from 2.5-year-old W H H L rabbits. These findings were similar or identical to those reported previously by us and others [1-3]. 4. Discussion
In major arteries, elastin normally does not undergo measurable turnover [11]. Consequently, the relative decrease in insoluble elastin in aorta from 6-month-old
120
K.M. Dalessandri et a l . / Mech. Ageing Dev. 74 (1994) 117-120
W H H L rabbits (vs. NZW rabbits) and the further reduction of elastin in older W H H L rabbits was taken as evidence for elastrolysis. Moreover, soluble elastin peptides were only observed in arteries in which medial degeneration and loss of insoluble elastin were obvious. The size range of such peptides was 20-55 kDa, i.e. considerably smaller in size than that of the elastin precursor, tropoelastin (mol. wt., 72 000, Refs. 10 and 11). With regard to lysyl oxidase, an enzyme involved in elastin crosslinking, several hundred micrograms/gram of tissue were detected by ELISA. Assuming that a significant quantity, i.e. one-third or more, was enzymatically functional, then clearly sufficient lysyl oxidase was present in the aorta of 6-month-old rabbits for the efficient crosslinking of any newly synthesized elastin (cf. Ref. 11 for the basis of this assumption). However, whether the five- to sixfold reduction in lysyl oxidase in extracts of older W H H L rabbits is of sufficient magnitude to seriously compromise crosslinking remains to be determined. Nevertheless, it can be speculated that the decrease should lessen the potential for the repair of elastin fibers. Moveover, since lysyl oxidase is also suspected to be a structural component of the elastin fiber matrix [11], the relative decrease with aging deserves further attention as it is related to the maintenance of elastic fiber integrity. In summary, the genetic defects that lead to hyperlipidemia and vascular tissue degeneration in the aged W H H L rabbit have as components, loss of elastin and decreased levels of lysyi oxidase. The changes in elastin and lysyl oxidase in aged WHHL rabbits are easily detected and are consistent with morphologic findings 11-31. 5. References 1
K.M. Dalessandri, H. Bogren, S. Bjorkerud et al., Watanabe hyperlipidemic rabbit as a model of aortic degeneration of the medial lamellar elastin unit. J. Invest. Surg., 5 (1992) 19-23. 2 E. Esper, E.K. Chan and H. Buchwald, Natural history of atherosclerosis and hyperlipidemia in heteroygous WHHL (WHHL-Hh) rabbits. J. Lab. Clin. Meal, 121 (1993) 97-102. 3 E. Esper, W.J. Runge, R. Gunther and H. Buchwald, Natural history of atherosclerosis and hyperlipidemia in heterozygous WHHL (WHHL-Hh) rabbits. 1I. Morphologic evaluation of spontaneously occurring aortic and coronary lesions. J. Lab. Clin. Med., 121 (1993) 103-110. 4 D.K. Stoller, C.T. Campos, C.B. Grorud, V. Michalek and H. Buckwald, Reduction ofatherosclerosis with nonsteroidal anti-inflammatory drugs. J. Surg. Res., 54 (1993) 7-11. 5 D. Steinberg, S. Parthasarthy and T. Carew, In vivo inhibition of foam cell development by probucol in Watanabe rabbits. Am. J. Cardiol., 62 (1988) 6B-12B. 6 J.M. Reilly, C.M. Brophy and D. Tilson, Characterization of an elastase from aneurysmal aorta which degrades intact aortic elastin. Ann. Vasc. Surg., 6 (1992) 495-502. 7 J.S. Campa, R.M. Greenhalgh and J.T. Powell, Elastin degradation in abdominal aortic aneurysms. Alherosclerosis, 65 (1987) 13-21. 8 J.R. Cohen, I. Darfato, L. Ratner and D. Tilson, Alpha 1 antitrypsin phenotypes in patients with abdominal aortic aneurysms. J. Surg. Res., 49 (1990) 319-321. 9 N. Romero-Chapman, K. Reiser, D. Hyde and R. Rucker, Purification properties and influence of dietary copper on accumulation of lysyl oxidase in rat skin. Biochem. J., 275 (1991) 657. 10 D. Tinker, N. Romero-Chapman, K. Reiser, D. Hyde and R. Rucker, Elastin metabolism during recovery from impaired cross-link information. Arch. Biochem. Biophys., 278 (1990) 326. 11 K. Reiser, R.J. McCormick and R.B. Rucker, Enymatic and nonenymatic cross-linking of collagen and elastin. FASEB J., 6 (1992) 2439-2449.