Normal elastin content of aorta in bovine Marfan syndrome

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

57,

145-152 (1992)

Normal Elastin Content of Aorta in Bovine Marfan Syndrome’ J.C.

PARSONS,Y.HOFFMAN, K. A. POTTER,AND T.E.

BESSER

Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, 99164-7040 Received May 25, 1992, and in revised form July 27, 1992 Samples from the ascending aortae from two calves affected by bovine Marfan syndrome were subjected to biochemical analyses of the connective tissue and were compared to age-matched controls. Elastin was extracted from the aortic samples with 5 M guanidineHCl, bacterial collagenase digestion, and dithiothreitol reduction. Amino acid analysis revealed that desmosine and isodesmosine levels were the same in Marfan calves as in control animals. Gravimetric measurements of elastin, amino acid composition, soluble protein, and uranic acid values also showed no significant difference between Marfan and control tissue. In contrast to elastin, collagen in aortae of Marfan calves was significantly higher than the mean of several controls. These findings, along with other observations of this animal model, support the conclusion that the microscopic and biochemical lesions of aortic elastin in bovine Marfan syndrome likely result from defective microfibrillar metabolism. Absence of cystic medial necrosis in bovine Marfan aortae may explain normal elastin content in the animal model. 0 1992 Academic Press. Inc.

INTRODUCTION Marfan syndrome is a life-threatening genetic disease that affects 4-10 per 100,000 people (Pyeritz and McKusick, 1979). Manifestations of the syndrome include elongated extremities, scoliosis, lens dislocation, aortic dilation, and mitral valve prolapse. Premature death of Marfan patients is often due to progression of the cardiovascular lesions to dissecting aortic aneurysm or cardiac failure (Pyeritz, 1986). The typical histologic aortic lesion in human Mat-fan patients is disruption and fragmentation of elastic fibers, and mutinous degeneration of aortic media (cystic medial necrosis) (Takebayashi et al., 1973). Cystic medial necrosis, though commonly seen in human Mat-fan aortae, is also associated with a variety of other cardiovascular diseases of man (Abraham et al., 1982). Biochemical alterations in elastin and desmosine crosslinks within elastin have been described in human Marfan aortae (Abraham et al., 1982; Perejda et al., 1985; Halme et al., 1985). Although these data pointed to a genetic defect in elastin, genetic linkage studies have excluded the elastin gene as the mutation site (Blanton et al., 1990). The microfibrillar protein fibrillin, abundant in aorta and lens ciliary processes (Sakai et al., 1986), has been implicated in the disease by immunofluorescent (Hollister et al., 1990) and linkage studies (Lee et al., 1991; Dietz et al., 1991). Additionally, a point mutation in the fibrillin gene has been described in two human Marfan patients (Dietz et al., 1991). ’ Supported in part by NIH Grant RR00515, by the Adler fund and the Robert R. Fast fund of the College of Veterinary Medicine, Washington State University, and by a Grant-in-Aid from Washington State University. The authors thank Dr. S. Gurusiddaiah of the Washington State University Bioanalytical Laboratory for amino acid analysis. 145 0014-4800/92 $5.00 Copyright 0 1992 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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Diagnosis, treatment, and pathogenesis of Marfan syndrome have been hampered by the lack of a spontaneous animal model. Copper-deficient diets in chicks (Simpson et al., 1980) and pigs (Coulson and Carnes, 1962) produce dissecting aortic aneurysms, but multisystemic lesions of the human syndrome are not seen. These induced models, while useful in hemodynamic studies, have limited value in determining lesion pathogenesis in the genetic disease. In 1990, a group of related calves was noted to have many skeletal, ocular, and cardiovascular features of human Marfan syndrome, including histologic evidence of disrupted aortic elastic fibers (Besser et al., 1990). Cystic medial necrosis, however, has not been seen in bovine Mat-fan aortae. We have shown that these cattle have decreased fibrillin immunoreactivity in dermal fibroblast tissue cultures, and decreased incorporation of tibrillin into extracellular matrix (Potter et al., 1992). These findings are similar to those in human Marfan patients (Hollister et al., 1990; Milewicz et al., 1992) and suggest that bovine Marfan syndrome is also caused by a fibrillin gene mutation. Fibrillin is the major component of the microfibrillar scaffolding upon which elastin is deposited. Thus microscopic and biochemical lesions of aortic elastin in human and bovine Mat-fan syndromes likely result from defective microfibrillar metabolism. We wanted to know if a similar genetic defect in a different species would result in the same biochemical alterations. To that end, we have examined biochemically the aortic connective tissue from two calves with Mat-fan syn’drome. Concentrations of elastin and elastin-associated crosslinks were not significantly decreased in bovine Marfan aortae. We did, however, find increased collagen concentrations in bovine Marfan aortic tissue, suggesting a mechanism for decreased aortic compliance in the bovine syndrome. METHODS Description

of the Model

Calves used in this study were from a group of seven related animals with a congenital syndrome of long, thin limbs, severe joint and tendon laxity, microspherophakia, ectopia lentis, cardiac murmurs, and aortic dilation. All affected calves were sired by a phenotypically normal bull suspected of having germline mosaicism for a new mutation resulting in this disease (Besser et al., 1990). Histologic and ultrastructural studies of aortic media from affected calves demonstrated disorganized elastin and narrowed elastic lamina separated by widened spaces (Fig. 1). Mucopolysaccharides were not noted within the media by either light microscopy (with alcian blue staining) or electron microscopy. Tissues used in this study came from a steer euthanized at 12 months and from a steer that died unexpectedly of a ruptured aorta at 16 months. Control animals were all between 12 and 24 months of age and exhibited no pathologic conditions. Assay

Procedures

Tissue samples were prepared following the methods of Abraham et al. (1982), with some modifications. To control for variations in elastic composition along the aorta, samples were taken from the ascending aorta just distal to the aortic valve. This site consistently showed histologic lesions of elastic fiber disruption in affected animals (Fig. 1). The tissue was cleaned of all but the intima and media, minced into approximately 3-mm3 pieces, and weighed. Wet weight was approx-

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147

FIG. 1. Photomicrographs of bovine aortae stained with Vierhoeff-Von Gieson for elastin. Both sections were taken from the ascending aorta just above the aortic valve. (A) Normal age-matched control. (B) 12-month-old steer with Marfan syndrome but no clinical signs. Only mild aortic dilation was seen grossly. Elastin fibers from the Marfan animal were irregular in size and disoriented when compared to the control animal. Tissue between elastic fibers in the affected calf is composed of hypertrophic smooth muscle cells and collagen. Cystic medial necrosis was not present in this animal, nor in four affected cattle examined subsequently. 520x.

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imately 1 g per sample. Minced aortae were lyophilized and further minced into pieces approximately 1 mm3, then weighed. The tissue was homogenized using a Polytron tissue homogenizer (Brinkmann Industries). Aliquots of the supernatant were taken for assay of soluble protein, hydroxyproline, and uranic acid. The insoluble residue was extracted with 5 M guanidine-HCl, further extracted twice with Tris-HCl, then digested with bacterial collagenase (Sigma, type V). Preparations were dialyzed against distilled water, lyophilized, and submitted for amino acid analyses. The supematant of the above precipitation was saturated with ammonium sulfate (390 mg/ml), centrifuged, and dialyzed against 0.1 M ammonium formate containing the protease inhibitors used earlier. After being centrifuged, the supernatants were evaporated and prepared for SDS-PAGE. The proteins were treated with silver stain (Bio-Rad, Richmond, CA) and compared with a standard which had the molecular weight of tropoelastin (72,000 Da). SDS-PAGE was carried out as specified by Laemmli (1970). Hydroxyproline was determined by a calorimetric assay according to Kivirikko et al. (1967). The method of Bitter and Muir (1962) was used to assay uranic acid. Proteins were determined using the Pierce BCA protein assay (Pierce, Rockford, IL). Amino acid concentrations were determined from hydrolysates obtained by heating 5 mg of the extracted elastin in a known quantity of double distilled 6 N HCl in evacuated and sealed ampules at 110°C for 4&72 hr. After removal of solvents from hydrolysates by vacuum evaporation, residues were dissolved in 0.2 N sodium citrate buffer, pH 2.2. Analyses were done in Beckman 121 MB automatic amino acid analyzer (Beckman Instrument Co., Fullerton, CA) equipped with a Hewlett Packard Model HP 3396A integrator. Data were analyzed by comparing the peak area for each amino acid in unknown protein samples to the peak area for the same amino acid in a standard mixture. Hydroxyproline determination was done similarly; however, an additional run of unextracted aortic tissue was needed to calculate collagen content. Desmosine and isodesmosine standards were obtained from Elastin Products Co. (Owensville, MO). Desmosine content was used to calculate the concentration of elastin in the aorta (Abraham et al., 1982). Nanomoles desmosine per dry weight of unextracted aortic tissue was divided by nanomoles per dry weight obtained for extracted preparations for the same aortic sample. Gravimetric measurement of elastin content was obtained by dividing the dry weight of the extracted samples by the dry weight of the same sample before extraction and multiplying by 100. Aortic collagen concentrations were calculated from the hydroxyproline values. The amount of hydroxyproline in elastin, as determined by amino acid analyses, was subtracted from the amount found in the crude tissue. The concentration of collagen was then obtained by multiplying the hydroxyproline value by 8.2. All values for control animals were expressed as the mean + standard error. Marfan tissue values were considered statistically different if they were outside of the mean + two standard deviations. RESULTS Table I summarizes the results of biochemical analyses of samples from the ascending aortae of affected and control cattle. Soluble protein and uranic acid values varied widely with no differences between affected and control animals.

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Specifically, the concentration of soluble proteins in the two Marfan calves was 5.22 and 3.58 mg/lOO mg dry weight of aorta, as compared with 4.39 + 1.79 in seven controls. The concentration of extractable uranic acid was 2.00 and 0.57 mg/lOO mg dry weight of aorta in the Marfan calves and 1.39 + 0.49 in seven controls. Protein in bovine aortae is slightly lower than in human aortae analyzed similarly (Abraham et al., 1982). Total elastin in ascending aortae of two Marfan affected calves, 47.1 and 55.0 mg/lOO mg dry weight of aorta, was not significantly different than the mean of five controls (31.4 -t 13.2). Percentage elastin, measured gravimetrically, in the Marfan-affected calves, 40.8 and 43.6%, was also not significantly different than the mean of seven controls (34.3 + 10.85). Collagen in aortae of Marfan calves was 78 and 71 mg/lOO mg, which was significantly higher than the mean of three controls (41.3 + 4.6). No tropoelastin was identified by SDS-PAGE of the solubilized protein fraction in any calf. Elastin amino acid analysis results are found in Table II. There was no difference between the normal and affected animals. All values are consistent with those values for bovine aorta (Starcher and Gallione, 1976), except lysine and desmosines, which are lower for all animals in this study. DISCUSSION Bovine Mat-fan syndrome has all of the pathognomonic clincopathologic features of the human syndrome (Pyeritz and McKusick, 1979), including strong evidence for a Iibrillin mutation (15). Two biochemical studies of human Marfan aortae showed decreased total elastin and decreased elastin-associated crosslinks (Perejda et al., 1985; Halme et al., 1985). In contrast, our study of bovine Marfan aortae showed normal elastin and desmosine concentrations, but increased collagen concentrations. Contrary to the aforementioned studies on human Mar-fan aortae, Halme et al. (1985) evaluated total elastin concentrations in aortae from people with annuloaortic ectasia, including Marfan syndrome. In that study, elastin concentrations varied considerably, correlating roughly with the amount of cystic medial necrosis. Indeed, 1 Mat-fan aorta with minimal lesions had normal elastin concentrations. All patients examined by Abraham et al. (1982) and Perejda et al. (1985) had cystic medial degeneration with accumulation of metachromatic extracellular matrix. Marfan cattle have no cystic medial necrosis, even adjacent to rupture or aneurysm sites, thus total aortic elastin concentrations might be expected to be normal. TABLE I Biochemical Analyses of Control and Marfan Bovine Ascending Aortae

Soluble protein (mg/lOO mg dry wt)

M2

M3

5.22

3.58

Total elastin (mg/lOO mg dry wt)

47.1

55.0

Total collagen (mg/lO0 mg dry wt)

78

71

Uranic Acid (100 mg dry wt)

2.00

0.57

Control x + SD 4.39 (n 31.4 (n 41 (n 1.39 (n

f = ” = f = ” =

1.79 7) 13.2 5) 4.6 3) 0.49 7)

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TABLE II Acid Composition of Elastin Preparation from Control and Marfan Bovine Ascending Aortae

Amino Acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine/alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Isodesmosine

Control” (n = 5) 9.6 8.0 5.8 14.8 100.4 574.6 117 0.8 21.0 98.2 7.2 25.8 1.8 0.4 5.3 1.6 1.0

f 2 f f + k k + 2 f f k 2 2 k + k

2.5 2.4 3.1 3.1 12.5 50.5 9.6 0.4 9.8 21.8 1.3 11.0 0.4 0.5 1.7 0.9 0.0

Marfan M2

M3

14 10 7 20 115 556 137 0 27 63 7 30 2 1 8 2 1

9 8 6 15 114 576 133 0 25 70 7 29 1 2 5 2 1

Note. Values are expressed as residues/lOOO. 0 The values are means + SD of five separate tissues.

Also in contrast to studies on human Mat-fan tissues, we found no decrease in desmosine residues in purified elastin from bovine Marfan aortae. Desmosine residues per 1000 amino acids were lower in all animals in our study than previously reported for bovine aortic elastin (Starcher and Gallione, 1976). Unexpectedly low lysine residue levels were found as well, suggesting that either our extraction scheme or amino acid analysis procedure did not favor preservation of lysine derivatives in bovine tissue. However, comparisons with age-matched control animals in this study remain valid. Increased total hydroxyproline in bovine Mat-fan tissue reflected increased collagen. Halme et al. (1985) also found increased collagen in human Mat-fan aortae with cystic medial necrosis, although Abraham et al. (1982) and Perejda et al. (1985) did not. Decreased tensile strength of aortic tissue, as reported by Perejda et al. (1985), could result from increased collagen, decreased elastin, or both. Indeed, copper-deficient swine also have decreased aortic tensile strength (Coulson and Carnes, 1962), most likely due to abnormal collagen metabolism. We also have found decreased compliance to increasing pressure loads in one bovine Mat-fan aorta (TE Besser, unpublished data). Our results suggest that decreased compliance in Marfan cattle is due to increased collagen in the absence of measurable decrease in elastin content. The fibrillin molecule is the major component of elastin-associated microfibrils (Sakai et al., 1986). Interactions of fibrillin molecules to form microfibrils, and microfibrils and tropoelastin to form insoluble elastin, are not understood completely. Fibrillin metabolic studies of human Marfan patients suggest that these patients have a variety of different mutations in the same gene (Hollister et al., 1990). Correlation of specific mutations with metabolic defects and clinical disease awaits additional sequence analysis, but it is possible that different mutations

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could result in various aortic biochemical abnormalities. We expect bovine Marfan syndrome to be associated with a Iibrillin mutation similar to those recognized in man, thus another explanation of biochemical differences is variation in response to aortic disease among species. Differences in body mass and aortic diameter could affect physiologic response to aortic stress, including fibrosis, in the ox compared to man. Species variation may also explain the absence of cystic medial necrosis in the bovine disease. In vitro studies of elastin biosynthesis and crosslinking in people and cattle with the same tibrillin mutation may be necessary to determine if species variation exists. This study demonstrates the utility of the bovine Marfan model in elucidating important pathogenetic mechanisms of the human Marfan syndrome. Future in vitro and in vivo investigations of elastin biosynthesis in Mar-fan cattle may lead to better therapeutic and prognostic evaluation of human Marfan patients. BIBLIOGRAPHY ABRAHAM, P. A., PEREJDA, A. J., CARNES, W. H. and UITTO, J. (1982) Marfan’s syndrome: Demonstration of abnormal elastin in aorta. .Z. Clin. Invest. 70, 1245-1252. BESSER, T. E., POTTER, K. A., BRYAN, Cl. M., and KNOWLEN, G. A. (1990). An animal model of the Marfan syndrome. Am. J. Med. Genet. 37, 15%165. BITTER, T., and MUIR, H. M. (1962). A modified uranic acid carbazole reaction. Anal. Biochem. 4, 330-334. BLANTON, S. H., SARFARAZI, M., EIBERG, H., DE GROOTE, J., FARNDON, P. A., KILPATRICK, M. W., CHILD, A. H., POPE, F. M., PELTONEN, L., FRANCOMANO, C. A., BOILEAU, C., KESTON, M. and TSIPOURAS, P. (1990). An exclusion map of Marfan syndrome. J. Med. Genet. 27, 73-77. COULSON, W. F., and CARNES, W. H. (1962). Cardiovascular studies in copper deficient swine II. Mechanical properties of the aorta. Lab. Invest. 11, 1316-1321. DIETZ, H. C., CUTTING, G. R., PYERITZ, R. E., MASLEN, C. L., SAKAI, L. Y., CORSON, G. M., PUFFENBERGER, E. G., HAMOSH, A., NANTHAKUMAR, E. J., CURRISTIN, S. M., STETTEN, G., MEYERS, D. A., and FRANCOMANO, C. A. (1991). Marfan syndrome caused by a recurrent de ~OVO missense mutation in the tibrillin gene. Nature. 352, 337-339. HALME, T., SAVUNEN, T., HEIKKI, A., VIHERSAAR~, T., and PENTINEN, R. (1985). Elastin and collagen in the aortic wall: Changes in the Marfan syndrome and annuloaortic ectasia. Exp. Mol. Pathol. 43, l-12. HOLLISTER, D. W., GODFREY, M. P., SAKAI, L Y., and PYERITZ, R. E. (1990). Marfan syndrome: Immunohistologic abnormalities of the microfibrillar fiber system in the Marfan Syndrome. N. Engl. J. Med. 323, 152-159. KIVIRIKKO, K. I., LAITENEN, O., and PROCKOP, D. J. (1967). Modifications of a specific assay for hydroxyproline in urine. Anal. Biochem. 19, 249-255. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227, 68-85. LEE, B., GODFREY, M., VITALE, E., HORI, H., MATTEI, M., SARFARAZI, M., TSIPOURAS, P., RAMIREZ, F., and HOLLISTER, D. W. (1991). Linkage of Marfan syndrome and a phenotypically related disorder to two different tibrillin genes. Nature. 352, 330-334. MILEWICZ, D. M., PYERITZ, R. E., CRAWFORD, S., and BYERS, P. H. (1992). Marfan Syndrome: Defective synthesis, secretion, and extracellular matrix formation of tibrillin by cultured dermal tibroblasts. J. Clin. Invest. 89, 79-86. PEREJDA, A. J., ABRAHAM, P. A., CARNES, W. H., COULSON, W. F., and UITTO, J. (1985). Marfan’s syndrome: Structural, biochemical, and mechanical studies of the aortic media. J. Lab. C/in. Med. 106, 376-383. POTTER, K. A., HOFFMAN, Y., SAKAI, L. Y., BYERS, P. H., BESSER, T. E., and MILEWICZ, D. M. (1992). Abnormal fibrillin metabolism in bovine Marfan syndrome. Submitted for publication. PYERITZ, R. E. (1986). The Marfan syndrome. Am. Fam. Physician 34, 83-94. PYERITZ, R. E., and MCKUSICK, V. A. (1979). Marfan syndrome: Diagnosis and management. N.

Eng. J. Med. 300, 772-777. SAKAI, L. Y., KEENE, D. R., and ENGVALL, component of extracellular microfibrils. J.

E. (1986).

Fibrillin,

a new

Cell Biol. 103, 2499-2500.

350 kD glycoprotein,

is a

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C. F., BOUCEK, R. J., and NOBLE, N. L. (1980). Similarity of aortic pathology in Marfan’s syndrome, copper deficiency in chicks, and @uninoproprionitrile toxicity in turkeys. Exp. Mol. Pathol. 32, 81-90. STARCHER, B. C., and GALLIONE, M. J. (1976). Purification and comparison of elastins from different animal species. Anal. Biochem. 74, 441-447. TAKEBAYASHI, S., KUBOTA, I., and TAKAGI, T. (1973). Ultrastructural and histochemical studies of the vascular lesions in Marfan’s syndrome, with report of four autopsy cases. Acta Pathol. Jpn. 23, 847-866. SIMPSON,