Morphological and biochemical investigations of mitral valve endocardiosis in pigs

Researchin VeterinaryScience1997,62, 121-125

~~Ir~

Morphological and biochemical investigations of mitral valve endocardiosis in pigs M. CASTAGNARO, S. AMEDEO, Department of Animal Pathology, School of VeterinaryMedicine, State University of Torino, Torino, A. BERTOLOTTO, E. MANZARDO, A. RICCIO, S. Luigi Gonzaga Hospital, Neurology Division, State University of Torino, F. GUARDA, Department of Animal Pathology, School of Veterinary Medicine,

State University of Torino, Torino, Italy

SUMMARY Pig endocardiosis is a pathological process affecting cardiac valves that is characterised by the accumulation of glycosaminoglyCallS (GAG) in the extracellular matrix. To investigate the involvement of GAG in the condition, the morphologyof the mitral valves from 23 affected pigs and seven normal controls was studied and qualitative and quantitative biochemical analyses of GAG were made. Gross and histopathological lesions were characterised by valve enlargement, collagen disorganisation and myxoid degeneration. No differences between normal and diseased valves were detected by lectin histochemistry. Electron microscopy revealed myofibroblast differentiation of many fibroblasts. A statistically significant increase of total GAG and hyaluronan was detected in the mitral valves of the pigs with endocardiosis by spectrophotometric, electrophoretic and densitometric analysis of the extracted GAG.Although it is not known whether the change in hyaluronan is a primary event or a result of other changes in the extracellular matrix, its accumulation in association with myofibroblast differentiation suggests that it plays a pathogenetic role in pig endocardiosis.

ENDOCARDIOSIS is a degenerative and dystrophic process affecting cardiac valves which has been described in many mammalian species including human beings, dogs and pigs (Savage et al 1983, Guarda et al 1988). The prevalence and the severity of the condition in pigs is strongly correlated with age: the incidence in young pigs is low and the valvular changes are mild, whereas the prevalence in three- to four-year-old pigs may reach 90 per cent or more. The condition is generally associated with other cardiac lesions such as prolapse of the mitral valve and jet impact lesions (Guarda et al 1992a). These age-dependent features of the disease have also been observed in dogs (Buchanan 1977, Kogure 1980, Whitney 1974). Another common and constant feature of endocardiosis in man, dog and pig is the accumulation of glycosaminoglycans (GAG) in the extracellular matrix (Schole 1973, Sokkar and Trautwein 1970, 1971, Trautwein et al 1973, Braunwald 1988, Guarda et al 1993, 1994). Although this is thought to be an important step in the development, progression and clinical manifestation of the disease, there is little information about the type and extent of involvement of specific GAG in the condition. GAG are unbranched sugar chains characterised by repeating disaccharide units which contain hexosamine (either D-glucosamine or D-galactosamine) and uronic acid (either D-glucuronic acid or L-iduronic acid) (Fransson 1987). With the exception of hyaluronan, all GAG are Osulphated to different degrees, giving the molecules a very high charge density and contributing to their typical biological properties (H66k et al 1984). All GAG, with the exception of hyaluronan, occur in the native state covalently bound to protein to form proteoglycans. The linkage between the protein core and the GAG is made through a serine residue and three additional linking sugars, one 0034-5288/971020121 + 05 $18.00/0

xylose and two galactose molecules, present at the nonreducing end of the GAG (Ruoslahti 1988). Proteoglycans are now considered not as passive, inert macromolecules but as dynamic components that influence fundamental biological processes, including cell proliferation, cell recognition and interactions and cytodifferentiation (Iozzo 1985, Hynes et al 1989). The characteristic composition of proteoglycans therefore contributes to the biological properties of a given tissue. In order to characterise further the type and extent of involvement of GAG in endocardiosis, the morphology of GAG was studied in pigs with different degrees of endocardiosis and a qualitative and quantitative biochemical evaluation was made.

MATERIALS AND METHODS

Tissues Mitral valves were collected at a slaughterhouse from 30 male, large white x landrace pigs aged eight to nine months. A macroscopical examination revealed that 23 of the pigs were affected by mitral valve endocardiosis and the other seven pigs were normal. The mitral valves were isolated, weighed and a small representative portion (1 c m x 0.3 cm) of each valve was fixed for histological and electron microscopical studies. The remaining part of the valve was frozen and stored at -20°C for biochemical evaluation.

Histology and lectin histochemistry For histology, the valves were fixed in formalin, embedded in paraffin wax and sections were cut at 4 ~tm and © 1997W. B. Saunders CompanyLtd

M. Castagnaro, S. Amedeo, A. Bertolotto, E. Manzardo, A. Riccio, F. Guarda

122

stained with haematoxylin and eosin and periodic acidSchiff-Alcian blue. For lectin histochemistry, sections were deparaffinised, rehydrated and any endogenous peroxidase activity was blocked by incubation in a 3 per cent hydrogen peroxide solution at 40°C for eight minutes. The slides were immersed in 0.1 per cent trypsin-calcium chloride solution for 20 minutes at 37°C, followed by incubation with nine different biotinylated lectins (Sigma Chemical, St Louis, MO) (Table 1) for one hour at room temperature. The concentration at which each lectin was used was 10 mg m1-1 for Con-A, 20 mg m1-1 for PNA, SBA, LCA and UEA-I and 50 mg m1-1 for GS-L PWM, RCA-I and WGA. Tissue sections were incubated with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) for 30 minutes at room temperature, following the instructions of the manufactttrer, then covered with a 0.01 per cent solution of 3,3'diaminobenzidine-hydrogen peroxide for four to 10 minutes. Two controls were used. To control binding specificity, each lectin was incubated with an 0.2M solution of the corresponding blocking sugar (Sigma Chemical, St Louis, MO) (Table 1), before the application of the solution to the tissue sections. In addition, incubation with avidin-biotinperoxidase alone served as a non-specific negative control. The sections were counterstained with Harris' haematoxylin. Positive staining was demonstrated by the golden, dark-brown 3,3'-diaminobenzidine tetrahydrochloridehydrogen peroxide reaction product.

Transmission electron microscopy For the electron microscopical studies, representative portions of the 30 mitral valves were cut into 1 to 2 m m 3 blocks, fixed in 2.5 per cent glutaraldehyde buffered with 0.2M sodium cacodylate (pH 7.4), post-fixed in 1 per cent osmium tetroxide in 0.1M sodium cacodylate buffer for two hours at 20°C, dehydrated in a series of graded ethanols, stained en bloc with a 3 per cent solution of uranyl acetate in ethanol and embedded in a mixture of Epon-812/Araldite (2:1). Semi-thin sections (1 Jxm) were stained with toluidine blue. Thin sections were cut on a Reichert-Jung Ultracut E (Reichert-Jung, Vienna, Austria), stained with uranyl acetate and lead citrate and examined with a Zeiss EM 109 with trans-fibre-optic photography (Carl Zeiss, Oberkochen, Germany).

Total glycosaminoglycan (GAG) extraction Samples were homogenised in two volumes of distilled water, using a Polytron (Kinematica) for three minutes at maximum speed. Proteins were digested twice by incuba-

tion on each occasion with papain (1 mg ml q of homogenised sample) (Boehringer) for 48 hours at 40°C. A few drops of octanoic acid were added to prevent bacterial growth. After centrifugation at 12,000 rpm for 20 minutes, the supernatant was brought to 7 per cent trichloroacetic acid, held for one hour at 4°C and centrifuged at 12,000 rpm for 20 minutes. The supernatant was diluted with four volumes of cold ethanol containing 1 per cent potassium acetate, stored overnight at -20°C and centrifuged at 12,000 rpm for 10 minutes. The precipitated GAG were finally dissolved in a volume of distilled water proportional to the wet weight of the mitral valves (1 ml g-l).

Quantitative analysis of GAG The GAG were analysed quantitatively by the carbazole method (Bitter and Muir 1962), using glucttronolactone as a standard. The concentration was expressed as ~tg uronic acid g q .

Electrophoresis of GAG The GAG were electrophoresed in 1M barium acetate buffer (pH 5.0) (Capelletti et al 1979a, b). Cellulose acetate sheets (Helena Labs) were stained with a 0.15 per cent solution of Alcian Blue 8GX for two minutes and destained in 0.05M sodium acetate (pH 3.7) for 45 minutes. The GAG reference standards used were chondroitin-6-sulphate (cc), chondroitin-4-sulphate (cs), hyaluronan, heparan sulphate (HS), dermatan sulphate (DS), and keratan sulphate (KS1 and KS2) (Sigma Co).

Identification of electrophoretic band The bands of cc, hyaluronan CA and DS were identified by the co-migration with standard GAG and by enzymatic digestion with chondroitinases ABC (ICN pharmaceutical), and chondroitin sulphate B (ICN Pharmaceutical). Five samples and a reference standard were electrophoresed on the same sheet.

Densitometric analysis Electrophoretic sheets stained with Alcian Blue 8GX were analysed with a Densitometer Appraise Junior (Beckman Instruments). The concentration of the total GAG and that of the single fractions were determined by comparing the electrophoretic bands of the samples with the reference band. The values of GAG obtained were expressed as percentages.

Table 1: Details of the lectins used in the study, their sugar specificity and binding inhibitors

Lectin origin Arachis hypogea Concanavalia ensiformis Glycine maximum Griffonia simplicifolia Lens culinaris Phytolacca americana Ricinus communis Triticum vulgaris Ulex europaeus *Gal Galactose, GalNAc Acetyl-neuraminic acid

Acronym

Major specific sugar*

Binding inhibitor*

PNA Con-A SBA GS-I LCA PWM RCA-I WGA UEA-I

GaI-~-(1-3)-GalNAc (z-D-GIc, {z-D-Man o~-D-GalNAc, c(-D-Gal cc-D-Gal o~-D-GIc, oc-D-Man (GIcNAc)n 13-Gal (,8-GIcNAC)n NeuNAc c~-L-fucose

Lactose c~-D-methyI-Man c~-D-GalNAc Lactose (z-D-methyl-Man GIcNAc Lactose NeuNAc c~-L-fucose

N-acetyi-galactosamine,

GIcNAc

N-acetyl-glucosamine,

Man

Mannose,

NeuNAc

Morphological and biochemical studies of pig endocardiosis Statistical analysis of biochemical data The biochemical data were analysed statistically by using the analysis of variance test for normally distributed data (with 95 per cent confidence) and the Mann-Whitney test for non-parametric data.

RESULTS

Gross and histopathological changes The gross lesions varied from a few small discrete nodules to larger nodules that had coalesced, giving the valve a lumpy appearance (Fig 1). The most severe lesions were plaque-like thickening of the valves, free borders with rolled edges and in few cases prolapse of the mitral valve. The most prominent light microscopical feature was the destruction of the normal collagen fibres and their replacement by a myxoid tissue readily stained with the periodic acid-Schiff-Alcian blue (Fig 2). Fibroblasts in the myxoid tissue were immature and polygonal to stellate in shape.

123

not stained in sections incubated with PNA, SBA, GS-I and UEA-I.

Transmission electron microscopy Electron microscopical examination of myxoid areas of the diseased valves revealed the loss of the typical collagen morphology. Collagen fibrils occurred either singly or they were disarranged and often associated with electron-dense, irregularly shaped structures (Figs 3 and 4). The morphology of the fibroblasts in the myxoid areas was changed from the normal spindle shape to a more polygonal form, by the indentation of the nuclear profile and occasionally by the presence of scattered cytoplasmic pinocytic vesicles. In addition, the cytoplasm of most fibroblasts contained filaments with focal electron densities, or electron-dense foci were visible on the cell membrane (Fig 5). These morphological features were not visible in fibroblasts from normal mitral valves.

Biochemical findings and statistical analysis Lectin histochemistry In the control and diseased valves, moderate to intense staining of the extracellular matrix was obtained with ConA, LCA, PWM, RCA-I and WGA. The extracellular matrix was

cc, hyaluronan, CA and DS were detected by electrophoresis: Fig 6 shows the electrophoretic pattern of GAG extracted from the mitral valves of five normal control pigs and Fig 7 presents the relative densitometric tracing.

FIG 1: Photograph of a pig mitral valve with endocardiosis; note the nodular appearance

FIG 3: Electron micrograph of a mitral valve from a normal pig. The collagen bundles (arrows) close to the cytoplasm of a fibroblast (F) are arranged regularly. Bar = 0-9 IJm

FIG 2: Photomicrograph of a pig mitral valve with endocardiosis. Myxoid changes (M) involve the spongiosa layer (S) while the fibrosa layer (F) is not affected. Haematoxylin and eosin. Bar = 300 t~m

FIG 4: Electron micrograph of a mitral valve from a pig with endocardiosis. The collagen is disarranged and is associated with many electron dense structures (arrows). Bar = 0.5 pm

124

M. Castagnaro, S. Amedeo, A. Bertolotto, E. Manzardo, A. Riccio, F. Guarda

FIG 5: Electron micrograph of a mitral valve from a pig with endocardiosis. Several bundles of filaments with focal electron densities are visible in the cytoplasm of a fibroblast (arrows). Bar = 0-5 pm

HA

CA

D$

1

'i ,s

/

;,*o.

FIG 6: Electrophoretic migration pattern of glycosaminoglycans extracted from the mitral valves of normal pigs. S = mixture of glycosaminoglycans reference standard; 1 to 5 = normal control samples; DS = Dermatan sulphate, HS = Heparan sulphate, HA = Hyaluronan, CA = Chondroitin sulphate

~1-----

CA

~l--

--

HA

~1--

--

HS

~l----

1

2

3

4

5

DS

S

FIG 7: Densitometric scanning of electrophoretic sheet with extracted glycosaminoglycans from a normal control pig. DS = Dermatan sulphate, HS = Heparan sulphate, HA = Hyaluronan, CA = Chondroitin sulphate

Although KS1 and KS2 were included as GAG standards, KS was not detected in either the normal or abnormal valves. The weights of the valves, the results of scanning the air-dried electrophoretic sheets and the results of the statistical analysis of the biochemical data (P values) were given in Table 2. The mitral valves with endocardiosis contained significantly higher concentrations of total GAG (P=0.006) and hyaluronan (P=0.003) than the control valves.

DISCUSSION The mitral valves with endocardiosis were enlarged, their collagen architecture was abnormal and collagen had been replaced by myxoid tissue. Alcian Blue staining of the myxoid tissue revealed GAG, and lectin histochemistry was used to investigate the terminal carbohydrate composition of the glyconconjugates. This technique was chosen because it had previously been used to detect specific carbohydrate changes in several pathological conditions (Castagnaro et al 1987, Castagnaro and Canese 1991, Biolatti et al 1994). Although no differences in staining were observed between the normal and diseased mitral valves, the results of staining with Alcian Blue and the biochemical data clearly indicated a difference in carbohydrate composition and it is concluded that the lack of discrimination between normal and diseased values by lectin histochemistry was due to the inadequate sensitivity of this technique. This view is also supported by recent findings on changes in sialic acid in endocardiosis of pig mitral valves which were detected by lectin-blot (D'Andrea et al 1995). Electron microscopy of the diseased valves confirmed the observations by light microscopy that their collagen structure was changed. Changes in collagen fibres have also been reported in human endocardiosis, referred to as mitral valve prolapse syndrome (MVPS) (Kern and Tucker 1972). In this study, however, the ultrastructural observations revealed a modification of fibroblast morphology characterised by a more polygonal shape, nuclear indentation and intracytoplasmic filaments with electron-dense structures. These features indicate myofibroblast differentiation (Ghadially 1988, Eyden 1993). Myofibroblasts have been detected in normal and diseased tissues where they have contractile or secretory functions (Ghadially 1988, Dominguez-Malagon 1993). Parry (1970) showed that Warthon's jelly, another tissue that is very rich in extracellular GAG, contains only myofibroblasts. Thus, it is possible that myofibroblasts are involved in GAG modification, but whether the presence of myofibroblasts in endocardiosis is the cause or the consequence of myxoid degeneration is uncertain. Further studies are needed to elucidate their possible pathogenic relevance in endocardiosis in pigs. A difference was detected between the total and specific GAG content of the normal and diseased mitral valves, in agreement with previous studies of dog endocardiosis, in which an increase in the content of hyaluronan and cs has been detected (Sokkar and Trautwein 1971). In this study, however, significant differences were detected only in the total GAG and hyaluronan contents. One of the most important functions of hyaluronan is to retain water, to regulate its flow and to be a resilient buffer against mechanical stress (Iozzo 1985). The biosynthesis of hyaluronan is under the control of factors that include hormones, inflammatory and cellular mediators and environmental factors (Lanrent and Fraser 1986). In brain tumours, an increase in hyaluronan content is thought to play a role in oedema and infiltration (Bertolotto et al 1986). Furthermore, its interaction with the cell surface can influence the synthesis and secretion of proteoglycans (Handley and Lowther 1976). Although these data indicate that an increase in hyaluronan is an important factor leading to the valve swelling, it is not clear whether the increase is a primary factor or a result of other events involving other components of the extracellular matrix. In human pathology, on the basis of a striking decrease in collagens type III and V in MVPS, it was suggested that the primary defect was a change in collagen (Hammer et al 1979, Jaffe et al 1981). However, a distur-

Morphological and biochemical studies of pig endocardiosis

125

TABLE 2: Mean (SD) weight of mitral valves (g), and weight Og) and concentration (~g g-l) of glycosaminoglycans (GAG) and the concentration (%) and weight (gg) of different GAG in mitral valves from normal pigs and from pigs with endocardiosis Mitral valves Normal Diseased P value

Weight (g)

GAG (#.g g-l)

1.58 (0.35) 1.87 (0.87) 0.687 a

1367 (176) 1683 (265) 0.006 b

Total GAG (pg) 2127 (409) 3154 (1635)

DS (%)

DS (~g)

14.4 (3.7) 191-8 (71.7) 11.7 (4.3) 195.6 (85.7) 0.135 b 0-91 lb

HS (%)

HS 0xg)

HA (%)

HA (pg)

CA (%)

CA (Itg)

7.2 (3) 6 (3.1) 0.621b

74-6 (35.2) 97.3 (52.9) 0.297 b

25.2 (2.2) 28.8 (5-2) 0.124 a

347.8 (42.6) 472-7 (104.8) 0.003 a

53-1 (5.2) 52-2 (5.6) 0-712 b

745.1 (77-5) 865.7 (190-7) 0.130 a

DS Dermatan sulphate, HS Heparan sulphate, HA Hyaluronan, CA Chondroitin sulphate a Data obtained by the Mann-Whitney Test; b Data obtained by the ANOVAtest

bance of collagen types I and III could not be detected by immunohistochemical and molecular techniques (Spoendlin et al 1992). In a recent investigation on MVPS, Tamura et al (1995) concluded that the changes in collagen fibres and accumulations of proteoglycans were non-specific and might have been caused by the abnormal mechanical forces to which the mitral valves are subjected because of their excessively large surface area. They also concluded that the presence of excessive amounts of proteoglycans might interfere with the normal assembly of collagen. Further studies on extracellular matrix components in early lesions such as those described in two-month-old pigs (Guarda et al 1992b) may give a new insight into the pathogenesis of this lesion in pigs. ACKNOWLEDGEMENTS The authors thank Drs O. Abate and M. Dacasto for their technical assistance and Mr Mario Monaco for the photographic work.

REFERENCES BERTOLOTTO, A., MAGRASSI, M. L., ORSI, L., SITIA, C. & SCHIFFER, D. (i986) Glycosaminoglycan changes in human gliomas. A biochemical study. Journal of Neuro-Oncology 4, 43-48 BITTER, T. & MUIR, H. M. (1962) A modified uronic acid carbazole reaction. Analytical Biochemistry 4, 330-334 BIOLATTI, B., CASTAGNARO, M., BOLLO, E., APPINO, S. & RE, G. (1994) Genital lesions following long-term administration of clenbuterol in female pigs. Veterinary Pathology 31, 82-92 BRAUNWALD, E. (1988) Valvular heart disease. In Heart Disease. Ed E. Braunwald. Philadelphia, W. B. Saunders Co. pp 1023-1092 BUCHANAN, J. W. (1977) Chronic valvular disease (endocardiosis) in dogs. Advances in Veterinary Science and Comparative Medicine 21, 75-106 CAPPELLETTI, R., DEL ROSSO, M. & CHIARUGI, V. P. (1979a) Rapid multisample separation method of the five most widespread animal glycosaminoglycans. Analytical Biochemistry 93, 37-40 CAPPELLETTI, R., DEL ROSSO, M. & CHIARUGI, V. P. (1979b) A new electrophoretic method for the complete separation of all known animal glycosaminoglycans in a monodimensional run. Analytical Biochemistry 99, 311-315 CASTAGNARO, M., ALROY, J., UCCI, A. A. & GLEW, R. H. (1987) Lectin histochemistry and ultrastructure of feline kidneys from six different storage diseases. Virchows Archive B 54, 16-26 CASTAGNARO, M. & CANESE, M. G. (1991) Lectin histochemistry of squamous metaplasia in different epithelial tumors of dogs. Veterinary Pathology 28, 8-15 D'ANDREA, G., AMEDEO, S., GUARDA, F. & CASTAGNARO, M. (1995) Biochemical analysis on gIycoprotein changes in cardiac valves of pig with endocardiosis. Proceedings of the 13th European Congress of European Society of Veterinary Pathology, Edinburgh. p 7A2 DOMINGUEZ-MALAGON, H. (1993) Proliferative disorders of myofibroblasts. Ultrastructural Pathology 17, 211-220 EYDEN, B. P. (1993) Brief review of the fibronexus and its significance for myofibroblastic differentiation and tumor diagnosis. Ultrastructural Pathology 17, 611-622 FRANSSON, L-/~. (1987) Structure and function of cell-associated proteoglycans. Trends in Biochemical Science 12, 406-411 GHADIALLY, F. N. (1988) Ultrastructural Pathology of the Cell and Matrix. 3rd edn. London, Butterworths. pp 872-881 GUARDA, F., NEGRO, M. & AMEDEO, S. (1988) Sulla patologia del prolasso della valvoIa mitrale deI suino. SchweizerArchivfiir Tierheilkunde 130, 583-590

GUARDA, F., GRIGLIO, B., NEGRO, M. & AMEDEO, S. (1992a) Osservazioni preliminari sull'endocardiosi dei suini riprodtutori a fine carriera. Selezione Veterinaria 33, 899-907 GUARDA, F., GRIGLIO, B. & NEGRO, M. (1992b) Sull'endocardiosi dei suinetti di eth inferiore ai due mesi. Obiettivi e Documenti Veterinari 12, 75-78 GUARDA, F., GRIGLIO, B. & ROSSIGNOLI, M. (1993) Herzmi~bildungen und Endokardiose beim Schwein. Deutsche Tierdrztliche Wochenschrift 100, 443445 GUARDA, F., GRIGLIO, B., AMEDEO, S., PASSARINO, G. & MOLLO, F. (1994) Patologia comparata dell'endocardiosi nel cane, nel suino e oeIl'nomo. Obiettivi e Documenti Veterinari 12, 57-64 HAMMER, D., LEIBER, C. V., BABA, N., VASKO, J. S., WOOLEY, C. F. & PINNELL, S. R. (1979) Altered collagen composition in a prolapsing mitral valve with ruptured chordae tendineae. American Journal of Medicine 67, 863-866 HANDLEY, C. & LOWTHER, D. A. (1976) Inhibition of proteoglycans synthesis by hyaluronan in chondrocytes in cell culture. Biochemical and Biophysical Acta 444, 69-75 HOOK, M., KJELLEN, L., JOHANSSON, S. & ROBINSON, J. (1984) Cell-surface glycosaminoglycans.Annual Reviews in Biochemistry 53, 847-869 HYNES, M. A., DODD, J. & JESSEL, T. M. (1989) Carbohydrate recognition, cell interactions, and vertebrate neural development. In Neurobiology of Glycoconjugates. Eds R. U. Margolis and R. K. Margolis. New York, Plenum Press Co. pp 337-365 IOZZO, R. V. (1985) Proteoglycans: structure, function, and role in neoplasia. Laboratory Investigation 53, 373-396 JAFFE, A. S., GELTMAN, E. M., RODEY, G. E. & UITTO, J. (1981) Mitral valve prolapse: a consistent manifestation of type IV Ehlers-Danlos syndrome. The pathogenetic role of the abnormal production of type III collagen. Circulation 64, 121-125 KERN, W. H. & TUCKER, L. B. (1972) Myxoid changes in cardiac valves: pathologic, clinical, and ultrastructural studies. American Heart Journal 84, 294-301 KOGURE, K. (1980) Pathology of chronic mitral valvular disease in the dog. Japanese Journal of Veterinary Science 42, 323-335 LAURENT, T. C. & FRASER, R. E. (1986) The properties and turnover of hyainronan. In Functions of the Proteoglycans. Eds D. Evered and J. Whelan. New York, John Wiley & Sons. pp 9-24 PARRY, E. W. (1970) Some electron microscope observations on the mesenchymal structures of full-term umbilical cord. Journal of Anatomy 107, 505-518 RUOSLAHTI, E. (1988) Structure and biology of proteoglycans. Annual Review in Cell Biology 4, 229-255 SAVAGE, D., DEVEREUX, R. B., GARRISON, R. J., CASTELLI, W. P., ANDERSON, S. T., LEVY, D., THOMAS, H. E., KANNEL, W. B. & FEINLEIB, M. (1983) Mitral valve prolapse in the general population. 2. Clinical features: the Framingham study. American Heart Journal 106, 577-594 SCHOLE, J. (1973) Untersuchungen zur Atiologie und Pathogenese tier Endokardiose und Endokarditis des Hundes. Teil IV: Biochemische Unterschungen. Deutsche Tieriirztliehe Wochenschrift 80, 472-473 SOKKAR, S. M. & TRAUTWEIN, G. (1970) Die Endokardiose der Atrioventrikularklappen des Hundes. I. Morphologische und histochemische Untersuchungen. Zentralblattfiir Veteriniirmedizin A 17, 757-779 SOKKAR, S. M. & TRAUTWEIN, G. (1971) Die Endokardiose der Atrioventrikuiarklappen des Hundes. II. Papier- und immunelektrophoretische Untersuchungen. Zentralblattfiir Veterindrmedizin A 18, 1-14 SPOENDLIN, B., GEORGULIS, J., EPPER, R., LITZISTORF, Y. & MIHATSCH, M. J. (1992) Pathologic der myxoiden Mitraiklappendegeneration: Literaturiibersicht und eigene Resuitate. Schweizer Rundschau und Medical Praxis 81, 1420-1426 TAMURA, K., FUKUDA, Y., ISHIZAKI, M., MASUDA, Y., YAMANAKA, N. & FERRANS, V. J. (1995) Abnormalities in elastic fibers and other connective-tissue components of floppy mitral valve. American Heart Journal 129, 11491158 TRAUTWEIN, G., BRASS, W., KERSTEN, U., ERNST, E., SCHNEIDER, P., SCHULZ, L. C. L., AMTSBERG, G., BISPING, W., KIRCHOFF, H. & SCHOLE, I. (1973) Uutersuchungen zur Atiologie und Pathogenese der Endokardiose and Endokarditis der Hundes. Teil V. Synopsis tier Ergbnisse. Deutsche Tiertirztliche Wochenschrift 80, 507-511 WHITNEY, J. C. (1974) Observations on the effect of age on the severity of heart valve lesions in the dog. Journal of Small Animal Practice 15, 511-522

Received February 26, 1996 Accepted August 21, 1996