Identification And Localization Of Proteoglycans In Normal And

							5. Identification and Localization of Proteoglycans in
Normal and Diseased Valves and Valvular Cell Cultures
K. Jane Grande-Allen1, Vishal Gupta1, Vincent C. Hascall1, Thomas N. Wight2, Norman B.
Ratliff1, Brian P. Griffin1, Delos M. Cosgrove III1, Ivan Vesely1
1
  Cleveland Clinic Foundation, Cleveland, Ohio, United States

OBJECTIVES: Heart valves contain a variety of glycosaminoglycans (GAGs), predominantly
hyaluronan, chondroitin 6-sulfate, and dermatan 4-sulfate. Except for hyaluronan, these GAGs
are present as proteoglycans with diverse functional roles. The specific identities of valvular
proteoglycans, however, have only been inferred from GAG sulfation patterns and chain
lengths. Therefore, we characterized the chondroitin/dermatan sulfate proteoglycans via
Western blotting and immunohistochemistry.
METHODS: Intact proteoglycans from normal and diseased mitral valves and valvular cell
cultures were extracted, purified, Western blotted, and probed for decorin, biglycan, and
versican. Valve tissues were also fixed, paraffin-embedded, sectioned, and probed for
hyaluronan, decorin, biglycan, and versican.
RESULTS: The core proteins for decorin and biglycan were found in blots from all valve
tissues. Versican core protein was present as V0 and degraded V1 isoforms.
Immunohistochemistry demonstrated hyaluronan almost everywhere in leaflets but minimally
in the highly collagenous regions (chordae and anterior leaflet clear zone). Decorin was located
in chordae (rich in collagen) and leaflet atrialis (rich in elastin). Biglycan was diffusely present
in valves, but absent from the leaflet free edge. Versican was present in the atrialis, spongiosa,
and chordal sheath, and was distributed widely throughout myxomatous mitral valves.
CONCLUSIONS: Our findings confirm the presence of the large compression-resistant
proteoglycan, versican, and small interstitial proteoglycans, decorin and biglycan, in the valve
tissues, as suggested by our previous GAG analyses. Interestingly, hyaluronan was far more
diffusely distributed throughout the different leaflet layers than we anticipated, and is probably
present in both a freely mobile form as well as a component of versican-hyaluronan
aggregates.

Proteoglycan  Molecular Weight of PG                        Molecular Weight of Core
Versican      Barely entered 3.5% stacking gel              V0 at ~450 kDa, V1 at ~ 400 kDa
Decorin       Smear from ~100 to 200 kDa                    43 and 50 kDa doublet
Biglycan      Smear from ~200 to 300 kDa                    50 kDa
Summary of SDS-PAGE and Western blot analyses