Valve proteoglycan content and glycosaminoglycan fine structure are unique to microstructure, mechanical load and age: Relevance to an age-specific tissue-engineered heart valve

Abstract

This study characterized valve proteoglycan and glycosaminoglycan composition during development and aging. This knowledge is important for the development of age-specific tissue-engineered heart valves as well as treatments for age-specific valvulopathies. Aortic valves and mitral valves from first-third trimester, 6-week, 6-month and 6-year-old pigs were examined using immunohistochemistry for versican, biglycan, decorin and hyaluronan, as well as elastin and fibrillin. The fine structure of glycosaminoglycans was examined by fluorophore-assisted carbohydrate electrophoresis. Decorin expression was strongest in the 6-year-old valves, particularly in the aortic valve spongiosa. The quantity of iduronate was also highest in the 6-year-old valves. The central tensile-loading region of the anterior mitral leaflet demonstrated reduced glycosaminoglycan content, chain length and hydration and a larger fraction of 4-sulfated iduronate and lower fraction of 6-sulfation. With age, the anterior leaflet center showed a further increase in 4-sulfated iduronate and decrease in 6-sulfation. In contrast, the anterior leaflet free edge showed decreased iduronate and 4-sulfated glucuronate content with age. The young aortic valve was similar to the mitral valve free edge with a higher concentration of glycosaminoglycans and 6-rather than 4-sulfation, but aged to resemble the mitral anterior leaflet center, with an increase in 4-sulfated iduronate content and a decrease in the 6-sulfation fraction. Elastin and fibrillin often co-localized with the proteoglycans studied, but elastin co-localized most specifically with versican. In conclusion, composition and fine structure changes in valve proteoglycans and glycosaminoglycans with age are complex and distinct within valve type, histological layers and regions of different mechanical loading.

Fig. 1.

A) Histological anatomy of the valve. B) Examples of grading rubrics for the characteristics staining intensity and delineation. For the examples of staining intensity the arrow points at the area demonstrating the stated grade. For the characteristic delineation, “1” indicates little differentiation in intensity between layers. The delineation rubric shown is for the mid-leaflet region (there was a rubric for each region of the leaflet including annulus, mid-leaflet, and free edge). “4” indicates maximum for each characteristic.

Fig. 2.

A) 1^st trimester valves largely consisted of an amorphous mass of proteoglycans (PGs) and glycosaminoglycans (GAGs). Over the course of fetal development rudimentary layers became apparent. B) In 1^st trimester valves, the PG expression was largely ubiquitous. Expression became more localized over the course of fetal development. Scale bars indicate 100 μm. C) Movat stain showed maximum PGs and GAGs in the spongiosa, but staining for decorin (DCN) (and versican and biglycan) was often stronger in other layers. In contrast, staining for hyaluronan (HA) was often strongest in the spongiosa. Sections are from the aortic valve and are oriented so that the ventricularis is at the top and fibrosa is at the bottom. Scale bars indicate 100 μm.

Fig. 3.

A) Strong expression of elastin within both atrialis and ventricularis layers of 1^st trimester mitral valve. Scale bar indicates 200 μm. B) Out of the three proteoglycans (PGs) examined, versican (VC) co-localized most closely with elastin, shown here in an aortic valve. In this figure the ventricularis is on the left and the fibrosa is on the right. Scale bars indicate 200 μm.

Fig. 4.

A) Percent hydration examined by valve and age. *=p<0.001 between different valves. α=p≤0.003 between different ages. Error bars are standard error of the mean. B) Percent of total glycosaminoglycan (GAG) composed of hyaluronan (HA) (as measured by FACE) examined by valve and age. *=p<0.001 MVF > AV > MVAC. α=p<0.001 between different age groups. MVF= mitral valve (MV) free edge; MVAC=MV anterior center; AV=aortic valve. Error bars are standard error of the mean.

Fig. 5.

Proportion of glycosaminoglycans (GAGs) in valves of different ages, as calculated from FACE data. Statistical differences are marked between different ages of a given valve and between the MVAC and MVF regions of the same age. (Differences between AV and the MV regions for a given age have not been marked. Differences between MVAC and MVF in fractions when considering all ages have not been marked, but are discussed in the text.). †=MVAC significantly different than MVF for the 6-week-old, 6-month-old, or 6-year-old. *=significant change in a given GAG across ages in a given valve region (MVF, MVAC, and AV). All marked differences are p≤0.05 except α=p<0.03R. MVF= mitral valve (MV) free edge; MVAC=MV anterior center; AV=aortic valve. G0S=unsulfated glucuronate, G4S=4-sulfated glucuronate, G6S=6-sulfated glucuronate, I4S=4-sulfated iduronate, I6S=6-sulfated iduronate, and XS=di- and tri-sulfated glucuronate/iduronate. Only percentages >10% are labeled.

Fig. 6.

Glycosaminoglycan (GAG) composition for each valve and each age group, as calculated from the FACE analysis. Note that the middle graph has a different scale on the y-axis. Statistically significant differences (p≤0.05) are marked between different ages of a given valve and between the MVAC and MVF regions of the same age. (Differences between AV and MVF as well as AV and MVAC for a given age have not been marked. Differences between MVAC and MVF in concentrations when considering all ages have not been marked, but are discussed in the text.). Error bars are standard error of the mean. α=significant change in a given GAG in a given valve across ages, β=p<0.03R. †= significant difference between MVAC and MVF in the 6-week-old, 6-month-old, or 6-year-old. MVF= mitral valve (MV) free edge; MVAC=MV anterior center; AV=aortic valve. G0S, G4S, G6S, I4S, I6S, and XS are all defined in the legend for Fig. 5.

Fig. 7.

Chain length of valve regions by age, as calculated from FACE data. Overall p=0.059 by ANOVA. MVF= mitral valve (MV) free edge; MVAC=MV anterior center; AV=aortic valve. Error bars are standard error of the mean.