Extracellular matrix remodeling in wound healing of critical size defects in the mitral valve leaflet

Abstract

The details of valvular leaflet healing following valvuloplasty and leaflet perforation from endocarditis are poorly understood. In this study, the synthesis and turnover of valvular extracellular matrix due to healing of a critical sized wound was investigated. Twenty-nine sheep were randomized to either CTRL (n = 11) or HOLE (n = 18), in which a 2.8-4.8 mm diameter hole was punched in the posterior mitral leaflet. After 12 weeks, posterior leaflets were harvested and histologically stained to localize extracellular matrix components. Immunohistochemistry was also performed to assess matrix components and markers of matrix turnover. A semi-quantitative grading scale was used to quantify differences between HOLE and CTRL. After 12 weeks, the hole diameter was reduced by 71.3 ± 1.4 % (p < 0.001). Areas of remodeling surrounding the hole contained more activated cells, greater expression of proteoglycans, and markers of matrix turnover (prolyl 4-hydroxylase, metalloproteases, and lysyl oxidase, each p ≤ 0.025), along with fibrin accumulation. Two distinct remodeling regions were evident surrounding the hole, one directly bordering the hole rich in versican and hyaluronan and a second adjacent region with abundant collagen and elastic fiber turnover. The remodeling also caused reduced delineation between valve layers (p = 0.002), more diffuse staining of matrix components and markers of matrix turnover (p < 0.001), and disruption of the collagenous fibrosa. In conclusion, acute valve injury elicited distinct, heterogeneous alterations in valvular matrix composition and structure, resulting in partial wound closure. Because these changes could also affect leaflet mechanics and valve function, it will be important to determine their impact on healing wounds.

Keywords: Collagen; Mitral regurgitation; Mitral valve; Proteoglycans; Wound healing.

Fig. 1.

A) Diagram showing location from which PML cross-sections were taken. AML indicates anterior leaflet of MV. Sections were cut from annulus to free edge, as indicated by the dashed lines. B) Anatomy of the normal MV posterior leaflet illustrating valve regions and histological layers in a Movat-stained section. In Movat pentachrome stain black=elastic fibers, blue=PGs/GAGs, yellow=collagen, red=muscle or fibrin. C) Examples of grading rubrics for the characteristics staining intensity and delineation. For the characteristic delineation, “1” indicates little differentiation in intensity between layers. “4” indicates maximum for each characteristic. Separate rubrics were made for the delineation of different regions of the valve (annulus, mid-leaflet, and free edge).

Fig. 2.

A) Substantial areas of remodeling significantly decreased hole diameter (Movat pentachrome stain). Red block markers spanned by arrows indicate initial diameter of hole punch; the black dashed line on the right tissue section indicates the measurement made to determine the final hole diameter. B) Areas surrounding hole punch show significant thickening as indicated by arrows (Movat pentachrome stain). Thickening was not always symmetric around the hole, as demonstrated by right image. C). Masson’s trichrome stain demonstrating accumulation of fibrin around hole. Scale bars indicate 200 μm. D) CD31-positive cells surrounding a rare demonstration of microvasculature within a HOLE leaflet. Scale bar indicates 50 μm. E-F) Masson’s trichrome stain illustrating examples of fibrin accumulation adjacent to hole. Scale bars indicate 200 μm.

Fig. 3.

A) Initial and final hole diameters for HOLE samples. B) Cell density was increased in areas of remodeling compared to the annulus of the same HOLE leaflet as well as compared to CTRL. *=p<0.001. Error bars indicate the standard error of the mean.

Fig. 4.

A) Intensity of PG and GAG staining in region around hole-punch in HOLE PML versus comparable regions in CTRL..B) Intensity of collagen-related marker staining in regions around hole-punch in HOLE PML versus comparable regions in CTRL. C) Average delineation of markers between layers in HOLE PML compared to CTRL. Error bars indicate the standard error of the mean. In each of A, B, and C, the ANOVA for the overall comparison of all related markers was p<0.001; the p-values above bars are from the post-hoc analyses.

Fig. 5.

A) Two regions of remodeling (R1, R2) were evident as indicated by the red arrows: R1=immediately adjacent to the hole, rich in VC, HA, and demonstrating alcian-blue staining in Movat-stained sections; R2=located interior to R1 relative to the hole-punch, rich in DCN, MMPs, Col III, and elastin. B) Decreased delineation (indicated by red arrow) in HOLE PML compared to CTRL. C) Visualization of picrosirius red-stained tissue under polarized light demonstrates disruption of collagen backbone in HOLE PML. Scale bars for all images represent 1 mm.