The online version of this article (doi:10.1186/1471-2261-14-29) contains supplementary material, which is available to authorized users.
The authors have no competing interests to declare.
KR and LP participated in leaflet characterization and phenotype analyses. AP participated in leaflet characterization. KR, LP, and AP participated in the design of the study, performed statistical analyses, and participated in data interpretation. KM conceived of the study and participated in its design and coordination and in data interpretation. KR and KM drafted the manuscript. All authors read and approved the final manuscript.
Extracellular matrix (ECM) disarray is found in calcific aortic valvular disease (CAVD), yet much remains to be learned about the role of individual ECM components in valvular interstitial cell (VIC) function and dysfunction. Previous clinical analyses have shown that calcification is associated with decreased collagen content, while previous in vitro work has suggested that the presence of collagen attenuates the responsiveness of VICs to pro-calcific stimuli. The current study uses whole leaflet cultures to examine the contributions of endogenous collagen in regulating the phenotype and calcification of VICs.
A “top-down” approach was used to characterize changes in VIC phenotype in response to collagen alterations in the native 3D environment. Collagen-deficient leaflets were created via enzymatic treatment and cultured statically for six days in vitro. After culture, leaflets were harvested for analysis of DNA, proliferation, apoptosis, ECM composition, calcification, and gene/protein expression.
In general, disruption of collagen was associated with increased expression of disease markers by VICs in whole organ leaflet culture. Compared to intact control leaflets, collagen-deficient leaflets demonstrated increased VIC proliferation and apoptosis, increased expression of disease-related markers such as alpha-smooth muscle actin, alkaline phosphatase, and osteocalcin, and an increase in calcification as evidenced by positive von Kossa staining.
These results indicate that disruption of the endogenous collagen structure in aortic valves is sufficient to stimulate pathological consequences in valve leaflet cultures, thereby highlighting the importance of collagen and the valve extracellular matrix in general in maintaining homeostasis of the valve phenotype.
Additional file 1: Figure S1: Leaflets were depleted of elastin using 20 U/mL elastase following procedures similar to those outlined for collagen depletion. After 6 days of culture, elastin-depleted leaflets were histologically analyzed for: (A) mineralization via von Kossa staining, (B) α-SMA expression, and (C) ALP expression. Detectable mineralization, α-SMA, and ALP were not found in any of the elastin-depleted leaflets. Scale bar = 100 μm. (PDF 291 KB)
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- Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification
Karien J Rodriguez
Laura M Piechura
Ana M Porras
Kristyn S Masters
- BioMed Central
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