Manipulation of valve composition to elucidate the role of collagen in aortic valve calcification

Abstract

BackgroundExtracellular 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.MethodsA “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.ResultsIn 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.ConclusionsThese 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.

Changes in Structural-Mechanical Properties and Degradability of Collagen during Aging-associated Modifications

“…Altogether, our study indicates that mineralization of collagen at both fiber and fibril levels reduces their intrinsic flexibility, which is an asset in bones. However, this is a drawback in arteries and other soft tissues, where excessive stiffness reduces the functionality of the tissue and leads to a range of disorders (19,20). Appreciable concentrations of calcium, phosphorus, silicium, and sulfur in the calcified lesions of spherical forms have been reported previously (51,62).…”

“…A typical feature of age-related diseases is the ectopic mineralization of ECM. This abnormal mineralization reduces the structural integrity and elasticity of arteries, which impairs cardiovascular function (19,20). Moreover, alterations in the mechanisms of calcium and phosphorous homeostasis are responsible for both skeletal and vascular disorders (21).…”

The presence of fructosamine in human aortic valves is associated with valve stiffness

“…As the extracellular matrix contents, particularly collagen, are determinants for maintaining valve integrity and mechanical strength,20 26 non-enzymatic collagen crosslinks (AGEs) induce a decreased valve extensibility with an increased valve stiffness 2 6 21. Both AGEs and calcium deposits have been detected on the extracellular aortic heart valve sites 19.…”

“…Some experimental studies have reported that aortic valve calcification is an active collagen-dependent regulated mechanism integral to aortic stenosis 20 28. Furthermore, AGEs could enhance the affinity of extracellular matrix components for calcium by the receptor of advanced glycation end products pathway 27 29.…”

“…N ε -(carboxymethyl) lysine deposits, the best-known AGEs, have been detected on the calcification sites of human aortic heart valves 19. As collagen is the major extracellular matrix component of the aortic valve,20 crosslinking of extracellular valve glycated collagen alters the tissue function as well as the mechanical properties of the valve 2 6 21. As valve stiffness reflects the mechanical properties of the valve tissue, it represents a risk factor of valve disease and can predict the need for heart valve replacement 13…”