Regulation of valvular interstitial cell calcification by components of the extracellular matrix

Abstract

The composition of the extracellular matrix (ECM) is believed to play a role in heart valve disease, and is highly relevant to the design of heart valve tissue engineering scaffolds, yet the interaction of valvular interstitial cells (VICs) with the ECM environment has not been well characterized. Thus, the transformation of VICs to an osteoblast-like phenotype was quantified in VICs cultured on different types of ECM coatings. The results show that the number and size of calcific nodules formed in VIC cultures, as well as the expression of the mineralization markers alkaline phosphatase (ALP) and CBFa1, were highly dependent upon the composition of the culture surface. In fact, VICs cultured on certain ECM components, namely collagen and fibronectin, were resistant to calcification, even upon treatment with several mineralization-inducing growth factors. Meanwhile, cultures of VICs on fibrin, laminin, and heparin coatings not only had a high number of calcified nodules, but also elevated levels of ALP and CBFa1. Nodule composition analysis revealed the presence of multiple types of mineralization, including hydroxyapatite. Although apoptotic and necrotic cells were more concentrated in nodules than in other parts of the VIC cultures, the nodules contained a strong majority population of viable cells. By demonstrating this ECM-dependence of VIC calcification, we aim to identify appropriate biomaterial environments for heart valve tissue engineering as well as elucidate mechanisms of valvular disease.

Multilayer three-dimensional filter paper constructs for the culture and analysis of aortic valvular interstitial cells

“…In single layers, SMαA expression was much lower by 4 days, indicating a VIC transition from an activated state to a more quiescent state that is often found in healthy valves in vivo. Reduction in VIC SMαA expression likely occurred due to culture on collagen-I substrates, as shown by collagen's ability to reduce VIC calcification found in earlier studies with collagen [11].…”

“…These studies have shown that VIC activation and calcification potential are measurably influenced by the substrate stiffness of the culture ECM or biomaterial [9]. Stiffer substrates such as polystyrene promote VIC calcification whereas collagen, hyaluronan, and fibronectin gels or surface coatings reduce calcification [11][12][13]. Production of calcific nodules by VICs is a complicated process, however, involving multiple cell signaling pathways that are influenced by inflammatory factors, growth factors such as TGF-β, substrate stiffness, biological activity of the substrate, and oxygen and nutrient availability [12][13][14][15][16][17].…”

Extracellular Matrix Organization, Structure, and Function

“…Furthermore, myofibroblast differentiation from VICs and calcification in vitro have been shown to be dependent on ECM composition [19].…”

“…In vitro studies were unable to induce calcification in VICs cultured on collagen proteins in standard media. It is believed that collagen actively inhibits VIC calcification [19]. Other studies have shown that scaffold collagen content also affects VIC proliferation.…”

“…In vivo and in vitro studies have shown that laminin interacts with endothelial and epithelial cells, and can help maintain physiological functionality of the cells [97,100,101]. However, VICs cultured on laminin have been found to support high quantities of calcific nodule formation in the presence of TGF-β, when compared to subendothelial ECM components collagen type I and fibronectin [19,102]. The various regions of laminin protein have been reported to mediate specific cell responses.…”