Regulation of valvular interstitial cell phenotype and function by hyaluronic acid in 2-D and 3-D culture environments

Abstract

Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2D) and 3D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC-HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC-HA interactions are crucial in maintaining a healthy VIC phenotype. Identification of ECM components that can regulate VIC phenotype and function has significant implications for understanding of native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.

Differential cell-matrix responses in hypoxia-stimulated aortic versus mitral valves

“…However, whole aortic valve leaflets cultured in this study showed no apparent angiogenic or calcific changes in AVIC protein expression. Cell-ECM interactions within the leaflets may have affected the VIC response to hypoxia and have previously been shown to regulate calcification in the aortic valve [45,46]. ECM changes during valve disease are also associated with upregulation of the receptor for hyaluronan mediated motility, a receptor linked to angiogenesis [47].…”

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

“…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].…”

“…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]. 2D culture systems, such as bioactive surface coatings on tissue culture polystyrene, have been used to successfully isolate many of these factors for analysis but yield limited results when used to replicate the complex array of factors involved in CAVD.…”

The effect of hyaluronan on airway mucus transport and airway epithelial barrier integrity: Potential application to the cytoprotection of airway tissue

“…Hyaluronan is a large glycosaminoglycan that is abundant in the extracellular matrix of many tissues and is involved in vertebrate tissue morphogenesis and cellular processes (Noble, 2002;Spicer and Tien, 2004;Rosines et al, 2007;Garcia-Fuentes et al, 2009;Rodriguez et al, 2011). In the airways, it is an important component of the extracellular matrix in the lung parenchyma and plays a key role during lung development (Calvitti et al, 2004).…”