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 (2-D) and 3-D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2-D 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 2-D 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 3-D 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 ECM components that can regulate VIC phenotype and function has significant implications for understanding native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.

Figure 1

Higher levels of hyaluronic acid (HA) were found when VICs were cultured on collagen (Coll) when compared to fibrin (FB) and tissue culture polystyrene (TCPS). *P<0.05 compared to FB and TCPS.

Figure 2

Culture of VICs upon 2-D substrates modified with immobilized HA in a range of molecular weights led to decreases in: (A) nodule number, (B) average size of individual nodules, (C) total calcified area, and (D) calcium content. Treatment with anti-CD44 reversed the suppressive effects of immobilized HA on nodule number (A, hatched bars). Treatment with an isotypic control antibody did not impact nodule formation (A, empty bars). *P<0.05 compared to TCPS for all panels.

Figure 3

VICs in 2-D culture were treated with different molecular weights of exogenous HA in solution and evaluated for: (A) nodule number, (B) average size of individual nodules, (C) total calcified area, and (D) calcium content. Treatment with anti-CD44 reversed the suppressive effects of soluble HA on nodule number (A, hatched bars). Treatment with an isotypic control antibody did not impact nodule formation (A, empty bars). P<0.05 compared to TCPS for all panels.

Figure 4

Immunohistochemical detection of the expression of CD44 in native valve leaflets; CD44 (left), DAPI (center), merged pictures (right). Scale bar = 100 µm.

Figure 5

Native valve leaflets were treated with anti-CD44 over 6 days of in vitro culture and then analyzed for: (A) VIC proliferation, and (B) VIC apoptosis. n=6 samples per condition, *P<0.01 compared to untreated control.

Figure 6

Treatment of native valve leaflets with anti-CD44 for a period of 6 days resulted in significantly elevated expression of several phenotypic markers of valve disease, as indicated by: (A) immunohistochemical detection of alpha smooth muscle actin (α-SMA; left), alkaline phosphatase (ALP; center) and core binding factor alpha 1 (Cbfa-1; right), (B) western blot for α-SMA, ALP, and CBFa-1, and (C) Von Kossa staining to detect mineralization (black). *P<0.05 compared to control. Scale bar = 100 µm.

Figure 7

Native leaflets were treated with two concentrations of hyaluronidase (HY-ase), followed by biochemical and mechanical characterization of the treated leaflets to validate this HA-depletion method. Quantitative assays were performed on the leaflets to measure: (A) HA, (B) collagen, (C) sulfated GAGs, (D) DNA, and (E) Young’s Modulus. *P<0.05 compared to untreated leaflet.

Figure 8

Effect of HA removal from native leaflets on: (A) VIC proliferation, and (B) VIC apoptosis after 6 days of in vitro culture. n=6 samples per condition, *P<0.01 compared to no enzyme control.

Figure 9

Effect of HA depletion on the expression of phenotypic markers of valve disease following 6 days of in vitro leaflet culture, as evaluated by immunohistochemical detection of: α-SMA (left), ALP (middle), and Cbfa-1 (right). Top to bottom: Day 0, no enzyme, HY-ase 0.1 U, and HY-ase 1000 U. Scale bar = 100 µm.

Figure 10

Effect of HA depletion on the expression of phenotypic markers of valve disease following 6 days of in vitro leaflet culture, as evaluated by western blot detection of: (A) α-SMA, (B) ALP, and (C) Cbfa-1. All results are normalized to β-tubulin. *P<0.05 compared to untreated leaflet.

Figure 11

Effect of HA depletion on VIC mineralization following 6 days of in vitro leaflet culture, as determined by: (A) von Kossa staining for mineralization; calcium deposits are stained in black, and (B) calcium content. *P<0.05 compared to untreated leaflet. Scale bar = 100 µm.