Figure 7.

Role of NF-κB in tumor necrosis factor (TNF)-α–induced retraction, stiffness, and osteogenic differentiation. A: Immunofluorescence of NF-κB activation. Valvular interstitial cells (VICs) were seeded in three-dimensional hydrogels and treated with control vehicle or 25 ng/mL of TNF-α for 8 days, embedded in OCT, and cryosectioned. Slides were immunostained with antibody specific to phosphorylated IκB (red), the images were acquired with Olympus IX71, and the immunofluorescence was quantified using image analysis software (Metamorph Advanced version 7.7). Nuclei were counterstained with DAPI (blue). B: Western analysis of NF-κB activation. VICs were seeded in 6-well culture dishes, treated with control vehicle or 25 ng/mL of TNF-α for the indicated time points, and probed with antibody specific to phosphorylated or total IκB. C and D: Retraction and stiffness in response to an inhibitor of NF-κB. VICs were seeded in three-dimensional hydrogels and treated with control vehicle, 25 ng/mL of TNF-α, and/or 10 μmol/L pyrrolidine dithiocarbamate (PDTC). The diameters of the hydrogels were normalized to the respective diameters at day 0. Instron compressive testing was performed at day 8. E: VICs were seeded in 96-well plates and treated with control vehicle, 25 ng/mL of TNF-α, and/or 10 μmol/L PDTC. Alkaline phosphatase (ALP) cytochemistry, normalized to cell count, was assessed after 7 days of treatment. ^∗P < 0.05 versus control vehicle; ^†P < 0.05 versus TNF-α. Scale bar = 100 μm (A).