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Alterations In Substrate Stiffness And Composition Effect The Progression Of Calcific Aortic Valve Disease
Lauren Baugh1, Irene Georgakoudi2, Phillip Hinds3, Gordon Higgins4, Lauren Black2.
1Tufts University, Medford, MA, USA, 2Department of Biomedical Engineering, Tufts University, Medford, MA, USA, 3Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA, USA, 4Molecular Cardiology research institute Center forTtranslational Genomics, Tufts Medical Center, Boston, MA, USA.

Objective: To test an in vitro model of calcific aortic valve nodule growth that modulates stiffness and substrate composition in polyacrylamide (PAAM) gels to understand progression of calcific aortic valve disease (CAVD). Methods: Rat aortic valves were isolated and used in an explant culture to obtain valve interstitial cells (VICs). The isolated VICs were used to seed PAAM gels created at three levels of stiffness: 5 kPa, 20 kPa, and 35 kPa. These stiffness values were chosen to model normal valve stiffness (5 kPa), a valve with extensive CAVD (35 kPa), and a transitional valve stiffness (20 kPa). We also incorporated either collagen I or hyaluronic acid (HA) as binding sites to distinguish compositional effects that occur with remodeling. To induce calcification, transforming growth factor - β1 (TFG-β1) was added to the culture medium. Additional murine leaflets were isolated and placed in culture media with TFG-β1.

Results: Our model system accurately recapitulates the development of calcified nodules in both human and murine CAVD as demonstrated through two-photon excited fluorescence (TPEF) spectral data. As demonstrated in Figure 1, nodule formation and collagen I reorganization were measured using TPEF and the second harmonic generation (SHG) signal from the collagen fibers, respectively (Figure 1 A); in Figure 1 B, elastic modulus was measured in the PAAM gels and the isolated murine leaflets using atomic force microscopy (AFM); and nodule growth was analyzed using the calcium fluorescent indicator fluo-4. Stiffness had a significant (p<0.05) impact on nodule size (Figure 1 C). Additionally, the development of nodules exacerbated nodule growth by further increasing matrix stiffness and altering the matrix structure indicated through disorganized collagen fibers demonstrated with SHG. Conclusions: VIC activation to myofibroblasts after TFG-β1 stimulation was enhanced with increasing substrate stiffness and alterations in substrate composition. Our model allows for identification of calcification in real time and provides a visual representation of changes in the surrounding substrate in response to nodule formation.

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