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Aortic Valve Knockout of pRb Increases Calcific Aortic Valve Disease Characteristics

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•Calcific aortic valve disease (CAVD) affects over 8 million people in the US [1]; currently, the only treatment for severe disease is total valve replacement surgery.
•The lack of basic disease mechanisms underlying CAVD has limited the number of pharmaceutical targets available for treatment; better understanding may lead to better therapies.
•Changes in valve tissue mechanics, composition, and fiber orientation have all been measured in the end stages of the disease, but it is unclear how these properties are altered during disease progression.
•In vitro models that allow for controlled experimental manipulation of environments relevant to the aortic valve may help parse out specific disease mechanisms.
•The loss of retinoblastoma protein (pRb) has been shown to produce cells that mineralized more heavily in vitro [2].
•pRb also regulates bone formation through interaction with runx2 [3].
•We created a knockout mouse model to study the effects of pRb.
•Porcine aortic valve interstitial cells (aVICs) were also used to create an in vitro model using viral transfection to knockdown Rb.
•aVICs were seeded on 2D, polyacrylamide (PAAM) gels at physiology stiffnesses and dosed for 24 hours with TGF-β1 to induce activation.
•Second harmonic generation (SHG) images of collagen showed an increase in directional variance (E) in the Rbfl/fl mice compared to the Rbfl/- leaflets. Figures (A) and (B) are representative SHG images while (C) and (D) show zoomed in sections of collagen fibers.
•Atomic force microscopy (AFM) was used to measure the Young’s Modulus of the valve leaflets. A representative heat map of leaflet tissue is shown in (F). There was a significant increase in leaflet stiffness in the Rbfl/fl mice (G).
•PAAM gels were created to model changing tissue stiffness associated with CAVD  and designed to incorporate different binding proteins to assess the effect of specific ECM components  in the fibrosa and spongiosa (A).
•5 kpa (healthy valve tissue), 20 kPa (beginning of disease), and 35 kPa (diseased valve tissue)  [4] stiffnesses were tested with either collagen or hyaluronic acid (HA) as a binding motif. Stiffness was confirm using AFM. Calcific nodules were stained with fluo-4 AM, a calcium dye (B).
•Over a 3 week culture period, significant changes were seen in the average nodule size of differing conditions. The shRb knockdown aVICs demonstrated increased nodule growth compared to the shNT aVICs  after 1 week in culture with HA and collagen gels (C-F).
•Valve leaflets from both Rbfl/- and Rbfl/fl mice were analyze with LC-MS/MS.
•Normalized spectral counts were compared between groups.
•Differences included a significant increase in both collagen IIIα1 and collagen VIα2 in the Rbfl/fl.
•The Rbfl/fl  valves also showed an increase in latent TGF-β1 binding protein.
•The knockout model further demonstrated a decrease in nestin and HA/proteoglycan link protein.
•Knocking out Rb decreases collagen fiber organization and increases aortic leaflet stiffness.
•Knockdown of Rb in aVICs demonstrates an increased propensity for calcification on lower stiffness PAAM gels.
•Proteomics analysis of Rbfl/fl valves shows KO mice have more, less mature collagen and proteoglycans.
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