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bioRxiv · 10.1101/2023.02.21.529461

In-vivo subdermal implant in the alpha-Gal knockout mice model for the assessment of bioprosthetic heart valve calcification rate before and after a polyphenols-based treatment

Naso, F.·Colli, A.·Zilla, P.·Calafiore, A. M.·Lotan, C.·Padalino, M.·Sturaro, G.·Gandaglia, A.·Spina, M.
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Abstract

RationaleThe presence of preformed antibodies against Gal in the human lead to opsonization of the implanted bioprosthetic heart valve (BHV), leading to deterioration and calcification. Murine subcutaneous implantation of BHVs leaflets has been widely used for testing the efficacy of anti-calcification treatments, however, unlike the situation in humans, leaflets implanted into a murine model will not be able to elicit an Gal immune response because both donor and recipient species constitutively express the epitope. ObjectiveThis study evaluates the calcium deposition on commercial BHV using a new humanized murine Gal knockout (KO) animal model. Furtherly, the anti-calcification efficacy of a polyphenol-based treatment was deeply investigated. Methods and ResultsBy using CRISPR/Cas9 approach an Gal KO mouse was created and used for the evaluation of the calcific propensity of original and polyphenols-treated BHV by subcutaneous implantation. The calcium quantification was carried out by plasma analysis; the immune response evaluation was performed by histology and immunological assays. Anti-Gal antibodies level in KO mice increases at least double after 2 months of implantation of original commercial BHV compared to WT mice, conversely, the polyphenols-based treatment seems to effectively mask the antigen to the KO mices immune system. Commercial leaflets explanted after 1 month from KO mice showed a four-time increased calcium deposition than what was observed on that explanted from WT. Polyphenol treatment prevents calcium deposition by over 99% in both KO and WT animals. ConclusionsThe implantation of commercial BHV leaflets significantly stimulates the KO mouse immune system resulting in massive production of anti-Gal antibodies and the exacerbation of the Gal-related calcific effect if compared with the WT mouse. The polyphenol-based treatment applied in this investigation showed an unexpected ability to inhibit the recognition of BHV xenoantigens by circulating antibodies completely preventing calcific depositions compared to the untreated counterpart.

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Naso, F., Colli, A., Zilla, P., Calafiore, A. M., Lotan, C., Padalino, M., Sturaro, G., Gandaglia, A., Spina, M.. 2023-02-22. In-vivo subdermal implant in the alpha-Gal knockout mice model for the assessment of bioprosthetic heart valve calcification rate before and after a polyphenols-based treatment. https://doi.org/10.1101/2023.02.21.529461

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