AUTHOR=Chakrabarti Mrinmay , Bhattacharya Aniket , Gebere Mengistu G. , Johnson John , Ayub Zeeshan A. , Chatzistamou Ioulia , Vyavahare Narendra R. , Azhar Mohamad 

TITLE=Increased TGFβ1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification

JOURNAL=Frontiers in Cardiovascular Medicine

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/cardiovascular-medicine/articles/10.3389/fcvm.2022.770065

DOI=10.3389/fcvm.2022.770065

ISSN=2297-055X

ABSTRACT=<sec><title>Aims</title><p>Calcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways.</p></sec><sec><title>Methods and Results</title><p>The <italic>klotho</italic> gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The <italic>klotho</italic> knockout (<italic>Kl</italic><sup>–/–</sup>) mice have shorter life span (8–12 weeks) and develop severe AV calcification. Here, we showed that increased TGFβ1 and TGFβ-dependent SMAD3 signaling were associated with AV calcification in <italic>Kl</italic><sup>–/–</sup> mice. Next, we generated <italic>Tgfb1</italic>- and <italic>Smad3</italic>-haploinsufficient <italic>Kl</italic><sup>–/–</sup> mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in <italic>Kl</italic><sup>–/–</sup> mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in <italic>Kl</italic><sup>–/–</sup>; <italic>Tgfb1</italic><sup>±</sup> mice compared to <italic>Kl</italic><sup>–/–</sup> mice. <italic>Smad3</italic> heterozygous deletion was observed to be more potent in reducing AV calcification in <italic>Kl</italic><sup>–/–</sup> mice compared to the <italic>Kl</italic><sup>–/–</sup>; <italic>Tgfb1</italic><sup>±</sup> mice. We observed significant inhibition of <italic>Tgfb1</italic>, <italic>Pai1</italic>, <italic>Bmp2, Alk2</italic>, <italic>Spp1</italic>, and <italic>Runx2</italic> mRNA expression in <italic>Kl</italic><sup>–/–</sup>; <italic>Tgfb1</italic><sup>±</sup> and <italic>Kl</italic><sup>–/–</sup>; <italic>Smad3</italic><sup>±</sup> mice compared to <italic>Kl</italic><sup>–/–</sup> mice. Western blot analysis confirmed that the inhibition of TGFβ canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both <italic>Kl</italic><sup>–/–</sup>; <italic>Tgfb1</italic><sup>±</sup> and <italic>Kl</italic><sup>–/–</sup>; <italic>Smad3</italic><sup>±</sup> mice.</p></sec><sec><title>Conclusion</title><p>Overall, inhibition of the TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in <italic>Kl</italic><sup>–/–</sup> mice. This information is useful in understanding the signaling mechanisms involved in CAVD.</p></sec>