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=Volume 9 - 2022

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=Aims: Calcific aortic valve disease (CAVD) is a progressive heart disease which 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 of CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways. 
Methods and results: The klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in the AV calcification. The klotho knockout (Kl-/-) 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 Kl-/- mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl-/- mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in Kl-/- mice.  Histological and morphometric evaluation suggested significant reduction of AV calcification in Kl-/-;Tgfb1+/- mice compared to Kl-/- mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl-/- mice compared to the Kl-/-;Tgfb1+/- mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl-/-;Tgfb1+/- and Kl-/-;Smad3+/- mice compared to Kl-/- mice. Western blot analysis confirmed the inhibition of TGFβ canonical and non-canonical signaling pathways was associated with the rescue of AV calcification of both Kl-/-;Tgfb1+/- and Kl-/-;Smad3+/- mice. 
Conclusion: Overall, inhibition of TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl-/- mice. This information is useful in understanding the signaling mechanisms involved in CAVD.