Atypical Cadherin FAT1 Protects Against Phosphate-Induced Calcification in Vascular Smooth Muscle Cells

Background: The Vascular smooth muscle cell (VSMC) is the key cell type in the pathogenesis of vascular calcification, a common problem that contributes to mortality in patients with CKD. The Atypical cadherin FAT1, a transmembrane protein, regulates proliferation and migration of VSMCs, but whether FAT1 affects vascular calcification is unexplored. We hypothesized that FAT1 limits phosphate-induced calcification in VSMCs. Methods: First, we assessed calcification using von Kossa staining in brachial artery biopsies from patients with ESKD who underwent arterio-venous access surgery and compared FAT1 expression between calcified (n=4) and non-calcified arteries (n=3) using immunofluorescence. Then, we treated mouse aortic ring ex vivo with high phosphate (3mM and 3.5mM) and evaluated calcification and FAT1 expression. Lastly, we compared phosphate-induced calcification in FAT1 knockout (KO) mouse VSMCs vs. controls. FAT1KO VSMCs were harvested from the aorta of FAT1loxP/loxPTagln-cre mice. VSMCs in culture were exposed to normal phosphate (0.9 mM) and high phosphate (3 mM and 3.5 mM) and calcification was quantified using Alizarin Red staining. Results: FAT1 expression was lower in calcified brachial arteries from ESKD patients compared to non-calcified controls (p=0.04; fig. a). In ex-vivo mouse aortic rings, treatment with high phosphate induced calcification (p=0.03) and resulted in lower FAT1 expression compared to those treated with normal phosphate, with a statistically notable trend (p for trend =0.07; fig. b). Western Blot confirmed the loss of FAT1 in FAT1KO mouse VSMCs (fig. c); and compared to controls, FAT1KO mouse VSMCs had higher phosphate-induced calcification with p=0.03 and p=0.007 for 3mM and 3.5 mM phosphate, respectively (fig. d). Conclusion: Our findings support the clinical relevance of FAT1 in vascular calcification associated with CKD and suggest a protective role of FAT1 against phosphate-induced vascular calcification. Our future work will involve validating these results in vivo and studying underlying mechanisms. Funding: Other NIH Support - NHLBI