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  • The present study determined the effect

    2023-11-30

    The present study determined the effect of DCA on VSMC calcification in vitro and in atherosclerotic ApoE-/- mice in vivo. We found that Carbetocin receptor non-toxic concentrations of DCA, did not affect VSMC viability, induced calcification of VSMC in culture and increased atherosclerotic vascular calcification in atherosclerosis in mice. Furthermore, DCA induced AKT-independent activation of p38 MAPK, which directly associated with Runx2 and induced Runx2 transactivity to promote VSMC calcification. These results offer new insights into the signaling mechanisms underlying DCA-induced vascular calcification; and provide opportunities to identify new therapeutic targets for preventing calcification.
    Materials and methods
    Results
    Discussion DCA has been shown to inhibit AKT activation and induce oxidative stress that lead to apoptosis of cancer cells. The potent effects of DCA on inhibiting activation of AKT, a key signaling pathway that promotes VSMC calcification, prompted us to evaluate the function of DCA in regulating vascular calcification. Unexpectedly, we found that DCA did not inhibit but induced VSMC calcification in vitro and in atherosclerosis mice in vivo. Similar to previous observations in other cells, we found that DCA also induced oxidative stress in VSMC (Supplemental Figure IA). Inhibition oxidative stress attenuated DCA-induced VSMC calcification (Supplemental Figure IB), supporting that DCA-activated oxidative stress contributes to DCA-induced VSMC calcification. As activation of AKT is required for oxidative stress-induced VSMC calcification [5] and activated AKT is sufficient to induce VSMC calcification [9], [10], one would speculate that inhibition of AKT by DCA may block calcification. Unexpectedly, while activation of AKT was inhibited by DCA, vascular calcification of VSMC was still markedly increased. These data suggest that oxidative stress-activated signals, but not AKT, play a major role in DCA-induced vascular calcification. Among many signaling pathways, we identified a novel function of DCA-induced activation of p38 MAPK signaling pathway in mediating DCA-induced Runx2 upregulation and calcification of VSMC, which is independent of AKT signaling pathway. Activation of p38 MAPK is a downstream signaling pathway that has shown to be induced by oxidative stress in many cells, including cancer cells, cardiac cells and neurons [36], [37]. Different from our previous observation with hydrogen peroxide-induced oxidative stress that induces AKT activation and VSMC calcification [5], DCA-induced oxidative stress was associated with AKT inhibition while activation of p38 MAPK signaling. The mechanisms underlying the differential activation of AKT and p38 MAPK signaling pathways by oxidative stress awaits further investigation. It is likely that the nature of oxidative stress, its microenvironment, and immediate targeting partners may be different, thereby inducing distinct signaling pathways, such as AKT or p38 MAPK, that lead to complex and dynamic outputs. Our studies have revealed a unique AKT-independent interplay of p38 MAPK and Runx2 signaling axis in promoting osteogenic differentiation and calcification of VSMC. Activation of p38 MAPK signaling has been shown to promote bone cell differentiation and bone formation [38], our observation of the role of p38 MAPK activation in DCA-induced VSMC osteogenic differentiation has further supported an important function of p38 MAPK activation in promoting osteogenesis. Importantly, our studies have uncovered a previous unknown link between p38 MAPK and Runx2 in VSMC, an interaction of p38 MAPK with Runx2. Although the expression of Runx2 and p38 MAPK was not co-dependent, we found the expression of both p38 MAPK and Runx2 were indispensable for DCA-induced VSMC calcification. Our findings of increased binding of Runx2 to DCA-induced phosphorylated p38 MAPK and that p38 MAPK knockdown attenuated DCA-induced Runx2 transactivity support that DCA-induced activation of p38 MAPK contributes to increased Runx2 transactivity, thus promoting VSMC calcification. A previous study showed that MAPK is capable of phosphorylating Runx2 [39]. Therefore, it is conceivable that binding of DCA-activated phosphorylated p38 MAPK to Runx2 may contribute to increased Runx2 transactivity and VSMC calcification. Accordingly, the DCA-induced p38 MAPK/Runx2 signaling axis may bypass the activation of AKT seen in hydrogen peroxide-induced VSMC calcification, which would explain why reduced activation of AKT by DCA did not interfere with the development of vascular calcification.