Dongxing Zhu

BACKGROUND: FTO (fat mass and obesity-associated) protein, a key N6-methyladenosine demethylase, plays a critical role in cardiovascular disease. However, its function in vascular calcification remains poorly understood. METHODS: High phosphate (Pi) was used to induce calcification in vascular smooth muscle cells (VSMCs) and aortic rings, and high-dose vitamin D ₃ (vD ₃ ) was used to induce vascular calcification in mice. FTO expression was modulated using adenovirus, adeno-associated virus, and siRNA. To identify FTO targets, we performed integrated MeRIP sequencing and RNA sequencing analyses. RESULTS: FTO expression was significantly downregulated in calcified VSMCs, mouse arteries, and human arteries relative to their noncalcified counterparts. Silencing of Fto increased high Pi–induced osteogenic gene expression and calcification in VSMCs. Conversely, overexpression of Fto resulted in the opposite effect. These results were confirmed in high Pi–induced rat aortic ring calcification and a vD ₃ -induced mouse model of vascular calcification through treatment with the FTO inhibitor F,B-2,3. Intriguingly, the ALP (alkaline phosphatase) inhibitor TNAP-IN-1 or levamisole hydrochloride partially reduced FTO inhibition-induced increases in VSMC calcification, rat aortic ring calcification, and vascular calcification in mice. Adeno-associated virus–mediated VSMC-specific overexpression of Fto significantly attenuated vascular calcification in mice with overloaded vD ₃ . Integrated MeRIP sequencing and RNA sequencing identified that Pcsk9 is a potential target of FTO. Knockdown of FTO increased N6-methyladenosine methylation and mRNA stability of Pcsk9 , and its secretion by VSMCs. In addition, knockdown of Fto enhanced the activation of NF-κB (nuclear factor-κB) signaling and increased the expression of inflammatory cytokines, including IL-6 , Icam1 , and Vcam1 , which were attenuated by concomitant knockdown of Pcsk9 . Finally, knockdown of Pcsk9 or p65 abolished silencing of the Fto -induced increase in osteogenic transition and calcification in VSMCs. CONCLUSIONS: Our results suggest that FTO may inhibit vascular calcification at least in part through Pcsk9 -mediated modulation of NF-κB signaling and altered ALP activity. FTO may therefore be an attractive target for the treatment of vascular calcification.

Aims: Calcific aortic valve disease (CAVD) is a common heart valve disease with significant clinical consequences. The mechanisms that drive the pathogenesis of CAVD remain to be fully elucidated. N6-methyladenosine (m6A), the most prevalent RNA epigenetic regulator, has recently been implicated in cardiovascular disease, but its role in CAVD has yet to be investigated. In this study, we investigated the potential function of m6A modification in CAVD. Methods and results: Using clinical samples from CAVD patients in combination with human valve interstitial cell (hVIC) calcification model, we screened the expression of m6A modulators and discovered that alkB homolog 5, RNA demethylase (ALKBH5), a key m6A demethylase, was significantly down-regulated in calcified hVICs and human aortic valves. Consistently, increased m6A levels were seen in calcified hVICs, and treatment with 3-deazaadenosine (DAA), an inhibitor of m6A modification, significantly reduced hVIC osteogenic differentiation and calcification. In addition, we showed that silencing of ALKBH5 expression increased global m6A levels and accelerated hVIC osteogenic differentiation and calcification, whereas overexpression of ALKBH5 resulted in the opposite effect. We demonstrated that ALKBH5 directly modulate m6A levels of TGFBR2 and its mRNA stability, leading to altered TGFBR2 expression and SMAD2 signalling in hVICs. We further showed that inhibition of TGFBR2 or knockdown of SMAD2 attenuated ALKBH5 knockdown-induced hVIC osteogenic differentiation and calcification. The expression of the m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) was up-regulated during the process of hVIC calcification. Intriguingly, we revealed that the ALKBH5 silencing-induced increased hVIC osteogenic differentiation and calcification were abolished after knockdown of YTHDF1. These data suggest a potential role YTHDF1 in aortic valve calcification. Conclusion: This study showed that ALKBH5 attenuated aortic valve calcification through the TGFBR2/SMAD2 signalling pathway via direct m6A modification of TGFBR2.

A link between increased glycolysis and vascular calcification has recently been reported, but it remains unclear how increased glycolysis contributes to vascular calcification. We therefore investigated the role of PFKFB3, a critical enzyme of glycolysis, in vascular calcification. We found that PFKFB3 expression was upregulated in calcified mouse VSMCs and arteries. We showed that expression of miR‐26a‐5p and miR‐26b‐5p in calcified mouse arteries was significantly decreased, and a negative correlation between Pfkfb3 mRNA expression and miR‐26a‐5p or miR‐26b‐5p was seen in these samples. Overexpression of miR‐26a/b‐5p significantly inhibited PFKFB3 expression in VSMCs. Intriguingly, pharmacological inhibition of PFKFB3 using PFK15 or knockdown of PFKFB3 ameliorated vascular calcification in vD ₃ ‐overloaded mice in vivo or attenuated high phosphate (Pi)‐induced VSMC calcification in vitro. Consistently, knockdown of PFKFB3 significantly reduced glycolysis and osteogenic transdifferentiation of VSMCs, whereas overexpression of PFKFB3 in VSMCs induced the opposite effects. RNA‐seq analysis and subsequent experiments revealed that silencing of PFKFB3 inhibited FoxO3 expression in VSMCs. Silencing of FoxO3 phenocopied the effects of PFKFB3 depletion on Ocn and Opg expression but not Alpl in VSMCs. Pyruvate or lactate supplementation, the product of glycolysis, reversed the PFKFB3 depletion‐mediated effects on ALP activity and OPG protein expression in VSMCs. Our results reveal that blockade of PFKFB3‐mediated glycolysis inhibits vascular calcification in vitro and in vivo. Mechanistically, we show that FoxO3 and lactate production are involved in PFKFB3‐driven osteogenic transdifferentiation of VSMCs. PFKFB3 may be a promising therapeutic target for the treatment of vascular calcification.

Tanshinone IIa is a key ingredient extracted from the traditional Chinese medicine Salvia miltiorrhiza (Danshen), and is widely used to treat various cardiovascular diseases. Vascular calcification is a common pathological change of cardiovascular tissues in patients with chronic kidney disease, diabetes, hypertension and atherosclerosis. However, whether Tanshinone IIa inhibits vascular calcification and the underlying mechanisms remain largely unknown. This study aims to investigate whether Tanshinone IIa can inhibit vascular calcification using high phosphate-induced vascular smooth muscle cell and aortic ring calcification model, and high dose vitamin D₃ (vD₃)-induced mouse models of vascular calcification. Alizarin red staining and calcium quantitative assay showed that Tanshinone IIa significantly inhibited high phosphate-induced vascular smooth muscle cell and aortic ring calcification. qPCR and Western blot showed that Tanshinone IIa attenuated the osteogenic transition of vascular smooth muscle cells. In addition, Tanshinone IIa also significantly inhibited high dose vD₃-induced mouse aortic calcification and aortic osteogenic transition. Mechanistically, Tanshinone IIa inhibited the activation of NF-κB and β-catenin signaling in normal vascular smooth muscle cells. Similar to Tanshinone IIa, inhibition of NF-κB and β-catenin signaling using the chemical inhibitors SC75741 and LF3 attenuated high phosphate-induced vascular smooth muscle cell calcification. These results suggest that Tanshinone IIa attenuates vascular calcification at least in part through inhibition of NF-κB and β-catenin signaling, and Tanshinone IIa may be a potential drug for the treatment of vascular calcification.

Sex differences in cardiovascular disease (CVD), including aortic stenosis, atherosclerosis and cardiovascular calcification, are well documented. High levels of testosterone, the primary male sex hormone, are associated with increased risk of cardiovascular calcification, whilst estrogen, the primary female sex hormone, is considered cardioprotective. Current understanding of sexual dimorphism in cardiovascular calcification is still very limited. This review assesses the evidence that the actions of sex hormones influence the development of cardiovascular calcification. We address the current question of whether sex hormones could play a role in the sexual dimorphism seen in cardiovascular calcification, by discussing potential mechanisms of actions of sex hormones and evidence in pre-clinical research. More advanced investigations and understanding of sex hormones in calcification could provide a better translational outcome for those suffering with cardiovascular calcification.