Alamandine suppresses vascular calcification through inhibition of ferroptosis.

Repression of the antiporter SLC7A11/glutathione/glutathione peroxidase 4 axis drives ferroptosis of vascular smooth muscle cells to facilitate vascular calcification

Vascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

Hyaluronan negatively regulates vascular calcification involving BMP2 signaling

Klotho/FGF23 axis mediates high phosphate‐induced vascular calcification in vascular smooth muscle cells via Wnt7b/β‐catenin pathway

Vascular calcification is very common in patients with chronic kidney disease and contributes to the increased risk of cardiovascular events. NAMPT (nicotinamide phosphoribosyltransferase), the rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide, has been shown to exert an antiaging effect on vascular smooth muscle cells. However, whether NAMPT is involved in the regulation of vascular calcification remains unclear. ELISA, immunofluorescence, and Western blot were used to detect NAMPT levels in human blood and tissues. Alizarin red staining, calcium content assay, and microcomputed tomography were used to investigate the role of NAMPT in vascular calcification. Gene expression analysis and coimmunoprecipitation were performed to elucidate the underlying mechanism. ELISA, immunofluorescence, and Western blot showed that NAMPT levels were increased in the blood of patients with chronic kidney disease and human calcified arterial tissues. Alizarin red staining and calcium content assay revealed that pharmacological inhibition or knockdown of NAMPT exacerbated vascular smooth muscle cell calcification, whereas overexpression of NAMPT reduced mineral deposition under osteogenic conditions. Similarly, ex vivo studies revealed that NAMPT inhibited calcification of rat and human arterial rings. Moreover, administration of NAMPT inhibitor FK866 promoted aortic calcification of chronic kidney disease rats, and smooth muscle cell-specific NAMPT knockout mice exhibited aggravated aortic calcification. Furthermore, pharmacological inhibition and knockdown of SIRT1 (sirtuin 1) abrogated the inhibitory effect of NAMPT on vascular calcification. In addition, smooth muscle cell-specific SIRT1 deficiency abrogated the protective effect of recombinant NAMPT on mouse aortic calcification. Coimmunoprecipitation and immunofluorescence assay further revealed that NAMPT inhibited the acetylation of NICD (Notch intracellular domain) and reduced the expression of HES1 (hairy and enhancer of split-1) in a SIRT1-dependent pathway. Our study unveils that NAMPT could serve as a novel endogenous inhibitor of vascular calcification via modulation of SIRT1-mediated deacetylation of NICD.

Vascular calcification is considered to be a killer of the cardiovascular system, involved inflammation and immunity. There is no approved therapeutic strategy for the prevention of vascular calcification. Sinomenine exhibited anti-inflammatory and immunosuppressive effects. Objective of this study was to investigate the effect of sinomenine in vascular calcification and its potential molecular mechanism. Adenine-induced uremic rats were constructed and administrated with sinomenine. Optical clearing of aortas, alizarin red staining, von Kossa staining, calcification quantification, micro-CT analyses of vascular calcification were performed to analyze calcification in aortas. Administration of 40 mg/kg/d sinomenine effectively alleviated vascular calcification in uremic rats. The miRNA sequencing revealed differentially expressed miRNAs in aortas and bioinformatic analysis assisted with miRNA screening. We screened 9 differential expressed miRNAs and their predicted target genes. By qRT-PCR, we validated that the expression of rno-miR-143-5p was corresponding to our prediction. Sinomenine inhibited vascular smooth muscle cells (VSMCs) calcification, accompanied with miR-143-5p upregulation. MiR-143-5p mimic decreased VSMCs calcification in high phosphate condition. On the contrary, miR-143-5p inhibitor increased VSMCs calcification in high phosphate condition, which was inhibited by sinomenine. In chronic kidney disease patients with vascular calcification, the expression level of circulating miR-143-5p was lower than those without vascular calcification. Sinomenine significantly inhibited vascular calcification in VSMCs and uremic rat. MiR-143-5p was one of the collection of miRNAs modified by sinomenine in vascular calcification. Reduction of miR-143-5p in VSMCs was not only a concomitant phenomenon in pro-calcification condition but also contribute to VSMCs calcification. Circulating miR-143-5p was supposed to be a potential biomarker for vascular calcification in chronic kidney disease patients. In conclusion, sinomenine effectively alleviated vascular calcification, which was attributed to miR-143-5p regulation partly.

MicroRNAs that target Ca2+ transporters are involved in vascular smooth muscle cell calcification

4-Octyl itaconate inhibits vascular calcification partially via modulation of HMOX-1 signaling

Vascular calcification is a notable risk factor for cardiovascular system. High phosphate can induce calcification in vascular smooth muscle cells (VSMCs), but the detail mechanism underlying this process remains unclear. In the present study, we determined the relationship between high phosphate and bone morphogenetic protein 9 (BMP9) in VSMCs, the effect of BMP9 on calcification in VSMCs and the effect of COX-2 on BMP9 induced calcification in VSMCs, as well as the possible mechanism underlying this biological process. We found that high phosphate obviously up-regulates the expression of BMP9 in VSMCs. Over-expression of BMP9 decreases the level of alpha-smooth muscle cell actin (α-SMA) apparently, but increases the level of Runx-2, Dlx-5, and ALP in VSMCs. Meanwhile, BMP9 increases the level of OPN and OCN, promotes mineralization in VSMCs and induces calcification in thoracic aorta. High phosphate and over-expression of BMP9 increases the level of COX-2. Over-expression of COX-2 enhances the inhibitory effect of BMP9 on α-SAM and increases the level of OPN and OCN induced by BMP9. However, inhibition of COX-2 decreases the BMP9-induced calcification in VSMCs and thoracic aorta. For mechanism, we found that high phosphate or BMP9 increases the level of β-catenin and p-GSK3β in VSMCs, but no substantial effect on GSK3β. However, COX-2 inhibitor decreases the expression of β-catenin induced by BMP9. Our findings indicated that BMP9 is involved in the phosphate-induced calcification in VSMCs and COX-2 partly mediates the BMP9-induced calcification in VSMCs through activating Wnt/β-catenin pathway.

Vascular calcification is a major risk factor for cardiovascular disease and accounts for a large proportion of deaths from cardiovascular disease in patients with chronic kidney disease. The high incidence, rapid progression and irreversibility of vascular smooth muscle cell (VSMC) calcification in patients has attracted attention. In the present study, the effect of intermedin1‑47 (IMD1‑47), an important isoform of intermedin, was investigated on the calcification of rat cardiovascular VSMCs induced by high phosphate (HP). To stimulate osteoblast‑like differentiation and calcification in rat VSMCs, 10mM β‑sodium glycerophosphate was used. The VSMCs were then treated with three doses of IMD1‑47 and the effects of IMD1‑47 on VSMC calcification, on the expression of osteogenic markers [osteoprotegerin, Runt‑related transcription factor2 (Runx2) and osteopontin (OPN)] and on alkaline phosphatase (ALP) activity were assessed. HP treatment significantly enhanced the cellular calcium content of VSMCs, the expression of osteogenic markers, and ALP activity, while IMD1‑47 significantly reversed these effects in a dose‑dependent manner. The protein expression levels of Wnt1, Wnt3a and active β‑catenin were determined and it was found that IMD1‑47 significantly inhibited their expression. Following β‑catenin silencing, the protein expression levels Runx2 and OPN were increased compared with the IMD1‑47 treatment alone, indicating a role for the Wnt/β‑catenin pathway in the effects of IMD1‑47 on osteogenic markers. The present study suggested that IMD1‑47 inhibited HP‑induced VSMC calcification by regulating the Wnt/β‑catenin signaling pathway.

Vascular calcification is an important risk factor related to all-cause mortality of cardiovascular events in patients with chronic kidney disease (CKD). Vascular extracellular matrix (ECM) proteins have been demonstrated to regulate vascular calcification. ECM protein thrombospondin 1 (THBS1/TSP-1) plays a critical role in the regulation of vascular diseases. However, whether THBS1 is involved in vascular calcification in CKD patients remains unclear. In this study, RNA sequencing datasets from the Gene Expression Omnibus (GEO) database GSE146638 showed that THBS1 was upregulated in the aortas of CKD rats. Enzyme-linked immunosorbent assay (elisa) revealed that serum THBS1 levels were increased in CKD patients with thoracic calcification. Western blotting and immunofluorescence analysis showed that THBS1 expression was increased in calcified vascular smooth muscle cells (VSMCs) and arteries. THBS1 knockdown exacerbated rat VSMC calcification induced by high phosphorus and calcium, as shown by Alizarin red staining and calcium content assays. Conversely, THBS1 overexpression attenuated VSMC calcification and abdominal aortic calcification in rats with CKD. Moreover, addition of recombinant THBS1 protein inhibited calcification of VSMCS and human arterial rings. Smooth muscle cell-specific knockout of THBS1 mice treated with vitamin D3 displayed aggravated aortic calcification. Mechanistically, the protein-protein interaction database STRING (http://string-db.org/) analysis and coimmunoprecipitation assays revealed THBS1 bound to integrin β3. Reduction of integrin β3 levels abrogated the protective effect of THBS1 on vascular calcification. RNA-seq analysis revealed that THBS1 overexpression modulated the nuclear factor-kappa B (NF-κB) signaling pathway. Of note, the inhibitory effect of THBS1 overexpression on the NF-κB signal was abolished by knockdown of integrin β3. In conclusion, THBS1 interacts with integrin β3 to inhibit vascular calcification through suppression of NF-κB signal, suggesting a promising therapeutic target for vascular calcification in CKD. © 2025 The Pathological Society of Great Britain and Ireland.

Vascular calcification is a common pathologic condition in patients with chronic kidney disease (CKD) and aging individuals. It has been established that vascular calcification is a gene‐regulated biological process resembling osteogenesis involving osteogenic differentiation. However, there is no efficient treatment available for vascular calcification so far. The natural polyamine spermidine has been demonstrated to increase life span and protect against cardiovascular disease. It is unclear whether spermidine supplementation inhibits vascular calcification in CKD. Alizarin red staining and quantification of calcium content showed that spermidine treatment markedly reduced mineral deposition in both rat and human vascular smooth muscle cells (VSMCs) under osteogenic conditions. Additionally, western blot analysis revealed that spermidine treatment inhibited osteogenic differentiation of rat and human VSMCs. Moreover, spermidine treatment remarkably attenuated calcification of rat and human arterial rings ex vivo and aortic calcification in rats with CKD. Furthermore, treatment with spermidine induced the upregulation of Sirtuin 1 (SIRT1) in VSMCs and resulted in the downregulation of endoplasmic reticulum (ER) stress signaling components, such as activating transcription factor 4 (ATF4) and CCAAT/enhancer‐binding protein homologous protein (CHOP). Both pharmacological inhibition of SIRT1 by SIRT1 inhibitor EX527 and knockdown of SIRT1 by siRNA markedly blocked the inhibitory effect of spermidine on VSMC calcification. Consistently, EX527 abrogated the inhibitory effect of spermidine on aortic calcification in CKD rats. We for the first time demonstrate that spermidine alleviates vascular calcification in CKD by upregulating SIRT1 and inhibiting ER stress, and this may develop a promising therapeutic treatment to ameliorate vascular calcification in CKD.

Ectopic calcification of the vasculature comes with age and is also prevalent in patients with chronic kidney disease (CKD). Klotho, a protein highly expressed in the kidney, has been identified as a novel anti-aging factor,[1][1],[2][2] and Klotho-deficient animals develop medial vascular

Vascular calcification (VC) is a common pathophysiological process of chronic kidney disease (CKD). Sirtuin 3 (Sirt3), a major NAD+-dependent protein deacetylase predominantly in mitochondria, is involved in the pathogenesis of VC. We previously reported that intermedin (IMD) could protect against VC. In this study, we investigated whether IMD attenuates VC by Sirt3-mediated inhibition of mitochondrial oxidative stress. A rat VC with CKD model was induced by the 5/6 nephrectomy plus vitamin D3. Vascular smooth muscle cell (VSMC) calcification was induced by CaCl2 and β-glycerophosphate. IMD1-53 treatment attenuated VC in vitro and in vivo, rescued the depressed mitochondrial membrane potential (MMP) level and decreased mitochondrial ROS levels in calcified VSMCs. IMD1-53 treatment recovered the reduced protein level of Sirt3 in calcified rat aortas and VSMCs. Inhibition of VSMC calcification by IMD1-53 disappeared when the cells were Sirt3 absent or pretreated with the Sirt3 inhibitor 3-TYP. Furthermore, 3-TYP pretreatment blocked IMD1-53-mediated restoration of the MMP level and inhibition of mitochondrial oxidative stress in calcified VSMCs. The attenuation of VSMC calcification by IMD1-53 through upregulation of Sirt3 might be achieved through activation of the IMD receptor and post-receptor signaling pathway AMPK, as indicated by pretreatment with an IMD receptor antagonist or AMPK inhibitor blocking the inhibition of VSMC calcification and upregulation of Sirt3 by IMD1-53. AMPK inhibitor treatment reversed the effects of IMD1-53 on restoring the MMP level and inhibiting mitochondrial oxidative stress in calcified VSMCs. In conclusion, IMD attenuates VC by improving mitochondrial function and inhibiting mitochondrial oxidative stress through upregulating Sirt3.

Vascular calcification (VC) is highly associated with increased morbidity and mortality in patients with advanced chronic kidney disease. Paracrine/autocrine factors such as vasoactive peptides are involved in VC development. Here, we investigated the expression of the novel peptide C-type natriuretic peptide (CNP) in the vasculature, tested its ability to prevent VC in vivo and in vitro, and examined the mechanism involved. Rat aortic VC was induced by vitamin D3 plus nicotine (VDN). CNP (500ng/kg/h) was administered by mini-osmotic pump. Calcification was examined by von Kossa staining; CNP and cyclic guanosine monophosphate (cGMP) contents were detected by radioimmunoassay, and mRNA and protein levels were examined by real-time PCR and Western blot analysis in aortas and calcified vascular smooth muscle cells (VSMCs). VDN-treated rat aortas showed higher CNP content and decreased expression of its receptor natriuretic peptide receptor B, along with increased vascular calcium deposition and alkaline phosphatase (ALP) activity. Low CNP levels were accompanied by increased vascular calcium deposition and ALP activity in VDN-treated rats when compared to vehicle treatment, which was further confirmed in cultured VSMCs. Administration of CNP greatly reduced VC in VDN-treated aortas compared with controls, which was confirmed in calcified VSMCs. The decrease in alpha-actin expression was ameliorated by CNP in vitro. Moreover, protein expression levels of osteopontin (OPN) were significantly up-regulated in calcified aortas, and CNP increased OPN expression in calcified aortas. Furthermore, CNP downregulated OPN and bone morphogenic protein 2 (BMP-2) expression in calcified aortas and VSMCs. Modulation of OPN and BMP-2 expression by CNP and the beneficial effects of CNP on calcified VSMCs were blocked significantly by protein kinase G inhibitor H7. Impaired local endogenous CNP and its receptor system may be associated with increased mineralization in vivo in rat aortas with VC, and administration of CNP inhibits VC development in vivo and in vitro, at least in part, via a cGMP/PKG pathway.