BMPs are members of the transforming growth factor beta (TGF-β) superfamily and have been reported to play a causal role in osteogenesis and VC (32). BMP-2 has been widely studied in vivo and in vitro for its procalcification properties. The main BMP-2 signaling axis involved in osteogenic differentiation and VC is the BMP-2/Smad1/Runx2 axis, in which BMP-2 binds to the corresponding receptor to activate the intracellular BMP effector proteins Smad1/5/8. The activated Smad complex then enters the nucleus to positively regulate Runx2, the transcription factor controlling bone formation (33). In addition, BMP-2 can activate the Wnt/β-catenin pathway, thereby inducing VC-associated processes in VSMCs (34). Multiple experiments have shown that the expression of BMP2 is upregulated in the setting of VC. When BMP2 was used to stimulate human coronary artery smooth muscle cells, the expression of Runx2 was upregulated within 24 h and intracellular calcium deposition was significantly increased (35). Treatment with a BMP antagonist (LDN-193189 or ALK3-Fc20) to inhibit BMP signaling attenuated VC in low density lipoprotein receptor-deficient (LDLR^-/-) mice (36). A report indicating that atherosclerotic intimal calcification is significantly accelerated in BMP-2-transgenic mice with high expression of BMP-2 provided further support for a role of BMP in VC (37). Runx2 is a transcription factor involved in osteoblast differentiation and bone formation. The expression level of Runx2 is low in normal blood vessels but high in calcified blood vessels (38). Runx2 can induce VSMC-mediated calcification in vitro and plays a key role in oxidative stress-induced VSMC-mediated calcification (39). Decreased expression of Runx2 was found to strongly inhibit intimal VC in atherosclerosis-susceptible mice and medial VC in mice with CKD, further supporting the critical role of Smooth muscle cell-specific Runx2 in the development of VC (Fig. 1) (40,41).

OPG can directly inhibit the differentiation and maturation of osteoclasts and induce the differentiation of osteoblasts, which are key inhibitors of calcification (42). OPG has been shown to be highly expressed in blood vessels, but its expression is low in calcified blood vessels in mice. OPG knockout mice were found to develop early and severe VC (43) and treatment of atherogenic mice with OPG resulted in a reduced calcified lesion area (44). However, the results of numerous clinical studies are opposite those reported in animal studies. For example, a meta-analysis involving 26,442 patients in the general population revealed that an elevated OPG level is associated with an increased risk of cardiovascular disease (45). In addition, Morena et al (46) reported that high expression of OPG is closely associated with coronary artery calcification.

OPN is a phosphoglycoprotein adhesion molecule found primarily in mineral-rich teeth and bone and is found to alleviate VC by inhibiting the formation and growth of mineral crystals (47). OPN expression is increased in calcified plaques, but OPN is not expressed in normal arteries (48). OPN deficiency does not cause calcification in wild-type mice but increases VC in mice deficient in the matrix protein Gla, suggesting that OPN plays an inducible inhibitory role in VC (Fig. 1) (49).

The Wnt/β-catenin pathway plays a crucial role in VC (28). When Wnt ligands bind to the frizzled (FZD)/low-density lipoprotein-related receptor (LRP) coreceptor complex, the canonical Wnt signaling pathway is activated. Dishevelled interacts with the intracellular tail of FZD and the integrated AXIN1/glycogen synthase kinase 3 (GSK3)/Adenomatous polyposis coli degradation complex binds to the phosphorylated tail of LRP, resulting in dissociation of the degradation complex. After phosphorylated β-catenin saturates the degradation complex, newly synthesized β-catenin translocates to the nucleus after attaining a certain concentration in the cytoplasm. β-Catenin subsequently forms a complex with T cytokine/Lymphoid enhancer factor (TCF/LEF), which activates downstream calcification genes such as Runx2. When the Wnt pathway is inactive, β-catenin is phosphorylated by the degradation complex and subsequently undergoes ubiquitination and proteasomal degradation (Fig. 2A and B) (50). In addition, previous cellular and animal studies have shown that the activation of AKT signaling plays an important role in upregulating Runx2 and promoting VC (39,51,52). Byon et al (39) reported that AKT phosphorylation can upregulate Runx2 expression and promote H₂O₂-induced calcification of VSMCs. Moreover, Heath et al (51) reported that AKT O-linked N-acetylglucosaminylation (O-GlcNAcylation) at two newly identified sites-threonine (T) 430 and T479-promoted the phosphorylation of AKT, thereby inducing the VMSC-mediated calcification. Deng et al (52) confirmed that phosphorylation of AKT promotes the loss of FOXO1/3 in the nucleus, inhibits Runx2 ubiquitination and promotes VSMC-mediated calcification (Fig. 3).

Apoptosis is another common factor that affects VC. Reports have indicated that initial calcium phosphate precipitation could be associated with apoptosis via the release of phosphorous via intracellular metabolism and calcification-promoting membrane phospholipid-rich microparticles (53,54). Further studies revealed that after cell death, cells can release free DNA to precipitate calcium phosphate crystals on blood vessel walls, leading to VC (55,56). In addition, in vitro experiments have suggested that calcification can be blocked by inhibiting apoptosis and that apoptosis induction can increase the incidence of calcification by 10-fold (53).

Mitochondria are important bioenergetic powerhouses and biosynthetic centers in the cell and various stimuli, such as hyperphosphataemia, hyperglycaemia and increased mitochondrial outer membrane permeability (57), can induce mitochondrial dysfunction. Mitochondrial dysfunction results in reduced production of adenosine triphosphate (ATP), abnormal production of active oxides, abnormal regulation of apoptosis and changes in the autophagic ability of cells, all of which are involved in the occurrence of VC (58). Previous studies have shown that inhibiting mitochondrial fission with melatonin can abrogate β-GP-induced VSMC-mediated calcification (59,60).

Ubiquitination is a process by which target proteins are degraded through the ubiquitin-proteasome pathway via a process regulated by a cascade comprising an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin ligase that binds to specific target proteins (61,62). Ubiquitination is widely involved in physiological processes such as DNA damage repair, transcriptional regulation, cell cycle progression, apoptosis and vesicle transport by regulating the stability, localization, activity and interactions of proteins (63,64).

The osteogenic transcription factor Runx2 plays a key role in regulating VC. Studies in osteocytes have shown that the degradation and stability of the Runx2 protein are regulated via the ubiquitin-proteasome pathway. The molecular mechanisms that promote Runx2 protein degradation involve Smad ubiquitination regulatory factor 1 (Smurf1) and cyclin D1. Smurf1 binds to Runx2 and mediates the conjugation of ubiquitin to Runx2, leading to its degradation (65,66). Cyclin D1 induces the C-terminal phosphorylation of Runx2 through cyclin D1/Cdk4 and promotes its proteasome-dependent degradation (67). Deng et al (52) demonstrated in vitro and in vivo that the phosphatase and tensin homologue (PTEN)/AKT/forkhead box O1/3 (FOXO1/3) signaling axes regulate VC by regulating Runx2 ubiquitination. Specifically, PTEN deficiency activates AKT, phosphorylates FOXO1/3, inhibits Runx2 ubiquitination, upregulates Runx2 and promotes VC without affecting the expression of common regulators of Runx2 ubiquitination, such as Smurf1 and cyclin-D1. Knockdown of FOXO1/3 in VSMCs mimics the aforementioned effects of PTEN deficiency, including the inhibition of Runx2 ubiquitination, the upregulation of Runx2 and the promotion of VC. In addition, a recent study revealed that kynurenine, a major product of indoleamine 2,3-dioxygenase 1 (IDO1)-mediated tryptophan metabolism, promoted Runx2 ubiquitination and delayed the progression of intimal calcification in transgenic apolipoprotein E-/- mice. Moreover, ubiquitination-mediated proteasomal degradation of Runx2 was found to be associated with the kynurenine-mediated aryl hydrocarbon receptor-dependent nongenomic pathway (68). Therefore, the molecular mechanisms regulating Runx2 ubiquitination in VSMCs may be novel and worthy of further exploration.

In cell models, the neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) E3 ubiquitin ligase was found to negatively regulate VC induced by a high-phosphorus environment through the ubiquitination of Smad1 and the development of VC is induced by the specific inhibition of Nedd4(69). Paradoxically, another study showed that Murine double minute 2 (MDM2) can promote VC through the ubiquitination of histone deacetylase 1 (HDAC1) via its E3 ligase activity. In both in vitro and in vivo models of VC, the HDAC1 protein level is significantly reduced, a phenomenon related to the ubiquitination of HDAC1 at lysine (K) 73. Coimmunoprecipitation assays revealed that the E3 ubiquitin ligase MDM2 induces the polyubiquitination of HDAC1. The overexpression of MDM2 significantly increases VC in a dose-dependent manner, whereas the knockdown of MDM2 inhibits VC (70). In addition, that study confirms that blocking proteasomal degradation by treatment with various ubiquitination inhibitors ameliorates VC in both in vitro and in vivo models (70).

The nuclear factor-κB (NF-κB) family plays important roles in inflammation and atherosclerosis. In unstimulated cells, NF-kB is present in the cytoplasm and binds to inhibitor of κB (IκB). In response to various stimuli, including inflammation, specific kinases, such as IκB-kinase β (IκKβ), mediate the phosphorylation of IκB. This leads to its ubiquitination and degradation, which are followed by the translocation of NF-κB to the nucleus and the activation of transcription (71). Knockdown of IκKβ in VSMCs reduces the ubiquitination of β-catenin, upregulates β-catenin and Runx2 signaling and accelerates calcification. By contrast, persistent activation of IκKβ inhibits calcification by increasing the ubiquitination of β-catenin (72). Dysregulation of the autophagy-lysosome pathway in VSMCs mediates VC induced by a high-phosphorus environment and this pathway has become a new target for VC therapy. Transcription factor EB (TFEB) is considered the master regulator of lysosomal biogenesis. A high-phosphorus environment can result in the degradation of TFEB through the ubiquitin-proteasome system and subsequently promote VC in vitro and in vivo (73).

The results of studies on the effects of ubiquitination on VC are inconsistent. Moreover, the related mechanisms are complex and need further study. Identifying the ubiquitination sites in calcification-related factors and targeting these sites is expected to lead to approaches for suppressing VC (Fig. 4).

Protein acetylation is a reversible modification that is catalyzed by acetyltransferases (N-terminal acetyltransferases/lysine acetyltransferases, NATs/KATs), which transfer acetyl groups from acetyl-CoA to amino acid residues in proteins such as histones and transcription factors (74,75). The reverse process, deacetylation, is catalyzed by lysine deacetylases. Histone acetylation is the most common type of acetylation. The main histone acetyltransferases are the members of the P300/CBP, Gcn5-related N-acetyltransferase, Steroid receptor coactivator and MYST (MOZ-YBF2/SAS3-SAS2-TIP60) families. There are four subfamilies of histone deacetylases (HDACs): Classes I, II and IV, which are Zn²⁺ dependent and class III (for example, SIRTs), which are NAD+ dependent. In addition, acetyltransferases catalyze the acetylation of nonhistone proteins, such as transcription factors and regulate transcription factor stability and DNA binding (31). In some cases, ubiquitination competes with acetylation for the same lysine residues (75).

The levels of histone 3 and 4 (H3 and H4) acetylation are elevated in the aortic valve calcification model. However, in this model, treatment with an inhibitor of the histone acetyltransferase P300, C646, was found to attenuate calcification in vivo and in vitro by significantly decreasing the acetylation levels of H3 and H4 (76,77). Studies have shown that β-catenin and Runx2 are also targets of P300-mediated acetylation and that acetylation of β-catenin by P300 can enhance the activation of β-catenin signaling (78,79). The acetylation of Runx2 can increase its stability and its osteogenic transcriptional activity, as well as osteoblast differentiation (80,81). By contrast, HDACs can directly bind to Runx2 and act as core inhibitors of its transcriptional activity. HDACs catalyze the deacetylation of Runx2, mediate its ubiquitination and degradation and inhibit osteoblast differentiation (80,82). HDAC inhibitors have been shown to promote Runx2 acetylation, thereby promoting osteoblast differentiation in vitro and osteogenesis in vivo (80,83). However, no evidence indicates that the aforementioned acetylation events are directly related to VC.

The class III HDAC sirtuin 1 (SIRT1) is an important factor in the regulation of VC, with three main related functions. i) SIRT1 can regulate Wnt/β-catenin signaling. Under physiological conditions, SIRT1 binds to p300 and the acetylation and nuclear translocation of β-catenin are reduced, thereby inhibiting osteogenic activity and VC. However, in a high-glucose environment, the low abundance of SIRT1 cannot prevent P300-mediated acetylation of β-catenin, thus promoting VC (84). Similarly, hyperphosphataemia can downregulate SIRT1 expression (85), allow constitutive acetylation of downstream proteins and accelerate calcification by promoting hyperacetylation of β-catenin and Runx2 via P300 (80,86). ii) In a high-glucose environment, the decreased expression of SIRT1 results in an increase in Runx2 expression and the promotion of VC via a direct increase in the acetylation level of the Runx2 promoter (87). iii) Other studies have shown that high mobility group box 1 (HMGB1), an activator of BMP-2 and protein kinase RNA-like ER kinase (PERK) are targets of SIRT1 for deacetylation. SIRT1 can inhibit the inflammatory response through the deacetylation of HMGB1, thereby inhibiting VC (88,89). Moreover, in both in vitro and in vivo models, SIRT1 was found to ameliorate endoplasmic reticulum (ER) stress-induced VC by deacetylating K889 in PERK (90). In addition to SIRT1, SIRT3 also plays an important role in VC. Sun et al (91) report that SIRT3 inhibits VC by regulating Nε-carboxymethyl-lysine (CML).

SIRT1 and SIRT3 play important roles in VC. Identifying their targets will help us to understand the mechanism of VC and develop protective agents against VC that will benefit the majority of patients (Fig. 5).

Carbamylation is a stable PTM in which a carbamoyl moiety (-CONH₂) binds to a functional group in a proteins, peptide and or free amino acid (92). Carbamylation is common in patients with CKD and can occur nonenzymatically in the presence of high concentrations of urea. Medial VC is common in patients with CKD with increased protein carbamylation (93). Mori et al (94) established a VC model of CKD in vitro and in vivo and confirmed that protein carbamylation aggravated VC. Specifically, carbamylation of ATP synthase aggravated VC in human vascular smooth muscle cells (hVSMCs) by impairing mitochondrial function and inhibiting the expression of ectonucleotide pyrophosphate/phosphodiesterase 1 (ENPP1). ENPP1 catalyzes the production of pyrophosphate, which is a potent inhibitor of ectopic calcification. Proteins sensitive to carbamylation were identified via proteomic mass spectrometry and various mitochondrial and cytoskeletal proteins were found to be carbamylated in hVSMCs exposed to uremic conditions. In vivo studies further confirmed the presence of evident protein carbamylation and mitochondrial dysfunction in the aortas of heminephrectomized rats fed a urea-rich diet. In addition, carbamylation of ATP synthase is found in the tunica media of patients with end-stage renal disease, supporting the existence of this mechanism in human VC.

Alesutan et al (95) reported that uromodulin can inhibit VC by inhibiting cytokine-dependent procalcification signaling. However, carbamylation of uromodulin interferes with its ability to bind to proinflammatory cytokines, resulting in the loss of its anticalcification properties in vitro. Carbamylated uromodulin can be detected in the serum of dialysis patients and the levels of proinflammatory cytokines that interact with uromodulin are reduced by uromodulin carbamylation, suggesting that the function of the cytokine trap is impaired (95).

Sortilin is an intracellular sorting receptor that has been identified as a cardiovascular risk factor in humans (96). Compared with control individuals with normal renal function, patients with CKD have increased serum levels of sortilin and harbor carbamylated sortilin in the circulation (93), localized to calcified areas. By generating hVSMCs and establishing an isolated rat aortic ring model of calcification, Jankowski et al (97) found that carbamylated sortilin can promote VC. Further studies revealed that sortilin carbamylation promotes VC mediated by hVSMCs, possibly due to an increase in their binding affinity for leukocytes. Similarly, sortilin carbamylation was found to be associated with the volume and progression of coronary artery calcification in patients with CKD (97,98).

Carbamylation may play a role in VC by affecting mitochondrial function (94) or the inflammatory response (95). The aforementioned studies provide valuable new insights into the pathogenesis of VC and the findings have implications for other complications associated with elevated concentrations of circulating uremic toxins (Fig. 6A).

Glycosylation is one of the most important PTMs in vivo and can occur on 50-70% of proteins in cells (99). There are four major types of glycosylation: O-glycosylation, N-glycosylation, C-glycosylation and glycosylphosphatidylinositol anchoring (99).

O-GlcNAcylation, in which a single GlcNAc monosaccharide is transferred to serine and threonine residues in the target intracellular protein, is the most commonly studied type of O-glycosylation. This modification is catalyzed by O-GlcNAc transferase (OGT) and studies have reported that abnormal OGT activity can lead to cardiovascular complications (100). For instance, Xu et al (101) reported that OGT expression is significantly increased in both high-phosphorus diet-induced 5/6 nephrectomized rats and VSMC calcification models and that OGT knockdown inhibits VC induced by a high-phosphorus environment. Further study of the mechanism by which OGT promotes VC in CKD revealed that OGT overexpression increases the glycosylation of Kelch-like ECH-associated protein 1 (Keap1), which leads to the degradation of nuclear factor erythroid 2-related factor 2 and inhibits VSMC autophagy, in turn promoting VC in vitro and in vivo. Xu et al (102) also report that OGT promotes the glycosylation of Yes-associated protein (YAP) to increase its stability and upregulates the expression of YAP to inhibit autophagy, thus accelerating VC induced by a high-phosphorus environment. Therefore, OGT knockdown is expected to inhibit VC in CKD by reducing the glycosylation of target proteins and activating autophagy. In addition, Heath et al (51) report that activating the O-GlcNAcylation of AKT increases Runx2 activity and promotes the development of VC in diabetic mice.

The relationship between N-glycosylation and VC is another research hotspot. Insulin-like growth factor-I (IGF-I) has been identified as a major inhibitor of VC. Siddals et al (103) used statins to deplete the substrates required for N-glycosylation and used tunicamycin to inhibit N-glycosylation. They found that statins and tunicamycin disrupt the inhibitory effect of IGF-I on β-glycerophosphate-induced VC by altering the glycosylation of the IGF receptor (IGFR). TGF-β is a multifunctional cytokine that has been shown to regulate VC and the differentiation of VSMCs in vivo (104,105). Studies have shown that TGF-β1 is a target of N-glycosylation. Sha et al (106) reported that tunicamycin inhibits TGF-β1 secretion and leads to an increase in the level of the cell-associated non-glycosylated form of TGF-β1. In addition, the TGF-β receptor TGF-βR is regulated by N-glycosylation. The removal of glycosylation from TGF-βR affects its interaction with its ligand, resulting in failure to activate downstream signaling pathways (107). Although a few reports have addressed TGF-β or TGF-β receptor N-glycosylation, no relevant research has been conducted on the direct relationship between TGF-β or TGF-β receptor N-glycosylation and VC. Notably, blocking core fucosylation, a specific form of N-glycosylation, suppresses VC-associated processes in VSMCs. Inhibition of TGFβR fucosylation significantly dysregulates downstream TGFβ/Smad2/3 signaling (108).

Accumulated studies have shown that advanced glycation end products (AGEs), which are predictors of cardiovascular disease mortality, may play an important role in VC. Previous studies have shown that the local and circulating levels of AGEs in patients with diabetic nephropathy and nondialysis CKD are significantly elevated (109,110). Moreover, recent clinical studies show that the measured levels of AGEs in the skin of 122 patients with type 2 diabetes and in the radial arteries of 54 patients with CKD are positively correlated with the degree of arterial calcification (111,112). In addition, several animal studies show that the knockdown of the receptor for AGEs (RAGE) has therapeutic benefits in inhibiting the development of VC (113-115). B4GALNT3-mediated LacdiNAc (LDN) glycosylation of sclerostin may be a bone-specific osteoporosis target (116), suggesting that glycosylated sclerostin may have potential research value in VC.

In general, glycosylation contributes to the development of VC. Predicting glycosylation events and sites through machine learning models (117) and identifying inhibitors of glycosylation may lead to the development of a therapeutic strategy to target VC in patients with CKD and diabetes (Fig. 6B).