Vascular calcification is an active extraosseous ossification progress regulated by multiple cell types and cytokines (11). It manifests excessive osteoblast synthesis and the mineralization of the calcified matrix (12). Abnormal mineral deposition gradually spreads throughout the blood vessels because of an imbalance between calcification inhibitors and promoters.

Vascular calcification classification provides researchers with different angles to study the mechanisms and effects of VC. Based on their volume and density, vascular calcified plaques are divided into microcalcification and macrocalcification (13). Microcalcification (<0.5 mm) and M1 macrophages are found in unstable plaques, whereas large calcified (>0.5 mm) plaques are accompanied by many M2 macrophages in stable plaques (13, 14). Microcalcification is primarily formed by degenerative inflammatory plaques, while large calcification formation is an active phenomenon similar to bone mineralization (13). In DM, the receptor for advanced glycation end (RAGE) and galectin-3 form calcified plaques of different sizes by regulating downstream signaling molecules, such as sortilin (15). Large-sized plaques with low calcium density are the most likely to induce acute cardiovascular events (16). In addition, osteocalcin is used as a biomarker of calcification stability to predict adverse events (17). As calcification is highly complex and genetic diversity in individual cells affects plaque structure, composition, and stability, the effects of calcification on plaque stability remain inconclusive.

The anatomical classification of VC is divided into intimal calcification, media calcification, valvular calcific aortic stenosis, and calciphylaxis (18, 19) (Table 1). Intimal calcification results from the combination of osteobiology and chronic inflammatory plaques (18, 20). FSS leads to intimal calcified plaque rupture because of the abnormal vascular wall resulting from fibrous atherosclerotic plaque and punctate calcifications (21). In the DM environment, macrophage galectin-3 is upregulated and promotes the migration of extracellular vesicles (EVs) derived from VSMCs to the intima, inducing vascular intimal calcification (22). Inflammation and lipid accumulation are not required for media calcification formation (18, 23). It was previously hypothesized that media calcification did not mediate thrombosis and vascular stenosis due to its special location (24). As a matter of fact, media calcification induces the loss of vascular elasticity and leads to heart failure, stagnation of blood, and thrombosis (24). Valvular calcific aortic stenosis is also an important category of the VC (25, 26). Currently, the only treatment for severe aortic valve calcification is aortic valve replacement. Calciphylaxis is a small artery calcification syndrome in end-stage kidney disease and is characterized by ischemic necrosis of the skin, subcutaneous adipose tissue, skeletal muscle, and other organs. As such, its prognosis is considerably poor; however, the exact pathogenesis remains unclear (26–29). Importantly, interdisciplinary clinical management can effectively improve the survival rate and life quality of patients with calciphylaxis (30).

Vascular calcification classification shows that VC morbidity and calcification patterns are distinct for different driving factors. The study of different VC models can identify specific mechanisms and therapeutic targets of different pathogenic factors.

In recent years, breakthroughs in VC research have been mainly related to similarities between calcification and ossification mechanisms, and its active calcification regulation. Currently, the understanding of VC pathogenesis includes chronic inflammation, endoplasmic reticulum stress (ERS), cell senescence, autophagy, apoptosis, and genetic factors (26) (Figure 1). Different aspects of calcification mechanism interact with each other, perpetuate vascular damage, and eventually mediate VC (7, 31).

Vascular calcification is closely associated with inflammatory conditions. The most common intimal calcification mechanism occurs in inflammatory plaques, with lipid deposition as the core (32). Activated NOD-like receptor protein 3 (NLRP3) promotes the release of pro-inflammatory cytokines such as interleukin 1β (IL-1β), which induces the receptor activator of NF-κB ligand (RANKL) secretion and causes a series of inflammatory reactions to induce osteogenic differentiation in vascular cells (33). Plasma inflammatory markers, C-reactive protein (CRP), and IL-1β have been proved to be important predictors of VC (34). In view of harmful and irreversible effect of VC, predicting and preventing VC are also important for the treatment strategy.

Endoplasmic reticulum stress leads to cell death, when protein processing exceeds endoplasmic reticulum folding, and ubiquitin/proteasomal mechanisms fail to scavenge abnormal proteins (35). Stellate ganglion block has been reported to prevent ERS activation and improve VC, potentially presenting new ideas for VC treatment (36). Fibroblast growth factor 21 (FGF21) inhibits the upregulated ERS marker GRP78 in the calcified aorta, prevents apoptosis via the activation of CHOP and caspase-12 pathways, and improves VC in rats (37). Inflammation and ERS have been considered a defense response, but when they play an excessive role for an extended time, homeostatic balance becomes disrupted, and blood vessels are remodeled (38). ERS can interact with inflammatory cytokines and trigger pathways related to VC; this is one of the pathological basis of VC (39).

Cell senescence accelerates the progression of age-related diseases such as AS and DM, promoting calcification via inflammatory cascade reactions (40). Senescent cells demonstrate impaired DNA damage repair, loss of VSMC contractile phenotypes, and increased osteogenic differentiation (41). Autophagy degrades and recycles damaged cytoplasmic molecules and misfolded proteins via lysosomes (42). Specific reactive oxygen species (ROS) concentrations enhance the autophagy of arterial wall cells and reduce the release of matrix vesicles (MVs), thus inhibiting VC (43). Apoptosis is activated in AS plaques; the apoptosome, produced by apoptotic VSMCs, is similar to MVs, the nucleation sites for the formation of calcium crystals. When apoptotic cell phagocytosis becomes dysfunctional, necrosis may occur and potentially induce inflammation. Secondary necrosis of apoptotic VSMCs releases IL-1α and IL-β molecules (44).

A particular relationship exists between VC and genetic factors. Pseudoxanthoma elastosis is an inherited disease characterized by ABCC6 gene mutations. Progressive calcification of elastic fibers is observed in the skin, eyes, and vascular system (45). Generalized arterial calcification of infancy, an autosomal recessive disease, shows severe calcification of the aorta and coronary arteries (46). Phenotypic switching is often controlled by multiple VC loci, such as chromosome 9p21.3, 6p21.3, and 10q21.3 loci (47). Therefore, gene-susceptible phenotypes are often centrally expressed in a family. Patients with a family history of coronary heart disease have an increased likelihood of developing coronary calcification (48). Healthy femoral arteries with normal histological morphology exhibit an increased chance of developing VC in the absence of risk factors, which may be linked to the enrichment of genes related to bone development (49). Epigenetics consists of DNA methylation, histone modification, chromatin remodeling, and non-coding RNA. Epigenetics also affects calcification phenotypes without altering DNA sequences (50). Previous studies have demonstrated that disordered arterial blood flow regulates the promoter of mechanically sensitive gene hypermethylation to induce VC (51). An increase in histone 3 and 4 acetylation is one of the mechanisms of aortic valve calcification. C646, a histone acetyltransferase inhibitor, effectively reduces aortic valve calcification in mice (52).

The diversity of VC mechanisms provides several perspectives for the formulation of treatment strategies; nonetheless, no surgical methods are currently available to fully alleviate VC.

The triad of mesenchymal cells, monocytes, and ECs control in situ and ectopic mineral metabolism (66). This review focuses on the role of ECs in VC. Osteocalcin expression in ECs is increased in the area where the highest mechanical stretching occurs, suggesting that ECs have the potential for osteogenic differentiation (67). In calcified arteries, co-expression of endothelial and osteogenic markers is observed by immunofluorescence staining (6). The main mechanisms allowing ECs to promote VC include EPC activation, oxidative stress, inflammation, autocrine and paracrine functions, mechanotransduction, hyperphosphatemia, and EndMT (Figure 2). Different mechanisms inducing endothelial dysfunction normally occur concurrently or act as concomitant triggers. ECs secrete gas signaling molecules and vasoactive substances to regulate inflammation, while inflammation also facilitates endothelial secretory function (68). Similar crosstalk also occurs between hyperphosphatemia and inflammation, mechanotransduction, and EndMT (69, 70). This indicates an intricate interaction network among those mechanisms and eventually leads to VC.

After a series of molecular events, ECs gradually lose EC lineage markers and acquire the characteristics of other cell lineages; this cellular reprograming is called EndMT (1, 109, 116, 117). The expression levels of newly formed endothelial markers VE-cadherin and CD31 decreased, whereas those of mesenchymal markers αSMA, N-cadherin, calmodulin, and FSP-1 increased. At the same time, the expression of several trigger factors, including Slug, Twist, and KLF4 in EndMT, can be monitored (1, 6, 118). Currently, no consensus has been reached on the definition of EndMT at the molecular level, impeding the establishment of a standardized model system and reducing the comparability and repeatability of experiments.

During embryonic development, EndMT occurs at the neural crest, heart valve, and neovascularization (119); in adults, the process is often associated with CVD characterized by vascular degeneration and fibrosis. During EndMT, EC polarity disappears; barrier functions decrease; migration and invasion increase; and proliferation and contraction are strengthened (1, 109). EndMT not only participates in normal tissue development; it also promotes disease progression and depends on timing and location (1, 119). Undoubtedly, EndMT mechanisms in the normal physiological state have particular relevance for pathological state mechanisms.

People have a clearer understanding of epithelial-to-mesenchymal transition (EMT), including reversible changes between EMT and mesenchymal-to-epithelial transition, than EndMT (116). As ECs comprise an epithelial cell subtype, applying well-known EMT mechanisms and targeted therapies to EndMT provides a good way to understand EndMT.

Endothelial-to-mesenchymal transition generates mesenchymal cells that can potentially differentiate into bone lineage and cartilage lineage cells, which provide an important cell source of VC (1, 119, 120). Evidence suggests that cadherin-11 in interstitial tissue prompts mesenchymal cells to differentiate into osteoblasts and chondrocytes, thus promoting VC (121). The level of EndMT in ECs exposed to Y-channel disorder blood flow is significantly increased, and calcium deposition is detected (122). Thus, EndMT is induced and regulated by different cytokines, transcription factors, and microenvironment changes, thus mediating the inhibition of endothelial genes, providing osteoblasts, and promoting VC (Figure 3).

TGF-β and BMP are important factors inducing EndMT (119). BMP2/4/6/7/promotes EndMT and induces EC calcification (119). BMP transmits osteogenic signals by binding to activin receptor-like kinase (ALK)1/2/3/6 and phosphorylating Smad1/5/8. Smad1/5/8 and Smad4 form a quaternary complex and translocate to the nucleus to regulate osteogenic gene expression (14, 123). BMPI receptor ACVR1 mutation is the main reason for FOP ectopic calcification (124). In an investigation of animal model- and patient-derived tissues, the pro-inflammatory cytokines TNF-α and IL-1β downregulated BMPR2 in aortic ECs, rendering ECs sensitive to osteogenic differentiation promoted by BMP9; furthermore, EndMT was promoted by reducing JNK signaling (123). BMPR2 integrates TGF-β/BMP and EC inflammatory signals, exerting protective effects on VC. BMPR2 deletion increases bone mass in experimental mice. Therefore, drugs enhancing BMPR2 expression should be developed to limit EndMT (123).

Inflammation decreases FGFR1 expression levels in ECs. FGF signal interruption increases TGFβR1 expression and initiates TGF-β cascade reaction and EndMT (109, 125). ECs undergoing EndMT express high levels of leukocyte adhesion molecules and become strong pro-inflammatory cells (126). Inflammation and EndMT form a positive feedback loop that destroys vascular homeostasis. The inhibition of the TGF-β signaling pathway on the endothelium induces VC regression in mice (109). With the complexity and importance of TGF-β signaling considered, this pathway is worth exploring for the treatment of VC.

Several factors are important regulators of TGF-β and BMP signaling. MGP is a calcification inhibitor that suppresses BMP2, the main regulator of Runx2/Cbfa1 expression and an effective osteogenic inducing factor (127). BMP4 stimulates EndMT by activating ALK2 (6). Increased BMP4 expression also induces MGP expression, and this negative feedback inhibits BMP2/4 osteogenesis (127). When MGP is deleted, EndMT is generated, widespread BMP signaling is promoted, and VC rapidly develops (119). In MGP transgenic mice, MGP overexpression inhibits BMP activation by reducing ALK1 and vascular endothelial growth factor (6, 128). Notably, the formation of AS lesions is decreased in MGP-deficient mice may be due to a sharp reduction in the expression of endothelial adhesion molecules, resulting in decreased inflammatory cell infiltration in the arterial wall (6).

In vitro studies report that the expression of SOX2 and endothelial markers in embryonic stem cells is time-consistent. By contrast, when small interfering RNA (siRNA) molecules are used to deplete SOX2 transcripts in ECs, EC differentiation decreases, suggesting that SOX2 is closely related to this process and participates in EndMT (119, 129). BMP induces SOX2 expression by enhancing Notch signaling, and RBPJκ deletion inhibits SOX2 induction (129). SOX2 amplifies the osteogenic effect of the BMP4 signaling pathway (129). β-Blockers are suggested to eliminate SOX2 induction in the arterial endothelium and inhibit EndMT to further limit VC; this process is related to a significant decrease in RBPJκ expression and its subsequent binding to the SOX2 promoter (129).

The key role of the activation of specific serine proteases mediated by SOX2 in EndMT initiation has been proposed (130). Serine proteases degrade the internal elastic plate, which exposes VSMCs to harmful stimulation and increases the expression of two osteogenic EC markers, namely, Cbfa1 and osterix (130). Serine protease may regulate the conversion between endothelial and mesenchymal cells by regulating SOX2 (130, 131). Thus, the considered regulation of serine proteases can help prevent VC. Twist1 and SOX2 regulate each other and together promote EndMT. NOTCH signaling, hypoxia, and FGF also regulate the EndMT process in the TGF signaling pathway (132).

The WNT pathway also participates in mesenchymal transition, with the involvement of ligands Fz, LRP, GSK-3, and β-catenin in pathway activation. Once activated, twist transcription factors promote EndMT (14). These ligands can thus be targeted to inhibit the WNT pathway.

Gene expression patterns in EndMT cells differ from those in EC and mesenchymal cells, suggesting that EndMT is not a complete cellular transformation. Therefore, the existence of the intermediate state renders VC regression possible by reversing EndMT (94). Based on lineage-tracing studies, ECs express mesenchymal genes during embryonic development, which cannot be transformed into myofibroblasts; however, during adult cardiac fibrosis, mesenchymal genes are not even expressed (106). Therefore, the specific role of EndMT and the relationship between EC and mesenchymal cells require further investigation.

In a mouse model of heart failure, QishenYiqi inhibits inflammatory reactions, activates the NO-cGMP-PKG pathway, significantly reduces elevated EC adhesion molecules and EndMT markers, and critically reverses EndMT (133). BRD4 is the “reader” of lysine acetylation; the BRD4 inhibitor JQ1 downregulates EndMT-related transcription factors, attenuates EndMT, and induces EC migration by TGF-β (134). The DNA hydroxymethylase TET2 reversed EndMT-induced shear stress to a certain extent. Lentivirus transfection studies have shown that TET2 overexpression upregulates the EC markers, VE-cadherin, and CD31; downregulates the interstitial cell markers, vimentin, and α-SMA; and reduces atherosclerotic plaque synchronously (135). Icariin reverses ox-LDL-induced EndMT by regulating the expression of the H19/miR-148b-3p/ELF5 axis (136). Although EndMT research remains limited to the laboratory, the development of material science strategies (see below) transforms this research at the molecular and genetic levels.

Statins prevent CVD and reduce the incidence of coronary events in patients with DM; these therapeutic effects arise from improved endothelial function. Statins inhibit the synthesis of isoprene-like intermediates blocking the downstream Rho signaling pathway (137). The Rho family affects specific functions related to the shape, movement, secretion, and proliferation of ECs; thus, statins exert multiple effects on blood vessels (137).

Statins improve endothelium-dependent vasodilation functions that primarily depend on the upregulation of EC-based KLF2 levels to enhance eNOS activity (138), increase NOS, reduce oxidative stress, and inhibit the effect of endothelin (ET-1) (64, 137). Simvastatin significantly increases eNOS expression and activity and restores NO production (139).

Statins also inhibit the release of inflammatory cytokines and reduce inflammation stimulation toward ECs. However, frequent statin use in DM patients accelerates coronary artery calcification (137, 140). Low statin concentrations inhibit calcification, whereas harmful doses of simvastatin and lovastatin increase BMP2 expression and increase ectopic mineral deposition (141).

Briefly, statins exert certain protective effects on ECs; their overall benefits cannot be discounted. Therefore, considered doses should be selected as indicated, with a view to optimizing endothelium-targeting effects and improving VC outcomes.

Diabetes is a risk factor for CVD. HG and insulin resistance environments generate persistent inflammation, oxidative stress, ECD, and mineral metabolism disorder; they also increase bone progenitor cell release into the circulation (98). Within this context, antidiabetic drugs could benefit ECD and VC.

Insulin is a widely used hypoglycemic drug. It stimulates the eNOS cofactor tetrahydrobiopterin, increases PI3K pathway activity, increases endothelium-derived NO, and improves endothelial function (142). However, insulin glargine has been reported to downregulate osteoprotegerin and promote VC in vitro (143). This finding is contrary to reports suggesting that hypoglycemic drug alleviates VC. Therefore, further research is needed.

Metformin promotes endothelial function as it improves hyperglycemia. The drug reduces osteogenic factor expression and inhibits VC induced by hyperphosphatemia via AMPK-RANKL signal transduction (102). Additionally, metformin increases eNOS activity and prevents VC via the AMPK-eNOS-NO pathway (144). It alleviates VSMC calcification induced by β-glycerophosphate, which may be related to autophagy via the AMPK/mTOR signaling pathway (145). Endothelial protection and VC alleviation, mediated by metformin, include several AMPK-mediated interactions. Therefore, AMPK can be a potentially viable therapeutic target.

In addition, several hypoglycemic drugs such as glucagon-like peptide 1 receptor agonists, sodium–glucose cotransporter 2 inhibitors, and dipeptidyl peptidase-4 inhibitors exhibit different endothelial and cardiovascular protective mechanisms (145). For instance, evogliptin significantly reduces calcium deposition and inhibits inflammatory cytokine in eNOS^–/– mice (146). Weight loss and diet regulation are also important hypoglycemic therapies as they reduce blood pressure damage, EC mechanical stress, and the spread of VC (14).

Inflammatory factors exert a damaging effect on ECs. With this finding considered, the development of major inflammatory factor (IL-6) inhibitors can alleviate VC (108). DMARDs reduce AS by inhibiting systemic inflammatory responses (108). Clinical trials have reported that Canakinumab significantly reduces IL-6 and CRP levels and inhibits inflammatory cells and EC adhesion; however, it increases the risk of infection and septicemia to a certain extent (147).

Aspirin, a non-steroidal anti-inflammatory drug (NSAID), improves improve endothelial function in AS and DM (148). However, in an AS multi-ethnic study of 6814 patients, baseline NSAID use exerts no significant protective effect on VC (149). Cyclooxygenase 2 (COX2) is an important target of anti-inflammatory drugs, but research suggests that the COX inhibitor celecoxib promotes aortic valve calcification (150). Contrary results also indicate that COX2 expression is increased in valvular calcification areas in klotho-deficient mice; in addition, feeding celecoxib to mice reduces calcification and blocks osteogenic gene expression (151).

Colchicine therapy inhibits endothelial inflammation via theNLRP3/CRP pathway. Studies have confirmed the beneficial effect of colchicine in AS (152). Soybean isoflavones generate antioxidant and anti-inflammatory effects and induce NO production, which contributes to the recovery of damaged endothelial function (153). Human umbilical vein ECs treated with genistein tend to accelerate autophagy and delay aging, which processes are related to the SIRT1/LKB1/AMPK pathway (154). Natural antioxidants exhibit considerable therapeutic effects, but the effectiveness of new antioxidants has yet to be evaluated in further clinical trials.

Some drugs can be used to regulate mineral metabolism. Calcium-based phosphate binders effectively control hyperphosphatemia, but their side effects induce hypercalcemia and promote VC, which should not be ignored (155).

The calcium-free phosphate binder Sveram significantly reduces different inflammation-related biomarkers (IL-6, IL-8, IL-10, CRP, TNF-α, and IFN-γ) and the mineral metabolism marker FGF-23; in addition, it reduces total cholesterol and increases HDL cholesterol. However, Sveram increases the endothelial markers implicated in cardiovascular events, including ICAM-1, VCAM-1, E-selectin, and L-selectin, leading to ECD (156, 157). Sveram has also been reported to increase flow-mediated vasodilation—that is, it enhances endothelial function (158). Some countries have approved the use of Sveram to delay VC progression in patients with CKD; however, Sveram does not significantly reduce mortality rates in dialysis patients (91).

Other phosphate binders such as lanthanum carbonate, nicotinamide, iron compounds, and bisphosphonates may clinically improve VC; however, their effectiveness and safety have yet to be confirmed (115).

In personalized and accurate medical care, genome-based discoveries must be translated into clinical practice. ECs have powerful and diverse functions and complex pathogenic signals; thus, a desirable therapeutic outcome via the control of a single gene is difficult to achieve.

In mice, 7C1 nanoparticles efficiently transported RNAi molecules into ECs to simultaneously silence five endothelial genes (Tie1, Tie2, VEcad, VEGFR-2, and ICAM2). The procedure demonstrated high tolerance, selectivity, and efficiency (159). siRNAs were encapsulated into 7C1 nanoparticles and intravenously injected into HCHF-fed ApoE^–/–mice. ECs absorbed the nanoparticles, which selectively inhibited TGF-β receptors. Finally, plaques and macrophages in diseased blood vessels were significantly decreased (160). Therefore, 7C1 nanoparticles can be used to study endothelial function and VC remission.