The vascular endothelium plays a critical role in regulating homeostatic network of the cardiovascular system [31]. Endothelial cell dysfunction (ECD), which marks the initial alteration in the trajectory of atherosclerotic lesion progression, is typified by the activation of endothelial cells (ECs) and their shift from an anti-inflammatory to a pro-inflammatory state [32, 33]. Recent studies suggest that the hemodynamic change is the primary cause of ECD [34].

Under undisturbed laminar flow, ECs exhibit an up-regulation of Kruppel-like factor 2 (KLF2), a transcription factor integral to maintain vascular equilibrium. This upsurge in KLF2 expression precipitates an increase in endothelial nitric oxide synthase (eNOS) gene expression, catalyzing the synthesis of nitric oxide (NO). NO, a small lipid soluble molecule, traverses cell membranes to influence a wide array of cell functions within the bloodstream and the vascular endothelium [35–37]. NO’s effects include the inhibition of platelet activation, adhesion, and aggregation [38], a reduction in leukocyte adhesion to the endothelium [39], a facilitation of vasorelaxation through dephosphorylation of myosin light chain in vascular smooth muscle cells [40], and an enhancement of the oxygen delivery capacity of red blood cells [41].

Steady laminar flow promotes the alignment of ECs [34] and inhibits signal transduction by pro-inflammatory stimuli such as TNF and interleukin-1 (IL-1) [42]. This hemodynamic condition diminishes IL-6 induced progression of cell cycle in ECs [43], and protects against ECs apoptosis, ensuring the endothelial integrity [44].

Blood flow irregularities trigger ECs activation, leading to a consequential decrease in eNOS gene expression and the stimulation of nuclear factor kappa B (NF-κB) signaling pathway [45, 46]. NF-κB plays a pivotal role in the initiation of pro-inflammatory responses within the endothelium. These responses include up-regulation of ECs surface adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1); the release of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and Fractalkine; and the production of pro-thrombotic mediators including tissue factor (TF), von Willebrand factor (vWF), and plasminogen activator inhibitor-1 (PAI-1), in both their soluble and membrane-bound forms [47–49].

Activated ECs cause an increased production of reactive oxygen species (ROS) [50]. The resulting oxidative stress, in addition to prolonged inflammation, disrupts adherent junctions, such as VE-cadherin and gap junctions, primarily due to the reduction in NO levels [51]. This disruption leads to an increased accumulation of sub-endothelial atherogenic apolipoprotein B (ApoB)-containing lipoproteins, including LDL, VLDL and chylomicrons [52, 53]. Importantly, the increased ROS facilitates oxidative modification of ApoB-containing lipoproteins [54], which act as dual-function agents in the immune response: serving as antigens that initiate the adaptive immune response and as adjuvant molecules that stimulate the innate immune system [55–57].

Lifestyle modifications and pharmacological interventions can improve the blood microenvironment by lowering lipid levels, restoring EC’s function. If ECs function is not restored, the lipid deposition within the vessel wall will persist. The retention of oxidized low-density lipoprotein (oxLDL) in the intima of blood vessels acts as a constant source of inflammatory stimulus, continuously activating ECs and triggering inflammatory responses [58, 59]. Therefore, it is crucial to remove oxLDL to suppress the pathogenesis of atherosclerosis.

Macrophages are the major immune cells that respond to inflammation triggered by oxLDL, playing a pivotal role in the formation and progression of atherosclerotic lesions [55, 57]. Macrophages mainly originate from myeloid progenitor cells in the bone marrow. Myeloid progenitor cells develop into circulating monocytes, which can infiltrate into atherosclerotic lesions from the bloodstream or from the spleen that acts as a reservoir for monocytes in mice [60]. The recruitment of monocytes to atherosclerotic lesions is mediated by the activation of ECs.

Activated ECs initiate monocyte recruitment by enabling their initial rolling on the endothelium through P-selectin engagement, followed by the strengthening of monocyte adhesion via interactions with immunoglobulin-G family proteins, VCAM-1 and ICAM-1. Subsequent monocyte infiltration into the subendothelial layer is driven by chemokines such as MCP-1 and IL-8 [35, 61, 62]. Once penetrating the intima of blood vessels, monocytes encounter a pro-inflammatory milieu constructed by activated ECs and oxidized lipid particles. This environment fosters the transformation of monocytes into pro-inflammatory macrophages, actively up-taking lipids [63, 64].

Macrophages play a crucial role in regulating plasma lipoprotein content and metabolism [65]. Under normal conditions, macrophages recognize native LDL via the LDL receptor (LDLR). The LDL is then endocytosed and transported to lysosomes, where the cholesteryl ester (CE) is hydrolyzed to free cholesterol (FC) by acid lipase [66, 67]. The FC is then transferred to the endoplasmic reticulum (ER) to be esterified by acyl CoA:cholesterol acyltransferase 1 (ACAT1). The CE produced by ACAT1 is stored in cytoplasm as lipid droplets, undergoing a continual cycle of hydrolysis to FC by neutral cholesterol esterases and re-esterification by ACAT1 [68, 69].

Neutral cholesteryl ester hydrolase 1 (NCEH1) processes CE, releasing FC that is transported out of the cell through ATP-binding cassette (ABC) transporters, including ABCA1 and ABCG1 and scavenger receptor class B1 (SR-BI) [70]. Apolipoprotein A-1 (ApoA-1) acts as a receptor for cholesterol transported by ABCA1, while high-density lipoprotein (HDL) accepts cholesterol transferred by ABCG1 and SR-BI. This machinery is tightly regulated under normal conditions to maintain cholesterol homeostasis. An increase in FC in an ER regulatory pool triggers a signaling cascade that down-regulates the LDL receptor, preventing foam cell formation in hypercholesterolemia. Thus, these proteins ensure an effective control of LDL and cholesterol content in peripheral blood under normal conditions [71–73].

In atherosclerosis, the process of macrophage-dependent cholesterol handling is disrupted. In addition to an increase in the production of oxLDL, macrophages are stimulated by multiple inflammatory factors and express various scavenger receptors, including SR-A1, CD36, and lectin-like oxLDL receptor-1 (LOX-1), which all have an affinity for oxLDL [74, 75], leading to an excessive uptake of oxLDL transforming macrophages to foam cells [76]. Simultaneously, the activity of ACAT is elevated, resulting in an overproduction of CE that accumulate in the endoplasmic reticulum. Furthermore, the expression of NCEH, ABCA1, and ABCG1 is decreased in atherosclerosis, further exacerbating intracellular cholesterol accumulation and foam cells formation [77, 78].

In addition to monocyte-derived macrophages, vascular smooth muscle cells (VSMCs) also significantly contribute to the foam cell population [79]. Clinical studies found that over 50% of foam cells may be derived from VSMCs in human atherosclerotic lesions [80]. Intracellular cholesterol accumulation inhibits VSMC gene expression (including α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SMMHC), and smooth muscle 22α (SM22α) and induces the expression of pro-inflammatory and macrophage markers [81]. VSMCs uptake oxidized LDL mainly through LDLR family and SR family [75, 82, 83]. Compared to macrophages, VSMCs are inefficient at lysosomal processing and cholesterol trafficking, with a much low expression of ABCA1 [84], contributing to an impaired cholesterol efflux [85]. In addition, ECs and dendritic cells have also been reported to participate in the formation of foam cells [86].

In short, lipids infiltrated in the vascular intima are engulfed by macrophages, smooth muscle cells, and other cells, and eliminated through lipid metabolism pathways. However, in the pathogenesis of atherosclerosis, the balance between lipid engulfment and elimination by the effector cells is disrupted. The excessive engulfment of oxLDL leads to lipid accumulation in these cells transforming these cells to foam cells. This progression pushes the atherosclerosis lesion towards a more severe and uncontrollable direction.

During the process of intravascular adipose streaks formation or pathological intimal thickening, inflammatory ECs and macrophages attract more monocytes to infiltrate the intima by secreting chemokines such as CCR2, CCR5, CXCR1, and CXCR2 [87, 88]. In the early stage of vascular intimal lesions, monocyte infiltration and aggregation are the main driving factors, while in later stages, macrophage proliferation becomes more important [89]. Inhibition of macrophage proliferation has been shown to reduce the size of plaque [90].

Within the inflammatory milieu, macrophages and other immune cells further release inflammatory factors, such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF) isoforms, matrix metalloproteinases (MMPs), fibroblast growth factor (FGF), and heparin-binding epidermal growth factor (HB-EGF), activating VSMCs located in the arterial wall media [91, 92]. Activated VSMCs gain the ability to proliferate, migrate, and secrete various extracellular matrix (ECM) proteins, and are attracted to the lesion to form a fibrous cap that stabilize lipid plaques [93].

The formation of the fibrous cap makes the atherosclerotic lesion less reversible. The persistence of inflammation leads to the accumulation of apoptotic cells that cannot be cleared by macrophages in a timely manner, causing secondary necrosis. This in turn further promotes inflammation, oxidative stress, and death of adjacent cells, forming a necrotic core [94–96].

The growth of the necrotic core causes a thinning of the fibrous cap. The accumulation of inflammatory cytokines and oxidative products in the vascular lesion provoke uncontrolled accumulation of adjacent VSMCs and, consequently, decreased synthesis of ECMs [96–98]. The components of the ECMs are degraded by macrophage-derived MMPs [99, 100], elastase, and tissue protease [101]. Under this condition, the production of TGF-β is reduced, leading to a decrease in collagen production in healthy VSMCs [102, 103]. In combination, these factors accelerate the thinning of the fibrous cap.

Core necrosis and fibrous cap thinning transform stable fibrous plaques to vulnerable plaques. Plaque rupture abruptly exposes the plaque interior to circulating pro-coagulant factors and platelets, leading to thrombosis [104, 105]. Atherosclerosis-associated clinical events are mainly attributed to thrombus detachment, causing acute vascular occlusion in major organs, leading to myocardial infarction, pulmonary embolism, and stroke.

The involvement of Cu in angiogenesis has been known for more than 40 years [112, 113]. In 1980s, studies using rabbits demonstrated that Cu and Cu-binding proteins significantly induced angiogenesis in the cornea [113]. It was further found that CuSO₄ alone stimulated the expression of VEGF in human keratinocytes in a dose-dependent manner [114]. Following these observations, a series of studies revealed that Cu is instrumental in regulating various EC functions, including proliferation, migration, and tube formation [115–117]. Cu-binding proteins play a critical role in VSMCs migration [118–120] and blood vessel maturation [115, 121]. Mechanistic understanding of the role of Cu in angiogenesis during last two decades revealed that Cu promotion of angiogenesis acts through its regulation of hypoxia-inducible factor-1 (HIF-1) in multiple cell types [122, 123].

The processes of Cu trafficking between intracellular organelles are depicted in Figure 3, including the indications of Cu regulation of HIF-1 transcriptional activity for angiogenetic gene expression. The initial evidence of Cu interaction with HIF-1 was reported by Jiang et al., who explored how dietary Cu supplementation mitigates pressure overload-induced cardiac hypertrophy in mice [124]. Sustained cardiac pressure overload leads to reduced myocardial Cu and VEGF levels, and diminished angiogenesis. Cu replenishment increases VEGF and promotes angiogenesis in the hypertrophic hearts, leading to regression of cardiac hypertrophy. Further studies found that in cultured human cardiomyocytes, Cu chelation blocks insulin-like growth factor (IGF)-1- or Cu-stimulated VEGF expression, which is relieved by addition of excess Cu. Both IGF-1 and Cu activate HIF-1α. Consequently, HIF-1α gene silencing blocks IGF-1- or Cu-stimulated VEGF expression. In addition, HIF-1α coimmunoprecipitates with a Cu chaperone for superoxide dismutase-1 (CCS-1), and gene silencing of CCS-1 prevents IGF-1- or Cu-induced HIF-1α activation and VEGF expression [124]. Thus, Cu promotion of angiogenesis is mediated by HIF-1α activation of angiogenic gene expression with the aid of CCS-1.

Cu regulates HIF-1 transactivation of angiogenic gene expression in multiple mechanisms of action. Under hypoxic conditions, Cu enters the nucleus in both CCS-1-dependent and -independent processes [125]. In the cytoplasm, Cu stabilizes HIF-1α, the rate-limiting component of HIF-1, leading to its accumulation and promoting its entrance to the nucleus [126–128]. In the nucleus, Cu inhibits the activity of asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) and ensures the formation of HIF-1 transcriptional complex [129]. Importantly, Cu selectively regulates the binding of HIF-1 to the HRE elements of target angiogenic genes by affecting the interaction between the transcription factor and the promoter region of the angiogenic genes [130, 131].

Cu selectively regulates the process of HIF-1 transactivation of angiogenic gene expression. It is important to note that not all of the HIF-1 regulated genes require Cu for expression [130, 132]. This was first demonstrated by an in vitro study in which the treatment of HUVECs with a Cu chelator, tetraethylenepentamine (TEPA), suppressed the expression of a group of HIF-1 target genes such as BNIP3 and VEGF, but did not affect other HIF-1 target genes such as IGF-2 [132]. This Cu selective regulation of the expression of HIF-1-controlled genes was further defined in studies of monkey model of HIF-1 regulation of angiogenesis in ischemic myocardium [133]. During the acute phase of ischemic injury, angiogenesis was activated in the injured heart along with an increase in angiogenic factors. In the chronic phase of myocardial ischemia, sustained accumulation of HIF-1α was observed. However, the accumulation of HIF-1α was not accompanied by the expression of HIF-1-controlled angiogenic factors, including VEGF, tyrosine-protein kinase receptor Tie-2, angiopoietin-1 (Ang-1), and FGF-1 in the ischemic myocardium [133]. On the other hand, an up-regulation of HIF-1-controlled non-angiogenic gene expression such as IGF-2 was associated with HIF-1α accumulation [133].

This paradoxical phenomenon, HIF-1α accumulation being accompanied by suppression of HIF-1 target angiogenic gene expression, is now recognized to be ascribed to the reduced Cu concentrations in the ischemic heart [133]. In response to ischemic insult, Cu content in the heart is significantly decreased, along with a significant increase in serum Cu concentrations [134–136]. A recent study using ChIP-sequencing and RNA-sequencing identified 218 Cu-dependent and 10 Cu-independent HIF-1 target genes across the genome under hypoxic conditions [131]. Cu efflux from the heart under hypoxic conditions leads to suppressed expression of Cu-dependent HIF-1 target genes, but does not change the expression of Cu-independent HIF-1 target genes.

The mechanism by which Cu selectively regulates the binding site of the HIF-1 target genes was recently revealed by a study in HUVECs [130]. In this study, Cu deprivation by TEPA completely suppressed the binding of HIF-1α to HRE site of BNIP3 along with a complete inhibition of BNIP3 mRNA expression, but the binding of HIF-1α to the HRE site of IGF-2 or the expression of IGF-2 mRNA was not affected under hypoxic conditions. Furthermore, de novo motif analysis of all 218 Cu-dependent and 10 Cu-independent HIF-1 target genes further revealed that the core bases “GGAA” and “TTCC,” previously identified as the core motifs for E26-transformation-specific (ETS) family [130] constitute the critical motifs for the binding sites of Cu-dependent genes, while there is no specific motif found in Cu-independent genes except the motif for HIF-1α [130]. The difference in the binding loci and pattern between all the Cu-dependent and Cu-independent HIF-1 target genes indicate that Cu, by selectively affecting the binding of HIF-1α to the critical motifs in the promoter and putative enhancer regions of HIF-1-regulated genes, selectively regulates the expression of HIF-1-controlled angiogenic genes.

The regulatory action of Cu on ECs, as depicted in Figure 4, promotes angiogenesis. Cu at physiologically relevant levels stimulated vessel tube formation from HUVECs cultured in Matrigel [115], indicating the involvement of ECs proliferation, migration and integration in this process. ECs migration is a critical process in the vessel tube formation. Cu stimulation of ECs migration was confirmed using HUVECs for wound healing and transwell migration assays [115]. In the process of Cu stimulation of ECs migration, a Cu transporter-1 (CTR-1) is critically involved. Gene silencing by siRNA targeting CTR-1 in HUVECs significantly suppressed Cu entrance to the cells along with an inhibition of ECs migration [115].

Cu promotes ECs proliferation that is essential for the maturation of new blood vessels. Recent studies showed that Cu stimulates ECs proliferation in a concentration-dependent fashion within the effective concentration range [115–117]. NO generated by eNOS plays a critical role in regulating ECs proliferation, angiogenesis and vascular homeostasis [117]. Cu stimulation of ECs proliferation is eNOS-dependent [117]. Li et al found that Cu increased the expression of eNOS in HUVECs and that siRNA targeting eNOS blocked Cu stimulation of ECs proliferation [117].

Exposure of ECs to excessive Cu stimulates the expression of pro-inflammatory cytokines in the cells, leading to endothelial dysfunction [35, 137]. Excessive Cu accumulation in the blood was found in the pathogenesis of atherosclerosis. It is important to note that under the condition of atherosclerosis, Cu is deficient in the atherosclerotic lesion walls, but Cu concentrations are increased in the circulation [138–142]. The endothelial dysfunction is closely related to the disturbance in Cu homeostasis between the vascular wall and circulation, being the critical event in the initiation and progression of atherosclerosis [32, 33, 143].

Serum Cu elevation is closely associated with hyperhomocysteinemia [144]. Experimental and clinical studies over the last decade have shown that the elevation of blood homocysteine (Hcy) levels is linked to increased risk of atherosclerosis [145, 146]. Patients with homocystinuria were associated with high plasma Cu concentrations [144, 147, 148]. A correlation between plasma concentrations of total Cu and Hcy was identified [149, 150]. Addition of small amounts of Cu significantly enhanced the inhibitory effect of Hcy on ECs function, thus suppressing angiogenesis in isolated endothelial tissues in culture [151]. Cu and Hcy complexes have been identified in vitro and their exposure to cultured ECs elicited remarkable changes in relation to atherogenic activities [152–155].

There is increasing evidence that indicates a strong correlation between Cu homeostasis and lipid metabolism. Systemic Cu alterations appear to be inversely correlated to the level of lipids and lipid-transporting lipoproteins in the peripheral circulation [156, 157]. It was observed that Cu-deficient diet feeding induced Cu deficiency in organ systems in rats along with increased levels of circulating HDL and LDL, and an increase in total cholesterol, triglyceride and phospholipid levels [158, 159]. The total volume of HDL components in the blood was elevated in Cu deficient Sprague-Dawley rats, accompanied by a corresponding increase in the cholesterol and protein content of the HDL and LDL fractions [159, 160]. In Cu deficient rats, the triglyceride content of circulating LDL and VLDL was increased, as was the ApoE content of HDL [159, 160], however, the concentration of liver cholesterol was reduced [161]. Systemic Cu deficiency is associated with an increased production of HDL and an increased turnover of HDL cholesterol esters [159].

The polarity of macrophages affects the role that they play in the regulation of lipid metabolism [162]. Cu in its Cu²⁺ ionic form was found to play an important role in the regulation of macrophage polarization [163]. The concentrations of Cu²⁺ lower than 10 μM promoted the expression of M2 related genes in macrophages. However, higher concentrations of Cu²⁺ (100 μM) stimulated pro-inflammatory marker expression [163]. This bipolar regulation of macrophages by Cu ions is of a significant impact on the role of macrophages in the initiation and progression of atherosclerosis. In particular, the elevation of Cu concentrations in the blood in the progression phase of atherosclerosis would affect the severity of atherosclerotic lesions.

The oxidative modification of LDL is a key event in human atherosclerosis. Cu ions catalyze oxidative modification of LDL in vitro [164–166]. It was also reported that Cu participates in the oxidation of LDL in vivo [167]. It was found that high cholesterol feeding leads to Cu deficiency in the vessel tissue and Cu elevation in the plasma [138]. The increase in plasma Cu associated with high cholesterol feeding would promote oxidative modification of LDL, thus promoting atherosclerosis. HDL is more sensitive to oxidation by Cu than LDL [168, 169]. Dose-dependent oxidative damage to HDL and protective effect of vitamin E against oxidation of HDL was observed in the studies of Cu incubation with HDL [170].

Cuproptosis, a newly identified Cu-induced cell death, occurs via Cu binding to lipoylated enzymes in the tricarboxylic acid cycle, leading to subsequent protein aggregation, proteotoxic stress, and eventual cell death [171]. It is possible that high levels of plasma Cu under the condition of atherosclerosis cause cuproptosis of macrophages and other immune cells in the circulation, leading to unbalanced immunological responses in the circulation. However, cuproptosis may not take place in the endothelial cells because they cells are Cu deficient under the condition of atherosclerosis [138, 139]. This phenomenon, high circulation versus low tissue Cu levels, underscores the critical role of Cu homeostasis in health and diseases, as discussed in several recent reviews [172–174].

The maintenance of vascular homeostasis depends on the dynamic stability of the cells and ECM that constitute blood vessels. Cu is involved in the regulation of vascular cells and the ECM via many factors and enzymes, as depicted in Figure 5.

Oxidative stress disrupts vascular homeostasis by impairs ECs function and vascular wall. Cu is a constituent of superoxide dismutase (SOD) and ceruloplasmin (CP), both are importantly involved in preventing oxidative injury [175–178]. It was also shown that low dietary Cu intake reduces glutathione peroxidase activity [179, 180]. Cu is required for mitochondrial cytochrome c oxidase (CCO) activity, rendering it essential for oxidative phosphorylation [181]. Cu deficiency leads to depressed activity of CCO [124, 182, 183]. These abovementioned enzymes or proteins are so crucial for maintaining the homeostasis of vascular system [184–189].

Several in vitro studies have examined Cu redox activity [190, 191]. Cu is redox active and involved in ROS generation [191]. Exposure to elevated levels of Cu significantly decreases glutathione levels [192]. The depletion of glutathione may enhance the cytotoxic effect of ROS and allow the metal to be more catalytically active, thus producing higher levels of ROS [193]. However, it has been demonstrated that there is virtually no free Cu in the biological system [194]. Therefore, the redox injury generated from free Cu in vitro studies may not extrapolated to the in vivo conditions. A general belief is that Cu-related generation of ROS is more related to Cu overload [195–197], but it is Cu deficiency that causes severe oxidative stress partially resulting from mitochondrial respiration defects due to CCO depression [182].

The ECM is an important component of the blood vessel wall and is essential for maintaining the structural integrity of blood vessels. Cu is essential for the synthesis and maturation of ECM. Fibronectin, an avascular elongation promoter [198], was observed to be increased in cultured ECs in exposure to trace amounts of Cu [199]. Fibronectin mats were strengthened when a small amount of Cu was present [200]. Lysyl oxidase (LOX) is a critical enzyme involved in the ECM remodeling, and Cu is required for the LOX activity [201]. Elastin is required for the lumen formation and maintenance. Studies conducted in swine found that Cu is associated with aorta elastin and essential for the function of the vessels [202–204].

Therefore, Cu is essential for vascular homeostasis through its action on multiple enzymes or proteins involved in the regulation of vascular cells and ECM. In most cases, the disturbance of vascular homeostasis would result from Cu deficiency, although Cu overload also causes severe consequences in vascular homeostasis. In terms of atherosclerosis, Cu deficiency in the vascular tissue and Cu overload in circulation are often observed concomitantly, leading to a double damage to the vessel wall.