Copper transporters and copper chaperones: roles in cardiovascular physiology and disease

Abstract

Copper (Cu) is an essential micronutrient but excess Cu is potentially toxic. Its important propensity to cycle between two oxidation states accounts for its frequent presence as a cofactor in many physiological processes through Cu-containing enzymes, including mitochondrial energy production (via cytochrome c-oxidase), protection against oxidative stress (via superoxide dismutase), and extracellular matrix stability (via lysyl oxidase). Since free Cu is potentially toxic, the bioavailability of intracellular Cu is tightly controlled by Cu transporters and Cu chaperones. Recent evidence reveals that these Cu transport systems play an essential role in the physiological responses of cardiovascular cells, including cell growth, migration, angiogenesis and wound repair. In response to growth factors, cytokines, and hypoxia, their expression, subcellular localization, and function are tightly regulated. Cu transport systems and their regulators have also been linked to various cardiovascular pathophysiologies such as hypertension, inflammation, atherosclerosis, diabetes, cardiac hypertrophy, and cardiomyopathy. A greater appreciation of the central importance of Cu transporters and Cu chaperones in cell signaling and gene expression in cardiovascular biology offers the possibility of identifying new therapeutic targets for cardiovascular disease.

INTRODUCTION

In recent years it has become increasingly evident that the essential metal micronutrients play critical roles in a wide range of physiological processes. These metals include iron, zinc, manganese, and copper, and in the case of copper (Cu), its importance is evident throughout human physiology.¹ The special properties of Cu, particularly its ready conversion between two oxidation states, Cu(I) and Cu(II), have led to its utilization as a cofactor in many oxido-reductive enzymatic processes. However, its propensity to produce reactive oxygen species (ROS) with potentially toxic consequences, together with its promiscuous reactivity with a number of amino acid side-chains of proteins as well as lipids, has necessitated the development of finely regulated homeostatic mechanisms. Examples of enzymes of special relevance to cardiovascular physiology that require Cu as a cofactor include Cu, Zn-superoxide dismutases (SOD) (SOD1 and SOD3), lysyl oxidase, mitochondrial cytochrome c-oxidase, and many others. Accumulating evidence suggests that cellular functions of Cu transporters and chaperones are not limited to Cu trafficking and may include unexpected roles involved in (patho)physiological responses (Fig. 1). In this short review, we will focus on the cellular processes that are especially important to cardiovascular physiology and some of the pathophysiological consequences observed in the cardiovascular system when Cu acquisition and its utilization fail to be appropriately regulated.

Fig. 1.

Copper trafficking pathways in mammalian cells. Once transported by Cu uptake transporter CTR1, soluble cytosolic Cu carrier proteins termed “Cu chaperones” may obtain the Cu directly from the uptake transporter or from GSH and are required for trafficking Cu to specific Cu-containing enzymes through direct protein-protein interactions. Three main copper chaperone pathways have been characterized thus far: 1) various Cu chaperones (cytochrome c-oxidases Cox1, Cox2, Cox11, and Cox17 and synthesis of cytochrome c-oxidases Sco1 and Sco2) and unknown Cu ligands (L), which deliver Cu to cytochrome c-oxidase in the mitochondria; 2) Cu chaperone for SOD1 (CCS), which delivers copper to SOD1 in the cytosol and mitochondrial intermembrane space (IMS); 3) antioxidant-1 (Atox1), which delivers copper to the secretory copper enzymes such as extracellular superoxide dismutase (ecSOD, SOD3) and lysyl oxidase (LOX) via the copper transporter ATP7A (Menkes disease copper ATPase, MNK) or ATP7B (Wilson disease copper ATPase, WND) in the trans-Golgi network. In addition to its chaperone function, Atox1 also function as a Cu-dependent transcription factor for Atox1-responsive genes such as ecSOD and cyclin D1. Cytosolic concentrations of free Cu are typically maintained at exquisitely low levels (10⁻¹⁸ M) by metal scavenging systems, including metallothionein (MT).

The recommended dietary allowance for Cu in adult men and women is 0.9 mg/day and the total body Cu is around 100 mg (1, 108, 181, 190). Although these are small amounts, the principles that govern cellular Cu homeostasis are the same as those that govern the major metal cations such as Na, K, or Ca. Cellular Cu levels are regulated by dedicated uptake systems balanced by the actions of ion-activated P-type ATPases that are responsible for its removal. These uptake and efflux transporters play additional roles, which are vital to appropriate Cu homeostasis. Under physiological conditions, there are vanishingly low levels of free Cu in cells (157). To protect the cell from the damaging effects of free Cu, a network of proteins, the intracellular metallochaperones, have evolved, which obtain Cu from the protein(s) mediating uptake and distribute it to target sites in the cell via direct interaction with their target proteins (Fig. 1). In this way, the essential acquisition of cofactor Cu is achieved while avoiding the harmful consequences of appreciable levels of free Cu.

There is little systematic knowledge of the chronic occurrence of dietary Cu toxicity and Cu deficiency or its potential effects in human physiology and cardiovascular disease (88). The occurrence of several human genetic diseases has stimulated interest in the field. The most familiar human diseases of Cu homeostasis are Menkes disease and Wilson disease, which result from mutations in the P-type ATPases, ATP7A and ATP7B, respectively. The basis of the different manifestation of these diseases, as systemic Cu deficiency or Cu toxicity, respectively, despite the high sequence identity of the proteins and similarity of causative mutations, can be found in the different locations of the proteins. ATP7B is predominantly expressed in the liver and ATP7A ubiquitously, but particularly in intestinal and neuronal cells (see below).

TRANSMEMBRANE Cu TRANSPORT

Cu Uptake Mediated by CTR1

The human high-affinity Cu transporter hCTR1 (SLC31A1) was first identified by complementation in yeast, in 1997 (195) and the mouse protein, which is 95% identical, three years later (104) (Fig. 1). Gene deletion of the mouse Cu transporter is lethal in midgestation CTR1⁻/⁻ embryos (96, 105). hCTR1 is a highly conserved high-affinity (42) Cu uptake protein with a K_m of about 3 μM, which is highly specific for Cu⁺ and the only known alternate substrate is Ag⁺ (for recent review, see Ref. 83). hCTR1 is a relatively small polypeptide of 190 amino acid residues. Its membrane topology has been determined. It has an extracellular amino-terminus, an intracellular carboxy-terminus, and three transmembrane segments (42, 90, 153). It associates into a stable homotrimer in the plasma membrane, forming a central conical pore (7) with an extracellular access of about 8 Å diameter and an intracellular access diameter of about 22 Å (40). The pore is lined at its extracellular side by a ring of six methionine residues (M150, M154, from each monomer) that are believed to provide an initial coordinating site for the permeating Cu (183). The intracellular carboxy terminus of about 14 amino acids ends with an HCH trimer that is completely conserved among mammalian species. This site provides a putative Cu-binding site at the exit of the pore in the cytosol. The lack of measurable free Cu in the serum or in cells leaves many aspects of the energetics of Cu uptake unresolved. It has been proposed that the binding of Cu to the COOH-terminal HCH triad provides a Cu coordinating site prior to Cu exit from the pore to the cytosol. The change in free energy, as Cu is bound to this site of relatively higher affinity than the initial Met ring coordination, was suggested to provide energy necessary for Cu permeation (40). This, however, is probably not the case, as it has subsequently been shown that the COOH-terminal triad, HCH, is not only not essential for CTR1-mediated Cu transport, but its deletion, or replacement by AAA, results in an increase in the maximal rate, the V_max of Cu transport (125). Thus, the COOH-terminal Cu-binding motif plays an important role in lowering the rate of transport through the pore. It seems likely that conformational changes on Cu loading of CTR1 occur, but their role in transport remains uncharacterized (42). It is apparent that the essential and relatively low requirement that cells have for Cu is achieved by expressing hCTR1 in low copy numbers and by ensuring that the transporter has an intrinsically low turnover number for Cu ions transported per trimer per second (about 5–10) (125).

There are many unresolved questions that relate to the detailed mechanism of Cu permeation through CTR1. The initial step, that is, the transfer to CTR1 from a serum Cu-carrier, remains a mystery, as the identity of these protein(s) or small molecule(s) is still undetermined. Recent work suggests that His residues in the extracellular amino-terminal tail are important for the transfer to hCTR1 (63). In a similar fashion (see below) the precise mechanism of the transfer process, from the transporter to an intracellular chaperone, is the subject of current investigation. It is clear, however, that the entity transported by CTR1 is Cu⁺. The site of reduction of Cu²⁺, presumably in the extracellular medium, is not known with certainty, but there is some indication that the plasma membrane-bound STEAP proteins, which have been associated with Cu reductase activity (139), may play a role or perhaps other unidentified reductants in the serum.

The presence of posttranslational glycosylation of hCTR1 has been well characterized. Both N-linked (42) and O-linked modifications are present. The extensive N-linked glycosylation, at Asn15, results in a shift in SDS gels to an apparent mass of about 35 kDa. It seems likely that the N-linked glycosylation at N15 does not greatly influence transport. However, an O-linked modification containing several sugars attached to Thr27 serves to protect hCTR1 against intracellular cleavage by a cellular protease (127). The cleaved product is localized in late endosomal compartments containing Rab9 (128). The shortened transporter, lacking much of the extracellular tail, is a significantly (50%) less effective Cu transporter.

Cu-Dependent Relocalization of CTR1

An additional role of CTR1 that has recently been reported involves a Cu-dependent relocalization of the transporter. It has been appreciated for many years that the P-type ATPases (ATP7A, -7B) responsible for Cu efflux and for Cu delivery into the Golgi and the secretory pathway (see below) are sensitive to cellular Cu levels and relocate to the plasma membrane from the Golgi when cellular Cu is high. This is an important protection against elevated intracellular Cu levels. In a similar fashion, when extracellular Cu is elevated, the uptake transporter, hCTR1, is removed from the plasma membrane and internalized (132). This prevents the cell from acquiring elevated intracellular Cu. Initial studies demonstrating internalization of CTR1 in the face of elevated extracellular Cu claimed that the endocytosed transporter was degraded (148) (60). More recent studies have shown that when extracellular Cu is lowered, hCTR1 recycles back to the plasma membrane, to again facilitate Cu influx. Such “activity-regulated transport” has recently been suggested, in a computational analysis, to play a major role in efficient homeostatic systems (164). The mechanism of hCTR1 internalization and recycling has recently been described. The internalization is clathrin- and dynamin-dependent and takes place via compartments containing Rab5 and EEA1, but not late endosomal or lysosomal pathways, and the recycling occurs via a Rab11-mediated pathway (35). It is also significant that deletion of the putative Cu-binding COOH-terminal triad, ¹⁸⁸HCH, or its substitution causes a double phenotype. The internalization of hCTR1 at elevated extracellular Cu no longer occurs and the V_max for transport (as discussed above) is also increased (125). It seems likely that Cu binding at this site may cause slowing of the transport cycle and facilitate interaction with the internalization machinery.

Other Cu Entry Pathways

There are three other entry pathways in addition to hCTR1 that have received attention as potential mediators of Cu uptake across the plasma membrane in human cells. These are 1) hCTR2, a related protein; 2) DMT1, an unrelated divalent metal ion transporter; and 3) Cu(II) transport in an anionic complex, by an anion exchange transporter. We will briefly discuss the current status of each.

The first of these, CTR2, originally identified via its homology to CTR1 (195), but lacking a major part of the extracellular amino-terminal domain, has been suggested to be an alternate, lower-affinity, CTR1-like transport protein (18). However, recent studies suggest an interesting, alternate, and unexpected role for CTR2 in Cu homeostasis. In a thorough examination and comparison of metazoan CTR1 and CTR2 evolution and function it has been suggested that CTR2 serves as an intracellular vesicular transporter and also plays a significant role in stimulating the degradation of CTR1 (see Ref. 111 and references therein).

The second alternate Cu transporter, DMT1, the epithelial divalent metal ion transporter, was suggested to transport Cu(II) ions (11) following overexpression in HEK cells. However, recent studies bring into question the direct involvement of DMT1 in Cu transport (167).

The third candidate is on its face somewhat surprising. In the 1980s it was reported that in human red blood cells the uptake of Pb²⁺, Cd²⁺, and Cu²⁺ was mediated by AE1 or Band 3, the prototypical anion exchange transporter (5, 168, 169). The transport was inhibited by DIDS (4,4′-diisothyocyano-stilbene-2,2′-disulfonic acid), a well-characterized inhibitor of anion exchange transporters. These observations were largely lost to the metal transport field. More recently, in studies of Cu uptake across the apical membrane of intestinal monolayers, it was proposed that Cu(II) uptake was mediated via an apical anion exchange system, and the transport was inhibited by DIDS (197). There are several members of this anion exchange family in the apical membrane of intestinal and renal epithelia and many of the observed characteristics of these metal ion fluxes corresponded to the system seen in human erythrocytes. Interestingly, this transport of CuX₄ anions would also account for very high fluxes observed in early studies of heterologous overexpression of hCTR1, in the absence or presence of CTR1 (103). It was observed that these “background” fluxes of Cu were greatly diminished in the presence of histidine or serum, effects also seen and discussed in the later studies of Cu uptake in epithelial cells (197). The presence of peptides or Cu-coordinating amino acids in serum make this pathway of little relevance in most physiological situations. However, in the particular environment (pH and salts) in the early intestine (duodenum) this system may play a significant role in dietary acquisition. Interestingly, in a recent study it was proposed that an anion (phosphate) transporter in mitochondria, SLC25A3, also mediated Cu uptake and that, when reconstituted into phospholipid liposomes Cu, uptake was facilitated (25). These findings are in line with the earlier studies, discussed above, in red cells and in epithelia, as well as findings suggesting a role for the mitochondrial phosphate carrier in Cu import in yeast (188). They raise the possibility that Cu entry into mitochondria may also be facilitated via an anionic complex of Cu(II).

Cu Export Mediated by P-type ATPases

The export of Cu from cells is accomplished by the actions of ATP7A and ATP7B, which are Cu-activated ATPases and belong to the P-type ATPase family of ion pumps (74, 117, 143) (Fig. 1). These proteins catalyze the metal cation-activated phosphorylation (from cellular ATP) of an essential aspartate residue and couple conformational equilibria between two phosphoenzyme states to the transmembrane transport of the metal cation. The Cu-ATPases contain many of the usual signature sequences of P-type ATPases and an additional amino-terminal domain with multiple (six) Cu binding domains (118) that serve as sensors of cytosolic Cu levels that presumably trigger the events involved in Cu-dependent relocalization. The two Cu-activated P-type ATPases spend part of their time in the plasma membrane and represent an important aspect of cellular Cu homeostasis (116). Under normal conditions of cellular Cu loads, these proteins are found in the Golgi where they pump Cu from the cytosol (supplied by a Cu chaperone, Atox1, see below) into the lumen of the organelle to be incorporated into secretory cuproenzymes present in the lumen. A recent review comprehensively summarizes the current knowledge of Cu trafficking in the secretory pathway (114) and the role of kinase-dependent phosphorylation in the cellular localization of these proteins. The precise mechanisms whereby the ATPases activate the cuproenzymes in the secretory pathway is the subject of current investigation. Under conditions where cytosolic Cu is elevated, these Cu-ATPases pump excess Cu into vesicles of the Golgi that bud off and fuse with the plasma membrane, thus removing excess Cu and placing the pumps in the plasma membrane where they can continue to lower cell Cu. When Cu is lowered they recycle to the Golgi. This Cu-dependent relocalization is an essential homeostatic mechanism that protects cells against elevated cytosolic Cu (180). The malfunctioning of the pumps, in terms of ATPase activity or Cu-dependent relocalization, will result in elevated cell Cu and also potentially loss of the ability to synthesize and secrete active cuproenzymes. Together, these factors are played out in the symptoms of Wilson and Menkes disease, genetically inherited human diseases (115). Their disease outcomes in vascular biology are a consequence of both functions, and loss of appropriate activity of lysyl oxidase, for example, leads to a lack of integrity of connective tissue and skin, which is a common hallmark of Menkes disease. Many of the neurological effects occur via a loss of cuproenzymes that are important in neurotransmitter and neuropeptide biosynthesis. However, the consequences of loss of function or its disruption are multiple and complex when studied in animal models (114), affecting the expression of many proteins not obviously dependent on, or involved with, Cu balance.

INTRACELLULAR Cu DISTRIBUTION

The Metallochaperones

The demonstration that there is essentially no free Cu available in the intracellular milieu made it clear that intracellular Cu was not captured by collision and binding processes between free Cu ions and the target proteins (157). Experimental data appearing first about 20 years ago pointed to the existence of a new class of intracellular proteins, the metallochaperones, which bind Cu and deliver it to specific proteins (152). Although much of the early work on Cu transport and the roles of metallochaperones was carried out in yeast, for the purposes of this review we will focus on mammalian systems. There are separate and specific chaperones that deliver Cu to their target proteins (Fig. 1): 1) ATOX1 (human antioxidant protein), which delivers Cu to the Cu-activated ATPases, ATP7A, ATP7B, and thus to cuproenzymes in the secretory pathway; 2) CCS (Cu chaperone for superoxide dismutase, SOD), which delivers Cu to Cu, Zn-SOD (71); and 3) various Cu chaperones for mitochondrial cytochrome c-oxidase (COX), which requires Cu for its maturation and function. For the delivery of Cu to COX in the mitochondrion, Cox11 delivers Cu to the Cu(B) site of the oxidase, whereas the delivery of Cu to the Cu(A) site involves a number of associated proteins, including Cox17, Cox19, Cox23 and Sco1, Sco2 (144, 162) (Fig. 1). However, the mechanisms that regulate IMS copper translocation and transfer to Cox17 and CCS at IMS remains elusive (102). The details of overall synthesis of COX and the regulation of the many assembly proteins has been discussed in a recent review (102).

Antioxidant-1

Antioxidant-1 (ATOX1) is a small (68 amino acid residues, 7.4 kDa) cytosolic protein that is highly conserved among mammalian species and is responsible for transferring Cu(I) to the Cu-ATPases for delivery to cuproenzymes in the trans-Golgi system and hence to the secretory pathway (Fig. 1). ATOX1 has a ferredoxin-like βαββαβ-fold. Cu(I) is bound via a MXCXXC motif and is transferred to the ATPase metal binding domain via a ligand transfer mechanism (20). It has recently been suggested that ATOX1 acquires its Cu(I) via direct transfer from the COOH-terminal HCH motif of hCTR1 the uptake protein (47). This proposal has ATOX1 associating with the membrane via coulombic interactions and obtaining Cu via direct interaction with the COOH terminus of the transporter. The lack of free Cu in the cytosol supports the idea that Cu(I) does not dissociate from the transporter, so a direct transfer to an acceptor is an appealing mechanism. However, extensive depletion of ATOX1 (or indeed CCS), or its overexpression had no effect on the rate of Cu uptake (126). The depletion of cellular glutathione (GSH) did, however, lower the rate of Cu uptake (126), suggesting that GSH is more likely to be the initial acceptor of Cu(I) from the transporter. Indeed, the GSH/GSSG pair is proposed to be involved in the metallochaperone function of Atox1, thereby regulating Cu homeostasis (reviewed in Refs. 19, 66). An additional important role for ATOX1 has been reported, which integrates Cu homeostasis with another critical cellular process where ATOX1 can serve as a transcriptional regulator in a Cu-dependent fashion (77). This has led to a series of interesting questions that remain and relate to the mechanisms of trafficking of ATOX1 to the nucleus from the cytosol and the regulation of its return.

Cu chaperone for SOD1

Cu chaperone for SOD1 (CCS) is the Cu chaperone for superoxide dismutase; it delivers Cu(I) to the major cytosolic cuproenzyme SOD1 (152), which is also present in mitochondria (Fig. 1). CCS is a 54-kDa protein composed of three domains. The crystal structure of the homodimeric complex of CCS and the heterodimeric CCS-SOD show the importance of matching protein-protein interactions in the chaperone-SOD metal-loading (97, 98). The precise steps in SOD metallation by CCS have been studied in great detail (see, e.g., Refs. 16, 173, 174). In many eukaryotes, including humans, metallation can also occur through CCS-independent pathways (107) and GSH may play the key Cu delivery role (31). Cellular Cu status regulates CCS levels at the posttranslational level, through effects on the 26S proteasome. When Cu is low, CCS levels are high, and when Cu is elevated, degradation of CCS occurs (17, 149).

Cu TRANSPORT PROTEINS AND CELL GROWTH

Cu stimulates cell proliferation and migration involved in physiological repair processes such as wound healing, angiogenesis, and neurogenesis, as well as in various pathophysiologies including tumor growth, atherosclerosis, and neurodegenerative diseases (82, 84, 115). Cancer cells and cells in atherosclerosis have higher Cu levels and more predominant nuclear localization of Cu in some cells than normal tissues (9, 51). Cu chelation prevents tumor growth and neointimal thickening after vascular injury (27, 58, 65, 119). Furthermore, nuclear accumulation of Cu is strongly associated with proliferation of hepatocytes in ATP7B^−/− mice, an animal model of Wilson disease, before the appearance of histopathological alterations (72). Because dysregulation of cell proliferation and migration is frequently associated with a number of diseases, an understanding of the molecular mechanism by which Cu stimulates cell proliferation/migration will potentially provide the targets for new therapeutic strategies.

Cu stimulates, while CTR1 deficiency or Cu chelation inhibit, cell proliferation and migration in endothelial cells (70, 135) and vascular smooth muscle cells (21, 198). Several mechanisms for Cu-dependent cell proliferation and/or migration have been proposed. Insulin or fibroblast growth factor (FGF) induces ERK phosphorylation via interaction of MEK1 with ERK in a Cu-dependent manner in flies and mouse fibroblasts, and mouse cardiac tissues (184). Furthermore, Brady et al. (26) showed that decreasing the levels of CTR1, or introducing mutations in MEK1 that disrupt Cu binding, decrease BRAFV600E-driven signaling and tumorigenesis in mice and human cells. In support of this, the Cu chelator tetrathiomolybdate (TTM) decreased tumor growth of human or murine cells transformed by BRAFV600E (26), and fibroblasts lacking CTR1 showed impaired MAPK signaling (182). Thus, these findings suggest that CTR1 plays an important role in cell proliferation in a number of cell types. It was previously demonstrated that Atox1 also has an unexpected function as a Cu-dependent (and sequence-specific transcription factor) involved in cyclin D1 expression and cell proliferation in vitro (77) and in vivo in a number of cell types and in wounded tissue (32, 39). Importantly, nuclear-targeted Atox1 rescued the impairment of wound healing seen in Atox1^−/− mice. This indicates that the nuclear function of Atox1 plays a critical role in cell proliferation and wound healing in vivo. Moreover, it has been proposed that Cu-dependent cell proliferation is mediated through enhanced binding of growth factors to cell surface (13), increased secretion of proangiogenic peptides such as FGF1 and IL-1α (151), or stabilization of the three-dimensional architecture of extracellular matrix such as fibronectin (4). It has also been demonstrated that ATP7A plays an important role in PDGF-induced vascular smooth muscle cell (VSMC) migration (12), as discussed below.

Cu TRANSPORT PROTEINS AND ANTITUMOR DRUGS

An interesting correlation between the cytotoxic actions of the antitumor drug, cisplatin, and cellular expression levels of hCTR1 has been reported and hCTR1-mediated cisplatin uptake has been proposed as its basis (76). Furthermore, a role for the Cu transporters in drug resistance has also been suggested (69). However, the lack of effect of overexpressing or knocking down CTR1 on cellular drug levels does not support the proposal that CTR1 mediates (or directly influences) Pt drug entry (78). This conclusion was recently confirmed and extended in studies of CTR1, CTR2, Atox1 and CCS and drug sensitivity utilizing CRISPR-cas9 genome editing techniques (22). How the proteins of the Cu homeostasis system might influence Pt drug sensitivity in cancer cells awaits characterization.

Cu VASCULAR REMODELING AND ATHEROSCLEROSIS

Cu levels play an important role in atherosclerosis, and inflammation. Several prospective epidemiological studies have shown that high serum Cu levels are associated with an increased future risk of coronary heart disease (CHD) in Finnish (163) and Dutch men (93) and in the US population (48). Moreover, previous studies have shown that high plasma levels of ceruloplasmin (CP), which is the major (60–70%), Cu binding protein in plasma, are associated with CHD risk in prospective studies in both men and women (161). Tissue Cu levels are significantly increased in pathological inflammatory conditions such as atherosclerosis and aortic aneurysm (94, 171). Implanting a Cu cuff promotes neointima thickening in response to vascular injury (189), whereas Cu chelators inhibit vascular inflammation and atherosclerotic lesion development in ApoE^−/− mice (14, 147, 171, 191–193) as well as neointimal formation in response to vascular injury (119, 120). Cu also plays an important role in inflammatory responses involved in innate and adaptive immunity (43, 146), which is in part via activating NF-κB (145). Cu deficiency alters intravascular adhesion of leukocytes to the activated endothelial cells (ECs) and expression of adhesion molecules, such as ICAM-1/VCAM-1 (165).

There are several potential mechanisms by which Cu might promote atherosclerosis in vivo. Cu may play an important role in oxidation of LDL. It has been shown that tissue homogenates prepared from atherosclerotic lesions contain detectable levels of catalytically active metal ions (100, 178). Furthermore, the appearance of the marker for protein oxidation (o-tyrosine) induced by Cu has been detected by mass spectrometric analysis in advanced human atherosclerotic lesions (50, 106). Mandinov and colleagues (119, 120) proposed that Cu chelation inhibits neointimal thickening by inhibiting stress-induced release of IL-1α and fibroblast growth factor 1, which have been implicated as regulators of vascular injury in vivo. The polypeptide Cu²⁺-binding proteins, which lack signal peptides, IL-1α and FGF1, are exported into the extracellular compartment in a stress-dependent manner by using intracellular Cu²⁺ to facilitate the formation of S100A13 heterotetrameric complexes (101, 121). Furthermore, Cu induces the expression of genes involved in LDL uptake and de novo cholesterol biosynthesis such as 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and LDL receptor in human macrophages (177). In addition, homocysteine, a risk factor for cardiovascular disease, interacts with Cu to enhance hydrogen peroxide (172). In agreement with this, there seems be a correlation among serum levels of Cu or Cu binding protein ceruloplasmin and homocysteine levels in patients with peripheral vascular disease (122) and ischemic heart disease (80). An optimal Cu level is apparently essential for preventing cardiovascular disease, including inflammatory atherosclerosis. Indeed, Cu deficiency has been proposed as a risk factor for coronary heart disease (88). Moreover, in experimental animal models, it is suggested that optimal dietary Cu intake (1–3 mg/day) in the cholesterol-fed rabbit is associated with a reduced susceptibility to aortic atherosclerosis, whereas Cu deficiency and an excessive daily Cu supplement (20 mg/day) are associated with increased atherogenesis (99). These findings suggest that optimal level of Cu is essential for preventing cardiovascular disease, including inflammatory atherosclerosis.

Cu Transport Proteins, Vascular Remodeling, and Atherosclerosis

The Cu exporter ATP7A and its chaperone Atox1 are highly expressed and colocalized in the intimal lesions of atherosclerotic vessels or in neointimal vascular smooth muscle cells (VSMCs) where Cu is highly accumulated in wire-injured vessels (12, 79, 92, 155). Atox1^−/− mice show inhibition of neointimal formation and extracellular matrix (ECM) expansion in response to vascular injury, which is associated with decreased VSMC accumulation within neointima and lowered LOX activity (92) (Fig. 2). In cultured VSMC, depletion of Atox1 or ATP7A inhibits VSMC migration in response to PDGF or wound scratch in a CTR1/Cu-dependent manner (12, 92). PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts at the leading edge, where it colocalized with PDGF receptor and Rac1 in migrating VSMCs. Knockdown of ATP7A or CTR1 prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. Atox1 depletion prevents PDGF-induced translocation of ATP7A, causes decrease in secretory Cu enzyme precursor prolysyl oxidase (Pro-LOX) in lipid rafts as well as lowering LOX activity. Atox1^−/− mice show decreased perivascular macrophage infiltration in wire-injured vessels as well as thioglycollate-induced peritoneal macrophage recruitment. These findings strongly suggest that ATP7A and Atox1 play an important role in PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge as well as regulating LOX activity. Furthermore, Atox1 is involved in neointimal formation after vascular injury through promoting VSMC migration and inflammatory cell recruitment in injured vessels (Fig. 2). Qin et al. (156) reported that ATP7A downregulation attenuates cell-mediated oxidation of LDL in THP-1 macrophages, which is associated with decreased expression and enzymatic activity of cPLA2 (a key intracellular enzyme involved in cell-mediated LDL oxidation). Moreover, they showed that ATP7A downregulation reduces macrophage infiltration into dermal wounds (86). The role of ATP7A in inflammatory response/immune defenses is further emphasized by the increased susceptibility to infection in patients with Menkes disease who lack a functional ATP7A Cu transporter (59, 185). In in vitro systems, White et al. reported that proinflammatory agents increased expression of CTR1, Cu uptake, and Cu-stimulated trafficking of ATP7A from the Golgi to cytoplasmic vesicles that overlap with the phagosomal compartment (68, 194). Not surprisingly, silencing of ATP7A impaired bactericidal activity, suggesting that Cu transport via ATP7A is required for bacterial killing. The significant role of macrophage ATP7A in inflammatory disease is in need of more detailed investigation

Fig. 2.

Role of Cu transporters and Cu chaperones in platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) migration and vascular remodeling. PDGF promotes interaction of ATP7A with antioxidant-1 (Atox1) and translocation from the trans-Golgi network (TGN) to the lipid rafts at the leading edge. This stimulates lamellipodia formation via recruiting Rac1, which in turn promotes directional VSMC migration. This is associated with a decrease in the cellular copper level and secretory copper enzyme prolysyl oxidase (Pro-LOX) at the lipid rafts, which is processed following secretion and activated by proteolysis to a mature lysyl oxidase (LOX) and a propeptide (LOX-PP), which may promote extracellular matrix (ECM) remodeling. Secreted copper may also contribute to PDGF-induced cell migration. Atox1 also regulates inflammatory cell recruitment. Thus, Cu transporters and Cu chaperones may play an important role in vascular remodeling by regulating inflammatory cell recruitment, VSMC migration, and ECM remodeling. CTR1, copper transporter 1; PDGFR, PDGF receptor.

HYPERTENSION

Cu Transport Proteins and Hypertension

The importance of Cu homeostasis in hypertension has been reported (88). Tissue levels of Cu are altered in hypertensive rats, which are associated with the severity of the hypertension (34, 55). Serum Cu concentration is increased in hypertensive patients (3, 140) and in rodents of various hypertension models (34). A Cu-deficient diet changes blood pressure in an age-dependent manner (89, 131). Several lines of evidence support a role for vascular Cu transport proteins, which regulate not only activation of Cu-containing enzymes such as SOD1 and SOD3, but also total intracellular Cu levels. In angiotensin II (ANG II)-induced hypertension, it was demonstrated that Atox1 functions to prevent ANG II-induced endothelial dysfunction and hypercontraction in resistant vessels, as well as in hypertension, in vivo by reducing extracellular O₂⁻ levels via increasing vascular SOD3 expression and activity (142). Furthermore, ANG II infusion into mice significantly decreases vascular Cu levels, which is inhibited in Atox1^−/− mice. This may be due to ANG II-induced increase in secretion of Cu-loaded SOD3 to the circulation. This is consistent with the decrease in levels of Cu in some tissues such as liver and kidney in hypertensive rodents (34).

Full activation of SOD3 requires not only Cu chaperone/transcription factor Atox1 but also the Cu transporter ATP7A, which localizes at the trans-Golgi network where ATP7A delivers Cu to SOD3 (53). Indeed, it was reported that ATP7A plays an important role in modulating ANG II-induced hypertension and endothelial function by regulating SOD3 activity and vascular superoxide anion production (154). In contrast, norepinephrine-induced hypertension, which is not associated with an increase in vascular O₂^·− production (158), is not affected in ATP7A^mut mice with reduced Cu transport, which is consistent with findings in SOD3 knockout mice (57). Basal and ANG II infusion-induced increase in vascular SOD3-specific activity is significantly inhibited in ATP7A^mut mice. ANG II stimulation promotes association of ATP7A with SOD3 in cultured vascular smooth muscle cells and in mouse aortas, which may contribute to a SOD3-specific activity by increasing Cu delivery to SOD3 through ATP7A (154).

It is also possible that Cu transport proteins in the kidney and central nervous system regulate blood pressure. Atox1 expression is observed in several regions of the brain including the choroid plexus that belongs to the circumventricular organ (CVO), where ATP7A is also expressed (137). It has been reported that deletion of SOD3 in the CVO increases blood pressure in the basal state and after ANG II infusion, in part by modulating sympathetic outflow (109). In particular, O₂^·− production in the brain is required for the genesis of hypertension (109, 110). It is conceivable that Atox1 expressed in brain may regulate blood pressure either by regulating SOD3 activity or other secretory Cu enzymes via its Cu chaperone function in the brain. Both Atox1 and ATP7A are also highly expressed in the kidney (115, 133). Gene transfer of SOD3 reduces renal sodium retention in hypertensive rats (33). Thus, Atox1 and ATP7A may play an important role in hypertension, in part, by regulating renal and brain function (196).

Cu Transport Proteins and Pulmonary Hypertension

Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes of pulmonary hypertension (PH). Zimnicka et al. (198) demonstrated that hypoxia increases Cu fluxes by upregulating CTR1 and ATP7A in pulmonary arteries and pulmonary arterial smooth muscle cells, which is associated with an increase in LOX activity and cross-linking of ECM, eventually leading to the increase in pulmonary arterial stiffness and development of pulmonary hypertension. Consistent with this, Nave et al. (136) showed that LOX is dysregulated in clinical and experimental PH. LOX plays a causal role in experimental PH and represents a candidate therapeutic target, suggesting that modulation of lung matrix cross-linking can affect pulmonary vascular remodeling associated with PH. Furthermore, Bogaard et al. (21) showed that a Cu-depleted diet prevented, and Cu chelation with TTM reversed, the development of PH and inhibited the proliferation of human pulmonary microvascular endothelial cells isolated from PH patients. The Cu chelation-induced reopening of obstructed vessels is caused by caspase-independent apoptosis, reduced vessel wall cell proliferation, and a normalization of vessel wall structure, while neither SOD1 inhibition nor LOX-1 inhibition prevents cell proliferation.

DIABETES

Tissue Cu levels are increased in streptozotocin (STZ)-induced diabetes, which is rescued by insulin treatment (44). Furthermore, Cu chelation therapy has been shown to mitigate various pathogenic states of diabetes, such as left ventricular hypertrophy in diabetic patients (37), diabetic neuropathy (30), and diabetic nephropathy (56).

Cu Transport Proteins and Diabetes

We recently found that ATP7A protein expression, but not Atox1, CCS, or COX17 (Cu chaperone for cytochrome c-oxidase), is selectively and significantly decreased in vessels of type 1 diabetes mellitus (T1DM) mice including STZ-induced and genetically induced (i.e., Ins2^Akita) mice (176) as well as those of type T2DM mice including high-fat diet (HFD)-induced and genetically induced (i.e., db/db) mice (Fig. 3). These data imply that Cu transport systems coupled to distinct Cu enzymes can be differentially regulated in response to diabetes, as reported for hypoxia (194). Given that ATP7A is required for activation of extracellular SOD (SOD3), but not that of SOD1 (53), the ATP7A downregulation in diabetic vessels contributes to the decrease in specific activity of SOD3, but not that of SOD1, which may result in increased superoxide production and endothelial dysfunction in both T1DM and T2DM mice (175, 176). We found that ATP7A protein expression is significantly reduced in blood vessels from T1DM mice, in part due to the insulin deficiency, but not high glucose, and that insulin treatment of VSMCs increases ATP7A protein expression without regulating its transcription (176). Consistent with this, insulin-induced ATP7A expression is also observed in human placental Jeg-3 cells (64). It is reported that ATP7A protein downregulation in T2DM mice vessels with hyperinsulinemia and insulin resistance is due to impaired insulin/Akt2 pathway in VSMCs (175). Akt2, activated by insulin in VSMCs, promotes ATP7A stabilization by preventing its ubiquitination/degradation that contributes to activation of SOD3 that in turn protects against T2DM-induced endothelial dysfunction (Fig. 3). Downregulation of ATP7A in T2DM vessels is restored by constitutively active Akt or in PTP1B^−/− (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhances insulin-Akt signaling. Immunoprecipitation, in vitro kinase assays, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce its phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2^−/− or T1DM or T2DM vessels, which is rescued by ATP7A overexpression. Endogenous roles for SOD3 and ATP7A are demonstrated by enhanced endothelial dysfunction and vascular O₂⁻ production in the SOD3^−/− or ATP7A mutant DM mice. These findings suggest a protective role for the endogenous insulin-Akt2/ATP7A/SOD3 pathway in vascular dysfunction in diabetes (Fig. 3).

Fig. 3.

Protective role of the insulin-Akt2/ATP7A/superoxide dismutase 3 (SOD3) pathway against diabetes mellitus (DM)-induced endothelial dysfunction. Decreased ATP7A expression in diabetic vessels with either hypoinsulinemia (type1 DM) or selective impairment of insulin/Akt signaling (type 2 DM) contributes to decreased SOD3 activity, resulting in increased O₂^·− production and endothelial dysfunction. Mechanistically, insulin increases Akt2 binding to ATP7A to induce ATP7A phosphorylation, which may increase ATP7A protein expression via preventing proteasomal degradation as well as ATP7A translocation to the plasma membrane, which contributes to full activation of SOD3 in vascular smooth muscle cells (VSMCs) and preserves endothelial function. ECs, endothelial cells; TGN, trans-Golgi network.

ANGIOGENESIS

Cu and Angiogenesis

Cu plays an important role in physiological and pathological angiogenesis (27, 28, 46, 58, 65, 193). Cu or Cu complexes directly stimulate angiogenesis in a number of model systems including the rabbit corneal system (159) and various source of endothelial cells (6, 129). Furthermore, Cu chelators inhibit tumor growth and angiogenic responses (27, 28, 58, 65) in numerous animal and xenograft models (58). Cu metabolism appears to be altered in tumors (8, 75), while angiogenic lesions in cancer have higher Cu levels in cell nuclei than normal tissues and serum Cu levels themselves appear to be elevated in a number of tumor types (36) including breast cancer (61). Thus, systemic removal of Cu may have promise in tumor treatment in an antiangiogenic strategy. Indeed, a number of clinical trials for the treatment of solid tumors by Cu chelation showed efficacy in disease stabilization (29, 112, 160). There are several possible mechanisms for Cu-mediated angiogenesis. It has been shown that expression of VEGF is induced by the treatment of cells with Cu salts, partly by activation of hypoxia-inducible factor-1α (HIF-1α) or other factors (81, 166, 186). FGF1, angiogenic growth factor, is a Cu-binding protein and its secretion requires the Cu-dependent formation of a complex consisting of FGF1, S100A13, and p40 synaptotagmin 1, due to the lack of a signal peptide (101, 121). Moreover, Cu binds to the receptor for FGF, angiogenic growth factor, and enables it to interact with heparin (65, 186). Cu also increases the affinity with which angiogenin, a potent angiogenic molecule, binds to high-affinity endothelial receptors (170). This array of findings suggests multiple other mechanisms whereby Cu may act as an essential cofactor in angiogenesis. Other relevant potential targets for Cu chelation approaches include various Cu containing enzymes such as SOD1, cytochrome c-oxidase (COX), tyrosinase, dopamine β-hydroxylase, LOX, and ceruloplasmin, because their levels are associated with various tumor progressions (65, 112, 186).

Cu Transport Proteins and Angiogenesis

We reported that Atox1 plays an essential role in inflammatory neovascularization via regulating ROS/NF-κB pathway (32) (Fig. 4). Atox1 expression is significantly upregulated in patients and mice with critical limb ischemia. Mice lacking Atox1 exhibit impaired ischemia-induced arteriogenesis/angiogenesis and infiltration of inflammatory cells that produce angiogenic cytokines including VEGF and TNF-α. Bone marrow transplantation experiments reveal that Atox1 in endothelial cells, but not bone marrow cells, is required for post-ischemic revascularization. In cultured ECs, Atox1 functions as a Cu chaperone to promote VEGF-induced angiogenesis via increasing LOX activity in a Cu/ATP7A-dependent manner. In response to TNF-α, but not VEGF, Atox1 translocates to the nucleus, resulting in binding to the p47^phox promoter, thereby promoting the ROS/NF-κB-dependent ICAM-1/VCAM-1 expression in ECs, in an ATP7A-independent manner, which in turn recruits inflammatory cells. This study provides a novel linkage between Atox1 and NADPH oxidase as well as positive feedback loops that promote inflammatory neovascularization organized by the Cu chaperone and transcription factor functions of Atox1 (Fig. 4). Furthermore, Finney et al. (45) showed endothelial Cu secretion in FGF2- and VEGF-activated microvascular ECs by using X-ray fluorescent microscopy that allows for the quantitative imaging of numerous metals at the submicron scale. It is possible that Cu exporter ATP7A may be involved in this response; however, its functional significance remains unclear. ATP7A is highly expressed in the vasculature (endothelial cells and VSMCs), macrophages, and fibroblasts (2, 12, 86, 155, 156, 194). The involvement of ATP7A in angiogenesis is implicated by the etiological role of mutant transporters, responsible for the human Menkes syndrome, in the onset of congenital cardiovascular abnormalities (67). Further investigation into the roles of ATP7A and CTR1 in postnatal angiogenesis is needed.

Fig. 4.

Role of antioxidant-1 (Atox1) in inflammatory neovascularization in response to ischemia and wound injury, which is dependent on arteriogenesis/angiogenesis and inflammation. In response to proinflammatory cytokine TNF-α, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer, p47^phox, to increase the reactive oxygen species (ROS)/NF-κB/VCAM-1/ICAM-1 axis in endothelial cells. This in turn promotes recruitment of inflammatory cells that secrete TNF-α and VEGF. In response to VEGF, Atox1 functions as a Cu chaperone for ATP7A, to increase lysyl oxidase (LOX) activity involved in angiogenesis. This process represents a novel positive feedback loop whereby the Cu chaperone protein Atox1 promotes inflammatory neovascularization. CTR1, copper transporter 1; TNFR, TNF receptor.

WOUND HEALING

The earliest recorded application of Cu in wounds can be traced to around 2000 BC in ancient Egypt (24, 41). Indeed, Cu-accelerated wound healing has been demonstrated in various clinical and experimental settings (23, 123, 166), including diabetic ulcers (134). Wound healing proceeds in three overlapping but functionally distinct phases: an inflammatory phase, the cell proliferation phase, and a maturation phase (62, 187). Little information is available regarding the mechanism of how exogenous or endogenous Cu promotes wound angiogenesis and an inflammation response required for tissue repair.

Endothelial Atox1 plays an essential role to sense Cu levels that accelerate wound angiogenesis and healing (39). Using a cutaneous wound healing model, it was shown that the Cu content and nuclear Atox1 are increased after wounding, while wound healing is impaired in global and EC-specific Atox1^−/− mice. Expression of Atox1 transcription factor-target proteins, such as p47^phox, NADPH oxidase, and cyclin D1, as well as extracellular matrix LOX activity in wound tissues are decreased in Atox1^−/− mice. This decrease, in turn, inhibits angiogenesis, macrophage recruitment, and ECM maturation by reducing O₂⁻ production in ECs, NF-κB activity, cell proliferation and collagen formation, resulting in impaired wound healing (Fig. 4). This Atox1-mediated wound angiogenesis may occur through several alternate mechanisms. Cu treatment markedly increases Atox1 and VEGF expression in wound tissues, which are abolished in Atox1^−/− mice. Consistent with this, earlier studies showed that Cu addition increases VEGF expression in part via activation of HIF-1α (81, 124, 166). We previously reported that Atox1 functions as a Cu-dependent transcription factor (77) and found that HIF-1α promoter has Atox1-responsive elements (GAAAGA), which raises the possibility that the Cu/Atox1/HIF-1α pathway may upregulate VEGF expression, thereby promoting wound angiogenesis (53, 54, 91, 113, 179). We found that Atox1^−/− mice exhibit reduced LOX-specific activity and collagen deposition in wounded skin compared with wild-type mice (32). Furthermore, Atox1 also delivers Cu to the extracellular superoxide dismutase (ecSOD), which plays an important role in ischemia- and wounding-induced angiogenesis, thereby promoting wound healing (52, 87, 141). Taken together, these results suggest that in vivo Atox1 is involved in Cu-dependent wound healing via promoting ECM maturation and angiogenesis by regulating VEGF expression, activity of LOX, and ecSOD.

Cu, CARDIAC HYPERTROPHY, AND CARDIOMYOPATHY

Cardiac tissue requires high levels of Cu to meet large amounts of energy demands by sustaining mitochondrial function (130). Animal models with a Cu-deficient diet induce cardiac hypertrophy and severe cardiovascular dysfunction (119, 150). The loss of CTR1 in the intestinal epithelium (genetically imposed Cu deficiency) also resulted in cardiac hypertrophy (138). Furthermore, a cardiac-specific Ctr1 knockout mouse showed a cardiac-specific reduction in Cu levels and cardiac hypertrophy, supporting a cardiac-intrinsic requirement to prevent hypertrophy (85). Interestingly, these cardiac-specific Ctr1 knockout mice showed increased Cu exporter ATP7A expression in both liver and intestinal enterocytes, which is associated with reduced Cu levels in the liver and concomitant increase in circulating serum Cu (85). These results suggest that the reduction in Cu levels in the heart in cardiac-specific Ctr1 knockout mice is signaled to the liver, as a major Cu storage organ, to express elevated levels of ATP7A (or ATP7B) to mobilize Cu into the periphery. Furthermore, the relevance of ATP7A to cardiac development is underscored by the congenital cardiovascular abnormalities shown in Menkes disease (67).

Cu supplementation can reverse hypertrophic cardiomyopathy in some patients. For example, the Cu supplement therapy in patients with mutations of Sco2 with severe hypertrophic cardiomyopathy reverses the hypertrophic cardiomyopathy along with significant improvement in all parameters of heart function and normalization of ECG signs and blood pressure (49). Consistent with this, in rodent models of cardiac dysfunction due to prolonged pressure overload, Cu supplementation can rescue cardiac function and improve electrical conduction (81). This Cu supplementation rescue effect is associated with restoration of VEGF expression and angiogenesis, and inhibited by anti-VEGF antibody (81). Moreover, using cardiac-specific Sco1-deficient mice, it was shown that the mitochondrial metallochaperone SCO1 maintains CTR1 at the plasma membrane to preserve Cu homeostasis in the murine heart (15).

CONCLUSIONS AND FUTURE DIRECTIONS

It is apparent that although much has been learned in recent years about the major players that are important for Cu homeostasis, the involvement of this trace metal in so many fundamental cellular processes ensures that perturbations of this system will have widespread physiological consequences. We have summarized current findings regarding biochemical aspects of Cu transporters and Cu chaperones and their roles in cardiovascular physiology and disease. It is clear that dysregulation of these Cu transport systems is likely a causative or contributing factor in many disease states. Much has been learned from studies on isolated proteins, in vitro cell culture systems, and global knockout or mutant mice. The next significant advances will rely on the development of new methods and approaches, including CRISPR Cas9 technology, cell type-specific and temporal gene targeting for each Cu transporter and Cu chaperone using the Cre-loxp system, and the new optical and physical methods of imaging Cu to find out where it is and what it does when it gets there (10, 38, 46). Understanding the roles of Cu transporters and Cu chaperones in Cu homeostasis, as well as those in redox signaling, inflammatory and immune responses, tumor progression, and cardiovascular and metabolic diseases (73, 95), will doubtlessly provide new targets for emerging therapeutic strategies.

GRANTS

Research carried out in the author’s laboratories was supported by National Institutes of Health National Heart, Lung, and Blood Institute (NHLBI) Grant R01 HL-070187 (to T. Fukai), Department of Veterans Affairs Merit Review Grant 2I01BX001232-05 (to T. Fukai), and NHLBI Grants R01 HL-133613 and R01 HL-116976 (to T. Fukai and M. Ushio-Fukai) and R01 HL-135584 (to M. Ushio-Fukai).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

T.F., M.U.-F., and J.H.K. prepared figures; T.F., M.U.-F., and J.H.K. drafted manuscript; T.F., M.U.-F., and J.H.K. edited and revised manuscript; T.F., M.U.-F., and J.H.K. approved final version of manuscript.

Glossary

ANG II

Angiotensin II

AE1

Anion exchanger 1

Atox1

Antioxidant-1

ApoE

Apolipoprotein E

BRAF

B-rapidly accelerated fibrosarcoma

CP

Ceruloplasmin

CVO

Circumventricular organ

Cu

Copper

ATP7A

ATP7B Copper-transporting p-type ATPases

CHD

Coronary heart disease

COX

Cytochrome c-oxidase

CCS

Cu chaperone for SOD1

CTR1

Copper transporter 1 (SLC31A1)

CRISPR

Clustered regulatory interspaced short palindromic repeats

Cas9

CRISPR-associated protein 9

cPLA2

Calcium-dependent phospholipase A2

DMT1

Divalent metal transporter 1

DIDS

4,4′-diisothyocyano-stilbene-2,2′-disulfonic acid

ERK

Extracellular signal-related kinase

MEK1

MAPK/ERK kinase

EEA1

Early endosome antigen 1

ECG

Electrocardiogram

ECs

Endothelial cells

ECM

Extracellular matrix

FGF

Fibroblast growth factor

GSH

Glutathione

HMG CoA

3-Hydroxy-3-methylglutaryl coenzyme A

HEK

Human embryonic kidney cells

hCTR1

Human CTR1

HFD

High-fat diet

IL-1α

Interleukin-1α

IMS

Mitochondrial intermembrane space

MAPK

Mitogen-activated protein kinase

LOX

Lysyl oxidase

MT

Metallothionein

Pt

Platinum

Pro-LOX

Proenzyme of lysyl oxidase

LOX-PP

Lysyl oxidase propeptide

PH

Pulmonary hypertension

PDGF

Platelet-derived growth factor

RAF

Rapidly accelerated fibrosarcoma

RASMs

Rat aortic smooth muscle cells

Rab

Ras-associated binding proteins

ROS

Reactive oxygen species

Sco1

Synthesis of cytochrome c-oxidase 1

SLC25A3

Solute carrier family 25 member 3

SOD

Superoxide dismutase

STEAP

Six-transmembrane epithelial antigen of the prostate

STZ

Streptozotocin

TTM

Tetrathiomolybdate

T1DM

Type 1 diabetes mellitus

T2DM

Type 2 diabetes mellitus

TGN

Trans-Golgi network

VEGF

Vascular endothelial growth factor

VSMC

Vascular smooth muscle cell