ArticleLiterature Review

A Requirement for Copper in Angiogenesis

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Abstract

Although two decades have passed since copper was shown to stimulate blood vessel formation in the avascular cornea of rabbits, only recently have clinical trials established that Cu privation by diet or by Cu chelators diminishes a tumor's ability to mount an angiogenic response. These data have shed new light on the functional role of Cu in microvessel development and, of equal importance, stimulated new nutritional models of cancer therapeutic intervention.

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... Copper (Cu) has important roles in angiogenesis through unknown mechanisms [20][21][22][23] . Cellular Cu entry occurs mainly through the Cu transporter CTR1 (ref. ...
... Furthermore, mCtr1-KO ECs showed almost complete inhibition of VEGF-induced signalling events (Fig. 2e). As Cu entry is required for activating the Cu-dependent enzyme LOX, which is involved in angiogenesis [20][21][22]33,41,42 , and for Cu binding to MEK1/2, which increases p-ERK1/2 (ref. 32 ), we examined the role of Cu in VEGF-induced signalling. ...
... How the newly identified CTR1-VEGFR2 complex interacts with VEGFR2-binding proteins to facilitate internalization is worthy of future investigation. Cu entry is involved in direct activation of MEK by Cu binding, stimulating phosphorylation of ERK 32 , growth factor signalling by FGF and insulin, and activation of the Cu enzyme LOX, which is involved in angiogenesis [20][21][22]33,41,42 . We found that VEGF-induced CTR1-Cys 189 OH formation promotes the phosphorylation of MEK1/2 and ERK1/2 in a Cu-transport-independent manner in ECs. ...
Article
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Vascular endothelial growth factor receptor type 2 (VEGFR2, also known as KDR and FLK1) signalling in endothelial cells (ECs) is essential for developmental and reparative angiogenesis. Reactive oxygen species and copper (Cu) are also involved in these processes. However, their inter-relationship is poorly understood. Evidence of the role of the endothelial Cu importer CTR1 (also known as SLC31A1) in VEGFR2 signalling and angiogenesis in vivo is lacking. Here, we show that CTR1 functions as a redox sensor to promote angiogenesis in ECs. CTR1-depleted ECs showed reduced VEGF-induced VEGFR2 signalling and angiogenic responses. Mechanistically, CTR1 was rapidly sulfenylated at Cys189 at its cytosolic C terminus after stimulation with VEGF, which induced CTR1–VEGFR2 disulfide bond formation and their co-internalization to early endosomes, driving sustained VEGFR2 signalling. In vivo, EC-specific Ctr1-deficient mice or CRISPR–Cas9-generated redox-dead Ctr1(C187A)-knockin mutant mice had impaired developmental and reparative angiogenesis. Thus, oxidation of CTR1 at Cys189 promotes VEGFR2 internalization and signalling to enhance angiogenesis. Our study uncovers an important mechanism for sensing reactive oxygen species through CTR1 to drive neovascularization.
... In 2023, Chunmei He et al. found that 11 out of 12 CRGs were up-regulated in endothelial cell expression, suggesting that CRGs may play a potential role in angiogenesis through lncRNA (177). Cuproptosis, as a landmark sign of cell death due to copper overload in vivo, is promising in inhibiting tumor angiogenesis and delaying tumor growth and metastasis and deserves the deep attention of researchers ( Figure 5). ...
... Besides affecting cancer cell metabolism directly, copper is also involved in cancer therapy by regulating hypoxia within the TME (191,192). Copper reverses the poor responsiveness of conventional cancer immunotherapy by inducing a redox reaction with simultaneous oxygen production (177). Until TME is fully formed, the tumor is noticed by the immune system. ...
Article
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As a biologically essential transition metal, copper is widely involved in various enzymatic reactions and crucial biological processes in the body. It plays an increasingly important role in maintaining normal cellular metabolism and supporting the growth and development of the human body. As a trace element, copper maintains the dynamic balance of its concentration in body fluids through active homeostatic mechanisms. Both excess and deficiency of copper ions can impair cell function, ultimately leading to cell damage and death. Cuproptosis is a novel form of cell death where copper ions cause cell death by directly binding to the lipoylated components of the citric acid cycle (CAC) in mitochondrial respiration and interfering with the levels of iron-sulfur cluster (Fe-S cluster) proteins, ultimately causing protein toxic stress. Its primary characteristics are Cu2+ concentration dependence and high expression in mitochondrial respiratory cells. Recent research has revealed that, compared to other forms of programmed cell death such as apoptosis, necrosis, and autophagy, cuproptosis has unique morphological and biochemical features. Cuproptosis is associated with the occurrence and development of various diseases, including cancer, neurodegenerative diseases, and cardiovascular diseases. This article focuses on a review of the relevance of cuproptosis in gastric cancer (GC).
... Beyond antimicrobial/antibacterial properties, bone scaffolds could advantageously benefit from other functionalities in view of favoring bone healing, which also covers the stimulation of osteogenesis (the activation of osteoblast cells and the promotion of osteointegration), as well as neo-angiogenesis (the activation of endothelial cells and the formation of new blood capillaries). Cu 2+ ions have proven to exhibit an interesting angiogenic behavior since at least 1980 [23,24] and the interest in them is extremely vivid [25][26][27][28]. The use of pro-angiogenic/osteogenic ions such as Cu 2+ appears as a relevant strategy compared to the use of growth factors, which is debated in the scientific community, as it is linked with reported side effects such as their potential carcinogenicity [29,30]. ...
... The triple cell staining ( Figure 8C(c)) showed well-organized cultures of interacting healthy cells, with elongated morphology, a prominent nucleus and a perinuclear localization of the mitochondria. The results confirm the negligible toxicity of hapCu, which is in line with a variety of previous studies addressing the biological performance of Cu-incorporated biomaterials [17,21,22,65], which further demonstrates an improvement of the angiogenic and osteogenic properties of the substituted materials [23][24][25][26][27][28]66,67]. ...
Article
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Biomimetic apatites exhibit a high reactivity allowing ion substitutions to modulate their in vivo response. We developed a novel approach combining several bioactive ions in a spatially controlled way in view of subsequent releases to address the sequence of events occurring after implantation, including potential microorganisms’ colonization. Innovative micron-sized core-shell particles were designed with an external shell enriched with an antibacterial ion and an internal core substituted with a pro-angiogenic or osteogenic ion. After developing the proof of concept, two ions were particularly considered, Ag+ in the outer shell and Cu2+ in the inner core. In vitro evaluations confirmed the cytocompatibility through Ag-/Cu-substituting and the antibacterial properties provided by Ag+. Then, these multifunctional “smart” particles were embedded in a polymeric matrix by freeze-casting to prepare 3D porous scaffolds for bone engineering. This approach envisions the development of a new generation of scaffolds with tailored sequential properties for optimal bone regeneration.
... In order to achieve the appropriate mechanical properties values and to address the problems of mechanical properties instability and strain rate sensitivity simultaneously, suitable alloying strategy is one of the solutions. Copper (Cu) is an essential trace element in the human body, and Cu ions have the functions of promoting endothelialization and antibacterial [19,29]. Besides, Cu has a medium solid solubility in Zn alloys (~2.75 wt% at 425 • C), and its solubility decreases with decreasing the temperature [30], so it is feasible Zn-Cu alloy can achieve solid solution strengthening and precipitation strengthening. ...
... mg/L that inhibited cell viability (endothelial and smooth muscle cells) was in line with previous studies [32,46,47], and tolerance of endothelial cells to Zn 2+ content is better than that of smooth muscle cells. The trace Cu 2+ release has a positive effect on endothelial cell viability [29,32], but accelerated Zn 2+ releases in 0Mn and 0.4Mn alloys could weaken or offset this effect, as shown in Fig. 12a and c. Although in vitro cytotoxicity deteriorated in 100% concentration extracts, suitable diluted extracts were recommended for in vitro cytotoxicity evaluation of pure Mg [48], and a similar method should be applicable to biodegradable Zn-based alloys. ...
Article
Recently, zinc (Zn) alloy has been considered as a promising biodegradable material due to its excellent physiological degradable behavior and acceptable biocompatibility. However, poor mechanical performance limits its application as vascular stents. In this study, novel biodegradable Zn-2.2Cu-xMn (x = 0.4, 0.7, and 1.0 wt%) alloys with suitable mechanical performance were investigated. The effects of Mn addition on microstructure, mechanical properties, and in vitro degradation of Zn-2.2Cu-xMn alloys were systematically investigated. After adding Mn, dynamic recrystallization (DRX) during hot extrusion was promoted, resulting in slightly finer grain size, higher DRXed regions ratio, and weaker texture. And volume fraction and number density of second phase precipitates (micron, submicron, and nano-sized ε and MnZn13 phase) and the concentration of (Cu, Mn) in the matrix were increased. Therefore, Zn-2.2Cu-xMn alloys exhibited suitable mechanical performances (strength >310 MPa, elongation >30%) mainly due to the combination effects of grain refinement, solid solution strengthening, second phase precipitation hardening, and texture weakening. Moreover, the alloys maintained good stability of mechanical properties within 18 months and good elongation over 15% even at a high strain rate of 0.1 s⁻¹. In addition, the alloys presented appropriate in vitro degradation rates in a basically uniform degradation mode and acceptable in vitro cytocompatibility. The above results indicated that the newly designed biodegradable Zn-2.2Cu-0.4Mn alloy with suitable comprehensive mechanical properties, appropriate degradation behavior, and acceptable cytocompatibility is a promising candidate for vascular stents.
... Increasing copper-containing biomaterials have been developed and explored in the field of medical implants in recent years [11][12][13]. Related studies suggested that Cu could increase the expression of VEGF, thereby stimulating the proliferation and migration of endothelial cells to promote the formation of new blood vessels [14,15]. The addition of copper in bioactive glass nanofibers spontaneously improves the vascularization during new bone formation by promoting osteoblastic and endothelial cell activity [16]. ...
Article
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Copper is an essential trace element for the human body. Abnormalities in copper metabolism can lead to bone defects, mainly by directly affecting the viability of osteoblasts and osteoclasts and their bone remodeling function, or indirectly regulating bone metabolism by influencing enzyme activities as cofactors. Copper ions released from biological materials can affect osteoblasts and osteoclasts, either directly or indirectly by modulating the inflammatory response, oxidative stress, and rapamycin signaling. This review presents an overview of recent progress in the impact of copper on bone metabolism. Translational potential of this article: The impact of copper on bone metabolism can provide insights into clinical application of copper-containing supplements and biomaterials.
... Copper's therapeutic spectrum comprises by antipathogenic (antibacterial [41][42][43][44][45][46][47][48][49][50], antiviral [51][52][53][54][55][56][57][58][59][60], and antifungal [61][62][63][64][65][66][67][68][69][70] activity), anticancer [71][72][73][74][75][76][77][78][79][80], hemostatic [81][82][83][84][85][86][87][88][89][90], angiogenic [91][92][93][94][95][96][97][98][99][100], and osteogenic [96,[101][102][103][104][105][106][107][108][109] applications. ...
Article
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The paper presents the study concerning the preparation and physio-chemical and biological properties of wool–copper (WO-Cu) materials obtained by the sputter deposition of copper onto the wool fibers. The WO-Cu material was subjected to physio-chemical and biological investigations. The physio-chemical investigations included the elemental analysis of materials (C, N, O, S, and Cu), their microscopic analysis, and surface properties analysis (specific surface area and total pore volume). The biological investigations consisted of the antimicrobial activity tests of the WO-Cu materials against colonies of Gram-positive (Staphylococcus aureus) bacteria, Gram-negative (Escherichia coli) bacteria, and fungal mold species (Chaetomium globosum). Biochemical–hematological tests included the evaluation of the activated partial thromboplastin time and pro-thrombin time. The tested wool–copper demonstrated the ability to interact with the DNA in a time-dependent manner. These interactions led to the DNA’s breaking and degradation. The antimicrobial and antifungal activities of the WO-Cu materials suggest a potential application as an antibacterial/antifungal material. Wool–copper materials may be also used as customized materials where the blood coagulation process could be well controlled through the appropriate copper content.
... 40 The agent also has anti-angiogenic properties. [41][42][43] We tested the effect of the tetrathiomolybdate salt on soft agar colony formation. The drug inhibited the colony size of MDA-MB-231 cells by 43%, 75%, 93%, and 94% at 0.5, 1, 2, and 4 μg/mL, respectively. ...
Article
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Background We previously reported that metastases are generally characterized by a core program of gene expression that activates tissue remodeling/vascularization, alters ion homeostasis, induces the oxidative metabolism, and silences extracellular matrix interactions. This core program distinguishes metastases from their originating primary tumors as well as from their destination host tissues. Therefore, the gene products involved are potential targets for anti‐metastasis drug treatment. Methods Because the silencing of extracellular matrix interactions predisposes to anoiks in the absence of active survival mechanisms, we tested inhibitors against the other three components. Results Individually, the low‐specificity VEGFR blocker pazopanib (in vivo combined with marimastat), the antioxidant dimethyl sulfoxide (or the substitute atovaquone, which is approved for internal administration), and the ionic modulators bumetanide and tetrathiomolybdate inhibited soft agar colony formation by breast and pancreatic cancer cell lines. The individual candidate agents have a record of use in humans (with limited efficacy when administered individually) and are available for repurposing. In combination, the effects of these drugs were additive or synergistic. In two mouse models of cancer (utilizing 4T1 cells or B16‐F10 cells), the combination treatment with these medications, applied immediately (to prevent metastasis formation) or after a delay (to suppress established metastases), dramatically reduced the occurrence of disseminated foci. Conclusions The combination of tissue remodeling inhibitors, suppressors of the oxidative metabolism, and ion homeostasis modulators has very strong promise for the treatment of metastases by multiple cancers.
... angiogenesis (e.g., angiogenin). 22,23 A previous study has shown significantly increased serum Cu levels in pancreatic cancer patients. 13 Although we observed an increase in the Cu levels in serum and tumor tissue, these changes were not statistically significant. ...
Article
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Pancreatic ductal adenocarcinoma (PDAC) patients have late presentation at the time of diagnosis and a poor prognosis. Metal dyshomeostasis is known to play a role in cancer progression. However, the blood and tissue metallome of PDAC patients has not been assessed. This study aimed to determine the levels of essential and toxic metals in the serum and pancreatic tissue from PDAC patients. Serum samples were obtained from PDAC patients before surgical resection. Tissue (tumor and adjacent normal pancreas) were obtained from the surgically resected specimen. Inductively coupled plasma–mass spectrometry (ICP‐MS) analysis was performed to quantify the levels of 10 essential and 3 toxic metals in these samples. Statistical analysis was performed to identify dysregulated metals in PDAC and their role as potential diagnostic and prognostic biomarkers. Significantly decreased serum levels of magnesium, potassium, calcium, iron, zinc, selenium, arsenic, and mercury and increased levels of molybdenum were shown to be associated with PDAC. There were significantly decreased levels of zinc, manganese and molybdenum, and increased levels of calcium and selenium in the pancreatic tumor tissue compared with the adjacent normal pancreas. Notably, lower serum levels of calcium, iron, and selenium, and higher levels of manganese, were significantly associated with a poor prognosis (i.e., overall survival) in PDAC patients. In conclusion, this is the first study to comprehensively assess the serum and tissue metallome of PDAC patients. It identified the association of metals with PDAC diagnosis and prognosis.
... Copper is involved in iron oxidation and is frequently co-localized with iron within mitochondria [24]. Previously, copper has been shown to stimulate blood vessel formation in the avascular cornea of rabbits [25]. Copper-chitosan treatments have been demonstrated to promote corneal epithelial wound healing via nitric oxide metabolism [26]. ...
Article
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Introduction: To investigate the molecular alterations of cuproptosis-related genes and to construct the cuproptosis-related circRNA-miRNA-mRNA networks in neovascular age-related macular degeneration (nAMD). Methods: The transcriptional profiles of laser-induced choroid neovascularization (CNV) mouse models and nAMD patient samples were obtained from sequencing and from the GEO database (GSE146887), respectively. The expression levels of ten cuproptosis-related genes (FDX1, DLAT, LIAS, DLD, PDHB, MTF1, CDKN2A, GLS, LIPT1, and PDHA1) were extracted and verified in both mouse CNV models and patient peripheral blood mononuclear cells (PBMCs) samples. The cuproptosis-related circRNA-miRNA-mRNA network was further constructed based on miRNet database, the dataset GSE131646 of small RNA expression profile, and the dataset GSE140178 of circRNA expression profile in mouse CNV models. Results: The significant upregulation of Cdkn2a and Mtf1 and the downregulation of other 5 cuproptosis-related genes were verified in the mouse CNV model, but only CDKN2A significantly upregulated in PBMCs of patients with nAMD. Four miRNAs were detected in the intersection between miRNet prediction and sequencing data: miR-129-5p, miR-129-2-3p, miR-182-5p, and miR-615-3p. There were 9 circRNAs at the intersection of hsa-miR-182-5p and hsa-miR-615-3p predictions, one circRNA predicted by hsa-miR-129-5p and GSE140178 (hsa-circASH1L), and one circRNA predicted by hsa-miR-182-5p and hsa-miR-615-3p (hsa-circNPEPPS). Conclusion: This study suggested the repression of cuproptosis in nAMD pathologies and constructed a cuproptosis-related network of 8 cuproptosis-related genes, 4 miRNAs, and 11 circRNAs.
... Alloying is a typical process for improving the mechanical property of materials [16], and copper (Cu) is an effective alloying element to increase the mechanical property of pure Zn. Meanwhile, Cu is a trace element required for metabolism in the human body and has an indispensable physiological function, such as playing an essential role in bone growth, vascular endothelial cell proliferation, and reconstruction [17]. Cu deficiency affects glucose and cholesterol metabolism, increases oxidative damage, cause fetal stunting, causes electrophysiological abnormalities in the heart, impairs the contractile function of the heart muscle, and causes fetal stunting during pregnancy. ...
... For instance, Cu ions have been shown to play a crucial role in the angiogenic response [63] through control of the expression of hypoxia-inducible factor (HIF-1α), thereby simulating hypoxia, that notably contributes to formation of blood vessels [62]. Cu 2+ ions provoke the proliferation of endothelial cells and thus angiogenesis through mediation of the release of cytokines and VEGF [64][65][66]. Additionally, the release of such ions can upregulate growth factor-β (TGFβ), as a pro-angiogenic factor, in diabetic wounds [67,68], which reduces the risk of ischemia in skin flaps [69]. The presence of borate ions in human keratinocyte cultures, even in millimolar concentration, can upregulate matrix metalloproteinases MMP-2 and MMP-9, thereby driving the migration of these cells and promoting remodeling of granulation tissue [70]. ...
Article
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Since the discovery of silicate bioactive glass (BG) by Larry Hench in 1969, different classes of BGs have been researched over decades mainly for bone regeneration. More recently, validating the beneficial influence of BGs with tailored compositions on angiogenesis, immunogenicity and bacterial infection, the applicability of BGs has been extended to soft tissue repair and wound healing. Particularly, fibrous wound dressings comprising BG particle reinforced polymer nanofibers and cotton-candy-like BG fibers have been proven to be successful for wound healing applications. Such fibrous dressing materials imitate the physical structure of skin’s extracellular matrix and release biologically active ions e.g. regenerative, pro-angiogenic and antibacterial ions, e.g. borate, copper, zinc, etc., that can provoke cellular activities to regenerate the lost skin tissue and to induce new vessels formation, while keeping an anti-infection environment. In the current review, we discuss different BG fibrous materials meant for wound healing applications and cover the relevant literature in the past decade. The production methods for BG-containing fibers are explained and as fibrous wound dressing materials, their wound healing and bactericidal mechanisms, depending on the ions they release, are discussed. The present gaps in this research area are highlighted and new strategies to address them are suggested.
... Cu is a trace element that is an important component of many enzymes in the human body. Cu also plays an important role in the immune system [169,170], restoring the rate of bone resorption to normal levels [171] and promoting the deposition of collagen fibres [172]. A lack of Cu affects osteoinduction and osteoclast activity [173], in addition to Cu's ability to stimulate endothelial cell proliferation and promote angiogenesis [174,175]. ...
Article
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Magnesium and magnesium alloys have great application potential in the field of orthopaedics. Compared with traditional inorganic nonmetallic materials and medical polymer materials, magnesium alloys have many advantages, such as better strength, toughness, fatigue resistance, and easy processing. Its mechanical properties are suitable and controllable. It can meet the same elastic modulus, cell compatibility, and biodegradability as human cortical bone. There are also some drawbacks for biodegradability, as magnesium and its alloys, with their high degradation rate, can cause insufficient integrity of the mechanical properties. This paper summarises the research on magnesium and its magnesium alloy materials in the field of bone implantation, looking at what magnesium and its magnesium alloys are, the history of magnesium alloys in bone implant materials, the manufacturing of magnesium alloys, the mechanical properties of magnesium alloys, the bio-compatibility and clinical applications of magnesium alloys, the shortcomings, and the progress of research in recent years.
... Lysyl oxidase is an enzyme that catalyses the formation of aldehyde-based crosslinks in collagen [33]. Copper has also been found to be an essential element for angiogenesis, leading to hypotheses regarding its value in promoting the vascularisation of bone [629][630][631][632][633]. It has been shown that copper-containing scaffolds in vivo are able to promote blood vessel formation due to superior collagen formation. ...
... This suggested that MG63/HUVECs coculture facilitated VEGFA secretion and the interactive effects caused by Ce-Cu co-incorporation further strengthened it, which in turn upregulated VEGFR1 expressions of the HUVECs. We subsequently conducted tube formation assay and analyzed relevant statistics (Fig. 5E and 5F), which corroborated our deduction because HUVECs movement was the reflection of VEGFA consequence [59,60] and the curve variant in Fig. 5F was generally in accordance with VEGFA (Fig. 5B). ...
Article
Endowing biomaterials with functional elements enhances their biological properties effectively. However, improving bioactivity and biosafety simultaneously is still highly desirable. Herein, cerium (Ce) and copper (Cu) are incorporated into silicocarnotite (CPS) to modulate the constitution and microstructure for degradability, bioactivity and biosafety regulation. Our results demonstrated that introducing Ce suppressed scaffold degradation, while, co-incorporation of both Ce and Cu accelerated degradability. Osteogenic effect of CPS in vitro was promoted by Ce and optimized by Cu, and Ce-induced angiogenic inhibition could be mitigated by cell coculture method and reversed by Ce–Cu co-incorporation. Ce enhanced osteogenic and angiogenic properties of CPS in a dose-dependent manner in vivo, and Cu–Ce coexistence exhibited optimal bioactivity and satisfactory biosafety. This work demonstrated that coculture in vitro was more appropriately reflecting the behavior of implanted biomaterials in vivo. Interactive effects of multi-metal elements were promising to enhance bioactivity and biosafety concurrently. The present work provided a promising biomaterial for bone repair and regeneration, and offered a comprehensive strategy to design new biomaterials which aimed at adjustable degradation behavior, and enhanced bioactivity and biosafety.
... The most notorious examples are genetic disorders (Menkes disease, occipital horn syndrome, Wilson's disease, MEDNIK-syndrome, aceruloplasminemia, and others) but at present many neurodegenerative, oncological and cardiovascular diseases are attributed to copper-related disorders. [26][27][28][29] Ag(I) ions can effectively interact with Cu(I)-binding motifs of copper transport proteins and engage in copper metabolic pathways. They can be inserted into cuproenzymes that contain cysteine residues in their active centers, 30 bind to metallothionein 31 and glutathione, 32 displace copper in odorant receptors, 33 form stable complexes with cysteine residues in classic zinc fingers and zinc fingers of soluble hormone receptors, 34 and accumulate in mitochondria and cell nuclei. ...
Article
Silver nanoparticles (AgNPs) of various size and shape are widely used because of their antibacterial properties. However, their bioactivity to mammals is insufficiently investigated. The aim of the present study...
... However, the aberrant compartmentalization of extracellular copper pools has been implicated in various disease processes, such as in promoting the aggregation of amyloid-beta peptides in Alzheimer's disease and angiogenesis in cancer progression. 6 In Wilson's disease ( WD ) , which is caused by a mutation of the copper transporter ATP7B, an elevated amount of non-CP-bound copper is observed, but the composition of the ligands for this extracellular copper pool remains unclear. 7 , 8 Elucidating extracellular components associated with copper under both healthy and disease states may expand our mechanistic understanding of extracellular copper signaling as well as contribute to the pool of potential biomarkers that report on copper status. ...
Article
Copper is essential in a host of biological processes, and disruption of its homeostasis is associated with diseases including neurodegeneration and metabolic disorders. Extracellular copper shifts in its speciation between healthy and disease states, and identifying molecular components involved in these perturbations could widen the panel of biomarkers for copper status. While there have been exciting advances in approaches for studying the extracellular proteome with mass-spectrometry-based methods, the typical workflows disrupt metal-protein interactions due to the lability of these bonds either during sample preparation or in gas-phase environments. We sought to develop and apply a workflow to enrich for and identify protein populations with copper-binding propensities in extracellular fluids using an immobilized metal affinity chromatography (IMAC) resin. The strategy was optimized using human serum to allow for maximum quantity and diversity of protein enrichment. Protein populations could be differentiated based on protein load on the resin, likely on account of differences in abundance and affinity. The enrichment workflow was applied to plasma samples from patients with Wilson disease (WD) and protein IDs and differential abundancies compared to healthy subjects were compared to those yielded from a traditional proteomic workflow. While the IMAC workflow preserved differential abundance and protein ID information from the traditional workflow, it identified several additional proteins being differentially abundant including those involved in lipid metabolism, immune system, and antioxidant pathways. Our results suggest the potential for this IMAC workflow to identify new proteins as potential biomarkers in copper-associated disease states.
... Lysyl oxidase is an enzyme that catalyses the formation of aldehyde-based crosslinks in collagen [33]. Copper has also been found to be an essential element for angiogenesis, leading to hypotheses regarding its value in promoting the vascularisation of bone [629][630][631][632][633]. It has been shown that copper-containing scaffolds in vivo are able to promote blood vessel formation due to superior collagen formation. ...
Thesis
One of the greatest requirements of modern medicine is the ability to treat patients suffering from osteoarthritis (OA) and bone fractures. Currently, there is no long-term therapy for OA; symptoms can be managed with anti-inflammatories and analgesics until they worsen to the extent that the damage becomes debilitating, and joint arthroplasty, is necessitated. However, these replacements are not perfect; firstly, there is the need for surgery and secondly, if the patient is young, the prosthetic can deteriorate, engendering further surgery. Bone fractures are regularly seen in orthopaedic clinics and are commonly repaired using fixation techniques or biomaterials. After any intervention, the fracture site can remain compromised, potentially engendering re-fracture and/or further surgical involvement. Regenerative strategies for both OA and bone fracture aim to alleviate pain, whilst maintaining or restoring damaged tissues to healthy states. Mesenchymal stem/stromal cells (MSC) are thought to facilitate tissue repair via either progenitor or secreaome functions. BM-MSC have, in previous work, been investigated as a therapy for OA via either their direct application or through their secreted Extracellular Vesicles (EV). In this study, MSC have been successfully isolated from bone marrow, and from these isolated cells, EV have been captured and characterised. The isolated EV have been shown to be readily internalised by chondrocytes and, in order to determine the method of EV internalisation by chondrocytes, in vitro drug inhibition studies were performed on labelled EV. Via inhibition of the caveolin dependent endocytosis pathway, EV uptake was prevented, thus indicating that this method of endocytosis is the method of EV internalisation. In regenerative medicine for knee OA, it is likely that MSC and EV would be injected into the knee. In order to determine if the MSC and EV would reside in the joint, both were labelled with gold nanostars and Supra Magnetic Iron Oxide Nanoparticles (SPION). These labelled cells and EV were then injected into a sheep stifle 1 week post creation of an OA model (meniscal transection model). These labelled cells and EV could then be seen within the knee for up to 4 weeks post injection, as ascertained via Magnetic Resonance Imaging (MRI) and MultiSpectral Optoacoustic Tomography (MSOT). Upon evaluating the regenerative effects of the MSC and EV, no difference in cartilage damage could be seen. During bone fracture, MSC and osteoblasts are recruited to the site of injury. Bioglasses have been used previously as a material to improve bone repair through the release of ions and conditioning the local environment. Our work has shown that conditioned media from bioglasses can influence both MSC and osteoblasts to augment the bone repair process. Through screening bioglasses on MSC and osteoblasts, the potential for bioglasses to alter MSC derived EV to promote osteogenesis has been shown. As a conclusion, this study has shown that the BM-MSC are a source of EV, and that both the MSC and EV can potentially be used in a musculoskeletal scenario of regenerative medicine.
... Angiogenesis plays a crucial role in efficient wound healing and tissue regeneration, especially in cardiovascular patch implanting position. Copper has previously been suggested to be proangiogenic by multiple mechanisms that include releasing various angiogenic factors and stimulating endothelial cell proliferation [50,51]. In this study, we found that Cu@TP-dBPs had an improved proangiogenic effect both in vitro and in vivo. ...
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Decellularization is a promising technique to produce natural scaffolds for tissue engineering applications. However, non-crosslinked natural scaffolds disfavor application in cardiovascular surgery due to poor biomechanics and rapid degradation. Herein, we proposed a green strategy to crosslink and functionalize acellular scaffolds via the self-assembly of copper@tea polyphenol nanoparticles (Cu@TP NPs), and the resultant nanocomposite acellular scaffolds were named as Cu@TP-dBPs. The crosslinking degree, biomechanics, denaturation temperature and resistance to enzymatic degradation of Cu@TP-dBPs were comparable to those of glutaraldehyde crosslinked dBPs (Glut-dBPs). Furthermore, Cu@TP-dBPs were biocompatible and had abilities to inhibit bacterial growth and promote the formation of capillary-like networks. Subcutaneous implantation models demonstrated that Cu@TP-dBPs were free of calcification and allowed for host cell infiltration at day 21. Cardiac patch graft models confirmed that Cu@TP-dBP patches showed improved ingrowth of functional blood vessels and remodeling of extracellular matrix at day 60. These results suggested that Cu@TP-dBPs not only had comparable biomechainics and biostability to Glut-dBPs, but also had several advantages over Glut-dBPs in terms of anticalcification, remodeling and integration capabilities. Particularly, they were functional patches possessing antibacterial and proangiogenic activities. These material properties and biological functions made Cu@TP-dBPs a promising functional acellular patch for cardiovascular applications.
... Cu, a key microelement essential to organisms and a cofactor of many enzymes, is involved in angiogenesis and lymphangiogenesis, such as a stimulator of angiogenesis and endothelial cell migration in the vascular system and a local regulator for the growth or regression of new blood vessels [14][15][16][17][18]. Cu depletion has been shown to inhibit angiogenesis in a wide variety of cancer cells and xenograft systems [19][20][21]. ...
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Molecular transport and cell circulation between tissues and organs through blood and lymphatic vessels are essential for physiological homeostasis in vertebrates. Despite the report of its association with vessel formation in solid tumors, the biological effects of Copper (Cu) accumulation on angiogenesis and lymphangiogenesis during embryogenesis are still unknown. In this study, we unveiled that intersegmental blood circulation was partially blocked in Cu²⁺-stressed zebrafish embryos and cell migration and tube formation were impaired in Cu²⁺-stressed mammalian HUVECs. Specifically, Cu²⁺-stressed embryos showed down-regulation in the expression of amotl2 and its downstream pERK1/2-foxm1-MMP2/9 regulatory axis, and knockdown/knockout of foxm1 in zebrafish embryos phenocopied angiogenesis defects, while FOXM1 knockdown HUVECs phenocopied cell migration and tube formation defects, indicating that excessive Cu²⁺-induced angiogenesis defects and blocked cell migration via down-regulating amotl2-pERK1/2-foxm1-MMP2/9 regulatory axis in both embryos and mammalian cells. Additionally, thoracic duct was revealed to be partially absent in Cu²⁺-stressed zebrafish embryos. Specifically, Cu²⁺-stressed embryos showed down-regulation in the expression of ccbe1 (a gene with pivotal function in lymphangiogenesis) due to the hypermethylation of the E2F7/8 binding sites on ccbe1 promoter to reduce their binding enrichment on the promoter, contributing to the potential mechanisms for down-regulation of ccbe1 and the formation of lymphangiogenesis defects in Cu²⁺-stressed embryos and mammalian cells. These integrated data demonstrate that Cu²⁺ stress impairs angiogenesis and lymphangiogenesis via down-regulation of pERK1/2-foxm1-MMP2/9 axis and epigenetic regulation of E2F7/8 transcriptional activity on ccbe1 expression, respectively.
... New blood vessels are formed in a multistep coordinated sequence, essentially comprising the breakdown of the vessel's basement membrane, the migration and proliferation of the endothelial cells within the ECM, and the tube's formation. Copper activates many components involved in angiogenesis, like VEGF, angiogenin, ceruloplasmin, SPARC, NFkB; conversely, decreased copper availability impairs their function [127]. Several studies on in vitro tumor models and xenografts address copper availability in blood vessel growth [76]. ...
Article
Copper homeostasis is strictly regulated by protein transporters and chaperones, to allow its correct distribution and avoid uncontrolled redox reactions. Several studies address copper as involved in cancer development and spreading (epithelial to mesenchymal transition, angiogenesis). However, being endogenous and displaying a tremendous potential to generate free radicals, copper is a perfect candidate, once opportunely complexed, to be used as a drug in cancer therapy with low adverse effects. Copper ions can be modulated by the organic counterpart, after complexed to their metalcore, either in redox potential or geometry and consequently reactivity. During the last four decades, many copper complexes were studied regarding their reactivity toward cancer cells, and many of them could be a drug choice for phase II and III in cancer therapy. Also, there is promising evidence of using ⁶⁴Cu in nanoparticles as radiopharmaceuticals for both positron emission tomography (PET) imaging and treatment of hypoxic tumors. However, few compounds have gone beyond testing in animal models, and none of them got the status of a drug for cancer chemotherapy. The main challenge is their solubility in physiological buffers and their different and non-predictable mechanism of action. Moreover, it is difficult to rationalize a structure-based activity for drug design and delivery. In this review, we describe the role of copper in cancer, the effects of copper-complexes on tumor cell death mechanisms, and point to the new copper complexes applicable as drugs, suggesting that they may represent at least one component of a multi-action combination in cancer therapy.
... However, the ductility significantly declined with respect to pure Zn, while Zn-Cu alloys have elongation values above 30%, which successfully accomplishes the criteria for cardiovascular stents applications [27]. Moreover, Cu is well-known for its antibacterial properties [1,[28][29][30] and it is reported to stimulate endothelial cell migration and angiogenesis [31][32][33]. Alloying with peritectic-forming elements such as Cu or eutectic-forming elements such as Mg, refines the microstructure of pure Zn and enhances its mechanical properties [34,35]. The importance of controlling topography, chemistry, and mechanical properties at micro and nanoscale is fundamental for obtaining the desired cellular response [28]. ...
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In the recent decades, zinc (Zn) and its alloys have been drawing attention as promising candidates for bio-resorbable cardiovascular stents due to its degradation rate more suitable than magnesium (Mg) and iron (Fe) alloys. However, its mechanical properties need to be improved in order to meet the criteria for vascular stents. This work investigates the mechanical properties, biodegradability and biocompatibility of Zn-Mg and Zn-Cu alloys in order to determine a proper alloy composition for optimal stent performance. Nanoindentation measurements are performed to characterize the mechanical properties at the nanoscale as a function of the Zn microstructure variations induced by alloying. The biodegradation mechanisms are discussed and correlated to microstructure, mechanical performance and bacterial/cell response. Addition of Mg or Cu alloying elements refined the microstructure of Zn and enhanced yield strength (YS) and ultimate tensile strength (UTS) proportional to the volume fraction of secondary phases. Zn-1Mg showed the higher YS and UTS and better performance in terms of degradation stability in Hanks' solution. Zn-Cu alloys presented an antibacterial effect for S. aureus controlled by diffusion mechanisms and by contact. Biocompatibility was dependent on the degradation rate and the nature of the corrosion products.
... However, unbound or "free" Cu ions are not generally encountered in cells in either valence state (for a detailed discussion of the chemical speciation of Cu in cells, see Fahrni [1]). Cu is essential for many physiological processes including iron homeostasis [2], angiogenesis [3], neurotransmitter biosynthesis [4], immune function (including neutrophil activation and macrophage function) [5] and energy metabolism [6]. ...
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Essential metals such as copper (Cu) and zinc (Zn) are important cofactors in diverse cellular processes, while metal imbalance may impact or be altered by disease state. Cu is essential for aerobic life with significant functions in oxidation-reduction catalysis. This redox reactivity requires precise intracellular handling and molecular-to-organismal levels of homeostatic control. As the central organ of Cu homeostasis in vertebrates, the liver has long been associated with Cu storage disorders including Wilson Disease (WD) (heritable human Cu toxicosis), Idiopathic Copper Toxicosis and Endemic Tyrolean Infantile Cirrhosis. Cu imbalance is also associated with chronic liver diseases that arise from hepatitis viral infection or other liver injury. The labile redox characteristic of Cu is often discussed as a primary mechanism of Cu toxicity. However, work emerging largely from the study of WD models suggests that Cu toxicity may have specific biochemical consequences that are not directly attributable to redox activity. This work reviews Cu toxicity with a focus on the liver and proposes that Cu accumulation specifically impacts Zn-dependent processes. The prospect that Cu toxicity has specific biochemical impacts that are not entirely attributable to redox may promote further inquiry into Cu toxicity in WD and other Cu-associated disorders.
... Early in 1980, McAuslan and Gole showed that Cu can stimulate blood vessel formation by introducing CuSO 4 into anterior chamber implants at micromolar levels [34]. Later studies further suggested that Cu could not only stimulate the proliferation and migration of endothelial cells to promote the formation of new blood vessels, but also inhibit the proliferation of vascular smooth muscle cells and thrombosis [22,35]. These results prompted more intense research on Cu in vessels. ...
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Copper is an indispensable trace metal element in the human body, which is mainly absorbed in the stomach and small intestine and excreted into the bile. Copper is an important component and catalytic agent of many enzymes and proteins in the body, so it can influence human health through multiple mechanisms. Based on the biological functions and benefits of copper, an increasing number of researchers in the field of biomaterials have focused on developing novel copper-containing biomaterials, which exhibit unique properties in protecting the cardiovascular system, promoting bone fracture healing, and exerting antibacterial effects. Copper can also be used in promoting incisional wounds healing, killing cancer cells, Positron Emission Tomography (PET) imaging, radioimmunological tracing and radiotherapy of cancer. In the present review, the biological functions of copper in the human body are presented, along with an overview of recent progress in our understanding of the biological applications and development of copper-containing materials. Furthermore, this review also provides the prospective on the challenges of those novel biomaterials for future clinical applications.
... The addition of these alloying element, particularly, Cu and Mn can improve the mechanical properties, not only strength, but also elongation. Cu is an essential trace element required for bone growth and connectivity, and enhances the proliferation of vascular endothelial cells, and revascularization [184,185]. In addition, Cu deficiency leads to normocytic cholesterol metabolism and neutropenia [186]. ...
Article
Biodegradable metals (BMs) gradually degrade in vivo by releasing corrosion products once exposed to the physiological environment in the body. Complete dissolution of biodegradable implants assists tissue healing, with no implant residues in the surrounding tissues. In recent years, three classes of BMs have been extensively investigated, including magnesium (Mg)-based, iron (Fe)-based, and zinc (Zn)-based BMs. Among these three BMs, Mg-based materials have undergone the most clinical trials. However, Mg-based BMs generally exhibit faster degradation rates, which may not match the healing periods for bone tissue, whereas Fe-based BMs exhibit slower and less complete in vivo degradation. Zn-based BMs are now considered a new class of BMs due to their intermediate degradation rates, which fall between those of Mg-based BMs and Fe-based BMs, thus requiring extensive research to validate their suitability for biomedical applications. In the present study, recent research and development on Zn-based BMs are reviewed in conjunction with discussion of their advantages and limitations in relation to existing BMs. The underlying roles of alloy composition, microstructure, and processing technique on the mechanical and corrosion properties of Zn-based BMs are also discussed.
... [30][31][32][33][34][35] Alternatively, copper ions (Cu 2+ ) are the bioactive components with the specific capability for promoting angiogenesis, which is originated from their performance on stabilizing the expression of hypoxia-inducible factor (HIF-1 ) and secretion of vascular endothelial growth factor (VEGF), further enhancing the recruitment and differentiation of cells during the blood vessel-producing procedure. [36][37][38][39][40][41][42] It has been fully revealed that Cu ions can facilitate the cell migration, angiogenesis, and collagen deposition for the specific biomedical implementation on accelerating wound healing. [43][44][45][46] As the necessary component for maintaining human health, the adults typically require the highest safe intake amount of Cu of 10 mg per day. ...
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As an essential trace element in the human body, transitional metal copper (Cu) ions are the bioactive components within the body featuring dedicated biological effects such as promoting angiogenesis and influencing lipid/glucose metabolism. The recent substantial advances of nanotechnology and nanomedicine promote the emerging of distinctive Cu‐involved biomaterial nanoplatforms with intriguing theranostic performances in biomedicine, which are originated from the biological effects of Cu species and the physiochemical attributes of Cu‐composed nanoparticles. Based on the very‐recent significant progresses of Cu‐involved nanotheranostics, this work highlights and discusses the principles, progresses, and prospects on the elaborate design and rational construction of Cu‐composed functional nanoplatforms for a diverse array of biomedical applications, including photonic nanomedicine, catalytic nanotherapeutics, antibacteria, accelerated tissue regeneration, and bioimaging. The engineering of Cu‐based nanocomposites for synergistic nanotherapeutics is also exemplified, followed by revealing their intrinsic biological effects and biosafety for revolutionizing their clinical translation. Finally, the underlying critical concerns, unresolved hurdles, and future prospects on their clinical uses are analyzed and an outlook is provided. By entering the “Copper Age,” these Cu‐involved nanotherapeutic modalities are expected to find more broad biomedical applications in preclinical and clinical phases, despite the current research and developments still being in infancy.
Article
Copper (Cu) is a vital micronutrient necessary for proper development and function of mammalian cells and tissues. Cu mediates the function of redox active enzymes that facilitate metabolic processes and signaling pathways. Cu levels are tightly regulated by a network of Cu-binding transporters, chaperones, and small molecule ligands. Extensive research has focused on the mammalian Cu homeostasis (cuprostasis) network and pathologies, which result from mutations and perturbations. There are roles for Cu-binding proteins as transcription factors (Cu-TFs) and regulators that mediate metal homeostasis through the activation or repression of genes associated with Cu handling. Emerging evidence suggests that Cu and some Cu-TFs may be involved in the regulation of targets related to development—expanding the biological roles of Cu-binding proteins. Cu and Cu-TFs are implicated in embryonic and tissue-specific development alongside the mediation of the cellular response to oxidative stress and hypoxia. Cu-TFs are also involved in the regulation of targets implicated in neurological disorders, providing new biomarkers and therapeutic targets for diseases such as Parkinson's disease, prion disease, and Friedreich's ataxia. This review provides a critical analysis of the current understanding of the role of Cu and cuproproteins in transcriptional regulation.
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The Zn0.6Cu wires are fabricated into stents for the potential biodegradable application of nasal wound healing. The degradation behavior of Zn0.6Cu stents in 0.9 wt% NaCl at 36.5 °C is evaluated. It shows that the untreated Zn0.6Cu stent experiences severe crevice corrosion with acceleration and autocatalytic effects within the micro-cracks and ruptures at 4.67 ± 1.15 d, with the average corrosion rate of 0.28 mm y−1. Fortunately, the anodic polarization (AP) + hydrothermal (H) conversion coating, consisting of ZnCO3, Zn(OH)2 and ZnO, could inhibit the crevice corrosion significantly by reducing the cathode/anode ratio, extending the rupture time up to 16.50 ± 2.95 d, with the average corrosion rate of 0.14 mm y−1. This research indicates that the biodegradable Zn-based stent has some potential applications in nasal wound recovery area.
Article
In-stent restenosis can be caused by the activation, proliferation and migration of vascular smooth muscle cells (VSMCs), which affects long-term efficacy of interventional therapy. Copper (Cu) has been proved to accelerate the endothelialization and reduce thrombosis formation, but little is known about its inhibition effect on the excessive proliferation of VSMCs. In this study, 316L-Cu stainless steel and L605-Cu cobalt-based alloy with varying Cu content were fabricated and their effects on surface property, blood compatibility and VSMCs were studied in vitro and in vivo. CCK-8 assay and EdU assay indicated that the Cu-bearing metals had obvious inhibitory effect on proliferation of VSMCs. Blood clotting and hemolysis tests showed that the Cu-bearing metals had good blood compatibility. The inhibition effect of the Cu-bearing metals on migration of cells was detected by Transwell assay. Further studies showed that Cu-bearing metals significantly decreased the mRNA expressions of bFGF, PDGF-B, HGF, Nrf2, GCLC, GCLM, NQO1 and HO1. The phosphorylation of AKT and Nrf2 protein expressions in VSMCs were significantly decreased by Cu-bearing metals. Furthermore, it was also found that SC79 and TBHQ treatments could recover the protein expressions of phospho-AKT and Nrf2, and their downstream proteins as well. Moreover, 316L-Cu stent proved its inhibitory action on the proliferation of VSMCs in vivo. In sum, the results demonstrated that the Cu-bearing metals possessed apparent inhibitory effect on proliferation and migration of VSMCs via regulating the AKT/Nrf2/ARE pathway, showing the Cu-bearing metals as promising stent materials for long-term efficacy of implantation.
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Chronic wounds have become the leading cause of death, particularly among diabetic patients. Chronic wounds affect ~6.5 million patients each year, according to statistics, and wound care and management incur significant financial costs. The rising prevalence of chronic wounds, combined with the limitations of current treatments, necessitates the development of new and innovative approaches to accelerate wound healing. Copper has been extensively studied for its antibacterial and anti‐inflammatory activities. Copper in its nanoparticle form could have better biological properties and many applications in health care.
Article
As a copper (Cu) transport ATPase, ATP7A plays an important role in maintaining Cu homeostasis in the body, but the developmental and physiological roles of atp7a in zebrafish embryogenesis are rarely studied. In this study, normal morphological phenotypes of atp7a−/− homozygous zebrafish were observed at both embryonic and adult stages, however, atp7a−/− larvae exhibited delayed touch response and obvious transcriptome changes. Compared with the WT (wild type), differentially expressed genes (DEGs) in atp7a−/− larvae showed the enrichment in gene ontology (GO) terms related to several processes including ATPase activity, oxidoreductase activity, active transmembrane transporter activity, ion binding, and the citrate cycle. Furthermore, decreases in both ATP content and Na+/K+-ATPase activity in atp7a−/− embryos and larvae were unveiled. 57 overlapping DEGs were found both in WT stressed with Cu and in WT mutated with atp7a, and GO term analysis indicated the enrichment in the genes related to neurexin family protein binding and neuronal cell-cell adhesion. Moreover, 42 overlapping DEGs in Cu stressed WT and Cu stressed atp7a−/− were identified. GO term analysis showed an enrichment in the genes related to heme binding, implying that Cu was independent of the integral function of atp7a to affect heme binding. In addition, genes involved in the negative regulation of angiogenesis were down-regulated in atp7a−/− mutants with and without Cu stress, which failed to occur in WT, implying that the integral function of atp7a is required for maintaining the normal expression of angiogenesis genes. The integrative data in this study demonstrated that atp7a is required for ion transport and angiogenesis, and for Cu-induced neurexin family protein binding defects, rather than for Cu-induced heme binding defects, during zebrafish embryogenesis. These findings provide possible clues for human diseases with ATP7A dysfunction and imbalanced Cu homeostasis.
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A microdots array-based fluoremetric method with superwettability profile has been developed for the simultaneous and separate detection of Fe3+ and Cu2+ ions in red wine samples. A wettable micropores array was initially designed with high density by using polyacrylic acid (PAA) and hexadecyltrimethoxysilane (HDS), followed by the NaOH etching route. Zinc metal organic frameworks (Zn-MOFs) were fabricated as the fluorescent probes to be immobilized into the micropores array to obtain the fluoremetric microdots array platform. It was found that the fluorescence of Zn-MOFs probes could decrease significantly in the presence of Fe3+ and/or Cu2+ ions towards their simultaneous analysis. Yet, the specific responses to Fe3+ ions could be expected if using histidine to chelate Cu2+ ions. Moreover, the developed Zn-MOFs-based microdots array with superwettability profile can enable the accumulation of targeting ions from the complicated samples without any tedious pre-processing. Also, the cross-contamination of different samples droplets can be largely avoided so as to facilitate the analysis of multiple samples. Subsequently, the feasibility of simultaneous and separate detection of Fe3+ and Cu2+ ions in red wine samples was demonstrated. Such a design of microdots array-based detection platform may promise the wide applications in analyzing Fe3+ and/or Cu2+ ions in the fields of food safety, environmental monitoring, and medical diseases diagnostics.
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The ratio of ligand to Cu(II) ions has an essential effect on the geometrical configuration and anti-tumour activity of metal-based complexes. In this work, we synthesised two Cu(II) thiosemicarbazone complexes, namely, [Cu(L)(Cl)] (C1) and [Cu3(L)2(Cl)4] (C2), by controlling the ratio of Cu(II) ion to ligand, to evaluate their anti-tumour activity. The ability of C1 to catalyze hydrogen peroxide to produce reactive oxygen species (ROS) was significantly higher than that of Cu(II) ion. Moreover, the bridge of Cu(II) and two molecules generated a new complex (C2), which, in contrast to C1, enhanced the generation of Fenton-like-triggered ROS. Consequently, the produced ROS depleted reduced glutathione, caused oxidative cell stress and promoted apoptosis through mitochondrial apoptotic pathways. In addition, C2 exhibited better tumour suppression than C1 in a nude mouse tumour xenograft model.
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Intrauterine adhesions (IUAs) caused by mechanical damage or infection increase the risk of infertility in women. Although numerous physical barriers such as balloon or hydrogel are developed for the prevention of IUAs, the therapeutic efficacy is barely satisfactory due to limited endometrial healing, which may lead to recurrence. Herein, a second near‐infrared (NIR‐II) light‐responsive shape memory composite based on the combination of cuprorivaite (CaCuSi4O10) nanosheets (CUP NSs) as photothermal conversion agents and polymer poly(d,l‐lactide‐co‐trimethylene carbonate) (PT) as shape memory building blocks is developed. The as‐prepared CUP/PT composite possesses excellent shape memory performance under NIR‐II light, and the improved operational feasibility as an antiadhesion barrier for the treatment of IUAs. Moreover, the released ions (Cu, Si) can stimulate the endometrial regeneration due to the angiogenic bioactivity. This study provides a new strategy to prevent IUA and restore the injured endometrium relied on shape memory composite with enhanced tissues reconstruction ability. A second near‐infrared light‐responsive shape memory composite, which is composed of cuprorivaite (CaCuSi4O10) nanosheets as photothermal conversion agents and polymer poly(d,l‐lactide‐co‐trimethylene carbonate) as shape memory building blocks, is developed for intrauterine adhesions prevention and endometrial regeneration.
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Chronic bacterial infection, local inflammation, and insufficient angiogenesis contribute to poor healing of diabetic wounds. Here, Cu2O/Pt nanocubes (CPN) are successfully developed with good biocompatibility for treatment of diabetic wounds in rats. The synthesized CPN are characterized using SEM, XPS, and XRD. CPN exhibit triple-enzyme mimetic activity: oxidase-like, peroxidase-like, and catalase-like activities. Moreover, CPN show significant antibacterial activity against Gram-negative and Gram-positive bacteria when combined with low concentration of H2O2, via generation of highly reactive ROS. CPN also exhibit significantly accelerated wound healing in a full-layer deprivation rat model infected by Staphylococcus aureus, which is ascribed to the constant release of copper ions, subsequently activating the VEGF/AKT/ERK1/2 signaling pathway and promoting angiogenesis. CPN are able to catalyze H2O2 to generate O2 for local hypoxia alleviation. Furthermore, in vivo results indicate that treatment with CPN promotes the expression of transforming growth factor and matrix metalloproteinases, causing enhanced cell proliferation and collagen deposition, as well as extracellular matrix remodeling. In contrast, CPN decrease the expression of proinflammatory cytokines, such as TNF-ɑ and IL-1β, which are induced by bacterial infection and hyperglycemia. These results suggest a novel strategy for the treatment of diabetic wound healing.
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In this work, mechanical properties, degradation behavior and biocompatibility of as-extruded and solid solution treated Mg-Cu binary alloy with 0.22 and 0.45 wt.% Cu additions have been investigated, and the effects of main microstructural parameters (grain size, dislocation, second phase distribution and content) on these properties are analyzed. Mechanical tests revealed that as-solutionized Mg-Cu alloys (with larger grain size and less content of Mg2Cu phase) exhibited better mechanical properties than the as-extruded alloys. Strengthening effects of the microstructural parameters have been analyzed and quantified. The results suggested that the inverse elevation of the yield strength was mainly related to the over-solubility Cu atom in the matrix. Immersion tests showed that the solid solution treatment improved the corrosion resistance of Mg-0.2Cu alloy (with 0.22 wt.% Cu) but deteriorated it seriously for Mg-0.5Cu alloy (with 0.45 wt.% Cu). The enhanced corrosion resistance of Mg-0.2Cu alloy was owing to the apparent decrease of Mg2Cu phase content. In contrast, the deterioration of corrosion resistance for Mg-0.5Cu alloy was attributed to the distribution change of the Mg2Cu phase from diffuse to local, because large localized Mg2Cu particles can lead to severer corrosion of the matrix. Cytotoxicity experiments revealed that the as-solutionized Mg-0.2Cu alloy with the least Mg2Cu phase content exhibited the best biocompatibility due to the slowest degradation rate.
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Near-infrared (NIR) light-responsive shape-memory composites have shown great potential in various biomedical applications due to their amazing remote actuation property. However, most of them are bioinert and restricted in NIR-I biological window. Here, we developed a new biodegradable NIR-II-responsive shape memory composite by the incorporation of CaSiCu4O10 nanosheets (CUP NSs) into shape memory polymer of poly (D, L-lactide-co-trimethylene carbonate) (PDLLA-co-TMC, denoted as PT). The as-prepared CUP/PT composites possessed outstanding shape memory performance upon low power NIR-II laser irradiation (1064 nm, 0.2 W·cm⁻²), offering the possibility of deep-tissue applications. Moreover, due to the sustained release of bioactive Cu²⁺ and SiO4⁴⁻ ions, the composites exhibited strong stimulation of angiogenesis both in vitro and in vivo. Our work demonstrated the promising potential of CUP/PT composites as NIR-II light-responsive biomaterials with enhanced tissue reconstruction ability, especially for wound healing.
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Traditional bone wax has lots of shortcomings such as the risk of infection and inflammation and the ability to hinder osteogenesis that limit its clinical applications. In this study, we designed a novel biodegradable bone wax with desirable angiogenic and antibacterial ability and low foreign body reaction by mixing calcium sulfate, poloxamer, and cupric ions. To evaluate its biocompatibility and angiogenetic effect in vitro, we cultured human umbilical vein endothelial cells (HUVECs) with the indicated bone wax to observe cell viability and vessel-like tubular formation. The bone wax was then implanted in a critical-sized bone defect rat model for 4 and 8 weeks to successfully stimulate angiogenesis in vivo. Finally, the bone wax extract was incubated with Gram-positive Staphylococcus aureus to confirm its antibacterial ability. The copper-loaded biodegradable bone wax overcomes the drawbacks of traditional bone wax and provides a new approach for the treatment of bone injuries.
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In this study, multifunctional tantalum copper composite nanotubes (TaCu-NTs) were coated on titanium for enhanced bacteriostatic, angiogenic and osteogenic properties. Three coatings of Ta, TaCu1 (Ta: Cu = 4:1 at.%), and TaCu2 (Ta: Cu = 1:1 at.%) were deposited on titanium by magnetron sputtering. The bare titanium and the three coatings were subsequently anodized into four kinds of nanotubes (NT) of TNT, Ta-NT, TaCu1-NT, and TaCu2-NT, respectively. The released copper ions measured by inductively coupled plasma atomic emission spectroscopy (ICP/AES) presented that TaCu2-NT coating released the highest amount of copper ions, which led to the best bacteriostasis against Escherichia coli and Staphylococcus aureus. Potentiodynamic polarization tests clarified that Ta-NT showed the highest corrosion resistance, followed by TaCu1-NT and TaCu2-NT. TaCu2-NT showed not only the best angiogenic property in terms of cell migration, tube formation, and real-time quantitative polymerase chain reaction (RT-qPCR) of human umbilical vein endothelial cells (HUVECs), but also the best osteogenic property in terms of cell viability, alkaline phosphatase activity, and mineralization of MC3T3-E1 cells. Therefore, TaCu2-NT coating has a greater potential than the other coatings of TNT, Ta-NT and TaCu1-NT in promoting bacteriostasis, angiogenesis and osteointegration for titanium implants.
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Bone related-bacterial diseases including wound infections and osteomyelitis (OM) remain a serious problem accompanied with amputation in most severe cases. In this work, we report an exceptional effective antibacterial alginate aerogel, which consists of tigecycline (TGC) and octahedral Cu crystal as an organo-inorganic synergy platform for antibacterial and local infection therapy applications. The alginate aerogel could greatly prolong the release of copper ions and maintain effective antibacterial concentration over 18 days. The result of in-vitro experiments demonstrated that the alginate aerogel has an exceptional effective function on antibacterial activity. Cytotoxicity tests indicated that the alginate aerogel has low biological toxicity (average cell viability >75%). These remarkable results suggested that the alginate aerogel exhibits great potential for the treatment of OM, and has a prosperous future of application in bone tissue engineering.
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Caffeic acid phenethyl ester (CAPE) is an active polyphenol of propolis from honeybee hives, and exhibits antioxidant and interesting pharmacological activities. However, in this study, we found that in the presence of Cu(II), CAPE exhibited pro-oxidative rather than antioxidant effect: synergistic DNA damage was induced by the combination of CAPE and Cu(II) together as measured by strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, which is dependent on the molar ratio of CAPE:Cu(II). Production of Cu(I) and H2O2 from the redox reaction between CAPE and Cu(II), and subsequent •OH formation was found to be responsible for the synergistic DNA damage. DNA sequencing investigations provided more direct evidence that CAPE/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Interestingly, we found there are competitive binding between CAPE and DNA with Cu(II)/Cu(I), which changed the redox activity of Cu(II)/Cu(I), via complementary applications of different analytical methods. The observed DNA damage was mainly attributed to the formation of DNA-Cu(II)/Cu(I) complexes, which is still redox active and initiated the redox reaction near the binding site between copper and DNA. Based on these data, we proposed that the synergistic DNA damage induced by CAPE/Cu(II) might be due to the competitive binding between CAPE and DNA with Cu, and site-specific production of •OH near the binding site of copper with DNA. Our findings may have broad biological implications for future research on the pro-oxidative effects of phenolic compounds in the presence of transition metals.
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The effect of dietary copper deficiency on tumour growth, neovascularisation and microvascular integrity was studied in the rat cremaster muscle. Male, weanling Sprague-Dawley rats were fed purified diets which were copper deficient (< 0.5 micrograms g-1 of diet) or copper adequate (5 micrograms g-1 of diet). Seven days after initiation of diets, a chondrosarcoma was implanted in the cremaster muscle of each rat. Five, 10 or 20 days after tumour implantation, rats were anesthetised and their cremasters prepared for observation by intravital microscopy. Intraarterial injection of fluorescein isothiocyanate-conjugated albumin and subsequent observation of fluorescence in the perivascular space indicated no difference in microvascular albumin leakage between the tumour vasculature of copper deficient and copper adequate rats. Neither tumour growth (assessed by wet weight), vascular density (assessed by light microscopy), nor any ultrastructural characteristics of the tumour or its vasculature (assessed by electron microscopy) were affected by copper deficiency. In view of findings by others which indicate changes in tumour characteristics with copper deficiency, we conclude that the copper dependency of tumour growth and vascularisation is a function of the type of tumour, the host tissue, or the conditions of copper depletion.
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Although the angiogenic proteins acidic fibroblast growth factor (FGF-1) and basic fibroblast growth factor (FGF-2) both interact with the transition metal copper, itself a putative modulator of angiogenesis, a role for copper in FGF function has not been established. Using nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we detect the complete conversion of recombinant forms of human FGF-1 monomer protein to FGF-1 homodimers after exposure to copper ions. In contrast, not all forms of bovine FGF-1 isolated from bovine brain or a recombinant preparation of human FGF-2 completely formed homodimers after exposure to copper ions under similar conditions. Since the copper-induced FGF-1 homodimers reverted to the monomer form in the presence of dithiothreitol, specific alkylation of cysteine residues by pyridylethylation prevented FGF-1 homodimer formation, and preformed FGF-1 homodimers could not be dissociated by the metal chelator EDTA, FGF-1 dimer formation appeared to result from the formation of intermolecular disulfide bonds by copper-induced oxidation of sulfhydryl residues. FGF-1 homodimers bound with similar apparent affinity as FGF-1 monomers to immobilized copper ions, both eluting at 60 mM imidazole. Both human FGF-1 monomer and dimer forms had a 6-fold higher apparent affinity for immobilized copper ions, as compared with human FGF-2, which eluted in the monomer form at 10 mM imidazole. Further, in contrast to FGF-1 monomers, which dissociate from immobilized heparin in 1.0 M NaCl, preformed FGF-1 homodimers had reduced apparent affinity for immobilized heparin and eluted at 0.4 M NaCl. In contrast, the apparent affinity of human FGF-2 for immobilized heparin was unaffected after exposure to copper ions. Heparin appeared to modulate the formation of copper-induced intermolecular disulfide bonds for FGF-1 but not FGF-2, since co-incubation of heparin and copper with FGF-1 monomers resulted in dimers and other oligomeric complexes. FGF-1 copper-induced homodimers failed to induce mitogenesis in [3H]thymidine incorporation assays, an effect which could be reversed by treatment with dithiothreitol, whereas FGF-2-induced mitogenic activity was relatively unaffected by pretreatment with copper. The differences between human FGF-1 and FGF-2 in protein-copper interactions may be due to differing free thiol content and arrangement between the two proteins. A recombinant human FGF-1 mutant containing the two cysteines conserved throughout the FGF family of proteins but lacking a cysteine residue (Cys 131) present in wild-type human FGF-1 but not human FGF-2 readily formed copper-induced dimers.(ABSTRACT TRUNCATED AT 400 WORDS)
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Microvascular proliferation, a hallmark of malignant brain tumors, represents an attractive target of anticancer research, especially because of the quiescent nonproliferative endothelium of the normal brain. Cerebral neoplasms sequester copper, a trace metal that modulates angiogenesis. Using a rabbit brain tumor model, normocupremic animals developed large vascularized VX2 carcinomas. By contrast, small, circumscribed, relatively avascular tumors were found in the brains of rabbits copper-depleted by diet and penicillamine treatment (CDPT). The CDPT rabbits showed a significant decrease in serum copper, copper staining of tumor cell nuclei, microvascular density, the tumor volume, endothelial cell turnover, and an increase in the vascular permeability (breakdown of the blood-brain barrier), as well as peritumoral brain edema. In non-tumor-bearing animals, CDPT did not alter the vascular permeability or the brain water content. CDPT also inhibited the intracerebral growth of the 9L gliosarcoma in F-344 rats, with a similar increase of the peritumoral vascular permeability and the brain water content. CDPT failed to inhibit tumor growth and the vascularization of the VX2 carcinoma in the thigh muscle or the metastases to the lung, findings that may reflect regional differences in the responsiveness of the endothelium, the distribution of copper, or the activity of cuproenzymes. Metabolic and pharmacologic withdrawal of copper suppresses intracerebral tumor angiogenesis; angiosuppression is a novel biologic response modifier for the in situ control of tumor growth in the brain.
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To investigate the effects of D-penicillamine (D-Pen) on angiogenesis, we have studied the effects of this drug on in vitro proliferation of human endothelial cells (EC) and in vivo corneal neovascularization. D-Pen, in the presence of copper sulfate, suppressed tritiated thymidine ([3H]TdR) incorporation into EC in a dose-dependent manner. Significant inhibition was observed with D-Pen concentrations attainable in the serum and tissues of treated patients. Neither D-Pen nor copper ion alone significantly affected [3H]TdR incorporation into EC. The inhibition by D-Pen and copper was blocked by catalase (CAT) or horseradish peroxidase but not by boiled CAT or SOD. When rabbits were daily injected intravenously with D-Pen at the per kilogram dosage administered to rheumatoid patients, neovascularization as quantitated by the proliferation of corneal new blood vessels was significantly inhibited. These results suggest that hydrogen peroxide generated by D-Pen and copper exerts a pronounced antiangiogenic effect through inhibition of EC proliferation. It is, therefore, considered that D-Pen may suppress rheumatoid synovitis by reducing the number of small blood vessels available for the emigration of chronic inflammatory cells, and the proliferation of the synovial tissue.
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Salts of CuI, II, NiII, SnII, InIII and a sub-fraction of the cupoprotein ceruloplasmin induced phagokinesis of cultured aortal endothelial cells. A variant aortal endothelial cell line was highly sensitive; cells travelled up to 1000 μm in 24 h in response to 2× 10−6 M SnCl2. Other metal ions tested (ZnII, CoII, MnII, CrII, FeIII, AlIII, SbIII and MoII) were not active. The motility response of endothelial cells to Cu ions in vitro is proposed as a model system for studying early events in neovascularization and as a sensitive assay for detecting angiogenic activity in fractions from cells and tissues.
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SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113-130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo.
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High affinity fibroblast growth factor (FGF) receptors contain a cluster of acidic amino acids in their extracellular domains that is reminiscent of the calcium binding domains of some cell adhesion molecules. Based on this observation, we used a calcium blotting technique to show that FGFR-1 binds calcium and that calcium binding is not observed in a mutagenized form of the receptor that lacks the acidic box region. The acidic box also binds other divalent cations, including copper. This latter interaction appears unique since the binding of copper to FGFR-1 mediates the binding of the receptor to immobilized heparin. While this observation may help explain the angiogenic properties of copper, divalent cation binding to FGF receptors may also mediate the interaction between FGF receptors, cell adhesion molecules and other proteoglycan components of the extracellular matrix.
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Preclinical and in vitro studies have determined that copper is an important cofactor for angiogenesis. Tetrathiomolybdate (TM) was developed as an effective anticopper therapy for the initial treatment of Wilson's disease, an autosomal recessive disorder that leads to abnormal copper accumulation. Given the potency and uniqueness of the anticopper action of TM and its lack of toxicity, we hypothesized that TM would be a suitable agent to achieve and maintain mild copper deficiency to impair neovascularization in metastatic solid tumors. Following preclinical work that showed efficacy for this anticopper approach in mouse tumor models, we carried out a Phase I clinical trial in 18 patients with metastatic cancer who were enrolled at three dose levels of oral TM (90, 105, and 120 mg/day) administered in six divided doses with and in-between meals. Serum ceruloplasmin (Cp) was used as a surrogate marker for total body copper. Because anemia is the first clinical sign of copper deficiency, the goal of the study was to reduce Cp to 20% of baseline value without reducing hematocrit below 80% of baseline. Cp is a reliable and sensitive measure of copper status, and TM was nontoxic when Cp was reduced to 15-20% of baseline. The level III dose of TM (120 mg/ day) was effective in reaching the target Cp without added toxicity. TM-induced mild copper deficiency achieved stable disease in five of six patients who were copper deficient at the target range for at least 90 days.
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Fibroblast growth factor (FGF) 1 is known to be released in response to stress conditions as a component of a multiprotein aggregate containing the p40 extravescicular domain of p65 synaptotagmin (Syt) 1 and S100A13. Since FGF1 is a Cu2+-binding protein and Cu2+ is known to induce its dimerization, we evaluated the capacity of recombinant FGF1, p40 Syt1, and S100A13 to interact in a cell-free system and the role of Cu2+ in this interaction. We report that FGF1, p40 Syt1, and S100A13 are able to bind Cu2+ with similar affinity and to interact in the presence of Cu2+ to form a multiprotein aggregate which is resistant to low concentrations of SDS and sensitive to reducing conditions and ultracentrifugation. The formation of this aggregate in the presence of Cu2+ is dependent on the presence of S100A13 and is mediated by cysteine-independent interactions between S100A13 and either FGF1 or p40 Syt1. Interestingly, S100A13 is also able to interact in the presence of Cu2+ with Cys-free FGF1 and this observation may account for the ability of S100A13 to export Cys-free FGF1 in response to stress. Lastly, tetrathiomolybdate, a Cu2+ chelator, significantly represses in a dose-dependent manner the heat shock-induced release of FGF1 and S100A13. These data suggest that S100A13 may be involved in the assembly of the multiprotein aggregate required for the release of FGF1 and that Cu2+ oxidation may be an essential post-translational intracellular modifier of this process.
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Angiogenesis plays a central role in wound healing. Among many known growth factors, vascular endothelial growth factor (VEGF) is believed to be the most prevalent, efficacious, and long-term signal that is known to stimulate angiogenesis in wounds. Whereas a direct role of copper to facilitate angiogenesis has been evident two decades ago, the specific targets of copper action remained unclear. This report presents first evidence showing that inducible VEGF expression is sensitive to copper and that the angiogenic potential of copper may be harnessed to accelerate dermal wound contraction and closure. At physiologically relevant concentrations, copper sulfate induced VEGF expression in primary as well as transformed human keratinocytes. Copper shared some of the pathways utilized by hypoxia to regulate VEGF expression. Topical copper sulfate accelerated closure of excisional murine dermal wound allowed to heal by secondary intention. Copper-sensitive pathways regulate key mediators of wound healing such as angiogenesis and extracellular matrix remodeling. Copper-based therapeutics represents a feasible approach to promote dermal wound healing.
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The release of signal peptideless proteins occurs through nonclassical export pathways and the release of fibroblast growth factor (FGF)1 in response to cellular stress is well documented. Although biochemical evidence suggests that the formation of a multiprotein complex containing S100A13 and Synaptotagmin (Syt)1 is important for the release of FGF1, it is unclear where this intracellular complex is assembled. As a result, we employed real-time analysis using confocal fluorescence microscopy to study the spatio-temporal aspects of this nonclassical export pathway and demonstrate that heat shock stimulates the redistribution of FGF1 from a diffuse cytosolic pattern to a locale near the inner surface of the plasma membrane where it colocalized with S100A13 and Syt1. In addition, coexpression of dominant-negative mutant forms of S100A13 and Syt1, which both repress the release of FGF1, failed to inhibit the stress-induced peripheral redistribution of intracellular FGF1. However, amlexanox, a compound that is known to attenuate actin stress fiber formation and FGF1 release, was able to repress this process. These data suggest that the assembly of the intracellular complex involved in the release of FGF1 occurs near the inner surface of the plasma membrane and is dependent on the F-actin cytoskeleton.
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Copper plays an essential role in promoting angiogenesis. Tumors that become angiogenic acquire the ability to enter a phase of rapid growth and exhibit increased metastatic potential, the major cause of morbidity in cancer patients. We report that copper deficiency induced by tetrathiomolybdate (TM) significantly impairs tumor growth and angiogenesis in two animal models of breast cancer: an inflammatory breast cancer xenograft in nude mice and Her2/neu cancer-prone transgenic mice. In vitro, TM decreases the production of five proangiogenic mediators: (a) vascular endothelial growth factor; (b) fibroblast growth factor 2/basic fibroblast growth factor; (c) interleukin (IL)-1alpha; (d) IL-6; and (e) IL-8. In addition, TM inhibits vessel network formation and suppresses nuclear factor (NF)kappaB levels and transcriptional activity. Our study suggests that a major mechanism of the antiangiogenic effect of copper deficiency induced by TM is suppression of NFkappaB, contributing to a global inhibition of NFkappaB-mediated transcription of proangiogenic factors.
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Tetrathiomolybdate (TM), a copper-lowering agent, has been shown in preclinical murine tumor models to be antiangiogenic. We evaluated the antitumor activity of TM in patients with advanced kidney cancer in a Phase II trial. Fifteen patients with advanced kidney cancer were eligible to participate in this trial. TM was initiated p.o. at 40 mg three times a day with meals and 60 mg at bedtime to deplete copper. A target serum ceruloplasmin (CP) level of 5-15 mg/dl was defined as copper depletion. Doses of TM were reduced for grade 3-4 toxicity and to maintain a CP level in the target range. Once copper depletion was attained, patients underwent baseline tumor measurements and then again every 12 weeks for response assessment. Patients not exhibiting progressive disease at 12 weeks after copper depletion continued on treatment. Serum levels of Interleukin (IL)-6, IL-8, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assayed pretreatment and at various time points on treatment. Dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI) was performed on selected patients in an attempt to assess changes in tumor vascularity. All of the patients rapidly became copper depleted. Thirteen patients were evaluable for response. No patient had a complete response or PR. Four patients (31%) had stable disease for at least 6 months during copper depletion (median, 34.5 weeks). TM was well tolerated, with dose reductions most commonly occurring for grade 3-4 granulocytopenia of short duration not associated with febrile episodes. Serum levels of IL-6, IL-8, VEGF, and bFGF did not correlate with clinical activity. Serial DCE-MRI was performed only in four patients, and a decrease in vascularity seemed to correlate with necrosis of a tumor mass associated with tumor growth. TM is well tolerated and consistently depletes copper as measured by the serum CP level. Clinical activity was limited to stable disease for a median of 34.5 weeks in this Phase II trial in patients with advanced kidney cancer. Serum levels of proangiogenic factors IL-6, IL-8, VEGF, and bFGF may correlate with copper depletion but not with disease stability in this small cohort. TM may have a role in the treatment of kidney cancer in combination with other antiangiogenic therapies.
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The induction of an acute inflammatory response followed by the release of polypeptide cytokines and growth factors from peripheral blood monocytes has been implicated in mediating the response to vascular injury. Because the Cu2+-binding proteins IL-1alpha and fibroblast growth factor 1 are exported into the extracellular compartment in a stress-dependent manner by using intracellular Cu2+ to facilitate the formation of S100A13 heterotetrameric complexes and these signal peptideless polypeptides have been implicated as regulators of vascular injury in vivo, we examined the ability of Cu2+ chelation to repress neointimal thickening in response to injury. We observed that the oral administration of the Cu2+ chelator tetrathiomolybdate was able to reduce neointimal thickening after balloon injury in the rat. Interestingly, although immunohistochemical analysis of control neointimal sections exhibited prominent staining for MAC1, IL-1alpha, S100A13, and the acidic phospholipid phosphatidylserine, similar sections obtained from tetrathiomolybdate-treated animals did not. Further, adenoviral gene transfer of the IL-1 receptor antagonist during vascular injury also significantly reduced the area of neointimal thickening. Our data suggest that intracellular copper may be involved in mediating the response to injury in vivo by its ability to regulate the stress-induced release of IL-1alpha by using the nonclassical export mechanism employed by human peripheral blood mononuclear cells in vitro.
Article
SPARC is a transiently expressed extracellular matrix-binding protein that alters cell shape and regulates endothelial cell proliferation in vitro. In this study, we show that SPARC mRNA and protein are synthesized by endothelial cells during angiogenesis in vivo. SPARC and peptides derived from a cationic region of the protein (amino acids 113-130) stimulated the formation of endothelial cords in vitro; moreover, these peptides stimulated angiogenesis in vivo. Mapping of the active domain demonstrated that the sequence KGHK was responsible for most of the angiogenic activity; substitution of the His residue decreased the effect. We found that proteolysis of SPARC provided a source of KGHK, GHK, and longer peptides that contained these sequences. Although the Cu(2+)-GHK complex had been identified as a mitogen/morphogen in normal human plasma, we found KGHK and longer peptides to be potent stimulators of angiogenesis. SPARC113-130 and KGHK were shown to bind Cu2+ with high affinity; however, previous incubation with Cu2+ was not required for the stimulatory activity. Since a peptide from a second cationic region of SPARC (SPARC54-73) also bound Cu2+ but had no effect on angiogenesis, the angiogenic activity appeared to be sequence specific and independent of bound Cu2+. Thus, specific degradation of SPARC, a matrix-associated protein expressed by endothelial cells during vascular remodeling, releases a bioactive peptide or peptides, containing the sequence (K)GHK, that could regulate angiogenesis in vivo.
Article
Objective: To determine whether tetrathiomolybdate (TM), a powerful chelator of copper, is capable of lowering the body stores of copper and suppressing the growth of head and neck squamous cell carcinoma in an orthotopic murine model. Study design: In vivo, murine model. Methods: Twelve 8-week-old male C3H/HeJ mice were assigned to either a TM treatment group (n = 7) or a control group (n = 5). Serum samples were obtained from a single mouse in each group to measure the level of ceruloplasmin as a surrogate marker of total body copper on days 0, 4, and 7. Mice in both groups received a floor-of-mouth injection of 1.5 x 105 SCC VII/SF cells. After 7 to 10 days of tumor growth the treatment group received fresh water daily, to which TM was added to achieve an oral intake of 50 mg per mouse. The control group received only fresh drinking water daily. Tumor volume measurements were obtained every other day. Microvessel density counts were assessed in the tumors by Factor VIII analysis. Results: Measurable tumor growth was achieved in 100% of the mice by the tenth day. Total body copper was reduced by 28% from baseline levels in mice in the treatment group. The difference in mean tumor volume in the control group was 4.7 times greater than the TM-treated group at the completion of treatment (3004 mm3 and 633mm3, respectively). This accounted for an overall suppression rate of 79% (P =.008; two-tailed Student t test). In addition, microvessel density was reduced by 50% in the TM-treated group. Conclusion: In this initial study, the first of its kind in head and neck squamous cell carcinoma, we have demonstrated the ability of TM to significantly suppress both the growth of squamous cell carcinoma and tumor vascularity in this orthotopic murine model, suggesting its potential for efficacy in the treatment of this disease in humans.
Article
Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 μM CuSO4 in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-α, transforming growth factor-β, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1α. Moreover, despite the previous observations that copper increased total specific binding of 125I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis. J. Cell. Biochem. 69:326–335, 1998. © 1998 Wiley-Liss, Inc.
Article
Copper, cadmium, and disulfiram (an ionophore for copper) modulate the synthesis of several polypeptides in two clonal lines of bovine aortal endothelial cells. After treatment of type 1 endothelial cells with 10 ⁻³ M CuSO 4 or 10 ⁻⁵ M CdCl 2 four cell‐associated polypeptides (M r = 28,000, 32,000, 73,000, and 83,000 daltons) were induced. In contrast, in Type 2 endothelial cells, which have cultural characteristics distinct from Type 1, only one new cell‐associated protein (M r = 32,000 and 40,000 daltons) was induced. Other differences are revealed by analyses of proteins secreted into the growth medium. In particular low levels of only CuSO 4 (10 ⁻⁶ M) enhanced the synthesis in Type 2 cells of a protein (M r = 220,000 daltons) identified as fibronectin. Since only copper ions induced fibronectin, we propose that the mechanism of induction of fibronectin synthesis, in contrast to the induction of cell−associated polypeptides, does not involve a sulphydryl−containing receptor molecule. It is suggested that the specific enhancement of fibronectin synthesis by copper ions may be a controlling event in the stimulation by copper ions of endothelial cell migration and angiogenesis.
Article
Low concentrations of copper sulphate, Dispirin, or Walker carcinoma extract elicit intraocular vascularization when tested by anterior chamber implants in rats. The response is markedly depressed by pre-treatment of animals with methylprednisolone acetate, suggesting that such induced vascularization is mediated by leucocytes. Since many agents inducing vascularization also induce migration of cultured endothelial cells, it raises the problem of how to isolate and study the action of possible leucocyte-derived angiogenic factors. Regardless of the identity of the natural angiogenic factor or factors, it is proposed that during blood vessel formation specialized endothelial cells migrate in response to an angiogenic signal and deposit fibronectin on which other cells can track and subsequently adhere, forming an endothelium.
Article
The involvement of leukocytes in corneal neovascularization has been known for a long time. Recent observations suggest that collagenase from leukocytes may be a common mediator for prostaglandin E1 (PGE1)- and copper-induced corneal neovascularization. This study was designed to investigate the effect of copper ion on collagenase activity from leukocytes and other sources and leukocyte infiltration in the corneal angiogenic process induced by PGE1. These results demonstrated that collagenase production from leukocytes was stimulated in a dose-dependent manner by copper ion but not by PGE1. Copper chloride 0.2 mM produced the highest stimulation. Copper ion had no effect on collagenase release from corneal fibroblasts and capillary endothelium. There were more polymorphonuclear leukocytes (PMN) in the prostaglandin E1 treated corneas than in the control. The time-course study showed that the appearance of PMN reached a peak on day 2 and new vessel growth could not be identified until day 4. These results supported an earlier suggestion that leukocytes play a role in corneal neovascularization and further suggested that copper in corneal neovascularization can stimulate the release of collagenase from leukocytes.
Article
The principal objective of our work is to sufficiently understand the mechanism of angiogenesis in the adult organism to allow interference with the process on a rational basis. It is apparent that several "factors" can trigger angiogenesis. To test these, we used the rabbit cornea mostly because it is avascular (i.e., the background is zero) and transparent (i.e., the newly formed capillaries that invade the cornea are easily visible in the unanaesthetized animal). Under these conditions, it was found that the cornea ready to be colonized by capillaries under the action of an angiogenesis effector becomes rich in copper ions and sialic acid. Motility of bovine capillary endothelium was utilized to analyze the angiogenesis process on the ground that mobilization of capillary endothelium is the first morphological event observed during angiogenesis in vivo and the methods to measure cell motility are reasonably accurate. With this approach it was found that heparin, fibronectin, and gangliosides are involved in the mobilization of capillary endothelium. The precise interaction among these three components is not yet clear.
Article
The purpose of this study was to determine the copper deposition and localization during the evolution of two murine mammary adenocarcinomas. In the normal tissue, the copper was located within the cytoplasm, whereas it was intra- and perinuclear in the tumors. The more angiogenic and metastatic tumor showed the higher percentage of copper-positive cells. In the tumor, copper deposits correlated well with its angiogenic and metastatic ability, but additional factors would be required for the process to be induced.
Article
64Cu was injected in the form of CuCl2 either by subcutaneous or by intraperitoneal route, and its distribution inside different organs was analyzed in 5 different tumor models, 4 in mice and 1 in rats. In all organs tested (blood, liver, kidneys, spleen, intestine, muscle, and tumor) no significant differences were observed in the results obtained after either injection route. All tumors analyzed (Krebs ascite, intestinal Leiomyo sarcoma, human tumor, mammary adenocarcinoma, either spontaneous or chemically induced) contained a relatively high concentration of 64Cu. For all tumor models tested, the 64Cu distribution was altered as compared with that of the corresponding control animals.
Article
The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.
Article
This article has no abstract; the first 100 words appear below. THE growth of solid neoplasms is always accompanied by neovascularization. This new capillary growth is even more vigorous and continuous than a similar outgrowth of capillary sprouts observed in fresh wounds or in inflammation.¹ Many workers have described the association between growing solid malignant tumors and new vessel growth.²³⁴⁵⁶ However, it has not been appreciated until the past few years that the population of tumor cells and the population of capillary endothelial cells within a neoplasm may constitute a highly integrated ecosystem. In this ecosystem the mitotic index of the two cell populations may depend upon each other. Tumor cells . . . Supported by a grant (5 RO1 CA08185–06) from the National Cancer Institute, a grant from the American Cancer Society, National Chapter (IC-28), and gifts from the Merck Company and the Alza Corporation. Source Information From the Department of Surgery, Children's Hospital Medical Center and Harvard Medical School, Boston, Massachusetts 02115.
Article
Copper's role in connective tissue is linked to the enzyme lysyl oxidase. From a biochemical perspective, copper is a cofactor for the enzyme and a determinant of its activity in connective tissues. Lysyl oxidase catalyses a post-translational oxidation of certain lysine and hydroxylysine residues. The peptidyl aldehydes so formed become active centres for the formation of cross-links in collagen and elastin. Less well understood is how copper controls the steady-state activity of lysyl oxidase; the enzyme fails in copper deficiency. Giving copper to a deprived animal increases lysyl oxidase activity in aortic tissue. Such activation in vivo appears to require caeruloplasmin. Suspending aortic tissue in a copper-enriched growth medium also activates lysyl oxidase provided that tissue structure is kept intact. Activation in vitro occurs with the binding of copper to a large-molecular-weight component, presumably the enzyme. Binding will not occur if protein synthesis is blocked. These studies clearly show that the synthesis of mature elastin and collagen can be controlled by the availability of copper. They further suggest that transport of copper to aortic tissue and its engagement to lysyl oxidase are linked to the synthesis or lysyl oxidase, an extracellular carrier, or both.
Article
The interstitial fluid of MTW9A and Walker carcinomas and their ethanol extract induced strong angiogenic response in the rabbit (New Zealand White) corneal test. The fluid collected in vivo was rich in E-type prostaglandins, and prostaglandin E1 (PGE1) in particular was strongly angiogenic at the lowest dose as compared with the angiogenic responses of prostaglandins E2, I2, and F2 alpha. Neoplastic fibroblasts also induced a strong angiogenic response, but in indomethacin-treated rabbits neovascularization failed to occur. Copper was concentrated in the cornea during PGE1-induced neovascularization, and copper-deficient rabbits were unable to mount an angiogenic response in the corneal test. Ceruloplasmin, the copper carrier of plasma, was found to be angiogenic at high doses. In indomethacin-treated rabbits, however, ceruloplasmin at the same high doses failed to induce angiogenesis. The experiments are interpreted to indicate that angiogenesis is the end result of a sequence of events, two of which are PGE1 production and copper mobilization in the tissue where neovascularization occurs.
Article
The ability to induce new formation of capillaries in the cornea was tested for ceruloplasmin, the copper carrier of serum, for fragments of the ceruloplasmin molecule with and without copper, for heparin, and for glycyl-L-histidyl-L-lysine, bound or not bound to copper ions. Male or female 2- to 3-kg New Zealand White rabbits were used. These experiments were prompted by the previous observation of copper accumulation in the cornea during angiogenesis and by the inability of copper-deficient rabbits to mount an angiogenic response. The results showed that the three different molecules were all able to induce angiogenesis provided that they were bound to copper. Fragments of the ceruloplasmin molecule also induced angiogenesis but only when copper was bound to the peptides. The data are interpreted to indicate that copper ions are involved in the sequence of events leading to angiogenesis and that the carrier molecules may be of quite a different nature.
Article
The objective of this work was to contribute to the interpretation of the mechanisms of capillary formation in the adult tissues. We have observed that the heparin-copper complex is angiogenic in vivo and stimulates migration of capillary endothelium in vitro. This effect is specific for capillary endothelium since aortic endothelium or fibroblasts from the rabbit cornea or human skin were unresponsive.
Article
Angiogenin is a potent inducer of blood-vessel formation with ribonucleolytic activity. Angiogenin binds to high affinity endothelial cell receptors and with lower affinity to extracellular matrix components. Here we report the effect of copper and zinc on these interactions. There was a 4.3-fold increase in angiogenin binding to calf pulmonary artery endothelial cells in the presence of Cu2+ in vitro. A 3.8-fold increase was observed with Zn2+, whereas Ni2+, Co2+, or Li+ had no effect. Specific angiogenin binding to the lower affinity matrix sites was increased by 2.7- and 1.9-fold in the presence of Cu2+ and Zn2+ respectively. Metal ion affinity chromatography and atomic absorption spectrometry were used to show the direct interaction of angiogenin with copper and zinc ions. Angiogenin bound 2.4 mol of copper per mole of protein. We suggest that copper, a modulator of angiogenesis in vivo, may be involved in the regulation of the biological activity of angiogenin.
Article
Copper ions stimulate proliferation of human umbilical artery and vein endothelial cells but not human dermal fibroblasts or arterial smooth muscle cells. Incubation of human umbilical vein endothelial cells for 48 h with 500 microM CuSO4 in a serum-free medium in the absence of exogenous growth factors results in a twofold increase in cell number, similar to the cell number increase induced by 20 ng/ml of basic fibroblast growth factor under the same conditions. Copper-induced proliferation of endothelial cells is not inhibited by 10% fetal bovine serum or by the presence of antibodies against a variety of angiogenic, growth, and chemotactic factors including angiogenin, fibroblast growth factors, epidermal growth factor, platelet-derived growth factor, tumor necrosis factor-alpha, transforming growth factor-beta, macrophage/monocyte chemotactic and activating factor, and macrophage inflammatory protein-1alpha. Moreover, despite the previous observations that copper increased total specific binding of 125I-angiogenin to endothelial cells, binding to the 170 kDa receptor is not changed; hence, the mitogenic activity of angiogenin is not altered by copper. Copper-induced proliferation, along with early reports that copper induces migration of endothelial cells, may suggest a possible mechanism for the involvement of copper in the process of angiogenesis.
Article
Fibronectin, a large extracellular matrix cell adhesion glycoprotein has diverse functions in wound repair including organization of matrix deposition and promotion of angiogenesis. We have previously shown that purified plasma fibronectin can be made into three-dimensional, fibrous materials, termed fibronectin mats (Fn-mat). The aim of this study was to examine means of increasing the stability of Fn-mats using a novel treatment with micromolar concentrations of copper ions which may be used to improve wound healing/nerve repair. Cytotoxicity of incorporated copper was determined using rat Schwann cells, rat tendon fibroblasts and human dermal fibroblasts. Dissolution of protein from the Fn-mat showed that treatment with the lowest concentration of copper used (1 microM) increased the stability of mats by 3-4 times at room temperature relative to control mats and twofold at 37 degrees C. Copper mediated increase in stability was dose dependent. Orientation of the Fn-fibres (within mats), monitored by scanning electron microscopy was retained with 1 microM copper but disappeared with higher concentrations. Schwann cells grew in culture with mats stabilized by 1 microM copper treatment without reduction in cell number but growth was inhibited at 10-200 microM Cu. All types of fibroblasts were unaffected by copper treatment upto 200 microM. Fn-mats can be successfully stabilized by this technique producing longer survival in vitro. The differential effects of copper on these cell types suggests that CuFn-mats may be used to select the type of cells which colonize these materials.
Article
Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, can be removed from the MT with tetrathiomolybdate (TTM). However, the insoluble Cu/TTM complex formed with excess TTM is known to be deposited in the liver. The metabolic fate of the insoluble Cu/TTM complex was investigated in the present study. LEC rats were injected with TTM at the dose of 10 mg/kg body weight for 8 consecutive days and were fed with a standard or low Cu diet for 14 days after the last injection. About 95% of the Cu in the liver became insoluble together with Mo. The concentration of Cu in the liver supernatants of rats fed with the standard diet increased significantly compared with that in rats dissected 24 h after the last injection (control rats), while the concentration in rats fed with the low Cu diet remained at a comparable level to that in the controls. The rate of Cu accumulation in the livers of rats fed with the standard diet did not differ before and after the treatment, suggesting that there was no rebound effect by treatment with TTM. These results suggest that the insoluble Cu/TTM complex is resolubilized in the liver, and that the solubilized complex is excreted into the bile and blood, i.e., the insoluble Cu/TTM complex is not the source of Cu re-accumulation in the form bound to MT in the liver after TTM treatment. It was concluded that, once Cu is complexed with TTM, the metal is excreted either immediately in the soluble form or slowly in the insoluble form into the bile and blood.
Article
Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti-angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)-chelating agent for patients with Wilson's disease of penicillamine intolerance. In our study, we examined the effect of Cu-chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu-deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu-chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.
Article
Recent studies have revealed that copper is an important cofactor for several angiogenic agents. We examined the antiangiogenic effect against hepatocellular carcinoma (HCC) of the copper chelator trientine, especially focusing on the relationship between copper and interleukin-8 (IL-8), a potent angiogenic factor produced by hepatoma cells. HuH-7 hepatoma cells were transplanted into nude mice and the growth of xenograft tumors was compared to and without administration of trientine. Using the resected tumor, microvessel density, apoptotic potential and proliferative activity were analyzed histologically and IL-8 mRNA was semiquantified by RT-PCR. In addition, HuH-7 cells were cultured in control medium, medium supplemented with copper, medium supplemented with trientine and medium supplemented with both copper and trientine. Human IL-8 levels were measured in the supernatants by ELISA. Using the extracts from cultured cells, IL-8 mRNA was semiquantified by RT-PCR. Trientine suppressed the growth of xenograft tumors significantly. Histologically, apoptotic potential was increased significantly and microvessel density, decreased. The production of IL-8 from the tumor was suppressed by trientine. In vitro, IL-8 production by HuH-7 cells in copper-containing medium was significantly greater than that in copper-free medium, and this effect was weakened when trientine was added. However, no significant change was apparent when trientine was added to the medium alone. In conclusion, the chelating effect of trientine prevented copper from functioning as a cofactor in angiogenesis, which resulted in reduced IL-8 production from HuH-7 cells.
Article
The need for agents to lower body copper in Wilson's disease, a disease which results from copper toxicity has been the driving force for the development of the effective anticopper drugs penicillamine, trientine, zinc, and now tetrathiomolybdate (TM). Because of its rapid action, potency, and safety, TM is proving to be a very effective drug for initial treatment of acutely ill Wilson's disease patients. Beyond this, TM has antiangiogenic effects, because many proangiogenic cytokines require normal levels of copper. This has led to use of TM in cancer, where it is generally effective in animal tumor models, and has shown efficacy in preliminary clinical studies. Most recently, it has been found that TM has antifibrotic and antiinflammatory effects through inhibition of profibrotic and proinflammatory cytokines.
Effect of copper ion on collage-nase release
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Lin MT, Chen YL. Effect of copper ion on collagenase release. Invest Opthomol Vis Sci. 1992;33: 558 –563.
Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells Hu GF. Copper stimulates proliferation of human endothelial cells under culture
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