Counteract of bone marrow of blotchy mice against the increases of plasma copper levels induced by high-fat diets in LDLR-/- mice.

BACKGROUND: Bone marrow of blotchy mouse (blotchy marrow) reflects the function of transmembrane domain and relevant intramembrane sites of ATP7A in myeloid cells. By chronic infusion of angiotensin II, we previously found that blotchy marrow plays a minor role in regulating plasma copper. Moreover, the recipients of blotchy marrow presented a moderate reduction of plasma lipids and inflammatory mediator production. Little is known about whether these changes are a specific response to angiotensin II or reveal a more general role of ATP7A. OBJECTIVE AND DESIGN: We investigated if blotchy marrow reduces plasma lipids and inflammatory mediators induced by high-fat diets. To test this hypothesis, blotchy and control marrows were reconstituted to the recipient mice (irradiated male LDLR-/- mice), followed by high-fat-diet feeding for 4 months. At the end points, plasma metals (copper, zinc and iron), lipid profiling (cholesterol, triglyceride, phospholipids and lipoprotein) and six inflammatory mediators (lymphotacin, MCP3, MCP5, TIMP1, VEGF-A and IP-10) were measured. Parallel experiments were performed using male LDLR-/- mice fed either high-fat diets or chow diets for 4 months. RESULTS: In addition to hyperlipidemia and low-grade inflammation, high-fat diets selectively increased plasma copper concentration compared to chow diets in LDLR-/- mice. After high-fat-diet feeding, the recipients with blotchy marrow showed a decrease in plasma copper (p < 0.01) and an increase in plasma iron (p < 0.05). The recipients with blotchy marrow also presented decreases in cholesterol (p < 0.01) and phospholipids (p < 0.05) in plasma. Surprisingly, plasma levels of MCP3 (p < 0.05), MCP5 (p < 0.05), TIMP1 (p < 0.01), VEGF-A (p < 0.01) and IP-10 (p < 0.01) were significantly increased in the recipients with blotchy marrow compared to controls; the increased levels of MCP3, MCP5 and TIMP1 were more than 50%. CONCLUSION: Our studies showed that blotchy marrow counteracts the increased copper levels induced by high-fat diets, indicating that circulating myeloid cells can regulate blood copper levels via ATP7A. Moreover, transplantation of blotchy marrow followed by high-fat diets leads to a decrease in lipid profile and an increase in inflammatory mediator production. Overall, blotchy marrow mediates divergent responses to angiotensin II and high-fat diets in vivo.

Soluble elastin peptides in cardiovascular homeostasis: Foe or ally.

Elastin peptides, also known as elastin-derived peptides or elastokines, are soluble polypeptides in blood and tissue. The blood levels of elastin peptides are usually low but can increase during cardiovascular diseases, such as atherosclerosis, aortic aneurysm and diabetes with vascular complications. Generally, elastin peptides are derived from the degradation of insoluble elastic polymers. The biological activities of elastin peptides are bidirectional, e.g., a pro-inflammatory effect on monocyte migration induction vs. a protective effect on vasodilation promotion. However, recent in vivo studies have demonstrated that elastin peptides promote the formation of atherosclerotic plaques in hypercholesterolemic mice and induce hyperglycemia and elevations in plasma lipid levels in fasted mice. More important, the detrimental effects induced by elastin peptides can be largely inhibited by genetic or pharmacological blockade of the elastin receptor complex or by neutralization of an antibody against elastin peptides. These studies indicate new therapeutic strategies for the treatment of cardiovascular diseases by targeting elastin peptide metabolism. Therefore, the goal of this review is to summarize current knowledge about elastin peptides relevant to cardiovascular pathologies to further delineate their potential application in cardiovascular disease.

Bone marrow from blotchy mice is dispensable to regulate blood copper and aortic pathologies but required for inflammatory mediator production in LDLR-deficient mice during chronic angiotensin II infusion.

BACKGROUND: The blotchy mouse caused by mutations of ATP7A develops low blood copper and aortic aneurysm and rupture. Although the aortic pathologies are believed primarily due to congenital copper deficiencies in connective tissue, perinatal copper supplementation does not produce significant therapeutic effects, hinting additional mechanisms in the symptom development, such as an independent effect of the ATP7A mutations during adulthood. METHODS: We investigated if bone marrow from blotchy mice contributes to these symptoms. For these experiments, bone marrow from blotchy mice (blotchy marrow group) and healthy littermate controls (control marrow group) was used to reconstitute recipient mice (irradiated male low-density lipoprotein receptor -/- mice), which were then infused with angiotensin II (1,000 ng/kg/min) for 4 weeks. RESULTS: By using Mann-Whitney U test, our results showed that there was no significant difference in the copper concentrations in plasma and hematopoietic cells between these 2 groups. And plasma level of triglycerides was significantly reduced in blotchy marrow group compared with that in control marrow group (P < 0.05), whereas there were no significant differences in cholesterol and phospholipids between these 2 groups. Furthermore, a bead-based multiplex immunoassay showed that macrophage inflammatory protein (MIP)-1ß, monocyte chemotactic protein (MCP)-1, MCP-3, MCP-5, tissue inhibitor of metalloproteinases (TIMP)-1, and vascular endothelial growth factor (VEGF)-A production was significantly reduced in the plasma of blotchy marrow group compared with that in control marrow group (P < 0.05). More important, although angiotensin II infusion increased maximal external aortic diameters in thoracic and abdominal segments, there was no significant difference in the aortic diameters between these 2 groups. Furthermore, aortic ruptures, including transmural breaks of the elastic laminae in the abdominal segment and lethal rupture in the thoracic segment, were observed in blotchy marrow group but not in control marrow group; however, there was no significant difference in the incidence of aortic ruptures between these 2 groups (P = 0.10; Fisher's exact test). CONCLUSIONS: Overall, our study indicated that the effect of bone marrow from blotchy mice during adulthood is dispensable in the regulation of blood copper, plasma cholesterol and phospholipids levels, and aortic pathologies, but contributes to a reduction of MIP-1ß, MCP-1, MCP-3, MCP-5, TIMP-1, and VEGF-A production and triglycerides concentration in plasma. Our study also hints that bone marrow transplantation cannot serve as an independent treatment option.

Inflammatory mediator profiling reveals immune properties of chemotactic gradients and macrophage mediator production inhibition during thioglycollate elicited peritoneal inflammation.

Understanding of spatiotemporal profiling of inflammatory mediators and their associations with MΦ accumulation is crucial to elucidate the complex immune properties. Here, we used murine thioglycollate elicited peritonitis to determine concentrations of 23 inflammatory mediators in peritoneal exudates and plasma before (day 0) and after (days 1 and 3) thioglycollate administration to peritoneal cavities; these mediators included TNF-α , FGF-9, IFN-γ , IP-10, RANTES, IL-1α , IL-6, IL-7, IL-10, IL-11, IL-12p70, IL-17A, lymphotactin, OSM, KC/GRO, SCF, MIP-1ß , MIP-2, TIMP-1, VEGF-A, MCP-1, MCP-3, and MCP-5. Our results showed that concentrations of most mediators in exudates and plasma reached peak levels on day 1 and were significantly reduced on day 3. Conversely, MΦ numbers started to increase on day 1 and reached peak levels on day 3. Moreover, LPS treatment in vitro significantly induced mediator productions in cell culture media and lysates from MΦ isolated on day 3. Our results also showed that on day 0, concentrations of many mediators in plasma were higher than those in exudates, whereas on day 1, the trend was reversed. Overall, the findings from thioglycollate elicited peritonitis reveal that reversible chemotactic gradients between peritoneal exudates and blood exist in basal and inflamed conditions and the inflammatory mediator production in vivo is disassociated with macrophage accumulation during inflammation resolution.

Production of LPS-induced inflammatory mediators in murine peritoneal macrophages: neocuproine as a broad inhibitor and ATP7A as a selective regulator.

Copper chelation regulates the production of inflammatory mediators in vivo during vascular inflammation and atherogenesis. Little is known about how the copper egress pump ATP7A regulates the production of these mediators. In this study, we isolated ATP7A deficient macrophages (MΦ) from the peritoneal cavity of blotchy mice and identified the lipopolysaccharide (LPS)-induced inflammatory mediators that were altered by ATP7A deficiency. These results were compared with the effect of neocuproine (a copper chelator) treatment on both ATP7A deficient and control MΦ. Seven of the 24 inflammatory mediators examined in this study had significant changes in expression in the ATP7A deficient MΦ compared to controls; 16 of these mediators were significantly reduced in MΦ treated with neocuproine compared to controls. Both neocuproine treatment and ATP7A deficiency reduced IFN-γ, MCP-1, MCP-3, and VEGF-A levels. Interestingly, the production of KC/GRO was upregulated by ATP7A deficiency but downregulated by neocuproine treatment. Neocuproine, but not ATP7A deficiency, reduced the production of FGF-9, IL-1α, IL-12p70, IL-2, IL-3, IL-4, IL-6, MIP-1ß, MIP-2, RANTES, and TNFα. ATP7A deficiency but not neocuproine treatment reduced IP-10 and MCP-5 levels. In addition, both ATP7A deficiency and neocuproine treatment had no effect on GM-CSF, IL-10, IL-11, IL-7, OSM, and SCF. Together, these findings provide evidence that MΦ ATP7A selectively regulates LPS-induced inflammatory mediators, in part, via modulation of cellular copper availability, whereas neocuproine generally inhibits the production of inflammatory mediators. These results also imply that although copper chelation and ATP7A downregulation may result in different copper concentrations, gradients, and/or distribution in the cells, they may not lead to opposite biological effects on inflammatory mediator production.

CD14 directs adventitial macrophage precursor recruitment: role in early abdominal aortic aneurysm formation.

BACKGROUND: Recruitment of macrophage precursors to the adventitia plays a key role in the pathogenesis of abdominal aortic aneurysms (AAAs), but molecular mechanisms remain undefined. The innate immune signaling molecule CD14 was reported to be upregulated in adventitial macrophages in a murine model of AAA and in monocytes cocultured with aortic adventitial fibroblasts (AoAf) in vitro, concurrent with increased interleukin-6 (IL-6) expression. We hypothesized that CD14 plays a crucial role in adventitial macrophage precursor recruitment early during AAA formation. METHODS AND RESULTS: CD14(-/-) mice were resistant to AAA formation induced by 2 different AAA induction models: aortic elastase infusion and systemic angiotensin II (AngII) infusion. CD14 gene deletion led to reduced aortic macrophage infiltration and diminished elastin degradation. Adventitial monocyte binding to AngII-infused aorta in vitro was dependent on CD14, and incubation of human acute monocytic leukemia cell line-1 (THP-1) monocytes with IL-6 or conditioned medium from perivascular adipose tissue (PVAT) upregulated CD14 expression. Conditioned medium from AoAf and PVAT induced CD14-dependent monocyte chemotaxis, which was potentiated by IL-6. CD14 expression in aorta and plasma CD14 levels were increased in AAA patients compared with controls. CONCLUSIONS: These findings link CD14 innate immune signaling via a novel IL-6 amplification loop to adventitial macrophage precursor recruitment in the pathogenesis of AAA.

Zinc, copper, and blood pressure: Human population studies.

Copper and zinc are essential trace biometals that regulate cardiovascular homeostasis, and dysregulation of these metals has been linked to vascular diseases, including hypertension. In this article, we review recent human population studies concerning this topic, focusing on: 1) the relationship between blood pressure and levels of zinc and copper; 2) correlations between trace metals, the renin-angiotensin system, obesity, and hypertension; 3) the relationship between environmental metal pollution and the development of hypertension; and 4) methods commonly employed to assay zinc and copper in human specimens. Moreover, based on the findings of these studies, we suggest the following topics as the basis for future investigations: 1) the potential role of environmental metal pollution as a causal factor for hypertension; 2) metal profiles within specific pathogenic subsets of patients with hypertension; 3) standardizing the experimental design so that the results between different studies are more comparable; and 4) the requirement for animal experiments as complementary approaches to address mechanistic insight that cannot be studied in human populations.

Coupling transmission electron microscopy with synchrotron radiation X-ray fluorescence microscopy to image vascular copper.

Recently, using synchrotron radiation X-ray fluorescence microscopy (SRXRF), the copper accumulation in rat aortic elastin and copper topography in human THP-1 cell monolayer have been described. However, it is necessary to locate more accurately cellular copper in the vascular cells and tissues. In the current study, SRXRF coupling with transmission electron microscopy (TEM) was used to image copper in sections of human THP-1 cells and mouse aorta. The results showed that sections of 1 µm thickness are required for SRXRF producing a correlative image with TEM between copper topography and cellular ultrastructure. As compared with SRXRF alone, coupling TEM with SRXRF can clearly identify the location of copper in the nucleus and nucleolus in non-dividing THP-1 cell sections, and can differentiate the copper location at elastic laminae from collagen in mouse aortic sections. Thus, these results revealed new information about the copper topography in vascular cells and tissues and highlighted the potential of TEM-SRXRF to investigate the role of copper in macrophage and aortic homeostasis.

Tissue markers in human atherosclerotic carotid artery plaque.

Carotid artery stenosis predisposes to thrombo-embolization and stroke. Established tissue markers such as osteopontin, nitric oxide synthases, myeloperoxidases, and matrix metalloproteinases have been examined within stenotic plaques and their impact upon plaque stability discussed. However, a new generation of tissue markers is being discovered, and their role in atherosclerotic development and plaque stability is being debated. Prostaglandin synthase, 15-lipoxygenase-2, myeloid-related proteins 8 and 14, and protease nexin-1 have recently been shown to correlate with carotid artery atherosclerosis. These proteins highlight new areas of interest in the role of macrophages in atherosclerotic development, plaque formation, and rupture. Additionally, these new molecules raise the possibility of new screening and treatment techniques.

Human macrophage ATP7A is localized in the trans-Golgi apparatus, controls intracellular copper levels, and mediates macrophage responses to dermal wounds.

The copper transporter ATP7A has attracted significant attention since the discovery of its gene mutation leading to human Menkes disease. We previously reported that ATP7A is highly expressed in the human vasculature and identified a novel vascular function of ATP7A in modulation of the expression and activity of extracellular superoxide dismutase. We recently identified that ATP7A expression in THP-1 cells (a monocyte/macrophage model cell line) plays a role in the oxidation of low density lipoproteins, indicating that it is necessary to further investigate its expression and function in monocytes/macrophages. In the current study, we demonstrated the protein and mRNA expression of ATP7A in human peripheral blood mononuclear cell (PBMC)-derived macrophages and alveolar macrophages. ATP7A was strongly co-localized with the trans-Golgi apparatus in PBMC-derived macrophages. Intracellular copper, detected by synchrotron X-ray fluorescence microscopy, was found to be distributed to the nucleus and cytoplasm in human THP-1 cells. To confirm the role of endogenous ATP7A in macrophage copper homeostasis, we performed inductively coupled plasma mass spectrometry in murine peritoneal macrophages, which showed markedly increased intracellular copper levels in macrophages isolated from ATP7A-deficient mice versus control mice. Moreover, the role of ATP7A in regulating macrophage responses to dermal wounds was studied by introduction of control and ATP7A-downregulated THP-1 cells into dermal wounds of nude mice. Infiltration of THP-1 cells into the wounded area (detected by expression of human macrophage markers MAC2 and CD68) was reduced in response to downregulation of ATP7A, hinting decreased macrophage accumulation subsequent to dermal wounds. In summary, alongside our previous studies, these findings indicate that human macrophage ATP7A is localized in the trans-Golgi apparatus, regulates intracellular copper levels, and mediates macrophage responses to a dermal wound.

The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature.

Since their establishment thirty years ago, THP-1 cells have become one of most widely used cell lines to investigate the function and regulation of monocytes and macrophages in the cardiovascular system. However, because this cell line was derived from the blood of a patient with acute monocytic leukemia, the extent to which THP-1 cells mimic monocytes and macrophages in the vasculature is not entirely known. This article serves as a meaningful attempt to address this question by reviewing the recent publications. The interactions between THP-1 cells and various vascular cells (such as endothelial cells, smooth muscle cells, adipocytes, and T cells) provide insight into the roles of the interconnection of monocytes-macrophages with other vascular cells during vascular inflammation, particularly atherogenesis and obesity. Transcriptome, microRNA profile, and histone modifications of THP-1 cells shed new light on the regulatory mechanism of the monocytes-macrophages in response to various inflammatory mediators, such as oxidized low density lipoprotein, lipopolysaccharide, and glucose. These studies hint that under certain defined conditions, THP-1 cells not only resemble primary monocytes-macrophages isolated from healthy donors or donors with disease, such as diabetes mellitus, but also mimic the in situ alteration of macrophages in the adipose tissue of obese subjects and in atherosclerotic lesions. A potential trajectory is to use this cell line to study the novel molecular mechanisms in monocytes and macrophages in relation to the physiology and pathophysiology of the cardiovascular system, however, the conclusion of studies employing THP-1 cells requires further verification using primary cells and/or in vivo models to be generalized to monocytes and macrophages.

Synchrotron radiation X-ray fluorescence microscopy reveals a spatial association of copper on elastic laminae in rat aortic media.

Copper, an essential trace metal in humans, plays an important role in elastic formation. However, little is known about the spatial association between copper, elastin, and elastin producing cells. The aorta is the largest artery; the aortic media is primarily composed of the elastic lamellae and vascular smooth muscle cells, which makes it a good model to address this issue. Synchrotron radiation X-ray fluorescence microscopy (SRXRF) is a new generation technique to investigate the spatial topography of trace metals in biological samples. Recently, we utilized this technique to determine the topography of copper as well as other trace elements in aortic media of Sprague Dawley rats. A standard rat diet was used to feed Sprague Dawley rats, which contains the normal dietary requirements of copper and zinc. Paraffin embedded segments (4 µm of thickness) of thoracic aorta were analyzed using a 10 keV incident monochromatic X-ray beam focusing on a spot size of 0.3 µm × 0.2 µm (horizontal × vertical). The X-ray spectrum was measured using an energy-dispersive silicon drift detector for elemental topography. Our results showed that phosphorus, sulfur, and zinc are predominately distributed in the vascular smooth muscle cells, whereas copper is dramatically accumulated in elastic laminae, indicating a preferential spatial association of copper on elastic laminae in aortic media. This finding sheds new light on the role of copper in elastic formation. Our studies also demonstrate that SRXRF allows for the visualization of trace elements in tissues and cells of rodent aorta with high spatial resolution and provides an opportunity to study the role of trace elements in vasculature.

Trace metal imaging with high spatial resolution: applications in biomedicine.

New generations of analytical techniques for imaging of metals are pushing hitherto boundaries of spatial resolution and quantitative analysis in biology. Because of this, the application of these imaging techniques described herein to the study of the organization and dynamics of metal cations and metal-containing biomolecules in biological cell and tissue is becoming an important issue in biomedical research. In the current review, three common metal imaging techniques in biomedical research are introduced, including synchrotron X-ray fluorescence (SXRF) microscopy, secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). These are exemplified by a demonstration of the dopamine-Fe complexes, by assessment of boron distribution in a boron neutron capture therapy cell model, by mapping Cu and Zn in human brain cancer and a rat brain tumor model, and by the analysis of metal topography within neuromelanin. These studies have provided solid evidence that demonstrates that the sensitivity, spatial resolution, specificity, and quantification ability of metal imaging techniques is suitable and highly desirable for biomedical research. Moreover, these novel studies on the nanometre scale (e.g., of individual single cells or cell organelles) will lead to a better understanding of metal processes in cells and tissues.

Bioimaging of metals and biomolecules in mouse heart by laser ablation inductively coupled plasma mass spectrometry and secondary ion mass spectrometry.

Bioimaging mass spectrometric techniques allow direct mapping of metal and biomolecule distributions with high spatial resolution in biological tissue. In this study laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) was used for imaging of transition metals (Fe, Cu, Zn, Mn, and Ti), alkali and alkaline-earth metals (Na, K, Mg, and Ca, respectively), and selected nonmetals (such as C, P, and S) in native cryosections of mouse heart. The metal and nonmetal images clearly illustrated the shape and the anatomy of the samples. Zinc and copper were inhomogeneously distributed with average concentrations of 26 and 11 µg g(-1), respectively. Titanium and manganese were detected at concentrations reaching 1 and 2 µg g(-1), respectively. The highest regional metal concentration of 360 µg g(-1)was observed for iron in blood present in the lumen of the aorta. Secondary ion mass spectrometry (SIMS) as an elemental and biomolecular mass spectrometric technique was employed for imaging of Na, K, and selected biomolecules (e.g., phosphocholine, choline, cholesterol) in adjacent sections. Here, two different bioimaging techniques, LA-ICPMS and SIMS, were combined for the first time, yielding novel information on both elemental and biomolecular distributions.

Absorption of CH330331, a novel 4-anilinoquinazoline inhibitor of epidermal growth factor receptor tyrosine kinase: comparative studies using in vitro, in situ and in vivo models.

CH330331 is a prototype of a new class of synthetic small molecule tyrosine kinase inhibitors (TKIs). In vitro Caco-2 cell monolayers, the in situ single-pass rat intestinal perfusion (SPIP) technique with mesenteric vein cannulated and an in vivo animal model were employed to investigate its permeability and transepithelial transport mechanisms. The Caco-2 model showed that the transport of CH330331 across the monolayers from the apical (AP) to basolateral (BL) side was 6- to 10-fold higher than that from the BL to AP side. The apparent permeability coefficient (P(app) ) values of CH330331 at 5-20 µg/ml from the AP to BL and from BL to AP side were 5.30-2.21 × 10(-6) cm/s, with a decrease in P(app) values from the AP to BL side at increased CH330331 concentrations. In the perfused rat intestinal model, a concentration dependent change in permeability was detected where P(blood) at 5 µg/ml (1.66 ± 0.69 × 10(-6) cm/s) and 10 µg/ml (1.80 ± 0.45 × 10(-6) cm/s) was significantly different from P(blood) at 20 µg/ml (0.98 ± 0.31 × 10(-6) cm/s, p<0.05). Some inhibitors could also change the transepithelial transport of CH330331. Moreover, the in vivo study showed that the oral bioavailability of CH330331 was 82.7% in the rat. All the results confirmed that the transepithelial transport of CH330331 was rapid and saturable, which might involve an active mechanism. The oral bioavailability of CH330331 was relatively high in vivo.

Participation of ATP7A in macrophage mediated oxidation of LDL.

ATP7A primarily functions to egress copper from cells, thereby supplying this cofactor to secreted copper-accepting enzymes. This ATPase has attracted significant attention since the discovery of its mutation leading to human Menkes disease and the demonstration of its distribution in various tissues. Recently, we reported that ATP7A is expressed in the human vasculature. In the present study, we investigated the cellular expression of ATP7A in atherosclerotic lesions of LDL receptor (-/-) mice. Subsequently, we examined the role of ATP7A in regulating the oxidation of LDL in a macrophage cell model. We observed that ATP7A is expressed in atherosclerotic murine aorta and colocalizes with macrophages. To investigate the function of ATP7A, we downregulated ATP7A expression in THP-1 derived macrophages using small interfering RNA (siRNA). ATP7A downregulation attenuated cell-mediated oxidation of LDL. Moreover, downregulation of ATP7A resulted in decreased expression and enzymatic activity of cytosolic phospholipase A(2) alpha (cPLA(2)alpha), a key intracellular enzyme involved in cell-mediated LDL oxidation. In addition, cPLA(2)alpha promoter activity was decreased after downregulation of ATP7A, suggesting that ATP7A transcriptionally regulates cPLA(2)alpha expression. Finally, cPLA(2)alpha overexpression increased LDL oxidation, which was blocked by coadministration of ATP7A siRNA oligonucleotides. These findings suggest a novel mechanism linking ATP7A to cPLA(2)alpha and LDL oxidation, suggesting that this copper transporter could play a previously unrecognized role in the pathogenesis of atherosclerosis.

Copper egress is induced by PMA in human THP-1 monocytic cell line.

Copper egress is an essential regulator of the kinetics of cellular copper and is primarily regulated by ATP7A, a copper-transporting P-type ATPase. However, little is known under which physiological condition copper egress is induced and its molecular consequence. In current manuscript, using THP-1 cells, a human monocytic cell line, we found that ATP7A expression was increased in cells exposed to phorbol-12-myristate-13-acetate (PMA), a potent inducer of neovascularization and cancer. Inductively coupled plasma mass spectrometry revealed that PMA also induced copper egress. Inhibition of ATP7A expression using small interfering RNA abrogated PMA induced copper egress. PMA treatment in THP-1 cells resulted in increased expression of matrix metalloproteinase (MMP) 9 and vascular endothelial growth factor receptor 1 (VEGFR1), whereas inhibition of ATP7A resulted in suppression of PMA-induced expression of VEGFR1, but not MMP9. Finally, addition of exogenous copper into the conditioned medium did not change VEGFR1 expression in THP-1 cells. Collectively, we demonstrate that PMA induces copper egress in THP-1 cells, which is regulated by ATP7A, and ATP7A regulates VEGFR1 expression. Considering the involvement of copper in neovascularization, our current finding provides the potential evidence to interpret the molecular mechanism.

Transepithelial transport characteristics of the antihypertensive peptide, Lys-Val-Leu-Pro-Val-Pro, in human intestinal Caco-2 cell monolayers.

An antihypertensive peptide, Lys-Val-Leu-Pro-Val-Pro (KVLPVP), can reduce blood pressure in hypertensive rats after being orally administered. In this study, the transepithelial transport of intact KVLPVP was examined by Caco-2 monolayers. The results were as follows: (i) The flux was not saturable for apical (AP) to basolateral (BL) or BL-AP transport when the concentration of KVLPVP was 1-8 mM. (ii) Sodium deoxycholate loosened the tight junction in the Caco-2 cells and significantly improved the transport process. (iii) Phenylarsine oxide, a transcytotic process inhibitor, had little effect on the transport process. (iv) The influx and eflux of KVLPVP remained unchanged in the presence of the ATP inhibitor sodium azide. (v) This transport was not inhibited by the peptide transporter substrates Gly-Pro or arphanine A. All these data indicate that paracellular transport diffusion was the major flux mechanism for the intact KVLPVP.

Role of Menkes ATPase in angiotensin II-induced hypertension: a key modulator for extracellular superoxide dismutase function.

The extracellular superoxide dismutase (SOD3), a secretory copper-containing enzyme, regulates angiotensin II (Ang II)-induced hypertension by modulating levels of extracellular superoxide anion. The present study was designed to determine the role of the copper transporter Menkes ATPase (MNK) in Ang II-induced SOD3 activity and hypertension in vivo. Here we show that chronic Ang II infusion enhanced systolic blood pressure and vascular superoxide anion production in MNK mutant (MNK(mut)) mice as compared with those in wild-type mice, which are associated with impaired acetylcholine-induced endothelium-dependent vasorelaxation in MNK(mut) mice. These effects in MNK(mut) mice are rescued by infusion of the SOD mimetic Tempol. By contrast, norepinephrine-induced hypertension, which is not associated with an increase in vascular superoxide anion production, is not affected in MNK(mut) mice. Mechanistically, basal and Ang II infusion-induced increase in vascular SOD3-specific activity is significantly inhibited in MNK(mut) mice. Coimmunoprecipitation analysis reveals that Ang II stimulation promotes association of MNK with SOD3 in cultured vascular smooth muscle cell and in mouse aortas, which may contribute to SOD3-specific activity by increasing copper delivery to SOD3 through MNK. In summary, MNK plays an important role in modulating Ang II-induced hypertension and endothelial function by regulating SOD3 activity and vascular superoxide anion production and becomes a potential therapeutic target for oxidant stress-dependent cardiovascular diseases.

Newly developed angiotensin II-infused experimental models in vascular biology.

Angiotensin II is a major vasoactive peptide in the renin-angiotensin system (RAS). In vitro evidence demonstrates that this peptide can modulate the function of various adhesion molecules, chemokines, cytokines and growth factors, and ultimately contributes to cell proliferation, hypertrophy and inflammation. Moreover, in vivo studies further support that angiotensin II induces several vascular alterations including sustained elevations of blood pressure, enhanced inflammatory response, increased medial thickness of the aortas, and formation of aortic dissection and aneurysms. Thus, it has been a long time that angiotensin II-induced hypertension, atherosclerosis and abdominal aortic aneurysms emerge as important experimental models with respect to vascular biology. Applications of these models to investigate the vascular diseases have dramatically improved our understanding in the pathogenesis of these diseases. However, the pathophysiology of angiotensin II in vivo remains to be determined in many other vascular diseases where angiotensin II has been implicated as the detrimental factor, at least in part due to the limit availability of animal models. Recently some new exciting experimental models based on angiotensin II infusion have been reported to replicate the human diseases, such as postmenopausal hypertension, preeclampsia, vascular remodeling, vascular aging and neovascularization. In this review, we will focus on the rationales and anticipated applications of these newly developed models, with special emphasis placed on those relevant to the vascular biology. We will also discuss the limitations of the method of chronic angiotensin II infusion and additional approaches to overcome these limitations. These experimental models will provide great opportunity for us to investigate the molecular mechanisms of angiotensin II and evaluate therapeutic approaches, particularly to finely tune the potential role of RAS activation in various vascular events using genetically engineered mice.