Photoacoustic in vivo 3D imaging of tumor using a highly tumor-targeting probe under high-threshold conditions.

Three-dimensional (3D) representation of a tumor with respect to its size, shape, location, and boundaries is still a challenge in photoacoustic (PA) imaging using artificial contrast agents as probes. We carried out PA imaging of tumors in mice using 800RS-PMPC, which was obtained by coupling of 800RS, a near-infrared cyanine dye, with PMPC, a highly selective tumor-targeting methacrylate polymer having phosphorylcholine side chains, as a probe. The conjugate 800RS-PMPC forms compact nanoparticles (dDLS = 14.3 nm), retains the biocompatibility of the parent polymer (PMPC) and exhibits unprecedented PA performance. When applied to mice bearing a 6 × 3 × 3 mm3 tumor buried 6 mm beneath the skin, the probe 800RS-PMPC selectively accumulates in the tumor and emits PA signals that are strong enough to be unambiguously distinguished from noise signals of endogenous blood/hemoglobin. The PA image thus obtained under high-threshold conditions allows 3D characterization of the tumor in terms of its size, shape, location, and boundaries.

Biological reduction of nitroimidazole-functionalized gold nanorods for photoacoustic imaging of tumor hypoxia.

Tumor-selective accumulation of gold nanorods (GNR) has been demonstrated for visualization of tumor hypoxia by photoacoustic imaging. We prepared GNRs with hypoxia-targeting nitroimidazole units (G-NI) on their surface. Biological experiments revealed that G-NI produced a strong photoacoustic signal in hypoxic tumor cells and tissues.

Aggregate Formation of Oligonucleotides that Assist Molecular Imaging for Tracking of the Oxygen Status in Tumor Tissue.

The use of DNA aggregates could be a promising strategy for the molecular imaging of biological functions. Herein, phosphorescent oligodeoxynucleotides were designed with the aim of visualizing oxygen fluctuation in tumor cells. DNA-ruthenium conjugates (DRCs) that consisted of oligodeoxynucleotides, a phosphorescent ruthenium complex, a pyrene unit for high oxygen responsiveness, and a nitroimidazole unit as a tumor-targeting unit were prepared. In general, oligonucleotides have low cell permeability because of their own negative charges; however, the DRC formed aggregates in aqueous solution due to the hydrophobic pyrene and nitroimidazole groups, and smoothly penetrated the cellular membrane to accumulate in tumor cells in a hypoxia-selective manner. The oxygen-dependent phosphorescence of DRC in cells was also observed. In vivo experiments revealed that aggregates of DRC accumulated in hypoxic tumor tissue that was transplanted into the left leg of mice, and showed that oxygen fluctuations in tumor tissue could be monitored by tracking of the phosphorescence emission of DRC.

Human thioredoxin-1 attenuates the rate of lipopolysaccharide-induced preterm delivery in mice in association with its anti-inflammatory effect.

BACKGROUND: Maternal intrauterine infection/inflammation represents the major etiology of preterm delivery and the leading cause of neonatal mortality and morbidity. The aim of this study was to investigate the anti-inflammatory properties of thioredoxin-1 in vivo and its potential ability to attenuate the rate of inflammation-induced preterm delivery. METHODS: Two intraperitoneal injections of lipopolysaccharide from Escherichia coli were administered in pregnant mice on gestational day 15, with a 3-h interval between the injections. From either 1 h before or 1 h after the first lipopolysaccharide injection, mice received three intravenous injections of either recombinant human thioredoxin-1, ovalbumin, or vehicle, with a 3-h interval between injections. RESULTS: Intraperitoneal injection of lipopolysaccharide induced a rise of tumor necrosis factor-α, interferon-γ, monocyte chemotactic protein 1, and interleukin-6 in maternal serum levels and provoked preterm delivery. Recombinant human thoredoxin-1 prevented the rise in these proinflammatory cytokine levels. After the inflammatory challenge, placentas exhibited severe maternal vascular dilatation and congestion and a marked decidual neutrophil activation. These placental pathological findings were ameliorated by recombinant human thioredoxin-1, and the rate of inflammation-induced preterm delivery was attenuated. CONCLUSION: Thioredoxin-1 may thus represent a novel effective treatment to delay inflammation-induced preterm delivery.

Confined Singlet Oxygen in Mesoporous Silica Nanoparticles: Selective Photochemical Oxidation of Small Molecules in Living Cells.

Chemical conversion of specific bioactive molecules by external stimuli in living cells is a powerful noninvasive tool for clarification of biomolecular interactions and to control cellular functions. However, in chaotic biological environments, it has been difficult to induce arbitrary photochemical reactions on specific molecules because of their poor molecular selectivity. Here we report a selective and nontoxic photochemical reaction system utilizing photoactivated mesoporous silica nanoparticles to control biological functions. Methylene blue modification within nanoparticle pores for photosensitization produced singlet oxygen confined to the pore that could mediate selective oxidation of small molecules without any damage to living cells. This intracellular photochemical system produced bioactive molecules in situ and remotely controlled the cell cycle phase. We also confirmed that this photoreaction could be applied to control cell cycle phase in tumor tissue transplanted in mice. The cell cycle phase in the cells in mice, to which our system was administered, was arrested at the G2/M phase upon photoirradiation. We demonstrate a simple and promising method for the exogenous conversion of an intracellular biomolecule to another functional compound.

Water-soluble phosphorescent ruthenium complex with a fluorescent coumarin unit for ratiometric sensing of oxygen levels in living cells.

Dual emission was applied to a molecular probe for the ratiometric sensing of oxygen concentration in a living system. We prepared ruthenium complexes possessing a coumarin unit (Ru-Cou), in which the (3)MLCT phosphorescence of the ruthenium complex was efficiently quenched by molecular oxygen, whereas the coumarin unit emitted constant fluorescence independent of the oxygen concentration. The oxygen status could be determined precisely from the ratio of phosphorescence to fluorescence. We achieved the molecular imaging of cellular oxygen levels using Ru-Cou possessing an alkyl chain, which provided appropriate lipophilicity to increase cellular uptake.

Phosphorescent ruthenium complexes with a nitroimidazole unit that image oxygen fluctuation in tumor tissue.

Understanding oxygen fluctuation in a cancerous tumor is important for effective treatment, especially during radiotherapy. In this paper, ruthenium complexes bearing a nitroimidazole group are shown to report the oxygen status in tumor tissue directly. The nitroimidazole group was known to be accumulated in hypoxic tumor tissues. On the other hand, the ruthenium complex showed strong phosphorescence around 600 nm. The emission of ruthenium is quenched instantaneously by molecular oxygen due to energy transfer between triplet states of oxygen and ruthenium complex, but the emission is then recovered by the removal of oxygen. Thus, we could observe oxygen fluctuation in tumor tissue in a real-time manner by monitoring the phosphorescence of the ruthenium complex. The versatility of the probe is demonstrated by monitoring oxygen fluctuation in living cells and tumor tissue planted in mice. The ruthenium complex promptly penetrated plasma membrane and accumulated in cells to emit its oxygen-dependent phosphorescence. In vivo experiments revealed that the oxygen level in tumor tissue seems to fluctuate at the sub-minute timescale.

The protective role of the transmembrane thioredoxin-related protein TMX in inflammatory liver injury.

AIMS: Accumulating evidence indicates that oxidative stress is associated with inflammation, and the cellular redox status can determine the sensitivity and the final outcome in response to inflammatory stimuli. To control the redox balance, mammalian cells contain a variety of oxidoreductases belonging to the thioredoxin superfamily. The large number of these enzymes suggests a complex mechanism of redox regulation in mammals, but the precise function of each family member awaits further investigations. RESULTS: We generated mice deficient in transmembrane thioredoxin-related protein (TMX), a transmembrane oxidoreductase in the endoplasmic reticulum (ER). When exposed to lipopolysaccharide (LPS) and d-(+)-galactosamine (GalN) to induce inflammatory liver injury, mutant mice were highly susceptible to the toxicants and developed severe liver damage. LPS-induced production of inflammatory mediators was equivalent in both wild-type and TMX(-/-) mice, whereas neutralization of the proinflammatory cytokine tumor necrosis factor-α suppressed the toxic effects of LPS/GalN in the mutant mice. Liver transcriptional profiles revealed enhanced activation of the p53-signaling pathway in the TMX(-/-) mice after LPS/GalN treatment. Furthermore, TMX deficiency also caused increased sensitivity to thioacetamide, which exerts its hepatotoxicity through the generation of reactive oxygen species. INNOVATION: The present study is the first to address the role of the oxidoreductase TMX in inflammatory liver injury. The phenotype of mice deficient in TMX suggests a functional link between redox regulation in the ER and susceptibility to oxidative tissue damage. CONCLUSION: We conclude that TMX plays a major role in host defense under the type of inflammatory conditions associated with oxidative stress.

Interleukin-13 damages intestinal mucosa via TWEAK and Fn14 in mice-a pathway associated with ulcerative colitis.

BACKGROUND & AIMS: TWEAK, a member of the tumor necrosis factor (TNF) superfamily, promotes intestinal epithelial cell injury and signals through the receptor Fn14 following irradiation-induced tissue damage and during development of colitis in mice. Interleukin (IL)-13, an effector of tissue damage in similar models, has been associated with the pathogenesis of ulcerative colitis (UC). We investigated interactions between TWEAK and IL-13 following mucosal damage in mice. METHODS: We compared patterns of gene expression in intestinal tissues from wild-type and TWEAK knockout mice following γ-irradiation. Intestinal explants from these mice were used to detect cell damage induced by IL-13 and TNF-α. Levels of messenger RNA for IL-13, TWEAK, and Fn14 were measured in mucosal samples from patients with UC. RESULTS: Based on gene expression analysis, TWEAK mediates γ-irradiation-induced epithelial cell cycle arrest and apoptosis. However, TWEAK alone did not induce damage or apoptosis of primary intestinal epithelial cells. On the other hand, exogenous IL-13 activated caspase-3 in naïve intestinal explants; this process required TWEAK, Fn14, and secretion of endogenous TNF-α which was mediated by ADAM17. Conversely, activation of caspase by exogenous TNF-α required IL-13, TWEAK, and Fn14. In mucosa from patients with UC, messenger RNA levels of IL-13, TWEAK, and Fn14 increased with level of disease severity. CONCLUSIONS: IL-13-induced damage of intestinal epithelial cells requires TWEAK, its receptor (Fn14), and TNF-α. IL-13, TNF-α, TWEAK, and Fn14 could perpetuate and aggravate intestinal inflammation in patients with UC.

Differential roles of Annexin A1 (ANXA1/lipocortin-1/lipomodulin) and thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP) in glucocorticoid signaling of HTLV-I-transformed T cells.

Glucocorticoid (GC) is widely used for therapeutic purposes in immunological and hematological disorders. Annexin A1 (ANXA1/lipocortin-1/lipomodulin), a GC-inducible molecule, was regarded as a vital anti-inflammatory mediator of GC. Thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP), a regulator of redox reactions, cell growth and lipid metabolism, was also reportedly induced by GC. HTLV-I infected T cells undergo the transition from the IL-2 dependent to IL-2 independent growth during the long-term culture in vitro. We found that these T cells responded to GC with growth arrest and apoptosis in the IL-2 dependent growth stage, whereas they failed to respond to GC after their growth had shifted into the IL-2 independent stage. Here we employed these T cell lines and studied the roles of ANXA1 and TBP-2 in mediating GC-induced apoptosis. In GC-sensitive T cells, ANXA1 expression was negligible and unaffected by GC treatment, whereas TBP-2 was expressed and induced by GC treatment. In GC-resistant T cells, however, ANXA1 was highly expressed regardless of GC treatment and promoted cellular proliferation. In contrast, TBP-2 expression was lost and could not mediate the GC-induced apoptosis. In conclusion, these results suggest that TBP-2, but not ANXA1, is directly involved in the switching of GC sensitivity and GC resistance in HTLV-I infected T cell lines, whereas ANXA1 may be a biomarker indicative of the advanced stage of the transformation.

Direct association of thioredoxin-1 (TRX) with macrophage migration inhibitory factor (MIF): regulatory role of TRX on MIF internalization and signaling.

Thioredoxin-1 (TRX) is a small (14 kDa) multifunctional protein with the redox-active site Cys-Gly-Pro-Cys. Macrophage migration inhibitory factor (MIF) is a 12 kDa cytokine belonging to the TRX family. Historically, when we purified TRX from the supernatant of ATL-2 cells, a 12 kDa protein was identified along with TRX, which was later proved to be MIF. Here, we show that TRX and MIF form a complex in the cell and the culture supernatant of ATL-2 cells. Using a BIAcore assay, we confirmed that TRX has a specific affinity with MIF. We also found that extracellular MIF was more effectively internalized into the ATL-2 cells expressing TRX on the cell surface, than the Jurkat T cells which do not express surface TRX. Moreover, anti-TRX antibody blocked the MIF internalization, suggesting that the cell surface TRX is involved in MIF internalization into the cells. Furthermore, anti-TRX antibody inhibited MIF-mediated enhancement of TNF-alpha production from macrophage RAW264.7 cells. These results suggest that the cell surface TRX serves as one of the MIF binding molecules or MIF receptor component and inhibits MIF-mediated inflammatory signals.

Dendritic cells derived from TBP-2-deficient mice are defective in inducing T cell responses.

Thioredoxin-binding protein-2 (TBP-2), also known as vitamin D3-up-regulated protein 1 (VDUP1), was identified as an endogenous molecule interacting with thioredoxin (TRX). Here, we show that dendritic cells (DC) derived from TBP-2-deficient mice are defective in the function of T cell activation. To compare TBP-2(-/-) DC function with wild-type (WT) DC, we stimulated DC with lipopolysaccharide (LPS). Although TBP-2(-/-) DC and WT DC expressed comparable levels of MHC class II and costimulatory molecules such as CD40, CD80 and CD86, the IL-12p40, IL-12p70 and IL-6 productions of TBP-2(-/-) DC were attenuated. In a mixed leukocyte reaction (MLR), the concentrations of IL-2, IFN-gamma, IL-4 and IL-10 in the culture supernatant of MLR with TBP-2(-/-) DC were significantly lower than those in the cultures with WT DC. In MLR also, as with LPS stimulation, IL-12p40 and IL-12p70 production from TBP-2(-/-) DC was less than that from WT DC. Proliferation of T cells cultured with TBP-2(-/-) DC was poorer than that with WT DC. In vivo delayed-type hypersensitivity responses in TBP-2(-/-) mice immunized with ovalbumin were significantly reduced compared to WT mice. These results indicate that TBP-2 plays a crucial role in DC to induce T cell responses.

Lipid raft-mediated uptake of cysteine-modified thioredoxin-1: apoptosis enhancement by inhibiting the endogenous thioredoxin-1.

Thioredoxin-1 (TRX) plays important roles in cellular signaling by controlling the redox state of cysteine residues in target proteins. TRX is released in response to oxidative stress and shows various biologic functions from the extracellular environment. However, the mechanism by which extracellular TRX transduces the signal into the cells remains unclear. Here we report that the cysteine modification at the active site of TRX promotes the internalization of TRX into the cells. TRX-C35S, in which the cysteine at residue 35 of the active site was replaced with serine, was internalized more effectively than wild-type TRX in human T-cell leukemia virus-transformed T cells. TRX-C35S bound rapidly to the cell surface and was internalized into the cells dependent on lipid rafts in the plasma membrane. This process was inhibited by wild-type TRX, reducing reagents such as dithiothreitol, and methyl-beta-cyclodextrin, which disrupts lipid rafts. Moreover, the internalized TRX-C35S binds to endogenous TRX, resulting in the generation of intracellular reactive oxygen species (ROS) and enhanced cis-diamine-dichloroplatinum (II) (CDDP)-induced apoptosis via a ROS-mediated pathway involving apoptosis signal-regulating kinase-1 (ASK-1) activation. These findings suggest that the cysteine at the active site of TRX plays a key role in the internalization and signal transduction of extracellular TRX into the cells.

Redox regulation of mast cell histamine release in thioredoxin-1 (TRX) transgenic mice.

Thioredoxin-1 (TRX) is a stress-inducible redox-regulatory protein with antioxidative and anti-inflammatory effects. Here we show that the release of histamine from mast cells elicited by cross-linking of high-affinity receptor for IgE (FcepsilonRI) was significantly suppressed in TRX transgenic (TRX-tg) mice compared to wild type (WT) mice. Intracellular reactive oxygen species (ROS) of mast cells stimulated by IgE and antigen was also reduced in TRX-tg mice compared to WT mice. Whereas there was no difference in the production of cytokines (IL-6 and TNF-alpha) from mast cells in response to 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) stimulation in TRX-tg and WT mice. Immunological status of TRX-tg mice inclined to T helper (Th) 2 dominant in primary immune response, although there was no difference in the population of dendritic cells (DCs) and regulatory T cells. We conclude that the histamine release from mast cells in TRX-tg mice is suppressed by inhibition of ROS generation. As ROS are involved in mast cell activation and facilitate mediator release, TRX may be a key signaling molecule regulating the early events in the IgE signaling in mast cells and the allergic inflammation.

Thioredoxin-1 ameliorates myosin-induced autoimmune myocarditis by suppressing chemokine expressions and leukocyte chemotaxis in mice.

BACKGROUND: Cardiac myosin-induced myocarditis is an experimental autoimmune myocarditis (EAM) model used to investigate autoimmunological mechanisms in inflammatory heart diseases and resembles fulminant myocarditis in humans. We investigated the therapeutic role of thioredoxin-1 (TRX-1), a redox-regulatory protein with antioxidant and antiinflammatory effects, in murine EAM. METHODS AND RESULTS: EAM was generated in 5-week-old male BALB/c mice by immunization with porcine cardiac myosin at days 0 and 7. Recombinant human TRX-1 (rhTRX-1), C32S/C35S mutant rhTRX-1, or saline was administered intraperitoneally every second day from day 0 to 20. In addition, rabbit anti-mouse TRX-1 serum or normal rabbit serum was administered intraperitoneally on days -1, 2, and 6. Animals were euthanized on day 21. Histological analysis of the heart showed that TRX-1 significantly reduced the severity of EAM, whereas mutant TRX-1 failed to have such an effect, and anti-TRX-1 antibody enhanced the disease markedly. Immunohistochemical analysis showed that TRX-1 significantly suppressed cardiac macrophage inflammatory protein (MIP)-1alpha, MIP-2, and 8-hydroxydeoxyguanosine expression and macrophage infiltration into the heart in EAM. Although serum levels of MIP-1alpha were not suppressed by TRX-1 until day 21, both an in vitro chemotaxis chamber assay and an in vivo air pouch model showed that TRX-1 significantly suppressed MIP-1alpha- or MIP-2-induced leukocyte chemotaxis. However, real-time reverse transcription-polymerase chain reaction showed that TRX-1 failed to decrease chemokine receptor expression increased in the bone marrow cells of EAM mice. CONCLUSIONS: TRX-1 attenuates EAM by suppressing chemokine expressions and leukocyte chemotaxis in mice.

Cysteine-dependent immune regulation by TRX and MIF/GIF family proteins.

Thioredoxin (TRX) superfamily proteins that contain a conserved redox-active site -Cys-Xa.a.-Xa.a.-Cys- includes proinflammatory cytokine, macrophage migration inhibiting factor (MIF) and the immune regulatory cytokine, glycosylation inhibiting factor (GIF) in which Cys-60 is cysteinylated. In this report, we have analyzed the functional interaction between TRX and MIF/GIF. The stable Jurkat T cell line transfected with human TRX gene (TRX-transfectant) was highly resistant to hydrogen peroxide-induced apoptosis, but not the cell line transfected with vector (mock-transfectant). The expression level of MIF/GIF protein of TRX-transfectant was lower than that of mock-transfectant. Conversely, the expression level of intracellular TRX protein in CD4(+)-T cells derived from MIF -/- mice were significantly higher than that from background BALB/c mice. These findings collectively suggest that oxidative stress-induced apoptosis on T lymphocytes might be protected by the reciprocal regulation of TRX and MIF/GIF expression.

TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum.

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.