Rapid binding of electrostatically stabilized iron oxide nanoparticles to THP-1 monocytic cells via interaction with glycosaminoglycans.

Magnetic resonance imaging (MRI) with contrast agents that target specific inflammatory components of atherosclerotic lesions has the potential to emerge as promising diagnostic modality for detecting unstable plaques. Since a high content of macrophages and alterations of the extracellular matrix are hallmarks of plaque instability, these structures represent attractive targets for new imaging modalities. In this study, we compared in vitro uptake and binding of electrostatically stabilized citrate-coated very small superparamagnetic iron oxide particles (VSOP) to THP-1 cells with sterically stabilized carboxydextran-coated Resovist(®). Uptake of VSOP in both THP-1 monocytic cells and THP-derived macrophages (THP-MΦ) was more efficient compared to Resovist(®) without inducing cytotoxicity or modifying normal cellular functions (no changes in levels of reactive oxygen species, caspase-3 activity, proliferation, cytokine production). Importantly, VSOP bound with high affinity to the cell surface and to apoptotic membrane vesicles. Inhibition of glycosaminoglycan (GAG) synthesis by glucose deprivation in THP-MΦ was associated with a significant reduction of VSOP attachment suggesting that the strong interaction of VSOP with the membranes of cells and apoptotic vesicles occurs via binding to negatively charged GAGs. These in vitro experiments show that VSOP-enhanced MRI may represent a new imaging approach for visualizing high-risk plaques on the basis of targeting pathologically increased GAGs or apoptotic membrane vesicles in atherosclerotic lesions. VSOP should be investigated further in appropriate in vivo experiments to characterize accumulation in unstable plaque.

Protection of vascular cells from oxidative stress by proteasome inhibition depends on Nrf2.

AIMS: Increased levels of reactive oxygen species cause oxidative stress and severely damage lipids, proteins, and DNA. We have previously shown that partial proteasome inhibition induces an antioxidative gene pattern in endothelial cells. Here, we elucidate the mechanisms of proteasome inhibitor-mediated upregulation of antioxidative enzymes and cytoprotection. METHODS AND RESULTS: Non-toxic proteasome inhibition upregulated mRNA and protein expression of superoxide dismutase 1 (SOD1) and haem oxygenase 1 (HO1) in several human endothelial and vascular smooth muscle cell types. Transcriptional activation of these enzymes was shown by inhibition of RNA polymerase II and nuclear run-on assays. Transfection of endothelial cells with luciferase reporter constructs revealed that upregulation can be largely confined to an antioxidant response element (ARE), which proved to be sufficient for transcriptional activation of SOD1 and HO1. Co-transfection studies and bandshift analyses confirmed binding of the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition as shown by immunoblots-to the ARE site of HO1. Experiments with aortic endothelial and smooth muscle cells from Nrf2 wild-type and knockout mice revealed an essential role of Nrf2: in wild-type cells, proteasome inhibitor-mediated induction of SOD1 and HO1 was accompanied by protection of vascular cells against oxidative stress as determined by lactate dehydrogenase release assays. In contrast, proteasome inhibitor-mediated induction of antioxidative enzymes and cytoprotection were completely lost in cells from Nrf2 knockout mice. CONCLUSION: Nrf2-dependent transcriptional activation of antioxidative enzymes is crucial for proteasome inhibitor-mediated protection of vascular cells against oxidative stress.

Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitor-mediated cardioprotection.

AIMS: We have shown previously that non-toxic inhibition of the ubiquitin-proteasome system upregulates antioxidative defence mechanisms and protects endothelial cells from oxidative stress. Here, we have addressed the question whether the induction of antioxidative enzymes contributes to cardioprotection by non-toxic proteasome inhibition. METHODS AND RESULTS: Treatment with 0.5 micromol/L MG132 for 48 h proved to be non-toxic and protected neonatal rat cardiac myocytes against H(2)O(2)-mediated oxidative stress in lactate dehydrogenase assays. This correlated with reduced levels of intracellular reactive oxygen species as determined by loading myocytes with dichlorofluorescein. Immunoblots showed significant upregulation of superoxide dismutase 1 (SOD1), haem oxygenase 1, and catalase upon proteasome inhibition. Luciferase assays using a reporter driven by the SOD1 promoter revealed proteasome inhibitor-mediated induction of luciferase activity. Deletion and mutation analyses identified an antioxidant response element (ARE) in the SOD1 promoter to be not only essential but also sufficient for transcriptional upregulation by proteasome inhibition. An essential role for the antioxidative transcription factor NF-E2-related factor 2 (Nrf2)-which was stabilized by proteasome inhibition-in ARE-mediated transcriptional activation was revealed in cardiac myocytes from Nrf2 wild-type and knockout mice: proteasome inhibition upregulated antioxidative enzymes and conferred protection against H(2)O(2)-mediated oxidative stress in Nrf2 wild-type cells. In contrast, the induction of antioxidative enzymes and cytoprotection were completely abolished in cardiac myocytes from Nrf2 knockout mice. CONCLUSION: Non-toxic proteasome inhibition upregulates antioxidative enzymes via an Nrf2-dependent transcriptional activation of AREs and confers cardioprotection.

Human-specific induction of glutathione peroxidase-3 by proteasome inhibition in cardiovascular cells.

Glutathione peroxidase-3 (GPx-3) is a key antioxidant enzyme in the plasma. GPx-3 was previously identified as the major antioxidative enzyme that was induced upon nontoxic proteasome inhibition in endothelial cells. Here, we investigated the determinants of the proteasome inhibitor-induced expression of GPx-3. Nontoxic proteasome inhibition massively upregulates GPx-3 RNA and protein in human umbilical cord vein cells within 24 h. Surprisingly, induction of GPx-3 was species-specific for human cells. The exponential upregulation of GPx-3 is mediated by transcriptional activation of the human GPx-3 promoter and, in addition, stabilization of GPx-3 mRNA: in reporter gene assays with full-length and deleted variants of the human GPx-3 promoter we identified a putative antioxidative response element (ARE) as essential and also sufficient for transcriptional activation of GPx-3 by proteasome inhibition. However, the ARE-specific antioxidative transcription factor Nrf2 is not involved in the activation of GPx-3. UV-crosslinking using the 3'UTR of GPx-3 revealed an altered protein binding pattern in the presence of proteasome inhibitors, thus indicating regulation of mRNA stability of human GPx-3. As GPx-3 is secreted into the plasma, our data point toward a borderline defense mechanism of endothelial cell-derived GPx-3 to protect the vasculature from oxidative stress.