ABSTRACT
Understanding the mechanism of toxicity of nanomaterials remains a challenge with respect to both mechanisms involved and product regulation. Here we show toxicity of ultrasmall gold nanoparticles (AuNPs). Depending on the ligand chemistry, 1.4-nm-diameter AuNPs failed electrophysiology-based safety testing using human embryonic kidney cell line 293 cells expressing human ether-á-go-go-Related gene (hERG), a Food and Drug Administration-established drug safety test. In patch-clamp experiments, phosphine-stabilized AuNPs irreversibly blocked hERG channels, whereas thiol-stabilized AuNPs of similar size had no effect in vitro, and neither particle blocked the channel in vivo. We conclude that safety regulations may need to be reevaluated and adapted to reflect the fact that the binding modality of surface functional groups becomes a relevant parameter for the design of nanoscale bioactive compounds.
Subject(s)
Ether-A-Go-Go Potassium Channels/physiology , Gold/chemistry , Metal Nanoparticles/chemistry , Animals , ERG1 Potassium Channel , Electrocardiography/methods , Electrophysiology/methods , Ether-A-Go-Go Potassium Channels/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C57BL , Nanotechnology/methods , Patch-Clamp Techniques , Potassium Channel Blockers/chemistry , Protein Binding , TemperatureABSTRACT
The serum protein fetuin-A is essential for mineral homeostasis and shows immunomodulatory functions, for example by binding to TGF superfamily proteins. It proved neuroprotective in a rat stroke model and reduced lethality after systemic lipopolysaccharide challenge in mice. Serum fetuin-A concentrations are highest during intrauterine life. Different species show intrauterine cerebral fetuin-A immunoreactivity, suggesting a contribution to brain development. We therefore aimed at specifying fetuin-A immunoreactivity in brains of newborn rats (age P0-P28) and human neonates (20-40 weeks of gestation). In humans and rats, fetuin-A was found in cortex, white matter, subplate, hippocampus, subventricular zone, and ependymal cells which supports a global role for brain function. In rats, overall fetuin-A immunoreactivity decreased with age. At P0 fetuin-A immunoreactivity affected most brain structures. Thereafter, it became increasingly restricted to distinct cells of the hippocampus, cingular gyrus, periventricular stem cell layer, and ependyma. In ependymal cells the staining pattern complied with active transependymal transport from cerebrospinal fluid. Double immunofluorescence studies revealed colocalization with NeuN (mature neurons), beta III tubulin (immature neurons), GFAP (astrocytes), and CD68 (activated microglia). This points to a role of fetuin-A in different brain functional systems. In human neonatal autopsy cases, frequently affected from severe neurological and non-neurological diseases, fetuin-A immunoreactivity was heterogeneous and much less associated with age than in healthy tissues studied earlier, suggesting an impact of exogeneous noxious factors on fetuin-A regulation. Further research on the role of fetuin-A in the neonatal brain during physiological and pathological conditions is recommended.
Subject(s)
Brain Chemistry , Brain/embryology , Nerve Tissue Proteins/analysis , alpha-2-HS-Glycoprotein/analysis , Animals , Animals, Newborn , Brain/growth & development , Female , Gestational Age , Humans , Immunoenzyme Techniques , Male , Microscopy, Fluorescence , Nerve Tissue Proteins/physiology , Organ Specificity , Rats , Rats, Wistar , alpha-2-HS-Glycoprotein/physiologyABSTRACT
Fetuin-A is a liver-derived plasma protein involved in calcified matrix metabolism. Fetuin-A mediates the formation and stabilization of calciprotein particles (CPPs), soluble colloids made of fetuin-A, further serum proteins, and calcium phosphate mineral. CPP formation ensures mineral solubilization and rapid clearance from circulation by macrophages of the mononuclear phagocyte system, thus preventing pathological calcification. Accordingly, low levels of free serum fetuin-A and high serum CPPs are associated with pathological calcification in patients suffering from chronic kidney disease.
Subject(s)
Calcinosis/metabolism , Fetuins/metabolism , Minerals/metabolism , Animals , Blood Proteins/metabolism , Calcium Phosphates/metabolism , Humans , Mononuclear Phagocyte System/metabolism , Osteopontin/metabolism , Phosphorylation , Protein BindingABSTRACT
RATIONALE: Fetuin-A is a liver-derived plasma protein involved in the regulation of calcified matrix metabolism. Biochemical studies showed that fetuin-A is essential for the formation of protein-mineral complexes, called calciprotein particles (CPPs). CPPs must be cleared from circulation to prevent local deposition and pathological calcification. OBJECTIVE: We studied CPP clearance in mice and in cell culture to identify the tissues, cells, and receptors involved in the clearance. METHODS AND RESULTS: In mice, fetuin-A-containing CPPs were rapidly cleared by the reticuloendothelial system, namely Kupffer cells of the liver and marginal zone macrophages of the spleen. Macrophages from scavenger receptor-AI/II (SR-A)-deficient mice cleared CPPs less efficiently than macrophages from wild-type mice, suggesting that SR-AI/II is involved in CPP binding and endocytosis. Accordingly, we found reduced clearance of CPPs in SR-A/MARCO-deficient mice. CONCLUSIONS: We could demonstrate that fetuin-A-containing CPPs facilitate the clearance of mineral debris by macrophages via SR-A. Since the same receptor also contributes to the uptake of modified low-density lipoprotein particles in atherosclerosis, defective endocytosis of both types of particle may impinge on lipid as well as mineral debris clearance in calcifying atherosclerosis.
