The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating and addressing the complexities of heterogeneous nanoparticle subpopulations.

Citrate is an iron chelator and it has been shown to be the major iron ligand in the xylem sap of plants. Furthermore, citrate has been demonstrated to be an important ligand for the non-transferrin bound iron (NTBI) pool occurring in the plasma of individuals suffering from iron-overload. However, ferric citrate chemistry is complicated and a definitive description of its aqueous speciation at neutral pH remains elusive. X-Ray crystallography data indicates that the alcohol function of citrate (Cit4-) is involved in Fe(III) coordination and that deprotonation of this functional group occurs upon complex formation. The inability to include this deprotonation in the affinity constant calculations has been a major source of divergence between various reports of iron(III)-citrate affinity constants. However the recent determination of the alcoholic pKa of citric acid (H4Cit) renders the reassessment of the ferric citrate system possible. The aqueous speciation of ferric citrate has been investigated by mass spectrometry and EPR spectroscopy. It was observed that the most relevant species are a monoiron dicitrate species and dinuclear and trinuclear oligomeric complexes, the relative concentration of which depends on the solution pH value and the iron : citric acid molar ratio. Spectrophotometric titration was utilized for affinity constant determination and the formation constant for the biologically relevant [Fe(Cit)2]5- is reported for the first time.

Aims Whether and how iron affects the progression of atherosclerosis remains highly debated. Here, we investigate susceptibility to atherosclerosis in a mouse model (ApoE−/− FPNwt/C326S), which develops the disease in the context of elevated non-transferrin bound serum iron (NTBI). Methods and results Compared with normo-ferremic ApoE−/− mice, atherosclerosis is profoundly aggravated in iron-loaded ApoE−/− FPNwt/C326S mice, suggesting a pro-atherogenic role for iron. Iron heavily deposits in the arterial media layer, which correlates with plaque formation, vascular oxidative stress and dysfunction. Atherosclerosis is exacerbated by iron-triggered lipid profile alterations, vascular permeabilization, sustained endothelial activation, elevated pro-atherogenic inflammatory mediators, and reduced nitric oxide availability. NTBI causes iron overload, induces reactive oxygen species production and apoptosis in cultured vascular cells, and stimulates massive MCP-1-mediated monocyte recruitment, well-established mechanisms contributing to atherosclerosis. NTBI-mediated toxicity is prevented by transferrin- or chelator-mediated iron scavenging. Consistently, a low-iron diet and iron chelation therapy strongly improved the course of the disease in ApoE−/− FPNwt/C326S mice. Our results are corroborated by analyses of serum samples of haemochromatosis patients, which show an inverse correlation between the degree of iron depletion and hallmarks of endothelial dysfunction and inflammation. Conclusion Our data demonstrate that NTBI-triggered iron overload aggravates atherosclerosis and unravel a causal link between NTBI and the progression of atherosclerotic lesions. Our findings support clinical applications of iron restriction in iron-loaded individuals to counteract iron-aggravated vascular dysfunction and atherosclerosis.

Invasive bladder tumours express the cell-surface Sialyl-Tn (STn) antigen, which stems from a premature stop in protein O-glycosylation. The STn antigen favours invasion, immune escape, and possibly chemotherapy resistance, making it attractive for target therapeutics. However, the events leading to such deregulation in protein glycosylation are mostly unknown. Since hypoxia is a salient feature of advanced stage tumours, we searched into how it influences bladder cancer cells glycophenotype, with emphasis on STn expression. Therefore, three bladder cancer cell lines with distinct genetic and molecular backgrounds (T24, 5637 and HT1376) were submitted to hypoxia. To disclose HIF-1α-mediated events, experiments were also conducted in the presence of Deferoxamine Mesilate (Dfx), an inhibitor of HIF-1α proteasomal degradation. In both conditions all cell lines overexpressed HIF-1α and its transcriptionally-regulated protein CA-IX. This was accompanied by increased lactate biosynthesis, denoting a shift toward anaerobic metabolism. Concomitantly, T24 and 5637 cells acquired a more motile phenotype, consistent with their more mesenchymal characteristics. Moreover, hypoxia promoted STn antigen overexpression in all cell lines and enhanced the migration and invasion of those presenting more mesenchymal characteristics, in an HIF-1α-dependent manner. These effects were reversed by reoxygenation, demonstrating that oxygen affects O-glycan extension. Glycoproteomics studies highlighted that STn was mainly present in integrins and cadherins, suggesting a possible role for this glycan in adhesion, cell motility and invasion. The association between HIF-1α and STn overexpressions and tumour invasion was further confirmed in bladder cancer patient samples. In conclusion, STn overexpression may, in part, result from a HIF-1α mediated cell-survival strategy to adapt to the hypoxic challenge, favouring cell invasion. In addition, targeting STn-expressing glycoproteins may offer potential to treat tumour hypoxic niches harbouring more malignant cells.