Characterization and heme oxygenase-1 content of extracellular vesicles in human biofluids

Heme oxygenase-1 (HO-1), a highly inducible stress protein that degrades heme to biliverdin, carbon monoxide, and free ferrous iron, is increased in blood and other biofluids of subjects with various systemic and neurological disorders. HO-1 does not contain an N-terminal signal peptide and the mechanism responsible for its secretion remains unknown. Extracellular vesicles (EVs) are membrane-bound inclusions that transport microRNAs, messenger RNAs, lipids, and proteins among diverse cellular and extracellular compartments. The objective of the current study was to determine whether EVs in human biofluids contain HO-1, and whether the latter may be transported in EVs from brain to periphery. Total, L1 cell adhesion molecule protein (L1CAM)-enriched (neuron-derived), and glutamate aspartate transporter 1 (GLAST)-enriched (astrocyte-derived) EVs were purified from five different human biofluids (saliva [n = 40], plasma [n = 14], serum [n = 10], urine [n = 10], and cerebrospinal fluid [n = 11]) using polymer precipitation and immuno-affinity-based capture methods. L1CAM-enriched, GLAST-enriched, and L1CAM/GLAST-depleted (LGD) EV, along with EV-depleted (EVD), fractions were validated by nanoparticle tracking analysis, enzyme-linked immunosorbent assay (ELISA), and western blot. HO-1 was assayed in all fractions using ELISA and western blot. The majority of HO-1 protein was localized to LGD, L1CAM-enriched, and GLAST-enriched EVs of all human biofluids surveyed after adjusting for age and sex, with little HO-1 protein detected in EVD fractions. HO-1 protein in human biofluids is predominantly localized to EV compartments. A substantial proportion of EV HO-1 in peripheral human biofluids is derived from the central nervous system and may contribute to the systemic manifestations of various neurological conditions.