Changes in the proteome of extracellular vesicles shed by rat liver after subtoxic exposure to acetaminophen

Abstract

To identify changes in extracellular vesicles (EVs) secreted by the liver following druginduced liver injury (DILI), rats were treated with a subtoxic dose (500 mg/kg) of the analgesic drug, acetaminophen (APAP). EVs were collected by liver perfusion of sham and APAP-treated rats. Changes in EVs morphology were examined by transmission electron microscopic analysis of negatively stained vesicles. Results from morphometric analysis of EVs revealed striking differences in their size and distribution. Proteome composition of EVs collected by liver perfusion was determined by mass spectrometry using methods of sample preparation that enabled better detection of both highly hydrophobic proteins and proteins with complex post-translational modifications. The collection of EVs after liver perfusion is an approach that enables the isolation of EVs shed not only by isolated hepatocytes, but also by the entire complement of hepatic cells. EVs derived after DILI had a lower content of alpha-1-macroglobulin, ferritin, and members of cytochrome 450 family. Fibronectin, aminopeptidase N, metalloreductase STEAP4, integrin beta, and members of the annexin family were detected only in APAP-treated samples of EVs. These results show that the present approach can provide valuable insights into the response of the liver following drug-induced liver injury.

Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen

“…The presented results show important changes in the composition of EVs shed by the liver after APAP treatment. These results had already been noticed after our preliminary experiments were recently published [ 19 ]. In order to achieve better interpretation of these results, and to elucidate the biological function of proteins shed by liver EVs before and after APAP treatment, additional bioinformatic analysis was performed.…”

“…In our present work, several proteins that are components of the extracellular matrix and non-parenchymal cells, such as Kupffer, stellate, and endothelial cells, were detected only in EVs of APAP-treated livers [ 19 , 20 , 21 , 22 , 23 ]. In contrast, some hepatocyte-derived proteins were not detected, or detected in relatively lower concentrations in EVs after APAP treatment, but high concentrations of these proteins were detected in EVs shed by normal rat livers or cultured hepatocytes [ 12 , 13 , 14 , 19 ]. These findings suggest that EVs collected after perfusion of APAP-treated livers are secreted not only by hepatocytes as parenchymal cells, but also by non-parenchymal liver components like stellate and endothelial cells, Kupffer cells, and non-resident macrophages.…”

“…Raw data are given in Tables S4 and S5 in the Supplementary Materials . Our preliminary data have been presented by Kovač-Peić et al [ 19 ].…”

“…Changes to an EVs proteome after this kind of treatment indicate significant perturbation in the whole organ, even before other symptoms of toxic effects are observed. Already our preliminary experiments demonstrated striking differences in the size and distribution of EVs, as well as their protein composition [ 19 ]. In our present work, several proteins that are components of the extracellular matrix and non-parenchymal cells, such as Kupffer, stellate, and endothelial cells, were detected only in EVs of APAP-treated livers [ 19 , 20 , 21 , 22 , 23 ].…”

“…Already our preliminary experiments demonstrated striking differences in the size and distribution of EVs, as well as their protein composition [ 19 ]. In our present work, several proteins that are components of the extracellular matrix and non-parenchymal cells, such as Kupffer, stellate, and endothelial cells, were detected only in EVs of APAP-treated livers [ 19 , 20 , 21 , 22 , 23 ]. In contrast, some hepatocyte-derived proteins were not detected, or detected in relatively lower concentrations in EVs after APAP treatment, but high concentrations of these proteins were detected in EVs shed by normal rat livers or cultured hepatocytes [ 12 , 13 , 14 , 19 ].…”