PS-B12-5: THE INFLUENCE OF CELLULAR AGING ON THE MOLECULES CLEARANCE THAT UNDERGO ENDOCYTOSIS VIA AN ANGIOTENSIN II TYPE 1 RECEPTOR-DEPENDENT PATHWAY.

Abstract

Objective: We recently reported that ligand binding to the receptor for advanced glycation end-products (RAGE) or oxidized LDL receptor (LOX-1) activates the G protein-coupled angiotensin II (AII) type 1 receptor (AT1) via a membrane complex of the receptor (2021 Sci Rep, 2021 iScience). The intracellular events triggered by this AT1 activation include signal transduction in a G-protein-dependent pathway and endocytosis of the ligand-receptors complex in a β;-arrestin-dependent pathway. Particularly, little is known about the significance of AT1-induced endocytosis of ligands. In this study, we used amyloid β; (A β;), a known ligand for RAGE, to examine whether cell aging affects the fate of ligands undergoing AT1-dependent endocytosis. Design and Methods: Human unvelical vascular endothelial cell (HUVEC) and human brain microvascluar endothelial cell (HBMEC) were continuously cultured to induce replicative senescence. Fluorescent A β; (FA β;) was used to visualize cellular uptake of A β;. Fluorescence/cell number was measured after 24hour application of FA β; (F0) or after 24hour application followed by 24hour washout of FA β; (F1). Clearance of A β; was culculated by (F0-F1)/F0. Cellular aggeragation of A β; was assessed by PROTEOSTAT∨ reagent. Pretreatment with lysosomal inhibitors (Chloroquine, Bafilomycin A), siRNA knockdown of AT1, or trehalose as a lysosomal activator was used if applicable. β;galactosidase assay and real time PCR of p16 mRNA were performed to quantify cellular senescence. Results: In HUVECs, pretreatment with Chloroquine or Bafilomycin A markedly decreased A β; clearance. Older HUVEC with passage 12 (P12) and older HBMEC with P20 showed higher β;-galactosidase activity and p16 expression compared with younger cells with P3 and P5, respectively. Clearance of A β; was decreased in older cells compared to younger cells in both cell types. A β; aggregation was higher in older cells than younger cells in both cell types. siRNA of AT1 prominently decreased uptake and aggregation of A β; but did not influence clearance rate of Aβ; in both types of cells. Pretreatment of trehalose significantly restored clearance of Aβ; in older HBMEC. Conclusions: These findings indicate that cellular senescence reduces the clearance of molecules that undergo endocytosis via an AT1-dependent pathway. Further investigation will be required, but impaired lysosomal function due to aging might be involved in this mechanism.