Characterizing ANGPTL5 and RCASBP vector transfection-mediated ANGPTL5 induction in normoxic chicken embryo fibroblasts

Abstract

Extracellular vesicles (EVs) are a heterogeneous group of phospholipid bilayer-enclosed structures containing protein, lipid, and nucleic cargo originating from any of a cell’s membranes. EVs play critical roles in intercellular communication, culminating in signalling pathway activation and eliciting behavioural changes in recipient cells. Preliminary Western blot and immunofluorescence analyses on hypoxic and contact-inhibited chicken embryo fibroblasts (CEFs) confirmed the presence of pro-angiogenic ANGPTL5. Furthermore, ANGPTL5 was shown to be upregulated in EVs isolated from hypoxic and contact-inhibited CEF tissue culture medium, suggesting that ANGPTL5 may have pro-survival roles under oxidative stress conditions. Further investigation is needed to elucidate whether supplementing serum-starved CEF culture with hypoxic-CEF-derived EVs can relieve ER stress and prompt cells to re-enter the cell cycle. Current literature implicates EVs in quiescence signalling pathway(s), suggesting the EVs have a molecular and mechanistic influence on cellular decision-making between proliferation and quiescence and possibly cell survival. Results from RCASBP ANGPTL5 cDNA vector transfection experiments showed that the RCASBP ANGPTL5 cDNA vector is an effect tool for inducing ANGPTL5 expression in CEF under normoxic conditions (21% O2). The robustness of the inductive effect also appeared to increase with time post-transfection. Furthermore, treating normal CEF in normoxia with EV isolated from ANGPTL5-CEF alone is sufficient to induce ANGPTL5 expression in normoxic CEF. The strength of ANGPTL5 induction with ANGPTL5-CEF-derived EV also seemed to increase with longer treatment time. The results suggest that ANGPTL5 could relay information from the cell of origin to recipient cells via exosomal transmission, highlighting the potential role of ANGPTL5 in intercellular communication during cellular quiescence.