William Antoniades (University of Georgia)| Neil Grimsey (University of Georgia)
A key mediator of angiogenesis and inflammatory response in chronic lung disease is the mitogen-activated protein kinase (MAPK), p38α. The typical activation pathway for p38α uses MAP kinase kinase 3 (MKK3), and MKK6 to phosphorylate and activate p38α. However, an atypical p38α signaling pathway activated by a family of pro-inflammatory G-protein coupled receptors (GPCRs) induces a direct interaction between transforming growth factor β activated kinase 1 binding protein-1 (TAB1) and p38α, bypassing MKK3/6-dependent p38α activation. The binding of TAB1 to p38α induces the autophosphorylation of p38α, triggering proinflammatory signaling in the vasculature. Recent studies have highlighted the use of cell-penetrating inhibitors that specifically block atypical p38α signaling, leaving MKK3/6-dependent signaling intact. Using the Xpack Lentiviral expression system, we have generated a stable cell line that expresses red fluorescent protein (RFP) labeled peptide inhibitors in endothelial cells. The Xpack system contains an exosomal packaging motif enabling RFP-linked inhibitor peptides to be targeted for exosomes. Using filter centrifugation and ultracentrifugation we have isolated RFP peptide loaded exosomes/extracellular vesicles. These inhibitor loaded extracellular vesicles can successfully deliver inhibitors to vascular endothelial cells in vitro. Our current studies focus on assessing whether cellular uptake of peptide loaded exosomes can efficiently attenuate GPCR-induced proinflammatory signaling. To investigate this we are using a combination of immunoblotting and live-cell Forster’s Resonance Energy Transfer (FRET) assays. We predict that exosomally delivered peptides will inhibit p38 signaling by GPCR ligands in endothelial cells providing a tool to produce large quantities of bioavailable inhibitors that can be developed into therapeutics. These studies demonstrate the potential for exosomal nanocarriers to deliver peptide inhibitors to block GPCR inflammation.