The mitogen-activated protein kinase (MAPK) p38 is an essential family of kinases, regulating responses to environmental stress and inflammation. There is an ever-increasing plethora of physiological and pathophysiological conditions attributed to p38 activity, ranging from cell division and embryonic development to the control of a multitude of diseases including retinal, cardiovascular, and neurodegenerative diseases, diabetes, and cancer. Despite the decades of intense investigation, a viable therapeutic approach to disrupt p38 signaling remains elusive. A growing body of evidence supports the pathological significance of an understudied atypical p38 signaling pathway. Atypical p38 signaling is driven by a direct interaction between the adaptor protein TAB1 and p38α, driving p38 autophosphorylation independent from the classical MKK3 and MKK6 pathways. Unlike the classical MKK3/6 signaling pathway, atypical signaling is selective for just p38α, and at present has only been characterized during pathophysiological stimulation. Recent studies have linked atypical signaling to dermal and vascular inflammation, myocardial ischemia, cancer metastasis, diabetes, complications during pregnancy, and bacterial and viral infections. Additional studies are required to fully understand how, when, where, and why atypical p38 signaling is induced. Furthermore, the development of selective TAB1-p38 inhibitors represents an exciting new opportunity to selectively inhibit pathological p38 signaling in a wide array of diseases.

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.

A key mediator of angiogenesis and inflammatory response in chronic lung disease is the mitogen‐activated kinase (MAP), 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 was discovered in a family of pro‐inflammatory G‐protein coupled receptors (GPCRs). The atypical pathway uses transforming growth factor β activated kinase 1 binding protein‐1 (TAB1) as opposed to MKK3/6 to interact with p38α. The binding of TAB1 to p38α induces the autophosphorylation of p38α which leads to increased proangiogenic signaling. Recent studies have highlighted the use of cell‐penetrating inhibitors that specifically block atypical p38 signaling, leaving MKK3/6 dependent signaling intact to maintain normal cellular function. We have shown that we can label peptide inhibitors with a red fluorescent protein which can be transiently expressed in endothelial cells. Using a lentivirus we are generating endothelial cell lines stably expressing an array of RFP linked inhibitor peptides targeted for exosomal packaging. Purified peptide loaded exosomes/nanocarriers are then used to deliver inhibitors to vascular cells in vitro. Our current studies focus on assessing the cellular uptake of peptide loaded exosomes by observing the mRFP using confocal microscopy and immunoblotting. We predict that exosomally delivered peptides inhibit p38 signaling by GPCR ligands in endothelial cells. These studies will demonstrate the potential for exosomal nanocarriers to deliver peptide inhibitors to block GPCR inflammation.