Bagwell, Monique

Introduction: Platelets are heavily implicated in angiogenesis; however, the underlying mechanism remains relatively uncharacterized. The goal of this study was to evaluate the angiogenic potency of a platelet-derived regenerative exosome product (PEP) in an effort to develop a novel treatment for peripheral arterial disease. Here, we evaluated the mechanistic basis of PEP in angiogenesis and it’s use In Vivo in a rodent hind limb ischemia model. Methods/Results: Under Current Good Manufacturing Practices, conditioned medium from apheresis platelet cultures was purified to acquire the exosomes. PEP, enriched in exosome markers CD63, CD9 and Flotillin, induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and vascular tube formation In Vitro. Proteomic evaluation of PEP highlighted receptor tyrosine kinase (RTK) pathways and western blot validation demonstrated induction of phospho-MAPK and phospho-AKT following PEP treatment of HUVECs. Systematic evaluation of RTK receptor involvement was performed using small molecule inhibitors and receptor knock-down in HUVECs, ruling out RTK activation as a mode of action. However, small molecule inhibition of PI3 kinase demonstrated elimination of PEP-induced AKT phosphorylation and reduction in PEP-induced angiogenesis In Vitro measured by HUVEC proliferation and migration. In Vivo, evaluation of angiogenesis was performed in a rodent model of hindlimb ischemia treated with PEP embedded in fibrin glue (PEP/TISSEEL). Blood flow was evaluated by SPY angiography on day 0 pre- and post-operation, day 21, and day 28. Animals treated with PEP/TISSEEL showed a significant increase in blood flow compared to sham and TISSEEL treated animals (percent perfusion at 28 days: sham 40.9%, TISSEEL 44.3%, PEP/TISSEEL 100.2%), with significant increase in vascular area on histology (at 28 days vascular area (μm 2 )/muscle area(μm 2 ): healthy control 12.71, sham 4.66, TISSEEL 4.76, PEP/TISSEEL 15.54). Conclusions: Exosome driven activation of PI3 kinase can induce angiogenesis both In Vitro and In Vivo. PEP provides a novel, clinical-grade, off-the-shelf therapy to promote angiogenesis for the treatment of peripheral arterial disease.

Objectives: Peripheral arterial disease is a significant cause of morbidity and mortality. The goal of this study was to develop a novel treatment to promote angiogenesis. Platelets have been previously implicated in angiogenic events; however, platelet activation rather than presence of growth factors has been associated with new blood vessel formation. This study evaluated exosomes purified from activated platelets to determine their potential in promoting angiogenesis both in vitro and in vivo. Methods & Results: Under Current Good Manufacturing Practices, conditioned medium processed from apheresis platelet cultures yielded a purified exosome product (PEP) enriched for CD63, CD9 and Alix. In vitro, PEP drove HUVEC proliferation, migration, and vascular tube formation. Proteomic evaluation showed Flk1 (VEGFR2) was one of the key angiogenic proteins in PEP. HUVECs treated with Flk1 antibody neutralized PEP (anti-Flk1-PEP) showed reduced vascular tube formation compared to PEP. To evaluate in vivo angiogenesis, a rodent model of hindlimb ischemia was treated with sustained release PEP embedded in fibrin glue (PEP/TISSEEL) with blood flow evaluated by SPY angiography at 28 days post-treatment. Animals treated with PEP/TISSEEL showed improved blood flow with significant difference (p<0.001) as compared to sham, TISSEEL, and anti-Flk1-PEP/TISSEEL. Muscle tissue stained with α smooth muscle actin showed a significant increase (p<0.001) in vascular area in PEP/TISSEEL treated animals compared to TISSEEL alone. To further evaluate clinical translatability, a rabbit ear ischemia wound model was then treated with PEP/TISSEEL. Rabbit ears showed accelerated wound healing and restored blood flow on SPY angiography. Histologic evaluation of vascular area showed a significant increase in vascular area (p<0.05) compared to sham and TISSEEL alone animals. Conclusions: Exosome driven donation of Flk1 here promoted angiogenic events and was successfully translated into healing outcomes in the setting of two animal models of peripheral ischemia. This novel insight into biotherapy achievable pro-angiogenesis establishes a clinical-grade off-the-shelf exosome technology for the treatment of peripheral vascular disease.