Li, Jialun

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.

Background: Nerve injuries can result in detrimental functional outcomes. Currently, autologous nerve graft offers the best outcome for segmental peripheral nerve injury. Allografts are alternatives, but do not have comparable results. This study evaluated whether plasma-derived exosome can improve nerve regeneration and functional recovery when combined with decellularized nerve allografts. Methods: The effect of exosomes on Schwann cell proliferation and migration were evaluated. A rat model of sciatic nerve repair was used to evaluate the effect on nerve regeneration and functional recovery. A fibrin sealant was used as the scaffold for exosome. Eighty-four Lewis rats were divided into autograft, allograft, and allograft with exosome groups. Gene expression of nerve regeneration factors was analyzed on postoperative day 7. At 12 and 16 weeks, rats were subjected to maximum isometric tetanic force and compound muscle action potential. Nerve specimens were then analyzed by means of histology and immunohistochemistry. Results: Exosomes were readily taken up by Schwann cells that resulted in improved Schwann cell viability and migration. The treated allograft group had functional recovery (compound muscle action potential, isometric tetanic force) comparable to that of the autograft group. Similar results were observed in gene expression analysis of nerve regenerating factors. Histologic analysis showed no statistically significant differences between treated allograft and autograft groups in terms of axonal density, fascicular area, and myelin sheath thickness. Conclusions: Plasma-derived exosome treatment of decellularized nerve allograft may provide comparable clinical outcomes to that of an autograft. This can be a promising strategy in the future as an alternative for segmental peripheral nerve repair. Clinical Relevance Statement: Off-the-shelf exosomes may improve recovery in nerve allografts.

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.

Background: Tendons are primarily acellular, limiting their intrinsic regenerative capabilities. This limited regenerative potential contributes to delayed healing, rupture, and adhesion formation after tendon injury. Purpose: To determine if a tendon’s intrinsic regenerative potential could be improved after the application of a purified exosome product (PEP) when loaded onto a collagen scaffold. Study Design: Controlled laboratory study. Methods: An in vivo rabbit Achilles tendon model was used and consisted of 3 groups: (1) Achilles tenotomy with suture repair, (2) Achilles tenotomy with suture repair and collagen scaffold, and (3) Achilles tenotomy with suture repair and collagen scaffold loaded with PEP at 1 × 10 12 exosomes/mL. Each group consisted of 15 rabbits for a total of 45 specimens. Mechanical and histologic analyses were performed at both 3 and 6 weeks. Results: The load to failure and ultimate tensile stress were found to be similar across all groups ( P ≥ .15). The tendon cross-sectional area was significantly smaller for tendons treated with PEP compared with the control groups at 6 weeks, which was primarily related to an absence of external adhesions ( P = .04). Histologic analysis confirmed these findings, demonstrating significantly lower adhesion grade both macroscopically ( P = .0006) and microscopically ( P = .0062) when tendons were treated with PEP. Immunohistochemical staining showed a greater intensity for type 1 collagen for PEP-treated tendons compared with collagen-only or control tendons. Conclusion: Mechanical and histologic results suggested that healing in the PEP-treated group favored intrinsic healing (absence of adhesions) while control animals and animals treated with collagen only healed primarily via extrinsic scar formation. Despite a smaller cross-sectional area, treated tendons had the same ultimate tensile stress. This pilot investigation shows promise for PEP as a means of effectively treating tendon injuries and enhancing intrinsic healing. Clinical Relevance: The production of a cell-free, off-the-shelf product that can promote tendon regeneration would provide a viable solution for physicians and patients to enhance tendon healing and decrease adhesions as well as shorten the time required to return to work or sports.

Introduction: Counteracting the pandemic of peripheral vascular disease necessitates development of proangiogenic treatments. As stem cell benefit has increasingly been associated with paracrine influence on diseased tissue, a purified extracellular vesicle population (PEP) was established to promote tissue regeneration and neovascularization. The mechanistic basis for PEP-induced proangiogenic effects was here probed and validated in two separate in vivo models of vascular ischemia. Hypothesis: PEP drives pro-angiogenic effects though donation of VEGFR. Methods: Nanosight and immunoblotting were utilized to characterize PEP purity and identity. OMICs profiling identified VEGFR as highly prioritized, with downstream signaling was tested in human umbilical vascular endothelial cells using Matrigel assay, qPCR and immunoblotting. In vivo, blood perfusion (Fluorescent imaging - SPY) was quantified in a rodent model of hind-limb ischemia and in a rabbit model of critical ischemic ear, each treated with sham or PEP. Rabbit tissues were next examined by whole transcriptome analysis to assess PEP-induced proangiogenic signaling. Immunohistochemical staining with smooth muscle actin and von Willebrand factor probed de novo blood vessel formation. Results: PEP was characterized by size (mean: 128.6 +/-1 nm) and VEGFR expression. VEGFR knock-down diminished endothelial cell angiogenic activity in vitro, rescued with PEP (5.3x1010+/-109 vesicles/ml) was sufficient enhance total and phosphor-VEGFR expression, along with amplification of downstream VEGFR targets (MAPK, AKT, CXCL8, NOTCH4, MMP9) and enhanced tube formation. In both hind-limb (n=5) and critical ear (n=4) ischemia models, local delivery of PEP to ischemic tissue resulted in restoration of blood perfusion and prioritized proangiogenic signaling in tissue transcriptome pathway analysis, further confirmed by immunohistochemistry. Conclusions: Taken together, our study establishes a new regenerative paradigm documenting exosome-based delivery of VEGFR as a key driver for angiogenesis. This PEP-based platform broadens potential options available for treatment of peripheral vascular disease and may have therapeutic implications in restoration of microvasculature.

AbstractTargeted muscle reinnervation enables native muscles to send electromyographic signals to myoelectric receptors, which drive movements in a prosthesis. This system requires voluntary contracture of muscles for sequential control of powered prosthetic joints. This report describes a surgical solution for cases where the chest wall is depleted of muscle targets. A 13‐year‐old boy with left forequarter amputation and pectoralis major resection as a result of extended necrotizing facilities 8 years prior received a neurotized free Vertical Rectus Abdominus Mycocutaneous (VRAM) flap (28 × 10 cm) designed to produce myoelectric signals, reduce pain, and provide stability for prosthetic fitting. Five intercostal nerves from the VRAM were coapted to portions of the brachial plexus to create a myoelectric interface for targeted muscle reinnervation. The postoperative course was uneventful. At 39 months of follow‐up, the patient gained control of the transferred VRAM and was able to operate a custom‐fitted myoelectric prosthesis together with contraction of the ipsilateral infraspinatus muscle. The neurotized VRAM transfer created a neural interface in an area with depleted neuromuscular targets while decreasing pain and adding tissue bulk for proper prosthesis fitting. Such a surgical strategy may have applications in other areas of the body.