Behfar, Atta

BACKGROUND: Ventricular fibrillation (VF) remains a leading cause of sudden cardiac death with an incompletely characterized electrophysiological substrate. Traditional large-animal VF models are limited by hemodynamic collapse and myocardial ischemia. This study establishes a reproducible swine model using dual mechanical circulatory devices to enable high-density electroanatomic mapping under nonischemic conditions. METHODS: Five Yorkshire swine (55–65 kg) underwent sustained VF supported by venoarterial extracorporeal membrane oxygenation (2.5–3.0 L/min) and percutaneous left ventricular unloading (Impella CP, P-2–P-4). High-density electroanatomic mapping using CARTO 7 was performed at baseline and after defibrillation. Bipolar voltage was acquired from 7 predefined ventricular regions (21 measurements per animal per time point; n=210 paired observations). Unipolar voltage mapping and continuous surface ECG monitoring were used to assess acute ischemic injury. Systemic perfusion was maintained at systolic arterial pressure ≥80 mm Hg. Linear mixed-effects modeling was used to analyze voltage changes from pre-VF to post-VF. RESULTS: Sustained VF (10±1 minutes) was reproducibly induced and maintained with consistent hemodynamics (systolic arterial pressure, 90±5 mm Hg; extracorporeal membrane oxygenation flow, 2.5±0.5 L/min). All animals were successfully defibrillated. High-density mapping was feasible in all experiments (1000±300 points per chamber). Bipolar voltage showed no change from pre-VF (5.86±2.56 mV) to post-VF (6.53±2.22 mV; mean difference, 0.67 mV [95% CI, –0.31 to 1.65]; P =0.15). Both left ( P =0.17) and right ventricles ( P =0.08) demonstrated voltage preservation. Regional analysis showed no significant changes (all P >0.05); post-VF voltages remained above viability thresholds (>1.5 mV). Unipolar voltage maps recovered toward baseline, with no ECG injury pattern identified after defibrillation. CONCLUSIONS: Dual mechanical circulatory support with venoarterial extracorporeal membrane oxygenation and Impella CP enables reproducible, high-fidelity electroanatomic mapping and preserves myocardial substrate after sustained VF in a large-animal model. This platform overcomes key technical barriers in VF research, providing a nonischemic foundation for future mechanistic and translational studies.

TRAIL is a TNF family ligand that trimerizes TRAIL-R1 (DR4) or TRAIL-R2 (DR5) to induce apoptosis, necroptosis, and/or NF-κB activation in receptor-bearing cells. We previously identified TRAILshort as a splice variant of TRAIL that lacks cysteine 230, cannot trimerize, and acts as a dominant-negative ligand that blocks TRAIL-mediated apoptosis. TRAILshort is expressed on cell surfaces and within extracellular vesicles, enabling it to confer TRAIL resistance to both producing and bystander cells. In this study, we showed that elevated TRAILshort levels were associated with chronic viral infections, cancer, and autoimmune diseases, suggesting a link to impaired immune regulation. Using unbiased phosphoproteomics and mechanistic studies, we demonstrated that TRAILshort binding to DR5 recruited and activated the phosphatase Src homology region 2 domain–containing phosphatase 1 (SHP-1), leading to zeta-chain-associated protein kinase 70 (ZAP-70) dephosphorylation, disruption of ZAP-70–CD3ζ interactions, and impaired T cell receptor signaling, thereby reducing T cell activation, proliferation, and cytokine production in response to antigen or CD3/CD28 ligation. Genetic or pharmacologic SHP-1 inhibition reverses these effects. In humanized mouse models, TRAILshort promoted the persistence of transformed mouse embryonic fibroblasts (MEFs) and L428 and antagonized CD19-directed CAR T cell activity, revealing TRAILshort as an immunomodulator of T cell function with therapeutic implications, including blocking TRAILshort to restore T cell immunity or delivering TRAILshort to enforce tolerance. TRAILshort, a variant of TRAIL, suppresses T cell function and immune responses in cancer and infectious diseases, highlighting its potential as a therapeutic target.

Stem cell-derived β (SC-β) cells are a promising therapy for type 1 diabetes (T1D), but their long-term efficacy is limited by immune-mediated graft rejection. Platelet-derived exosome product (PEP) has emerged as a novel immunomodulatory agent, although its effect on SC-β cell xenograft rejection remains unclear. Here, we evaluated PEP in stimulated human peripheral blood mononuclear cells (PBMCs) and in an immunocompetent mouse model of SC-β cell transplantation under the kidney capsule. Immune-related gene expression was assessed by quantitative PCR, immune cell infiltration by immunofluorescence, and graft function by circulating human insulin. In vitro, PEP reduced NK-cell-associated gene expression, including NK1.1, EOMES, and KLRK1, and increased IL-10 expression. In vivo, untreated SC-β cell xenografts showed progressive immune infiltration and loss of detectable human insulin by day 14, whereas PEP-treated grafts retained detectable insulin through day 14 across co-transplantation, pretreatment, and systemic administration strategies. PEP co-transplantation delayed, but did not prevent, xenograft rejection, with graft loss observed by day 21. This delay was associated with reduced NK1.1+ cell infiltration and lower expression of selected inflammatory and rejection-associated markers, including NK1.1, Nos2, and Nlrp3. These findings suggest that PEP modulates graft-associated immune responses and may serve as an adjunct immunomodulatory strategy for stem cell-based therapies for type 1 diabetes. Further studies are needed to evaluate sustained graft durability, safety, and translational efficacy.

Abstract Introduction The inhalation of aerosolized therapeutics is a promising strategy for the targeted delivery of drugs and biologics to the lungs. We have previously demonstrated the efficacy and feasibility of nebulized exosomes using a platelet-derived exosome product (PEP: Rion, Inc.), which significantly attenuated emphysema in a murine model (PMID: 38325750). However, the PEP aerosol’s physical properties—such as size, tissue deposition, anatomic location, and stability remain incompletely characterized. To address this, we utilized a cascade impactor to define aerosol particle size and deposition patterns and employed immunofluorescence staining to assess the regional and anatomic localization of nebulized PEP within the murine lung. Methods PEP particle size and tissue deposition were determined by passing the aerosol through a Marple cascade impactor (CI, DSI), which was connected to an inhalation tower (DSI). PEP aerosols were generated by a vibrating mesh nebulizer (Aerogen, Cat No AG-AL1100) at a controlled velocity and humidity and delivered to the CI at a flow rate of 2L/min. Aerosols were separated by size and allowed to deposit at the corresponding stages as they passed through the pinholes of the stage, by design. The stages were weighed after nebulization, and the percentage of aerosol deposition was calculated to simulate the human respiratory tree, according to the manufacturer’s specifications. In parallel, fluorescence DiR-labeled PEP was nebulized to spontaneously breathing mice, and confocal microscopy was used to determine intrapulmonary distribution. Results A total of 2.25 mL of PEP reconstituted in 0.9% normal saline (11.4 mg/mL final concentration) was nebulized for 35 minutes at a flow rate of 2 L/min, yielding 3.5 mg of total aerosol deposition, which is 13.6% of the total PEP mass (25.65mg) being nebulized, across the CI stages. Of this, 74% of the aerosolized particles with a particle size ≤ 1µm and deposited in Stages 7, 8 & F, corresponding to the deep regions of the lungs, specifically regions beyond the alveolar ducts and within the alveoli. In vivo 3D confocal imaging corroborated these findings, revealing abundant DiR-labeled PEP deep into the alveolar space within the peripheral regions of all lung lobes. Conclusion The study result demonstrates the feasibility and capability of delivering aerosolized PEP deeply into the alveolar regions of the lung at the given pressure, humidity, flow rate and nebulizer settings. This study establishes a methodological framework for characterizing and optimizing aerosolized exosome therapeutics for precise, region-specific delivery within the respiratory tract. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Abstract Rationale Alpha1-antitrypsin (AAT; gene name SERPINA1) is a critical antiprotease that protects the lung from neutrophil elastase-mediated injury. AAT deficiency predisposes to emphysema, which is further exacerbated by cigarette smoke (CS) exposure. We previously reported (PMID: 38325750) that a nebulized human platelet-derived exosome product (PEP) attenuated CS-induced emphysema in C57BL/6 mice. Given the presence of AAT protein in these platelet-derived exosomes (identified by proteomics), we hypothesized that PEP nebulization would significantly attenuate both spontaneous and CS-induced emphysema in SERPINA1 knockout (KO) mice. Methods Transgenic SERPINA1 KO C57BL/6 mice (both sexes) exposed to CS (10 cigarettes/day and 5 days/week) for 2 months were then randomized to receive nebulized PEP or 0.9% saline (placebo) twice weekly for 4 additional weeks, with continued CS exposure. PEP was reconstituted in 0.9% saline (22.7mg/mL; ∼3 × 10¹0 vesicles/mL) and administered via pressure- and humidity-controlled nose-only inhalation tower (2.25 mL of PEP or saline over 35 minutes). Lung static compliance (Cst), cutaneous O2 saturation, mean linear intercepts (Lm), and plethysmographic parameters were measured during and after the study. Results Histological analysis of the lung tissue sections revealed that PEP significantly attenuated the CS-induced increase in alveolar size, as determined by measurement of the mean linear intercepts (Lm, p < 0.001). Lung static compliance (Cst), which was increased in CS-exposed mice, was also significantly decreased in the PEP-treated group (p < 0.001) compared to placebo in the male and female combined group. Functional respiratory parameters further supported the protective effect of PEP in this AAT-deficient model. In female mice, PEP nebulization significantly improved several CS-impaired pulmonary function parameters, including minute volume (MV, p = 0.024), the flow at the point 50% of tidal volume is expired (EF50, p = 0.021), Peak Inspiratory Flow (PIF, p = 0.009), Peak Expiratory Flow (PEF, p = 0.018), and O2 saturation (p = 0.035). However, these improvements were not observed in male mice. Conclusions These findings extend prior observations and underscore the therapeutic potential of nebulized AAT-enriched PEP directly to the alveolar region, effectively attenuating injury and improving pulmonary function in AAT deficiency-related emphysema. This abstract is funded by: Department of Defense, Mayo Clinic Funding, The Young and Middle-aged Medical High-end Talents in Chongqing Program

Abstract Introduction We previously demonstrated that nebulized platelet-derived exosome product (PEP) alleviates cigarette smoke (CS)-induced murine emphysema. To elucidate the underlying molecular mechanisms, we conducted single-nucleus RNA sequencing of lung tissue, which initially revealed a shift in the cell source for TNF signaling (a critical regulator of inflammation in COPD lungs) from innate lymphoid cells (ILC) to alveolar macrophages (aMac) after chronic CS exposure. In this study, we conducted a more in-depth analysis of cellular communication to determine the mechanism by which PEP modulates CS-induced TNF signaling in the lung microenvironment. Methods Single-cell transcriptomics and cell-cell communication analyses were performed between CS-exposed placebo control lungs vs. sham control lungs and CS-exposed PEP-treated vs. placebo control lungs using R packages, including Seurat, CellChat, Patchwork, Future, and NMF. A comparative analysis of ligand-receptor interactions across these datasets was conducted using CellChat to infer intercellular signaling networks. Results Single-nuclear RNA-seq analysis revealed that the Thy1-ITGAX_ITGB2 signaling pathway was significantly upregulated by CS, acting as the adhesive and activating bridge that facilitates the switch in TNF production from ILC to aMac. Thy1 is a GPI-anchored ligand expressed on ILCs, and ITGAX_ITGB2 is its cognate receptor on aMacs. Concurrently, Ptprc-Mrc1 signaling, an inhibitory pathway in ILC-aMac crosstalk, was downregulated, contributing to sustained aMac activation by CS. Furthermore, analysis of aMac-aMac interactions revealed upregulated GRN/Sort1 signaling, which promotes the internalization of progranulin, an anti-inflammatory glycoprotein that competes for TNF/TNFR signaling. Ptprc-Mrc1 signaling was also downregulated within the aMac autocrine/paracrine network, further perpetuating a pro-inflammatory state. In contrast, treatment with PEP downregulated Thy1-ITGAX_ITGB2 signaling and upregulated Lamc1-Cd44 signaling, a hallmark of aMac deactivation, thereby inducing a phenotypic switch in inflammatory macrophages toward a reparative M2 state, promoting the resolution of CS-induced chronic inflammation. Conclusions These data suggest that the transition of TNF production from ILCs to aMacs under chronic CS stress is driven by increased Thy1-ITGAX_ITGB2 interactions and decreased Ptprc-Mrc1 signaling between ILCs and aMacs crosstalk within the local tissue. Enhancing the crosstalk between Lamc1-Cd44 and Ptprc-Mrc1 may suppress TNF production and attenuate CS induced aMac activation. Targeting these pathways, as demonstrated by nebulized PEP treatment, holds therapeutic potential to deactivate inflamed aMacs and promote homeostasis in the inflamed lung. This abstract is funded by: Department of Defense, Mayo Clinic Funding

Importance Rectovaginal fistulas (RVFs) are challenging to treat, often requiring multiple surgical interventions. Research into novel therapies is limited by the lack of a sizeable and representative animal model. Given the similarities in porcine and human vaginal and rectal tissue, pigs are an ideal species for translation to human patients. Objective The objective of this study was to develop a feasible and reproducible RVF model in pigs. Study Design Six female pigs underwent RVF creation and were divided into 3 groups according to the following methods: (1) placement of 1-inch disc magnets across the rectovaginal septum, (2) a 1-inch round defect created using electrocautery and placement of a 1-inch stainless-steel gauge stent, (3) a 1-inch round defect using electrocautery without subsequent stenting. Fistulas were evaluated at 6 weeks with measurements and photographic documentation. The animals were euthanized between 12 and 14 weeks. The primary outcome of this study was fistula patency. Secondary outcomes were fistula dimension and histologic appearance of the fistula tract. Results All pigs did well with no infection or complications related to fistula creation. Both surgical methods reliably resulted in a patent fistula at 14 weeks: cautery alone and cautery with gauge stenting. Only 1 patent fistula in the magnet group was noted at the time of euthanasia. All patent fistulas showed histologic evidence of complete epithelialization of the fistula tract. Conclusions This study demonstrates 3 safe and feasible methods for RVF creation in pigs, with 2 methods showing increased reliability.

ABSTRACT Extracellular vesicles (EVs) are non‐replicating, lipid membrane‐bound nanoparticles released by most eukaryotic cells into the extracellular space. When derived from platelets, a subset of EVs can exhibit remarkable anti‐inflammatory and antioxidant properties, making them attractive candidates to attenuate sterile inflammatory states such as limb skeletal muscle ischaemia‐reperfusion injury (IRI). Peripheral extremity IRI complicating acute limb revascularisation procedures, replantation of major traumatic amputations and limb allotransplants can trigger a life‐threatening reperfusion syndrome that leads to multi‐organ system failure and death. In this study, we used a platelet‐derived EVs‐enriched preparation as a therapeutic strategy for extremity IRI. To allow for a clinically translatable drug delivery strategy, we established and validated a catheter‐directed regional intra‐arterial limb infusion (RLI) delivery approach that permitted selective and effective targeting of pEVs to limb skeletal muscles and soft tissues in the vascularly isolated rodent extremity simulating pre‐reperfusion preservation and revascularisation conditions of the ischaemic or amputated extremity. In addition to being safe and feasible, we demonstrated robust retention of EVs by skeletal muscles of the operative limb during the early critical phase of reperfusion injury following RLI. We also show that pretreatment of ischaemic skeletal muscles using an pEVs‐enriched formulation just before reestablishing extremity blood flow alleviated metabolic derangements and suppressed the systemic and localised proinflammatory cytokine response associated with skeletal muscle reperfusion and reduced histologic markers of myofibre injury without adverse effects on serum chemistries. This work opens a new perspective to investigate novel approaches for regional delivery of therapeutic pEVs to skeletal muscle to mitigate limb IRI in vivo, and to achieve both in situ and ex vivo targeted delivery of nanocarriers to the isolated limb tissue for other therapeutic applications.

Chronic wounds remain a major unmet clinical challenge, often failing to progress to normal healing due to persistent inflammation, impaired angiogenesis, and cellular senescence. Exosomes have recently been investigated as promising acellular therapeutics capable of restoring intercellular communication and promoting tissue regeneration. Among these, the Purified Exosome Product (PEP) represents a next-generation, platelet-derived exosome formulation manufactured under Good Manufacturing Practice (GMP) conditions with high purity, stability, and reproducibility. This review summarizes the current advances in exosome-based chronic wound therapeutics and PEP delivery systems and their translational potentials. Incorporation of PEP into bioengineered carriers such as fibrin sealant, collagen scaffolds, and hyaluronic acid (HA) hydrogels enables localized and sustained exosome release, significantly prolonging therapeutic effects and improving regenerative outcomes. Fibrin-based PEP delivery achieved complete wound closure and functional skin regeneration in animal models, while collagen and HA-based systems showed promising results for injectable and dermatologic applications. Beyond its intrinsic healing effects, PEP may also serve as a nanocarrier for other drugs, offering a future direction in chronic wound management.

Background: Tendon-bone healing after rotator cuff injury is, or remains, a clinical challenge. Purpose: To examine the role of intraoperative use of purified exosome products (PEPs) in the treatment of rotator cuff injuries and to assess the effect of PEP on supraspinatus tendon-bone healing after rotator cuff repair in a rat model. Study Design: Controlled laboratory study. Methods: This study used 120 Sprague-Dawley rats that underwent an acute supraspinatus tendon transection from the insertion, followed by repair using a single transosseous suture. They were divided into 3 groups: a control group that received only sutures, a TISSEEL group that used a commercially available fibrin sealant patch (TISSEEL) of approximately 0.5 cm 2 , and a PEP-TISSEEL group that used a similarly sized TISSEEL patch combined with PEP. Autopsies were performed at 1, 3, 6, and 8 weeks after surgery. Analyses were performed using molecular biological, biomechanical, and histological techniques. Results: At 6 weeks, the PEP-TISSEEL group achieved greater failure load (mean, 27.5 ± 5.3 N) than the control (mean, 16.4 ± 4.2 N; P = .012) and TISSEEL (mean, 23.4 ± 5.0 N; P = .041) groups, with superiority persisting at 12 weeks (27.2 ± 6.5 N vs 13.1 ± 2.6 N [ P = .002] for the control group and 21.1 ± 5.8 N [ P = .037] for the TISSEEL group). DigiGait showed earlier recovery of stride length and paw angle. Histology revealed organized collagen and reduced scarring, while molecular assays demonstrated early inflammatory modulation with subsequent upregulation of collagen type 1 or 3 and TGF-β, supporting accelerated tendon-bone healing. Conclusion: Intraoperative application of PEP-TISSEEL enhanced tendon-bone healing in a rat model by improving biomechanical strength, promoting enthesis-specific histological regeneration, and accelerating functional recovery as evidenced by gait analysis. Clinical Relevance: The intraoperative application of PEP combined with TISSEEL significantly enhances rotator cuff tendon healing by modulating early inflammation, promoting collagen synthesis, and improving biomechanical properties in a rat model. These findings suggest that PEP-based therapies could offer a promising adjunct in surgical repair, potentially reducing retear rates and improving functional outcomes in patients with rotator cuff injuries. Further clinical studies are warranted to translate these benefits into human applications.