Targeted Intra-Atrial Delivery of GMP-Grade Human Extracellular Vesicles Prevents Inflammation-Driven Postoperative Atrial Fibrillation in Pigs.

Figures

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  Figure 1. GMP-Grade Cardiac EV Manufacturing and Bioactivity (A) Schematic of Good Manufacturing Practice–compatible production of human cardiac extracellular vesicles (EVs) from right atrial tissue through working cell bank expansion and vesicle isolation. (B) Cell yield and viability across early passages demonstrate reproducible expansion. (C) Nanoparticle tracking analysis confirms EV size distribution. (D) Flow cytometry demonstrates expression of canonical EV surface markers with absence of cytochrome C. (E) Dose-dependent acetylcholinesterase activity confirms EV identity. (F) EVs suppress lipopolysaccharide (LPS)–induced interleukin (IL)-1β, IL-18, and caspase-1 production, indicating inhibition of inflammasome signaling. Data are mean ± SD from 3-6 biological replicates; statistical comparisons were made using analysis of variance with Šídák post hoc testing. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. AChE = acetylcholinesterase.

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  Figure 2. Selective Atrial Biodistribution of Cardiac EVs Rats underwent sham surgery or talc-induced pericarditis followed by intra-atrial injection of heart-derived extracellular vesicles (EVs), with untreated animals as controls. One hour later, atria and peripheral organs were harvested for quantitative polymerase chain reaction (qPCR) detection of human neurotrophin-4 (NT4). EV signal was greatest in atrial tissue with minimal off-target distribution to liver, lungs, kidneys, or spleen, and atrial retention was enhanced in pericarditis. Data are presented as mean ± SD from 4 biological replicates. Group differences were analyzed using unpaired 2-tailed Student’s t-tests. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 3. Systemic Safety of Cardiac EVs in Rats Immune-competent rats underwent sternotomy with epicardial talc application and were randomized to intra-atrial injection of vehicle or human extracellular vesicles (EVs). Blood and urine were collected at baseline and serially through 30 days for assessment of renal injury, liver function, and inflammatory cytokines. Biomarkers of kidney injury, hepatic function, and systemic inflammation remained stable over time, indicating absence of systemic toxicity or immunogenicity following EV administration. Data are mean ± SD and were analyzed using a mixed-effects model with Šídák correction for multiple comparisons. ∗P < 0.05 vs EV-treated animals. ARG = arginase; KIM = kidney injury molecule; TIMP = tissue inhibitor of metalloproteinase; VEGF = vascular endothelial growth factor.

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  Figure 4. Development of Transepicardial Atrial Injection in Pigs (A) Experimental timeline showing pacemaker implantation, pericardial talc application, intra-atrial injection, continuous rhythm monitoring, and terminal electrophysiological testing. (B) Fluoroscopy confirming right atrial lead placement. (C) Sequential intraoperative images demonstrating methylene blue retention within atrial myocardium after injection. (D) Gross pathology showing localized epicardial staining with transmural diffusion to the endocardium. (E) Transesophageal echocardiography confirming preserved atrial geometry and absence of thrombus following injection. These data establish the feasibility and structural safety of targeted intra-atrial delivery in pigs.

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  Figure 5. Spontaneous and Inducible AF in Control Pigs (A) Continuous pacemaker recordings from a representative vehicle-treated pig demonstrate spontaneous atrial fibrillation (AF) episodes during the 7-day postoperative monitoring period, with summary metrics of AF burden, episode duration, onset time, and atrial tachycardia/nonsustained events. (B) Representative electrophysiological testing shows failure of single extrastimulus pacing to induce AF, while double extrastimuli and 50-Hz burst pacing induce brief, self-terminating AF episodes. AT = atrial tachycardia; NS = nonsustained.

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  Figure 6. EVs Suppress AF, Fibrosis, and Inflammation in Pigs (A) Representative surface electrocardiograms during 50-Hz burst pacing demonstrate inducible atrial fibrillation (AF) in vehicle-treated but not extracellular vesicle (EV)–treated pigs, with summary of spontaneous and inducible AF incidence. (B) Maximal AF duration during burst stimulation. (C) Atrial effective refractory period (AERP). (D) Heart rate and conduction parameters. (E) Quantification of atrial fibrosis by hydroxyproline content and histology with representative Masson’s trichrome images. (F) Quantification of inflammatory infiltrates with representative hematoxylin and eosin sections. EV therapy reduced AF burden, fibrosis, and inflammatory infiltration while preserving conduction. Data are mean ± SD and were analyzed using 2-tailed Student’s t-tests. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 7. Systemic Cytokines and End-Organ Safety in Pigs (A) Plasma cytokines and vascular biomarkers measured at baseline and 7 days after surgery in vehicle- and extracellular vesicle (EV)–treated pigs. (B) Renal function markers, including creatinine and urea. (C) Hepatic function markers, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and bilirubin. EV treatment was associated with normalization of selected inflammatory mediators without adverse effects on renal or hepatic function. Data are presented as mean ± SD and were analyzed using 1-way analysis of variance with Šídák’s correction for multiple comparisons. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. IL = interleukin.