Rizzo, Skylar A.

ABSTRACT Cytotoxic T lymphocytes (CTLs) are known to eliminate target cells through perforin-mediated, contact-dependent killing - a process limited by the number of effector T cells despite its serial nature. CTLs likely also possess a mass killing mechanism that can eliminate target cells more efficiently. Using CAR-T cells against B7H3 present on B16 tumor targets, we demonstrate that activated T cells can also kill targets in a perforin-independent manner by releasing diffusible TNF and IFNγ capable of killing nearby targets in a paracrine fashion even if they bear no antigen. Against an unperturbed target, paracrine killing is inefficient but is enhanced by the deletion of TNFR1 signaling molecules such as TAK1, HOIP, or TBK1/IKKε. Notably, these molecules are inhibited naturally by pathogen-encoded antagonists. Expression of a microbial-encoded antagonist, such as the Yersinia -encoded YopJ that antagonizes TAK1 or Ebola-encoded VP35 that antagonizes TBK1/IKKε, alters the target cell response to these cytokines from non-lethal to lethal. We propose that target cells are inherently resistant to killing by TNF and IFNγ, but the presence of a microbial factor alters this sensitivity, providing for the selective elimination of the infected cell while minimizing harm to uninfected bystander cells. We propose the term ‘pathogen-restriction’ to describe this discriminatory mechanism. Potentially, any microbial-derived factor that crosstalks with the TNF and IFNγ signaling pathways can function as a pathogen-restriction element. GRAPHICAL ABSTRACT Short Summary T cells use TNF and IFNγ to kill target cells in a paracrine manner where target elimination is restricted by the presence of pathogen-derived factors.

Background The purpose of this study was to investigate a therapeutic approach targeting the inflammatory response and consequent remodeling from ischemic myocardial injury. Methods and Results Coronary thrombus aspirates were collected from patients at the time of ST‐segment–elevation myocardial infarction and subjected to array‐based proteome analysis. Clinically indistinguishable at myocardial infarction (MI), patients were stratified into vulnerable and resilient on the basis of 1‐year left ventricular ejection fraction and death. Network analysis from coronary aspirates revealed prioritization of tumor necrosis factor‐α signaling in patients with worse clinical outcomes. Infliximab, a tumor necrosis factor‐α inhibitor, was infused intravenously at reperfusion in a porcine MI model to assess whether infliximab‐mediated immune modulation impacts post‐MI injury. At 3 days after MI (n=7), infliximab infusion increased proregenerative M2 macrophages in the myocardial border zone as quantified by immunofluorescence (24.1%±23.3% in infliximab versus 9.29%±8.7% in sham; P <0.01). Concomitantly, immunoassays of coronary sinus samples quantified lower troponin I levels (41.72±7.34 pg/mL versus 58.11±10.75 pg/mL; P <0.05) and secreted protein analysis revealed upregulation of injury‐modifying interleukin‐2, ‐4, ‐10, ‐12, and ‐18 cytokines in the infliximab‐treated cohort. At 4 weeks (n=12), infliximab treatment resulted in significant protective influence, improving left ventricular ejection fraction (53.9%±5.4% versus 36.2%±5.3%; P <0.001) and reducing scar size (8.31%±10.9% versus 17.41%±12.5%; P <0.05). Conclusions Profiling of coronary thrombus aspirates in patients with ST‐segment–elevation MI revealed highest association for tumor necrosis factor‐α in injury risk. Infliximab‐mediated immune modulation offers an actionable pathway to alter MI‐induced inflammatory response, preserving contractility and limiting adverse structural remodeling.

The purpose of this study was to evaluate the biodistribution of a platelet-derived exosome product (PEP), previously shown to promote regeneration in the setting of wound healing, in a porcine model delivered through various approaches. Exosomes were labeled with DiR far-red lipophilic dye to track and quantify exosomes in tissue, following delivery via intravenous, pulmonary artery balloon catheter, or nebulization in sus scrofa domestic pigs. Following euthanasia, far-red dye was detected by Xenogen IVUS imaging, while exosomal protein CD63 was detected by Western blot and immunohistochemistry. Nebulization and intravenous delivery both resulted in global uptake of exosomes within the lung parenchyma. However, nebulization resulted in the greatest degree of exosome uptake. Pulmonary artery balloon catheter-guided delivery provided the further ability to localize pulmonary delivery. No off-target absorption was noted in the heart, spleen, or kidney. However, the liver demonstrated uptake primarily in nebulization-treated animals. Nebulization also resulted in uptake in the trachea, without significant absorption in the esophagus. Overall, this study demonstrated the feasibility of pulmonary delivery of exosomes using nebulization or intravenous infusion to accomplish global delivery or pulmonary artery balloon catheter-guided delivery for localized delivery.

The purpose of this study was to evaluate left ventricular (LV) unloading strategies in patients supported with peripheral venoarterial extracorporeal membrane oxygenation (VA-ECMO). A retrospective review was conducted of all consecutive patients requiring VA-ECMO support for any indication, who underwent novel LV unloading strategies with either direct left atrial venoarterial (LAVA) cannulation or pulmonary artery venoarterial (PAVA) venting, in comparison to Impella and intra-aortic balloon pump (IABP). The primary outcome was successful bridge to transplant, LV assist device, or myocardial recovery. Forty-six patients (63% male, mean age 52.8 ± 17.6 years) were included. Fourteen patients (30%) underwent novel unloading with either LAVA or PAVA, 11 patients (24%) underwent IABP placement, and 21 patients (46%) underwent Impella insertion. In the novel LV unloading cohort, 10 patients (71%) survived to hospital discharge. Four patients (29%) were weaned from ECMO and eight patients (57%) underwent cardiac transplantation. Although a trend favoring cannula-based unloading for the primary outcome was noted, the cohort was too small for statistical significance (79% LAVA/PAVA, 57% Impella, 45% IABP; p = 0.21). However, probability of survival was greater in the LAVA/PAVA cohort compared to Impella and IABP (p < 0.05). Thus, we demonstrate the efficacy of LA and PA cannulation as an alternative LV unloading strategy for patients supported with peripheral VA-ECMO.

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.

Introduction: Patients with coronary artery disease (CAD) not amenable to revascularization are reported to have a 3-year mortality >14%. Advances in treatment modalities including medical management, rotational atherectomy, balloon lithotripsy, and drug-eluting stents work for focal disease but poorly treat diffuse CAD. In a porcine model of in-stent restenosis, we here evaluated whether a pro-angiogenic platelet-derived exosome product (PEP) could be delivered into the perivascular space to foster collateral vessel formation. Methods/Results: Cobalt chromium stents were placed and were oversized by 1 mm in the left anterior descending (LAD) and posterior descending arteries (PDA) of sus scrofa domesticus pigs. In-stent restenosis was angiographically visualized at the 2-month timepoint, with hemodynamic significance documented with fractional flow reserve (FFR) of <0.8. To document feasibility of PEP delivery into the perivascular space, exosomes were labeled with a far-red fluorescent tag and delivered into the perivascular bed. Here, the Bullfrog® micro-infusion catheter was positioned either in the great cardiac vein or the middle cardiac vein and PEP was delivered with the microneedle pointed towards the LAD or the PDA. Biodistribution demonstrated exosome retention and uptake around cardiac veins. Using the Bullfrog® technique, PEP was delivered in the perivascular space adjacent to areas of significant coronary stenosis. Initial results 1-month post-treatment demonstrated improved FFR of treated stenotic vessels (from 0.68 to 0.85), while untreated vessels demonstrate no change from previous FFR measurement (from 0.79 to 0.78). Conclusions: Here, exosome-mediated angiogenesis in the perivascular space improved coronary blood flow, distal to areas of stenosis. In patients with diffuse CAD, delivery of biotherapy in cardiac veins running anatomically parallel to stenosed epicardial arteries provides a novel therapeutic opportunity to supplement the existing armamentarium of therapies available for in-stent restenosis.

Coronary angiography is the gold standard for the diagnosis and treatment of epicardial coronary artery disease. Further, it is an essential tool for conduction of translational cardiac research on large animal models, in the development of novel cardiac therapies. While in humans coronary angiography has an exceedingly low mortality rate; in pigs, procedural mortality has been reported as high as 15%, likely due to unsuitable variations in angiographic techniques. Porcine models are used due to the resemblance of cardiac function and anatomy to humans. However, procedural guidelines delineating proper equipment to be used for angiography in this model has yet to be systematically assessed. Aims: To evaluate clinically common catheters for their utility in the safe engagement of the coronary vessels in pigs. Establish optimal workflow and fluoroscopy views for safe coronary artery engagement and angiography. Method/Results: Percutaneous superficial femoral artery access was acquired via ultrasound guidance and subsequent cardiac catheterization was performed in sus domesticus weighing 35-50 kg. Catheters that were tested included the JL 2.5, JL3.0, JR 3.0, AR1, AL1, MPA1, Champ1, 3DRC, hockey stick and RCB (5-7 french) operated over a standard .035 J tip wire. AP views were optimum to engage both the left and right coronary vessels. Only AR1, 3DRC, and hockey stick catheters successfully engaged the RCA, while all catheters tested could successfully engage the LCA. AR1 was noted to be the best for RCA engagement and AL1 for Left system engagement. AR1 was also the most succesful in engagement of both LM and RCA in same pig. AL1 provided the most stable engagement for Left coronary system. AR1/JL were better for LAD intervensions. AL1 is best for LCX intervensions Conclusion: We present a systematic description of percutaneous coronary angiographic procedures highlighting that AL for the LCA and AR for the RCA are the best choice of catheters for fastest and safest engagement of the coronary arteries limiting both radiation time and contrast use. While variations in anatomy can mandate changes in angiographic angles and catheter techniques, AP visualization and use of AL and AR catheters provided the most consistent and effective approach for porcine angiography.

For patients in cardiogenic shock, percutaneous mechanical circulatory support devices (MCSD) have emerged as critical tool to improve hemodynamics and optimize end-organ perfusion; successfully bridging patients to recovery, decision or transplantation. The most commonly utilized MCSD is the femoral intra-aortic balloon pump (IABP). However, femoral access hampers the patient's mobility, quality of life, and capacity to participate in rehabilitation. The advent of the axillary artery approach has served to circumvent limitations associated with the femoral artery approach. However, optimum anatomical placement of IABP within the axillary artery (axIABP) is a topic of debate considering its proximity to the brachial plexus. Here, we report our experince with percutaneous access of the third poriton of the axillary artery which showed promising results with minimal device related complications or adverse events. Methods/Results: Between 2017-2023, a total of 89 patients in cardiogenic shock underwent percutaneous axIABP placement at the Mayo Clinic, MN. All demographic, clinical, and periprocedural data were obtained from hospital chart review and recorded in a database under IRB approval. Indications for axIABP support was determined by multidisciplinary assessment. Complications encountered in the cohort included infection at site 6.7% (n=6), device malfunction [rupture, fracture, leak] 8.98%(n=8), and balloon migration 5.61% (n=5). One patient developed local erythema, 4 (4.49%) patients developed significant vascular complications, including access site bleeding in two patients 2.24%, arterial thrombosis in three patients 3.37%, and self-limited intracranial bleeding in one patient 1.12%. Of note, no nerve injury or damage was observed in the entire patient cohort. Conclusion: Our study highlights the feasibility of percutaneous axIABP in the third portion of the axillary artery, while assessing safety profile and ease of removal. Notably in our review, we did not encounter any brachial nerve damage. Ultrasound was pivotal in identifying and avoiding nerves and nerve bundles during initial access. In conclusion, this approach of axIABP insertion is feasible and can be leveraged for delivery of future innovation in MCSD

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.