Witt, Tyra A.

Introduction: The Impella 5.5 is frequently employed to provide temporary mechanical circulatory support as bridge to recovery or durable therapy. Optimization of device management relies on understanding ventricular pressure-volume (PV) relationships. Aim: The aim of this study was to evaluate the biventricular (BiV) relationship with PV loop assessment during Impella 5.5 support in non-myopathic ventricles. Methods: Six domestic Yorkshire Pigs were used for this study. The Impella 5.5 (J&J MedTech, NJ) was inserted percutaneously under ultrasound and fluoroscopic guidance through either the carotid artery or aortic arch, using a 26Fr DrySeal Sheath (Gore, AZ). BiV PV loop conductance catheters (Sigma-M, CD Leycom, Hengelo, The Netherlands) were inserted via femoral venous access. The LV PV loop catheter is positioned via transeptal puncture and with intracardiac echocardiogram guidance. BiV PV loops are generated for each level of Impella 5.5 support. Results: With increasing levels of Impella 5.5 support, left ventricular (LV) PV loops shifted increasingly to the left and right ventricular (RV) loops to the right. Accordingly, LV end-diastolic volume (EDV) decreased (110±4 ml to 90±8 ml; p<0.001) and RV EDV increased (121±7 ml to 147±13 ml; p<0.001), from baseline to P9. Native LV systolic function decreased with increasing Impella 5.5 support, as demonstrated by a reduction in stroke volume (SV) (56±5 ml to 49±5 ml; p<0.001), cardiac output (CO) (3.4±0.8 ml/min to 2.0±0.3 ml/min; p<0.001), ejection fraction (EF) (51±4% to 44±6%; p<0.001), stroke work (SW) (4483±1023 ml*mmHg to 2383±719 ml*mmHg; p<0.001), and LV peak positive dP/dt (825 ± 184 mmHg/s to 523 ± 99 mmHg/s; p<0.001). Conversely, the native RV experienced preload-mediated increase in SV (65±7 ml to 86±20 ml; p<0.001), CO (3.7±0.8 ml/min to 4.3±0.9 ml/min; p<0.001), EF (53±4% to 58±11%; p<0.001), SW (918±143 ml*mmHg to 1529±365 ml*mmHg; p<0.001), and RV dP/dt (164±16 mmHg/s to 228±29 mmHg/s, p<0.001). Load independent contractility was assessed in the LV (1.64±0.33 mmHg/ml to 1.71±0.41 mmHg/ml; p=0.2) and RV (0.25±0.04 mmHg/ml to 0.29±0.05 mmHg/ml; p<0.001). Conclusions: This study highlights ventricular interdependence with Impella 5.5 support with opposing BiV trends. In a healthy heart, the RV can adequately compensate for increased preload offered by the device.

Introduction: Microaxial flow pumps reduce mortality in myocardial infarction (MI) related cardiogenic shock (CS). The Impella 5.5 generates increased flow to provide circulatory support, and optimization relies on mechanistic understanding of pressure-volume (PV) relationships. Aim: Using a porcine model of acute MI in tandem with biventricular (BiV) PV loop conductance catheters, this study aimed to understand the impact of Impella 5.5 on BiV hemodynamics in MI CS. Methods: In 6 Domestic Yorkshire Pigs, an Impella 5.5 (J&J MedTech, NJ) was inserted percutaneously under fluoroscopic guidance via carotid or aortic access. An angioplasty balloon catheter was advanced past the first diagonal and inflated to above nominal pressure with blood flow obstruction for 90 minutes. BiV PV loop conductance catheters (Sigma-M, CD Leycom, Hengelo, The Netherlands) were inserted via femoral venous access. The left ventricle (LV) PV loop catheter is positioned via transeptal puncture using intracardiac echocardiography. BiV PV loops are generated for each level of Impella support from P1 to P7. Results: With increasing Impella 5.5 support, LV PV loops shifted to the left and right ventricular (RV) loops to the right. Without statistical significance, LV end diastolic volume (EDV) decreased by a mean of 12.3 ± 10.7% from baseline and RV EDV increased by 32 ± 40%. Native LV function decreased with increasing Impella 5.5 support, demonstrated by a reduction in stroke volume (SV) (35±13 ml to 27±14 ml; p<0.001), cardiac output (CO) derived from SV&heart rate (2.0±0.5 ml/min to 1.7±0.7 ml/min; p<0.001), and ejection fraction (EF) (37±7% to 29±12%; p<0.001). Less consistent trends were noted in peak positive dP/dt and end-systolic pressure volume relationship. Native RV function increased with higher levels of Impella 5.5 support, noted by increasing SV (27±24 ml to 41±28 ml; p=0.004), CO (1.6±1.2 ml/min to 2.3±1.4 ml/min; p=0.001), and EF (42±16% to 45±9%; p=0.04). Arterial elastance and ventriculo-arterial coupling increased in the LV from baseline to P7 (2.2±1.4 mmHg/ml to 2.8±1.0 mmHg/ml and 1.8±0.5 to 3.2±1.9, respectively; p<0.001) and decreased in the RV (1.3±0.7 mmHg/ml to 0.9±0.7 mmHg/ml (p<0.001) and 2.1±2.2 to 1.3±0.6 (p=0.004)). Conclusions: Impella 5.5 speed modulation highlights significant ventricular interdependence in the setting of acute MI. PV loop derived parameters here visualized parameters that may be utilized to improve optimization of hemodynamic support in CS.

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.

Peter J. Psaltis, 2011 Finalist and Presenting Author

Background: Hematopoietic stem cells and the vasculature share a unique relationship during development, in the adult bone marrow and in the setting of extramedullary hematopoiesis within diseased calcified vessels. We hypothesized that the adult vasculature may contain progenitor cells with the capacity to reconstitute multi-lineage hematopoietic cells. Methods: Aortas from normal and ApoE −/− adult mice were disaggregated with collagenase and elastase. Disaggregates were cultured in methylcellulose for quantification of short and long term hematopoietic colony forming units (CFU). Flow cytometry assessed various populations of hematopoietic progenitor cells. Aortic disaggregates (or bone marrow or blood) from ROSA26 and GFP mice were transplanted into sublethally irradiated C57BL/6 recipients (n=6) via tail vein injections. Bone marrow cells from the primary recipients were transplanted at 4 months to secondary C57BL/6 recipients (n=6). Mice were sacrificed 3 months later. Blood and bone marrow were analyzed. Results: In short term culture in methylcellulose, aortic disaggregates generated erythroid, macrophage, granulocyte and megakaryocyte CFU, with predominance of macrophage colonies, primarily from adventitia (p<0.05) and a greater frequency was found in Apo E −/− mice (p<0.05). Long term culture defined that the combined frequency of the hematopoietic precursors approximates the frequency found in bone marrow (1:47,277). Flow cytometry of the aortic disaggregates indicates that among lin-/c-kit+/sca-1+ cells a small population of hematopoietic stem cells (CD150+endoglin+) and a population of multipotent progenitor cells (CD150− endoglin−) exist. Infusion of aortic disaggregates generated bone marrow, blood and spleen chimerism in primary and secondary transplanted mice at 3– 4 months. Flow cytometry of peripheral cells demonstrated multilineage hematopoietic cells. Conclusions: For the first time, we have identified a variety of progenitor cell populations in the aorta capable of hematopoiesis. Infusion of these cells into sublethally irradiated mice results in stable chimeras. These data support the presence of novel aortic hematopoietic progenitor cells.

Background: Tissue factor pathway inhibitor (TFPI) is a key regulator of the tissue factor/factor VIIa complex. Systemic genetic disruption of TFPI exon 4 results in intrauterine lethality in mice. As such, the ability to study the biologic and tissue specific effects of TFPI deficiency has been limited. To define the tissue specific roles of TFPI we utilized a Cre-lox strategy and generated mice with flanking loxP sites surrounding exon 4 (encodes for the K1 domain). Homozygous floxed mice (TFPI Flox ) were crossed with the Tie2 Cre + or LysM Cre + mice and subsequently inbred to create homozygously floxed mice expressing Cre (TFPI Tie2 or TFPI LysM ). Methods and Results: The presence and efficiency of the deleted mRNA product was determined by RT-PCR. TFPI Tie2 and TFPI LysM mice were phenotypically similar to TFPI Flox littermates including weight, blood counts, and prothrombin and activated partial thromboplastin times. TFPI Tie2 mice have complete deletion in endothelial cells, blood cells and bone marrow cells. TFPI LysM mice show partial deletion in blood cells and complete deletion in macrophages. TFPI Tie2 mice had a 60% reduction (P < 0.0001) in circulating TFPI activity compared to TFPI Flox littermate controls. TFPI LysM mice did not have a significant reduction in circulating TFPI activity levels compared to their TFPI Flox counterparts. Neither TFPI Tie2 nor TFPI LysM mice had significant changes in hemostasis compared to TFPI Flox mice as determined by a tail bleeding assay. In an ADP-induced model of thromboembolism there were no significant changes in mortality between the TFPI Tie2 or TFPI LysM and their TFPI Flox littermates. In a ferric chloride model of arterial thrombosis, TFPI Tie2 mice had a significant reduction (P < 0.05) in time to occlusion while TFPI LysM mice did not differ compared to TFPI Flox mice. Conclusions: Deletion of TFPI from cells expressing Tie2 or LysM is consistent with normal development and hemostasis. TFPI Tie2 mice have decreased circulating levels of TFPI and an enhanced thrombotic response to ferric chloride. These data support an important regulatory role for EC TFPI in arterial thrombosis.

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.

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.

Introduction: At the forefront of post-heart transplant care is early detection of transplant rejection. However, the current gold standard for detecting acute cellular transplant rejection is recurrent invasive endomyocardial biopsies (EMB). We aimed at developing a non-invasive biomarker for acute cellular heart transplant rejection (ACR) using a plasma extracellular vesicle (EV) platform. Methods: Peripheral and coronary sinus plasma samples were prospectively collected from 183 (31.7% female) patients from 2020 to 2023 during routine surveillance EMB. A discovery cohort comprised five patients who displayed ISHLT 0-1R biopsy followed by an ISHLT 2R grade rejection event. Plasma EVs were isolated from the baseline (0-1R) and rejection (2R) time points. Subsequently, integrative analysis of plasma EV proteomic, miRNomic, and metabolomics was performed pairwise with the samples. Results: The study collected 943 sample time points from 183 patients. Of these sample collections, 43 (4.5%) cases were 2R rejections. Our proteomic analysis of the plasma EVs identified 28 proteins found explicitly during a grade 2R rejection and 87 differentially expressed proteins during a 2R rejection event. An Ingenuity Pathway Analysis (IPA) analysis of the upregulated proteins highlighted EVs mediated mTOR signaling and antigen presentation as central to rejection events. Although all patients in the discovery cohort were negative for antibody-mediated rejection (AMR), the proteomic findings also revealed increased B-cell receptor signaling, implying a mechanistic intersection of ACR and AMR. Conclusions: A distinct set of proteins were found in plasma EVs during ACR events, providing potentially specific and sensitive biomarkers to reduce the need for EMB that could be exploited for ACR diagnosis. The proteins identified formed an immune signaling network that suggests a novel role for EVs in ACR pathogenesis.