Donor heart ischemic time can be extended beyond 9 hours using hypothermic machine perfusion in sheep.

BACKGROUND: The global shortage of donor hearts available for transplantation is a major problem for the treatment of end-stage heart failure. The ischemic time for donor hearts using traditional preservation by standard static cold storage (SCS) is limited to approximately 4 hours, beyond which the risk for primary graft dysfunction (PGD) significantly increases. Hypothermic machine perfusion (HMP) of donor hearts has been proposed to safely extend ischemic time without increasing the risk of PGD. METHODS: Using our sheep model of 24 hours brain death (BD) followed by orthotopic heart transplantation (HTx), we examined post-transplant outcomes in recipients following donor heart preservation by HMP for 8 hours, compared to donor heart preservation for 2 hours by either SCS or HMP. RESULTS: Following HTx, all HMP recipients (both 2 hours and 8 hours groups) survived to the end of the study (6 hours after transplantation and successful weaning from cardiopulmonary bypass), required less vasoactive support for hemodynamic stability, and exhibited superior metabolic, fluid status and inflammatory profiles compared to SCS recipients. Contractile function and cardiac damage (troponin I release and histological assessment) was comparable between groups. CONCLUSIONS: Overall, compared to current clinical SCS, recipient outcomes following transplantation are not adversely impacted by extending HMP to 8 hours. These results have important implications for clinical transplantation where longer ischemic times may be required (e.g., complex surgical cases, transport across long distances). Additionally, HMP may allow safe preservation of "marginal" donor hearts that are more susceptible to myocardial injury and facilitate increased utilization of these hearts for transplantation.

A clinically relevant sheep model of orthotopic heart transplantation 24 h after donor brainstem death.

BACKGROUND: Heart transplantation (HTx) from brainstem dead (BSD) donors is the gold-standard therapy for severe/end-stage cardiac disease, but is limited by a global donor heart shortage. Consequently, innovative solutions to increase donor heart availability and utilisation are rapidly expanding. Clinically relevant preclinical models are essential for evaluating interventions for human translation, yet few exist that accurately mimic all key HTx components, incorporating injuries beginning in the donor, through to the recipient. To enable future assessment of novel perfusion technologies in our research program, we thus aimed to develop a clinically relevant sheep model of HTx following 24 h of donor BSD. METHODS: BSD donors (vs. sham neurological injury, 4/group) were hemodynamically supported and monitored for 24 h, followed by heart preservation with cold static storage. Bicaval orthotopic HTx was performed in matched recipients, who were weaned from cardiopulmonary bypass (CPB), and monitored for 6 h. Donor and recipient blood were assayed for inflammatory and cardiac injury markers, and cardiac function was assessed using echocardiography. Repeated measurements between the two different groups during the study observation period were assessed by mixed ANOVA for repeated measures. RESULTS: Brainstem death caused an immediate catecholaminergic hemodynamic response (mean arterial pressure, p = 0.09), systemic inflammation (IL-6 - p = 0.025, IL-8 - p = 0.002) and cardiac injury (cardiac troponin I, p = 0.048), requiring vasopressor support (vasopressor dependency index, VDI, p = 0.023), with normalisation of biomarkers and physiology over 24 h. All hearts were weaned from CPB and monitored for 6 h post-HTx, except one (sham) recipient that died 2 h post-HTx. Hemodynamic (VDI - p = 0.592, heart rate - p = 0.747) and metabolic (blood lactate, p = 0.546) parameters post-HTx were comparable between groups, despite the observed physiological perturbations that occurred during donor BSD. All p values denote interaction among groups and time in the ANOVA for repeated measures. CONCLUSIONS: We have successfully developed an ovine HTx model following 24 h of donor BSD. After 6 h of critical care management post-HTx, there were no differences between groups, despite evident hemodynamic perturbations, systemic inflammation, and cardiac injury observed during donor BSD. This preclinical model provides a platform for critical assessment of injury development pre- and post-HTx, and novel therapeutic evaluation.

Combined Mesenchymal Stromal Cell Therapy and Extracorporeal Membrane Oxygenation in Acute Respiratory Distress Syndrome. A Randomized Controlled Trial in Sheep.

Rationale: Mesenchymal stromal cell (MSC) therapy is a promising intervention for acute respiratory distress syndrome (ARDS), although trials to date have not investigated its use alongside extracorporeal membrane oxygenation (ECMO). Recent preclinical studies have suggested that combining these interventions may attenuate the efficacy of ECMO.Objectives: To determine the safety and efficacy of MSC therapy in a model of ARDS and ECMO.Methods: ARDS was induced in 14 sheep, after which they were established on venovenous ECMO. Subsequently, they received either endobronchial induced pluripotent stem cell-derived human MSCs (hMSCs) (n = 7) or cell-free carrier vehicle (vehicle control; n = 7). During ECMO, a low Vt ventilation strategy was employed in addition to protocolized hemodynamic support. Animals were monitored and supported for 24 hours. Lung tissue, bronchoalveolar fluid, and plasma were analyzed, in addition to continuous respiratory and hemodynamic monitoring.Measurements and Main Results: The administration of hMSCs did not improve oxygenation (PaO2/FiO2 mean difference = -146 mm Hg; P = 0.076) or pulmonary function. However, histological evidence of lung injury (lung injury score mean difference = -0.07; P = 0.04) and BAL IL-8 were reduced. In addition, hMSC-treated animals had a significantly lower cumulative requirement for vasopressor. Despite endobronchial administration, animals treated with hMSCs had a significant elevation in transmembrane oxygenator pressure gradients. This was accompanied by more pulmonary artery thromboses and adherent hMSCs found on explanted oxygenator fibers.Conclusions: Endobronchial hMSC therapy in an ovine model of ARDS and ECMO can impair membrane oxygenator function and does not improve oxygenation. These data do not recommend the safe use of hMSCs during venovenous ECMO.

Effect of ex vivo extracorporeal membrane oxygenation flow dynamics on immune response.

BACKGROUND: Extracorporeal membrane oxygenation is a life-saving support for heart and/or lung failure patients. Despite technological advancement, abnormal physiology persists and has been associated with subsequent adverse events. These include thrombosis, bleeding, systemic inflammatory response syndrome and infection. However, the underlying mechanisms are yet to be elucidated. We aimed to investigate whether the different flow dynamics of extracorporeal membrane oxygenation would alter immune responses, specifically the overall inflammatory response, leukocyte numbers and activation/adhesion surface antigen expression. METHODS: An ex vivo model was used with human whole blood circulating at 37°C for 6 hours at high (4 L/minute) or low (1.5 L/minute) flow dynamics, with serial blood samples taken for analysis. RESULTS: During high flow, production of interleukin-1ß (p < 0.0001), interleukin-6 (p = 0.0075), tumour necrosis factor-α (p = 0.0013), myeloperoxidase (p < 0.0001) and neutrophil elastase (p < 0.0001) were significantly elevated over time compared to low flow, in particular at 6 hours. While the remaining assessments exhibited minute changes between flow dynamics, a consistent trend of modulation in leukocyte subset numbers and phenotype was observed at 6 hours. CONCLUSION: We conclude that prolonged circulation at high flow triggers a prominent pro-inflammatory cytokine response and activates neutrophil granule release, but further research is needed to better characterize the effect of flow during extracorporeal membrane oxygenation.

Mesenchymal stem cells may ameliorate inflammation in an ex vivo model of extracorporeal membrane oxygenation.

INTRODUCTION: Mesenchymal stem cells exhibit immunomodulatory properties which are currently being investigated as a novel treatment option for Acute Respiratory Distress Syndrome. However, the feasibility and efficacy of mesenchymal stem cell therapy in the setting of extracorporeal membrane oxygenation is poorly understood. This study aimed to characterise markers of innate immune activation in response to mesenchymal stem cells during an ex vivo simulation of extracorporeal membrane oxygenation. METHODS: Ex vivo extracorporeal membrane oxygenation simulations (n = 10) were conducted using a commercial extracorporeal circuit with a CO2-enhanced fresh gas supply and donor human whole blood. Heparinised circuits (n = 4) were injected with 40 × 106-induced pluripotent stem cell-derived human mesenchymal stem cells, while the remainder (n = 6) acted as controls. Simulations were maintained, under physiological conditions, for 240 minutes. Circuits were sampled at 15, 30, 60, 120 and 240 minutes and assessed for levels of interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tumour necrosis factor-α, transforming growth factor-ß1, myeloperoxidase and α-Defensin-1. In addition, haemoglobin, platelet and leukocyte counts were performed. RESULTS: There was a trend towards reduced levels of pro-inflammatory cytokines in mesenchymal stem cell-treated circuits and a significant increase in transforming growth factor-ß1. Blood cells and markers of neutrophil activation were reduced in mesenchymal stem cell circuits during the length of the simulation. As previously reported, the addition of mesenchymal stem cells resulted in a reduction of flow and increased trans-oxygenator pressures in comparison to controls. CONCLUSIONS: The addition of mesenchymal stem cells during extracorporeal membrane oxygenation may cause an increase in transforming growth factor-ß1. This is despite their ability to adhere to the membrane oxygenator. Further studies are required to confirm these findings.

Administration of mesenchymal stem cells during ECMO results in a rapid decline in oxygenator performance.

Mesenchymal stem cells (MSCs) have attracted attention as a potential therapy for Acute Respiratory Distress Syndrome (ARDS). At the same time, the use of extracorporeal membrane oxygenation (ECMO) has increased among patients with severe ARDS. To date, early clinical trials of MSCs in ARDS have excluded patients supported by ECMO. Here we provide evidence from an ex-vivo model of ECMO to suggest that the intravascular administration of MSCs during ECMO may adversely impact the function of a membrane oxygenator. The addition of clinical grade MSCs resulted in a reduction of flow through the circuit in comparison to controls (0.6 ±0.35 L min-1vs 4.12 ± 0.03 L min-1, at 240 minutes) and an increase in the transoygenator pressure gradient (101±9 mmHg vs 21±4 mmHg, at 240 minutes). Subsequent immunohistochemistry analysis demonstrated quantities of MSCs highly adherent to membrane oxygenator fibres. This study highlights the potential harm associated with MSC therapy during ECMO and suggests further areas of research required to advance the translation of cell therapy in this population.

Inhibition of proliferation of small intestinal and bronchopulmonary neuroendocrine cell lines by using peptide analogs targeting receptors.

BACKGROUND: Currently, no consistently effective therapy is available to inhibit cell proliferation or metastasis of neuroendocrine tumor (NET) disease. The effects of 4 novel peptides were analyzed: a targeted cytotoxic analog of luteinizing hormone-releasing hormone (LH-RH) analog (AN-152), a targeted cytotoxic analog of somatostatin (AN-238), and 2 antagonists of growth hormone-releasing hormone (GH-RH) on 3 NET (carcinoid) cell lines that expressed respective peptide receptors. METHODS: The effects of the compounds were evaluated on cell proliferation in vitro using MTT uptake and Ki67 expression, apoptosis (caspase 3 expression and activity), and cell cycle parameters (DNA distribution). RESULTS: Proliferation of the LH-RH receptor-expressing lung NET, NCI-H720 line, was inhibited 2-fold by AN-152 containing doxorubicin compared with the chemotherapy alone (IC50 of 9.1 nM vs 24 nM). This was associated with a reduction in Ki67 transcript and an increase in both caspase 3 mRNA levels and activity. Proliferation of the GH-RH receptor expressing lung NET, NCI-H727 line, was inhibited by both GH-RH antagonists, the effects being mediated through changes in Ki67 expression, but not in caspase 3-mediated apoptosis. The small intestinal NET, KRJ-I line, was 8x more sensitive to inhibition by AN-238 than to 2-pyrolino-doxorubicin, reflected by increased caspase 3 transcript as well as activity. AN-238-mediated growth inhibition culminated in complete G1 arrest. CONCLUSIONS: The data demonstrate GH-RH antagonists or peptide-linked antineoplastic agents such as AN-152 and AN-238 are effective inhibitors of NET proliferation in vitro. Because peptide receptors such as those for GH-RH, LH-RH, and SST subtypes are commonly expressed by NETs, the development of antineoplastic agents targeted to specific tumor receptors may provide a more efficacious strategy than systemic chemotherapeutic agents currently in use.