Lars Burdorf

Fluorescent Cardiac Imaging A Novel Intraoperative Method for Quantitative Assessment of Myocardial Perfusion During Graded Coronary Artery Stenosis

Background— The purpose of the present study was to examine whether the effect of coronary stenoses of variable severity on myocardial perfusion can be quantitatively assessed in vivo by analysis of fluorescent cardiac imaging (FCI) compared with the gold standard, the fluorescent microsphere method. FCI is a novel technology to visualize coronary vessels and myocardial perfusion intraoperatively using the indocyanine green dye with an infrared-sensitive imaging device. Methods and Results— Graded stenoses and total vessel occlusion of the left anterior descending coronary artery were created in 11 open-chest pigs. Stenoses were graded to reduce resting left anterior descending coronary artery flow by 25%, 50%, 75%, and 100% of baseline flow measured by transit-time flowmeter. FCI images were analyzed with a digital image processing system. The impairment of myocardial perfusion was quantified by background-subtracted peak fluorescence intensity and slope of fluorescence intensity obtained with FCI and compared with myocardial blood flow assessed by fluorescent microsphere. All stenoses resulted in an impairment of myocardial perfusion visualized by FCI. Occlusion of the left anterior descending coronary artery resulted in a total perfusion defect (no fluorescence intensity) of the corresponding anterior myocardial wall. During graded stenosis and total vessel occlusion, normalized background-subtracted peak fluorescence intensity and slope of fluorescence intensity decreased significantly (P<0.0001). Both background-subtracted peak fluorescence intensity (r=0.92, P<0.0001) and slope of fluorescence intensity (r=0.93, P<0.0001) analyzed by FCI demonstrated good linear correlation with fluorescent microsphere–derived myocardial blood flow. Conclusions— The impairment of myocardial perfusion in response to increased coronary stenosis severity and total vessel occlusion can be quantitatively assessed by FCI and correlates well with results obtained by fluorescent microsphere.

State of Art: Clinical ex vivo lung perfusion: Rationale, current status, and future directions

In recent years, ex vivo lung perfusion has emerged as an effective tool for increasing the number of available lungs accepted for transplant. As ex vivo lung perfusion use becomes more widespread, questions have arisen regarding the metabolic activity of the donor lung during ex vivo lung perfusion, optimal perfusion-ventilation strategy, and which parameters best define organ improvement or deterioration. Answers to many of these questions can be found in the published experience with the isolated perfused lung in the study of lung mechanics, pulmonary metabolism, and the effects of various interventions on lung quality. The purpose of this review is to summarize past and present evidence and to provide important background for clinicians and investigators using the ex vivo lung perfusion/isolated perfused lung system.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway‐regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life‐supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri‐transplant platelet activation (as β‐thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

Meta‐analysis of the independent and cumulative effects of multiple genetic modifications on pig lung xenograft performance during ex vivo perfusion with human blood

Genetically modified pigs are a promising potential source of lung xenografts. Ex vivo xenoperfusion is an effective platform for testing the effect of new modifications, but typical experiments are limited by testing of a single genetic intervention and small sample sizes. The purpose of this study was to analyze the individual and aggregate effects of donor genetic modifications on porcine lung xenograft survival and injury in an extensive pig lung xenoperfusion series.

Expression of human CD46 modulates inflammation associated with GalTKO lung xenograft injury

Evaluation of lungs from GalTKO.hCD46 pigs, genetically modified to lack the galactose‐α(1,3)‐galactose epitope (GalTKO) and to express human CD46, a complement regulatory protein, has not previously been described. Physiologic, hematologic and biochemical parameters during perfusion with heparinized fresh human blood were measured for 33 GalTKO.hCD46, GalTKO (n = 16), and WT pig lungs (n = 16), and 12 pig lungs perfused with autologous pig blood. Median GalTKO.hCD46 lung survival was 171 min compared to 120 for GalTKO (p = 0.27) and 10 for WT lungs (p < 0.001). Complement activation, platelet activation and histamine elaboration were significantly reduced during the first 2 h of perfusion in GalTKO.hCD46 lungs compared to GalTKO (ΔC3a at 120′ 812 ± 230 vs. 1412 ± 1047, p = 0.02; ΔCD62P at 120′ 9.8 ± 7.2 vs. 25.4 ± 18.2, p < 0.01; Δhistamine at 60′ 97 ± 62 vs. 189 ± 194, p = 0.03). We conclude that, in addition to significant down‐modulation of complement activation, hCD46 expression in GalTKO lungs diminished platelet and coagulation cascade activation, neutrophil sequestration and histamine release. Because GalTKO.hCD46 lung failure kinetics correlated directly with platelet and neutrophil sequestration, coagulation cascade activation and a rise in histamine levels within the first hour of perfusion, further progress will likely depend upon improved control of these pathways, by rationally targeted additional modifications to pigs and pharmacologic interventions.

Xenogeneic Lung Transplantation Models

Study of lung xenografts has proven useful to understand the remaining barriers to successful transplantation of other organ xenografts. In this chapter, the history and current status of lung xenotransplantation are briefly reviewed and two different experimental models, the ex vivo porcine-to-human lung perfusion and the in vivo xenogeneic lung transplantation, are presented. We focus on the technical details of these lung xenograft models in sufficient detail, list the needed materials, and mention analysis techniques to allow others to adopt them with minimal learning curve.

Lung xenotransplantation: recent progress and current status.

Xenotransplantation has undergone important progress in controlling initial hyperacute rejection in many preclinical models, with some cell, tissue, and organ xenografts advancing toward clinical trials. However, acute injury, driven primarily by innate immune and inflammatory responses, continues to limit results in lung xenograft models. The purpose of this article is to review the current status of lung xenotransplantation—including the seemingly unique challenges posed by this organ—and summarize proven and emerging means of overcoming acute lung xenograft injury.

Pig-to-baboon liver xenoperfusion utilizing GalTKO.hCD46 pigs and glycoprotein Ib blockade

Although transplantation of genetically modified porcine livers into baboons has yielded recipient survival for up to 7 days, survival is limited by profound thrombocytopenia, which becomes manifest almost immediately after revascularization, and by subsequent coagulopathy. Porcine von Willebrands factor (VWF), a glycoprotein that adheres to activated platelets to initiate thrombus formation, has been shown to constitutively activate human platelets via their glycoprotein Ib (GPIb) receptors. Here, we report our pig‐to‐primate liver xenoperfusion model and evaluate whether targeting the GPIb‐VWF axis prevents platelet sequestration.

Transgenic expression of human leukocyte antigen-E attenuates GalKO.hCD46 porcine lung xenograft injury.

Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3‐αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA‐E and β2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models.

Reduced Fibrin Deposition and Intravascular Thrombosis in hDAF Transgenic Pig Hearts Perfused With Tirofiban

Background. Solid organ xenograft rejection is associated with vascular injury resulting at least in part in platelet activation, and rejected xenografts invariably demonstrate intravascular thrombosis and interstitial hemorrhage. Complement activation plays a prominent role in platelet-endothelial interaction. We tested the effects of platelet GPIIb/IIIa inhibitor tirofiban during perfusion of hDAF pig hearts. Methods. Using a working-heart model, nontransgenic and hDAF pig hearts were perfused with tirofiban or human blood only. Myocardial damage was determined by hemodynamic parameters (cardiac output, stroke work index) and creatine phosphokinase. Further monitoring included the assessment of complement factors (C3, C4), platelets, fibrinogen, ATIII, and graft histology. Results. Tirofiban increased cardiac output (CO) and stroke work index (SWI) of nontransgenic pig hearts and improved superior CO and SWI of hDAF pig hearts. Although perfusion time of nontransgenic pig hearts was prolonged by tirofiban (196±65 min vs. 162±122 min), a similar effect in hDAF pig hearts (218±116 min vs. 222±30 min) could not be demonstrated. Tirofiban reduced consumption of C3 and C4 independently from hDAF. Depletion of fibrinogen was equally diminished by tirofiban and hDAF; the combination of both agents obtained no further reduction. ATIII consumption was most effectively inhibited by this combination. Intravascular fibrin deposition was reduced by tirofiban and hDAF, but particularly by the combination of the two agents. Conclusions. Improvement of heart performance and reduction of myocardial damage and intravascular thrombosis confirm a role of the GPIIb/IIIa inhibitor tirofiban for the prevention of hDAF pig heart rejection and xenograft function.