Atli Eyjolfsson

Clinical transplantation of initially rejected donor lungs after reconditioning ex vivo.

BACKGROUND A major problem in clinical lung transplantation is the shortage of donor lungs. Only about 20% of donor lungs are accepted for transplantation. A method to evaluate and recondition lungs ex vivo has been tested on donor lungs that have been rejected for transplantation. METHODS The donor lungs were reconditioned ex vivo in an extracorporeal membrane oxygenation (ECMO) circuit with STEEN solution (Vitrolife AB, Kungsbacka, Sweden) mixed with erythrocytes. The hyperoncotic solution dehydrates edematous lung tissue. Functional evaluations were performed with deoxygenated perfusate by varying the inspired fraction of oxygen. After the reconditioning, the lungs were kept immersed at 8 degrees C in extracorporeal membrane oxygenation until transplantation was performed. RESULTS Six of nine initially rejected donor lungs were reconditioned to acceptable function, and in six recipients, double lung transplantation was performed. Three-month survival was 100%. One patient has since died due to sepsis after 95 days, and one due to rejection after 9 months. Four recipients are alive and well without any sign of bronchiolitis obliterans syndrome 24 months after the transplantation. CONCLUSIONS The result from the present study is promising, and we continue to transplant reconditioned lungs.

ADP Receptor P2Y12 Is Expressed in Vascular Smooth Muscle Cells and Stimulates Contraction in Human Blood Vessels

Objective—ADP plays an important role in platelet aggregation by activating P2Y12 receptors. We assessed the hypothesis that P2Y12 receptors are expressed in vascular smooth muscle cells (VSMC). Methods and Results—P2Y12 receptor mRNA was found to have a high expression among the P2 receptors in human VSMC, significantly higher than the other 2 ADP receptors (P2Y1 and P2Y13, real-time polymerase chain reaction). Western blots gave a band of 50 kD, similar to that in platelets. To unmask a P2Y12 receptor-mediated vasoconstriction by simulating the in vivo situation, vessels were precontracted to a submaximal level. 2-MeSADP stimulated contractions in vessel segments from internal mammary artery (IM), IM branches and small veins (Emax=15±6% of 60mmol/L K+ contraction, pEC50=5.6±0.6, Emax=21±1%, pEC50=6.8±0.1, and Emax=48±9%, pEC50=6.6±0.4). The selective P2Y12 antagonist AR-C67085 blocked 2-MeSADP contractions. The contraction was not reduced in patients using clopidogrel, a drug inhibiting ADP-induced platelet aggregation by blocking the P2Y12 receptor. This may be explained by the high instability of the active clopidogrel metabolite that never reaches the systemic circulation. Conclusion—ADP acting on P2Y12 receptors not only is important for platelet activation but also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm.

Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium-derived hyperpolarising factor.

The present study was aimed at examining P2 receptor‐mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 μM noradrenaline. Endothelial denudation abolished the dilator responses. The selective P2Y1 agonist, 2‐MeSADP, induced a potent vasodilatation (pEC50=6.9±0.1). The P2Y1 antagonist of 10 μM, MRS 2216, shifted the 2‐MeSADP concentration‐response curve 1.1 log units to the right. The combined P2Y1 and P2X agonist, 2‐MeSATP, stimulated a dilatation with a potency similar to that of 2‐MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2‐MeSATP and 2‐MeSADP indicating that P2X receptors do not mediate vasodilatation. Both the P2Y2/4 agonist, UTPγS and the P2Y6 agonist, UDPβS, stimulated potent dilatations (pEC50=7.8±0.4 for UTPγS and 8.4±0.2 for UDPβS). The 2‐MeSADP‐induced nitric oxide (NO)‐mediated dilatation was studied in the presence of 10 μM indomethacin, 50 nM charybdotoxin and 1 μM apamin. The involvement of the endothelium‐derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L‐NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L‐NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2‐MeSADP dilatation with similar efficacy (Emax=25±5% for NO, 25±6% for EDHF and 27±5% for prostaglandins). In conclusion, extracellular nucleotides induce endothelium‐derived vasodilatation in human vessels by stimulating P2Y1, P2Y2/4 and P2Y6 receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins

The reversible oral P2Y12 antagonist AZD6140 inhibits ADP-induced contractions in murine and human vasculature

OBJECTIVES The platelet ADP P2Y(12) receptor which is a target for the antithrombotic drug clopidogrel is also distributed on vascular smooth muscle cells and stimulate contraction. This study investigates whether AZD6140, in contrast to clopidogrel, can inhibit ADP-mediated arterial contractions. METHODS Mice were treated with clopidogrel, 50 mg/kg, 24 and 2 h before experiment. Thoracic aorta ring segments from both clopidogrel-treated (n=5) and untreated (n=4) mice were mounted in myograph baths. Contractions of human left internal mammary arteries (IMA) and small arteries were studied in an identical manner. RESULTS Clopidogrel treatment per os did not inhibit contractions by the stable ADP analogue 2-MeSADP (10 microM). However, addition of 1 microM AZD6140 in vitro inhibited ADP contraction (% of maximal contraction by 60 mM K(+)) both in the clopidogrel-treated, from 64% to 32% (P=0.002) and in the untreated group, from 59% to 33% (P=0.015). 2-MeSADP contractions in human IMA and small arteries were inhibited by AZD6140. CONCLUSIONS The antiplatelet drug AZD6140 blocks the contractile effects of ADP in both murine and human vasculature. These effects of AZD6140 could be beneficial in the management of conditions in which vasospasm may play a role.

How to Recondition Ex Vivo Initially Rejected Donor Lungs for Clinical Transplantation: Clinical Experience from Lund University Hospital

A major problem in clinical lung transplantation is the shortage of donor lungs. Only about 20% of donor lungs are accepted for transplantation. We have recently reported the results of the first six double lung transplantations performed with donor lungs reconditioned ex vivo that had been deemed unsuitable for transplantation by the Scandiatransplant, Eurotransplant, and UK Transplant organizations because the arterial oxygen pressure was less than 40 kPa. The three-month survival of patients undergoing transplant with these lungs was 100%. One patient died due to sepsis after 95 days, and one due to rejection after 9 months. Four recipients are still alive and well 24 months after transplantation, with no signs of bronchiolitis obliterans syndrome. The donor lungs were reconditioned ex vivo in an extracorporeal membrane oxygenation circuit using STEEN solution mixed with erythrocytes, to dehydrate edematous lung tissue. Functional evaluation was performed with deoxygenated perfusate at different inspired fractions of oxygen. The arterial oxygen pressure was significantly improved in this model. This ex vivo evaluation model is thus a valuable addition to the armamentarium in increasing the number of acceptable lungs in a donor population with inferior arterial oxygen pressure values, thereby, increasing the lung donor pool for transplantation. In the following paper we present our clinical experience from the first six patients in the world. We also present the technique we used in detail with flowchart.

Evaluation of cystatin C with iohexol clearance in cardiac surgery.

Background: Post‐operative renal dysfunction after cardiac surgery is not uncommon and can lead to adverse outcome. The ability to accurately monitor renal function is therefore important. Cystatin C is known to be a sensitive marker of the glomerular filtration rate (GFR), but it has not been fully evaluated in cardiac surgery. Iohexol clearance is considered a reliable reference method for the determination of GFR. The aim of this study is to, for the first time, evaluate the diagnostic accuracy of plasma cystatin C compared with iohexol clearance in cardiac surgery.

Characterization of lipid particles in shed mediastinal blood.

BACKGROUND Shed mediastinal blood is known to be a source of microemboli in cardiac surgery. The aim of this study was to characterize in detail the lipid particles found in this blood. METHODS Blood samples were collected from 24 patients undergoing routine cardiac surgery with cardiopulmonary bypass. Arterial and shed blood was analyzed using the Coulter counter technique to establish the number and size of particles. The composition of these lipid particles was compared with that of adipose tissue from the mediastinum using gas chromatography. Scanning electron microscopy was used to visualize the lipid particles in samples of shed blood. RESULTS Lipid particles in the size range of 10 to 60 microm were characterized in shed mediastinal blood, and more than 300,000 particles per milliliter of blood were found. Triglyceride profiles in these lipid particles and in adipose tissue were similar, suggesting that their origin is the mediastinum. Scanning electron microscopy showed spherical formations corresponding in size to the particles counted using the Coulter counter. CONCLUSIONS During the past decade attention has focused on microembolism in cardiac surgery, and this study has helped define the problem. Different strategies, such as eliminating the use of shed mediastinal blood or purifying the blood by different techniques, may improve the results of cardiac surgery in the future.

Functional and pharmacological characteristics of permeability transition in isolated human heart mitochondria.

The objective of the present study was to validate the presence and explore the characteristics of mitochondrial permeability transition (mPT) in isolated mitochondria from human heart tissue in order to investigate if previous findings in animal models of cardiac disorders are translatable to human disease. Mitochondria were rapidly isolated from fresh atrial tissue samples obtained from 14 patients undergoing Maze surgery due to atrial fibrillation. Human heart mitochondria exhibited typical mPT characteristics upon calcium overload such as swelling, evaluated by changes in light scattering, inhibition of respiration and loss of respiratory coupling. Swelling was a morphologically reversible event following transient calcium challenge. Calcium retention capacity (CRC), a quantitative measure of mPT sensitivity assayed by following extramitochondrial [Ca2+] and changes in respiration during a continuous calcium infusion, was significantly increased by cyclophilin D (CypD) inhibitors. The thiol-reactive oxidant phenylarsine oxide sensitized mitochondria to calcium-induced mPT. Release of the pro-apoptotic intermembrane protein cytochrome c was increased after, but not before, calcium discharge and respiratory inhibition in the CRC assay. From the present study, we conclude that adult viable heart mitochondria have a CypD- and oxidant-regulated mPT. The findings support that inhibition of mPT may be a relevant pharmacological target in human cardiac disease and may underlie the beneficial effect of cyclosporin A in reperfusion injury.

Circulating particles during cardiac surgery.

Shed blood is known to be a source of lipid micro-emboli in cardiac surgery. The aim of this study was to characterize the occurrence of these particles at different stages of the operation, and to study their occurrence in the circulation at multiple time-points after the retransfusion of shed blood. Forty-four patients undergoing routine surgery with cardiopulmonary bypass were included. Blood was sampled from the surgical field at different sampling locations during the operation. Shed blood was collected in a transfusion bag and retransfused. After which, blood was sampled from the arterial line of the heart-lung machine. A Coulter counter was used for particle determinion. The mean volume of shed blood collected was 340+/-215 ml. Particles in the size range 10-60 microm were found at varying concentrations, with the highest concentrations being found in blood collected after cannulation and from the pleura. After retransfusion of this blood, a biphasic response was seen in the blood drawn from the efferent line of the heart-lung machine. Particles are found in shed blood at all times during cardiac surgery, and when this blood was retransfused an increase was seen in particle concentration in the heart-lung machine.

Lipid emboli distribution in cardiac surgery is dependent on the state of emulsification

Objective. Lipid embolizations from retransfused shed blood during cardiac surgery have been shown to enter the circulation and end up in different organs. The purpose of this investigation was to evaluate differences in the kinetics and deposition between emulsified and non-emulsified lipid emboli in a porcine model. Design. Twelve animals were anesthetized and put on cardiopulmonary bypass. A shed-blood phantom (6 animals given emulsified and 6 given non-emulsified lipids) was produced from arterial blood, saline, and tritium-labeled triolein. The phantom was infused into the cardiopulmonary bypass circuit. Arterial and venous blood samples were taken at short intervals. Tissue samples were taken post-mortem from examined organs and prepared for scintillation counting. Levels of radioactivity were used to measure lipid emboli content in blood and tissue. Results. Emulsified lipid emboli generated a 5-fold higher embolic load in the arterial and a 12-fold higher in the venous circulation, compared with non-emulsified lipid emboli. Emulsified lipid micro emboli resulted in a 2–15-fold higher tissue deposition in investigated organs compared with non-emulsified lipid micro emboli. Conclusions. This study shows that the state of emulsion significantly alter the kinetics and tissue deposition of lipid emboli. Emulsified lipid emboli give higher embolic load in the arterial and venous circulation, and higher tissue deposition versus non-emulsified lipid emboli. In both groups, the embolic load was higher in the arterial circulation than on the venous side.