Christine L. Rothnie

Inhibition of Toll-like Receptor 4 With Eritoran Attenuates Myocardial Ischemia-Reperfusion Injury

Background— We previously reported that the functional mutation of Toll-like receptor 4 (TLR4) in C3H/HeJ mice subjected to myocardial ischemia-reperfusion (MI/R) injury resulted in an attenuation of myocardial infarction size. To investigate the ligand-activating TLR4 during MI/R injury, we evaluated the effect of eritoran, a specific TLR4 antagonist, on MI/R injury, with the goal of defining better therapeutic options for MI/R injury. Methods and Results— C57BL/6 mice received eritoran (5 mg/kg) intravenously 10 minutes before 30 minutes of in situ of transient occlusion of the left anterior descending artery, followed by 120 minutes of reperfusion. Infarct size was measured using triphenyltetrazoliumchloride staining. A c-Jun NH2-terminal kinase (JNK) activation was determined by Western blotting, nuclear factor (NF)-&kgr;B activity was detected by gel-shift assay, and cytokine expression was measured by ribonuclease protection assay. Mice treated with eritoran developed significantly smaller infarcts when compared with mice treated with vehicle alone (21.0±6.4% versus 30.9±13.9%; P=0.041). Eritoran pretreatment resulted in a reduction in JNK phosphorylation (eritoran versus vehicle: 3.98±0.81 versus 7.01±2.21-fold increase; P=0.020), less nuclear NF-&kgr;B translocation (2.70±0.35 versus 7.75±0.60-fold increase; P=0.00007), and a decrease in cytokine expression (P<0.05). Conclusions— We conclude that inhibition of TLR4 with eritoran in an in situ murine model significantly reduces MI/R injury and markers of an inflammatory response.

Antibody to CD18 reduces neutrophil and T lymphocyte infiltration and vascular cell adhesion molecule-1 expression in cardiac rejection.

Most antirejection therapies target immune activation but may not reduce leukocyte infiltration into the graft. The leukocyte integrin CD18 has been shown to be important for leukocyte migration in vitro. We postulated that antibody blockade of CD18 might reduce the migration of different leukocyte subpopulations into myocardium during rejection. Using a rabbit model, we evaluated the effect of a monoclonal antibody to CD18 on the infiltration of neutrophils (polymorphonuclear leukocytes [PMNs]), T lymphocytes, and macrophages into cardiac grafts. In addition, vascular cell adhesion molecule-1 (VCAM-1) expression was assessed to determine the relationship between leukocyte infiltration and VCAM-1 expression, an unblocked alternate adhesion pathway. Donor hearts from Stauffland rabbits were transplanted heterotopically into the cervical position of New Zealand White recipients. Recipient rabbits received either monoclonal antibody to CD18 daily without other immunosuppression (n=51), saline injections as placebo controls (n=52), or nonfunctional isotype-matched antibody (n=4). Recipient rabbits were killed at 1 hr, 6 hr, 24 hr, 3 days, and 7 days after transplantation (10-12 rabbits per group at each time point). PMNs, T lymphocytes, and macrophages were differentiated by routine staining and immunocytochemistry, respectively, and quantified as the number of cells per standardized field. VCAM-1 expression was examined immunocytochemically in 30 treated and 30 control transplanted hearts. Monoclonal antibody to CD18 significantly reduced the infiltration of PMNs and T lymphocytes into myocardium during rejection, but did not affect the infiltration of macrophages. Blocking the CD18/intercellular adhesion molecule-1 adhesion pathway also resulted in a decrease in VCAM-1 expression, which correlated in time with the reduction in T lymphocyte infiltration.

Left ventricular dysfunction during extracorporeal membrane oxygenation in a hypoxemic swine model

BACKGROUND Perfusion of the coronary circulation with hypoxemic blood from the left ventricle has been postulated to cause myocardial dysfunction during venoarterial extracorporeal membrane oxygenation for respiratory support. METHODS We investigated this hypothesis in 10 anesthetized open-chest piglets (7 to 9 kg) undergoing venoarterial extracorporeal membrane oxygenation after placement of minor-axis sonomicrometry crystals and left ventricular micromanometer. The left atrial partial pressure of oxygen was made hypoxemic (25 to 40 mm Hg) after initiation of extracorporeal membrane oxygenation by ventilation with a hypoxic gas mixture. Left ventricular contractile function, including peak LV pressure, shortening fraction, maximum rate of increase of left ventricular pressure, velocity of circumferential fiber shortening, end-systolic pressure-minor axis dimension relationship, and preload recruitable dimensional stroke work, was measured or calculated on extracorporeal membrane oxygenation before (baseline) and at 4 and 6 hours after rendering the left atrial blood hypoxemic. RESULTS Left ventricular shortening fraction and velocity of circumferential fiber shortening were significantly lower (p < 0.05) at 4 and 6 hours when compared with baseline. The slope of the end-systolic pressure-minor axis dimension relationship decreased but was not significantly different at 4 and 6 hours when compared with baseline owing to poor linear correlation (r = 0.30 to 0.93). The preload recruitable dimensional stroke work was more linear (r = 0.87 to 0.99), and the slope was significantly lower (p < 0.01) at 4 and 6 hours when compared with baseline. CONCLUSIONS Hypoxemic cardiac output from the left ventricle during venoarterial extracorporeal membrane oxygenation is associated with depression of left ventricular systolic function in this animal model. Current use of venoarterial extracorporeal membrane oxygenation for respiratory support may not provide adequate oxygen supply to the myocardium.

Regulation and actions of activin A and follistatin in myocardial ischaemia–reperfusion injury

Activin A, a member of the transforming growth factor-β superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia-reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia-reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30 min of ischaemia and 2h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia-reperfusion were absent in TLR4(-/-) mice. Pre-treatment with follistatin significantly reduced ischaemia-reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3h of hypoxia and 2h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia-reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.