Albert J. Chong

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.

Tissue factor and thrombin mediate myocardial ischemia-reperfusion injury

Reperfusion of the ischemic heart is necessary to prevent irreversible injury of the myocardium, which leads to permanent organ dysfunction. However, reperfusion in itself leads to myocardial ischemia/reperfusion (I/R) injury, which is characterized by an acute inflammatory response mediated by activated inflammatory cells, chemokines, cytokines, and adhesion molecules. The molecular mechanisms of myocardial I/R injury are not completely known. Tissue factor (TF) and thrombin, two potent procoagulant and proinflammatory mediators, are recognized to play significant roles in myocardial I/R injury. To investigate the role of TF and thrombin in myocardial I/R injury, we used rabbit and murine in situ coronary artery ligation models. Increased TF mRNA, antigen, and activity were found in ischemic cardiomyocytes. Administration of an inhibitory antirabbit TF monoclonal antibody before or during the onset of ischemia resulted in a significant reduction in infarct size. Functional inhibition of thrombin with hirudin also reduced the infarct size. However, defibrinogenating rabbits with ancrod had no effect on infarct size, suggesting a requirement of thrombin generation but not fibrin deposition in myocardial I/R injury.

Preservation of endothelium-dependent vasodilation with low-potassium university of wisconsin solution

University of Wisconsin solution has provided excellent myocardial preservation. However, the high potassium content of the currently available University of Wisconsin solution has been implicated in coronary artery endothelial damage. We placed 16 neonatal (age 1 to 3 days) Duroc piglet hearts on an isolated nonworking perfusion circuit. Endothelium-dependent and endothelium-independent vasodilation were tested by measuring coronary blood flow after intracoronary infusion of bradykinin (10(-6) mol/L) and nitroprusside (10(-6) mol/L), respectively. In addition, nitric oxide levels were measured after bradykinin infusion. The hearts were then arrested blindly with either a modified University of Wisconsin solution (group 1; n = 8, K+ = 25 mEq/L) or standard University of Wisconsin solution (group 2; n = 8, K+ = 129 mEq/L) by infusion of cardioplegic solution every 20 minutes for a total of 2 hours. After bradykinin infusion, the mean coronary blood flow increased by 237.1% +/- 14.0% of baseline valves before arrest and by 232.8% +/- 16.0% after arrest in group 1 (p = not significant). As in the first group, the mean coronary blood flow in group 2 increased by 231.1% +/- 13.7% before arrest; however, the increase in mean coronary blood flow after arrest was significantly attenuated (163.3% +/- 12.8%, p < 0.01). The loss of endothelium-dependent coronary blood flow response in group 2 correlated with a decreased capacity to release nitric oxide after arrest (prearrest 8.25 +/- 2.30 nmol/min per gram versus postarrest -2.46 +/- 2.29 nmol/min per gram, p < 0.01). Endothelium-independent vasodilatory response revealed no significant difference between groups before and after arrest. These results suggest that the low-potassium University of Wisconsin solution provides superior protection of the endothelium by preserving the endothelium-dependent vasodilatory response to nitric oxide release.

Microvascular Inflammatory Response in Cardiac Surgery

Cardiac surgical procedures, with or without cardiopulmonary bypass, elicit a systemic inflammatory response in patients that induces the elaboration of multiple cytokines, chemokines, adhesion molecules, and destructive enzymes. This inflammatory reaction involves multiple interdependent and redundant cell types and humoral cascades, which allows for amplification and positive feedback at numerous steps. This systemic inflammatory response ultimately results in a broad spectrum of clinical manifestations, with multiple organ failure being the most severe form. Investigative efforts have focused on understanding the mechanism of this systemic inflammatory response syndrome in order to develop potential therapeutic targets to inhibit it, thereby possibly decreasing postoperative morbidity and mortality. Multiple therapeutic methods have been investigated, including pharmacologic inhibitors and modifications of surgical technique and the cardiopulmonary bypass circuit. Although studies have demonstrated that the use of these therapies in experimental and clinical settings has attenuated the systemic inflammatory response, they have failed to conclusively show clinical benefit from these therapies. These therapies may be too specific to minimize the deleterious effects of a systemic inflammatory response that results from the activation of multiple, interdependent, and redundant inflammatory cascades and cell types. Hence, further studies that investigate the molecular and cellular events underlying the systemic inflammatory response syndrome and the resultant effects of anti-inflammatory therapies are warranted to ultimately achieve improvements in clinical outcome after cardiac surgical procedures.