Elizabeth N. Morgan

Inhibition of the Tissue Factor-Thrombin Pathway Limits Infarct Size after Myocardial Ischemia-Reperfusion Injury by Reducing Inflammation

Functional inhibition of tissue factor (TF) has been shown to improve coronary blood flow after myocardial ischemia/reperfusion (I/R) injury. TF initiates the coagulation protease cascade, resulting in the generation of the serine protease thrombin and fibrin deposition. Thrombin can also contribute to an inflammatory response by activating various cell types, including vascular endothelial cells. We used a rabbit coronary ligation model to investigate the role of TF in acute myocardial I/R injury. At-risk areas of myocardium showed increased TF expression in the sarcolemma of cardiomyocytes, which was associated with a low level of extravascular fibrin deposition. Functional inhibition of TF activity with an anti-rabbit TF monoclonal antibody administered either 15 minutes before or 30 minutes after coronary ligation reduced infarct size by 61% (P = 0.004) and 44% (P = 0.014), respectively. Similarly, we found that inhibition of thrombin with hirudin reduced infarct size by 59% (P = 0.014). In contrast, defibrinogenating the rabbits with ancrod had no effect on infarct size, suggesting that fibrin deposition does not significantly contribute to infarct size. Functional inhibition of thrombin reduced chemokine expression and inhibition of either TF or thrombin reduced leukocyte infiltration. We propose that cardiomyocyte TF initiates extravascular thrombin generation, which enhances inflammation and injury during myocardial I/R.

Inhibition Of Interleukin-8 Blocks Myocardial Ischemia-Reperfusion Injury

INTRODUCTION Interleukin-8 is thought to play a role in neutrophil activation and transcapillary migration into the interstitium. Because neutrophils are principal effector cells in acute myocardial ischemia-reperfusion injury, we postulated that the inhibition of interleukin-8 activity with a neutralizing monoclonal antibody directed against rabbit interleukin-8 (ARIL8.2) would attenuate the degree of myocardial injury encountered during reperfusion. METHODS In New Zealand White rabbits, the large branch of the marginal coronary artery supplying most of the left ventricle was occluded for 45 minutes, followed by 2 hours of reperfusion. Fifteen minutes before reperfusion, animals were given an intravenous bolus of either 2 mg/kg of ARIL8.2 or 2 mg/kg anti-glycoprotein-120, an isotype control antibody that does not recognize interleukin-8. At the completion of the 120-minute reperfusion period, infarct size was determined. RESULTS In the area at risk for infarction, 44.3% +/- 4% of the myocardium was infarcted in the anti-glycoprotein-120 group compared with 24.8% +/- 9% in the ARIL8.2 group (p < 0.005). In control animals, edema and diffuse infiltration of neutrophils were observed predominantly in the infarct zone and the surrounding area at risk. Tissue myeloperoxidase determinations did not differ significantly between groups, indicating that the cardioprotective effect of ARIL8.2 was independent of an effect on neutrophil infiltration. CONCLUSIONS A specific monoclonal antibody that neutralizes interleukin-8 significantly reduces the degree of necrosis in a rabbit model of myocardial ischemia-reperfusion injury.

Endothelial response to cardiopulmonary bypass surgery

BACKGROUND The vascular endothelium has been shown to actively participate in maintaining normal cardiovascular homeostasis by influencing the regulation of membrane permeability, lipid transport, vasomotor tone, coagulation, fibrinolysis, and inflammation. Endothelial cells are very responsive to a wide range of local and systemic stimuli that occur during cardiopulmonary bypass (CPB) operation. Major pathologic conditions result from impaired vascular function secondary to CPB, including vasospasm, coagulopathy, and widespread neutrophil adhesion secondary to a systemic inflammatory response. Additionally, more chronic responses to endothelial cell injury include the development of intimal hyperplasia and arteriosclerosis, both of which limit the long-term success of coronary artery bypass grafting. METHODS Because of the increasingly recognized role of the endothelium in the maintenance of normal cardiovascular function, this article will review the normal structure and function of the endothelium, as well as the major pathologic conditions that result in response to CPB. RESULTS Potential treatments to counteract endothelial cell dysfunction secondary to CPB are under active investigation. Strategies may be directed toward blocking single cytokines, integrins, or adhesion molecules involved in endothelial dysfunction or, alternatively, toward targeting a molecular event that governs the expression of these proinflammatory, procoagulant, and vasoactive genes. In our laboratory, we have used both strategies to study the pathologic response to CPB. We blocked neutrophil adhesion in subhuman primates with a monoclonal antibody. Alternatively, we targeted the transcriptional activation of multiple genes involved in the endothelial cells response to CPB. CONCLUSIONS Although both therapies help elucidate the multiple, redundant pathways involved in the pathologic response to CPB, it is through molecular biology that we are beginning to understand the mechanics of transcriptional control and translational expression that occurs in the endothelial cell in response to CPB. This knowledge will allow the development of therapies that inhibit not a single cytokine or adhesion molecule, but rather an array of substances that result in the endothelial cells pathologic response to CPB.

An essential role for NF-κB in the cardioadaptive response to ischemia

Abstract Background . Ischemic preconditioning (IP) is the phenomenon whereby brief episodes of ischemia protect the heart against a subsequent ischemic stress. We hypothesize that activation of the transcription factor NF-κB mediates IP. Methods . Rabbits were randomly allocated to one of three groups: (1) 45 minutes of myocardial ischemia followed by 2 hours of reperfusion (I/R); (2) three cycles of 5-minute ischemia and 5 minutes of reperfusion followed by I/R (IP + I/R); or (3) IP in the presence of ProDTC, a specific NF-κB inhibitor, followed by I/R (IP ProDTC + I/R). Infarct size, indices of regional contractility, and NF-κB activation were determined. Results . In preconditioned rabbits (IP + I/R), infarct size was reduced 83% compared with both I/R alone and IP ProDTC + I/R groups ( p ProDTC + I/R groups ( p ProDTC + I/R groups showed NF-κB activation with I/R that was absent in preconditioned animals. Conclusions . The cytoprotective effects induced by IP require activation of NF-κB.

Treating myocardial ischemia-reperfusion injury by targeting endothelial cell transcription

Exacerbation of, rather than improvement in, a hypoxic injury after reperfusion of ischemic tissues is recognized as the specific clinicopathologic entity referred to as ischemia/reperfusion (I/R) injury. Arguably, one of the most common forms of I/R injury occurs during cardiac surgery, which has a mandatory period of myocardial ischemia required to allow surgery in a bloodless, motionless field, followed by coronary artery reperfusion after removal of the aortic cross-clamp. In this review, we examine the endothelial cell activation phenotype that initiates and propagates myocardial I/R injury. Emphasis is given to the biology of one transcription factor, NF-kappaB, that has the principal role in the regulation of many endothelial cell genes expressed in activated endothelium. NF-kappaB-dependent transcription of endothelial cell genes that are transcribed in response to I/R injury may be a favorable approach to preventing tissue injury in the setting of I/R. Elucidating safe and effective therapy to inhibit transcription of endothelial cell genes involved in promoting injury after I/R injury may have wide applicability to the patients with heart disease and other forms of I/R injury.

Inhibition of the transcriptional activator protein nuclear factor κb prevents hemodynamic instability associated with the whole-body inflammatory response syndrome

BACKGROUND The transcription factor nuclear factor kappaB mediates the expression of a number of inflammatory genes involved in the whole-body inflammatory response to injury. We and others have found that dithiocarbamates specifically inhibit nuclear factor kappaB-mediated transcriptional activation in vitro. OBJECTIVE We hypothesized that inhibition of nuclear factor kappaB with dithiocarbamate treatment in vivo would attenuate interleukin 1 alpha-mediated hypotension in a rabbit model of systemic inflammation. METHODS New Zealand White rabbits were anesthetized and cannulated for continuous hemodynamic monitoring during 240 minutes. Rabbits were treated intravenously with either phosphate-buffered saline solution or 15 mg/kg of a dithiocarbamate, either pyrrolidine dithiocarbamate or proline dithiocarbamate, 60 minutes before the intravenous infusion of 5 micrograms/kg interleukin 1 alpha. Nuclear factor kappaB activation was evaluated by electrophoretic gel mobility shift assay of whole-tissue homogenates. RESULTS Infusion of interleukin 1 alpha resulted in significant decreases in mean arterial pressure and systemic vascular resistance, both of which were prevented by treatment with dithiocarbamate. Pyrrolidine dithiocarbamate induced a significant metabolic acidosis, whereas proline dithiocarbamate did not. Nuclear factor kappaB-binding activity was increased within heart, lung, and liver tissue 4 hours after interleukin 1 alpha infusion. Treatment with dithiocarbamate resulted in decreased nuclear factor kappaB activation in lung and liver tissue with respect to that in control animals. CONCLUSIONS These results demonstrate that nuclear factor kappaB is systemically activated during whole-body inflammation and that inhibition of nuclear factor kappaB in vivo attenuates interleukin 1 alpha-induced hypotension. Nuclear factor kappaB thus represents a potential therapeutic target in the treatment of hemodynamic instability associated with the whole-body inflammatory response.