Ischemia-reperfusion injury, which is characterized by the production of reactive oxygen species (ROS) and the activation of transcriptional factor NFkappaB, is often encountered in surgical conditions. There is a need to elucidate the molecular mechanisms by which tissues respond to the reperfusion to cause ROS production and NFkappaB activation. The Hypothesis underlying the proposed research plan is as follow: A sudden onset of reperfusion causes ROS production in vascular ECs, which in turn activates the IKK-NF)kappaB pathway to lead to the expression of genes encoding for inflammatory mediators. During this process, the sudden increases in perfusion rate, pressure, and oxygen content, individually or in combination, will lead to specific alterations in gene expression patterns to cause tissue damage. Using the DNA microarray technology to survey the perfusion-induced gene expression patterns will provide valuable information for the understanding of reperfusion injury. Integrins, serving as mechano-sensors, mediate these molecular events involved in reperfusion injury. Reducing the rate of application of reperfusion attenuates the response of integrins, decreases ROS production and IKK activation, and hence can alleviate the pathological consequences of reperfusion. In order to test the proposed hypotheses, three Specific Aims are proposed.
In Specific Aim 1, we will determine the effects of fluid perfusion, pressure and oxygen content on ROS production and IKK activation in endothelial cells (ECs). ROS inhibitors, scavengers, and mutants of small GTPases will be used to inhibit the Reperfusion-induced ROS-IKK pathway. The critical rate of perfusion and pressure application for the signal transduction will be determined by using a specially designed hydrostatic flow-ramping system.
In Specific Aim 2, we will elucidate the roles of integrins as sensors in the activation of ROS-IKK pathway. The blocking peptides and specific blocking antibodies for different integrins will be used to determine the type(s) of integrins that are involved in the perfusion-regulated ROS-IKK activation. In addition, signaling molecules linking integrins to the ROS- IKK will be investigated.
In Specific Aim 3, we will investigate the profiles of gene expression involved in the pathogenesis of reperfusion injury by using the DNA microarray technology. mRNA will be isolated from ECs under different reperfusion conditions and subjected to DNA microarray analysis to reveal the transcriptional program governed by reperfusion. The elucidation of these molecular mechanisms and gene expression profiles will have significant implications in the understanding of reperfusion injury and the development of novel treatment strategies.
