Calcineurin Inhibition: Lung Reperfusion Injury

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DESCRIPTION (provided by applicant): Approximately 25 percent of lung transplant recipients and 15 percent of patients treated for chronic pulmonary embolic disease will develop post- operative lung ischemia reperfusion injury (IRI). Treatment is conservative and this problem continues to account for significant morbidity and mortality. Requirements for several proinflammatory mediators have been characterized. However, antagonism of a single mediator rarely produces dramatic protection. Clinical treatment with directed antibodies is likely therefore to be expensive, impractical and marginally effective. More effective interventions would block several mediators at once, potentially by acting at the transcriptional level. Inhibition of calcineurin mediated activation of NF-kB can reduce expression of a variety of proinflammatory mediators. Since cyclosporin (CSA) and tacrolimus (TAC) are calcineurin inhibitors that are already available clinically, it would be desirable to examine their effects in experimental lung IRI. CSA and TAC reduce IRI in other tissues and can consistently reduce NF-kB activation both in vitro and in vivo. In preliminary studies, we have reliably established an animal model of lung IRI and shown that NF-kB activation follows lung IRI and that activation is associated with the appearance of a number of inflammatory cytokines and chemokines. Our hypothesis, therefore, is that calcineurin mediates NF-kB activation with lung IRI which results in transcriptional upregulation of critical pro-inflammatory mediators and the ultimate development of tissue injury. We will address this hypothesis systematically beginning with interventional studies in vivo and proceed to mechanistic analysis both in vivo and in vitro. We will pursue three related aims: Aim 1: To determine whether calcineurin inhibition is protective against the development of lung IRI. We will accomplish this using our animal model. Aim 2: To determine whether calcineurin inhibition results in transcriptional down-regulation of mediators involved in lung IRI through an NFkB-dependent mechanism. We will measure protection offered by CSA/TAC and assess associated changes in cytokine production and NFkB in nuclear protein. Aim 3: To define the cellular sources of critical mediators of lung IRI and their dependence on calcineurin driven transcription and cell-cell interactions. We will accomplish this by culturing alveolar macrophages , type II pneumocytes and pulmonary artery endothelial cells (individually and in combination) and subject them to hypoxia and reoxygenation.