Date on Master's Thesis/Doctoral Dissertation

5-2005

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Physiology and Biophysics

Committee Chair

Roberts, Andrew M.

Author's Keywords

Inducible nitric oxide synthase; P-selectin; Platelet-arteriolar wall adhesion; Arteriolar constriction; Lung reperfusion

Subject

Nitric oxide synthase; Nitric oxide--Physiological effect; Reperfusion injury

Abstract

Reperfusion of the ischemic lung causes pulmonary arteriolar vasoconstriction and reduces alveolar perfusion. Lung ischemia-reperfusion (IR) injury leads to platelet and leukocyte activation which could contribute to decreased alveolar perfusion during reperfusion by platelet-arteriolar wall interactions (rolling and adhesion) and subsequent microvascular constriction. During the oxidative stress of ischemia and reperfusion, formation of reactive nitrogen species (RNS) may promote platelet adhesion to the vascular wall with subsequent formation of microthrombi and release of vasoactive substances. Platelets play an important role in causing reperfusion injury in the systemic vasculature through accumulation and release of mediators, but information is lacking about consequences of their interactions with the arteriolar wall in the pulmonary microciculation over the time course of lung reperfusion. During IR induced oxidative stress, overproduction of nitric oxide (NO) could contribute to formation of harmful RNS by interactions with oxygen radicals. The emphasis of the present study was to investigate the relationship between inducible nitric oxide synthase (iNOS), RNS, P-selectin and platelet-arteriolar wall rolling/adhesion and microvascular constriction in the intact lung during the time course of pulmonary ischemia-reperfusion injury. We examined the hypothesis that pulmonary IR induces platelet-arteriolar wall rolling and adhesion (interactions) via a P-selectin dependent mechanism that contributes to pulmonary microvascular constriction during reperfusion. Increased P-selectin expression results from elevated iNOS activity and subsequent RNS generation. Subpleural arterioles, labeled platelets, and leukocytes were examined in anesthetized, open-chest rabbits by intravital fluorescence microscopy. Ischemia was caused by reversibly occluding the right pulmonary artery for 5 min, 1 h or 2 h (5minIR group, 1IR group and 2IR group, respectively). During reperfusion, postischemic platelet rolling and adhesion were independent from leukocyte-arteriolar wall interactions and correlated with pulmonary arteriolar constriction in proportion to the length of ischemia. After 1-h occlusion during reperfusion, platelet rolling was significantly greater than in the 5minIR or control group. Although arteriolar diameters decreased during the first 0.5 h of reperfusion, platelet adhesion was not increased and arteriolar diameters returned to the baseline by 1 h of reperfusion. However, after two hours of ischemia, platelet rolling increased and platelet adhesion was accompanied by arteriolar constriction that was correlated with the level of platelet adhesion after both 1-h and 2-h ischemia. Blockade of systemic P-selectin by Fucoidan (the selectin ligand) inhibited platelet rolling, adherence, and vasoconstriction. Pretreatment of only exogenously labeled platelets with monoclonal antibody (MoAb) to P-selectin prevented platelet rolling and adherence, but not vasoconstriction. In rabbits that were treated with an iNOS inhibitor (1400W) before occlusion (2IR+1400W group), platelet-arteriolar wall interactions and vasoconstriction were prevented. In lung tissue subjected to IR, iNOS activity and expression were markedly greater than control and were also dependent on ischemia duration. Immunochemically detected P-selectin and nitrotyrosine expression in ischemic lung tissue, and the plasma level of soluble P-selectin in 1IR and 2IR groups, were significantly higher than in non-ischemic lungs and were inhibited by pretreatment with 1400W. These results indicate that during reperfusion of the intact lung, pulmonary IR causes platelet rolling and adhesion along arteriolar walls and suggest that this process is mediated by platelet and endothelial P-selectin. The results show that platelet adhesion and arteriolar constriction during early reperfusion can result from increased iNOS activity and is highly correlated with RNS and P-selectin expres

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