The general focus of this laboratory has been the study of acute pulmonary insufficiency. Currently, our aims are to examine the mechanisms of injury following 2h of hindlimb ischemia, to define the mediators released and their role in the induction of hindlimb and pulmonary microvascular permeability. It is believed that reperfusion following ischemia will lead to synthesis of the chemoattractants and chemoativators leukotriene (LT)r and oxygen free radicals. These agents, directly or indirectly by stimulating polymorphonuclear leukocyte (PMN) entrapment and thromboxane (Tx) A2 synthesis are thought to moderate local endothelial cell (EC) permeability by causing disassembly of actin filaments. This alteration in the cellular cytoskeleton permits instability and enlargement of interendothelial junctions. If the ischemic stimulus to the hindlimb is sufficient to cause release of chemoattractants and chemoactivators into the circulation, pulmonary damage may be the consequence of the direct action of these agents ont he microvasculature as well as the secondary consequence of PMN entrapment with release of vasotoxic agents including TxA2 in the lungs. Hindlimb ischemia in the anesthetized rat or sheep will be produced by tourniquet occlusion for 2h. Plasma titers of the cicosanoids LTB4 and TxB2 will be measured by high pressure liquid chromatography and radioimmunoassay. In sheep with a hindlimb venous tourniquet or a left atrial balloon the degree of microvascular permeability in the hindlimb or lung will be assessed by measure of lymph flow and lymph/plasma protein concentration in lymph collected from the popliteal lymphatic or caudal mediastinal lymph node respectively. In order to test the location of eicosanoid synthesis, titer in plasma and lymph will be assayed and experiments conducted in animals depleted of circulating PMN using chemotherapy, anti-PMN antibodies or crystalloid perfusion of the hindlimb. Isolated PMN as well as the rat model will be used to assay PMN activation by flow cytometry and to quantitate pulmonary leukosequetration and edema following hindlimb ischemia. In order to document the importance of a mediator, experiments will be designed as often as possible to: (1)assay levels of the putative mediator and relate its appearance to pathophysiologic alterations (2) assess the ability of antagonists to prevent the pathophysiologic effect and (3) infuse a putative mediator such as LTB 4, oxygen free radical or Tx mimic and demonstrate the occurrence of the pathophysiologic effect. In vitro methods using pulmonary artery and pulmonary microvessel endothelial cells (EC) grown on an amnion membrane or microcarrier beads will be used to study mechanisms of PMN diapedesis and EC permeability. The emphasis will be on quantitating the ability of the eicosanoids by means of membrane cytoskletal modifications to mediate both diapedeses and macromolecular permeability. Taken as a whole these data should define a role of eicosanoids, oxygen free radicals and PMN in the local and pulmonary response to ischemia as well as demonstrate a role for actin filaments in modifying the microvascular barrier.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035141-06
Application #
3287327
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Project Start
1985-07-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
6
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
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Kyriakides, C; Austen Jr, W G; Wang, Y et al. (2001) Mast cells mediate complement activation after acid aspiration. Shock 16:21-4
Kyriakides, C; Favuzza, J; Wang, Y et al. (2001) Recombinant soluble P-selectin glycoprotein ligand 1 moderates local and remote injuries following experimental lower-torso ischaemia. Br J Surg 88:825-30
Kyriakides, C; Jasleen, J; Wang, Y et al. (2001) Neutrophils, not complement, mediate the mortality of experimental hemorrhagic pancreatitis. Pancreas 22:40-6
Kyriakides, C; Wang, Y; Austen Jr, W G et al. (2001) Moderation of skeletal muscle reperfusion injury by a sLe(x)-glycosylated complement inhibitory protein. Am J Physiol Cell Physiol 281:C224-30
Kyriakides, C; Wang, Y; Austen Jr, W G et al. (2001) Sialyl Lewis(x) hybridized complement receptor type 1 moderates acid aspiration injury. Am J Physiol Lung Cell Mol Physiol 281:L1494-9
Kyriakides, C; Austen Jr, W; Wang, Y et al. (2000) Endothelial selectin blockade attenuates lung permeability of experimental acid aspiration. Surgery 128:327-31
Kyriakides, C; Austen Jr, W G; Wang, Y et al. (2000) Neutrophil mediated remote organ injury after lower torso ischemia and reperfusion is selectin and complement dependent. J Trauma 48:32-8
Kyriakides, C; Woodcock, S A; Wang, Y et al. (2000) Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle. Am J Physiol Cell Physiol 279:C520-8
Woodcock, S A; Kyriakides, C; Wang, Y et al. (2000) Soluble P-selectin moderates complement dependent injury. Shock 14:610-5

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