Ischemia-reperfusion injury leads to the activation and deposition of serum complement on the vascular endothelium. Inhibition of complement activation or depletion of complement proteins prior to reperfusion has been shown to reduce tissue injury. However, the mechanisms regulating complement activation and deposition at the endothelial cell surface during ischemia (hypoxia) and reperfusion (reoxygenation) have not been fully elucidated. The general aim of this proposal is to investigate the mechanisms and effects of complement activation and deposition on human endothelial cells following hypoxia and reoxygenation. Recently, the existence of a third pathway of complement activation known as the lectin complement pathway has been demonstrated. This proposal will investigate whether: 1) reoxygenation of hypoxic human endothelial cells increases cell surface deposition of mannan-binding lectin leading to activation of the lectin complement pathway; 2) deposition of the terminal complement complex (C5b-9) on human endothelial cells decreases intracellular levels of cyclic guanosine monophosphate (cGMP) and increases cell surface expression of vascular cell adhesion molecules (VCAM-1) and intercellular adhesion molecules (ICAM-1); and 3) in vivo inhibition of the lectin complement pathway attenuates myocardial reperfusion injury. Experimental techniques to be used in this proposal include ELISA, flow cytometry, protein affinity chromatography, western blotting, and monoclonal antibody production. This award will enable the Applicant to further develop his investigative skills by attaining proficiency in both cellular and molecular techniques, and by expanding his knowledge of immunology, cell biology, biochemistry, and pathology. Moreover, the career development plan associated with this proposal will enable the Applicant to achieve his long-term goal of becoming an independent Clinician-Scientist capable of fostering the development of other future Clinician-Scientists. The findings to be gained from these experiments are likely to have significant clinical implications regarding complement-mediated vascular injury in humans. In addition to furthering our understanding of the basic mechanisms regulating complement activation, the findings of this proposal may also lead to the development of a novel therapeutic strategy against complement-mediated vascular injury in man. Finally, the results from this proposal may apply to other organ systems where complement activation is thought to play a significant role in mediating vascular injury, including the myocardial (myocardial ischemia), cerebral (stroke), gastrointestinal (inflammatory bowel disease), pulmonary (adult respiratory distress syndrome) and renal (glomerular nephritis) circulations.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL003854-02
Application #
6056113
Study Section
Special Emphasis Panel (ZHL1-CSR-Y (M2))
Project Start
1998-09-01
Project End
2003-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02115
Ziegeler, Stephan; Tsusaki, Byron E; Collard, Charles D (2003) Influence of genotype on perioperative risk and outcome. Anesthesiology 99:212-9
Pintar, Tatjana; Collard, Charles D (2003) The systemic inflammatory response to cardiopulmonary bypass. Anesthesiol Clin North America 21:453-64
Collard, Charles D; Park, Kellie A; Montalto, Michael C et al. (2002) Neutrophil-derived glutamate regulates vascular endothelial barrier function. J Biol Chem 277:14801-11
Lekowski, R; Collard, C D; Reenstra, W R et al. (2001) Ulex europaeus agglutinin II (UEA-II) is a novel, potent inhibitor of complement activation. Protein Sci 10:277-84
Collard, C D; Gelman, S (2001) Pathophysiology, clinical manifestations, and prevention of ischemia-reperfusion injury. Anesthesiology 94:1133-8
Montalto, M C; Collard, C D; Buras, J A et al. (2001) A keratin peptide inhibits mannose-binding lectin. J Immunol 166:4148-53
Collard, C D; Montalto, M C; Reenstra, W R et al. (2001) Endothelial oxidative stress activates the lectin complement pathway: role of cytokeratin 1. Am J Pathol 159:1045-54
Shernan, S K; Collard, C D (2001) Role of the complement system in ischaemic heart disease: potential for pharmacological intervention. BioDrugs 15:595-607
Collard, C D; Vakeva, A; Morrissey, M A et al. (2000) Complement activation after oxidative stress: role of the lectin complement pathway. Am J Pathol 156:1549-56
Collard, C D; Agah, A; Reenstra, W et al. (1999) Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues. Arterioscler Thromb Vasc Biol 19:2623-9

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