טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentJacobi Jeanna
SubjectThe impact of Primed Polymorphonuclear Leukocytes on
Endothelial Function
DepartmentDepartment of Medicine
Supervisor Ms. Batya Kristal


Abstract

Recent models of atherogenesis proposed that atherosclerosis begins as the endothelial responses to chronic minimal injury.  Previous studies from our laboratory have shown that peripheral polymorphonuclear leukocytes (PMNLs) are primed in hemodialysis patients (HD) contributing to systemic oxidative stress and inflammation. The continuous endothelial- leukocytes interaction in the bloodstream exposes the endothelial monolayers to chronic exogenous reactive oxygen species (ROS) and inflammatory mediators inducing vascular wall damage. Thereby we designed a study to evaluate the modulation of endothelial cells function exposed to primed HD PMNLs. Separated PMNLs (106) from HD patients and normal controls (NC) were co cultivated with primary human umbilical vein endothelial cells cultures (HUVEC,105) for 15 min. Injury was assessed by HUVEC morphology examined by immunohistochemical staining, by necrotic cell death estimated by neutral red uptake and  apoptosis by annexin-V. Proinflammatory changes were expressed by expression of HUVEC adhesion molecules measured by flow cytometry and by endothelial IL-8 and eNOS mRNA by RT-PCR. Endothelial procoagulable changes were reflected by intracellular tissue factor (TF) levels estimated by flow cytometry. HUVEC exposure to PMA-stimulated NC PMNLs and to non PMA-stimulated HD PMNLs caused decreased survival, increased apoptosis, increased expression of E-selectin and P-selectin and decreased expression of ICAM-1. Decreased levels of eNOS mRNA and increased expression of IL-8 mRNA were observed 6 hours after exposure.  Increased intracellular TF levels were detected 4 hours after exposure. PMA stimulation of HD PMNLs further aggravated all the above-mentioned results while addition of SOD and catalase partially prevented HUVEC death. Immunohistochemical staining exhibits morphological damage induced by primed HD PMNLs. Our results indicate that primed HD PMNLs via releasing ROS and inflammatory mediators play a critical role in triggering endothelial injury, increasing the proinflammatory and procoagulatory state of HUVEC, mechanisms underlying the development of atherosclerosis.