טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMazor Meital
SubjectDisruption of Endothelial Integrity by Primed
Polymorphonuclear Leukocytes - an Early Event
Leading to the Development of
Atherosclerosis
DepartmentDepartment of Medicine
Supervisors Ms. Batya Kristal
Assistant Professor Shifra Sela
Full Thesis textFull thesis text - English Version


Abstract

Background: Atherosclerosis and cardiovascular diseases are the primary causes of morbidity and mortality in patients with end stage renal disease on chronic hemodialysis (HD). PMNLs of HD patients are primed and induce activation/dysfunction of endothelial cells in-vitro.

Hypothesis: Primed PMNLs initiate the atherosclerotic process by: 1 ) Uncontrolled degranulation and release of granular enzymes into the blood stream, disrupting endothelial integrity and injuring the blood vessel wall. 2) Primed PMNLs promote monocyte activation still in the circulation, enhancing monocyte adhesion to the endothelium. This adhesion triggers differentiation of the adhered monocytes to macrophages, enabling the initiation of the atherosclerotic process. 3) Primed PMNLs are more sensitive to heparin induced apoptosis, a mechanism that will limit/regulate injury of adjacent tissue.

Methods: Blood was drawn from HD patients and healthy controls (HC). PMNLs were separated and the intracellular levels of the granular enzymes, heparanase, elastase and cathepsin G, were measured. We also measured the levels of these enzymes and the products of their activity, namely soluble heparan-sulfate (HS) and soluble vascular endothelial (VE)-cadherins, in plasma of all subjects. A co-cultivation system was used: human umbilical vein endothelial cells (HUVEC) were exposed to HD and HC PMNLs, followed by measuring HS and VE-cadherin on HUVEC. In order to study the PMNL effect on monocyte activation and differentiation, PMNLs were co-cultivated with THP-1 monocytes in the presence and absence of HUVEC for 72 hours, following determination of monocyte activation and differentiation.

Results: 1) Primed HD PMNLs contain lower intracellular levels of elastase and cathepsin G, while heparanase is over-expressed compared to HC PMNLs. The levels of these enzymes in plasma are higher in HD vs. HC. 2) Our co-cultivation studies revealed that primed PMNLs degraded HS from endothelial surface and increased binding of anti VE- cadherin antibody to the antigen. Soluble VE cadherin was not increased in plasma of HD patients, and soluble HS was even decreased. 3) Primed PMNLs induced monocyte activation, which in the presence of activated endothelium further differentiated to macrophages. 4) Primed PMNLs express high levels of CD11b and thus are more sensitive to the apoptotic effect of heparin.

Conclusions: Primed PMNLs constitute a source for high levels of proteolytic enzymes in HD plasma, disrupting endothelial integrity and initiating the atherosclerotic process by promoting monocyte adherence and differentiation into macrophage. We suggest that heparin and its less sulfated form, HS, can regulate and restrict activation of primed PMNLs.