title: Verotoxin-1-induced up-regulation of adhesive molecules renders microvascular endothelial cells thrombogenic at high shear stress.
abstract: Verotoxin-1 (VT-1)-producing Escherichia coli is the causative agent of postdiarrheal hemolytic uremic syndrome (D+HUS) of children, which leads to renal and other organ microvascular thrombosis. Why thrombi form only on arterioles and capillaries is not known. This study investigated whether VT-1 directly affected endothelial antithrombogenic properties promoting platelet deposition and thrombus formation on human microvascular endothelial cell line (HMEC-1) under high shear stress. Human umbilical vein endothelial cells (HUVECs) were used for comparison as a large-vessel endothelium. HMEC-1 and HUVECs were pre-exposed for 24 hours to increasing concentrations of VT-1 (2-50 pM) and then perfused at 60 dynes/cm(2) with heparinized human blood prelabeled with mepacrine. Results showed that VT-1 significantly increased platelet adhesion and thrombus formation on HMEC-1 in comparison with unstimulated control cells. An increase in thrombus formation was also observed on HUVECs exposed to VT-1, but to a remarkably lower extent. The greater sensitivity of HMEC-1 to the toxin in comparison with HUVECs was at least in part due to a higher expression of VT-1 receptor (20-fold more) as documented by FACS analysis. The HMEC-1 line had a comparable susceptibility to the thrombogenic effect of VT-1 as primary human microvascular cells of the same dermal origin (HDMECs). The adhesive molecules involved in VT-induced thrombus formation were also studied. Blocking the binding of von Willebrand factor to platelet glycoprotein Ib by aurintricarboxylic acid (ATA) or inhibition of platelet alpha(IIb)beta(3)-integrin by chimeric 7E3 Fab resulted in a significant reduction of VT-1-induced thrombus formation, suggesting the involvement of von Willebrand factor-platelet interaction at high shear stress in this phenomenon. Functional blockade of endothelial beta(3)-integrin subunit, vitronectin receptor, P-selectin, and PECAM-1 with specific antibodies was associated with a significant decrease of the endothelial area covered by thrombi. Confocal microscopy studies revealed that VT-1 increased the expression of vitronectin receptor and P-selectin and redistributed PECAM-1 away from the cell-cell border of HMEC-1, as well as of HDMECs, thus indicating that the above endothelial adhesion molecules are directly involved and possibly determine the effect of VT-1 on enhancing platelet adhesion and thrombus formation in microvascular endothelium. These results might help to explain why thrombi in HUS localize in microvessels rather than in larger ones and provide insights on the molecular events involved in the process of microvascular thrombosis associated with D+HUS.
authors.affiliation: Mario Negri Institute for Pharmacological Research, Bergamo, Italy. morigi@marionegri.it
authors.forename: M
authors.initials: M
authors.lastname: Morigi
authors.forename: M
authors.initials: M
authors.lastname: Galbusera
authors.forename: E
authors.initials: E
authors.lastname: Binda
authors.forename: B
authors.initials: B
authors.lastname: Imberti
authors.forename: S
authors.initials: S
authors.lastname: Gastoldi
authors.forename: A
authors.initials: A
authors.lastname: Remuzzi
authors.forename: C
authors.initials: C
authors.lastname: Zoja
authors.forename: G
authors.initials: G
authors.lastname: Remuzzi
mesh_terms: D001792:Blood Platelets / Q000502:physiology; D002196:Capillaries / Q000378:metabolism*; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000502:physiology; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000503:physiopathology*; D006801:Humans; D019007:P-Selectin / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000494:pharmacology*; D013314:Stress, Mechanical; D013917:Thrombin / Q000494:pharmacology; D013927:Thrombosis / Q000209:etiology* / Q000378:metabolism / Q000503:physiopathology; D015854:Up-Regulation; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D016207:Cytokines; D019007:P-Selectin; D019030:Receptors, Vitronectin; D022622:Shiga Toxin 1; D014841:von Willebrand Factor; D013917:Thrombin