Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome

Abstract

Summary. Background: The pathogenesis of Shiga toxin (Stx)‐mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. Materials and methods: We measured cell surface TF activity in response to tumor necrosis factor‐α (TNF‐α) (20 ng mL−1, 2–144 h), Stx‐1 (10−11 mol L−1, 4–144 h), or their combination (TNF‐α 22 h and Stx‐1 for the last 0.5–4 h of TNF‐α incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). Results and conclusions: We observed that while TNF‐α caused an increase in cell surface TF activity on both cell types, the combination of TNF‐α and Stx‐1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 ± 0.38‐fold (n = 38, P < 0.001), consistent with our parallel observation that Stx‐1 binds to HGECs but not to HUVECs. Anti‐TF antibody abolished functional TF while anti‐tissue factor pathway inhibitor antibody enhanced TF activity. Stx‐1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real‐time polymerase chain reaction demonstrated that exposure to TNF‐α markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF‐α and Stx‐1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine‐activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

Shiga Toxin Subtypes Display Dramatic Differences in Potency

“…The role of apoptosis in Stx-mediated disease is also unclear. Several studies utilizing mouse models have observed apoptosis in renal cells following treatment with Stx (23,42,47), while other studies have observed necrosis (10,29,37,46,58,60).…”

Role of the renin angiotensin system in TNF-α and Shiga-toxin-induced tissue factor expression

“…We have also observed that cytokine-activated human umbilical vein (macrovascular) endothelial cells (HUVECs) fail to demonstrate further enhancement of the cell-surface TF activity after exposure to Stx, which potentially may explain localization of thrombosis to the microvasculature and account for the specificity of organ involvement in HUS [13]. However, the potential pathogenic or modulatory role of locally produced Ang II in the prothrombotic state that constitutes HUS is largely unknown and has not been addressed in previous in vitro studies.…”

“…As we observed no differences between the effect of Stx-1 and Stx-2 in upregulating TF activity in HGECs, we subsequently focused on the effects of Stx-1, which has been used more often in cell culture experiments by other investigators. The concentration of Stx-1 as 10 −11 M was selected based both on the observations of Yagi et al [15], who detected Stx concentration of this order of magnitude in the plasma of patients with HUS, and on our observations that 10 −11 M Stx-1 enhanced TF on TNF-α stimulated HGECs [13].…”

“…We have recently demonstrated a role for Stx as a mediator of procoagulant activity by showing that Stx-1 can enhance the expression of functional TF on cytokine-activated human glomerular (microvascular) endothelial cells (HGECs) [13]. We have also observed that cytokine-activated human umbilical vein (macrovascular) endothelial cells (HUVECs) fail to demonstrate further enhancement of the cell-surface TF activity after exposure to Stx, which potentially may explain localization of thrombosis to the microvasculature and account for the specificity of organ involvement in HUS [13].…”

Multifaceted Hemolytic Uremic Syndrome in Pediatrics

“…Stx binds to specific receptors distributed throughout endothelial cells of the gastrointestinal tract, pancreas, brain, and kidney and is responsible for a multitude of cytotoxic, proinflammatory and prothrombotic effects [10]. Systemic complications resulting from Stx include cell death, platelet activation and deposition, cytokine release and increased tissue factor expression on the cells of the microvasculature [11]. Microthrombi formation in the microvasculature following Stx-induced platelet activation results in the abnormal shear stress and disturbances in blood flow that eventually leads to the production of the fragmented red blood cells or schistocytes representative of a microangiopathic anemia.…”