Molecular and functional analysis of Shiga toxin–induced response patterns in human vascular endothelial cells

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is the major cause of hemolyticuremic syndrome (HUS) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. EHEC produces one or more Shiga toxins (Stx1 and Stx2), and it was assumed that Stx's only relevant biologic activity was cell destruction through inhibition of protein synthesis. However, recent data indicate that in vivo the cytokine milieu may determine whether endothelial cells survive or undergo apoptosis/necrosis when exposed to Stxs. In this study, we analyzed the genome-wide expression patterns of human endothelial cells stimulated with subinhibitory concentrations of Stxs in order to characterize the genomic expression program involved in the vascular pathology of HUS. We found that Stxs elicited few, but reproducible, changes in gene expression. The majority of genes reported in this study encodes for chemokines and cytokines, which might contribute to the multifaceted inflammatory response of host endothelial cells observed in patients suffering from EHEC disease. In addition, our data provide for the first time molecular insights into the epidemiologically well-established higher pathogenicity of Stx2 over Stx1. (Blood. 2003;102: 1323-1332

The CXCR4/CXCR7/SDF-1 pathway contributes to the pathogenesis of Shiga toxin–associated hemolytic uremic syndrome in humans and mice

“…It is interesting that this low concentration of Stx had such potent and reproducible effects on endothelial phenotype. Previous expression profiling studies have been performed using Stx in HUVECs and in vivo (45,60). We identified many of the same genes in our work; however, modulation of the CXCR4/CXCR7/ SDF-1 pathway was not described in either previous study.…”

“…Total cellular RNA was extracted at 0, 5,15,30,60,120,180,270, and 360 minutes after the addition of actinomycin D. Levels of specific transcripts were determined by real-time RT-PCR using the primers listed above. Data were normalized to 18S rRNA.…”

Hemolytic Uremic Syndrome

“…Recently, however, it has been shown that treatment of endothelial cells with sublethal doses of Stx, exerting minimal influence on protein synthesis, leads to increased mRNA levels and protein expression of chemokines, such as IL-8 and monocyte chemoattractant protein-1 (MCP-1) and cell adhesion molecules, a process preceded by NF-B activation (71). Analysis of genome-wide expression pattern of human endothelial cells stimulated with sublethal doses of Stx evidenced 25 and 24 genes upregulated by Stx-1 and Stx-2, respectively, mostly encoding for chemokines and cytokines, cell adhesion molecules, including P-selectin and ICAM-1, and transcription factors (EGR-1, NF-B2, and NF-BIA) (72). Chemokines and cytokines are likely involved in the chemoattraction and activation of neutrophils.…”

Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils

“…The A chain is endowed with the enzymatic activity (15)(16)(17) and is responsible for an array of damage within intoxicated cells, as well as multiple cellular and host responses, culminating in the typical prothrombotic state and renal involvement seen in HUS. Among the toxin actions on endothelial cells involved in HUS pathogenesis, damage to DNA, ribotoxic stress, endoplasmic reticulum stress, activation of the apoptotic program, stimulation of the production of proinflammatory cytokines, and upregulation of adhesion molecules have been described (17)(18)(19)(20)(21)(22)(23). Moreover, activation of complement, formation of platelet-leukocyte complexes, and production of tissue factor-bearing microparticles in blood by Stx have been demonstrated in vitro and in the blood from patients with HUS (24)(25)(26).…”