Endothelial progenitor cells accelerate endothelial regeneration in an in vitro model of Shigatoxin-2a-induced injury via soluble growth factors.

Endothelial injury with consecutive microangiopathy and endothelial dysfunction plays a central role in the pathogenesis of the postenteropathic hemolytic uremic syndrome (D + HUS). To identify new treatment strategies, we examined the regenerative potential of endothelial progenitor cells (EPCs) in an in vitro model of Shiga toxin (Stx) 2a-induced glomerular endothelial injury present in D + HUS and the mechanisms of EPC-triggered endothelial regeneration. We simulated the proinflammatory milieu present in D + HUS by priming human renal glomerular endothelial cells (HRGECs) with tumor necrosis factor-α before stimulation with Stx2a. This measure led to a time- and concentration-dependent decrease of HRGEC viability of human renal glomerular endothelial cells as detected by a colorimetric assay. Coincubation with EPCs (104-105 cells/ml) under dynamic flow conditions led to a significant improvement of cell viability in comparison to untreated monolayers (0.45 ± 0.06 vs. 0.16 ± 0.04, P = 0.003). A comparable regenerative effect of EPCs was observed in a coculture model using cell culture inserts (0.41 ± 0.05 vs. 0.16 ± 0.04, P = 0.003) associated with increased concentrations of vascular endothelial growth factor, insulin-like growth factor I, fibroblast growth factor-2, and hepatocyte growth factor in the supernatant. Treatment of Stx2a-injured monolayers with a combination of these growth factors imitated this effect. EPCs did not show distinct sings of migration and angiogenic tube formation in functional assays. These data demonstrate that EPCs significantly improve endothelial viability after Stx2a-induced injury in vitro and that this effect is associated with the release of growth factors by EPCs.

Shiga Toxin 2a-Induced Endothelial Injury in Hemolytic Uremic Syndrome: A Metabolomic Analysis.

BACKGROUND: Endothelial dysfunction plays a pivotal role in the pathogenesis of postenteropathic hemolytic uremic syndrome (HUS), most commonly caused by Shiga toxin (Stx)-producing strains of Escherichia coli. METHODS: To identify new treatment targets, we performed a metabolomic high-throughput screening to analyze the effect of Stx2a, the major Stx type associated with HUS, on human renal glomerular endothelial cells (HRGEC) and umbilical vein endothelial cells (HUVEC). Cells were treated either with sensitizing tumor necrosis factor α (TNF-α) or Stx2a, a sequence of both or remained untreated. RESULTS: We identified 341 metabolites by combined liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry. Both cell lines exhibited distinct metabolic reaction profiles but shared elevated levels of free fatty acids. Stx2a predominantly altered the nicotinamide adenine dinucleotide (NAD) cofactor pathway and the inflammation-modulating eicosanoid pathway, which are associated with lipid metabolism. In HRGEC, Stx2a strongly diminished NAD derivatives, leading to depletion of the energy substrate acetyl coenzyme A and the antioxidant glutathione. HUVEC responded to TNF-α and Stx2a by increasing production of the counteracting eicosanoids prostaglandin I2, E1, E2, and A2, while in HRGEC only more prostaglandin I2 was detected. CONCLUSIONS: We conclude that disruption of energy metabolism and depletion of glutathione contributes to Stx-induced injury of the renal endothelium and that the inflammatory response to Stx is highly cell-type specific.