Andreas Oberbach1, Jochen Neuhaus2, Nico Jehmlich3, Nadine Schlichting4, Marco Heinrich5, Yvonne Kullnick5, Friedrich-Wilhelm Mohr5, Joachim Kugler6, Sven Baumann7, Uwe Völker8, Volker Adams9. 1. Department of Cardiac Surgery, Heart Center Leipzig, University of Leipzig, Leipzig, Germany; Department of Health Sciences/Public Health, University of Dresden, Dresden, Germany. 2. Department of Urology, University of Leipzig, Leipzig, Germany. 3. Ernst-Moritz-Arndt-University Greifswald, Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, Germany; Helmholtz Centre for Environmental Research, Department of Proteomics, Leipzig, Germany. 4. Department of Pediatric Surgery, University of Leipzig, Leipzig, Germany. 5. Department of Cardiac Surgery, Heart Center Leipzig, University of Leipzig, Leipzig, Germany. 6. Department of Health Sciences/Public Health, University of Dresden, Dresden, Germany. 7. Helmholtz Centre for Environmental Research, Department of Metabolomics, Leipzig, Germany. 8. Ernst-Moritz-Arndt-University Greifswald, Interfaculty Institute of Genetics and Functional Genomics, Department of Functional Genomics, Germany. 9. Department of Cardiology, University of Leipzig, Heart Center Leipzig, Leipzig, Germany. Electronic address: adav@medizin.uni-leipzig.de.
Abstract
BACKGROUND: Uric acid (UA) has been identified as one major risk factor for cardiovascular diseases. Lowering of serum UA levels improves endothelial function. The present study investigates for the first time concentration-dependent effects of UA on human aortic endothelial cells (HAEC) and the cellular pathways involved in global proteomic analysis. METHODS: The concentration dependent effects of UA on HAEC were investigated by nanoLC-MS/MS and ingenuity pathway analysis to reveal putative cellular pathways. For verification of the identified pathways the abundance or activity of key proteins was measured using ELISA or Western blotting. NO production was quantified by confocal laser microscopy. RESULTS: We identified ubiquitin-proteasome system (UPS) and eIF4 signaling as the major pathways regulated by UA. K-means clustering analysis revealed 11 additional pathways, of which NO, superoxide signaling and hypoxia were further analyzed. A complex regulatory network was detected demonstrating that 500μmol/L UA, which is well above the concentration regarded as pathological in clinical settings, led to diminishing of NO bioavailability. In addition a UA-dependent downregulation of eIF4, an upregulation of UPS and an increase in HIF-1α were detected. CONCLUSIONS: Here we show for the first time, that increasing UA levels activate different sets of proteins representing specific cellular pathways important for endothelial function. This indicates that UA may alter far more pathways in HAEC than previously assumed. This regulation occurs in a complex manner depending on UA concentration. Further studies in knockout and overexpression models of the identified proteins are necessary to prove the correlation with endothelial dysfunction.
BACKGROUND:Uric acid (UA) has been identified as one major risk factor for cardiovascular diseases. Lowering of serum UA levels improves endothelial function. The present study investigates for the first time concentration-dependent effects of UA on human aortic endothelial cells (HAEC) and the cellular pathways involved in global proteomic analysis. METHODS: The concentration dependent effects of UA on HAEC were investigated by nanoLC-MS/MS and ingenuity pathway analysis to reveal putative cellular pathways. For verification of the identified pathways the abundance or activity of key proteins was measured using ELISA or Western blotting. NO production was quantified by confocal laser microscopy. RESULTS: We identified ubiquitin-proteasome system (UPS) and eIF4 signaling as the major pathways regulated by UA. K-means clustering analysis revealed 11 additional pathways, of which NO, superoxide signaling and hypoxia were further analyzed. A complex regulatory network was detected demonstrating that 500μmol/L UA, which is well above the concentration regarded as pathological in clinical settings, led to diminishing of NO bioavailability. In addition a UA-dependent downregulation of eIF4, an upregulation of UPS and an increase in HIF-1α were detected. CONCLUSIONS: Here we show for the first time, that increasing UA levels activate different sets of proteins representing specific cellular pathways important for endothelial function. This indicates that UA may alter far more pathways in HAEC than previously assumed. This regulation occurs in a complex manner depending on UA concentration. Further studies in knockout and overexpression models of the identified proteins are necessary to prove the correlation with endothelial dysfunction.