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Published on: August 13, 2016
Proteomics investigation of protein expression changes in ouabain induced apoptosis in human umbilical vein
Jie Qiu1, Hai-Qing Gao, Bao-Ying Li
1Department of geriatrics, Qilu Hospital of Shandong University, 107 Wenhua Xi Road, Jinan 250012, China.
Abstract:
Ouabain is Na(+)/K(+)-ATPase inhibitor and an endogenous regulator of blood pressure, it has dual effect on vascular endothelial cells(VEC) cell growth and VEC apoptosis is contributed to vascular dysfunction involved in vascular remolding. However, the precise mechanisms of apoptosis induced by ouabain remained unclear. The objective of this study was to identify the differently expressed proteins involved in VEC apoptosis induced by ouabain in order to explore cellular and subcellular mechanisms related to ouabain actions. Human umbilical vein endothelial cells (HUVEC) were exposed to increasing concentrations (0.1 nM to 10 microM) of ouabain at 12-48 h intervals. Cell viability tests revealed that high concentrations of ouabain inhibited cell growth. Flow cytometry and caspase-3 activity analysis confirmed that apoptosis was primarily responsible for ouabain induced cell death. Two-dimensional electrophoresis in conjunction with mass spectrometry revealed that the ouabain-induced apoptosis was accompanied by regulated expression of programmed cell death protein 6, cytochrome C1, endothelin converting enzyme, claudin-1, reticulon-4, galectin-1, ras-related protein rab-11B, calnexin, profilin-1 and heat shock protein 60 (HSP60). Further study on cytochrome c and HSP60 demonstrated that levels of mitochondria and cytosol cytochrome c and HSP60 changed in response to ouabain treatment. Data showed that mitochondria proteins such as HSP60 interferes with HSP60-Bax interactions played an important role in ouabain induced apoptosis. These data bring new sights into physiological role for ouabain in VEC apoptosis and vascular remodeling, thus provide new strategies for new anti-cardiovascular disease drug development or the identification of biomarkers for vascular dysfunction in ouabain related hypertension.
Insights
Ouabain, a blood pressure regulator, induces vascular endothelial cell apoptosis through altered protein expression, particularly involving HSP60 and mitochondria. This reveals new insights into vascular remodeling and potential cardiovascular drug targets.
Area of Science:
- Cardiovascular Biology
- Cellular Mechanisms
- Molecular Biology
Background:
- Ouabain is a Na(+)/K(+)-ATPase inhibitor and endogenous blood pressure regulator.
- It exhibits dual effects on vascular endothelial cells (VEC), influencing cell growth and apoptosis, which contribute to vascular dysfunction and remodeling.
- The precise mechanisms of ouabain-induced VEC apoptosis were previously unclear.
Purpose of the Study:
- To identify differentially expressed proteins in VEC apoptosis induced by ouabain.
- To explore the cellular and subcellular mechanisms underlying ouabain's actions on VECs.
- To elucidate the role of specific proteins in ouabain-induced apoptosis and vascular remodeling.
Main Methods:
- Human umbilical vein endothelial cells (HUVEC) were treated with varying concentrations of ouabain (0.1 nM to 10 microM).
- Cell viability, flow cytometry, and caspase-3 activity assays were performed to confirm apoptosis.
- Two-dimensional electrophoresis coupled with mass spectrometry identified differentially expressed proteins.
- Mitochondrial and cytosolic levels of cytochrome c and heat shock protein 60 (HSP60) were analyzed.
Main Results:
- High ouabain concentrations inhibited HUVEC growth and induced apoptosis.
- Proteomic analysis revealed altered expression of proteins including programmed cell death protein 6, cytochrome c, endothelin converting enzyme, claudin-1, reticulon-4, galectin-1, rab-11B, calnexin, profilin-1, and HSP60.
- Changes in mitochondrial and cytosolic levels of cytochrome c and HSP60 were observed.
- Mitochondrial HSP60 was found to interfere with HSP60-Bax interactions, playing a role in apoptosis.
Conclusions:
- Ouabain induces VEC apoptosis through specific protein expression changes, involving mitochondrial pathways and HSP60.
- These findings provide new insights into the physiological role of ouabain in VEC apoptosis and vascular remodeling.
- The study suggests potential strategies for developing anti-cardiovascular disease drugs and identifying biomarkers for ouabain-related hypertension.
