Abstract WP442: Oxidative Stress And Cyclic Strain Mediated Smad6 Regulation In Vascular Endothelial Cells Correlates With Autophagy Induction And Endothelial Dysfunction
Background: Vascular endothelial cells (ECs) are subjected to enhanced-oxidative stress and -cyclic strain in hypertension, stroke and atherosclerotic vascular disease. The role for TGF-ß and bone morphogenic proteins (BMPs) has been implied in vascular development and vessel remodeling. Although endothelial specific regulation of their intracellular signaling protein Smad6 was observed under the stimuli of laminar shear stress, little is known about the function of Smad6 in adult vascular endothelium.
Methods: Human aortic ECs were treated with H202 (10 to 100umol/L) or exposed to 7% uniaxial cyclic strain. For transient knock down, ECs were transfected with siRNA constructs against Smad6 and Smad7. Expression levels of Smad6 and Smad7 were measured by quantitative RT-PCR and western blotting analysis. NO production and BrdU labeling for proliferative cells were measured by ELISA assay. In vitro angiogenesis was evaluated in tube-formed ECs. The process of autophagy was analyzed by electron microscopy. Cells in survival or apoptosis were detected by immunofluorescence cell staining of HMGB1 or cleaved- caspase-3.
Results: Exposure of ECs to 24 hours of oxidative stress or cyclic strain showed consistent reduction in Smad6 mRNA and protein, while no change in Smad7. Notably, electron microscopic analysis showed that knockdown of endogenous Smad6 expression induced “self-eating” autophagy after 24 hours of treatment, while silencing of Smad7 did not induce autophagy. Immunofluorescence cell staining showed that EC with silencing Smad6 were positive for HMGB1 but negative for cleaved caspase-3. Importantly, ECs with silencing Smad6 represented significant reduction in NO production, prolifereative activities and in vitro angiogenesis as compared to those with silencing Smad7. Furthermore, treatment with TGF-ß1, BMP-2, or BMP-4 did not show synergistic effects on autophagy induction and endothelial function after silencing Smad6.
Conclusions: These data demonstrate a novel mechanism for functional regulation of Smad6 in vascular ECs, and indicate that dysregulation of Smad6 may have important implications in endothelial autophagy and endothelial dysfunction in the settings of hypertension, stroke and vascular inflammation.
- © 2012 by American Heart Association, Inc.