Abstract 12381: Klf5 Mediates a New Regulatory Pathway of Pathologic Vascular Injury Through DNA Double-Strand Break/Repair System
Background: Transcription factor Krüppel-like factor 5 (KLF5) is a key element linking external stress and cardiovascular remodeling by up-regulating platelet derived growth factor (PDGF)-A chain gene activity. However, the underlying mechanisms remain to be elucidated. Therefore, the unambiguous and comprehensive identification of interacting proteins is crucial for understanding these mechanisms. In the present study, we identified interacting factors of KLF5 by proteomic analysis and characterized their regulation in the vascular pathogenic response.
Methods: Double-stranded oligonucleotide containing the binding sequence for KLF5 in the PDGF-A promoter was synthesized and attached to metal beads, to which cell nuclear extract was applied. SDS-PAGE visualized specific bands to the sequence which were subjected to in-gel digestion and peptide mass fingerprinting by MALDI-TOF/MS spectrometry. Protein-protein interaction study was performed using co-immunoprecipitation and confocal microscopy.
Results: Factors that are known to be important in the DNA damage/repair pathway were successively identified. We therefore examined the involvement of the complex in vascular pathologies. Double-strand break as determined by immunohistochemistry using γ-H2AX antibody, a marker of activation of the double-stranded DNA damage/repair response, was observed in pathogenically stimulated vascular endothelial cells (HUVEC) and neointimal tissues in rat carotid artery balloon injury model. KLF5 was shown to mediate the response on γ-H2AX as shown by co-immunoprecipitation and confocal microscopy.
Conclusions: We show a hitherto unknown regulatory mechanism by DNA double-strand break/repair system involving KLF5 in the vascular pathogenic response. Our findings might provide a clue to understanding the initiation of pathological cell proliferation observed in atherosclerosis or restenosis after percutaneous coronary intervention. Collectively, this new pathway might also be a tempting target for therapeutic intervention aimed at modulating the activity of KLF5 upon PDGF-A chain and its associated pathologies in the cardiovascular system.
- © 2011 by American Heart Association, Inc.