Abstract 11435: Coupling Factor 6 Promotes Endothelial Apoptosis and Inflammation by Attenuating Cxcr4 Expression Through Hif-1alpha and cSrc Pathways
Background: Vascular endothelial cells are exposed to an acidic pH, and CXC chemokine receptor type 4 (CXCR4) is a key protective molecule against acidosis. We investigated the effect of coupling factor 6 (CF6), a novel activator for a proton importer, on CXCR4 signaling pathway and its molecular mechanism.
Methods and Results: CF6 decreased CXCR4 expression in human umbilical vascular endothelial cells (HUVEC) in a time- and dose-dependent manner (65±14% mRNA and 41±10% protein reduction by CF6 at 10-7M after 24 hours, respectively). CXCR4 mRNA was decreased by hypoxia or CF6 at 10-7M to a similar degree, but no additive effect was found. Pretreatment with siRNA for hypoxia inducible factor (HIF)-1α, or PP1, a specific inhibitor of c-Src, attenuated CF6 induced decrease in CXCR4 mRNA without affecting CF6-induced intracellular acidosis. Chip assay revealed that CF6 enhanced the interaction between a repressor of HIF-1α and CXCR4 promoter at the hypoxia response element (HRE) located at position -29 to -25. CF6 enhanced protein-to-protein interaction between phospho-c-Src and histone deacetylase 3 (HDAC3) which is responsible for CXCR4 repression, but did not affect interaction between HADC3 to CXCR4 promoter at HRE. The percentage of apoptotic cells, measured by annexin-V propidium iodide, was increased from 4.3±0.7 % to 5.6±1.7 % and from 5.3±1.8 % to 7.0±1.3 % by CF6 at 10-7M in normoxia and hypoxia for 24 hours, respectively (both p<0.05). Pretreatment of the cells with siRNA for HIF-1α or administration of CXCR4 ligand, SDF-1, blocked CF6-induced increase in apoptotic cells in both normoxia and hypoxia. Masson’s trichrome staining and immunostaining for CD-16 and CD-206 showed that the increase in wall thickness and infiltration of CD16 (inflammatory macrophages) or CD206-positive cells (fibrosis-promoting macrophages) was detected in coronary small arteries and perivascular tissues of CF6-overexpressing transgenic mice showing the 25±4% lower expression of CXCR4.
Conclusion: CF6 decreases CXCR4 expression through HIF-1α and c-Src-mediated mechanisms in vascular endothelial cells. Since CXCR4 plays an important role in survival function, CF6 is implicated in the progression of arteriosclerosis by the complex mechanisms.
- © 2013 by American Heart Association, Inc.