Abstract 899: FHL-2 Suppresses VEGF-induced Angiogenesis via Sphingosine Kinase-1
Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 (SK1) is a bioactive sphingolipid as a potent angiogenic or arteriosclerotic mediator, and its modulator can be novel therapeutic target. Here we demonstrated that four-and-a-half LIM-only protein 2 (FHL-2) was a candidate gene through down-regulation of sphingosine kinase-1 activity. Although FHL-2 has been known to be specifically expressed in heart, we confirmed that FHL-2 was expressed in human aortic endothelial cells (HAEC) by Northern blot, Western blot, and histochemical analyzes. Interestingly, expression level of FHL-2 decreased in EC treated with tumor necrosis factor-α (TNF-α 10 ng/ml). FHL-2 can directly bind to SK-1 and confocal microscopic analyzes of human endothelial cells (HAEC) demonstrated that FHL-2 co-localized with SK1. Thin layer chromatographic analysis showed that VEGF activated SK1, whereas overexpressed FHL-2 inhibited synthesis of S1P by suppression of SK1 activity in EC. It has been reported that vascular endothelial growth factor (VEGF)-induced phosphorylation of Akt and eNOS was blocked by S1P inhibitor, but not MAPK. Similarly, phospholylation of Akt and eNOS induced by VEGF (50 ng/ml) in EC was suppressed by FHL-2 overexpression, but not MAPK, and reversed by the addition of S1P. We found that FHL-2 inhibited VEGF-induced EC growth by c-fos promoter assay (control: 1.00±0.04, control+VEGF 50 ng/ml: 2.17±0.11, FHL-2+VEGF 50 ng/ml 0.94±0.02, p<0.0001, control vs. control+VEGF 50 ng/ml, control+VEGF 50 ng/ml vs. FHL-2+VEGF 50 ng/ml, respectively) and MTS assay (control: 1.000±0.017, control+VEGF 50 ng/ml: 1.082±0.002, FHL-2+VEGF 50 ng/ml: 1.028±0.004, p<0.0001, control vs. control+VEGF 50 ng/ml, p<0.0005, control+VEGF 50 ng/ml vs. FHL-2+VEGF 50 ng/ml) in EC. Finally, we examined whether FHL-2 affect vasculogenesis in Xenopus embryo. Whole mount In situ hybridization assay indicated that angiogenesis was inhibited in Xenopus embryo injected FHL-2 mRNA compared to control mRNA. In conclusion, these dates suggested that FHL-2 regulated angiogenesis by down-regulation of SK1 activity. Therefore, FHL-2 is a novel therapeutic target of vascular diseases including ischemic disorder and arteriosclerosis.