Abstract 465: CEACAM-1 is a Key Regulator of Endothelial Proliferation and Migration in vitro and in vivo via FAK Dependent Proteasomal Degradation of FOXO Transcription Factors and Metalloproteinase Secretion
As recently demonstrated CEACAM-1 is both necessary and sufficient to induce collateral growth (JCI. doi:10.1172/JCI24340). To elucidate cellular and molecular mechanisms responsible for the arteriogenic effect of this cell adhesion molecule we determined the proliferative index (PI) of arteriolar anastomoses in the hind limb of mice with endothelial overexpression of CEACAM-1 long and in CEACAM-1 deficient mice after continuous infusion of BrdU. Furthermore, we compared the impact of CEACAM-1 expression on proliferation in non-confluent and confluent layers of endothelial cells expressing CEACAM-1 long (C-L), CEACAM-1 short (C-S) and wild type (WT) (MTT and BrdU assays). Protein expressions of key regulatory molecules were investigated via Western blotting (Phospho-FAK, SKP2, FOXO, p27). CEACAM-1 interactions with different extracellular matrix components were investigated after culturing C-L, C-S and WT cells on fibronectin, laminin and without matrices. Migration was investigated via videomicroscopy and boyden chamber assays. Expression of key regulatory elements (Phospho-FAK) as well as metalloproteinases were determined via western blotting and xymography. Endothelial overexpression of CEACAM-1 lead to a 4 times higher endothelial proliferative activity in vivo (PI tie-2 CEACAM vs. WT: 17±1% vs. 6±3%; p<0.001, n=6) . In vitro we detected a significantly higher proliferation in C-L cells as compared to C-S and WT cells (Doubling times on fibronectin: C-L vs. C-S vs. WT: 7±0.4h * vs. 13±0.7h* vs. 10±0.6 *,* p<0.01). CEACAM-1 long overexpression was associated with c-src and FAK phosphorylation counteracting increased expression of cell silencing proteins (FOXO, p27) in confluent cells. Migration was also significantly higher in C-L cells and dependent upon extracellular matrix type (cells/well C-L vs. C-S vs. WT: 223±22* vs. 61±0,9 *vs. 33±5*,*p<0.01). The increased migratory capacity of C-L cells was associated with increased MMP activity and increased FAK phosphorylation. Based upon these findings we propose that CEACAM-1 is a key regulator of endothelial migration and proliferation in close interaction with the focal adhesion kinase pathway during angiogenesis and collateral growth.