Abstract 3697: Expression and Function of Ephrin/Eph Family Genes in Atherosclerosis-Related Adult Human Cells: From Aspect of Chemotaxis
Background: Recent study shows that ephrin-B1 and its cognate receptor EphB2, the regulators for cell migration in embryogenesis, are overexpressed in human carotid atherosclerotic plaque and both modulate monocyte transmigration crucial to atherogenesis.
Hypotheis: Ephrin-B1 and EphB2 can also be upregulated in abdominal aortic aneurysm (AAA), a dilated form of atherosclerosis. Also, the other members of ephrin/Eph family might be expressed in atherosclerosis-related adult human cells.
Methods: Aortic wall strips in AAA graft-replacement surgeries (n=10) and infra-renal aortas as control were obtained. Real-time RT-PCR and immunohistochemistry determined expression and localization of ephrin-B1 and EphB2 in AAA, respectively. Their chemotactic effects were examined with Boyden chamber assay. For isolated adult human monocytes and T-lymphocytes and cultured arterial endothelial cells, expression of all the 21 members of ephrin/Eph family, which have high DNA sequence similarities, was profiled by RT-PCR with some technical considerations. We customized a primer set within one exon for all the target genes to use exogenous genomic DNA as positive PCR template. From the total RNA, contaminated endogenous genomic DNA was eliminated with gDNA Wipeout Buffer (Qiagen) before cDNA synthesis.
Results: Higher expression levels of ephrin-B1 (0.410 ± 0.046 vs 1.198 ± 0.252, P = 0.027) and EphB2 (0.764 ± 0.212 vs 1.272 ± 0.137) were observed in AAA and these molecules were expressed in macrophages, T-lymphocytes, and endothelial cells within the lesions. SDF-1-induced transmigration of mononuclear cells were inhibited by extracellular domains of ephrin-B1 or EphB2, coated on the porous membranes, down to 54.7 ± 12.7% (P = 0.01) or 50.7 ± 13.1% (P = 0.01), respectively. Monocytes, T-lymphocytes and endothelial cells expressed many ephrin/Eph family genes. For example, monocytes: ephrins-A1 and A3–5; EphsA1–2, A4 – 6 and A8; ephrins-B1–2; EphsB1– 4 and B6.
Conclusions: Ephrin-B1 and EphB2 seem to affect dilated as well as stenotic forms of atherosclerosis development. Both A- and B-subclasses of ephrin/Eph cell-surface proteins might modulate inflammatory-cell chemotaxis through cell-to-cell interaction, which should be further sought.