Figure 6. Uptake of CXCL12 by mouse heart valve. The aortic heart valves of wild type mice internalize CXCL12 tagged with a fluorescent protein (CXCL12-mCherry, upper right, red dots marked with arrowheads). Uptake of CXCL12-mCherry is partially competed with unlabeled 1.5 mM CXCL12 (lower left), and is partially resistant to administration of 10 mM AMD3100 (lower right). Pictures were taken in the indicated area (white frame upper left). doi:10.1371/journal.pone.0009175.g006 CXCR7 Scavenges CXCL12 CXCR7 expressed on these cells, early passage HUVECs were incubated with [125I]-CXCL12 for 3 h at 37uC. Figure 7A shows that HUVECs proteolyzed nearly 30% of the added chemokine. Degradation was marginally inhibited by AMD3100 (10 mM), but efficiently competed by 1 mM CXCL11, suggesting proteolysis is largely attributed to CXCR7-dependent scavenging. CXCR7 is expressed on human vascular endothelium and is further upregulated by inflammatory cytokines such as TNFa or IL-1b [7,40]. To reveal a potential scavenging activity of CXCR7 on primary human tissue we incubated fresh human umbilical cord slices with CXCL12 tagged at the C-terminus with fluorescent protein Venus. Figure 7B (upper left) shows the expression of CXCR7 at the lining endothelium of the umbilical veins. Incubation of the slices with 200 nM CXCL12-Venus causes the appearance of fluorescent endosomes, suggesting the uptake of the chemokines by surface receptors (upper right). Pretreatment of the slices with 10 mM AMD3100 for 10 min had no effect on the uptake. By contrast, addition of 1.5 mM CXCL11 fully competed the uptake of CXCL12. The data are in accordance with the observations made with HUVECs and substantiate the conclusion that CXCR7 acts as scavenger on human endothelial cells. Figure 7C summarizes data obtained with umbilical cords from different donors. Although a notable donor to donor variation is visible, the statistical analysis reveals CXCL11-sentive uptake of CXCL12, which is marginally affected by AMD3100, supporting the view that CXR7 acts as a scavenger in endothelial cells. Discussion The tight regulation of the surface expression of CXCR7 in the presence of CXCL12 suggests that the receptor functions a scavenger. Biochemical analysis in MDCK cells and HUVECs Figure 7. Scavenging activity of CXCR7 in primary human endothelium. (A) Human umbilical vein endothelial cells (HUVECs) were exposed for 3 h at 37uC to [125I]-CXCL12 in the presence of 1 mM CXCL11 or 10 mM of the CXCR4 inhibitor AMD3100. TCA resistant counts represent degraded chemokine and were plotted as percent of total added counts (cont). (B) Vein endothelial cells in fresh human umbilical cord slices express CXCR7 (purple upper left, blue nuclei DAPI), vein endothelial cells (CD31 marker red) internalize CXCL12 tagged with a fluorescent protein (CXCL12-venus, upper right, green dots marked with arrowheads), uptake of CXCL12-venus is not affected by 10 mM AMD3100 (lower left), but is competed with 1.5 mM CXCL11 (lower right). (C) Several images (5126512 pixels) from 3 independent experiments as shown in (B) were analyzed using Metamorph software. Total pixel areas showing fluorescence of the CXCL12-venus per image are shown. P,0.05 between cont. and CXCL11, not significant between cont. and AMD3100. doi:10.1371/journal.pone.0009175.g007 PLoS ONE | www.plosone.org 9 February 2010 | Volume 5 | Issue 2 | e9175