Figure 12.

Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target CXCR4 or angiopoietin-2 prior to adoptive transfer. Panels show immunohistology of consecutive sections through injured mouse carotid arteries harvested on day 28 post-injury. All sections stained with DAPI (4,6 diamidino-2-phenylindole) nuclear stain (blue) and anti-CXCL-12 (red). The green staining is light emitted by the EYFP cells themselves. Yellow indicates co-localization. The annotated white line defines the junction between neointima and media. (A,B) Injured wild-type (WT) mice were adoptively transferred with CD34+ cells on day of injury. In (A), the cells came from WT mice. In (B), the cells came from CD31-TFPI-Tg mice. 1 week later, both groups of mice received a second adoptive transfer of EYFP CD34+ cells that were pre-incubated with either isotype control antibody or anti-CXCR4 as indicated. Sections were harvested 1 week later (day 14 post-injury). The annotated white line defines the junction between neointima and media. The anti-CXCR4 prevented late recruitment of adoptively transferred CD34+ cells in (A), but in (B), there is no late recruitment of adoptively transferred EYFP cells, even those pre-incubated with control antibody. (C) Adoptive transfer of EYFP CD34+ cells to injured WT mice at the time of injury. Cells first incubated with an anti-TIE-2 or isotype control, or siRNA targeting angiopoietin-2 or scrambled control. Compared to the control cells and those incubated with isotype control antibody, the anti-TIE-2 antibody significantly reduced the neointimal area but did not inhibit angiopoietin-2 expression. Compared to the scrambled siRNA, the specific siRNA targeting angiopoietin-2 significantly reduced the neointimal area in association with abolition of angiopoietin-2 expression cells. Tables besides panels describe summary of staining from all mice (n = 6), showing the proportion of the neointimal area that is positive for CXCL-12, EYFP, or both on day of examination (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods). *cf isotype for anti-TIE-2 p < 0.001. ^†cf scrambled siRNA p < 0.001. Experiments repeated at least twice.