Figure 8.

Pre-incubation of enhanced yellow fluorescent protein (EYFP) CD34+ cells with reagents to target angiopoietin-2 prior to adoptive transfer. (A) Neointimal area (left axis, white bars) and neointima:media ratio (right axis, black bars) of vessels taken from wild-type (WT) animals 28 days post-injury after adoptive transfer of 1 × 10⁶ EYFP CD34+ cells that were either pre-incubated with isotype control antibody, an anti-TIE-2 antibody, control scrambled siRNA or specific siRNA targeting angiopoietin-2, and administered immediately post-injury. Data derived from examination of three random sections from six different vessels. *p < 0.001, ^†p < 0.001. (B) Cross sectional images of WT carotid artery 28 days post-injury stained with elastin van Gieson’s stain after adoptive transfer of cells pre-incubated with the same reagents as above. (C) Panels show immunohistology of 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-angiopoietin-2 (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. Compared to the cells 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 angiopooietin-2 expression by EYFP cells. Tables besides panel (C) describe summary of staining from all mice (n = 6), showing the proportion of the neointimal area that is positive for EYFP, angiopoietin-2, or both on day 28 (% ± SEM). Data derived from three random sections from each of the arteries from each mouse (see Materials and Methods). Subtracting the proportional area occupied by dual positive cells from the total area occupied by angiopoietin-2+ cells gives the proportional area occupied by non-adoptively transferred angiopoietin-2+ cells. *cf isotype for anti-TIE-2 p < 0.001. ^†cf isotype for anti-TIE-2 p = NS. ^#cf scrambled siRNA p < 0.001. Experiments repeated at least twice.