FIGURE 11.

Targeting of angiogenic ECs with CD44 or EphA2 siRNA blocks in vivo LMW-HA-mediated angiogenesis. Panel A, Matrigel plugs from mice with intravenous administration of anginex-conjugated liposomes containing either pCMV6 empty vector or pCMV6-A-GFP (Origene) were frozen in OCT embedding medium and cut in 12-μm-thick sections. Frozen sections were fixed, permeabilized, blocked, and incubated overnight with turboGFP antibody (OriGene). Alexa 488-conjugated secondary antibodies (Molecular Probes) were used to visualize GFP staining. Nuclear staining was performed with DAPI. The results of panel A indicate that our anginex-conjugated liposomes can successfully target cells in the Matrigel plug. Panel B, Matrigel plugs from mice with intravenous administration of anginex-conjugated liposomes containing either no siRNA (Control), control siRNA, EphA2 siRNA, or CD44 siRNA were depolymerized by BD Cell Recovery Solution (BD Biosciences), and each plug was dissolved in 300 μl of recovery solution as described in the manufacturer's protocol, run on SDS-PAGE, and immunoblotted (IB) for EphA2, CD44, and laminin. The results of panel B indicate that we can specifically silence the expression of proteins in Matrigel plugs. Panel C, Matrigel plugs ± LMW-HA (5 μg/ml) were implanted subcutaneously in mice that were subsequently injected (day 7) with anginex-labeled liposomes containing control, CD44, or EphA2 siRNA. The plugs were then harvested 14 days after the initial implantation, and images were obtained. Panel D, plugs from panel C were analyzed for angiogenesis histologically using Trichrome staining and cell counting (see “Experimental Procedures”). These experiments were repeated three times with five animals per treatment group. Results are expressed as the number of cells/plug. An asterisk indicates a statistically significant difference (p < 0.05) from Control.