Real-time reverse transcriptase–polymerase chain reaction data showing exogenous vascular endothelial growth factor-165 (VEGF₁₆₅)/green fluorescent protein (GFP) messenger ribonucleic acid (mRNA) transcript at days 17, 21, and 28 and week 8 after ligation (intramuscular [IM] gene therapy in solid bars, ultrasound-mediated [UM] therapy in open bars). Relative VEGF₁₆₅/GFP mRNA expression was significantly lower for UM delivery compared with IM delivery at all time points after delivery. *p < 0.05 compared with IM gene therapy.

(A) Normalized microvascular blood volume (MBV) in the ischemic muscle for the 3 treatment groups at baseline (pre-treatment) and days 17, 21, and 28 and week 8 after ligation. *p < 0.005 compared with corresponding data in control untreated animals, p < 0.05 compared with corresponding data in IM-treated animals, p < 0.001 compared with corresponding baseline (pre-delivery) data. (B) Normalized microvascular blood flow (MBF) in the ischemic muscle for the 3 treatment groups at baseline (pre-treatment) and at days 17, 21, and 28 and week 8 after ligation. *p < 0.01 compared with corresponding data in control untreated animals; p < 0.005 compared with corresponding data in IM-treated animals; p < 0.05 compared with corresponding baseline (pre-delivery) data. Abbreviations as in Figure 1.

(A to F) Representative stacked images of microvessels in ischemic hind-limb muscle after no treatment and IM and UM delivery of VEGF₁₆₅/GFP plasmid deoxyribonucleic acid, using fluorescent microangiography (FMA). Scale bar = 50 µm. (G) Quantitative microvessel density by FMA of ischemic hind-limb muscle at day 28 and week 8 following ligation, in all groups. At day 28 after ligation, FMA revealed a significant increase in the density of microvessels in the ischemic leg of both IM- and UM-treated animals, as compared with control untreated animals, with a trend for greater vessel density in UM-treated ischemic muscle. *p < 0.05 compared with corresponding data from control ischemic muscle. Abbreviations as in Figure 1.

Focality of transgene expression after IM delivery. The IM injection sites were marked with fluorescent microspheres (A, single white arrow) and GFP expression assessed in adjacent fields at day 3 following injection. Strong GFP signal is seen in close proximity to injection sites (A, double white arrows), declining with increasing distance from the needle tract. Remote hind-limb muscle is shown in B where there is minimal GFP expression. C shows the number of high power (20x) fields (out of 25) positive for GFP signal in IM- and UM-treated ischemic skeletal muscle. *p < 0.05 compared with corresponding data from IM-treated muscle. IV = intravenous; U/S = ultrasound; other abbreviations as in Figure 1.

Examples of immunofluorescent staining of IM-treated (A to C) and UM-treated (D to F) ischemic muscle, at day 17 after ligation. Red = CD31 staining; blue = nuclear staining (TO-PRO-3); yellow = colocalization of GFP and red CD31 staining. Within IM-treated muscle, there were areas without discernable GFP signal (A, yellow arrows), interspersed with regions of strong GFP signal localized predominantly within myocytes (B, white arrows) and surrounding capillaries, with little GFP signal within arterioles (C, red arrows). In comparison, within UM-treated muscle, a more diffuse GFP signal was seen, localized to the vascular endothelium of both capillaries (D, yellow arrows), their surrounding myocytes (E, white arrows), and within small- to medium-sized arterioles (F, red arrows). Scale bar = 50 µm. Abbreviations as in Figure 1.