The gamma variate function was used to model the first-pass kinetics of the contrast agent and to calculate blood flow ratios (i.e., perfusion ratios if tissue volume is equal) between transduced and contralateral intact limbs. The area under the curve represents blood volume in the region of interest. Blood flow is blood volume divided by the mean transit time (MTT) of the contrast agent. Alternatively, we found that a very good estimate of blood flow (perfusion) can be achieved with the ratio of peak signal intensities between the hind limbs (see Fig. 4). Times to arrival as well as MTT give important information on the structure of the vasculature (see Fig. 4). Ad = adenovirus; AdLacZ = adenoviral beta-galactosidase; CPS = contrast pulse sequence; VEGF = vascular endothelial growth factor.

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(A and B) Relative blood volume–magnetic resonance imaging (BV-MRI) performed with transversal plane contrast-enhanced T₂*-weighted MRI with intravenous superparamagnetic iron oxide particles (Resovist) 6 days after AdLacZ or AdVEGF gene transfer. Shown are R₂* maps that demonstrate highly increased blood volume in the AdVEGF-injected semimembranosus muscle (brackets). (C and D) Contrast-enhanced power Doppler ultrasound (CEU) can detect blood flow only in large vessels, whereas in panels E and F, CPS ultrasound imaging at 8.0 MHz shows blood flow with a very high spatial resolution and effective tissue subtraction. Arrowheads indicate the profound femoral artery, and asterisks denote free fluid between muscles owing to AdVEGF-induced vascular permeability. See Online Videos 1 and 2. Abbreviations as in Figure 1.

(A) Transversal CPS imaging at 8.0 MHz in AdVEGF-injected semimembranosus muscle 6 days after gene transfer. Note the dark area inside the muscle showing little CPS signal, owing to low basal perfusion typical for intact skeletal muscle (surrounded by a green line). (B) A histological section of the same location as in panel A, covering the whole muscle and immunostained with the CD31 endothelial marker. Normal-sized capillaries colocalize with very low CPS signal inside of the area marked by a green line, whereas enlarged capillaries outside of the area colocalize with a good signal in CPS. (C and D) Higher magnifications of the regions marked with black boxes in panel B demonstrate a clear demarcation between the areas with normal (black arrows) versus enlarged (diameter 10 to 20 µm, red arrows) capillaries. Asterisks denote enlarged veins, and arrowheads indicate the profound femoral artery in panels A to C. Scale bar = 200 µm. See Online Video 3. Abbreviations as in Figure 1.

(A) Blood volume in the muscle is increased 41- to 51-fold after AdVEGF (n = 9) gene transfer (GT) as measured by BV-MRI and CPS ultrasound, respectively. (B) Perfusion is increased 20- to 24-fold after AdVEGF treatment, depending on the method used. (C) Arrival time is shortened in AdVEGF-injected muscles, reflecting a decrease in peripheral resistance owing to vessel enlargement. In contrast, MTT is increased after AdVEGF GT. (D) Capillary mean size is 29-fold larger in AdVEGF-treated muscles than in AdLacZ (n = 10) muscles. (E) Capillary density does not change after AdVEGF GT in 6 days. (F) Of AdVEGF-treated muscles, 9.2% are covered by capillary lumens, whereas normal capillary total area in AdLacZ muscles is 0.3%. *p < 0.05, **p < 0.01, and ***p < 0.001 versus AdLacZ. Analyzed by the Kruskal-Wallis test, followed by Mann-Whitney U test. Abbreviations as in Figures 1 and 2.

(A to D) Normal power Doppler without contrast enhancement (left) and 14 MHz CPS imaging (right) 6 days after AdLacZ or AdVEGF gene transfer in mouse hind limbs. (A and B) AdVEGF induces angiogenesis and perfusion increases almost exclusively in the subcutis (brackets), probably owing to the low tropism of adenovirus in mouse skeletal muscle. The femoral artery is indicated by arrowheads. (C and D) Also in the calf, angiogenesis mostly occurs in the adipose tissue (brackets). Here, arrowheads denote the popliteal artery. Lectin capillary stainings of skeletal muscle (E and F) and subcutis (G and H) nearby. Although there are a few slightly enlarged capillaries in skeletal muscle (F, arrows), angiogenesis mostly takes place in the adipose tissue. Note red blood cells inside enlarged blood vessels (asterisks), indicating that the vessels are perfused. Scale bar = 100 µm. See also Online Video 4. Abbreviations as in Figures 1 and 2.

The AdVEGF (n = 6) increases perfusion by 2.7- and 2.5-fold in the thigh and calf muscles, respectively, 6 days after gene transfer. *p < 0.05 and **p < 0.01 versus AdLacZ (n = 6). Analyzed by analysis of variance followed by independent samples t test. Q1 = 25th percentile; Q2 = 50th percentile (median); Q3 = 75th percentile; other abbreviations as in Figure 1.