Files in this Data Supplement:

• Methods - (PDF) (94 kb).
• Figure - (PDF) (754 kb). Proposed summary of the effects of adenoviral VEGF overexpression in normal and ischemic muscles and factors involved in capillary arterialization and growth of arteries and veins. VEGFR-2 signaling via VEGF overexpression on ECs leads to prolonged NO production via the PI3K/Akt signaling pathway causing chronic relaxation of pericytes and EC proliferation via the MAPK pathway. Increased blood flow and pressure result in elevated shear stress and wall strain in enlarging capillaries. The direct effect of VEGF on vascular permeability together with increased capillary pressure enhance plasma protein extravasation which probably also contributes to pericyte and SMC proliferation via the establishment of stimulating ECM. The capillary transformation towards an arterial phenotype is rapid and, if VEGF expression is transient, bidirectional. In ischemic muscle perfusion increases less by AdVEGF and is uneven leading to more variable vascular growth response also consisting of sprouting angiogenesis and formation of blood lacunae. Secondarily to markedly elevated microvascular blood flow by VEGF, bigger arteries and veins undergo compensatory enlargement to meet strongly elevated blood flow and pressure. If there is a need for collateral-dependent blood flow, the enlarged collateral arteries persist while other vessels shrink to normal size after withdrawal of VEGF.
• Movie I - (Windows Media Video file) (239 KB). Native Power Doppler ultrasound imaging of normal rabbit semimembranosus muscle six days after AdLacZ GT. Rest perfusion in skeletal muscle is low and only the distal branch of PFA can be observed in the base of the triangle-shaped semimembranosus muscle.
• Movie II - (Windows Media Video file) (200 KB). Native Power Doppler ultrasound imaging of rabbit semimembranosus muscle six days after the ligation of PFA and AdLacZ GT. No signal from blood flow in semimembranosus muscle can be detected without contrast agent. A vessel outside the semimembranosus muscle can be seen.
• Movie III - (Windows Media Video file) (341 KB). Native Power Doppler ultrasound imaging of normal rabbit semimembranosus muscle six days after AdVEGF GT. Perfusion is strongly enhanced within the injected muscle. The semimembranosus and gracilis (on top) muscles are also notably edemic.
• Movie IV - (Windows Media Video file) (401 KB). Native Power Doppler ultrasound imaging of rabbit semimembranosus muscle six days after the ligation of PFA and AdVEGF GT. Perfusion is clearly increased but not as much as in the normal muscle treated with AdVEGF.
• Movie V - (Windows Media Video file) (4.09 MB). A tour over the cross-section of the whole semimembranosus muscle immunostained for CD31 six days after AdLacZ GT. Note that the same muscle is viewed in Power Doppler ultrasound imaging (Video 1). Capillaries are normal-sized and only some dilated capillaries can be observed as the result of trauma in the immediate vicinity of the needle track. Also the distal branch of PFA and accompanying vein, which give power Doppler signal in Video 1, are normal sized (in the end of the video clip).
• Movie VI - (Windows Media Video file) (3.34 MB). A tour over the cross-section of the whole semimembranosus muscle immunostained for CD31 six days after the ligation of PFA and AdLacZ GT. No necrosis outside the needle track, infiltrations of inflammatory cells or angiogenesis is detected.
• Movie VII - (Windows Media Video file) (4.19 MB). A tour over the cross-section of the whole semimembranosus muscle immunostained for CD31 six days after AdVEGF GT. Note efficient capillary vessel growth throughout the muscle. Arteries and veins in between the muscle bundles, which show increased blood flow in Video 2, are clearly enlarged (at the first stop of camera). A few necrotic myofibers are seen in the needle track which is partially visible in the middle of the muscle.
• Movie VIII - (Windows Media Video file) (5.91 MB). A tour over the cross-section of the whole semimembranosus muscle immunostained for CD31 six days after the ligation of PFA and AdVEGF GT. Some areas of the muscle are covered with large blood lacunae formed from capillaries while in some regions blood vessel growth is different involving sprouting angiogenesis. Note “islands” of skeletal myocytes in the lacunae surrounded by ECs and septas composed of ECs and SMCs separating different compartments of this extraordinary formation of blood vasculature.
• Movie IX - (Windows Media Video file) (714 KB). CEU imaging of normal rabbit semimembranosus muscle six days after AdLacZ GT. See Fig 4 for identification of different muscles. Contrast agent appears into the muscle approx. 5 s after i.v. injection. Signal enhancement i.e. perfusion is low.
• Movie X - (Windows Media Video file) (744 KB). CEU imaging of rabbit semimembranosus muscle six days after the ligation of PFA and AdLacZ GT. Signal enhancement i.e. perfusion is low. The contrast agent appears in the distal part before than in the proximal region of the muscle because collaterals grow into the muscle from the distal branches of the femoral artery after the main arterial supply (PFA) to the muscle has been blocked.
• Movie XI - (Windows Media Video file) (708 KB). CEU imaging of normal rabbit semimembranosus muscle six days after AdVEGF GT. Both semimembranosus and gracilis muscles are edemic. Contrast agent appears much quicker (at approx. 2 s) than in AdLacZ muscle and perfusion is increased dramatically. Even branching of the vessels can be observed.
• Movie XII - (Windows Media Video file) (720 KB). CEU imaging of rabbit semimembranosus muscle six days after the ligation of PFA and AdVEGF GT. The muscle fills from the distal end via enlarged collaterals but perfusion is lower than after AdVEGF in normal muscle.
• Movie XIII - (Windows Media Video file) (1.75 MB). 3D reconstruction of CEU data of normal rabbit semimembranosus muscle six days after AdLacZ GT. The proximal end of semimembranosus muscle and the origin of the distal branches of PFA entering the muscle are located in the upper end of the image (when the video clip is in the beginning).
• Movie XIV - (Windows Media Video file) (1.27 MB). 3D reconstruction of CEU data of rabbit semimembranosus muscle six days after the ligation of PFA and AdLacZ GT. Blood flow in the ligated PFA is not visible. A couple of collaterals grow in the distal end of the muscle but otherwise blood flow is low.
• Movie XV - (Windows Media Video file) (2.73 MB). 3D reconstruction of CEU data of normal rabbit semimembranosus muscle six days after AdVEGF GT. The whole vascular network has strongly elevated perfusion as shown by enhanced signal intensity and bigger size of the vessels.
• Movie XVI - (Windows Media Video file) (3.15 MB). 3D reconstruction of CEU data of rabbit semimembranosus muscle six days after the ligation of PFA and AdVEGF GT. The perfusion increase and collateral growth is concentrated in the distal end of the region as the preexisting anastomoses have enlarged and supply the proximal part of the muscle with blood.