Supporting Information

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SI Methods

The mouse corneal assay was performed as previously described [Cao R, Brakenhielm E, Li X, Pietras K, Widenfalk J, Ostman A, Eriksson U, Cao Y (2002) FASEB J 16:1575-1583]. A micropellet (0.35 ´ 0.35 mm) of sucrose aluminum sulfate (Bukh Meditec, Copenhagen, Denmark) coated with hydron polymer type NCC containing 160 ng of PDGF-AA, -AB, or -BB, 80 ng of FGF-2, 160 ng of PDGF-AA/80 ng of FGF-2, 160 ng of PDGF-AB/80 ng of FGF-2, or 160 ng of PDGF-BB/40 ng of FGF-2 was surgically implanted into a micropocket in the mouse cornea (one pellet per eye, one eye implanted per mouse). The corneal neovascularization was examined and quantified on day 5 after pellet implantation. Vascularization areas were calculated by measuring vessel lengths and clock-hours (the circumferential area of neovascularization if the eye is considered as a clock). For vascular stability analysis, the corneal neovascularization pattern/profile was examined on days 5, 12, 24, and 70 after pellet implantation. All animal studies were reviewed and approved by the Animal Care and Use Committee of the North Stockholm Animal Board.

At the end of the experiments, all animals were euthanized and the hearts were immediately collected. The treated areas of the left ventricle myocardium in the left anterior descending territory were free-dissected and fixed with 3% paraformaldehyde or were immediately frozen in liquid nitrogen. The frozen samples were kept at -70°C until microsphere perfusion histological analyses were performed according to the manufacturer's instruction. To determine vessel density, paraffin-embedded, 5-µm sections were incubated with a rabbit anti-von Willebrand factor (vWF) (1:800 dilution; ab6994 from Abcam) antibody or a mouse anti-a smooth muscle actin (αSMA) (1:1,600 dilution; ab7817 from Abcam) antibody, followed by incubation with secondary antibodies labeled with horseradish peroxidase (Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China). Positive signals were revealed by using diaminobenzidine substrate. Positive vessels were counted under a light microscope (´20) to determine vessel density (10 random fields per group). Ischemic myocardial sections were also used for detection of vWF/aSMA double positivity using immunofluorescent analysis. A mixture of a rabbit anti-vWF polyclonal antibody (1:400) and a mouse anti-aSMA monoclonal antibody (1:800) was applied to each tissue sample, followed by incubation (for 30 min at 37°C) with a TRITC-conjugated goat anti-rabbit IgG followed by a FITC-conjugated goat anti-mouse IgG. Vessels were examined under a fluorescent microscope (´20 magnification), and a maturation index (percentage of smooth^{muscle cell-positive vessels vs. total vessel numbers) was calculated.}