Volume 5, Issue 5, Pages 517-527 (May 2002) Delivery of FGF Genes to Wound Repair Cells Enhances Arteriogenesis and Myogenesis in Skeletal Muscle  John Doukas, Kate Blease, Darren Craig, Chenglie Ma, Lois A. Chandler, Barbara A. Sosnowski, Glenn F. Pierce  Molecular Therapy  Volume 5, Issue 5, Pages 517-527 (May 2002) DOI: 10.1006/mthe.2002.0579 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Activity and retention of matrix formulations in muscle wounds. (A) AdLuc formulated in either gelatin or gelatin–collagen admixtures was delivered to skeletal muscle wounds as 5 × 1010 virus particles/wound. At day 6 after delivery, muscle tissue lysates were prepared, and both luciferase activity and protein content determined. Data are presented as pg luciferase/µg protein (mean ± SD, n = 3). (B, C) Excisional defects created in rat rectus muscles were injected with DNALuc formulated in either saline (B) or 1% collagen–1% gelatin (C) (n = 4 per group). Bromphenol blue was included in both formulations to allow for visualization of treatments post-delivery. Muscles were processed 6 hours post-delivery, as hematoxylin/eosin-stained paraffin sections. Original magnifications, ×100. Molecular Therapy 2002 5, 517-527DOI: (10.1006/mthe.2002.0579) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Neovascularization following FGF gene delivery to muscle wounds. Excisional defects created in rat rectus muscles were injected with AdFGF2 (A, B), DNAFGF2 (C, D), or DNAFGF6 (E, F). All treatments were formulated in 1% collagen–1 % gelatin; adenovirus was delivered at 5 ×1010 virus particles and plasmids at 1 mg/wound. At day 21 after treatment, muscles were harvested and stained according to Masson's trichrome procedure (A, C, E) or an immunohistochemical procedure to detect α-actin expression (B, D, F). Arrows point to representative microvessels and arrowheads to representative muscular arterioles. Original magnification for all images, ×400. Molecular Therapy 2002 5, 517-527DOI: (10.1006/mthe.2002.0579) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Tissue remodeling following PDGFB gene or FGF2 protein delivery to muscle wounds. Excisional defects created in rat rectus muscles were injected with DNAPDGFB (A, B), DNALuc (C), or FGF2 protein (D). All treatments were formulated in 1% collagen–1% gelatin; plasmids were delivered at 1 mg and FGF2 protein at 60 µg/wound. At day 14 (A), 21 (C, D), or 38 (B) post-treatment, muscles were harvested and stained according to Masson's trichrome procedure. Arrow points to a representative microvessel. Original magnifications for all images, ×400. Molecular Therapy 2002 5, 517-527DOI: (10.1006/mthe.2002.0579) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 N-CAM expression following FGF gene or protein delivery to muscle wounds. (A–C) Excisional defects created in rat rectus muscles were injected with either DNALuc (A) or DNAFGF2 (B, C) formulated in 1% collagen–1% gelatin and delivered at 1 mg/wound. At day 21 post-treatment, muscles were harvested and stained to detect N-CAM expression. Original magnification, ×100 (A, B) or ×400 (C). (D) Tissue sections from Table 2 (study no. 3) were stained and analyzed so as to quantify N-CAM expression by skeletal myotubes. Data are presented as means ± SEM (n = 8); both the DNAFGF2 and DNAFGF6 groups differ from all others by P < 0.0001. Molecular Therapy 2002 5, 517-527DOI: (10.1006/mthe.2002.0579) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Influence of wounding on matrix remodeling and transgene expression. Rat rectus muscles were directly injected with DNAFGF2 formulated in 1% collagen–1% gelatin using a 27-gauge needle so as to induce minimal tissue injury (A). Alternatively, DNAFGF2HA was formulated in saline and directly injected into uninjured muscles by the same method (B, C). Finally, excisional defects created in rectus muscles were injected with either DNAFGF2HA (D, E) or DNAPDGFB (F) formulated in 1% collagen–1% gelatin. At day 2 (B), 3 (F), 4 (D), 8 (C, E), or 21 (A) post-treatment, muscles were harvested and stained according to Masson's trichrome procedure (A), or immunohistochemically to detect FCF2HA (B–E) or PDCF-BB protein (F) expression. Original magnifications, ×400 (A–D) or ×1000 (E, F). Molecular Therapy 2002 5, 517-527DOI: (10.1006/mthe.2002.0579) Copyright © 2002 American Society for Gene Therapy Terms and Conditions