Volume 12, Issue 2, Pages 328-336 (August 2005) Adenoviral-mediated delivery of early growth response factor-1 gene increases tissue perfusion in a murine model of hindlimb ischemia  Young-Sam Lee, Hyung-Suk Jang, Jeong-Min Kim, Jung-Sun Lee, Jae-Young Lee, Koung Li Kim, In-Soon Shin, Wonhee Suh, Jin-Ho Choi, Eun-Seok Jeon, Jonghoe Byun, Duk-Kyung Kim  Molecular Therapy  Volume 12, Issue 2, Pages 328-336 (August 2005) DOI: 10.1016/j.ymthe.2005.03.027 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Egr-1* upregulates multiple target genes important for vessel formation in skeletal myocytes. (A) Cotransfection experiments demonstrate that the functional activity of Egr-1* is not inhibited by NAB2. Human embryonic kidney (293) cells were transfected with 0.4 μg of human TF promoter/luciferase reporter plasmid (pGL3(b)-hTF0.8) and 0.2 μg of expression vectors as indicated. One day after transfection, cells were harvested and analyzed for luciferase activity. Data are presented as the mean values of relative light units (RLU) ± SEM. *P < 0.05 (one-way ANOVA); ns, not significant. (B) Adenovirus-mediated overexpression of Egr-1 and Egr-1*. Recombinant adenovirus expressing Egr-1 or Egr-1* under the control of the cytomegalovirus promoter was produced and used to infect skeletal myocytes at the indicated m.o.i. The transduced cells were harvested 24 h after infection and cellular lysates subjected to Western blotting probed with anti-Egr-1 antibody. The same blot was reprobed with anti-Sp1 antibody to normalize sample loading. (C) Adenovirus-mediated overexpression of Egr-1* induces multiple target genes related to angiogenesis. Skeletal myocytes were infected with Ad-LacZ (control), Ad-Egr-1, or Ad-Egr-1* at an m.o.i. of 250. One day after infection, 2 μg of total RNA from each sample was reverse-transcribed and the cDNA amplified by PCR using primers specific for the indicated genes (Table 2). The fold induction relative to that of the control-virus-infected cells is shown. The level of each mRNA was normalized to that of the β-actin control. (D) Ad-Egr-1* induces secretion of growth factors related to angiogenesis in skeletal myocytes. The conditioned media from the transduced cells were harvested and assayed for cytokine secretion by ELISA (R&D Systems). Results are from at least three separate experiments and each bar represents the mean ± SEM. *P < 0.05 (Student's t test). Molecular Therapy 2005 12, 328-336DOI: (10.1016/j.ymthe.2005.03.027) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 In vitro evaluation of the angiogenic potential of Egr-1*. (A) Ad-Egr-1*-transduced human skeletal myocytes enhance HUVEC proliferation in coculture. Cell proliferation was measured with a WST-1 assay 2 days after coculture. Results are from three separate experiments and each bar represents the mean ± SEM. *P < 0.05 (Student's t test). (B) Ad-Egr-1*-transduced human skeletal myocytes promote migration of HUVEC following scrape injury in a coculture assay. Migration of HUVEC into the denuded zone (middle box) was photographed 24 h after coculture. One of two independent experiments is shown (original magnification, ×40). Molecular Therapy 2005 12, 328-336DOI: (10.1016/j.ymthe.2005.03.027) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 Ex vivo evaluation of the angiogenic potential of Egr-1*. (A) Explant culture of skeletal muscle injected with Ad-LacZ or Ad-Egr-1*. A total of 1 × 108 PFU of recombinant adenovirus in 30 μl of saline was injected into the tibialis anterior muscle. Three days after injection, the muscle was retrieved and embedded in 0.2 ml of growth-factor-reduced Matrigel in a 24-well plate. Samples were then covered with 1.5 ml of M199 medium containing 2% FBS. Few sprouted cells are seen in the Ad-LacZ group, whereas extensive sprouting is observed in the Ad-Egr-1* group on day 7 (original magnification, ×12.5). (B) The areas of sprouting were measured from magnified images on day 7 and used for quantitative comparisons. The data are expressed as means ± SEM (n = 8). *P < 0.05 vs. LacZ group. Molecular Therapy 2005 12, 328-336DOI: (10.1016/j.ymthe.2005.03.027) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Ad-Egr-1* delivery enhances blood flow recovery in the ischemic hindlimb. (A) Representative laser Doppler imaging (LDI) of mouse hindlimb on day 7. Significant increase in blood flow recovery of the ischemic hindlimb (left leg) is seen in the Ad-Egr-1*-injected group. The perfusion signal is displayed in color codes ranging from dark blue (0) to white (1000). (B) Quantitative evaluation of blood flow recovery. The LDI flux ratio is the ratio of blood flow in the ischemic (left) limb to that in the normal (right) limb. Values are expressed as means ± SEM (n = 4–9). *P < 0.05 vs. LacZ or PBS group (one-way ANOVA). Molecular Therapy 2005 12, 328-336DOI: (10.1016/j.ymthe.2005.03.027) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Analysis of the injected hindlimb for gene expression and vessel density. (A) Time-course analysis of Egr-1* expression in the injected adductor muscle by RT-PCR. (−RT) No reverse transcription. 18S rRNA was used as an internal control (QuantumRNA 18S internal standards; Ambion, Austin, TX, USA). (B) Increased expression of Egr-1 target genes in vivo in the injected muscle as examined by RT-PCR on day 3. The number indicates fold increase over LacZ control. (C) Comparison of capillary density of adductor muscle harvested on day 7. The capillary density was normalized to myofiber density (the number of capillaries divided by the number of myofibers in the given area). Data are expressed as means ± SEM (n = 3–4). (D) Comparison of α-SMA-positive vessels in the adductor muscle on day 7. Immunostaining with α-SMA antibody revealed blood vessels that stained positive (brown color). Original magnification, ×200. Data are expressed as means ± SEM (n = 16). *P < 0.05 vs. LacZ group; **P < 0.01 vs. PBS group (one-way ANOVA). Molecular Therapy 2005 12, 328-336DOI: (10.1016/j.ymthe.2005.03.027) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions