Elevated serum bone morphogenetic protein 4 in patients with chronic kidney disease and coronary artery disease.

Chronic kidney disease (CKD) is associated with increased coronary artery disease (CAD) and coronary artery calcification. We hypothesized that the osteogenic factor, bone morphogenetic protein-4 (sBMP-4), is elevated in subjects with both CKD and CAD. Serum was collected from 79 subjects undergoing diagnostic angiography and stratified according to CAD and CKD status. Subjects with both CAD and CKD had significantly elevated sBMP-4 compared to those with only one or no disease. sBMP-4 continued to be associated with the presence of both diseases after adjustment for other risk factors. To determine if sBMP-4 is associated with coronary artery calcification, we compared coronary artery calcium scores (CAC) to sBMP-4 in 22 subjects. A positive correlation between CAC and sBMP-4 was seen. In conclusion, sBMP-4 is elevated in patients with both CAD and CKD and positively correlates with CAC, suggesting a role for sBMP-4 in the increased CAD seen in CKD patients.

Use of a constitutively active hypoxia-inducible factor-1alpha transgene as a therapeutic strategy in no-option critical limb ischemia patients: phase I dose-escalation experience.

BACKGROUND: Critical limb ischemia, a manifestation of severe peripheral atherosclerosis and compromised lower-extremity blood flow, results in a high rate of limb loss. We hypothesized that adenoviral delivery of a constitutively active form of the transcription factor hypoxia-inducible factor-1alpha (ie, Ad2/HIF-1alpha/VP16 or HIF-1alpha) into the lower extremity of patients with critical limb ischemia would be safe and might result in a durable clinical response. METHODS AND RESULTS: This phase I dose-escalation program included 2 studies: a randomized, double-blind, placebo-controlled study and an open-label extension study. In total, 34 no-option patients with critical limb ischemia received HIF-1alpha at doses of 1x10(8) to 2x10(11) viral particles. No serious adverse events were attributable to study treatment. Five deaths occurred: 3 in HIF-1alpha and 2 in placebo patients. In the first (randomized) study, 7 of 21 HIF-1alpha patients met treatment failure criteria and had major amputations. Three of the 7 placebo patients rolled over to receive HIF-1alpha in the extension study. No amputations occurred in the 2 highest-dose groups of Ad2/HIF-1alpha/VP16 (1x10(11) and 2x10(11) viral particles). The most common adverse events included peripheral edema, disease progression, and peripheral ischemia. At 1 year, limb status observations in HIF-1alpha patients included complete rest pain resolution in 14 of 32 patients and complete ulcer healing in 5 of 18 patients. CONCLUSIONS: HIF-1alpha therapy in patients with critical limb ischemia was well tolerated, supporting further, larger, randomized efficacy trials.

Ultrasound velocity criteria for carotid in-stent restenosis.

OBJECTIVE: To examine duplex ultrasound (US) criteria for carotid in-stent restenosis (ISR). BACKGROUND: Carotid artery stent (CAS) placement is an alternative to surgery for the treatment of carotid stenosis in high surgical risk patients. US is the primary method used to follow carotid stent patency. This study investigates US velocity measurements in carotid ISR. METHODS: Two hundred sixty consecutive patients with CAS placement from June 2000 to June 2004 were followed with serial US. ISR was determined by using the standard US velocity criteria for nonstented carotid artery using peak systolic velocity (PSV), end-diastolic velocity (EDV), and internal carotid artery to common carotid velocity ratio (ICA/CCA ratio). Patients suspected of having carotid ISR > or =50% by US, underwent invasive angiography with stenosis graded by NASCET criteria. Results were compared to patients with nonstented carotid artery stenosis using Two-tailed Student's t-test. RESULTS: PSV and ICA/CCA ratio increased to a greater degree in ISR. In 50-69% stenotic arteries, the mean ICA/CCA ratio was 2.76 +/- 0.7 in the ISR group compared to 2.04 +/- 0.3 in the nonstented carotid group (P < 0.05). In > or =70% stenotic arteries, there were increases in PSV (520 +/- 93 vs. 362 +/- 60, P < 0.05) and ICA/CCA ratio (7.58 +/- 2 vs. 4.51 +/- 1.3, P < 0.05) in ISR versus nonstented carotid arteries, respectively. CONCLUSION: PSV and ICA/CCA ratio in ISR increased to a greater extent for angiographic stenosis > or =50%. PSV 240 cm/sec and ICA/CCA ratio 2.45 are optimal thresholds for > or =50% ISR, and PSV 450 cm/sec and ICA/CCA ratio 4.3 are optimal thresholds for > or =70% ISR.

Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy.

BACKGROUND: The aim of the present study was to develop and characterize a novel in vivo cancer gene therapy model in which intra-arterial adenoviral gene delivery can be characterized. In this model, the rat cremaster muscle serves as the site for tumor growth and provides convenient and isolated access to the tumor parenchyma with discrete control of arterial and venous access for delivery of agents. RESULTS: Utilizing adenovirus encoding the green fluorescent protein we demonstrated broad tumor transfection. We also observed a dose dependent increment in luciferase activity at the tumor site using an adenovirus encoding the luciferase reporter gene. Finally, we tested the intra-arterial adenovirus dwelling time required to achieve optimal tumor transfection and observed a minimum time of 30 minutes. CONCLUSION: We conclude that adenovirus mediated tumor transfection grown in the cremaster muscle of athymic nude rats via an intra-arterial route could be achieved. This model allows definition of the variables that affect intra-arterial tumor transfection. This particular study suggests that allowing a defined intra-tumor dwelling time by controlling the blood flow of the affected organ during vector infusion can optimize intra-arterial adenoviral delivery.

Surveillance imaging for carotid in-stent restenosis.

Carotid artery stent placement is the procedure of choice in suitable candidates who require carotid revascularization and are at increased risk for surgical therapy. To ensure late patency of the stent, continued surveillance is required. We present three cases to illustrate the strengths and weaknesses of noninvasive imaging techniques for surveillance of carotid stents, ultimately validated with invasive contrast angiography.

Cellular, but not direct, adenoviral delivery of vascular endothelial growth factor results in improved left ventricular function and neovascularization in dilated ischemic cardiomyopathy.

OBJECTIVES: We sought to compare the effects on angiogenesis and left ventricular (LV) function of adenoviral vascular endothelial growth factor-165 (AdVEGF-165) gene delivery by direct injection of AdVEGF-165 to the transplantation of skeletal myoblasts (SKMB) transfected with AdVEGF-165 in a rat model of ischemic cardiomyopathy. BACKGROUND: Angiogenesis offers the potential for treating ischemic cardiomyopathy. However, the optimal method of delivering angiogenic factors for neovascularization remains undetermined. With the increased clinical interest in cell therapy for the treatment of LV dysfunction, SKMB transplantation may serve as a means of gene transfer. METHODS: Two months after left anterior descending coronary artery ligation, rats received either injection of an adenoviral construct encoding VEGF-165, or 1 million SKMB transfected with AdLuciferase (AdLuc) or AdVEGF-165. Cardiac function was assessed echocardiographically, and neovascularization was assessed histologically four weeks after therapy. RESULTS: Neovascularization was significantly increased by both AdVEGF delivery strategies (100 +/- 7% and 185 +/- 33% increase in vascular density compared with SKMB alone, respectively). However, cell-based delivery, but not direct injection of AdVEGF-165, resulted in increased cardiac function (73.5 +/- 12.6% and 1.5 +/- 8.8% increase in shortening fraction compared with saline control; AdLuc-transfected SKMB: 29.4 +/- 15.0%). The improved function was not due to increased engraftment of VEGF expressing SKMB. Rather, improved function correlated with less apoptosis in the border zone in those animals that received AdVEGF-165 expressing SKMB. CONCLUSION: Our data demonstrate that cell-based delivery of VEGF leads to an improved treatment effect over direct adenoviral injection, and suggest that already developed adenoviral vectors that encode secreted factors could potentially offer greater efficacy in combination with SKMB transplantation.

Protein kinase C mediates induced secretion of vascular endothelial growth factor by human glioma cells.

To understand how vascular endothelial growth factor (VEGF) production is activated in malignant glioma cells, we employed protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors to evaluate the extent to which these protein kinases were involved in signal transduction leading to VEGF production. PTK inhibitors blocked glioma proliferation and epidermal growth factor (EGF)-induced VEGF secretion, while H-7, a PKC inhibitor, inhibited both EGF-induced and baseline VEGF secretion. Phorbol 12-myristate 13-acetate (PMA), a non-specific activator of PKC, induced VEGF secretion by glioma cells, which was enhanced by calcium ionophore A23187, but completely blocked after prolonged treatment of cells with 1 microM PMA, by presumably depleting PKC. All inhibitors (genistein, AG18, AG213, H-7, prolonged PMA treatment) which inhibited EGF-induced VEGF secretion in glioma cells also inhibited cell proliferation at similar concentrations. However, PKC inhibition only blocked 50% of the VEGF secretion induced by growth factors (EGF, platelet-derived growth factor-BB, or basic fibroblast growth factor). This reserve capacity could be ascribed to a PKC-independent effect, or to PKC isoenzymes not down-regulated by PMA. These findings extend our previous assertion that VEGF secretion is tightly coupled with proliferation by suggesting that activation of convergent growth factor signaling pathways will lead to increased glioma VEGF secretion. Understanding of signal transduction of growth factor-induced VEGF secretion should provide a rational basis for the development of novel strategies for therapy.

Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy.

BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, p<0.02) resulting in greater left-ventricular mass (1.24 [0.29] vs 1.57 [0.27] g) and better cardiac function (shortening fraction 9.2 [4.9] vs 17.2 [4.2]%, n=8 per group, p<0.05). INTERPRETATION: These findings show that SDF-1 is sufficient to induce therapeutic stem-cell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication.

BACKGROUND: "Therapeutic angiogenesis" seeks to improve perfusion by the growth of new blood vessels. The Regional Angiogenesis with Vascular Endothelial growth factor (RAVE) trial is the first major randomized study of adenoviral vascular endothelial growth factor (VEGF) gene transfer for the treatment of peripheral artery disease (PAD). METHODS AND RESULTS: This phase 2, double-blind, placebo-controlled study was designed to test the efficacy and safety of intramuscular delivery of AdVEGF121, a replication-deficient adenovirus encoding the 121-amino-acid isoform of vascular endothelial growth factor, to the lower extremities of subjects with unilateral PAD. In all, 105 subjects with unilateral exercise-limiting intermittent claudication during 2 qualifying treadmill tests, with peak walking time (PWT) between 1 to 10 minutes, were stratified on the basis of diabetic status and randomized to low-dose (4x10(9) PU) AdVEGF121, high-dose (4x10(10) PU) AdVEGF121, or placebo, administered as 20 intramuscular injections to the index leg in a single session. The primary efficacy end point, change in PWT (DeltaPWT) at 12 weeks, did not differ between the placebo (1.8+/-3.2 minutes), low-dose (1.6+/-1.9 minutes), and high-dose (1.5+/-3.1 minutes) groups. Secondary measures, including DeltaPWT, ankle-brachial index, claudication onset time, and quality-of-life measures (SF-36 and Walking Impairment Questionnaire), were also similar among groups at 12 and 26 weeks. AdVEGF121 administration was associated with increased peripheral edema. CONCLUSIONS: A single unilateral intramuscular administration of AdVEGF121 was not associated with improved exercise performance or quality of life in this study. This study does not support local delivery of single-dose VEGF121 as a treatment strategy in patients with unilateral PAD.

Enhancement of muscle flap hemodynamics by angiopoietin-1.

Angiopoietin-1 (Ang-1) constitutes a novel family of endothelial cell-specific angiogenic factors. Ang-1 functions mainly in remodeling, maturation, and stabilization of blood vessels. Its direct role in the process of angiogenesis remains unknown. The authors designed an experimental study to investigate the angiogenic potential of Ang-1 and to determine its hemodynamic effects on the cremaster muscle flap model in the rat. Adenovirus-mediated gene therapy was used for delivery of Ang-1. The study sample included 45 male Sprague-Dawley rats weighing 200 to 250 g. After the cremaster muscle tube flaps were prepared, rats were randomized into three different groups of 15 animals. In group I (the control), the flaps received phosphate-buffered saline (PBS). In group II, flaps were treated with adenovirus vector encoding Ang-1 (Ad-Ang-1). In group III, flaps received a control gene encoding green fluorescein protein (Ad-GFP). All treatments were administered via intra-arterial injections of either viral particles (10(8) placque-forming units) or PBS. The external iliac artery was used for this purpose. The cremaster tube flap was then preserved in a subcutaneous pocket in the lower limb. The tube flap was withdrawn from the limb on days 3, 7, and 14 after intra-arterial injection to evaluate microcirculatory measurements such as red blood cell velocity, vessel diameter, capillary density, and microvascular permeability by intravital microscopy. Evaluations were performed by an investigator who was blinded to treatment groups. In a series of control experiments performed with Ad-GFP, adenoviral gene expression was evidenced by the observation of shiny GFP deposits along the vessel walls under fluorescence microscopy throughout the whole cremaster flap 2 days after transfection. At day 3 there was no evidence of any differences in capillary density and permeability index (PI). At day 7, the functional capillary density was significantly higher in the Ad-Ang-1-treated group compared with the control and the Ad-GFP groups (10/hpf +/- 2 vs. 7/hpf +/- 0.5, p = 0.006; 5/hpf +/- 1.6, p = 0.0001). The PI in the Ad-Ang-1-treated group was significantly lower compared with the Ad-GFP-treated group (1.1/hpf +/- 0.1% vs. 1.4/hpf +/- 0.1%, p = 0.0005). At 14 days, the number of the flowing capillaries was significantly higher in the Ad-Ang-1-treated group compared with the control and the Ad-GFP-treated groups (13/hpf +/- 1.7 vs. 9/hpf +/- 2 and 6/hpf +/- 1.3, p = 0.0001). The microvascular PI was significantly lower in the Ad-Ang-1-treated group compared with the Ad-GFP-treated group (1.3/hpf +/- 0.2% vs. 1.8/hpf +/- 0.5%, p = 0.004). Histologically, the cremaster flaps revealed focal and mild inflammation regardless of the treatment and time point of evaluation. There was evidence of vasculitis in muscles pretreated with Ad-GFP and Ad-Ang-1. In summary, in the Ad-Ang-1-treated cremaster flaps, functional capillary density increased from 46% at day 7 to 98% at day 14 when compared with the control group (p < 0.0001). In conclusion, in this experimental muscle flap model, Ad-Ang-1 treatment proved to be a successful method of angiogenic therapy, providing a long-lasting angiogenic effect over a period of 14 days. The increased capillary perfusion accompanied by the formation of more stable and mature vessels resistant to fluorescein isothiocyanate-conjugated albumin leakage may serve as in vivo evidence that Ang-1 therapy improves skeletal muscle flap hemodynamics. These exciting findings raise the possibility that Ang-1 may have implications for therapeutic angiogenesis. To the authors' knowledge, their study demonstrates for the first time the feasibility of intravascular gene therapy using a virus vector in an attempt to enhance muscle flap hemodynamics.

Gene therapy by adenovirus-mediated vascular endothelial growth factor and angiopoietin-1 promotes perfusion of muscle flaps.

An experimental study was conducted to investigate the potential use of intravascular gene therapy with adenovirus-mediated (Ad) vascular endothelial growth factor (VEGF) or angiopoietin-1 (Ang-1) for the enhancement of muscle flap perfusion and to evaluate the effect of therapy on microcirculatory hemodynamics and microvascular permeability in vivo by using a cremaster muscle flap model in the rat. The cremaster tube flap was left intact after isolation of the pudo-epigastric pedicle. A total of 90 male Sprague-Dawley rats were divided into five groups of 18 each, according to the type of intraarterial treatment. Control flaps received phosphate-buffered saline. Group 2 (the control gene encoding green fluorescent protein, Ad-GFP) served as the adenovirus control. In Groups 3, 4, and 5, flaps were pretreated with Ad-VEGF, Ad-Ang-1, and Ad-Ang-1 + Ad-VEGF, respectively. Flaps were preserved in a subcutaneous pocket in the hindlimb for evaluation of functional capillary density and microvascular permeability indices at 3, 7, and 14 days by intravital microscopy system. At day 7 and 14, Ad-VEGF, Ad-Ang-1, and combined treatment groups showed significantly higher numbers of capillary densities when compared with control and Ad-GFP groups (p < 0.05). At day 14, Ad-VEGF was the superior treatment group compared with Ad-Ang-1 and Ad-VEGF + Ad-Ang-1 (p < 0.05). Overall, there was a linear increase in the number of functional capillaries in all treatment groups (p < 0.05). At day 3 after Ad-Ang-1 therapy, a significantly lower permeability index was found when compared with Ad-VEGF + Ad-Ang-1 and Ad-VEGF alone treatment (p < 0.05). At day 7, the Ad-VEGF group had the highest score of permeability index compared with control, combined, and Ad-Ang-1 groups (p < 0.05). Histologic evaluation of muscle flaps demonstrated mild focal inflammation. There was evidence of mild vasculitis in all flaps except control muscles. Intravascular angiogenic therapy with Ad-VEGF or Ad-Ang-1 was technically feasible, as demonstrated by expression of the control gene, GFP, along the vascular tree. All treatment groups increased perfusion of the muscle flap over a period of 14 days, indicating a long-lasting effect of gene therapy. Ang-1 alone or in combination with VEGF was as effective as VEGF alone in augmenting muscle perfusion with more stable vessels 1 week after gene therapy.

Tumor-infiltrating endothelial cells and endothelial precursor cells in inflammatory breast cancer.

Inflammatory breast cancer (IBC) is a specific type of breast tumor that generally has a poor prognosis, in spite of recent advances in treatment. In the present study, semiquantitative reverse transcriptase polymerase chain reaction examination of resected specimens showed that angiogenic factors, not lymphangiogenic factors, are overexpressed in IBC tumors, compared with non-IBC tumors. Immunohistochemical analysis of the specimens revealed a significantly higher population of tumor-infiltrating (TI) endothelial cells (ECs) or endothelial precursor cells (EPCs) in tumor-associated stroma of IBC specimens than in non-IBC specimens. In a previous study, we examined the phenotype of host cells in response to transplanted IBC cells, using an established human IBC xenograft model (WIBC-9) (Shirakawa et al., Cancer Res 2001;61:445-51). The data obtained in that study are consistent with the findings of the present study. To explore the therapeutic potential of blocking vascular endothelial growth factor (VEGF) and angiopoietin (Ang) pathways in IBC, established vectors encoding soluble Flt-1 (sFlt-1) and soluble Tie2 (sTie2) were injected directly into WIBC-9. Both vectors produced growth inhibition ratios of WIBC-9 that were significantly higher than those of a non-IBC xenograft (MC-5). Also, both vectors suppressed WIBC-9 lung metastases. The efficacy correlated with the number of TI ECs/EPCs, which was determined by fluorescence-activated cell sorting. These ECs/EPCs incorporated acetylated lipoprotein and were integrated within a HUVEC monolayer in vitro culture on day 5.

Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo.

The capacity of an adenovirus encoding the mature form of vascular endothelial growth factor (VEGF)-D, VEGF-D Delta N Delta C, to induce angiogenesis, lymphangiogenesis, or both was analyzed in 2 distinct in vivo models. We first demonstrated in vitro that VEGF-D Delta N Delta C encoded by the adenovirus (Ad-VEGF-D Delta N Delta C) is capable of inducing endothelial cell proliferation and migration and that the latter response is primarily mediated by VEGF receptor-2 (VEGFR-2). Second, we characterized a new in vivo model for assessing experimental angiogenesis, the rat cremaster muscle, which permits live videomicroscopy and quantitation of functional blood vessels. In this model, a proangiogenic effect of Ad-VEGF-D Delta N Delta C was evident as early as 5 days after injection. Immunohistochemical analysis of the cremaster muscle demonstrated that neovascularization induced by Ad-VEGF-D Delta N Delta C and by Ad-VEGF-A(165) (an adenovirus encoding the 165 isoform of VEGF-A) was composed primarily of laminin and VEGFR-2-positive vessels containing red blood cells, thus indicating a predominantly angiogenic response. In a skin model, Ad-VEGF-D Delta N Delta C induced angiogenesis and lymphangiogenesis, as indicated by staining with laminin, VEGFR-2, and VEGFR-3, whereas Ad-VEGF-A(165) stimulated the selective growth of blood vessels. These data suggest that the biologic effects of VEGF-D are tissue-specific and dependent on the abundance of blood vessels and lymphatics expressing the receptors for VEGF-D in a given tissue. The capacity of Ad-VEGF-D Delta N Delta C to induce endothelial cell proliferation, angiogenesis, and lymphangiogenesis demonstrates that its potential usefulness for the treatment of coronary artery disease, cerebral ischemia, peripheral vascular disease, restenosis, and tissue edema should be tested in preclinical models.

Enhancement of epigastric skin flap survival by adenovirus-mediated VEGF gene therapy.

A novel approach to treat ischemic tissues by using gene therapy has recently been introduced on the basis of the angiogenic potential of certain growth factors. The authors investigated the effect of adenovirus-mediated gene therapy with vascular endothelial growth factor (VEGF) delivered into the subdermal space to treat compromised skin flaps. For this purpose, the epigastric skin flap model in rats, based solely on the right inferior epigastric vessels, was used. Thirty male Sprague-Dawley rats were divided into five groups of six rats each. Viral transfection with 108 plaque-forming units was performed 2 days before the epigastric flap elevation. Rats received subdermal injections of adenovirus encoding VEGF (Ad-VEGF) or green fluorescent protein (Ad-GFP) as treatment control. Another set of animals (n = 6) received no injections and were designated as control. To determine whether site of injection had an impact on flap viability, injections were given into the predicted local ischemic area (Ad-VEGF local, n = 6; Ad-GFP local, n = 6) and into the midline of the flap (Ad-VEGF midline, n = 6; Ad-GFP midline, n = 6). A flap measuring 8 x 8 cm was outlined on the abdominal skin extending from the xiphoid process proximally and the pubic region distally, to the anterior axillary lines bilaterally. Then, the epigastric flap was elevated as an island on the right inferior epigastric vessels and sutured back to its bed. Flap viability was evaluated at 7 and 14 days after the first operation. The epigastric flaps were scanned to the computer and areas of hypoxic and/or necrotic zones relative to total flap surface area were measured and expressed as percentages by using Image Pro Plus software. Specimens were taken for histologic evaluation at day 14 before the animals were killed. Combined area of necrotic and hypoxic zones as well as necrotic zone were decreased to 9.7 +/- 1.4 percent and 1.4 +/- 0.9 percent in Ad-VEGF local, and 11.8 +/- 1.9 percent and 3.5 +/- 1.64 percent in Ad-VEGF midline compared with the control and Ad-GFP treatment groups (control, 23 +/- 3.6 percent and 20.1 +/- 3.3 percent; Ad-GFP local, 24.8 +/- 4.8 percent and 16.2 +/- 5.9 percent; and Ad-GFP midline, 23.4 +/- 6.9 percent and 19.5 +/- 7.7 percent; p < 0.05). Histologic evaluation by light microscopy failed to demonstrate any quantitative difference in vascularity of skin flaps between the treatment groups. In this study, the authors demonstrated that adenovirus-mediated gene therapy using VEGF enhanced epigastric skin flap survival, as confirmed by the significant reduction in combined area of necrotic and hypoxic zones of the flap. Compared with the control, both local and midline subdermal injections of Ad-VEGF showed improvement in overall flap survival by 57.9 and 48.7 percent, respectively. The results of this study raise the possibility of using adenovirus-mediated therapeutic angiogenesis for safer flap surgery in high-risk patients.