Faculty Bibliography

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.

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.

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.

OBJECTIVE: The objective of this study was to evaluate the safety and tolerance of increasing single and repeated (n = 2) doses of intramuscular naked plasmid DNA encoding for fibroblast growth factor (FGF) type 1 (NV1FGF) administered to patients with unreconstructible end-stage peripheral arterial occlusive disease (PAD). The secondary objectives were to determine the biologic activity of NV1FGF on hemodynamic and clinical parameters associated with improved perfusion. METHODS: Fifty-one patients with unreconstructible peripheral arterial occlusive disease with rest pain or tissue necrosis underwent treatment with intramuscular NV1FGF. Increasing single (500, 1000, 2000, 4000, 8000, and 16,000 microg) and repeated (2 x 500, 2 x 1000, 2 x 2000, 2 x 4000, and 2 x 8000 microg) doses of NV1FGF were injected into the ischemic thigh and calf. Arteriography was performed before treatment and was repeated 12 weeks after treatment. Side effects and serious adverse events were monitored. Measurements of plasma and urine levels were performed to evaluate NV1FGF plasmid distribution. Serum FGF-1 was measured as an analysis of gene expression at the protein level. Transcutaneous oxygen pressure, ankle brachial index, toe brachial index, pain assessment with visual analog scale, and ulcer healing also were assessed. The safety results are presented for 51 patients, and the clinical outcomes are presented for the first 15 patients (500 to 4000 microg) who completed the 6-month follow-up study. RESULTS: NV1FGF was well tolerated. Sixty-six serious adverse events were reported; however, none were considered to be related to NV1FGF. Four patients had adverse events that were possibly or probably related to the study treatment: injection site pain, pain, peripheral edema, myasthenia, and paresthesia. No laboratory adverse events were related to the study treatment. Two deaths remote from the treatment were considered not related. Biodistribution of plasmid was limited and transient in plasma and absent in urine. No increase in the FGF-1 serum level was detected. A significant reduction in pain (P <.001) and aggregate ulcer size (P <.01) was associated with an increased transcutaneous oxygen pressure (P <.01) as compared with baseline pretreatment values. A significant increase in ankle brachial index (P <.01) was seen. CONCLUSION: NV1FGF is well tolerated and potentially could be effective for the treatment of patients with end-stage limb ischemia. Biologic parameters indicate improved perfusion after NV1FGF administration. Dose response is not yet evident. The safety of NV1FGF and the magnitude of improvement observed in this study encourage further investigation with a placebo-controlled, double-blind clinical trial.

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.

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.

Many similarities exist in the cellular responses elicited by VEGF and governed by integrins. Here, we identify a basis for these interrelationships: VEGF activates integrins. VEGF enhanced cell adhesion, migration, soluble ligand binding, and adenovirus gene transfer mediated by alphavbeta3 and also activated other integrins, alphavbeta5, alpha5beta1, and alpha2beta1, involved in angiogenesis. Certain tumor cells exhibited high spontaneous adhesion and migration, which were attributable to a VEGF-dependent autocrine/paracrine activation of integrins. This activation was mediated by the VEGFR2 receptor and regulated via phosphatidylinositol-3-kinase, Akt, and the PTEN signaling axis. Thus, integrin activation provides a mechanism for VEGF to induce a broad spectrum of cellular responses.

Previously, we induced vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) secretion in glioma cell lines by using physiologic concentrations of epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), or platelet-derived growth factor-BB (PDGF-BB). We hypothesized that VEGF/VPF might enhance the blood supply required for the unregulated growth of tumors, and that it acts as the central mediator of tumor angiogenesis. The objective of this study was to determine whether the expression of VEGF/VPF by meningiomas is regulated by growth factors or sex hormones. By means of an enzyme-linked immunosorbent assay of CH-157MN meningioma cell supernatants, we demonstrated that EGF and bFGF similarly induce VEGF secretion by CH-157MN meningioma cells. At the maximum concentrations of EGF (50 ng/mL) and bFGF (50 ng/mL) used in this study, VEGF secretion was induced to 140% to 160% above baseline constitutive secretion. PDGF-BB homodimer did not enhance VEGF secretion significantly. Estradiol (up to 10(-7) mol/L), progesterone (up to 10(-5) mol/L), or testosterone (up to 10(-5) mol/L) did not stimulate or inhibit VEGF secretion in CH-157MN meningioma cells (p > 0.05). Furthermore, we demonstrated that dexamethasone decreased VEGF secretion to 32% of baseline constitutive secretion. This might explain the effect of corticosteroids in alleviating peritumoral brain edema in meningiomas. These results suggest that VEGF secretion in CH-157MN meningioma cells is mainly regulated by growth factors and corticosteroids, but not by sex hormones. Understanding the regulation of VEGF/VPF secretion in meningiomas might contribute to the development of a new therapeutic strategy.

Vascular endothelial growth factor (VEGF) is a potent and selective vascular endothelial cell mitogen and angiogenic factor. VEGF expression is elevated in a wide variety of solid tumors and is thought to support their growth by enhancing tumor neovascularization. To block VEGF-dependent angiogenesis, tumor cells were transfected with cDNA encoding the native soluble FLT-1 (sFLT-1) truncated VEGF receptor which can function both by sequestering VEGF and, in a dominant negative fashion, by forming inactive heterodimers with membrane-spanning VEGF receptors. Transient transfection of HT-1080 human fibrosarcoma cells with a gene encoding sFLT-1 significantly inhibited their implantation and growth in the lungs of nude mice following i.v. injection and their growth as nodules from cells injected s.c. High sFLT-1 expressing stably transfected HT-1080 clones grew even slower as s.c. tumors. Finally, survival was significantly prolonged in mice injected intracranially with human glioblastoma cells stably transfected with the sflt-1 gene. The ability of sFLT-1 protein to inhibit tumor growth is presumably attributable to its paracrine inhibition of tumor angiogenesis in vivo, since it did not affect tumor cell mitogenesis in vitro. These results not only support VEGF receptors as antiangiogenic targets but also demonstrate that sflt-1 gene therapy might be a feasible approach for inhibiting tumor angiogenesis and growth.

This study examined the effects of full-length p16 gene transfer by recombinant adenovirus on cell growth and on sensitivity to CDDP or ACNU chemotherapies. We developed a recombinant adenovirus expressing the full-length human p16 gene (AxCA-hp16) by the COS-TPC method. AxCA-hp16 was infected into the p16-null human glioma cell line, U251MG. AxCA-hp16 infection inhibited proliferation of U251MG cells. A proliferation assay employing MTT showed that AxCA-hp16 infection induced chemoresistance, preventing CDDP-induced cell death (11- to 15-fold) and ACNU-induced cell death (80- to 92-fold). In the absence of AxCA-hp16, cell death was induced with CDDP or ACNU at 3 to 5 days after treatment, as demonstrated by Trypan-blue exclusion. Flow-cytometric analysis showed that CDDP or ACNU arrested cells in the G2 phase on day 1 and that cells re-entered the cycle on day 3. However, the cells infected with AxCA-hp16 after CDDP or ACNU treatment showed G1 arrest on day 5 after re-entering the cycle from G2 arrest on day 3. The cells infected with AxCA-hp16 before CDDP or ACNU treatment showed G1 arrest over the 5 days after the infection. This study demonstrated that G1 arrest induced with p16-gene expression prevents ACNU- or CDDP-induced cell death. The cell death induced by ACNU and CDDP therefore appears to occur in the phase after the G1/S check point.