Expression Of Angiostatin Research Articles

625. AAV Gene Therapy for Liver Metastatic Cancer

Metastatic liver cancer has a very poor prognosis and lacks effective therapy. We have previously demonstrated that intraportal injection of an adeno-associated-virus(AAV) vector led to long term and persistent expression of angiostatin and suppressed EL-4 tumors that metastasized to livers. In the present study, we seek strategies to improve the therapeutic efficacy of AAV mediated anti-angiogenic gene therapy in combating liver metastases. A recombinant adeno-associated-virus (AAV) vector encoding costimulator B7.1 was engineered. Incubation with AAV-B7.1 viruses resulted in rapid expression of B7.1 on the surface of 80% EL-4 cells. Transfected EL-4 cells vaccinated mice rejected the B7.1-engineered tumor cells, and resisted a secondary challenge with parental cells. Splenocytes from the vaccinated mice were highly cytotoxic towards parental EL-4 cells in vitro. However, the vaccinated mice failed to resist the challenge of a heavy burden of EL-4 cells. Combination of AAV-B7.1 vaccination and AAV-angiostatin therapy protected mice against the challenge of a heavy burden of EL-4 cells, and eradicated metastasized tumors that had disseminated to the liver. Furthermore, the combinational therapy promoted the survival rate of mice with advanced liver cancers. These encouraging results warrant future investigation of the use of AAV-mediated immunotherapy and anti-angiogenic gene therapy for targeting unresectable disseminated liver metastases.

Chronic hyperglycemia attenuates coronary collateral development and impairs proliferative properties of myocardial interstitial fluid by production of angiostatin.

Development of coronary collateral vessels is impaired in patients with diabetes mellitus. We tested the hypothesis that hyperglycemia alone attenuates collateral development and abolishes proliferative properties of myocardial interstitial fluid (MIF) by enhancing expression of matrix metalloproteinases (MMP) and angiostatin. Chronically instrumented dogs were randomly assigned to receive an infusion of normal saline (control; n=9) or 70% dextrose in water to increase blood glucose to 350 to 400 mg/dL for 8 h/d (hyperglycemia; n=7) in the presence or absence (sham; n=9) of brief (2 minutes), repetitive coronary artery occlusions (1/h; 8/d for 21 days). Collateral perfusion increased to 41+/-11% and 49+/-6% of normal zone flow in control dogs on days 14 and 21 (P<0.05) but remained unchanged over 21 days in hyperglycemic and sham dogs (12+/-3% and 13+/-3%, respectively). A progressive reduction of the postocclusive peak reactive hyperemic response was also observed in control dogs (16+/-1 to 10+/-1 Hz. 10(2) on days 1 and 21, respectively) but not in hyperglycemic (17+/-2 to 20+/-2) or sham (17+/-2 to 16+/-1) dogs. Endothelial cell tube formation was produced by MIF obtained from control dogs but not hyperglycemic or sham dogs. Coincubation of MIF from hyperglycemic dogs with an angiostatin antibody restored endothelial cell tube formation. MMP-9 activity and expression of angiostatin were increased in dogs receiving exogenous glucose compared with controls Chronic hyperglycemia abolishes development of coronary collateral vessels by increasing MMP-9 activity and angiostatin expression in dogs.

Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor.

Angiogenesis is characteristic of solid tumor growth and a surrogate marker for metastasis in many human cancers. Inhibition of tumor angiogenesis using antiangiogenic drugs and gene transfer approaches has suggested the potential of this form of therapy in controlling tumor growth. However, for long-term tumor-free survival by antiangiogenic therapy, the factors controlling tumor neovasculature need to be systemically maintained at stable therapeutic levels. Here we show sustained expression of the antiangiogenic factors angiostatin and endostatin as secretory proteins by recombinant adeno-associated virus 2 (rAAV)-mediated gene transfer. Both vectors provided significant protective efficacy in a mouse tumor xenograft model. Stable transgene persistence and systemic levels of both angiostatin and endostatin were confirmed by in situ hybridization of the vector-injected tissues and by serum ELISA measurements, respectively. Whereas treatment with rAAV containing either endostatin or angiostatin alone resulted in moderate to significant protection, the combination of endostatin and angiostatin gene transfer from a single vector resulted in a complete protection. These data suggest that AAV-mediated long-term expression of both endostatin and angiostatin may have clinical utility against recurrence of cancers after primary therapies and may represent rational adjuvant therapies in combination with radiation or chemotherapy.

Angiostatin and matrix metalloprotease expression following ischemic acute renal failure.

Ischemic injury to the kidney results in blood vessel loss and predisposition to chronic renal disease. Angiostatin is a proteolytic cleavage product of plasminogen that inhibits angiogenesis, promotes apoptosis of endothelial cells, and disrupts capillary integrity. A combination of lysine-Sepharose enrichment followed by Western blotting was used to study the expression of angiostatin in response to the induction of ischemic renal injury. No angiostatin products were readily detectable in kidneys of sham-operated control rats. In contrast, both 38- and 50-kDa forms of angiostatin were dramatically enhanced in the first 3 days following 45-min ischemia-reperfusion injury. Renal angiostatin levels declined but remained detectable at late time points postrecovery (8-35 days postischemia). Angiostatin-like immunoreactivity was also elevated in the plasma and in urine for up to 35 days following injury. Lysine-Sepharose extracts of either kidney or urine inhibited vascular endothelial cell growth factor-induced proliferation of human aortic endothelial cells in vitro; an effect that was blocked by coincubation with an angiostatin antibody. RT-PCR verified that mRNA of the parent protein plasminogen was produced in the liver, but it was not present in either sham-operated or postischemic kidney. Matrix metalloproteinase (MMP)-2 and MMP-9, which may mediate angiostatin generation, were enhanced in postischemic kidney tissue and were localized to the renal tubules, interstitial cells, and the tubulo-interstitial space. These data indicate the possible local synthesis of angiostatin following acute renal failure (ARF) and suggest a possible role for MMPs in this activity. Renal angiostatin generation following ARF may modulate renal capillary density postischemia and thereby influence chronic renal function.

Anti-Tumor Efficacy of Human Angiostatin Using Liver-Mediated Adeno-Associated Virus Gene Therapy

Angiostatin is a potent endogenous inhibitor of angiogenesis and tumor growth in vivo. The therapeutic potential of adeno-associated viral (AAV) gene delivery of angiostatin in modulating tumor growth in vivo was evaluated. Sustained levels of angiostatin were detected in the sera of mice for up to 6 months after they received a single injection of AAV-angiostatin. AAV-mediated stable expression of angiostatin inhibited tumor burden in the highly aggressive B16F10 melanoma and Lewis lung carcinoma (LLC) models of experimental metastasis. Moreover, AAV-angiostatin prolonged survival in B16F10 and LLC tumor-bearing mice compared to control groups. Anti-tumor efficacy was consistently observed when angiostatin serum levels of 15–50 ng/ml were detected following gene transfer, but the effect was minimal when the levels were lower or higher than this range. The combination of AAV-angiostatin gene therapy with chemotherapy was also shown to extend marginally the survival of mice bearing preestablished human tumors; however, the effect was evident only within a narrow dose of circulating angiostatin. These studies demonstrate the feasibility of using AAV antiangiogenic gene therapy as a cancer treatment modality and suggest that the optimal anti-tumor efficacy of angiostatin following gene transfer may be limited to a narrow dose range.

The effect of the angiogenesis inhibitor, angiostatin, on hypertrophic scarring

Introduction: Hypertrophic scarring is commonly seen by plastic surgeons in China. Since the etiology of hypertrophic scarring is still unknown, the only reliable treatment is surgical excision. To understand if angiogenesis plays an important role in the formation of hypertrophic scars, we investigated the effect of angiostatin, a potent angiogenesis inhibitor, on hypertrophic scar formation. Our hypothesis is that angiogenesis is required for increased scar formation and angiogenesis inhibition may be one of the methods that can be used to prevent the formation of hypertrophic scars. Methods: We have developed a reliable model in rabbits that results in hypertrophic scarring by creating a 6mm x 6mm full thickness skin wound on both ears. The cDNA for angiostatin is cloned into the pcDNA 3.1 mammalian expression vector. After the wounds re-epithelialized but prior to excessive scarring, the angiostatin expression vector was injected with Lipofectin 2000 once every two days. The expression of angiostatin was confirmed by RT-PCR. The scar tissue was harvested 14 days after injection and processed for histology and total protein. Histology was examined with routine stains, the amount of collagen deposition in the scar tissue was detected by proline assay, and TGF-β1 and VEGF expression was detected by western blot. Results: Compared to the control injection scar, the injection of angiostatin led to a much more normal-looking of scar in the rabbit ear. The proline assay demonstrated that the injection of the angiostatin expression vector resulted in much less collagen in the scar tissue. Western blot analysis showed there was less TGF-β1 and VEGF protein expression in the treated ear compared to the control. Conclusion: The introduction of a vector over-expression angiostatin can result in the decreased formation of hypertrophic scars in a rabbit ear model. This is corroborated by evidence of decreased collagen deposition, the primary extracellular matrix component of scars. In addition, we demonstrate the decreased expression of τηε pro-fibrosis growth factor, TGF-1, and the potent angiogenic factor, VEGF. These data suggest that angiogenesis inhibitors may have a potential role in the treatment of hypertrophic scarring.

Angiostatin expression in ovarian cancer

Angiostatin is a potent inhibitor of neovascularization, tumor growth and metastasis. We examined the expression of angiostatin and vascular endothelial growth factor (VEGF) through immunohistochemical analysis, along with microvessel density, in primary tumors obtained from 55 ovarian carcinoma patients. Angiostatin expression was not related to either stage of disease or histology. However, VEGF expression and microvessel density were related to stage of disease. Angiostatin expression did not correlate with VEGF expression. Microvessel density correlated with VEGF, but not angiostatin expression. Univariate analysis revealed that lack of angiostatin expression, VEGF expression, microvessel density and advanced stage of disease were significant risk factors for reduced survival. Multivariate analysis revealed that lack of angiostatin expression and advanced stage of disease were significant risk factors for reduced survival. Survival time was longer in patients with angiostatin-positive and VEGF-negative tumors than in patients with angiostatin-negative and VEGF-positive tumors. The presence of angiostatin expression and absence of VEGF expression are favorable prognostic factors with regard to survival in ovarian carcinoma patients.

Long-term expression of angiostatin suppresses metastatic liver cancer in mice

Metastatic liver cancer has a very poor prognosis and lacks effective therapy. Anti-angiogenic therapies, which starve tumors of blood supply, have proven to be effective in preclinical models because tumor growth is angiogenesis dependent. However, long-term, high-level, and sustained expression of angiogenesis inhibitors, such as angiostatin, is necessary to prevent dormant tumors from becoming active again. To achieve this objective, we engineered a recombinant adeno-associated virus (AAV) vector encoding mouse angiostatin, an endogenous inhibitor of tumor vascularization. After intraportal delivery of this vector, high-level, stable transgene expression of angiostatin lasting for at least 6 months was observed locally in hepatocytes. Gene transfer of AAV-angiostatin via the portal vein led to significant suppression of the growth of both nodular and metastatic EL-4 lymphoma tumors established in the liver and prolonged the survival time of the mice. The growth of neovessels was inhibited significantly, and extensive apoptosis of tumor cells was observed. The anti-angiogenic activity of angiostatin was independent of vascular endothelial growth factor (VEGF). The AAV-angiostatin viruses did not appear to be toxic to mice, and there was no detectable apoptosis of hepatocytes. In conclusion, these encouraging results warrant future investigation of the use of AAV-mediated anti-angiogenic gene therapy for targeting unresectable liver metastases, especially after surgical removal of primary tumors. (H epatology 2003;37:1451-1460.)

Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model.

Ischemic retinal diseases, such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, are a major cause of blindness worldwide. Angiostatin is an internal peptide fragment of plasminogen that inhibits endothelial proliferation in vitro and tumor growth in vivo. We now demonstrate that HIV vector encoding angiostatin (HIV-angiostatin) can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Intravitreal injections of HIV-angiostatin led to stable expression of the angiostatin gene in retinal tissue. Retinal neovascularization was histologically quantitated by a masked protocol. Retinal neovascularization in the eye injected with HIV-angiostatin was reduced in 90% (9/10; P=0.025) of animals, compared with the eye injected with phosphate-buffered saline. Reduction of histologically evident neovascular nuclei per 6-microm section averaged 68%, with maximal inhibitory effects of 87%. Neovascularization was not reduced in the eyes injected with HIV vector encoding enhanced green fluorescent protein. This is the first report that HIV-angiostatin can reduce neovascular cell nuclei in a murine proliferative retinopathy model. These data suggest that the anti-angiogenic activity of angiostatin has therapeutic potential for the treatment of retinal neovascularization.

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources.

A recombinant strain of Pichia pastoris with a phenotype of MutS was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O2 concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l(-1) after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.

Inhibitory efficacy of local angiostatin expression in treatment of human hepatic carcinoma cells

OBJECTIVE: To study the efficacy of local therapy using overexpression of the mouse angiostatin gene to treat human hepatic carcinoma. To investigate the mechanism of any therapeutic effect. METHODS: Full-length mouse angiostatin cDNA was directly cloned into the eukaryotic expression plasmid pcDNA3.1(+). The recombinant plasmid was used to transfect HCC7721 human hepatic carcinoma cells (experimental group). The vector control group cells were transfected with pcDNA3.1(+), and the mock control group was not exposed to the plasmid. Angiostatin expression at the mRNA and protein levels was detected by using RNA dot blots and fluorescence-activated cell sorter (FACS), respectively. Cell growth in vitro was assayed with thiazoyl blue tetrazolium bromide and cell cycle distributions were analyzed with FACS. Angiostatin-expressing cells and the vector control cells were transplanted subcutaneously into nude BALB/c mice and tumor volumes were measured after 30 days. Microvessel densities (MVD) and angiostatin expression in the tumor tissues were detected by using immunohistochemical staining with the primary anti-vWF and anti-HA antibodies, respectively. RESULTS: The pcDNA3.1(+)-angio recombinant eukaryotic expression plasmid was successfully constructed. No significant differences existed in cell morphology, cell growth curve and cell cycle distributions among the three study groups in vitro. However, markedly smaller tumor volumes, lower microvessel densities in tumor tissues, and stronger angiostatin-positive staining were observed in the experimental group relative to the control group, indicating reduced tumorigenesis associated with angiostatin overexpression in vivo. CONCLUSIONS: Mouse angiostatin did not directly inhibit human hepatic carcinoma cell growth and proliferation in vitro. However, local production of angio­statin effectively inhibited tumorigenesis of HCC7721 in vivo, probably because of inhibition of tumor angio­genesis via a paracrine pathway. Transfer of the angiostatin gene may be an effective new approach to the treatment of hepatic carcinoma.

Angiostatin expression in endometrial cancer

Angiostatin is a potent inhibitor of neovascularization, tumor growth and metastasis. The expressions of angiostatin and vascular endothelial growth factor (VEGF) were immunohistochemically examined, along with microvessel density, in primary tumors obtained from 57 endometrial carcinoma patients with stage I disease. Angiostatin expression was not related to depth of myometrial invasion, histological grade or lymph-vascular space involvement. VEGF expression also had no relation to depth of myometrial invasion or histological grade, however, it was enhanced in tumors with lymph-vascular space involvement. Angiostatin expression did not correlate with VEGF expression. Microvessel density correlated with VEGF, but not angiostatin expression. Disease-free survival was longer in patients with angiostatin-positive tumors than in patients with angiostatin-negative tumors, but did not differ among patients with VEGF-negative and VEGF-positive tumors. No patients with angiostatin-positive and VEGF-negative tumors had recurrent disease. We concluded that negative-expression of VEGF and positive-expression of angiostatin are significant prognostic factors in endometrial carcinoma patients.

Differential effects of angiostatin, endostatin and interferon-α1 gene transfer on in vivo growth of human breast cancer cells

The administration of different angiogenesis inhibitors by gene transfer has been shown to result in inhibition of tumor growth in animal tumor models, but the potency of these genes has been only partially evaluated in comparative studies to date. To identify the most effective anti-angiogenic molecule for delivery by retroviral vectors, we investigated the effects of angiostatin, endostatin and interferon(IFN)-alpha(1) gene transfer in in vivo models of breast cancer induced neovascularization and tumor growth. Moloney leukemia virus-based retroviral vectors for expression of murine angiostatin, endostatin and IFN-alpha(1) were generated, characterized, and used to transduce human breast cancer cell lines (MCF7 and MDA-MB435). Secretion of the recombinant proteins was confirmed by biological and Western blotting assays. Their production did not impair in vitro growth of these breast cancer cells nor their viability, and did not interfere with the expression of angiogenic factors. However, primary endothelial cell proliferation and migration in vitro were inhibited by supernatants of the transduced cells containing angiostatin, endostatin, and IFN-alpha(1). Stable gene transfer of the IFN-alpha(1) cDNA by retroviral vectors in both MCF7 and MDA-MB435 cells resulted in a marked and long-lasting inhibition of tumor growth in nude mice that was associated with reduced vascularization. Endostatin reduced the in vivo growth of MDA-MB435, but not MCF7 cells, despite similar levels of in vivo production, and angiostatin did not impair the in vivo growth of either cell line. These findings indicate heterogeneity in the therapeutic efficacy of angiostatic molecules delivered by viral vectors and suggest that gene therapy with IFN-alpha(1) and endostatin might be useful for treatment of breast cancer.

Expression of angiostatin cDNA in a murine renal cell carcinoma suppresses tumor growth in vivo

Objectives. To investigate the effectiveness of angiostatin gene therapy for renal cancer using a mouse model. The generally poor prognosis of advanced renal cancer indicates the need for new therapeutic modalities. The dependency of solid tumor growth on angiogenesis suggests that antiangiogenic therapy would be effective against renal cell carcinoma, which is generally a hypervascular tumor. Methods. Murine renal cancer cells (Renca) transfected with murine angiostatin cDNA (AST-Renca) were subcutaneously implanted in BALB/c mice. Subsequently, the macroscopic appearance and volume of tumors were evaluated once per week. Renca cells transfected with empty plasmid DNA (mock-Renca) were used as a control. In addition, histologic sections of tumor were analyzed for neovascularization on the basis of an immunohistochemical analysis for CD31. The antitumor effect of AST-Renca on a parental Renca tumor at a distant site was also evaluated. Results. The mean volume of AST-Renca tumors was significantly less than that of the control vector-transfected tumors 3 weeks after implantation. In the cell proliferation assay, the expression of angiostatin did not inhibit the proliferation of Renca cells in vitro. Immunohistochemical analysis of neovascularization by staining with anti-CD31 antibody revealed that angiostatin suppressed tumor vessel formation. Moreover, implantation of AST-Renca inhibited the growth of parental Renca implanted simultaneously at a distant site. Conclusions. Expression of an angiostatin transgene can suppress the growth of murine renal cancer through the inhibition of tumor-induced angiogenesis. Angiostatin gene therapy may be effective against renal cancer.

38. AAV-Mediated Expression of PEDF or Angiostatin (K1K3) Reduces Retinal Neovascularization in a Mouse Model of Ischemic Retinopathy

38. AAV-Mediated Expression of PEDF or Angiostatin (K1K3) Reduces Retinal Neovascularization in a Mouse Model of Ischemic Retinopathy

Angiostatin inhibits coronary angiogenesis during impaired production of nitric oxide.

The in vivo mechanism by which inhibition of NO synthase impairs ischemia-induced coronary vascular growth is unknown. We hypothesized that production of the growth inhibitor angiostatin increases during decreased NO production, blunting angiogenesis and collateral growth. Measurements were made in myocardial tissue or interstitial fluid (MIF) from dogs undergoing repetitive coronary occlusions under control conditions or during antagonism of NO synthase (N(G)-nitro-L-arginine methyl ester [L-NAME]) for 7, 14, or 21 days. A sham group was instrumented identically but received no occlusions. In controls, capillary density in the ischemic zone increased initially but returned to baseline at the later times. In the L-NAME group, capillary density was lower at 7 days compared with that of controls. MIF from control dogs induced in vitro endothelial tube formation and cell proliferation, significantly greater than that from the L-NAME group. MIF from shams did not stimulate tube formation. In controls or shams, tube formation or cell proliferation did not change after administration of antiangiostatin, but this antibody restored the responses to control levels in the L-NAME group. Angiostatin expression in MIF was increased in the L-NAME group compared with controls and shams. The activities of tissue matrix metalloproteinases (MMPs) MMP-2 and MMP-9, which generate angiostatin, were increased in the L-NAME group. Inhibition of NO synthase increased expression of angiostatin and activities of MMP-2 and MMP-9. Our findings indicate that angiostatin inhibits coronary angiogenesis during compromised NO production and may underscore the impairment of coronary angiogenesis during endothelial dysfunction.

Expression of Angiostatin Using DNA-Based Semliki Forest Virus Replicon

Angiogenesis is recognized as a critical factor in the growth of tumor cells and plays a key role in the tumor metastasis. Recent studies for antiangiogenic substances are getting popular. The angiostatin, one of the antiangiogenic substances, leads to the increased apoptosis of the tumor cells by inhibiting the neovascularization of the tumor. The angiostatin was identified as the internal fragments of the plasminogen which has no antiangiogenic activity. By hydrolysis of the plasminogen, the angiostatin can be produced. In this study, we constructed the SFV-derived DNA vector by employing the cytomegalovirus immediate early enhancer/ promoter (CMV). This vector makes it possible to transfect the cells with DNA without the in vitro transcription process. The C-myc epitope and polyhistidine residue sequences were placed in downstream of the angiostatin gene to make it eligible to detect the expressed protein. The murine Ig k-chain V-J2-C signal sequence was placed in upstream to secrete the expressed protein from the cells. We confirmed the expression of angiostatin in the BHK-21 cells using DNA-based SFV replicon.

Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression.

Tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. Angiostatin, a multiple kringle (1-4)-containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis. Here we show that gene transfer of angiostatin into small (0.1 cm in diameter) solid EL-4 lymphomas established in syngeneic C57BL/6 mice led to reduced tumor angiogenesis and weak inhibition of tumor growth. In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T-cell costimulator B7.1, large (0.4 cm in diameter) tumors were rapidly and completely eradicated, whereas B7.1 and angiostatin monotherapies were ineffective. Combined gene transfer of B7.1 and angiostatin generated potent systemic antitumor immunity that was effective in eradicating a systemic challenge of 10(7) EL-4 cells. Gene transfer of angiostatin expression plasmids led to overexpression of angiostatin in tumors, increased apoptosis of tumor cells, and decreased density of tumor blood vessels, which may allow the immune system to overcome tumor immune resistance. The latter effects were not the result of a decrease in vascular endothelial growth factor expression, as tumoral vascular endothelial growth factor expression increased slightly after angiostatin gene transfer, presumably in response to increasing hypoxia. These results suggest that combining immunogene therapy with a vascular attack by angiostatin is a particularly effective approach for eliciting antitumor immunity.

Adenoviral Vector Expressing Murine Angiostatin Inhibits a Model of Breast Cancer Metastatic Growth in the Lungs of Mice

Angiostatin, an internal fragment of plasminogen, has been shown to inhibit the process of angiogenesis or neovascularization. In this study, we have expressed the cDNA for murine angiostatin under the control of the human cytomegalovirus promoter from a human type-5 adenovirus and shown that this vector produces a protein which retains biological activity. Angiostatin expression was determined by Northern blot analysis and Western immunoblotting. Ad-angiostatin, but not a control vector Ad-dl70, significantly reduced the viability of infected human umbilical cord vein endothelial cells (HUVEC) in vitro. In an in vivo model of basic fibroblast growth factor-induced angiogenesis, Ad-angiostatin (1 x 10(9) pfu) could inhibit endothelial cell migration and the formation of capillaries within a Matrigel plug which had been implanted for one week subcutaneously into C57BL/6 mice. Endothelial cells in these plugs had an altered, rounded, phenotype with dark picnotic nuclei indicative of apoptosis, which was confirmed using transmission electron microscopy. In contrast, endothelial cells from bFGF alone or in combination with the control vector-treated plugs retained the long spindle shape characteristic of endothelial cells. Intranasal delivery of Ad-angiostatin into the lungs of FVB/n mice demonstrated comparable cellular infiltration in the recovered bronchoalveolar lavage fluid with no signs of abnormal pathology as compared to PBS or control vector-treated animals. In a pulmonary metastatic breast cancer model, the delivery of Ad-angiostatin (1 x 10(9) pfu) to the lung significantly delayed tumor growth as measured by the number of visible surface tumor nodules. This study has demonstrated that the specific targeting of tumors to inhibit angiogenesis using an adenovirus expressing angiostatin, may deliver localized concentrations of protein having a greater impact on inhibition of tumor growth.

Growth of human melanoma xenografts is suppressed by systemic angiostatin gene therapy.

The effect of local and systemic delivery of the angiostatin gene on human melanoma growth was studied in nude mice. Liposome-coated plasmids carrying the cDNA coding for murine and human angiostatin (CMVang and BSHang) were injected weekly, locally or systemically, in mice transplanted with melanoma cells. The treatment reduced melanoma growth by 50% to 90% compared to that occurring in control animals treated with liposome-coated plasmid carrying the lacZ gene or in untreated controls. The growth of both locally injected and controlateral uninjected tumors in mice bearing two melanoma grafts was significantly suppressed after intratumoral treatment. Tumor growth inhibition was also observed in mice treated by intraperitoneal delivery, suggesting that angiostatin gene therapy acts through a systemic effect. Both melanoma growth suppression and delay in the onset of tumor growth were observed in treated mice. PCR performed on tumors and normal tissues showed that the lipofected DNA was present in tissues from treated mice, and angiostatin expression was demonstrated by RT-PCR. Histopathological analysis of melanoma nodules revealed an increase in apoptotic cells and a reduction in vessel density in tumors from treated mice. Our results suggest that systemic, liposome-mediated administration of genes coding for antiangiogenic factors represents a promising strategy for melanoma treatment in humans.