Remodeling the homeostasis of pro- and anti-angiogenic factors by Shenmai injection to normalize tumor vasculature for enhanced cancer chemotherapy.

ETHNOPHARMACOLOGICAL RELEVANCE: Normalization of the tumor vasculature can enhance tumor perfusion and the microenvironment, leading to chemotherapy potentiation. Shenmai injection (SMI) is a widely used traditional Chinese herbal medicine for the combination treatment of cancer in China. AIM OF THIS STUDY: This study aimed to investigate whether SMI can regulate tumor vasculature to improve chemotherapy efficacy and identify the underlying mechanism. MATERIALS AND METHODS: The antitumor effect of SMI combined with 5-florouracil (5-FU) was investigated in xenograft tumor mice. Two-photon microscopy, laser speckle contrast imaging and immunofluorescence staining were used to investigate the effects of SMI on tumor vasculature in vivo. The mRNA and protein expression of pro- and anti-angiogenic factors were measured by Q-PCR and ELISA. Histone acetylation and transcriptional regulation were detected by Western blot and ChIP assay. RESULTS: SMI promoted normalization of tumor microvessels within a certain time window, which was accompanied by enhanced blood perfusion and 5-FU distribution in tumors. SMI significantly increased the expression of antiangiogenic factor angiostatin and decreased the pro-angiogenic factors VEGF, FGF and PAI-1 by day 10. SMI combined with neoadjuvant chemotherapy in colorectal cancer patients also showed a significant increase in angiostatin and decrease in VEGF and FGF in surgically resected tumors when compared to the neoadjuvant chemotherapy group. Further in vitro and in vivo studies revealed that SMI downregulated VEGF, FGF and PAI-1 mRNA expression by inhibiting histone H3 acetylation at the promoter regions. The enhanced production of angiostatin was attributed to the regulation of the plasminogen proteolysis system via SMI-induced PAI-1 inhibition. CONCLUSION: SMI can remodel the homeostasis of pro- and anti-angiogenic factors to promote tumor vessel normalization, and thus enhance drug delivery and anti-tumor effect. This study provides additional insights into the pharmacological mechanisms of SMI on tumors from the perspective of vascular regulation.

Is Angiostatin Involved in Physiological Foveal Avascularity?

Purpose: The primate central retina is characterized by an avascular fovea and well-defined perifoveal capillary plexus. Neither blood vessels nor their accompanying astrocytes enter the fovea during any stage of retinal development; a balance of angiogenic and angiostatic factors probably maintains foveal avascularity throughout life. The aim of this study was to identify potentially angiorepulsive factors involved in the development of the avascular primate retinal fovea. Methods: Retinas of newborn, juvenile, and adult Callithrix jacchus and Macaca fascicularis monkeys and control human retinas were studied to determine the localization of angiostatin relative to III ß-tubulin, glial fibrillary acidic protein, vascular endothelial growth factor (VEGF), platelet endothelial cell adhesion molecule-1 (PECAM), and the angiostatin receptor αvß3-integrin in the foveal, macular, and peripheral retina. Expression studies were performed using immunohistochemistry (IHC) on retinal whole-mount and paraffin sections, and Western blotting on frozen material. The complex network of the main retinal cell types was identified by IHC of retinal whole mounts. Results: In general, lifetime expression of angiostatin was found in all retinas. Colabeling with different markers revealed retinal ganglion cells as the main source of angiostatin expression in the primate retina, whereas PECAM-immunopositive blood capillaries expressed the angiostatin receptor αvß3-integrin, and capillary-associated astrocytes expressed VEGF. Conclusions: This study provides the first evidence of angiostatin expression in the primate retina; the expression of angiostatin in the avascular foveal region and the peripheral retina suggests that angiostatin may play a role in the regulation of retinal vascularization, providing a possible explanation for the development and persistence of an avascular fovea.

Enhanced antitumor efficacy of an oncolytic herpes simplex virus expressing an endostatin-angiostatin fusion gene in human glioblastoma stem cell xenografts.

Viruses have demonstrated strong potential for the therapeutic targeting of glioblastoma stem cells (GSCs). In this study, the use of a herpes simplex virus carrying endostatin-angiostatin (VAE) as a novel therapeutic targeting strategy for glioblastoma-derived cancer stem cells was investigated. We isolated six stable GSC-enriched cultures from 36 human glioblastoma specimens and selected one of the stable GSCs lines for establishing GSC-carrying orthotopic nude mouse models. The following results were obtained: (a) VAE rapidly proliferated in GSCs and expressed endo-angio in vitro and in vivo 48 h and 10 d after infection, respectively; (b) compared with the control gliomas treated with rHSV or Endostar, the subcutaneous gliomas derived from the GSCs showed a significant reduction in microvessel density after VAE treatment; (c) compared with the control, a significant improvement was observed in the length of the survival of mice with intracranial and subcutaneous gliomas treated with VAE; (d) MRI analysis showed that the tumor volumes of the intracranial gliomas generated by GSCs remarkably decreased after 10 d of VAE treatment compared with the controls. In conclusion, VAE demonstrated oncolytic therapeutic efficacy in animal models of human GSCs and expressed an endostatin-angiostatin fusion gene, which enhanced antitumor efficacy most likely by restricting tumor microvasculature development.

Plasminogen activators are involved in angiostatin generation in vivo in benign and malignant ovarian tumor cyst fluids.

In many tumor types, angiogenesis is the net result of pro- and anti-angiogenic mediators and correlated with metabolic activity, growth, and degree of malignancy. One of the first discovered anti-angiogenic compounds is angiostatin, a proteolytic fragment of plasminogen. The requirements for in vivo angiostatin generation have not yet been determined. We investigated the levels of plasminogen and angiostatin by western blotting and of components of the plasminogen activator complex by ELISA in cyst fluid derived from benign and malignant ovarian tumors. Fluid samples from functional ovarian follicles, dermoid cysts and endometriotic lesions were evaluated separately. When no or minimal amounts of plasminogen were present in the cyst fluids, angiostatin was generally absent as well, irrespective of plasminogen activator concentrations. When plasminogen was present, the degree of conversion of plasminogen to angiostatin was significantly correlated with the level of uPA, and, to a lesser extent, to the tPA level. However, angiostatin was also found in a number of cyst fluid samples with minimal or no plasminogen activators, suggesting the involvement of other angiostatin generating proteases in these samples. Conversely, no angiostatin was observed in a number of cyst fluid samples containing both plasminogen and plasminogen activators. The presence of an inhibitor of the enzymatic activity of uPA and/or tPA, like PAI-1, may explain this finding. Our data show that plasminogen activators are clearly involved in in vivo angiostatin formation in ovarian cysts. Most likely, however, other proteases, as well as inhibitors of plasminogen activators, are involved as well.

Effect of hypoxia on angiogenesis related factors in glioblastoma cells.

Pathological angiogenesis is a characteristic feature of glioblastoma multiforme (GBM) where the balance between pro-angiogenic and anti-angiogenic factors are shifted towards the pro-angiogenic phenotype. In this study we sought to determine whether angiostatins are expressed by GBM cells and whether their expression along with other related factors [matrix metalloproteinase (MMP)-2, MMP-9, and collagen type I α1 (COLIA1)] are altered by hypoxia and/or correlated with the levels of cancer stem cell marker CD133. Using qRT-PCR, western blotting, and gelatin zymography, we examined the expression of angiostatins, MMP-2, MMP-9, COLIA1 and CD133 in GBM cell lines cultured under aerobic conditions and hypoxia. Expression levels of MMP-2 and MMP-9 were significantly induced by hypoxia. Angiostatins were detected in all GBM cell lines and were increased by hypoxia while the angiostatin isoform of 38-kDa was the most abundant in GBM cells under aerobic and hypoxic conditions. COLIA1 and CD133 were significantly increased in several GBM cell lines under hypoxia. Despite expression and upregulation of anti-angiogenic factors (e.g. angiostatins) in GBM cells, they are overwhelmed by the overexpression of a larger number of angiogenic factors that shift the angiogenic balance towards the pro-angiogenic phenotype. Thus, an exogenous administration of anti-angiogenic factors may be required to improve the treatment of GBM tumors.

Targeted anti-vascular therapies for ovarian cancer: current evidence.

Ovarian cancer presents at advanced stage in around 75% of women, and despite improvements in treatments such as chemotherapy, the 5-year survival from the disease in women diagnosed between 1996 and 1999 in England and Wales was only 36%. Over 80% of patients with advanced ovarian cancer will relapse and despite a good chance of remission from further chemotherapy, they will usually die from their disease. Sequential treatment strategies are employed to maximise quality and length of life but patients eventually become resistant to cytotoxic agents. The expansion in understanding of the molecular biology that characterises cancer cells has led to the rapid development of new agents to target important pathways but the heterogeneity of ovarian cancer biology means that there is no predominant defect. This review attempts to discuss progress to date in tackling a more general target applicable to ovary cancer-angiogenesis.

Angiostatin production increases in response to decreased nitric oxide in aging rat kidney.

The development of interstitial fibrosis occurs with aging. Impaired angiogenesis, associated with progressive loss of the renal microvasculature, is thought to be a cause of age-related nephropathy. However, the mechanism of capillary loss in aging kidney has not been fully elucidated. Angiostatin is a kringle-containing fragment of plasminogen and is a potent inhibitor of angiogenesis in vivo. Whether angiostatin generation is increased in the aging kidney has not been investigated. We examined 4, 10, 16, and 24-month-old Sprague-Dawley rats for angiostatin production and found that angiostatin generation was increased in aged rats. The protein expression and the activity of cathepsin D-the enzyme for angiostatin production--were increased in aged rats. In the aging kidney, nitric oxide (NO) availability is decreased. To investigate the role of NO in angiostatin production, human umbilical vein endothelial cells were treated with L-NG-nitroarginine methyl ester (L-NAME). L-NAME-treated cells showed increased cathepsin D activity and angiostatin production. For in vivo experiments, 16- to 18-month-old rats were treated with L-NAME or molsidomine for 3 months. Angiostatin production was increased in L-NAME-treated kidney, accompanied by increased cathepsin D activity. In contrast, angiostatin production was decreased in molsidomine-treated kidney, accompanied by decreased cathepsin D activity. In conclusion, angiostatin generation by cathepsin D was increased in the aging rat kidney. Decreased NO production activated cathepsin D activity. Increased angiostatin production may be related to capillary loss and interstitial damage in the aging rat kidney.

Synergistic antitumoral effect of IL-12 gene cotransfected with antiangiogenic genes for angiostatin, endostatin, and saxatilin.

Previously, it was reported that the cotransfection of angiostatin K1-3, endostatin, and saxatilin genes using cationic liposomes significantly inhibited tumor progression. IL-12 is a well-known immune modulator that promotes Th1-type antitumor immune responses and also induces antiangiogenic effects. In this study, we have examined the antitumoral function of the IL-12 gene cotransfected with antiangiogenic genes for angiostatin K1-3, endostatin, and saxatilin by O,O'-dimyristyl-N-lysyl glutamate (DMKE) cationic liposomes in a mouse tumor model. According to our results, the administration of the IL-12 gene or the genes for angiostatin K1-3, endostatin, and saxatilin exhibited effective inhibition of B16BL6 melanoma growth in mice. In particular, intravenous administration of the IL-12 gene along with intratumoral administration of the three antiangiogenic genes synergistically inhibited the B16BL6 tumor growth. These results suggest that systemically expressed IL-12 enhances antitumoral efficacy of locally expressed antiangiogenic proteins.

Radio-sensitivities and angiogenic signaling pathways of irradiated normal endothelial cells derived from diverse human organs.

The purpose of the present investigation was to study the effects of ionizing radiation on endothelial cells derived from diverse normal tissues. We first compared the effects of radiation on clonogenic survival and tube formation of endothelial cells, and then investigated the molecular signaling pathways involved in endothelial cell survival and angiogenesis. Among the different endothelial cells studied, human hepatic sinusoidal endothelial cells (HHSECs) were the most radio-resistant and human dermal microvascular endothelial cells were the most radio-sensitive. The radio-resistance of HHSECs was related to adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase-mediated expression of MMP-2 and VEGFR-2, whereas the increased radio-sensitivity of HDMECs was related to extracellular signal-regulated kinase-mediated generation of angiostatin. These observations demonstrate that there are distinct differences in the radiation responses of normal endothelial cells obtained from diverse organs, which may provide important clues for protection of normal tissue from radiation exposure.

Safety and biodistribution of an equine infectious anemia virus-based gene therapy, RetinoStat(®), for age-related macular degeneration.

RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.

Antisense HIF-1alpha prevents acquired tumor resistance to angiostatin gene therapy.

Angiostatin is a naturally occurring inhibitor of angiogenesis that is being developed as a drug to fight cancer. In this study we reveal that EL-4 tumors established in mice rapidly develop resistance to angiostatin gene therapy by upregulating hypoxia-inducible pathways. Angiostatin initially delayed tumor growth for 6 days by reducing blood vessel density. However, tumors quickly responded by upregulating the production of hypoxia-inducible factor-1alpha (HIF-1alpha) and its effector vascular endothelial growth factor (VEGF) in response to increasing tumor hypoxia, leading to restored angiogenesis and rapid tumor growth. Theoretically, blockade of HIF-1 should prevent resistance to anti-angiogenic therapy by preventing a tumor from responding to induced hypoxia. Antisense HIF-1alpha inhibited the expression of HIF-1alpha and of the HIF-1 effectors VEGF, glucose transporter-1 and lactate dehydrogenase. As a monotherapy, it was effective in eradicating small 0.1 cm diameter tumors, but only delayed the growth of large 0.4 cm diameter tumors. In contrast, timed injection of a combination of angiostatin and antisense HIF-1alpha plasmids completely eradicated large EL-4 tumors within 2 weeks, and prevented upregulation of hypoxia-inducible pathways induced by angiostatin. The data indicate that blocking hypoxia-inducible pathways by antisense HIF-1alpha can circumvent hypoxia-induced drug resistance and thereby augment the efficacy of anti-angiogenic therapies.

The possible mechanism for impaired angiogenesis after transient focal ischemia in type 2 diabetic GK rats: different expressions of angiostatin and vascular endothelial growth factor.

BACKGROUND: The development of collateral vessels, which is important to prevent ischemic tissues from cell death, is impaired in patients with diabetes mellitus. The process is regulated by many positive and negative factors. The purpose of the study is to test the hypothesis that stroke patients with diabetes have angiogenesis deficiency and the possible mechanism is hyperglycemia attenuates neovascularization by downregulating proliferative properties of vascular endothelial growth factor (VEGF) and upregulating negative properties of angiostatin. METHODS: Diabetes groups [Goto-Kakizaki (GK)] and respective controls (Wistar rats) underwent 1.5h of middle cerebral artery occlusion (MCAO) and then reperfused for 24h and 7d. Immunohistochemistry was used to describe the change of vessel density. The expression levels of VEGF and angiostatin were estimated by western blot. RESULTS: Compared with the controls, the diabetes groups had lower vessel density, more expression of angiostatin, and lower level of VEGF. CONCLUSIONS: These results showed angiogenesis was deficient in diabetes groups after ischemic reperfusion (I/R) injury. And the possible mechanism is hyperglycemia attenuates neovascularization by downregulating proliferative properties of VEGF and upregulating of negative properties of angiostatin.

Mesalamine restores angiogenic balance in experimental ulcerative colitis by reducing expression of endostatin and angiostatin: novel molecular mechanism for therapeutic action of mesalamine.

Mesalamine (5-aminosalicylate acid, 5-ASA) is an effective treatment for ulcerative colitis (UC). The mechanisms of its actions are not fully understood. Because angiogenesis is critical for healing UC, we examined whether 5-ASA alters the angiogenic balance between angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and antiangiogenic factors (e.g., endostatin and angiostatin) in the colon in experimental UC. Rats were treated with saline or 5-ASA (100 mg/kg) twice daily and euthanized 3 or 7 days after iodoacetamide-induced UC. Clinical signs (e.g., lethargy, diarrhea) and UC lesions were measured. Expression of VEGF, endostatin, angiostatin, tissue necrosis factor alpha (TNF-alpha), and matrix metalloproteinases (MMPs) 2 and 9 was determined by Western blots, enzyme-linked immunosorbent assay, and zymography in the distal colon. 5-ASA treatment reduced lethargy and diarrhea and significantly decreased colonic lesions (by approximately 50%) compared with saline treatment in UC (both, P < 0.05). 5-ASA did not reverse the increased levels of VEGF, but it significantly reduced expression of endostatin and angiostatin in UC compared with vehicle treatment (both, P < 0.05). Furthermore, 5-ASA treatment significantly diminished increased activity of TNF-alpha and MMP9 in UC. This is the first demonstration that 5-ASA treatment reverses an imbalance between the angiogenic factor VEGF and antiangiogenic factors endostatin and angiostatin in experimental UC. The effect of 5-ASA in UC may be caused by the down-regulation of expression of endostatin and angiostatin by modulation of MMP2 and MMP9 via inhibition of TNFalpha. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).

[Treatment of intraperitoneal implanted human ovarian carcinoma of nude mice by angiostatin gene and chemotherapy in vivo].

OBJECTIVE: To observe the effects of angiostatin gene combined with chemotherapy on implanted human ovarian carcinoma of nude mouse. METHODS: The mice were randomly divided into four groups after 7 days of the intraperitoneal injection of tumor cells (4 x 10(6)), and injected respectively with empty plasmid pcDNA3.0, angiostatin plasmid, cisplatin, and angiostatin plasmid + cisplatin. For combinational treatment, reagents were delivered in a timed fashion, where angiostatin plasmid was injected first, followed by cisplatin 24h later. The tumor samples were prepared to be used in the examinations of the expression of angiostatin with immunohistochemistry, of MVD in the tumor with immunohistochemistry, and of cell apoptosis with TUNEL staining. RESULTS: Tumor growth and ascites formation were inhibited in all 3 groups except for the control group. The therapeutic effectiveness in the combined group was more significant than in the other two groups. In this group, MVD (32.5 +/- 4.3) was the lowest and apoptosis index (5.12 +/- 0.63) was the highest (P < 0.01). CONCLUSIONS: Angiostatin gene therapy combined with chemotherapy has a synergistic effect on the inhibition of ovarian cancer angiogenesis and ascites formation. Combining multiple therapies to treat ovarian cancer is an effective strategy.

[Constitutive expression of human angiostatin in Pichia pastoris using glycerol as only carbon source].

Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).

A glycolytic mechanism regulating an angiogenic switch in prostate cancer.

The generation of an "angiogenic switch" is essential for tumor growth, yet its regulation is poorly understood. In this investigation, we explored the linkage between metastasis and angiogenesis through CXCL12/CXCR4 signaling. We found that CXCR4 regulates the expression and secretion of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1). Overexpression of PGK1 reduced the secretion of vascular endothelial growth factor and interleukin-8 and increased the generation of angiostatin. At metastatic sites, however, high levels of CXCL12 signaling through CXCR4 reduced PGK1 expression, releasing the angiogenic response for metastastic growth. These data suggest that PGK1 is a critical downstream target of the chemokine axis and an important regulator of an "angiogenic switch" that is essential for tumor and metastatic growth.

Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture.

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 60 h at 30 degrees C. Dissolved oxygen level was maintained at 25-30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.

[Fusion expression and identification of angiostatin and endostatin in E. coli BL21(DE3)].

Angiostatin(AS) and endostatin(ES) are both potent endogenous angiogenesis inhibitors, and the combination of AS and ES has been shown to have synergistic antiangiogenic effects. Here we report the fusion protein AS-ES expressed in E. coli which has antiangiogenic effects. At first, AS and ES genes were cloned respectively through RT-PCR, then fusion gene was made through gene splicing ,finally pET-42 (b)/AS-ES expression plasmid was constructed and transduced in E. coli BL21 (DE3). Target protein was in form of inclusion body,the rate of expression was about 14%, and MW about 65KD. Western blotting assay showed expressed protein had specific immune reaction to both the antibodies of AS and ES. The expressed protein which was refolded and purified through heparin affinity chromatography had antiangiogenic effect to vessels on chicken embryo chorioallantoic membrane. The results show that fusion protein AS-ES was expressed successfully in E. coli, and the expressed protein,which was renatured and purified, had immuno-reactivity to anti-AS and anti-ES in Western blotting and angiogenesis inhibition activity.