Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). CNV is also generated in high myopia ; angioid streaks and some inflammatory diseases and rarely after laser photocoagulation. Current treatment strategies for ARMD include laser photocoagulation , photodynamic therapy , but neither treatment addresses underlying stimuli for blood vessel growth. Therefore, recurrent disease is a problem of clinical significant relevance. Surgical excision of submacular neovascular membrane lead to the additional damage of the retinal pigment epithelium (RPE). Consequently, use of therapeutic agents that directly inhibit the angiogenic stimuli may be able to provide a more effective and permanent treatment. Vasostatin (VS) , the N-terminal domain (amino acid 1-180) of a calcium-binding protein , is a potent angiogenesis inhibitor , isolated from culture supernatants of an Epstein-Barr virus-immortalized cell line. In previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin attenuated the corneal neovascularization in animals. The recombinant vasostatin also prevented or apparently reduced growth of human Burkitt lymphoma and human colon carcinoma in animal model. The primary objective of this study was to vasostatin attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. The above experiments would enable us to test the hypothesis that the topical application of VS delivery might be a promising strategy for the treatment of ARMD and other retinal disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.

The growth and metastasis of solid tumors are dependent on angiogenesis. An endogenous angiogenesis inhibitor, vasostatin, is the proteolytic fragment derived from the N-terminal 180 residues of calreticulin. Previous studies indicated that vasostatin specifically inhibits endothelial cell proliferation, angiogenesis and tumor growth. However, continuous administration of vasostatin is difficult and expensive to facilitate, thereby underscoring the need to develop gene delivery approach. Adenovirus vectors possess advantages for gene delivery including high titer, high infection efficiency and broad host range. The aim of the present study was to generate and characterize recombinant adenovirus vectors encoding vasostatin (Ad-VS) or Igk-fused vasostatin (Ad-Igk-VS), thereby to evaluate the efficacy of anti-angiogenesis gene therapy for tumor suppression. Recombinant Ad-VS and Ad-Igk-VS were generated and verified by PCR and western blot analysis. In addition, adenovirus encoding angiostatin was also produced as positive control for angiogenesis assays. Adenovirus-mediated vasostatin gene delivery specifically inhibited the proliferation of bovine aortic endothelial cells (BAEC), but not non-endothelial cells such as Hela or NIH3T3 cells. Moreover, vasostatin gene delivery potently inhibited the proliferation, migration and tube formation, but not secretion of matrix metalloproteinases (MMPs), in endothelial cells. Flow cytometry analysis indicated that vasostatin gene delivery induced apoptosis in BAEC. Using western blot analysis, it was revealed that gene delivery of vasostatin increased the levels of Fas and FADD in BAEC. In conclusion, adenovirus-mediated vasostatin gene delivery inhibited various angiogenesis processes at least via induction of Fas/FasL pathway and may hold potential for cancer therapy.

Abstract Vasostatin, the N-terminal 180 amino acids domain of calreticulin, induces apoptosis in endothelial cells and inhibits angiogenesis. However, the mechanism underlying the apoptosis induce by vasostatin remains elusive. In the present study, we investigated the role of (1) Fas /FasL pathway, (2) oxidative stress, and (3) nitric oxide (NO) in the apoptotic mechanism of vasostatin in endothelial cells. Recombinant vasostatin was generated and shown to induce apoptosis of bovine aortic endothelial cells (BAEC) as demonstrated by flow cytometry analysis, nucleus staining, and DNA fragmentation assay. Vasostatin elevated the levels of Fas and its adaptor, FADD, in BAEC. Furthermore, vasostatin treatment increased the activities as well as the expression of active form of caspase-8 and caspase-3 in BAEC. However, pretreatment with either caspase-3 inhibitor or caspase-8 inhibitor alone was not sufficient to blockade the vasostatin-mediated apoptosis, suggesting the involvement of other pathways. Extensive screening using an array of caspase inhibitors further supported such notion. Oxidative stress is frequently involved in the apoptosis of endothelial cells. Previous studies indicated that vasostatin enhanced WST-1-derived formazan formation despite its cytotoxic effect, suggesting vasostatin treatment might enhance the production of superoxide. By measuring the level of superoxide anion in cultured media by cytochrome c reducing test, it was found that vasostatin treatment increased the production of superoxide anion in endothelial cells. Antioxidants such as NAC, GSH, BHA partially attenuated the vasostatin-mediated cytotoxicity and cell death in endothelial cells. Noteworthingly, adding allopurinol, inhibitor of xanthine oxidase, but not other oxidase inhibitors abrogated the cytotoxicity of vasostatin, indicating that xanthine oxidase could be the source of ROS produced by vasostatin relate with apoptosis. The elecctrophoretic mobility shift assays (EMSA) suggested that vasostatin treatment increased the NF£eB DNA binding activity. Western blot analysis indicated vasostatin increased the levels of NF£eB but decreased I£eB level, which seemed to coincide with the EMSA findings. NO plays an important role in endothelial function. To investigate the role of NO in the cytotoxicity by vasostatin, analyzed the levels of NO metabolites in cultured media of endothelial cells and found that vasostatin treatment increased NO release in time- dependent manners. The expression of eNOS, but not iNOS, in endothelial cells was upregulated by vasostatin. Besides, vasostatin treatment also increased the AP-1 binding activities. Moreover, NOS inhibitor, L-NAME, or NO scavenger, carboxy-PTIO, slightly attenuated the cytotoxic effects of vasostatin in endothelial cells. In addition to direct cytotoxicity, NO may react with superoxide (O2-) to form peroxynitrite (ONOO-), which attacked the intracellular protein and caused the cell damage. Indeed, we also detected a dose-dependent increment in the nitrotyrosination of cellular protein by vasostatin treatment. Taking together, these results indicate that vasostatin induces apoptosis in endothelial cells via multiple pathways. The interactions between these distinct pathways remain to be elucidated in the future.

IFN-α has been applied in medical treatment of various neuroendocrine (NE) tumors, either alone or combination with somatostatin analogues. They can improve clinical symptoms in 50-70% of patients but a significant tumor reduction is only observed in 5-15% patients. Vasostatin (vaso) is believed to be an angiogenic inhibitor. The aim of this study is to evaluate the feasibility to use IFN-α and vasostatin gene therapy in NE tumors. We constructed plasmid vectors carrying human IFN-α2 (hIFN-α2) gene and human vasostatin gene, which were transfected into BON I cell to obtain stable gene-expressing cell lines. We found that in animal tumor model and cell experiments gene transfer of vasostatin caused a faster cell growth and tumor development via down-regulation of the tumor suppressor gene and p27. Cell adhesion, spreading, migration and invasion ability were increased in vaso-expressing BON I cells. Transfecting chicken vinculin could reverse the malignant behavior and restored expression of tumor suppressor genes. Moreover, vinculin knockdown could result in a faster cell growth and an increased colony formation. Condition medium taken from hIFN-α2-expressing BON I cells showed significant antiproliferative effects both on the NE tumor cells, BON I and LCC18, and the endothelial cells, PAE. It also suppressed cell adhesion and cell invasion and inhibited angiogenesis on CAM assay. Mice implanted with a mixture of WT BON cells and hIFN-α2-expressing BON cells (1:1) demonstrated significantly lower tumor incidence and longer tumor doubling time. Furthermore, long-acting IFN-α2b (PEGIntron®) demonstrated a better anti-tumor effect in contrast with IFN-α2b (IntronA®). Intratumoral injection of hIFN-α2 plasmids significantly inhibited NE tumor growth and caused tumor regression. We concluded that gene transfer of vasostatin into BON I cells might cause an enhanced malignant tumor behavior. Therefore, vasostatin therapy can not be recommended for patients with NE tumors. Vinculin might play an important role in NE tumor development and growth regulation. Gene therapy by using plasmid DNA carrying hIFN-α2 gene is feasible and promising in NE tumors.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Current therapeutic approaches for HCC including surgical resection and trans-arterial embolization (TAE) remain largely ineffective, underscoring the need for development of novel therapeutic strategies. Because HCC is high vascularized, continuous administration angiogenesis inhibitor using gene therapy approach may facilitate long-term blockade tumor vasculature, thereby perturbing the growth of HCC. Vasostatin 112 (VS112) encodes an alternatively spliced fragment of angiogenesis inhibitor vasostatin, which encompasses residues 1-64 and 133-180 of calreticulin. In this study, recombinant adenovirus encoding VS112 (Ad-VS112) was generated to evaluate its potential for suppression of orthotopic Novikoff hepatoma in syngenic Sprague-Dawley (SD) rats. Adenovirus-mediated VS112 overexpression significantly inhibited the migration and tube formation of endothelial cells, indicating the anti-angiogenic potency of VS112 gene delivery. However, VS112 overexpression had no influence on the viability of N1-S1 Novikoff hepatoma cells. To investigate the prophylactic effect of VS112 expression on hepatoma growth, N1-S1 cells were infected with Ad-VS112 or adenovirus encoding green fluorescent protein (Ad-GFP) then implanted into the liver of SD rats. After 14 days, rats implanted with VS112-expressing showed significantly reduced incidence and size of hepatoma compared with those implanted with Ad-GFP-infected cells. To investigate the therapeutic efficacy of VS112 gene delivery, the SD rats were implanted with N1-S1 cells on day 0, treated with adenovirus vectors (2 x 1010 plaques forming units) via intravenous route on day 1, then sacrificed on day 14 to monitor hepatoma growth. By measuring tumor weight, it was found that Ad-VS112-treated rats exhibited significantly decreased tumor burden compared with control groups, which was in accordance with their lower serum GOT level. Histological analysis revealed a significant reduction of vWF-positive blood vessels in Ad-VS112-treated tumors, which was accompanied with a decrease in Ki-67-positive proliferating cells and an increase in TUNEL-positive apoptotic cells. Moreover, the expression of pro-inflammatory nuclear factor kappa B (NF£eB) and cyclooxgenase II (COXII) was also effectively attenuated in Ad-VS112-treated hepatoma. In conclusion, prior or post VS112 gene delivery potently suppresses the growth of orthotopic hepatoma,thereby holding promises for future treatment of HCC.

Age-related macular degeneration (AMD) is the leading cause for visual impairment and blindness in the elder population of developed countries. The primary underlying cause for significant visual loss is the choroidal neovascularization (CNV). Current treatment strategies for AMD include laser photocoagulation, photodynamic therapy (PDT) and excision of neovascular membranes, but have met with limited success. In our previous studies, we demonstrated that gene delivery of angiogenesis inhibitor vasostatin (VS) attenuated the corneal neovascularization in animals. The primary objective of this study was to investigate gene delivery of vasostatin (VS) attenuated the choroidal neovascularization in animals. Retinal and visual function will be evaluated. However, systematic expression of angiogenesis inhibitor may bring adverse effects to physiological processes. The feasibility, efficiency and safety of gene delivery with systemic and local routes were evaluated. Intramuscular polymer-based gene delivery had no side effect such as virus vector and revealed the safety. Recombinant adenovirus (Ad) was used gene delivery system because of its high titer, wide host range, and transduction efficiency. Adeno-associated virus (AAV) represents highly efficient that can facilirate long-term transduction. We propose to improve the efficacy and safety of VS gene delivery, and to search for the effective delivery route and other adjuvant therapy in conjunction with VS for treatment of CNV. Recently, PDT with veteporfin is an established treatment for subfoveal CNV secondary to AMD. We tried to compare the effect and safety of standard and reduced-dose light application PDT in an animal mocel of CNV. The 180-residue VS and its 48-residue (VS48) inhibited the migration and tube formation in cultured endothelial cells. Topical VS application suppresses the progression of laser-induced CNV via angiogenesis ihhibition, as well as in VS48. VS-48 inhibited the growthof vessels in arota rings. Electroretinograms (ERG) analysis revealed that topical VS-48 application for 21 days had no effect on rat retinal functions. Topical VS-48 treatment significantly reversed the CNV-induced alterations in ERG. Transfection of pCMV3-VS into muscle cells resulted in increased production and release of exogenous VS, which specifically inhibited the proliferation of endothelial cells. Rats treated with intrmuscular injection with PVP-VS also showed a significant reduction in the CNV lesions for at least 42 days. Subconjunctival injection with Ad vector revealed no retinal toxicity in ERG. Ad-luciferase via subconjunctival injections showed ocular expression for as long as 112 days by using bioluminescence image analysis in rodent. AAV-luciferase via subconjunctival injections showed ocular expression for as long as 365 days by using bioluminescence image analysis in mice, and AAV serotype 5-luciferase even showed expression lasting for 2 years. Suppression of laser photocoagulation¡Vinduced CNV by Ad-VS was documented in rat model. Combination therapies are important as treatment options. We demonstrated that PDT could effectively attenuate CNV in a rat model, and reduced doses, worked just as well as the standard dose. In the preliminary study of PDT combined topical VS application, treatment led to CNV attenuation more than alone with PDT. The above experiments would enable us to demonstrate that the vasostatin delivery might be a promising strategy for the treatment of AMD and other retinal or ocular disorders. Furthermore, the results from animal studies might be extrapolated for future clinical application.