We assessed the antiangiogenic effects of subconjunctival injection of Fc-endostatin (FcE) using a human vascular endothelial growth factor-induced rabbit corneal neovascularization model. Angiogenesis was induced in rabbit corneas through intrastromal implantations of VEGF polymer implanted 2 mm from the limbus. NZW rabbits were separated into groups receiving twice weekly subconjunctival injections of either saline; 25 mg/mL bevacizumab; 2 mg/mL FcE; or 20 mg/mL FcE. Corneas were digitally imaged at 5 time points. An angiogenesis index (AI) was calculated (vessel length (mm) × vessel number score) for each observation. All treatment groups showed a significant decrease in the vessel length and AI compared to saline on all observation days (P<0.001). By day 15, FcE 2 inhibited angiogenesis significantly better than FcE 20 (P<0.01). There was no significant difference between FcE 2 and BV, although the values trended towards significantly increased inhibition by BV. BV was a significantly better inhibitor than FcE 20 by day 8 (P<0.01). FcE was safe and significantly inhibited new vessel growth in a rabbit corneal neovascularization model. Lower concentration FcE 2 exhibited better inhibition than FcE 20, consistent with previous FcE studies referencing a biphasic dose-response curve. Additional studies are necessary to further elucidate the efficacy and clinical potential of this novel angiogenesis inhibitor.

Antiangiogenesis therapy has become a vital part of the armamentarium against cancer. Hypertension is a dose-limiting toxicity for VEGF inhibitors. Thus, there is a pressing need to address the associated adverse events so these agents can be better used. The hypertension may be mediated by reduced NO bioavailability resulting from VEGF inhibition. We proposed that the hypertension may be prevented by coadministration with endostatin (ES), an endogenous angiogenesis inhibitor with antitumor effects shown to increase endothelial NO production in vitro. We determined that Fc-conjugated ES promoted NO production in endothelial and smooth muscle cells. ES also lowered blood pressure in normotensive mice and prevented hypertension induced by anti-VEGF antibodies. This effect was associated with higher circulating nitrate levels and was absent in eNOS-knockout mice, implicating a NO-mediated mechanism. Retrospective study of patients treated with ES in a clinical trial revealed a small but significant reduction in blood pressure, suggesting that the findings may translate to the clinic. Coadministration of ES with VEGF inhibitors may offer a unique strategy to prevent drug-related hypertension and enhance antiangiogenic tumor suppression.

Antiangiogenic inhibitors were shown to enhance antitumor effects of photon radiation therapy via at least two mechanisms, i.e., resensitizing tumor endothelium to radiation cell kill and “normalizing” tumor pathophysiology. We aimed to investigate the effect of antiangiogenic treatment combined with heavy ion (carbon) radiation therapy. The combination of fractionated and single dose carbon ion radiation therapy with angiogenesis inhibitor Fc-endostatin was investigated using s.c. human epidermoid carcinoma, A431, and human pancreatic adenocarcinoma, BxPC-3 xenograft models in NCr nude mice. In addition, the effects of the combined treatment on vascularity and vessel maturity of the respective tumors as well as on the angiogenic potential of HUVECs and HDMECs in vitro were assessed. The animal experiments were conducted in four arms, i.e., control (PBS), 100μg Fc-endostatin s.c. every 6 days, carbon irradiation (1x4Gy for A431 and 4x3GyE for BxPC-3) and dual combination regimen. Tumor volume was detected by caliper measurements. In vitro angiogenesis assay was performed using an endothelial-fibroblast 3D model and time-lapse microscopy. Apoptosis was detected by caspase-3/7 activity and evaluated 24h after irradiation. Tumor proliferation (Ki67) and microvessel density (MVD, anti-CD31/SMA) were assessed by IHC. In A431 models, the combination of Fc-endostatin with carbon ion therapy significantly prolonged time to progression as compared to radiation therapy alone (p < 0.05) or endostatin treatment alone (p < 0.01). Fc-endostatin combined with 4 daily fractions of 3 GyE carbon ion irradiation markedly enhanced tumor growth delay of BxPC-3 xenografts as compared to both monotherapy arms. Combined treatment exerted potent antiangiogenic effects in co-culture angiogenesis assay in vitro and reduced tumor MVD in vivo. Fc-endostatin also increased carbon irradiation -induced endothelial cell apoptosis. We report here the first preclinical data on beneficial effects of combined antiangiogenesis and carbon radiation therapy.

OBJECT:Brain tumors pose many unique challenges to treatment. The authors hypothesized that Fc-endostatin may be beneficial. It is a newly synthesized recombinant human endostatin conjugated to the Fc domain of IgG with a long half-life (weeks) and unknown toxicity. The authors examined the efficacy of Fc-endostatin using various delivery methods. METHODS:Efficacy was assessed using the intracranial 9L gliosarcoma rat model treated with Fc-endostatin for use in rodents (mFc-endostatin), which was administered either systemically or locally via different delivery methods. Oral temozolomide (TMZ) was administered in combination with mFc-endostatin to determine if there was a beneficial synergistic effect. RESULTS:Intracranial delivery of mFc-endostatin via a polymer or convection-enhanced delivery 5 days after tumor implantation increased median survival, compared with the control group (p = 0.0048 and 0.003, respectively). Animals treated weekly with subcutaneous mFc-endostatin (started 5 days post-tumor implantation) also had statistically improved survival as compared with controls (p = 0.0008). However, there was no statistical difference in survival between the local and systemic delivery groups. Control animals had a median survival of 13 days. Animals treated either with subcutaneous mFc-endostatin weekly or with polymer had a median survival of 18 and 15 days, respectively, and those treated with oral TMZ for 5 days (Days 5-9) had a median survival of 21 days. Survival was further increased with a combination of oral TMZ and mFc-endostatin polymer, with a median survival of 28 days (p = 0.029, compared with TMZ alone). Subcutaneous mFc-endostatin administered every week starting 18 days before tumor implantation significantly increased median survival when compared with controls (p = 0.0007), with 12.5% of the animals ultimately becoming long-term survivors (that is, survival longer than 120 days). The addition of TMZ to either weekly or daily subcutaneous mFc-endostatin and its administration 18 days before tumor implantation significantly increased survival (p = 0.017 and 0.0001, respectively, compared with TMZ alone). Note that 12.5% of the animals treated with weekly subcutaneous mFc-endostatin and TMZ were long-term survivors. CONCLUSIONS:Systemically or directly (local) delivered mFc-endostatin prolonged the survival of rats implanted with intracranial 9L gliosarcoma. This benefit was further enhanced when mFc-endostatin was combined with the oral chemotherapeutic agent TMZ.

Vascular endothelial growth factor VEGF165 is a critical element for development of the vascular system in physiological and pathological angiogenesis. VEGF isoforms have different affinities for heparan sulphate proteoglycan (HSPG) as well as for VEGF receptors; HSPGs are important regulators in vascular development. Therefore, inhibition of interactions between VEGF and HSPGs may prevent angiogenesis. Here, we demonstrate that an HSPG-binding synthetic peptide, corresponding to exon 6a-encoded domain of VEGF gene, has anti-angiogenic property. This 20 amino acids synthetic peptide prevents VEGF165 binding to several different cell types, mouse embryonic sections and inhibits endothelial cell migration, despite its absence in VEGF165 sequence. Our in vivo anti-tumor studies show that the peptide inhibits tumor growth in both mouse Lewis-Lung Carcinoma and human Liposarcoma tumor-bearing animal models. This is the first evidence that a synthetic VEGF fragment corresponding to exon 6a has functional antagonism both in vitro and in vivo. We conclude that the above HPSG binding peptide (6a-P) is a potent inhibitor of angiogenesis-dependent diseases.

Angiogenesis refers to growth of blood vessels from pre-existing ones. In 1971, Folkman proposed that by choking off the blood supply to tumors, they are starved, leading to their demise. A few years ago, the monoclonal antibody Avastin became the first antiangiogenic biological approved by FDA, for treatment of cancer patients. Two other antiangiogenic endogenous protein fragments were isolated in Folkman's laboratory more than a decade ago. Here, we present a short review of data demonstrating that angiostatin and endostatin display a biphasic antitumor dose-response. This behavior is common among a large number of antiangiogenic agents and the reduced effectiveness of antiangiogenic agents at high dose rates may be due to suppression of growth of new vessels carrying the agent into the critical region around the tumor.

Angiostatin, a proteolytic fragment of plasminogen, is a potent endogenous antiangiogenic agent. The molecular mechanisms governing angiostatin's antiangiogenic and antitumor effects are not well understood. Here, we report the identification of mitochondrial compartment as the ultimate target of angiostatin. After internalization of angiostatin into the cell, at least 2 proteins within the mitochondria bind this molecule: malate dehydrogenase, a member of Krebs cycle, and adenosine triphosphate synthase. In vitro and in vivo studies revealed differential regulation of key prosurvival and angiogenesis-related proteins in angiostatin-treated tumors and tumor-endothelium. Angiostatin induced apoptosis via down-regulation of mitochondrial BCL-2. Angiostatin treatment led to down-regulation of c-Myc and elevated levels of another key antiangiogenic protein, thrombospondin-1, reinforcing its antitumor and antiangiogenic effects. Further evidence is provided for reduced recruitment and infiltration of bone marrow-derived macrophages in angiostatin-treated tumors. The observed effects of angiostatin were restricted to the tumor site and were not observed in other major organs of the mice, indicating unique tumor specific bioavailability. Together, our data suggest mitochondria as a novel target for antiangiogenic therapy and provide mechanistic insights to the antiangiogenic and antitumor effects of angiostatin.

378 Angiostatin is a key endogenous angiogenesis inhibitor. Since its discovery in 1994, a number of studies have reported potent anti-tumor and antiangiogenic activities of this agent. However, the molecular mechanism of action, including definite receptors or binding partners and the consecutive intracellular signalling patterns are not completely understood. The aim of this study was to identify potential receptor and cellular binding partners of angiostatin. First, the Fc-fragment of immunoglobulin was linked to human angiostatin by recombinant expression of fused hFc-angiostatin. We used the Fc fragment as a tag to tract and detect potential membrane, cytosolic, nuclear and mitochondrial binding partners of angiostatin. A comprehensive protein array platform, containing ~ 27,648 unique protein fragments, was generated using cDNA expression clones in an E. coli expression vector. Proteins were verified for proper expression and printed in duplicates onto two PVDF membranes. This represents one of the largest protein sets interrogated in array-based protein-protein interaction studies to date. Differential binding of human Fc-angiostatin vs. human Fc fragment with spotted proteins on the array were detected using HRP-conjugated anti-Fc antibody. This technical platform enabled us to identify a series of novel angiostatin binding partners. Noteworthy, a substantial portion of angiostatin binding partners were located to the mitochondrial compartment and are functionally involved in the cellular metabolism and oxidative processes e.g. peroxiredoxins, glutathione S-transferase, NADH dehydrogenase or mitochondrial import receptor subunit (TOM20 homolog). In support to the quality of our array based approach, we also found several previously described angiostatin interacting proteins such as annexin A2. Angiostatin binding to a selected subset of identified proteins was further confirmed by immuno-precipitation analysis. The affinity and binding kinetics of angiostatin to the identified proteins are evaluated using label-free surface plasmon resonance based technology (Biacore). The in-vivo importance of the identified protein-protein interactions in angiostatin signalling is confirmed by cellular co-localization and functional gain (c-DNA transfection) and loss of function (RNAi) studies. Here, we describe a novel high-throughput screening strategy to detect potential protein-protein interactions. Using this method, we identified a series of potential targets of angiostatin. Functional validation of these proteins might not only enhance our understanding of angiostatin’s mechanism of action but also elucidate promising targets for the antiangiogenic cancer therapy.

Purpose: The half-life of the antiangiogenic molecule endostatin that has been used in clinical trial is short (∼2 h). In addition, ∼50% of the clinical grade endostatin molecules lack four amino acids at their NH2 termini. Lack of these amino acids gives rise to a molecule that is devoid of zinc, resulting in no antitumor activity. Our goal was to develop a new version of endostatin that does not show such deficiency. Experimental Design: A recombinant human endostatin conjugated to the Fc domain of IgG was constructed and expressed in mammalian cell culture. The presence of Fc has been shown by previous investigators to play a major role in increasing the half-life of the molecule. Fc-endostatin was tested in tumor-bearing mice, and its half-life was compared with the clinical grade endostatin. Results: The antitumor dose of Fc-endostatin was found to be ∼100 times less than the clinical grade endostatin. The half-life of Fc-endostatin in the circulation was found to be weeks rather than hours, as observed for endostatin alone. In addition, a U-shaped curve was observed for antitumor activity of endostatin as a function of endostatin concentration delivered to the animals. Conclusion: Fc-endostatin is a superior molecule to the original clinical endostatin. Due to its long half-life, the amount of protein required is substantially reduced compared with the clinically tested endostatin. Furthermore, in view of the U-shaped curve of efficacy observed for endostatin, we estimate that the requirement for Fc-endostatin is ∼700-fold less than endostatin alone. The half-life of endostatin is similar to that of vascular endothelial growth factor–Trap and Avastin, two other antiangiogenic reagents. We conclude that a new clinical trial of endostatin, incorporating Fc, may benefit cancer patients.

4598 Endostatin, a 187 amino acid fragment from the C-terminus of collagen 18 has been shown to be a potent angiogenic inhibitor. Recently, it was demonstrated that p53 upregulates prolyl-4-hydoxylase, the enzyme responsible for cleaving endostatin from collagen 18. Because, endostatin has a very short half-life, we have expressed this protein fused to Fc. The half-life of endostatin is approximately 4 hours whereas it is approximately two weeks for Fc-endostatin, following injection in mice. This difference in half-life makes it possible to employ significantly smaller doses of Fc-endostatin. Previously, our laboratory established that the efficacy of endostatin in mice follows a U-shaped curve when examined as a function of the protein concentration. Anti-tumor activities of endostatin and Fc-endostatin were investigated. We now report that the amount required for inhibition of tumors by Fc-endostatin is at least 150-fold less than that of endostatin. This finding will likely have an impact on future trials of this inhibitor. Endostatin has been administered to patients in a Phase I and II clinical trials. Employing Fc-endostatin should enable us to generate sufficient protein for a more meaningful clinical trial of endostatin. Both Avastin (anti-VEGF monoclonal antibody produced by Genentech) and VEGF-trap ( Regeneron Pharmaceuticals ) employ similar Fc constructs for increasing the half-lives of their anti-angiogenic proteins.

Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with Nand 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface.

Developing continuous systemic delivery of endostatin has been a goal of many laboratories. We have employed a method of gene therapy utilizing different viral constructs. Here, we report that a new serotype of adeno-associated viruses, which incorporates canine endostatin, provides dose-dependent transgene expression in the circulation after intramuscular injection in mice. Elevated levels of endostatin remained stable in the circulation for at least 4 months. In vitro assays determined that the protein expressed was biologically active. Antitumor activities of the above construct demonstrated a U-shape curve, where the maximum activity was observed within a certain critical concentration range. These data suggest that an optimum dose range may be required to achieve therapeutic efficacy in large animal models.