Recombinant Endostatin Research Articles

High-level synthesis of recombinant murine endostatin in Chinese hamster ovary cells

Endostatin, a carboxy-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent that specifically inhibits proliferation of endothelial cells and growth of various primary tumors. Here, we describe the expression by Chinese hamster ovary (CHO) cells of murine endostatin and of a tagged-fusion protein, (his) 6-met-endostatin. A dicistronic mRNA expression vector was utilized in which endostatin cDNA was inserted upstream of the amplifiable marker gene, dihydrofolate reductase (DHFR). After transfection of the expression vectors, stepwise increments in methotrexate levels in the culture medium were applied, promoting gene amplification and increasing expression levels of the proteins of interest. The expression level of secreted native endostatin was about 78 μg/mL while the one for secreted (his) 6-met-endostatin was about 114 μg/mL, for the best expressing clones. Characterization of physico-chemical and immunological activities of the proteins was performed using SDS–PAGE and Western blotting. The biological activities of recombinant endostatins were tested with a cow pulmonary artery endothelial (C-PAE) cell proliferation assay. Both recombinant endostatin and (his) 6-met-endostatin inhibited, in a dose-dependent fashion, growth of C-PAE cells stimulated by basic fibroblast growth factor (bFGF).

Characterization of Endostatin Binding to Heparin and Heparan Sulfate by Surface Plasmon Resonance and Molecular Modeling: ROLE OF DIVALENT CATIONS

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.

Inhibition of cytokine-induced microvascular arrest of tumor cells by recombinant endostatin prevents experimental hepatic melanoma metastasis.

We investigated effects of endostatin (ES) in the prometastatic microenvironment of inflammation occurring during the microvascular phase of cancer cell infiltration in the liver. We used a model of intrasplenic injection of B16 melanoma (B16M) cells leading to hepatic metastasis through vascular cell adhesion molecule-(VCAM-1)-mediated capillary arrest of cancer cells via interleukin-18 (IL-18)-dependent mechanism. We show that administration of 50 mg/kg recombinant human (rh) ES 30 min before B16M, plus repetition of same dose for 3 additional days decreased metastasis number by 60%. A single dose of rhES before B16M injection reduced hepatic microvascular retention of luciferase-transfected B16M by 40% and inhibited hepatic production of tumor necrosis factor alpha (TNF-alpha) and IL-18 and VCAM-1 expression by hepatic sinusoidal endothelia (HSE). Consistent with these data, rhES inhibited VCAM-1-dependent B16M cell adhesion to primary cultured HSE receiving B16M conditioned medium, and it abolished the HSE cell production of TNF-alpha and IL-18 induced by tumor-derived vascular endothelial cell growth factor (VEGF). rhES abrogated recombinant murine VEGF-induced tyrosine phosphorylation of KDR/flk-1 receptor in HSE cells, preventing the proinflammatory action of tumor-derived VEGF on HSE. rhES also abolished hepatic production of TNF-alpha, microvascular retention of luciferase-transfected B16M, and adhesion of B16M cells to isolated HSE cells, all of them induced in mice given 5 micro g/kg recombinant murine VEGF for 18 h. This capillary inflammation-deactivating capability constitutes a nonantiangiogenic antitumoral action of endostatin that decreases cancer cell arrest within liver microvasculature and prevents metastases promoted by proinflammatory cytokines induced by VEGF.

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Tsiridis et al. presented an unusual case of avascular necrosis in the peripheral bone, which was later found to be combined with a glioblastoma in the brain. They suspected the avascular necrosis was caused by an endogenous inhibitor of angiogenesis, such as endostatin or transforming growth factor β, secreted from the glioblastoma tissue. Their hypothesis is very attractive. However, it is necessary to show data supporting a direct causal relationship between a glioblastoma and avascular necrosis of the peripheral bone. Detection of the tissue levels or serum levels of endostatin or transforming growth factor β could support the hypothesis. Although we showed increased levels of tissue endostatin in glioblastomas (1), we unfortunately did not examine the levels of endostatin in the serum for these patients. Furthermore, there have been no reports in which glioblastomas or other tumors secreting high levels of endostatin are combined with avascular necrosis of the peripheral bone (2, 3). With regard to the systemic effects of antiangiogenic therapies suggested by Tsiridis et al., no side effects such as avascular necrosis of the peripheral bone have been reported in a Phase I clinical trial of recombinant endostatin (4). However, this case report by Tsiridis et al. will promote further careful survey for the systemic effects of antiangiogenic factors in tumors secreting high levels of endostatin as well as in clinical application of recombinant endostatin.

Disparate effects of endostatin on tumor vascular perfusion and hypoxia in two murine mammary carcinomas

Purpose Recent results in the literature have demonstrated that the antiangiogenic agent endostatin can enhance antitumor effects when administered before or during radiotherapy. To better understand the underlying pathophysiologic basis for this radiosensitization, the current study investigated whether short-term endostatin administration is linked to alterations in tumor vascular perfusion and oxygen delivery. Methods and materials Three daily doses of recombinant endostatin (20 mg/kg) were administered to two murine mammary carcinomas, the highly vascularized MCa-35 and the less vascularized MCa-4. Image analysis techniques were used to quantify ( 1) total and perfused vascular spacing, and ( 2) changes in tumor hypoxia as a function of distance from the nearest blood vessel. Results In MCa-35 tumors, endostatin had no effect on vessel spacing, tumor hypoxia, or tumor growth. In MCa-4 tumors, total and perfused vessel spacings were also unchanged, but tumor growth was inhibited, and tumor hypoxia significantly decreased. These tumors demonstrated an increased vascular functionality suggestive of an increase in the number of intermittently perfused vessels, without corresponding alterations in tumor oxygen consumption rate. Conclusions Poorly vascularized, hypoxic mammary carcinomas were much more responsive to short-term endostatin treatment than well-vascularized, more homogeneously oxygenated tumors. Oxygen levels in the responsive tumors were transiently improved after treatment, which could have substantial implications with respect to the therapeutic effectiveness of combining antiangiogenic agents with conventional therapies.

Inhibitory effect of recombinant endostatin on angiogenesis and tumor growth of hepatoma.

To study the influence of recombinant endostatin on angiogenesis and tumor growth of mice H22 hepatoma, tumor models were constructed by injecting H22 hepatoma cells into the leg muscle of mice. Recombinant endostatin was produced by gene engineering in E. coli. The recombinant protein was injected subcutaneously to treat transplanted hepatoma faraway. The weight of tumors was measured, and the changes of necrosis of tumor cells and vessel density were observed by immunohistochemistry. The results suggested that the growth of hepatoma models transplanted in the muscle of legs was suppressed by recombinant endostatin. The density of vascularity was decreased, but the necrosis of tumor cells increased. The inhibitory effect of recombinant endostatin on angiogenesis and tumor growth of hepatoma was not affected after chemotherapy.

Endostatin Associates with Lipid Rafts and Induces Reorganization of the Actin Cytoskeleton via Down-regulation of RhoA Activity

Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Observations that endostatin inhibits endothelial cell migration and induces disassembly of the actin cytoskeleton provide putative cellular mechanisms for this effect. To understand the mechanisms of endostatin-induced intracellular signaling, we analyzed the association of recombinant endostatin with endothelial cell lipid rafts and the roles of its heparin- and integrin-binding properties in this interaction. We observed that a fraction of cell surface-bound endostatin partitioned in low density membrane raft fractions together with caveolin-1. Heparinase treatment of cells prevented the recruitment of endostatin to the lipid rafts but did not affect the association of endostatin with the non-raft fraction, whereas preincubation of endostatin with soluble alpha5beta1 integrin prevented the association of endostatin with the endothelial cell membrane. Endostatin treatment induced recruitment of alpha5beta1 integrin into the raft fraction via a heparan sulfate proteoglycan-dependent mechanism. Subsequently, through alpha5beta1 integrin, heparan sulfate, and lipid raft-mediated interactions, endostatin induced Src-dependent activation of p190RhoGAP with concomitant decrease in RhoA activity and disassembly of actin stress fibers and focal adhesions. These observations provide a cell biological mechanism, which plausibly explains the anti-angiogenic mechanisms of endostatin in vivo.

Recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro

Endostatin is a fragment of collagen XVIII that acts as an endogenous inhibitor of tumor angiogenesis and tumor growth. Anti-tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study we have investigated the structure and cellular effects of insoluble endostatin. We found that insoluble endostatin shows all the hallmarks of amyloid aggregates. Firstly, it binds Congo red and shows the characteristic apple-green birefringe when examined under polarized light. Secondly, electron microscopy shows that endostatin forms short unbranched fibrils. Thirdly, X-ray analysis shows the abundant presence of cross-β sheets, the tertiary structure that underlies fibrillogenesis. None of these properties was observed when examining soluble endostatin. Soluble endostatin can be triggered to form cross-β sheets following denaturation, indicating that endostatin is a protein fragment with an inherent propensity to form amyloid deposits. Like β-amyloid, found in the brains of patients with Alzheimer’s disease, amyloid endostatin binds to and is toxic to neuronal cells, whereas soluble endostatin has no effect on cell viability. Our results demonstrate a previously unrecognized functional difference between soluble and insoluble endostatin, only the latter acting as a cytotoxic amyloid substance.

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells.

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 microg ml(-1). A maximum production of 4.6 microg recombinant tumstatin (10(7) cells)(-1) was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO4.

Gene therapy delivery of endostatin enhances the treatment efficacy of radiation.

To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation. Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29). Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05). The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.

The effect of human recombinant endostatin (rh-Endo) on blood flow and glucose metabolism in normal tissues

The effect of human recombinant endostatin (rh-Endo) on blood flow and glucose metabolism in normal tissues

Study of the media's potential influence on prospective research participants' understanding of and motivations for participation in a high-profile phase I trial.

To describe prospective participants' initial source of information about, understanding of, and motivation to participate in a phase I clinical trial of the antiangiogenesis agent human recombinant endostatin. We surveyed 100 of 130 persons referred to the endostatin trial between October 1999 and November 2000 and analyzed media coverage of the agent from 1997 to 2000. Forty-seven percent of survey respondents first heard about the trial from media reports. Fifty-one percent of these subsequently contacted their physicians. Thirty-three percent of respondents correctly understood the purpose of the trial. Seventy-nine respondents were interviewed before they met trial investigators to discuss the trial. Of these, those who first heard about endostatin from the media were five times more likely to understand correctly the trial's purpose than those who first heard from other sources. Seventy-four percent (70 of 95) of respondents cited hope for personal benefit as the main reason for their willingness to enroll. Those who first heard about endostatin from the media were no more motivated by hope of personal benefit (77%) than those who first heard from other sources (71%) (P =.46). Ninety-nine percent of all respondents cited "joining the study gives me hope" as a contributing factor in their decision making about the trial. Media coverage prompted prospective participants to contact their physicians but did not seem to hinder understanding nor could it be shown to heighten their hope for personal benefit.

Surface-enhanced laser desorption–ionization retentate chromatography™ mass spectrometry (SELDI–RC–MS): a new method for rapid development of process chromatography conditions

Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array’s functionalized surface, an interaction occurs between the array’s surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography™–mass spectrometry (RC–MS), the “retentate” components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention–elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC–MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC–MS method.

Antiangiogenic and antitumor effects of endostatin on follicular thyroid carcinoma.

Tumor growth and metastasis depend on blood supply and blood vessel formation. Angiogenesis, therefore, represents a promising target for cancer therapy. Endostatin is one of the most potent antiangiogenic factors and has been shown to effectively inhibit angiogenesis and tumor growth in a variety of in vivo models. In this study, we tested the effects of endostatin on xenografted human follicular thyroid carcinoma (FTC) in nude mice. Our result demonstrated that recombinant endostatin significantly inhibited the growth of FTC xenografts. Furthermore, we established an endostatin-expressing FTC cell line (FTC-BmEndo) using retrovirus-mediated gene transfer approach. We found that the in vivo growth of FTC-BmEndo cells was significantly inhibited, compared with the parental FTC cells, whereas both lines grew at the same rate in vitro. High-level expression of endostatin within the FTC-BmEndo tumors was evidenced by immunohistochemical staining, paralleled with a reduced microvessel density. The systemic level of vascular endothelial growth factor was significantly lower in mice bearing the FTC-BmEndo tumors than in those bearing parental FTC tumors. By using two different approaches, namely the recombinant endostatin protein and the gene therapy strategy, our study demonstrated that endostatin could be effective in suppressing the growth of human FTC in immunodeficient mice.

Endostatin Concentrations in Healthy Dogs and Dogs with Selected Neoplasms

Endostatin prevents angiogenesis and tumor growth by inhibiting endothelial cell proliferation and migration. The purpose of this study was to determine serum endostatin concentrations in 53 healthy dogs and in 38 dogs with confirmed malignant neoplasms. Endostatin concentration was determined with a competitive enzymatic immunoassay (EIA) with rabbit polyclonal antibody generated against a recombinant canine endostatin protein. Both the presence of cancer and increasing age were associated with increased serum concentration of endostatin. Endostatin concentration in healthy dogs was 87.7 ± 3.5 ng/mL. Upper and lower limits of the reference range for serum endostatin concentration in healthy dogs were 60 and 113 ng/mL. Dogs with lymphoma (LSA) and hemangiosarcoma (HSA) had endostatin concentrations of 107 ± 9.3 ng/mL. In conclusion, this study demonstrates that endostatin can be quantified in dogs and that endostatin concentrations are high in dogs with HSA and LSA.

Expression of human endostatin in larvae of silkworm (Bombyx mori) and in vitro activity assays.

Human endostatin is a novel antiangiogenic molecule, which can inhibit the proliferation and development of new blood vessels, and experimentally can cause nearly complete regression of established tumors. In this paper, the cDNA encoding human endostatin was cloned into a baculovirus shuttle vector pBacPAK8 and co-infected with linearized Bm-BacPAK6 DNA into and BmN cells. The recombinant virus was screened and identified by PCR, DNA and RNA dot hybridization, and ELISA assay. The recombinant endostatin was expressed in culture cells, and the larvae and pupa of silkworm by inoculation of recombinant virus. The biological activity assay showed that the expression product in larvae was over 150 microg/ml, about 50-fold higher than that expressed in cultured cells. SDS-PAGE and Western blotting analysis showed a pattern of molecular weight of about 20 kDa. The bio-activity of the protein product was determined by human umbilical vein endothelial cells (ECV304) proliferation test in vitro and the chick chorioallantoic membrane (CAM) vascular inhibition test. Endostatin showed significant inhibitory effect on endothelial cells in a dose-dependent manner. Silkworm-produced endostatin induced apoptosis of endothelial cells and also inhibited angiogenesis in the CAM assay. Combination regimen using angiostatin and endostatin showed more than additive effect in angiogenic inhibition and increasing apoptosis when compared with treatment with the individual antiangiogenic protein.

Endostatin gene transfer inhibits joint angiogenesis and pannus formation in inflammatory arthritis.

Rheumatoid arthritis is a prevalent example of an inflammatory angiogenic disease, which is mediated by pro-inflammatory and pro-angiogenic cytokines such as tumor necrosis factor (TNF). To evaluate the effect of the potent anti-angiogenic factor endostatin on TNF-induced inflammatory arthritis, we injected an endostatin-expressing lentiviral vector directly into the joints of human TNF-transgenic mice before the onset of disease. Histological analysis of the injected joints 8 weeks later revealed that endostatin reduced blood vessel density within the synovial tissues and an overall mean arthritis index. In vitro and in vivo examination of the potential mechanism by which endostatin inhibited the arthritis revealed that endostatin blocks TNF-induced activation of JNK and JNK-dependent pro-angiogenic gene expression. These data suggest a novel mechanism by which endostatin inhibits angiogenesis, and demonstrates the potential utility of anti-angiogenic gene therapy for treatment of inflammatory arthritis.

High-level expression and secretion of recombinant mouse endostatin by Escherichia coli.

The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase.

Continuous intravascular secretion of endostatin in mice from transduced hematopoietic stem cells.

Endostatin, a 20-kDa carboxy-terminal fragment of collagen XVIII, is the leading member of a class of physiologic inhibitors of angiogenesis with potent antitumor activity. Repeated subcutaneous administration of recombinant endostatin in mice led to permanent regression of established tumors to a microscopic dormant state and prompted the initiation of human clinical trials. However, a discrepancy remained unresolved: sustained tumor regression has only been observed with a non-soluble, precipitated form of recombinant endostatin produced in bacteria. To shed light on this question and establish a model of systemic anti-angiogenic gene therapy of cancer that may surmount obstacles in protein production and delivery, we transduced murine hematopoietic stem cells with a retrovirus encoding a secretable form of endostatin. Despite continuous, high-level secretion of endostatin in the vasculature of all transplanted mice, we detected neither inhibition of in vivo neoangiogenesis nor antitumor activity. Resolution of this paradox may come from human trials of endostatin now underway.

Dephosphorylation of endothelial nitric oxide synthase contributes to the anti-angiogenic effects of endostatin.

Endostatin is an anti-angiogenic factor that inhibits endothelial cell (EC) migration and induces EC apoptosis. Because nitric oxide (NO) plays a key role in vascular endothelial growth factor (VEGF)-induced angiogenesis, we hypothesized that endostatin interferes with the activation of the endothelial NO synthase (eNOS). Human recombinant endostatin significantly reduced VEGF-induced NO-release, which suggests that endostatin inhibits eNOS activation. Because the activation of eNOS by VEGF is associated with the Akt-dependent phosphorylation of eNOS at Ser1177, we investigated whether endostatin interferes with phosphorylation of eNOS. Endostatin reduced VEGF-induced phosphorylation of eNOS at Ser1177, whereas Akt phosphorylation was not affected. Coinciding with the inhibition of eNOS phosphorylation, endostatin completely blocked VEGF-induced EC migration. The NO-donor SNAP reversed the inhibitory effect of endostatin on EC migration. In addition, endostatin significantly inhibited VEGF-induced tube formation, whereas endostatin did not affect tube formation induced by NO. Finally, a non-dephosphorylatable constitutive active eNOS construct (S1177D), but not constitutive active Akt, abolished the inhibitory effect of endostatin on EC migration. Endostatin activated PP2A, which is known to directly dephosphorylate eNOS at Ser1177. Inhibition of PP2A prevented the inhibitory effect of endostatin. Thus, endostatin inhibits VEGF-induced EC migration and angiogenesis upstream of NO-synthesis via dephosphorylation of eNOS at Ser1177.