Kashi Javaherian

Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis

Plaque angiogenesis promotes the growth of atheromas, but the functions of plaque capillaries are not fully determined. Neovascularization may act as a conduit for the entry of leukocytes into sites of chronic inflammation. We observe vasa vasorum density correlates highly with the extent of inflammatory cells, not the size of atheromas in apolipoprotein E-deficient mice. We show atherosclerotic aortas contain activities that promote angiogenesis. The angiogenesis inhibitor angiostatin reduces plaque angiogenesis and inhibits atherosclerosis. Macrophages in the plaque and around vasa vasorum are reduced, but we detect no direct effect of angiostatin on monocytes. After angiogenesis blockade in vivo, the angiogenic potential of atherosclerotic tissue is suppressed. Activated macrophages stimulate angiogenesis that can further recruit inflammatory cells and more angiogenesis. Our findings demonstrate that late-stage inhibition of angiogenesis can interrupt this positive feedback cycle. Inhibition of plaque angiogenesis and the secondary reduction of macrophages may have beneficial effects on plaque stability.

A 27-Amino-Acid Synthetic Peptide Corresponding to the NH2-Terminal Zinc-Binding Domain of Endostatin Is Responsible for Its Antitumor Activity

The first recombinant endostatin that elicited strong antitumor activity was expressed in Escherichia coli and administered as a suspension. Under these conditions, the protein retained its full antiangiogenic activity. Lack of requirement for a folded structure prompted us to investigate antitumor properties of synthetic peptides corresponding to different regions of endostatin. Here, we show that the entire antitumor, antimigration, and antipermeability activities of endostatin are mimicked by a 27-amino-acid peptide corresponding to the NH2-terminal domain of endostatin. This peptide contains three histidines that are responsible for zinc binding. Mutations of the zinc-binding histidines abolished its antitumor and antimigration activities, but not antipermeability properties.

Endostatin therapy reveals a U-shaped curve for antitumor activity

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.

Linking Antibody Fc Domain to Endostatin Significantly Improves Endostatin Half-life and Efficacy

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.

Angiostatin regulates the expression of antiangiogenic and proapoptotic pathways via targeted inhibition of mitochondrial proteins

Angiostatin, a proteolytic fragment of plasminogen, is a potent endogenous antiangiogenic agent. The molecular mechanisms governing angiostatins 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.

Endostatin lowers blood pressure via nitric oxide and prevents hypertension associated with VEGF inhibition

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.

The Morphogenic Properties of Oligomeric Endostatin Are Dependent on Cell Surface Heparan Sulfate

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 N- and 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.

Improvement in the standard treatment for experimental glioma by fusing antibody Fc domain to endostatin

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.

Oligomeric collagen XVIII‐derived endostatin requires cell surface heparan sulfate in order to induce morphological changes in endothelial and epithelial cell lines in vitro

Introduction  Monomeric endostatin (HEM) has been extensively investigated as an endogenous anti‐angiogenic molecule. In contrast, oligomeric endostatin (HED) induces a promigratory phenotype in endothelial cells in vitro that can be inhibited by an EM in a putative negative feedback loop ( Kuo et al. 2001 ). Endostatin is heparin‐binding but the role of cell‐surface glycosaminoglycans (GAG) in regulating the activity of HEM/HED is unclear.