Amy E. Birsner

2-Methoxyestradiol Inhibits Hypoxia-Inducible Factor-1α and Suppresses Growth of Lesions in a Mouse Model of Endometriosis

Endometriosis, the presence of ectopic endometrial tissue outside the uterine cavity, is a common disease affecting women during their reproductive years. Current therapeutic success is often unsatisfactory because of limited insight into disease mechanisms. Nevertheless, angiogenesis plays an essential role in the pathogenesis of the disease, making it a potential novel target for therapy. In the current study, we demonstrate in an established mouse model of endometriosis that transient hypoxia in transplanted endometriosis-like lesions results in the up-regulation of hypoxia-inducible factor-1alpha (HIF-1alpha), leading to the expression of vascular endothelial growth factor (VEGF), a key player in endometriosis-associated angiogenesis. Systemic treatment with the angiogenesis inhibitor 2-methoxyestradiol suppressed HIF-1alpha expression in vivo, resulting in a decreased downstream expression of HIF-1alpha target genes, such as for VEGF, phosphoglycerate kinase, and glucose transporter-1. 2-Methoxyestradiol also suppressed VEGF-induced vascular permeability, as demonstrated in a modified Miles assay. Finally, systemic treatment with 2-methoxyestradiol significantly inhibited the growth of endometriosis-like lesions in a dose-dependent manner. In conclusion, hypoxia appears to play an important role in the pathogenesis of endometriosis and endometriosis-associated angiogenesis, and the angiogenesis inhibitor 2-methoxyestradiol may be a potential candidate for systemic treatment in the future.

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.

The mouse cornea micropocket angiogenesis assay

The mouse corneal micropocket angiogenesis assay uses the avascular cornea as a canvas to study angiogenesis in vivo. Through the use of standardized slow-release pellets, a predictable angiogenic response is generated over the course of 5 d and then quantified. Uniform slow-release pellets are prepared by mixing purified angiogenic growth factors such as basic fibroblast growth factor or vascular endothelial growth factor with sucralfate (a stabilizer) and Hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate)) to allow slow release). This mixture is applied to a mesh that controls unit size and then allowed to harden. A micropocket is surgically created in the mouse cornea and a pellet implanted. Five days later, the area of the cornea overgrown by the angiogenic response is measured using a slit lamp. A skilled investigator can implant and grade 40 eyes in about 2.5 h. The results of the assay are used to assess the ability of potential therapeutic molecules or genetic differences to modulate angiogenesis in vivo.

Targeted Deletion of the Calcineurin Inhibitor DSCR1 Suppresses Tumor Growth

The NF-AT transcription factors regulated by the phosphatase calcineurin play a role in breast cancer metastasis-promoting tumor cell invasion. Metastasis is a multistep process requiring angiogenesis and endothelial activation. NF-AT is also expressed in endothelial cells, and calcineurin-NF-AT signaling is an important downstream effector of the proangiogenic cytokine VEGF. One isoform of the endogenous calcineurin regulator, Down syndrome candidate region-1 (DSCR1.Ex4), suppresses calcineurin-NFAT signaling blocking endothelial proliferation. However, overexpression of the other DSCR1 isoform (DSCR1.Ex1) may promote angiogenesis. We report that targeted deletion of both isoforms leads to hyperactivated calcineurin and precocious endothelial apoptosis, inhibiting formation of an effective tumor vasculature and suppressing tumorigenesis. Treatment with the specific pharmacological calcineurin inhibitor cyclosporin A rescues this endothelial defect in DSCR1(-/-) mice, restoring tumor growth.

Genetic loci that control vascular endothelial growth factor-induced angiogenesis.

Angiogenesis is regulated by the balance between angiogenic stimulators and inhibitors. Numerous reports have demonstrated that tumors induce aggressive angiogenesis by up‐regulating the production of angiogenesis stimulating growth factors to overcome the baseline levels of endogenous inhibitors. However, the possibility of large differences in the hosts responsiveness to angiogenic factors has been largely overlooked. Using the corneal micropocket neovascularization assay, we have observed >10‐fold differences in responsiveness to either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) among various mouse strains. The inheritance pattern observed for these traits supported a QTL (quantitative trait locus) approach to mapping the genes responsible for the differences in angiogenic responsiveness. To overcome variability in the assay, we used recombinant inbred lines to map this phenotype. In the BXD series of recombinant inbred mouse strains, we have mapped the regions responsible for regulating VEGF‐induced angiogenesis using both composite interval mapping and multiple interval mapping. Both approaches link VEGF responsiveness to regions on chromosomes 2 (near D2Mit6) and 10 (near D10Mit20). Candidate angiogenesisrelated genes in these regions include those for collagen XVIII/endostatin, matrix metalloproteinase 11, integrin β2, prostaglandin D2 synthase, and interleukin‐1 receptor antagonist.

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.

Green tea epigallocatechin-3-gallate inhibits angiogenesis and suppresses vascular endothelial growth factor C/vascular endothelial growth factor receptor 2 expression and signaling in experimental endometriosis in vivo

OBJECTIVE To investigate the antiangiogenesis mechanism of epigallocatechin-3-gallate (EGCG) in an endometriosis model in vivo. DESIGN Animal studies. SETTING University laboratory. ANIMAL(S) Human endometrium from women with endometriosis (n = 10) was transplanted into immunocompromised mice. INTERVENTION(S) Mice (n = 30) were randomly treated with EGCG, vitamin E (antioxidant control), or vehicle (negative control) for microvessel imaging. MAIN OUTCOME MEASURE(S) Endometriotic implants were collected for angiogenesis microarray and pathway analysis. Differentially expressed angiogenesis molecules were confirmed by quantitative polymerase chain reaction, Western blot, and immunohistochemistry. Effects of EGCG on angiogenesis signal transduction were further characterized in a human endothelial cell line. Microvessel parameters and the angiogenesis signaling pathway in endometriotic implants and endothelial cells were studied. RESULT(S) EGCG, but not vitamin E, inhibited microvessels in endometriotic implants. EGCG selectively suppressed vascular endothelial growth factor C (VEGFC) and tyrosine kinase receptor VEGF receptor 2 (VEGFR2) expression. EGCG down-regulated VEGFC/VEGFR2 signaling through c-JUN, interferon-γ, matrix metalloproteinase 9, and chemokine (C-X-C motif) ligand 3 pathways for endothelial proliferation, inflammatory response, and mobility. EGCG also suppressed VEGFC expression and reduced VEGFR2 and ERK activation in endothelial cells. VEGFC supplementation attenuated the inhibitory effects by EGCG. CONCLUSION(S) EGCG inhibited angiogenesis and suppressed VEGFC/VEGFR2 expression and signaling pathway in experimental endometriosis in vivo and endothelial cells in vitro.

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.

Vascular endothelial growth factor C is increased in endometrium and promotes endothelial functions, vascular permeability and angiogenesis and growth of endometriosis

Endometriosis is an angiogenesis-dependent disease. Many studies demonstrated inhibition of angiogenesis leads to inhibition of endometriotic growth, however underlying mechanism is still not fully understood. Our previous study suggested vascular endothelial growth factor C (VEGF-C) as a target of anti-angiogenesis therapy for endometriosis. In this study, VEGF-C in endometrium and its role in angiogenesis of endometriosis were studied. Human endometrium were obtained from women with and without endometriosis for molecular studies. VEGF-A, VEGF-B, VEGF-C and VEGF-D mRNA and proteins in eutopic and ectopic endometrium were measured. Human endothelial cells were transfected with VEGF-C siRNA in vitro, effects of VEGF-C on endothelial cell migration, invasion and tube formation were investigated in vitro. Angiogenesis was inhibited in wild type mice, vascular permeability in dermal skin was determined in vivo. Transplanted endometrium were inhibited by VEGF-C siRNA in immunocompromised mice, development, growth and angiogenesis of the experimental endometriosis were compared in vivo. The results showed that VEGF-C mRNA and protein were increased in eutopic and ectopic endometrium of endometriosis patients. VEGF-C siRNA significantly inhibited endothelial cell migration and tube formation. VEGF-C siRNA significantly inhibited development and angiogenesis of the experimental endometriotic lesions in mice. Supplementation and over-expression of VEGF-C significantly reversed the inhibitory effects on the endothelial functions, vascular permeability and endometriotic growth. In conclusion, VEGF-C is increased in endometrium and it promotes endothelial functions, vascular permeability and development of experimental endometriosis. VEGF-C is important for angiogenesis in endometriosis.

Melanocyte-secreted fibromodulin promotes an angiogenic microenvironment

Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.