Weilan Ye

The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation

Vascular development is a complex but orderly process that is tightly regulated. A number of secreted factors produced by surrounding cells regulate endothelial cell (EC) differentiation, proliferation, migration and coalescence into cord-like structures. Vascular cords then undergo tubulogenesis to form vessels with a central lumen. But little is known about how tubulogenesis is regulated in vivo. Here we report the identification and characterization of a new EC-derived secreted factor, EGF-like domain 7 (Egfl7). Egfl7 is expressed at high levels in the vasculature associated with tissue proliferation, and is downregulated in most of the mature vessels in normal adult tissues. Loss of Egfl7 function in zebrafish embryos specifically blocks vascular tubulogenesis. We uncover a dynamic process during which gradual separation and proper spatial arrangement of the angioblasts allow subsequent assembly of vascular tubes. This process fails to take place in Egfl7 knockdown embryos, leading to the failure of vascular tube formation. Our study defines a regulator that controls a specific and important step in vasculogenesis.

Hepatocytes Express Nerve Growth Factor during Liver Injury: Evidence for Paracrine Regulation of Hepatic Stellate Cell Apoptosis

A key feature of recovery from liver fibrosis is hepatic stellate cell (HSC) apoptosis, which serves the dual function of removing the major source of neomatrix and tissue inhibitors of metalloproteinases thereby facilitating matrix degradation. The mechanisms regulating HSC apoptosis remain undefined but may include the interaction of nerve growth factor (NGF) with its receptor, p75, on HSC. In this study, by TaqMan polymerase chain reaction in situ hybridization and immunohistochemistry, we demonstrate that NGF is expressed by hepatocytes during fibrotic injury. Peak hepatocyte expression of NGF (48 hours after CCl(4) injection) coincides with maximal rate of apoptosis of HSC by terminal dUTP nick-end labeling staining. Addition of recombinant NGF to HSC in tissue culture causes a dose-dependent increase in apoptosis. NGF regulates nuclear factor (NF)-kappaB activity, reducing p50/p65 binding detected by electromobility shift assay and reduced NF-kappaB CAT reporter activities from both basal unstimulated levels and after NF-kappaB induction by tumor necrosis factor. In each case, a relative reduction in NF-kappaB binding was associated with a significant increase in caspase 3 activity. These data provide evidence that NGF is expressed during fibrotic liver injury and may regulate number of activated HSCs via induction of apoptosis.

Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

Tumor-associated lymphatics are postulated to provide a transit route for disseminating metastatic cells. This notion is supported by preclinical findings that inhibition of pro-lymphangiogenic signaling during tumor development reduces cell spread to sentinel lymph nodes (SLNs). However, it is unclear how lymphatics downstream of SLNs contribute to metastatic spread into distal organs, or if modulating distal lymph transport impacts disease progression. Utilizing murine models of metastasis, longitudinal in vivo imaging of lymph transport, and function blocking antibodies against two VEGF family members, we provide evidence that distal lymphatics undergo disease course-dependent up-regulation of lymph transport coincidental with structural remodeling. Inhibition of VEGF-C activity with antibodies against VEGF-C or NRP2 prevented these disease-associated changes. Furthermore, utilizing a novel model of adjuvant treatment, we demonstrate that antagonism of VEGF-C or NRP2 decreases post SLN metastasis. These data support a potential therapeutic strategy for inhibiting distant metastatic dissemination via targeting tumor-associated lymphatic remodeling.

Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.

Regulation of vascular endothelial junction stability and remodeling through Rap1-Rasip1 signaling

The ability of blood vessels to sense and respond to stimuli such as fluid flow, shear stress, and trafficking of immune cells is critical to the proper function of the vascular system. Endothelial cells constantly remodel their cell–cell junctions and the underlying cytoskeletal network in response to these exogenous signals. This remodeling, which depends on regulation of the linkage between actin and integral junction proteins, is controlled by a complex signaling network consisting of small G proteins and their various downstream effectors. In this commentary, we summarize recent developments in understanding the small G protein RAP1 and its effector RASIP1 as critical mediators of endothelial junction stabilization, and the relationship between RAP1 effectors and modulation of different subsets of endothelial junctions.

Randomized Phase II Trial of Parsatuzumab (Anti‐EGFL7) or Placebo in Combination with FOLFOX and Bevacizumab for First‐Line Metastatic Colorectal Cancer

Abstract Lessons Learned. These negative phase II results for parsatuzumab highlight the challenges of developing an agent intended to enhance the efficacy of vascular endothelial growth factor inhibition without the benefit of validated pharmacodynamic biomarkers or strong predictive biomarker hypotheses. Any further clinical development of anti‐EGFL7 is likely to require new mechanistic insights and biomarker development for antiangiogenic agents. Background. EGFL7 (epidermal growth factor‐like domain 7) is a tumor‐enriched vascular extracellular matrix protein that supports endothelial cell survival. This phase II trial evaluated the efficacy of parsatuzumab (also known as MEGF0444A), a humanized anti‐EGFL7 IgG1 monoclonal antibody, in combination with modified FOLFOX6 (mFOLFOX6) (folinic acid, 5‐fluorouracil, and oxaliplatin) bevacizumab in patients with previously untreated metastatic colorectal cancer (mCRC). Methods. One‐hundred twenty‐seven patients were randomly assigned to parsatuzumab, 400 mg, or placebo, in combination with mFOLFOX6 plus bevacizumab, 5 mg/kg. Treatment cycles were repeated every 2 weeks until disease progression or unacceptable toxicity for a maximum of 24 months, with the exception of oxaliplatin, which was administered for up to 8 cycles. Results. The progression‐free survival (PFS) hazard ratio was 1.17 (95% confidence interval [CI], 0.71–1.93; p = .548). The median PFS was 12 months for the experimental arm versus 11.9 months for the control arm. The hazard ratio for overall survival was 0.97 (95% CI, 0.46–2.1; p = .943). The overall response rate was 59% in the parsatuzumab arm and 64% in the placebo arm. The adverse event profile was similar in both arms. Conclusions. There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first‐line mCRC.

Abstract LB-312: Egfl7 is expressed in a broad range of human tumors with apparent restriction to the endothelium

EGFL7 is a secreted protein expressed by proliferating endothelial cells in growing vessels during normal organ development. Though expression is generally restricted to a small subset of vessels in the adult, elevated expression has been observed under physiological conditions requiring new blood vessel growth, such as wound healing, pregnancy and tumor growth. EGFL7 facilitates new blood vessel formation by supporting endothelial cell adhesion and migration. In addition, EGFL7 can protect endothelial cells from stress-induced apoptosis, such as hypoxia or potentially growth factor withdrawal. A blocking antibody to EGFL7 (anti-EGFL7) has demonstrated increased survival benefits in preclinical models when combined with a blocking antibody to the pro-angiogenic growth factor VEGFA. Anti-EGFL7 is currently being investigated in Phase II clinical trials in NSCLC and mCRC in combination with bevacizumab and chemotherapy. We present here a comprehensive assessment of Egfl7 expression in a broad range of human solid tumors using in situ hybridization (BC n=150, CRC n=134, RCC n=8, LC n=62, LN mets predom. BC n=27, OV n=25) and quantitative PCR (BC n=92 primary, n=20 LN mets synchronous collection, n=12 distant matched metastasis sequential collection; CRC n=85 primary, n=9 distant matched metastasis sequential collection; NSCLC n=77 primary, n=3 distant matched metastasis sequential collection). Egfl7 is expressed in all tumor types interrogated. With the methods utilized in this study, expression appears to be limited to endothelial cells, with no appreciable tumor cell expression. This data is supported by co-expression analysis in preclinical and clinical samples, showing correlation with vascular markers (n=91 xenograft models CD31 r=0.85, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-312. doi:1538-7445.AM2012-LB-312

Abstract 3284: Preclinical and clinical evaluation of pharmacodynamic biomarkers of a novel anti-angiogenesis agent, anti-EGFL7

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL In this study we report the use of pharmacodynamic (PD) biomarkers to guide the clinical development of a novel antibody targeting epidermal growth factor-like domain 7 (EGFL7). EGFL7 is a secreted protein produced by endothelial cells of nascent blood vessels in tumors and other proliferating tissues, but is expressed at low levels in healthy quiescent vessels and many non-vascular cell types (Campagnolo et al. 2005; Fitch et al. 2004; Parker et al. 2004; Soncin et al. 2003). Upon secretion, EGFL7 becomes tightly associated with the perivascular extracellular matrix (ECM), and supports endothelial cell adhesion and migration (Parker et al. 2004; Schmidt et al. 2007). EGFL7 protein also protects endothelial cells from stress-induced apoptosis (Xu et al. 2008). Anti-EGFL7 is a humanized monoclonal antibody that binds to EGFL7. In preclinical models, anti-EGFL7 when combined with anti-VEGF augments the survival benefit observed from anti-VEGF alone. Anti-EGFL7 is currently being investigated in Phase I trials (in solid tumors) in combination with bevacizumab. In addition to endothelial cells, circulating CD34Hi/CD31dim progenitor cells (CPCs) express high levels of EGFL7 transcript. This population of cells can differentiate into endothelial cells in vitro, and we found that the EGFL7 protein promotes proliferation and possibly survival of the undifferentiated CPCs in vitro. In preclinical xenograft models, administration of anti-EGFL7 resulted in delayed (day 15) reduction in CPCs, which based on the time course, is unlikely to be mediated simply by clearance of antibody-bound CPCs. Hence, evaluation of CPCs was incorporated into the dose escalation phase of anti-EGFL7 clinical development as a pharmacodynamic biomarker (PD) of drug activity. Biomarker analysis of CPCs in humans correlated with preclinical findings. For example, reduction in CPCs in response to anti-EGFL7 therapy was observed at a time point similar to that in mice. The treatment dependent decrease in CPCs may serve as a pharmacodynamic biomarker that may help guide dose selection as well as represent another potential mechanism for the anti-angiogenic properties of anti-EGFL7 Campagnolo L, Leahy A, Chitnis S, Koschnick S et al. (2005) Am J Pathol 167(1):275-84 Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H. (2004) Dev Dyn. 230(2):316-24 Parker LH, Schmidt M, Jin SW et al (2004) Nature 428(6984):754-8. Schmidt M, Paes K, De Maziere A, (2007) Development 134(16):2913-23. Soncin F, Mattot V, Lionneton F (2003) EMBO J. 22(21):5700-11 Xu D, Perez RE, Ekekezie II (2008) Am J Physiol Lung Cell Mol Physiol. 294(1):L17-23 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3284. doi:10.1158/1538-7445.AM2011-3284

Abstract 3295: Inhibiting vascular morphogenesis in tumors: EGFL7 as a novel therapeutic target

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL EGFL7 is a vascular restricted extracellular matrix protein that is up-regulated during angiogenesis (Campagnolo et al., 2005; Fitch et al., 2004; Parker et al., 2004; Soncin et al., 2003). EGFL7 supports endothelial cell adhesion (Parker et al., 2004; Schmidt et al., 2007) and protects endothelial cells from stress-induced apoptosis (Xu et al., 2008). Inhibition of Egfl7 expression in zebrafish embryos abolished vascular lumen formation and reduced sprouting angiogenesis (Parker et al., 2004). We developed a panel of anti-EGFL7 monoclonal antibodies that block the adhesive and pro-survival activities of EGFL7. Anti-EGFL7 in combination with anti-VEGF resulted in significant tumor regression in multiple murine xenograft models, whereas anti-VEGF alone only slowed tumor growth in the same models. Anti-EGFL7 monoclonal antibodies also demonstrated prolonged survival and anti-tumor angiogenesis effects in stringent murine genetic tumor models when used as a single agent, and enhancement of anti-VEGF therapy in the combination setting. An ongoing Phase I study is being conducted to evaluate clinical safety, PK, PD and efficacy of anti-EGFL7. References: Campagnolo, L., Leahy, A., Chitnis, S., et al. EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. Am J Pathol (2005) 167, 275-284. Fitch, M. J., Campagnolo, L., Kuhnert, F., et al. Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn (2004) 230, 316-324. Parker, L. H., Schmidt, M., Jin, S. W., et al. The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature (2004) 428, 754-758. Schmidt, M., Paes, K., De Maziere, et al. EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution. Development (2007) 134, 2913-2923. Soncin, F., Mattot, V., Lionneton, F., et al. VE-statin, an endothelial repressor of smooth muscle cell migration. Embo J (2003) 22, 5700-5711. Xu, D., Perez, R. E., Ekekezie, I., et al. Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxiainduced cell death. Am J Physiol Lung Cell Mol Physiol (2008) 294, L17-L23. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3295. doi:10.1158/1538-7445.AM2011-3295

Abstract 1669: A quantitative real-time RT-PCR gene signature to detect circulating CD34+ cells as a pharmacodynamic biomarker following anti-EGFL7 treatment in preclinical models of efficacy

EGFL7 is an extracellular matrix protein that is highly expressed in endothelial cells of angiogenic vessels, including embryonic, tumor and injured vasculatures. Inhibition of EGFL7 activity by anti-EGFL7 monoclonal antibodies results in a reduction of tumor microvascular density (MVD) and tumor growth in genetically engineered models of cancer such as RIP-TβAg. Anti-EGFL7 therapy also leads to a decrease of circulating CD34+ cells in tumor-bearing mice. Utilizing in-silico and murine in-vivo analyses, we have identified a gene signature that can specifically monitor circulating CD34+ cell populations by quantitative real-time PCR using volumes as low as 50μl of mouse whole blood. This PCR-based assay shows a limit of quantification of 0.1 cells/μl of murine whole blood. Experiments comparing CD34+ progenitor cell quantification by flow cytometry to qPCR have shown equivalent performance and good correlation. We propose that using this qPCR based approach to evaluate changes in CD34+ cells in mouse blood in response to anti-angiogenesis therapies, including anti-EGFL7, may be an easy and viable alternative to the current flow cytometry-based assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1669.