Gretchen Frantz

FIZZ1, a novel cysteine‐rich secreted protein associated with pulmonary inflammation, defines a new gene family

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine‐rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non‐neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)‐mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF‐induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.

The ubiquitin ligase COP1 is a critical negative regulator of p53

COP1 (constitutively photomorphogenic 1) is a RING-finger-containing protein that functions to repress plant photomorphogenesis, the light-mediated programme of plant development. Mutants of COP1 are constitutively photomorphogenic, and this has been attributed to their inability to negatively regulate the proteins LAF1 (ref. 1) and HY5 (ref. 2). The role of COP1 in mammalian cells is less well characterized. Here we identify the tumour-suppressor protein p53 as a COP1-interacting protein. COP1 increases p53 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent fashion, independently of MDM2 or Pirh2, which are known to interact with and negatively regulate p53. Moreover, COP1 serves as an E3 ubiquitin ligase for p53 in vitro and in vivo, and inhibits p53-dependent transcription and apoptosis. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Furthermore, we identify COP1 as a p53-inducible gene, and show that the depletion of COP1 and MDM2 by siRNA cooperatively sensitizes U2-OS cells to ionizing-radiation-induced cell death. Overall, these results indicate that COP1 is a critical negative regulator of p53 and represents a new pathway for maintaining p53 at low levels in unstressed cells.

Identification of an angiogenic mitogen selective for endocrine gland endothelium

The known endothelial mitogens stimulate growth of vascular endothelial cells without regard to their tissue of origin. Here we report a growth factor that is expressed largely in one type of tissue and acts selectively on one type of endothelium. This molecule, called endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), induced proliferation, migration and fenestration (the formation of membrane discontinuities) in capillary endothelial cells derived from endocrine glands. However, EG-VEGF had little or no effect on a variety of other endothelial and non-endothelial cell types tested. Similar to VEGF, EG-VEGF possesses a HIF-1 binding site, and its expression is induced by hypoxia. Both EG-VEGF and VEGF resulted in extensive angiogenesis and cyst formation when delivered in the ovary. However, unlike VEGF, EG-VEGF failed to promote angiogenesis in the cornea or skeletal muscle. Expression of human EG-VEGF messenger RNA is restricted to the steroidogenic glands, ovary, testis, adrenal and placenta and is often complementary to the expression of VEGF, suggesting that these molecules function in a coordinated manner. EG-VEGF is an example of a class of highly specific mitogens that act to regulate proliferation and differentiation of the vascular endothelium in a tissue-specific manner.

Impact of Vascular Endothelial Growth Factor-A Expression, Thrombospondin-2 Expression, and Microvessel Density on the Treatment Effect of Bevacizumab in Metastatic Colorectal Cancer

PURPOSE Bevacizumab is a monoclonal antibody to vascular endothelial growth factor-A (VEGF). In the pivotal trial in metastatic colorectal cancer (mCRC), addition of bevacizumab to first-line irinotecan, fluorouracil, and leucovorin (IFL) significantly prolonged median survival. The aim of these retrospective subset analyses was to evaluate VEGF, thrombospondin-2 (THBS-2), and microvessel density (MVD) as prognostic factors and/or predictors of benefit from bevacizumab. PATIENTS AND METHODS In the pivotal trial, 813 patients with untreated mCRC were randomly assigned to receive IFL plus bevacizumab or placebo. Of 312 tissue samples collected (285 primaries, 27 metastases), outcome data were available for 278 (153 bevacizumab, 125 placebo). Epithelial and stromal VEGF expression were assessed by in situ hybridization (ISH) and immunohistochemistry on tissue microarrays and whole sections. Stromal THBS-2 expression was examined by ISH on tissue microarrays. MVD was quantified by Chalkley count. Overall survival was associated with these variables in retrospective subset analyses. RESULTS In all subgroups, estimated hazard ratios (HRs) for risk of death were < 1 for bevacizumab-treated patients regardless of the level of VEGF or THBS-2 expression or MVD. Patients with a high THBS-2 score showed a nonsignificant improvement in survival following bevacizumab treatment (HR = 0.11; 95% CI, 0.02 to 0.51) compared to patients with a low score (HR = 0.65; 95% CI, 0.41 to 1.02); interaction analysis P = .22. VEGF or THBS-2 expression and MVD were not significant prognostic factors. CONCLUSION These exploratory analyses suggest that in patients with mCRC addition of bevacizumab to IFL improves survival regardless of the level of VEGF or THBS-2 expression, or MVD.

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.

VEGF-null cells require PDGFR α signaling-mediated stromal fibroblast recruitment for tumorigenesis

We generated VEGF‐null fibrosarcomas from VEGF‐loxP mouse embryonic fibroblasts to investigate the mechanisms of tumor escape after VEGF inactivation. These cells were found to be tumorigenic and angiogenic in vivo in spite of the absence of tumor‐derived VEGF. However, VEGF derived from host stroma was readily detected in the tumor mass and treatment with a newly developed anti‐VEGF monoclonal antibody substantially inhibited tumor growth. The functional significance of stroma‐derived VEGF indicates that the recruitment of stromal cells is critical for the angiogenic and tumorigenic properties of these cells. Here we identified PDGF AA as the major stromal fibroblast chemotactic factor produced by tumor cells, and demonstrated that disrupting the paracrine PDGFR α signaling between tumor cells and stromal fibroblasts by soluble PDGFR α‐IgG significantly reduced tumor growth. Thus, PDGFR α signaling is required for the recruitment of VEGF‐producing stromal fibroblasts for tumor angiogenesis and growth. Our findings highlight a novel aspect of PDGFR α signaling in tumorigenesis.

The endocrine-gland-derived VEGF homologue Bv8 promotes angiogenesis in the testis: Localization of Bv8 receptors to endothelial cells.

We recently identified an angiogenic mitogen, endocrine-gland-derived vascular endothelial growth factor (EG-VEGF), with selective activity for endothelial cells of endocrine tissues. Here we describe the characterization of a highly related molecule, Bv8, also known as prokineticin-2. Human Bv8 shares 60% identity and 75% similarity with EG-VEGF. The human and mouse Bv8 genes share a common structure. Like EG-VEGF, Bv8 is able to induce proliferation, survival and migration of adrenal cortical capillary endothelial cells. Bv8 gene expression is induced by hypoxic stress. Bv8 expression occurs predominantly in the testis and is largely restricted to primary spermatocytes. Adenoviral delivery of Bv8 or EG-VEGF to the mouse testis resulted in a potent angiogenic response. We have localized the expression of the Bv8/EG-VEGF receptors within the testis to vascular endothelial cells. The testis exhibits relatively high turnover of endothelial cells. Therefore, Bv8 and EG-VEGF, along with other factors such as VEGF-A, may maintain the integrity and also regulate proliferation of the blood vessels in the testis.

Antibody-Drug Conjugates for the Treatment of Non–Hodgkin's Lymphoma: Target and Linker-Drug Selection

Antibody-drug conjugates (ADC), potent cytotoxic drugs covalently linked to antibodies via chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Here, we systematically examine the potential targets and linker-drug combinations that could provide an optimal ADC for the treatment for non-Hodgkins lymphoma. We identified seven antigens (CD19, CD20, CD21, CD22, CD72, CD79b, and CD180) for potential treatment of non-Hodgkins lymphoma with ADCs. ADCs with cleavable linkers mediated in vivo efficacy via all these targets; ADCs with uncleavable linkers were only effective when targeted to CD22 and CD79b. In target-independent safety studies in rats, the uncleavable linker ADCs showed reduced toxicity, presumably due to the reduced release of free drug or other toxic metabolites into the circulation. Thus, our data suggest that ADCs with cleavable linkers work on a broad range of targets, and for specific targets, ADCs with uncleavable linkers provide a promising opportunity to improve the therapeutic window for ADCs in humans.

Differential expression of the angiogenic factor genes vascular endothelial growth factor (VEGF) and endocrine gland-derived VEGF in normal and polycystic human ovaries.

Angiogenesis is a key aspect of the dynamic changes occurring during the normal ovarian cycle. Hyperplasia and hypervascularity of the ovarian theca interna and stroma are also prominent features of the polycystic ovary syndrome (PCOS), a leading cause of infertility. Compelling evidence indicated that vascular endothelial growth factor (VEGF) is a key mediator of the cyclical corpus luteum angiogenesis. However, the nature of the factor(s) that mediate angiogenesis in PCOS is less clearly understood. Endocrine gland-derived (EG)-VEGF has been recently identified as an endothelial cell mitogen with selectivity for the endothelium of steroidogenic glands and is expressed in normal human ovaries. In the present study, we compared the expression of EG-VEGF and VEGF mRNA in a series of 13 human PCOS and 13 normal ovary specimens by in situ hybridization. EG-VEGF expression in normal ovaries is dynamic and generally complementary to VEGF expression in both follicles and corpora lutea. A particularly high expression of EG-VEGF was detected in the Leydig-like hilus cells found in the highly vascularized ovarian hilus. In PCOS ovaries, we found strong expression of EG-VEGF mRNA in theca interna and stroma in most of the specimens examined, thus spatially related to the new blood vessels. In contrast, VEGF mRNA expression was most consistently associated with the granulosa cell layer and sometimes the theca, but rarely with the stroma. These findings indicate that both EG-VEGF and VEGF are expressed in PCOS ovaries, but in different cell types at different stages of differentiation, thus suggesting complementary functions for the two factors in angiogenesis and possibly cyst formation.

Vascular Endothelial-Junctional Adhesion Molecule (VE-JAM)/JAM 2 Interacts with T, NK, and Dendritic Cells Through JAM 3

Screening expressed sequence tag databases for endothelial-specific homologs to human junctional adhesion molecule (JAM) and A33-Ag, we identified a protein of 298 aa that represents the recently described vascular endothelial-JAM (VE-JAM)/JAM 2. We confirmed VE-JAM/JAM 2 expression to be restricted to the high endothelial venule of tonsil and lymph nodes, and we further expanded the localization to the endothelium of arterioles in and around inflammatory and tumor foci. In our functional characterizations of VE-JAM/JAM 2, we discovered that it can function as an adhesive ligand for the T cell line J45 and can interact with GM-CSF/IL-4-derived peripheral blood dendritic cells, circulating CD56+ NK cells, circulating CD56+CD3+ NK/T cells, and circulating CD56+CD3+CD8+ cytolytic T cells. In the course of our studies, we also isolated and characterized the functional VE-JAM/JAM 2 receptor, which, upon cloning, turned out to be a submitted sequence representing JAM 3 (accession number NP 113658). With these understandings, we have characterized a protein-interacting pair that can be important in the role of T, NK, and dendritic cell trafficking and inflammation.