Gladys Ingle

Hepatocyte Growth Factor Enhances Vascular Endothelial Growth Factor-Induced Angiogenesis in Vitro and in Vivo

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Gene Expression Profiling in an in Vitro Model of Angiogenesis

In the present study we have used a novel, comprehensive mRNA profiling technique (GeneCalling) for determining differential gene expression profiles of human endothelial cells undergoing differentiation into tubelike structures. One hundred fifteen cDNA fragments were identified and shown to represent 90 distinct genes. Although some of the genes identified have previously been implicated in angiogenesis, potential roles for many new genes, including OX-40, white protein homolog, KIAA0188, a homolog of angiopoietin-2, ADAMTS-4 (aggrecanase-1), and stanniocalcin were revealed. Support for the biological significance was confirmed by the abrogation of the changes in the expression of angiogenesis inhibitors and in situ hybridization studies. This study has significantly extends the molecular fingerprint of the changes in gene expression that occur during endothelial differentiation and provides new insights into the potential role of a number of new molecules in angiogenesis.

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.

IGF-I deficient mice show reduced peripheral nerve conduction velocities and decreased axonal diameters and respond to exogenous IGF-I treatment.

Although insulin-like growth factor-I (IGF-I) can act as a neurotrophic factor for peripheral neurons in vitro and in vivo following injury, the role IGF-I plays during normal development and functioning of the peripheral nervous system is unclear. Here, we report that transgenic mice with reduced levels (two genotypes: heterozygous Igf1+/- or homozygous insertional mutant Igf1m/m) or totally lacking IGF-I (homozygous Igf1-/-) show a decrease in motor and sensory nerve conduction velocities in vivo. In addition, A-fiber responses in isolated peroneal nerves from Igf1+/- and Igf1-/- mice are impaired. The nerve function impairment is most profound in Igf1-/- mice. Histopathology of the peroneal nerves in Igf1-/- mice demonstrates a shift to smaller axonal diameters but maintains the same total number of myelinated fibers as Igf1+/+ mice. Comparisons of myelin thickness with axonal diameter indicate that there is no significant reduction in peripheral nerve myelination in IGF-I-deficient mice. In addition, in Igf1m/m mice with very low serum levels of IGF-I, replacement therapy with exogenous recombinant hIGF-I restores both motor and sensory nerve conduction velocities. These findings demonstrate not only that IGF-I serves an important role in the growth and development of the peripheral nervous system, but also that systemic IGF-I treatment can enhance nerve function in IGF-I-deficient adult mice.

Vascular Endothelial Growth Factor–Induced Genes in Human Umbilical Vein Endothelial Cells: Relative Roles of KDR and Flt-1 Receptors

Objective—This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF. Methods and Results—We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A). We report that activation of KDR was sufficient to upregulate all the genes induced by VEGFwt. In contrast, there were no genes selectively upregulated by the Flt-selective mutant. However, high concentrations of the Flt-selective mutant could augment the expression of some genes induced by submaximal concentrations of VEGFwt but not the KDR-selective mutant. Conclusions—The binding of VEGF to its receptor, KDR, is necessary and sufficient to induce the gene expression profile induced by this growth factor. Furthermore, in human umbilical vein endothelial cells, the Flt-1 receptor appears to act as a decoy receptor, tempering the response to lower concentrations of VEGF.

Branching Out: A Molecular Fingerprint of Endothelial Differentiation into Tube-Like Structures Generated by Affymetrix Oligonucleotide Arrays

The process of endothelial differentiation into a network of tube‐like structures with patent lumens requires an integrated program of gene expression. To identify genes upregulated in endothelial cells during the process of tube formation, RNA was prepared from several different time points (0, 4, 8, 24, 40, and 48 hours) and from three different experimental models of human endothelial tube formation: in collagen gels and fibrin gels driven by the combination of PMA (80), bFGF (40 ng/ml) and bFGF (40 ng/ml) or in collagen gels driven by the combination of HGF (40 ng/ml) and VEGF (40 ng/ml). Gene expression was evaluated using Affymetrix® Gene Chip® oligonucleotide arrays. Over 1000 common genes were upregulated greater than twofold over baseline at one or more time points in the three different models. In the present study, we discuss the identified genes that could be assigned to major functional classes: apoptosis, cytoskeleton, proteases, matrix, and matrix turnover, pumps and transporters, membrane lipid turnover, and junctional molecules or adhesion proteins.

High CD21 expression inhibits internalization of anti-CD19 antibodies and cytotoxicity of an anti-CD19-drug conjugate

CD19 and CD21 (CR2) are co‐receptors found on B‐cells and various B‐cell lymphomas, including non‐Hodgkin lymphoma. To evaluate their suitability as targets for therapy of such lymphomas using internalization‐dependent antibody‐drug conjugates [such as antibody‐4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate, (N2′‐deacetyl‐N2′‐(3‐mercapto‐1‐oxopropyl)‐maytansine) (MCC‐DM1) conjugates, which require lysosomal degradation of the antibody moiety for efficacy], we examined uptake of antibodies to CD19 and CD21 in a panel of B‐cell lines. Anti‐CD21 antibodies were not sufficiently internalized even in the highest CD21‐expressing Raji cells, resulting in lack of efficacy with anti‐CD21‐MCC‐DM1 conjugates. Anti‐CD19 antibody uptake was variable, and was unexpectedly negatively correlated with CD21 expression. Thus, high CD21‐expressing Raji, ARH77 and primary B‐cells only very slowly internalized anti‐CD19 antibodies, while CD21‐negative or low expressing cells, including Ramos and Daudi, rapidly internalized these antibodies in clathrin‐coated vesicles followed by lysosomal delivery. Anti‐CD19‐MCC‐DM1 caused greater cytotoxicity in the faster anti‐CD19‐internalizing cell lines, implying that the rate of lysosomal delivery and subsequent drug release is important. Furthermore, transfection of Ramos cells with CD21 impeded anti‐CD19 uptake and decreased anti‐CD19‐MCC‐DM1 efficacy, suggesting that CD21‐negative tumours should respond better to such anti‐CD19 conjugates. This may have possible clinical implications, as anti‐CD21 immunohistochemistry revealed only approximately 30% of 54 diffuse large B‐cell lymphoma patients lack CD21 expression.

Stanniocalcin 1 Is an Autocrine Modulator of Endothelial Angiogenic Responses to Hepatocyte Growth Factor

Stanniocalcin 1 (STC1) is a secreted glycoprotein originally described as a hormone involved in calcium and phosphate homeostasis in bony fishes. We recently identified the mammalian homolog of this molecule to be highly up-regulated in an in vitro model of angiogenesis, as well as focally and intensely expressed at sites of pathological angiogenesis (e.g. tumor vasculature). In the present study, we report that STC1 is a selective modulator of hepatocyte growth factor (HGF)-induced endothelial migration and morphogenesis, but not proliferation. STC1 did not inhibit proliferative or migratory responses to vascular endothelial growth factor or basic fibroblast growth factor. The mechanism of STC1 inhibitory effects on HGF-induced endothelial migration seem to occur secondary to receptor activation because STC1 did not inhibit HGF-induced c-met receptor phosphorylation, but did block HGF-induced focal adhesion kinase activation. In the mouse femoral artery ligation model of angiogenesis, STC1 expression closely paralleled that of the endothelial marker CD31, and the peak level of STC1 expression occurred after an increase in HGF expression. We propose that STC1 may play a selective modulatory role in angiogenesis, possibly serving as a “stop signal” or stabilizing factor contributing to the maturation of newly formed blood vessels. HGF is a mesenchyme-derived pleiotropic factor with mitogenic, motogenic, and morphogenic activities on a number of different cell types. HGF effects are mediated through a specific tyrosine kinase, c-met, and aberrant HGF and c-met expression are frequently observed in a variety of tumors. Recent studies have shown HGF to be a potent growth factor implicated in wound healing, tissue regeneration, and angiogenesis.

An Antimesothelin-Monomethyl Auristatin E Conjugate with Potent Antitumor Activity in Ovarian, Pancreatic, and Mesothelioma Models

Mesothelin (MSLN) is an attractive target for antibody–drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to nondividing mesothelia. We generated novel antimesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine–citrulline linker. The humanized (h7D9.v3) version, αMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3 nmol/L. Because the antitumor activity of an antimesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic, and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of αMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose-dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of αMSLN-MMAE in preclinical models of ovarian, mesothelioma, and pancreatic cancers justifies the ongoing phase I clinical trial. Mol Cancer Ther; 13(11); 2630–40. ©2014 AACR.

DropArray™, a Wall-Less 96-Well Plate for Uptake and Immunofluorescence Microscopy, Confirms CD22 Recycles

CD22 is a cell surface glycoprotein restricted to normal and malignant B‐cells and is the target of several anti‐CD22 antibody‐based cancer therapies. For therapeutic antibody‐payload conjugates, it is important to understand the subcellular trafficking of anti‐CD22 antibodies to optimize antibody and/or linker–drug properties to maximize antitumor efficacy. It is agreed that anti‐CD22 antibodies rapidly internalize, but controversial whether they recycle or are degraded in lysosomes, and it is unclear if trafficking is antibody or cell‐type dependent. No studies examined anti‐CD22 trafficking to either pathway in B‐cells over time by dual immunofluorescence microscopy, likely partly because multiple samples of suspension cells are tedious to stain. We overcame this by using DropArray™, a novel wall‐less 96‐well plate technology allowing rapid simultaneous staining of suspension or adherent cells in small (10–20 μL) volumes. We examined the time‐course of trafficking of five different anti‐CD22 antibodies in eight B‐cell lines representing four B‐cell cancer types and show that in all cases antibodies internalize within 5 min and recycle, with only small amounts eventually trafficking to lysosomes. CD22 also localizes to recycling endosomes at steady state in the absence of antibody. Our data may help explain the differential efficacies of anti‐CD22 antibodies conjugated to different therapeutic payloads.