Pierre Scotney

VEGF-B is dispensable for blood vessel growth but critical for their survival, and VEGF-B targeting inhibits pathological angiogenesis

VEGF-B, a homolog of VEGF discovered a long time ago, has not been considered an important target in antiangiogenic therapy. Instead, it has received little attention from the field. In this study, using different animal models and multiple types of vascular cells, we revealed that although VEGF-B is dispensable for blood vessel growth, it is critical for their survival. Importantly, the survival effect of VEGF-B is not only on vascular endothelial cells, but also on pericytes, smooth muscle cells, and vascular stem/progenitor cells. In vivo, VEGF-B targeting inhibited both choroidal and retinal neovascularization. Mechanistically, we found that the vascular survival effect of VEGF-B is achieved by regulating the expression of many vascular prosurvival genes via both NP-1 and VEGFR-1. Our work thus indicates that the function of VEGF-B in the vascular system is to act as a “survival,” rather than an “angiogenic” factor and that VEGF-B inhibition may offer new therapeutic opportunities to treat neovascular diseases.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved β-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

VEGF-B inhibits apoptosis via VEGFR-1–mediated suppression of the expression of BH3-only protein genes in mice and rats

Despite its early discovery and high sequence homology to the other VEGF family members, the biological functions of VEGF-B remain poorly understood. We revealed here a novel function for VEGF-B as a potent inhibitor of apoptosis. Using gene expression profiling of mouse primary aortic smooth muscle cells, and confirming the results by real-time PCR using mouse and rat cell lines, we showed that VEGF-B inhibited the expression of genes encoding the proapoptotic BH3-only proteins and other apoptosis- and cell death-related proteins, including p53 and members of the caspase family, via activation of VEGFR-1. Consistent with this, VEGF-B treatment rescued neurons from apoptosis in the retina and brain in mouse models of ocular neurodegenerative disorders and stroke, respectively. Interestingly, VEGF-B treatment at the dose effective for neuronal survival did not cause retinal neovascularization, suggesting that VEGF-B is the first member of the VEGF family that has a potent antiapoptotic effect while lacking a general angiogenic activity. These findings indicate that VEGF-B may potentially offer a new therapeutic option for the treatment of neurodegenerative diseases.

Transgenic Overexpression of Vascular Endothelial Growth Factor-B Isoforms by Endothelial Cells Potentiates Postnatal Vessel Growth In Vivo and In Vitro

Vascular endothelial growth factors (VEGFs) play significant roles in endothelial growth, survival, and function, and their potential use as therapeutic agents to promote the revascularization of ischemic tissues in being avidly explored. VEGF-A has received most attention, as it is a potent stimulator of vascular growth. Results in clinical trials of VEGF-A as a therapeutic agent have fallen short of high expectations because of serious edematous side effects caused by its activity in promoting vascular permeability. VEGF-B, a related factor, binds some of the VEGF-A receptors but not to VEGF receptor 2, which is implicated in the vascular permeability promoting activity of VEGF-A. Despite little in vitro evidence to date for the ability of Vegf-B to directly promote angiogenesis, recent data indicate that it may promote postnatal vascular growth in mice, suggesting that it may have potential therapeutic application. We have specifically studied the effects of VEGF-B on vascular growth in vivo and on angiogenesis in vitro by analyzing transgenic mice in which individual isoforms (VEGFB167Tg and VEGFB186Tg) of VEGF-B are overexpressed in endothelial cells. VEGFB167Tg and VEGFB186Tg mice displayed enhanced vascular growth in the Matrigel assay in vivo and during cutaneous wound healing. In the aortic explant assay, explants from VEGFB167Tg and VEGFB186Tg mice displayed elevated vascular growth, suggesting a direct effect of VEGF-B isoforms in potentiating angiogenesis. These data support the use of VEGF-B as a therapeutic agent to promote vascular growth, in part, by potentiating angiogenesis. Furthermore, the lack of vascular permeability activity associated with either transgenic overexpression of the VEGF-B gene in endothelial cells or application of VEGF-B protein to the skin of mice in the Miles assay indicates that use of VEGF-B as a therapy should not be associated with edematous side effects.

Human vascular endothelial growth factor B: Characterization of recombinant isoforms and generation of neutralizing monoclonal antibodies

1. The vascular endothelial growth factor (VEGF) family is a focus of interest with respect to novel therapies for cardiovascular disease. Members of this family bind differentially to three receptor tyrosine kinases, namely VEGF‐R1, ‐R2 and ‐R3, and to the semaphorin receptors neuropilin 1 and 2. The role of VEGF‐R1 and the factors that interact exclusively with this receptor (VEGF‐B and placenta growth factor) has remained controversial.

Endogenous IL-11 Signaling Is Essential in Th2- and IL-13–Induced Inflammation and Mucus Production

IL-11 and IL-11 receptor (R)alpha are induced by Th2 cytokines. However, the role(s) of endogenous IL-11 in antigen-induced Th2 inflammation has not been fully defined. We hypothesized that IL-11, signaling via IL-11Ralpha, plays an important role in aeroallergen-induced Th2 inflammation and mucus metaplasia. To test this hypothesis, we compared the responses induced by the aeroallergen ovalbumin (OVA) in wild-type (WT) and IL-11Ralpha-null mutant mice. We also generated and defined the effects of an antagonistic IL-11 mutein on pulmonary Th2 responses. Increased levels of IgE, eosinophilic tissue and bronchoalveolar lavage (BAL) inflammation, IL-13 production, and increased mucus production and secretion were noted in OVA-sensitized and -challenged WT mice. These responses were at least partially IL-11 dependent because each was decreased in mice with null mutations of IL-11Ralpha. Importantly, the administration of the IL-11 mutein to OVA-sensitized mice before aerosol antigen challenge also caused a significant decrease in OVA-induced inflammation, mucus responses, and IL-13 production. Intraperitoneal administration of the mutein to lung-specific IL-13-overexpressing transgenic mice also reduced BAL inflammation and airway mucus elaboration. These studies demonstrate that endogenous IL-11R signaling plays an important role in antigen-induced sensitization, eosinophilic inflammation, and airway mucus production. They also demonstrate that Th2 and IL-13 responses can be regulated by interventions that manipulate IL-11 signaling in the murine lung.

The combination of ISCOMATRIX adjuvant and TLR agonists induces regression of established solid tumors in vivo.

The development of therapeutic vaccines for treatment of established cancer has proven challenging. Cancer vaccines not only need to induce a robust tumor Ag-specific immune response but also need to overcome the tolerogenic and immunosuppressive microenvironments that exist within many solid cancers. ISCOMATRIX adjuvant (ISCOMATRIX) is able to induce both tumor Ag-specific cellular and Ab responses to protect mice against tumor challenge, but this is insufficient to result in regression of established solid tumors. In the current study, we have used B16-OVA melanoma, Panc-OVA pancreatic, and TRAMP-C1 prostate cancer mouse tumor models to test therapeutic efficacy of ISCOMATRIX vaccines combined with other immune modulators. The coadministration of an ISCOMATRIX vaccine with the TLR3 agonist, polyinosinic-polycytidylic acid, and TLR9 agonist, CpG, reduced tumor growth in all tumor models and the presence of ISCOMATRIX in the formulation was critical for the therapeutic efficacy of the vaccine. This vaccine combination induced a robust and multifunctional CD8+ T cell response. Therapeutic protection required IFN-γ and CD8+ T cells, whereas NK and CD4+ T cells were found to be redundant. ISCOMATRIX vaccines combined with TLR3 and TLR9 agonists represent a promising cancer immunotherapy strategy.

Reducing VEGF-B Signaling Ameliorates Renal Lipotoxicity and Protects against Diabetic Kidney Disease

Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD.

A robust robotic high-throughput antibody purification platform

Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US

Species Cross-Reactivity of Antibodies Used to Treat Ophthalmic Conditions.

in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant.