Julie Gavard

evaluation of iron oxide nanoparticle biocompatibility

Nanotechnology is an exciting field of investigation for the development of new treatments for many human diseases. However, it is necessary to assess the biocompatibility of nanoparticles in vitro and in vivo before considering clinical applications. Our characterization of polyol-produced maghemite γ-Fe2O3 nanoparticles showed high structural quality. The particles showed a homogeneous spherical size around 10 nm and could form aggregates depending on the dispersion conditions. Such nanoparticles were efficiently taken up in vitro by human endothelial cells, which represent the first biological barrier to nanoparticles in vivo. However, γ-Fe2O3 can cause cell death within 24 hours of exposure, most likely through oxidative stress. Further in vivo exploration suggests that although γ-Fe2O3 nanoparticles are rapidly cleared through the urine, they can lead to toxicity in the liver, kidneys and lungs, while the brain and heart remain unaffected. In conclusion, γ-Fe2O3 could exhibit harmful properties and therefore surface coating, cellular targeting, and local exposure should be considered before developing clinical applications.

Vascular permeability and drug delivery in cancers.

The endothelial barrier strictly maintains vascular and tissue homeostasis, and therefore modulates many physiological processes such as angiogenesis, immune responses, and dynamic exchanges throughout organs. Consequently, alteration of this finely tuned function may have devastating consequences for the organism. This is particularly obvious in cancers, where a disorganized and leaky blood vessel network irrigates solid tumors. In this context, vascular permeability drives tumor-induced angiogenesis, blood flow disturbances, inflammatory cell infiltration, and tumor cell extravasation. This can directly restrain the efficacy of conventional therapies by limiting intravenous drug delivery. Indeed, for more effective anti-angiogenic therapies, it is now accepted that not only should excessive angiogenesis be alleviated, but also that the tumor vasculature needs to be normalized. Recovery of normal state vasculature requires diminishing hyperpermeability, increasing pericyte coverage, and restoring the basement membrane, to subsequently reduce hypoxia, and interstitial fluid pressure. In this review, we will introduce how vascular permeability accompanies tumor progression and, as a collateral damage, impacts on efficient drug delivery. The molecular mechanisms involved in tumor-driven vascular permeability will next be detailed, with a particular focus on the main factors produced by tumor cells, especially the emblematic vascular endothelial growth factor. Finally, new perspectives in cancer therapy will be presented, centered on the use of anti-permeability factors and normalization agents.

Differential Proteomic Analysis of Human Glioblastoma and Neural Stem Cells Reveals HDGF as a Novel Angiogenic Secreted Factor

Presence in glioblastomas of cancer cells with normal neural stem cell (NSC) properties, tumor initiating capacity, and resistance to current therapies suggests that glioblastoma stem‐like cells (GSCs) play central roles in glioblastoma development. We cultured human GSCs endowed with all features of tumor stem cells, including tumor initiation after xenograft and radio‐chemoresistance. We established proteomes from four GSC cultures and their corresponding whole tumor tissues (TTs) and from human NSCs. Two‐dimensional difference gel electrophoresis and tandem mass spectrometry revealed a twofold increase of hepatoma‐derived growth factor (HDGF) in GSCs as compared to TTs and NSCs. Western blot analysis confirmed HDGF overexpression in GSCs as well as its presence in GSC‐conditioned medium, while, in contrast, no HDGF was detected in NSC secretome. At the functional level, GSC‐conditioned medium induced migration of human cerebral endothelial cells that can be blocked by anti‐HDGF antibodies. In vivo, GSC‐conditioned medium induced neoangiogenesis, whereas HDGF‐targeting siRNAs abrogated this effect. Altogether, our results identify a novel candidate, by which GSCs can support neoangiogenesis, a high‐grade glioma hallmark. Our strategy illustrates the usefulness of comparative proteomic analysis to decipher molecular pathways, which underlie GSC properties. STEM CELLS 2012;30:845–853

Remodeling of VE-cadherin junctions by the human herpes virus 8 G-protein coupled receptor

Kaposi Sarcoma (KS) are opportunistic tumors, associated with human herpes virus 8 (HHV8) infection. KS development is highly favored by immune-depression and remains the second most frequent tumor in acquired immune deficiency syndrome patients. Although it has been shown that experimental expression of the HHV8 G-protein-coupled receptor (vGPCR) in the endothelial compartment is alone sufficient to recapitulate the formation and progression of KS-like lesions, its functional effects on endothelial homeostasis are not fully understood. Here we show that vGPCR expression in endothelial cells induces an increase in paracellular permeability both in vivo and in vitro. By using pharmacological inhibitors and small interference RNA-based knockdown, we demonstrate an essential role for the PI(3)Kinase-γ/Rac nexus in vGPCR-mediated permeability. This was further accompanied by dramatic remodeling of VE-cadherin-dependent cell–cell junctions. Importantly, this in vitro vGPCR-initiated signaling signature was observed in a large panel of human KS. Altogether, our results support the hypothesis that endothelial vGPCR signaling is co-opted in KS, and unveil new key cellular targets for therapeutic intervention.

Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma.

Glioblastoma are malignant highly vascularized brain tumours, which feature large oedema resulting from tumour-promoted vascular leakage. The pro-permeability factor Semaphorin3A (Sema3A) produced within glioblastoma has been linked to the loss of endothelial barrier integrity. Here, we report that extracellular vesicles (EVs) released by patient-derived glioblastoma cells disrupt the endothelial barrier. EVs expressed Sema3A at their surface, which accounted for in vitro elevation of brain endothelial permeability and in vivo vascular permeability, in both skin and brain vasculature. Blocking Sema3A or its receptor Neuropilin1 (NRP1) hampered EV-mediated permeability. In vivo models using ectopically and orthotopically xenografted mice revealed that Sema3A-containing EVs were efficiently detected in the blood stream. In keeping with this idea, sera from glioblastoma multiforme (GBM) patients also contain high levels of Sema3A carried in the EV fraction that enhanced vascular permeability, in a Sema3A/NRP1-dependent manner. Our results suggest that EV-delivered Sema3A orchestrates loss of barrier integrity in glioblastoma and may be of interest for prognostic purposes.

Differential effects of Bartonella henselae on human and feline macro- and micro-vascular endothelial cells.

Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines. To this purpose, we have developed nine feline EC lines which allowed comparing the effects of Bartonella strains on human and feline micro-vascular ECs representative of the infection development sites such as skin, versus macro-vascular ECs, such as umbilical vein. Our model revealed intrinsic differences between human (Human Skin Microvascular ECs –HSkMEC and Human Umbilical Vein ECs – iHUVEC) and feline ECs susceptibility to Bartonella henselae infection. While no effect was observed on the feline ECs upon Bartonella henselae infection, the human ones displayed accelerated angiogenesis and wound healing. Noticeable differences were demonstrated between human micro- and macro-vasculature derived ECs both in terms of pseudo-tube formation and healing. Interestingly, Bartonella henselae effects on human ECs were also elicited by soluble factors. Neither Bartonella henselae-infected Human Skin Microvascular ECs clinically involved in bacillary angiomatosis, nor feline ECs increased cAMP production, as opposed to HUVEC. Bartonella henselae could stimulate the activation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) in homologous cellular systems and trigger VEGF production by HSkMECs only, but not iHUVEC or any feline ECs tested. These results may explain the decreased pathogenic potential of Bartonella henselae infection for cats as compared to humans and strongly suggest that an autocrine secretion of VEGF by human skin endothelial cells might induce their growth and ultimately lead to bacillary angiomatosis formation.

Role of Endothelial Cell–Cell Junctions in Endothelial Permeability

The endothelial barrier separates the inner blood compartment from the surrounding tissues. At the molecular level, adhesion molecules accumulate at the endothelial cell-cell junction and contribute to maintain vascular integrity. An increase in the endothelial permeability is frequently associated with the deregulation of junctional adhesion. Here, we review how to evaluate the in vitro functions of endothelial cell-cell contacts. We focus this chapter on cell imagery and biochemical analysis of VE-cadherin, the main constituent of adherens junction, and we also provide description of endothelial cell models and methods for studying tight junctions.

Thermosensitivity profile of malignant glioma U87-MG cells and human endothelial cells following γ-Fe2O3 NPs internalization and magnetic field application

In this study we evaluate the thermosensitivity of healthy endothelial cells (HUVEC) and malignant glioblastoma (U87-MG) to magnetic hyperthermia (ac-magnetic field of 700 kHz, 23.10 kA m−1) for 1 hour with and without the presence of superparamagnetic 10 nm sized polyol-made γ-Fe2O3 nanoparticles (NPs). Interestingly, despite their reduced size, NPs exhibit high magnetization, close to that of the bulk material, in relation to their high crystalline quality. In practice, they ensured an efficient heating capacity, leading to about 20% and more than 50% cell death of HUVEC and U87-MG lines, respectively, when hyperthermia assays were achieved in the presence of these NPs. Magnetophoresis and X-ray fluorescence spectrometry measurements evidenced a more important internalization of NPs in U87-MG than in HUVECs. Surprisingly both cell lines reached the same maximal temperature, namely 42 °C, after hyperthermia treatment suggesting a higher thermosensitivity of the former compared to the latter, establishing the fact that polyol-made γ-Fe2O3 NP assisted hyperthermia is a harmful agent to glioma treatment.

Desert Hedgehog/Patch2 Axis Contributes to Vascular Permeability and Angiogenesis in Glioblastoma

Glioblastoma multiforme (GBM) constitutes the most common and the most aggressive type of human tumors affecting the central nervous system. Prognosis remains dark due to the inefficiency of current treatments and the rapid relapse. Paralleling other human tumors, GBM contains a fraction of tumor initiating cells with the capacity to self-renew, initiate and maintain the tumor mass. These cells were found in close proximity to brain vasculature, suggesting functional interactions between brain tumor-initiating cells (BTICs) and endothelial cells within the so-called vascular niche. However, the mechanisms by which these cells impact on the endothelium plasticity and function remain unclear. Using culture of BTICs isolated from a cohort of 14 GBM patients, we show that BTICs secretome promotes brain endothelial cell remodeling in a VEGF-independent manner. Gene array analysis unmasked that BTICs-released factors drove the expression of Ptch2 in endothelial cells. Interestingly, BTICs produce desert hedgehog (DHH) ligand, enabling a paracrine DHH/Ptch2 signaling cascade that conveys elevated permeability and angiogenesis. Finally, DHH silencing in BTICs dramatically reduced tumor growth, as well as vascularization and intra-tumor permeability. Collectively, our data unveil a role for DHH in exacerbated tumor angiogenesis and permeability, which may ultimately favor glioblastoma growth, and thus place the DHH/Ptch2 nexus as a molecular target for novel therapies.

Pharmacological targeting of apelin impairs glioblastoma growth

Glioblastomas are aggressive brain tumours that contain a subpopulation of highly plastic self-renewing cancer cells. Harford-Wright et al. show that the vasoactive peptide apelin, secreted by brain endothelial cells, regulates glioblastoma patient-derived cells with stem-like properties. Pharmacological blockade of apelin hampers glioblastoma cell expansion and improves survival in xenografted mice.