Luisa Campagnolo

Low Doses of Pristine and Oxidized Single-Wall Carbon Nanotubes Affect Mammalian Embryonic Development

Several in vitro and in vivo studies suggest local and systemic effects following exposure to carbon nanotubes. No data are available, however, on their possible embryotoxicity in mammals. In this study, we tested the effect of pristine and oxidized single-wall carbon nanotubes (SWCNTs) on the development of the mouse embryo. To this end, SWCNTs (from 10 ng to 30 μg/mouse) were administered to female mice soon after implantation (postcoital day 5.5); 10 days later, animals were sacrificed, and uteri, placentas, and fetuses examined. A high percentage of early miscarriages and fetal malformations was observed in females exposed to oxidized SWCNTs, while lower percentages were found in animals exposed to the pristine material. The lowest effective dose was 100 ng/mouse. Extensive vascular lesions and increased production of reactive oxygen species (ROS) were detected in placentas of malformed but not of normally developed fetuses. Increased ROS levels were likewise detected in malformed fetuses. No increased ROS production or evident morphological alterations were observed in maternal tissues. No fetal and placental abnormalities were ever observed in control animals. In parallel, SWCNT embryotoxicity was evaluated using the embryonic stem cell test (EST), a validated in vitro assay developed for predicting embryotoxicity of soluble chemical compounds, but never applied in full to nanoparticles. The EST predicted the in vivo data, identifying oxidized SWCNTs as the more toxic compound.

Egfl7, a Novel Epidermal Growth Factor-Domain Gene Expressed in Endothelial Cells

We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino‐terminal signal peptide and two centrally located EGF‐like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF‐like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7. Developmental Dynamics 230:316–324, 2004.

EGFL7 Is a Chemoattractant for Endothelial Cells and Is Up-Regulated in Angiogenesis and Arterial Injury

The endothelium of the adult vasculature is normally quiescent, with the exception of the vasculature of the female reproductive system. However, in response to appropriate stimuli (ie, wound healing, atherosclerosis, tumor growth and metastasis, arthritis) the vasculature becomes activated and grows new capillaries through angiogenesis. We have recently identified a novel endothelial-restricted gene, Egfl7, that encodes a 41-kd secreted protein (Fitch MJ, Campagnolo L, Kuhnert F, Stuhlmann H: Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells. Dev Dyn 2004, 230:316-324). Egfl7 is expressed at high levels early during mouse embryonic development and is strictly associated with the vascular bed. In this study, we investigated Egfl7 expression in the quiescent adult vasculature, in the pregnant uterus, and in two different models of arterial injury, namely ballooning and ferric chloride injury. By RNA in situ hybridization, Egfl7 expression in the vasculature was found to be restricted to the endothelium of the capillaries and mature vessels. In the pregnant uterus, increased vascularization was accompanied by up-regulation of Egfl7. On arterial injury, Egfl7 expression was up-regulated in the regenerating endothelium, but not in the neointima. Importantly, the EGFL7 protein acted as a chemoattractant for embryonic endothelial cells and fibroblasts in a cell migration assay. Together, these results suggest that Egfl7 functions in the formation and maintenance of endothelial integrity and that its up-regulation may be a critical component in the reorganization of the vascular bed in response to angiogenic stimuli.

Comprehensive in vitro toxicity testing of a panel of representative oxide nanomaterials: First steps towards an intelligent testing strategy

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO2 NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

Interactions of Engineered Nanoparticles with Organs Protected by Internal Biological Barriers

Engineered nanomaterials may exert adverse effects on human health which, in turn, may be linked to their propensity to cross biological barriers in the body. Here, available evidence is discussed, based on in vivo studies for interactions of commercially relevant nanoparticles with critical internal barriers. The internal barriers in focus in this review are the blood-brain barrier, protecting the brain, the blood-testis barrier, protecting the male germ line, and the placenta, protecting the developing fetus. The route of exposure (pulmonary, gastro-intestinal, intravenous, intraperitoneal, dermal), and, hence, the portal of entry of nanoparticles into the body, is of critical importance. Different physico-chemical properties, not only size, may determine the ability of nanoparticles to breach biological barriers; the situation is further compounded by the formation of a so-called corona of biomolecules on the surfaces of nanoparticles, the composition of which may vary depending on the route of exposure and the translocation of nanoparticles from one biological compartment to another. The relevance of nanoparticle interactions with internal biological barriers for their impact on the organs protected by these barriers is also discussed.

Mouse matriptase-2: identification, characterization and comparative mRNA expression analysis with mouse hepsin in adult and embryonic tissues

We report the identification and characterization of mouse matriptase-2 (m-matriptase-2), an 811-amino-acid protein composed of an N-terminal cytoplasmic domain, a membrane-spanning domain, two CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains, three LDLR (low-density-lipoprotein receptor class A) domains and a C-terminal serine-protease domain. All m-matriptase-2 protein domain boundaries corresponded with intron/exon junctions of the encoding gene, which spans approx. 29 kb and comprises 18 exons. Matriptase-2 is highly conserved in human, mouse and rat, with the rat matriptase-2 gene ( r-maltriptase-2 ) predicted to encode transmembrane and soluble isoforms. Western-blot analysis indicated that m-matriptase-2 migrates close to its theoretical molecular mass of 91 kDa, and immunofluorescence analysis was consistent with the proposed surface membrane localization of this protein. Reverse-transcription PCR and in-situ -hybridization analysis indicated that m-matriptase-2 expression overlaps with the distribution of mouse hepsin (m-hepsin, a cell-surface serine protease identified in hepatoma cells) in adult tissues and during embryonic development. In adult tissues both are expressed at highest levels in liver, kidney and uterus. During embryogenesis m-matriptase-2 expression peaked between days 12.5 and 15.5. m-hepsin expression was biphasic, with peaks at day 7.5 to 8.5 and again between days 12.5 and 15.5. In situ hybridization of embryonic tissues indicated abundant expression of both m-matriptase-2 and m-hepsin in the developing liver and at lower levels in developing pharyngo-tympanic tubes. While m-hepsin was detected in the residual embryonic yolk sac and with lower intensity in lung, heart, gastrointestinal tract, developing kidney tubules and epithelium of the oral cavity, m-matriptase-2 was absent in these tissues, but strongly expressed within the nasal cavity by olfactory epithelial cells. Mechanistic insight into the potential role of this new transmembrane serine protease is provided by its novel expression profile in embryonic and adult mouse.

A perspective on the developmental toxicity of inhaled nanoparticles

This paper aimed to clarify whether maternal inhalation of engineered nanoparticles (NP) may constitute a hazard to pregnancy and fetal development, primarily based on experimental animal studies of NP and air pollution particles. Overall, it is plausible that NP may translocate from the respiratory tract to the placenta and fetus, but also that adverse effects may occur secondarily to maternal inflammatory responses. The limited database describes several organ systems in the offspring to be potentially sensitive to maternal inhalation of particles, but large uncertainties exist about the implications for embryo-fetal development and health later in life. Clearly, the potential for hazard remains to be characterized. Considering the increased production and application of nanomaterials and related consumer products a testing strategy for NP should be established. Due to large gaps in data, significant amounts of groundwork are warranted for a testing strategy to be established on a sound scientific basis.

Molecular basis of thyrotropin and thyroid hormone action during implantation and early development

BACKGROUND Implantation and early embryo development are finely regulated processes in which several molecules are involved. Evidence that thyroid hormones (TH: T4 and T3) might be part of this machinery is emerging. An increased demand for TH occurs during gestation, and any alteration in maternal thyroid physiology has significant implications for both maternal and fetal health. Not only overt but also subclinical hypothyroidism is associated with infertility as well as with obstetric complications, including disruptions and disorders of pregnancy, labor, delivery, and troubles in early neonatal life. METHODS We searched the PubMed and Google Scholar databases for articles related to TH action on ovary, endometrium, trophoblast maturation and embryo implantation. In addition, articles on the regulation of TH activity at cellular level have been reviewed. The findings are hereby summarized and critically discussed. RESULTS TH have been shown to influence endometrial, ovarian and placental physiology. TH receptors (TR) and thyrotropin (thyroid-stimulating hormone: TSH) receptors (TSHR) are widely expressed in the feto-maternal unit during implantation, and both the endometrium and the trophoblast might be influenced by TH either directly or through TH effects on the synthesis and activity of implantation-mediating molecules. Interestingly, due to the multiplicity of mechanisms involved in TH action (e.g. differential expression of TR isoforms, heterodimeric receptor partners, interacting cellular proteins, and regulating enzymes), the TH concentration in blood is not always predictive of their cellular availability and activity at both genomic and nongenomic level. CONCLUSIONS In addition to the known role of TH on the hormonal milieu of the ovarian follicle cycle, which is essential for a womans fertility, evidence is emerging on the importance of TH signaling during implantation and early pregnancy. Based on recent observations, a local action of TH on female reproductive organs and the embryo during implantation appears to be crucial for a successful pregnancy. Furthermore, an imbalance in the spatio-temporal expression of factors involved in TH activity might induce early arrest of pregnancy in women considered as euthyroid, based on their hormonal blood concentration. In conclusion, alterations of the highly regulated local activity of TH may play a crucial, previously underestimated, role in early pregnancy and pregnancy loss. Further studies elucidating this topic should be encouraged.

A plasmid-encoded VEGF siRNA reduces glioblastoma angiogenesis and its combination with interleukin-4 blocks tumor growth in a xenograft mouse model

Angiogenesis is required for the development and biologic progression of glioblastoma multiforme (GBM), which is the most malignant infiltrative astrocytoma. Vascular endothelial growth factor (VEGF) plays a predominant role in the increased vascularity and endothelial cell proliferation in GBMs driven by the expression of pro-angiogenic cytokines. In this study, we employed a vector-encoded VEGF siRNA to impair VEGF secretion from U87 human glioblastoma cells. The direct intra-tumor injection of siRNA-encoding plasmid complexed with linear polyethylenimine (PEI) efficiently reduced the vascularization of treated tumors in xenografts established in SCID mice by subcutaneous inoculation of U87 cells, but was not able to reduce tumor growth. We then sought to strengthen the in vivo action of our siRNA by coupling it to a well known direct antiangiogenic agent, mouse interleukin 4 (mIL4). We infected U87 cells with a retroviral vector co-expressing the VEGF siRNA and mIL4 and produced stable cell lines that we used for an in vivo experiment of subcutaneous injection in SCID mice. In this setting, the concomitant expression of mIL4 and siRNA totally abolished the growth of subcutaneous tumors. These results suggest that our retroviral vector might be employed as a potential tool in future antiangiogenic gene therapy trials for glioblastoma.

Changes in cardiac autonomic regulation after acute lung exposure to carbon nanotubes: implications for occupational exposure

Carbon nanotubes (CNTs) are among the most relevant engineered nanomaterials (ENMs). Given the expected rise of exposure to ENMs, there is concern that they may adversely affect health of exposed people. Aim of the study was to test the hypothesis that single wall carbon nanotubes (SWCNTs) pulmonary exposure acutely affect the autonomic cardiovascular regulation in conscious rats. We studied Wistar-Kyoto rats in which a telemetry transmitter for continuous arterial pressure (AP) and heart rate (HR) recordings was surgically implanted. SWCNTs dispersed in phosphate buffer saline (PBS) or PBS alone were randomly administered intratracheally. Immediately before, and 24 hours after each instillation a 30 min AP recording was performed. The sequence analysis was performed to evaluate the baroreflex function. In the control group, PBS instillation did not induce any significant changes. At variance the SWCNT exposure induced a significant reduction of baroreflex system (BRS) (3.5 ± 0.6 versus 2.6 ± 0.40 msec/mmHg) without significant changes in the occurrence of baroreflex sequences (7.5 ± 0.47% versus 7.4 ± 0.38%). Our results show that SWCNT pulmonary exposure might affect the cardiovascular autonomic regulation thus contributing to cardiac and arrhythmic events.