Hanane Derradji

Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells.

Many space missions have shown that prolonged space flights may increase the risk of cardiovascular problems. Using a three‐dimensional clinostat, we investigated human endothelial EA.hy926 cells up to 10 days under conditions of simulated microgravity (µg) to distinguish transient from long‐term effects of µg and 1g. Maximum expression of all selected genes occurred after 10 min of clinorotation. Gene expression (osteopontin, Fas, TGF‐β1) declined to slightly upregulated levels or rose again (caspase‐3) after the fourth day of clinorotation. Caspase‐3, Bax, and Bcl‐2 protein content was enhanced for 10 days of microgravity. In addition, long‐term accumulation of collagen type I and III and alterations of the cytoskeletal alpha‐ and beta‐tubulins and F‐actin were detectable. A significantly reduced release of soluble factors in simulated microgravity was measured for brain‐derived neurotrophic factor, tissue factor, vascular endothelial growth factor (VEGF), and interestingly for endothelin‐1, which is important in keeping cardiovascular balances. The gene expression of endothelin‐1 was suppressed under µg conditions at days 7 and 10. Alterations of the vascular endothelium together with a decreased release of endothelin‐1 may entail post‐flight health hazards for astronauts. J. Cell. Biochem. J. Cell. Biochem. 101: 1439–1455, 2007.

Effects of basic fibroblast growth factor on endothelial cells under conditions of simulated microgravity

Fibroblast growth factors interact with appropriate endothelial cell (EC) surface receptors and initiate intracellular signal cascades, which participate in modulating blood vessel growth. EC, upon exposure to basic fibroblast growth factors (bFGFs) undergo profound functional alterations, which depend on their actual sensitivity and involve gene expression and de novo protein synthesis. We investigated the effects of bFGF on signaling pathways of EA.hy926 cells in different environments. EC were cultured under normal gravity (1 g) and simulated microgravity (µg) using a three‐dimensional (3D) clinostat. Microgravity induced early and late apoptosis, extracellular matrix proteins, endothelin‐1 (ET‐1) and TGF‐β1 expression. Microgravity reduced eNOS mRNA within 24 h. Moreover, a six‐ to eightfold higher amount of IL‐6 and IL‐8 was secreted within 24 h µg. In addition, microgravity induced a duplication of NF‐kappaB p50, while p65 was quadrupled. At 1 g, bFGF application (4 h) reduced ET‐1, TGF‐β1 and eNOS gene expression. After 24 h, bFGF enhanced fibronectin, VEGF, Flk‐1, Flt‐1, the release of IL‐6, IL‐8, and TGF‐β1. Furthermore, bFGF promoted apoptosis, reduced NFkB p50, but enhanced NFkB p65. After 4 h µg, bFGF decreased TGF‐β1, eNOS, and ET‐1 gene expression. After 24 h µg, bFGF elevated fibronectin, Flk‐1 and Flt‐1 protein, and reduced IL‐6 and IL‐8 compared with vehicle treated µg cultures. In µg, bFGF enhanced NF‐KappaB p50 by 50%, Bax by 25% and attenuated p65, activation of caspase‐3 and annexin V‐positive cells. bFGF differently changes intracellular signals in ECs depending whether it is applied under microgravity or normal gravity conditions. In microgravity, bFGF contributes to protect the EC from apoptosis. J. Cell. Biochem. 104: 1324–1341, 2008.

Stress response and humoral immune system alterations related to chronic hypergravity in mice

Spaceflights are known to induce stress and immune dysregulation. Centrifugation, as hindlimb unloading, is a good ground based-model to simulate altered gravity which occurs during space missions. The aim of this study was to investigate the consequences of a long-term exposure to different levels of hypergravity on the stress response and the humoral immunity in a mouse model. For this purpose, adult C57Bl/6J male mice were subjected for 21 days either to control conditions or to 2G or 3G acceleration gravity forces. Corticosterone level and anxiety behavior revealed a stress response which was associated with a decrease of body weight, after 21-day of centrifugation at 3G but not at 2G. Spleen lymphocyte lipopolysaccharide (LPS) responsiveness was diminished by 40% in the 2G group only, whereas a decrease was noted when cells were stimulated with concanavalin A for both 2G and 3G groups (about 25% and 20%, respectively) compared to controls. Pro-inflammatory chemokines (MCP-1 and IP-10) and Th1 cytokines (IFNγ and IL2) were slightly decreased in the 2G group and strongly decreased in the 3G mouse group. Regarding Th2 cytokines (IL4, IL5) no further significant modification was observed, whereas the immunosuppressive cytokine IL10 was slightly increased in the 3G mice. Finally, serum IgG concentration was twice higher whereas IgA concentration was slightly increased (about 30%) and IgM were unchanged in 2G mice compared to controls. No difference was observed in the 3G group with these isotypes. Consequently, functional immune dysregulations and stress responses were dependent of the gravity level.

Medium-mediated DNA repair response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts.

Radiation induced bystander effects, either protective or adverse, have been identified in a variety of cells and for different endpoints. They are thought to arise from communication between cells through direct cell-cell contacts and via transmissible molecules secreted into the medium by targeted cells. We have investigated medium-mediated damage response in human dermal fibroblasts (HDF) after exposure to ionizing irradiation. We show that HDF experience an elevated level of double stranded DNA damage repair response when incubated with conditioned growth medium of irradiated cells. The magnitude of this response is much lower than observed for directly irradiated cells and is proportional to the radiation dose, as is its persistence across time. Since secretion of cytokines is one of the possible pathways linking targeted and non-targeted cells a multiplex analysis was performed. Four cytokines - IL6, IL8, MCP-1 and RANTES - were identified in the growth medium of irradiated cells after exposure to X-rays (2Gy). These cytokines were significantly upregulated and each cytokine showed differential upregulation kinetics. Finally we performed a functional analysis to see if IL6 and MCP-1 could induce gammaH2AX foci formation. IL6 caused a significant increase in spot occupancy compared to controls. Although only indicative MCP-1 appears to have the opposite effect as it caused a drop in spot occupancy. The combined addition of these two cytokines produced no significant response was observed. Both IL6 and MCP-1 have an effect on the gammaH2AX spot occupancy possibly linking these cytokines to the bystander response.

Low dose irradiation of thyroid cells reveals a unique transcriptomic and epigenetic signature in RET/PTC-positive cells

The high doses of radiation received in the wake of the Chernobyl incident and the atomic bombing of Hiroshima and Nagasaki have been linked to the increased appearance of thyroid cancer in the children living in the vicinity of the site. However, the data gathered on the effect of low doses of radiation on the thyroid remain limited. We have examined the genome wide transcriptional response of a culture of TPC-1 human cell line of papillary thyroid carcinoma origin with a RET/PTC1 translocation to various doses (0.0625, 0.5, and 4Gy) of X-rays and compared it to response of thyroids with a RET/PTC3 translocation and against wild-type mouse thyroids irradiated with the same doses using Affymetrix microarrays. We have found considerable overlap at a high dose of 4Gy in both RET/PTC-positive systems but no common genes at 62.5mGy. In addition, the response of RET/PTC-positive system at all doses was distinct from the response of wild-type thyroids with both systems signaling down different pathways. Analysis of the response of microRNAs in TPC-1 cells revealed a radiation-responsive signature of microRNAs in addition to dose-responsive microRNAs. Our results point to the fact that a low dose of X-rays seems to have a significant proliferative effect on normal thyroids. This observation should be studied further as opposed to its effect on RET/PTC-positive thyroids which was subtle, anti-proliferative and system-dependent.

Ionizing radiation-induced gene modulations, cytokine content changes and telomere shortening in mouse fetuses exhibiting forelimb defects

Several lines of evidence have linked limb teratogenesis to radiation-induced apoptosis and to the p53 status in murine fetuses. In previous reports, we studied the occurrence of various malformations after intrauterine irradiation and showed that these malformations were modulated by p53-deficiency as well as by the developmental stage at which embryos were irradiated. In this new study, we focused onto one particular phenotype namely forelimb defects to further unravel the cellular and molecular mechanisms underlying this malformation. We measured various parameters expected to be directly or indirectly influenced by irradiation damage. The mouse fetuses were irradiated at day 12 p.c. (post conception) and examined for forelimb defects on gestational days 15, 16, 17 and 19 of development. The release of inflammatory cytokines was determined in the amniotic fluid on day 16 p.c. and the mean telomere lengths assessed at days 12, 13 and 19 p.c. Differential gene expression within the forelimb bud tissues was determined using Real Time quantitative PCR (RTqPCR) 24 h following irradiation. Apoptosis was investigated in the normal and malformed fetuses using the TUNEL assay and RTqPCR. First, we found that irradiated fetuses with forelimb defects displayed excessive apoptosis in the predigital regions. Besides, overexpression of the pro-apoptotic Bax gene indicates a mitochondrial-mediated cell death. Secondly, our results showed overexpression of MKK3 and MKK7 (members of the stress-activated MAP kinase family) within the malformed fetuses. The latter could be involved in radiation-induced apoptosis through activation of the p38 and JNK pathways. Thirdly, we found that irradiated fetuses exhibiting forelimb defects showed a marked telomere shortening. Interestingly, telomere shortening was observed as the malformations became apparent. Fourthly, we measured cytokine levels in the amniotic fluid and detected a considerable inflammatory reaction among the irradiated fetuses as evidenced by the increase in pro-inflammatory cytokine levels. Altogether, our data suggest that transcriptional modulations of apoptotic, inflammation, stress, and DNA damage players are early events in radiation-induced forelimb defects. These changes resulted in harsh developmental conditions as indicated by a marked increase in cytokine levels in the amniotic fluid and telomere shortening, two features concomitant with the onset of the forelimb defect phenotype in our study.

Iodine-deficiency-induced long lasting angiogenic reaction in thyroid cancers occurs via a vascular endothelial growth factor-hypoxia inducible factor-1-dependent, but not a reactive oxygen species-dependent, pathway.

BACKGROUND In the thyroid, iodine deficiency (ID) induces angiogenesis via a tightly controlled reactive oxygen species (ROS)-hypoxia inducible factor-1 (HIF-1)-vascular endothelial growth factor (VEGF) dependent pathway (ROS-HIF-VEGF). Deficient iodine intake may be associated with increased thyroid cancer incidence. The hypothesis of this work is to test whether ID affects the angiogenic processes in thyroid malignant cells by altering the ROS-HIF-VEGF pathway. METHODS Goiters were obtained in RET/PTC3 transgenic and wild-type (wt) mice and ID was induced in three thyroid carcinoma cell lines (TPC-1, 8305c, and R082-w1). Thyroid blood flow, VEGF mRNA and protein, and HIF-1α protein expression were measured. The role of HIF-1 and of ROS was assessed using echinomycin and N-acetylcysteine (NAC), respectively. RESULTS The goitrogen treatment increased the thyroid blood flow in wt and RET/PTC3 mice. Compared with wt mice, basal VEGF expression was higher in RET/PTC3 mice and increased with goitrogen treatment. In the three cell lines, ID induced marked increases in VEGF mRNA, and moderate increases in HIF-1α protein expression that were not transient as in normal cells. ID-induced VEGF mRNA expression was fully (8305c), partially (TPC-1), or not (R082-w1) blocked by echinomycin. NAC had no effect on ID-induced VEGF mRNA and HIF-1α protein expression in the three cell lines. CONCLUSIONS ID induces a long lasting angiogenic phenotype in thyroid cancer cells that occurs through VEGF induction via a pathway partially mediated by HIF-1, but not by ROS. These results suggest that, in contrast with normal cells, ID-induced angiogenesis in cancer cells occurs via alternative and likely less controlled routes, thereby leading to uncontrolled growth.

Transgenerational developmental effects and genomic instability after X-irradiation of preimplantation embryos: studies on two mouse strains.

Recent results have shown that irradiation of a single cell, the zygote or 1-cell embryo of various mouse strains, could lead to congenital anomalies in the fetuses. In the Heiligenberger strain, a link between the radiation-induced congenital anomalies and the development of a genomic instability was also suggested. Moreover, further studies showed that in that strain, both congenital anomalies and genomic instability could be transmitted to the next generation. The aim of the experiments described in this paper was to investigate whether such non-targeted transgenerational effects could also be observed in two other radiosensitive mouse strains (CF1 and ICR), using lower radiation doses. Irradiation of the CF1 and ICR female zygotes with 0.2 or 0.4Gy did not result in a decrease of their fertility after birth, when they had reached sexual maturity. Moreover, females of both strains that had been X-irradiated with 0.2Gy exhibited higher rates of pregnancy, less resorptions and more living fetuses. Additionally, the mean weight of living fetuses in these groups had significantly increased. Exencephaly and dwarfism were observed in CF1 fetuses issued from control and X-irradiated females. In the control group of that strain, polydactyly and limb deformity were also found. The yields of abnormal fetuses did not differ significantly between the control and X-irradiated groups. Polydactyly, exencephaly and dwarfism were observed in fetuses issued from ICR control females. In addition to these anomalies, gastroschisis, curly tail and open eye were observed at low frequencies in ICR fetuses issued from X-irradiated females. Again, the frequencies of abnormal fetuses found in the different groups did not differ significantly. In both CF1 and ICR mouse strains, irradiation of female zygotes did not result in the development of a genomic instability in the next generation embryos. Overall, our results suggest that, at the moderate doses used, developmental defects observed after X-irradiation of female zygotes of these two sensitive mouse strains should not be transmitted to the next generation. Paradoxically, other studies would be needed to address the question of a potential increase of fertility after doses lower than 0.2Gy in both strains.

Iodine deficiency induces a VEGF-dependent microvascular response in salivary glands and in the stomach

Despite efforts to optimize iodine supply in iodine deficient countries, iodine deficiency (ID) remains a global problem worldwide. Activation of the local microvasculature by ID in the thyroid gland aims at improving the local supply of iodide. For this purpose, the thyrocytes secrete vascular endothelial growth factor (VEGF) that acts on adjacent capillaries, via a reactive oxygen species (ROS)/Hypoxia Inducible factor (HIF)-dependent pathway. Beside the thyroid, other organs including salivary glands and the stomach do express the sodium/iodide symporter (NIS) and are able to take iodide up, potentially rendering them sensitive to ID. To verify this hypothesis, ID-induced effects on the local microvasculature were studied in salivary glands and in the stomach. ID was induced by feeding young mice with an iodide-deficient diet and NIS inhibitor perchlorate in the drinking water. In salivary glands, ID induced a transient increase in HIF-1α protein expression accompanied by a transient, VEGF-dependent increase in blood flow. In the gastric mucosa, ID transiently increased VEGF expression in the mucin-secreting epithelium and in ghrelin-secreting endocrine cells. These observations suggest that microvascular changes in response to ID occur in NIS-expressing tissues other than the thyroid. NIS expressing cells could be viewed as iodide sensors that respond to ID by inducing vascular changes, probably to optimize iodide bioavailability at regional or systemic levels.

Acute iodine deficiency induces a transient VEGF-dependent microvascular response in mammary glands involving HIF-1, ROS, and mTOR

Iodine deficiency (ID), which affects almost two billion people worldwide, is associated with breast pathologies such as fibrosis in human and induces breast atypia in animal models. Because ID induces vascular activation in the thyroid, another iodide-uptaking organ, and as breast is also sensitive to ID, we aimed to characterize ID-induced effects on the breast microvasculature in vivo and in two different breast cell lines in vitro. Virgin and lactating NMRI mice received an iodide-deficient diet and a Na+/I- symporter inhibitor for 1 to 20 days. Some virgin mice were treated with vascular endothelial growth factor A (VEGF) or VEGF receptor inhibitors. In vitro, ID was induced in MCF7 and MCF12A cells by replacing the iodide-containing medium by an iodide-deficient medium. In vivo, VEGF expression was increased following ID in mammary tissues. Consequently, ID induced a transient increase in mammary gland blood flow, measured after anesthesia, in virgin and lactating mice, which was repressed by VEGF or VEGF receptor inhibitors. In MCF7 cells, ID induced a transient increase in reactive oxygen species, followed by an increase in hypoxia-inducible factor-1α (HIF-1α) protein and VEGF mRNA expression. Antioxidant N-acetylcysteine and mammalian target of rapamycin (mTOR) inhibitor blocked ID-induced HIF-1α protein increase and VEGF transcription. However, mTOR activity was not inhibited by N-acetylcysteine. Similar responses were observed in MCF12A cells. These data indicate that ID activates the canonical VEGF pathway and mTOR in breast tissues, which provides new insights to better understand the correlation between ID, vascular activation, and breast pathologies.