Claire Squiban

Late and persistent up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression by ionizing radiation in human endothelial cells in vitro.

Adhesion molecules play a key role in cellular traffic through vascular endothelium, in particular during the inflammatory response when leukocytes migrate from blood into tissues. Since inflammation is one of the major consequences of radiation injury, we investigated the effect of ionizing radiation on cell-surface expression of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in cultured human umbilical vein endothelial cells (HUVEC). Flow cytometry performed on irradiated HUVEC revealed both a time- (from 2 to 10 days) and dose- (from 2 to 10 Gy) dependent up-regulation of basal expression of ICAM-1, and no induction of VCAM-1 or E-selectin. The radiation-induced increase in ICAM-1 expression on HUVEC was correlated with augmented adhesion of neutrophils on irradiated endothelial cells. Interleukin-6 (Il-6) or other soluble factors released by irradiation were not involved in the enhanced ICAM-1 expression by irradiation. Northern blot analysis showed an overexpression of ICAM-1 mRNA from 1 to 6 days after a 10 Gy exposure. Our data suggest that ICAM-1 participates in the radiation-induced inflammatory reaction of the endothelium.

Pravastatin Limits Endothelial Activation after Irradiation and Decreases the Resulting Inflammatory and Thrombotic Responses

Abstract Gaugler, M. H., Vereycken-Holler, V., Squiban, C., Vandamme, M., Vozenin-Brotons, M. C. and Benderitter, M. Pravastatin Limits Endothelial Activation after Irradiation and Decreases the Resulting Inflammatory and Thrombotic Responses. Radiat. Res. 163, 479–487 (2005). Endothelial dysfunction has been implicated in the pathogenesis of atherosclerosis, fibrosis and vascular occlusion after radiation therapy. Statins have been reported to improve endothelial function; however, this beneficial effect on endothelial cells has never been investigated after irradiation. Therefore, using human microvascular endothelial cells from lung that had been irradiated with 5 or 10 Gy, we assessed the effect of pravastatin on endothelial activation by ELISA, cell-ELISA and electrophoretic mobility shift assay and increased blood-endothelial cell interactions by a flow adhesion assay. Pravastatin inhibited the overproduction of monocyte chemoattractant protein 1, IL6 and IL8 and the enhanced expression of intercellular adhesion molecule 1 but had no effect on platelet-endothelial cell adhesion molecule 1 expression. Moreover, pravastatin down-regulated the radiation-induced activation of the transcription factor activator protein 1 but not of nuclear factor-κB. Finally, an inhibition by pravastatin of increased adhesion of leukocytes and platelets to irradiated endothelial cells was observed. The effect of pravastatin was maintained up to 14 days after irradiation and was reversed by mevalonate. Pravastatin exerts persistent anti-inflammatory and anti-thrombotic effects on irradiated endothelial cells. Statins may be considered in therapeutic strategies for the management of patients treated with radiation therapy.

Pravastatin Limits Radiation-Induced Vascular Dysfunction in the Skin

About half of people with cancer are treated with radiation therapy; however, normal tissue toxicity still remains a dose-limiting factor for this treatment. The skin response to ionizing radiation may involve multiple inflammatory outbreaks. The endothelium is known to play a critical role in radiation-induced vascular injury. Furthermore, endothelial dysfunction reflects a decreased availability of nitric oxide. Statins have been reported to preserve endothelial function through their antioxidant and anti-inflammatory activities. In this study, wild type and endothelial nitric oxide synthase (eNOS)(-/-) mice were subjected to dorsal skin irradiation and treated with pravastatin for 28 days. We demonstrated that pravastatin has a therapeutic effect on skin lesions and abolishes radiation-induced vascular functional activation by decreasing interactions between leukocytes and endothelium. Pravastatin limits the radiation-induced increase of blood CCL2 and CXCL1 production expression of inflammatory adhesion molecules such as E-selectin and intercellular adhesion molecule-1, and inflammatory cell migration in tissues. Pravastatin limits the in vivo and in vitro radiation-induced downregulation of eNOS. Moreover, pravastatin has no effect in eNOS(-/-) mice, demonstrating that eNOS plays a key role in the beneficial effect of pravastatin in radiation-induced skin lesions. In conclusion, pravastatin may be a good therapeutic approach to prevent or reduce radiation-induced skin damage.

Thrombopoietin promotes hematopoietic recovery and survival after high-dose whole body irradiation

PURPOSE The therapeutic potential of thrombopoietin (TPO), the major regulator of platelet production, was evaluated for hematopoietic recovery and survival in mice following lethal and supralethal total body irradiation (TBI). METHODS AND MATERIALS Hematopoietic recovery was studied in C57BL6/J mice after 8 Gy TBI (gamma-rays). Survival experiments were performed with C57BL6/J and BCBA F1 mice. Two protocols of TPO administration were evaluated: treatment for 7 consecutive days (7 x 0.3 microg/mice) beginning 2 h after exposure, or a single dose (0.3 microg/mice) administered 2 h after irradiation. RESULTS TPO improved the platelet nadir and accelerated the platelet reconstitution of irradiated mice in comparison to placebo-treated mice. Recovery of neutrophils and erythrocytes was stimulated as well. TPO induced an accelerated recovery of hematopoietic progenitors and immature multilineage progenitors in bone marrow and spleen. In addition, TPO administration induced approximately 90% survival of 8 Gy irradiated C57BL6/J mice, a TBI dose which resulted in 100% mortality within 30 days for placebo-treated mice. Single TPO administration was as effective as repeated injections for hematopoietic recovery and prevention of mortality. Dose-effect survival experiments were performed in BCBA F1 mice and demonstrated that TPO shifted the LD50/30 from approximately 9.5 Gy to 10.5 Gy TBI given as a single dose, and from 14 Gy to as high as 17 Gy when TBI was given in three equal doses, each separated by 24 h. CONCLUSION These results demonstrate that the multilineage hematopoietic effects of TPO may be advantageously used to protect against lethal bone marrow failure following high dose TBI.

Abdominal Radiation Exposure Elicits Inflammatory Responses and Abscopal Effects in the Lungs of Mice

Abstract Van der Meeren, A., Monti, P., Vandamme, M., Squiban, C., Wysocki, J. and Griffiths, N. Abdominal Radiation Exposure Elicits Inflammatory Responses and Abscopal Effects in the Lungs of Mice. Radiat. Res. 163, 144–152 (2005). An inflammatory reaction is a classical feature of radiation exposure and appears to be a key event in the development of the acute radiation syndrome. We have investigated the radiation-induced inflammatory response in C57BL6/J mice after total abdominal or total-body irradiation at a dose of 15 Gy. Our goal was to determine the radiation-induced inflammatory response of the gut and to study the consequences of abdominal irradiation for the intestine and for the lungs as a distant organ. A comparison with total-body irradiation was used to take into account the hematopoietic response in the inflammatory process. For both irradiation regimens, systemic and intestinal responses were evaluated. A systemic inflammatory reaction was found after abdominal and total-body irradiation, concomitant with increased cytokine and chemokine production in the jejunum of irradiated mice. In the lungs, the radiation-induced changes in the production of cytokines and chemokines and in the expression of adhesion molecules after both abdominal and total-body irradiation indicate a possible abscopal effect of radiation in our model. The effects observed in the lungs after irradiation of the abdomino-pelvic region may be caused by circulating inflammatory mediators consequent to the gut inflammatory response.

Inflammatory Reaction and Changes in Expression of Coagulation Proteins on Lung Endothelial Cells after Total-Body Irradiation in Mice

Abstract Van der Meeren, A., Vandamme, M., Squiban, C., Gaugler, M-H. and Mouthon, M-A. Inflammatory Reaction and Changes in Expression of Coagulation Proteins on Lung Endothelial Cells after Total-Body Irradiation in Mice. Radiat. Res. 160, 637–646 (2003). Inflammatory reaction is a classical feature of radiation exposure, and pneumonitis is a dose-limiting complication in the handling of hematological disorders treated with total-body irradiation. In the present study, we first evaluated the inflammatory response in C57BL6/J mice exposed to lethal doses of γ rays treated with antibiotics or not. Both interleukin 6 and KC (also known as Gro1) were increased in the plasma 10 to 18 days after radiation exposure, independent of bacterial infection, whereas fibrinogen release was linked to a bacterial infection. Furthermore, both Il6 and KC were increased in the lungs of irradiated mice. Our second objective was to characterize the endothelial cell changes in the lungs of total-body-irradiated mice. For this purpose, a quantitative RT-PCR was used to determine the expression of genes involved in inflammatory and coagulation processes. We found that the adhesion molecules P-selectin and platelet endothelial cell adhesion molecule 1 were up-regulated, whereas E-selectin remained unchanged. Tissue factor expression was up-regulated as well, and thrombomodulin gene expression was down-regulated. The investigation by immunohistochemistry of adhesion molecules confirmed the increase in the basal expression of both P-selectin and platelet endothelial cell adhesion molecule 1 on pulmonary endothelial cells. All together, our results suggest the involvement of endothelial cells in the development of radiation-induced inflammatory and thrombotic processes.

Irradiation enhances the support of haemopoietic cell transmigration, proliferation and differentiation by endothelial cells

Endothelial cells (ECs) are a critical component of the bone marrow stroma in the regulation of haemopoiesis. Recovery of bone marrow aplasia after radiation exposure depends, in part, on the repair of radiation‐induced endothelial damage. Therefore, we assessed the ability of an irradiated human bone marrow EC line (TrHBMEC) to support transmigration, proliferation and differentiation of CD34+ bone marrow cells either irradiated or not in transendothelial migration or co‐culture models. Radiation‐induced EC damage was reflected by an increased release of soluble intercellular adhesion molecule (sICAM)‐1 and platelet endothelial cell adhesion molecule (PECAM)‐1. Irradiation of TrHBMECs with a 10 Gy dose strongly enhanced the transmigration of CD34+ cells, granulo‐monocytic progenitors (CFU‐GM) and erythroid progenitors (BFU‐E). While ICAM‐1 and PECAM‐1 expression on irradiated TrHBMECs was increased, only antibodies against PECAM‐1 inhibited the radiation‐induced enhanced transmigration of haemopoietic cells. Irradiation of TrHBMECs (5–15 Gy) also increased proliferation and differentiation towards the granulo‐monocytic lineage of co‐cultured CD34+ cells, as well as colony formation by those cells and the production of interleukin 6 (IL‐6), IL‐8, granulocyte colony‐stimulating factor (CSF) and granulocyte‐macrophage CSF. Irradiated TrHBMECs were more capable of stimulating irradiated (1,2 Gy) CD34+ cells and haemopoietic progenitors than non‐irradiated TrHBMECs. Together, these results suggest that, despite the radiation‐induced damage, irradiated ECs may favour haemopoietic reconstitution after radiation exposure.

Characterization of the response of human bone marrow endothelial cells to in vitro irradiation

Endothelial cell dysfunction is a classic consequence of radiation damage. Bone marrow endothelial cells (BMEC) are a critical component of the stroma in the regulation of haemopoiesis. In animal models, radiation‐induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested. However, functions of BMEC involved in the haemopoietic regeneration have not been assessed. Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line (TrHBMEC) irradiated with 2, 5 or 10 Gy. Our results showed a time‐ and a dose‐dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble ICAM‐1 and von Willebrand factor. 2 Gy irradiated TrHBMEC expressed more ICAM‐1 on their surface than non‐irradiated cells, whereas no change in VCAM‐1, E‐selectin and PECAM‐1 expression was observed. An increased production of G‐CSF, GM‐CSF, IL‐8, IL‐6, IL‐1α, IL‐11, MIP‐1α and SCF and no production of LIF, TNF‐α, TPO and IL‐3 by 2 Gy irradiated TrHBMEC was observed. The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure. These results suggest that although radiation induces endothelial cell damage, irradiated cells still support the proliferation and the differentiation of CD34+ haemopoietic cells.

Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage

Abstract Van der Meeren, A., Mouthon, M-A., Vandamme, M., Squiban, C. and Aigueperse, J. Combinations of Cytokines Promote Survival of Mice and Limit Acute Radiation Damage in Concert with Amelioration of Vascular Damage. Radiat. Res. 161, 549–559 (2004). Recovery from hematopoietic aplasia is a predominant factor in the survival of total-body-irradiated mice within 30 days after exposure. However, other radiation-induced pathophysiological events have been shown to play a role, among which an inflammatory reaction must be considered. In the present study, we evaluated the therapeutic potential of a hematopoietic growth factor (thrombopoietin, Tpo) and pleiotropic cytokines (Il4 or Il11), used alone or in combination, on the survival of mice, hematopoietic reconstitution, inflammatory reaction and vascular changes. All treatments including Tpo induced a higher level of survival than did treatment with a placebo, with combinations being the most efficient. The increased survival could not be explained solely by an improved hematopoietic recovery. Treatments with Tpo also reduced the level of the chemokine KC in plasma and the level of expression of mRNA for inflammatory and coagulation proteins in the lungs of irradiated mice. In addition, radiation- induced vascular hyperpermeability was reduced with the use of Tpo. In summary, our results show that Tpo may improve survival by limiting vascular leakage, which in turn could limit inflammatory reactions and the ensuing tissue damage.

Plasminogen activator inhibitor-1 controls bone marrow-derived cells therapeutic effect through MMP9 signaling: role in physiological and pathological wound healing.

We assessed the role of plasminogen activator inhibitor‐1 (PAI‐1) and matrix metalloproteinase 9 (MMP9) in wound healing process and in the bone marrow mononuclear cells (BMMNC)‐related effects on physiological and pathological wound healing. A full thickness excision wound was created by removal of the skin on the midback of irradiated and nonirradiated animals. Angiogenesis and re‐epithelialization were markedly increased in PAI‐1−/− mice compared to wild‐type (WT) animals. We revealed high MMP activity in tissue of PAI‐1−/− animals. Of interest, the wound healing process was reduced in PAI‐1−/−:MMP9−/− animals compared to PAI‐1−/− mice, suggesting a key role of MMP9 in beneficial effect of PAI‐1 deficiency on wound closure. To unravel the role of PAI‐1 in BMMNC relative effects, mice were treated with or without local injection of BMMNC isolated from WT, PAI‐1−/−, and PAI‐1−/−: MMP9−/− animals for 14 days (106 cells, n = 6 per group). In WT nonirradiated mice, transplantation of BMMNC isolated from PAI‐1−/− animals enhanced wound formation when compared with WT BMMNC. BMMNC differentiation into cells with endothelial phenotype was enhanced by PAI‐1 deficiency. These effects were abrogated in PAI‐1−/−:MMP9−/− and MMP9−/− BMMNC. In addition, using chimeric mice, we demonstrated that PAI‐1 deficiency environment increased the BMMNC‐GFP recruitment to the wound site, whereas this effect was abrogated when using PAI‐1−/−:MMP9−/− BMMNC. PAI‐1 deficiency, at least through MMP9 upregulation, enhanced wound healing and BMMNC therapeutic potential in irradiated and nonirradiated animals. Stem Cells2012;30:1436–1446