Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing.

OBJECTIVE: We hypothesized that adipose tissue may contain progenitors cells with cutaneous and angiogenic potential. METHODS AND RESULTS: Adipose tissue-derived stroma cells (ADSCs) were administrated to skin punched wounds of both nonirradiated and irradiated mice (20 Gy, locally). At day 14, ADSCs promoted dermal wound healing and enhanced wound closure, viscolesticity, and collagen tissue secretion in both irradiated and nonirradiated mice. Interestingly, GFP-positive ADSCs incorporated in dermal and epidermal tissue in vivo and expressed epidermal markers K5 and K14. Cultured ADSCs in keratinocyte medium have been shown to differentiate into K5- and K14-positive cells and produced high levels of KGF. At Day 7, ADSCs also improved skin blood perfusion assessed by laser Doppler imaging, capillary density, and VEGF plasma levels in both irradiated and nonirradiated animals. GFP-positive ADSCs incorporated into capillary structures in vivo and expressed the endothelial cell marker CD31. Finally, in situ interphase fluorescence hybridization showed that a small number of ADSCs have the potential to fuse with endogenous keratinocytes. CONCLUSIONS: ADSCs participate in dermal wound healing in physiological and pathological conditions by their ability to promote reepithelialization and angiogenesis. Hence, adipose lineage cells represent a new cell source for therapeutic dermal wound healing.

Abdominal radiation exposure elicits inflammatory responses and abscopal effects in the lungs of mice.

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.

PECAM-1 (CD31) is required for interactions of platelets with endothelial cells after irradiation.

Sustained adhesion of platelets to endothelial cells (EC) is believed to contribute to thrombosis and vascular occlusions following radiation exposure leading to organ functional impairment and even death. Our objective was to evaluate the role of platelet endothelial cell adhesion molecule (PECAM)-1 in the prothrombotic response of EC after irradiation. Endothelial PECAM-1 expression was determined by cell-enzyme linked immunosorbent assay (ELISA) on human microvascular EC from lung (HMVEC-L) up to 21 days after a 10 Gy irradiation. Platelet- and leukocyte-endothelial cell interactions were assessed using a flow adhesion assay with fluorescently labeled whole blood, and the function of PECAM-1 in these processes was measured by using blocking antibody. PECAM-1 expression was significantly increased on irradiated HMVEC-L and remained elevated at 21 days. Anti-PECAM-1 antibody significantly inhibited adhesion of single platelets and thrombi on irradiated HMVEC-L. This inhibitory effect persisted at day 21. Anti-PECAM-1 also reduced leukocyte adhesion to irradiated HMVEC-L. The up-regulation of endothelial PECAM-1 following radiation exposure is persistent. PECAM-1 plays a key role platelet adhesion/aggregation on irradiated EC. Therefore, strategies targeting this adhesion molecule may prevent the development of radiation pathologies.

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.

Interleukin 4 promotes survival of lethally irradiated mice in the absence of hematopoietic efficacy.

The therapeutic potential of Il4 in lethally irradiated mice was evaluated in C57BL6/J mice subjected to 7 to 10 Gy total-body irradiation (TBI) from a (60)Co gamma-ray source. Il4 was administered 2 h after TBI either in a single injection or for 5 consecutive days. Il4 treatment increased 30-day survival of mice irradiated with doses as high as 8.5 Gy, which caused 100% mortality in placebo-treated animals. By convention, hematopoietic failure would induce death over a period of up to 30 days. However, in our study, the Il4-enhanced survival of mice within this period could not be attributed to significantly accelerated hematopoietic reconstitution as shown by blood cell counts and progenitor cell contents in the bone marrow and spleen. Our data strongly suggest that aplasia is not the only cause of death of animals irradiated with doses around the LD(50) and that Il4-treated animals can survive in spite of a very poor hematopoietic activity.

Differential regulation by IL-4 and IL-10 of radiation-induced IL-6 and IL-8 production and ICAM-1 expression by human endothelial cells.

Radiation exposure results in an inflammatory reaction with acute as well as subacute consequences. Leukocyte infiltration is one of the predominant early histological changes and involves both cytokines and adhesion molecules. Endothelial cells play a key role in this reaction. We have previously shown the increased production of interleukin 6 (IL-6) and IL-8 and the upregulation in intercellular adhesion molecule 1 (ICAM-1) expression by HUVEC following gamma ray exposure. In the present study, we used the cytokines IL-4 and IL-10 to regulate these radiation-induced manifestations. Human umbilical vascular endothelial cells (HUVEC) were treated with IL-4 and IL-10 (50 pg/ml) either before or after 10- Gy irradiation. Three and seven days after irradiation, IL-6 and IL-8 production by HUVEC (either treated or non-treated) was assessed by enzyme-linked immunosorbent assay (ELISA). Our results show that IL-4, when added after irradiation, reversed the radiation-induced increase in IL-8 production, although slightly increased IL-6 production. IL-10 decreased both IL-8 and IL-6 production when added after irradiation. ICAM-1 expression was evaluated 3 days after irradiation by flow cytometry. The radiation-induced upregulation in ICAM-1 expression remained unaffected by the use of IL-4. Altogether, our results show that radiation-induced endothelial cell activation may be ameliorated by IL-4 and/or IL-10, which is of significance in designing strategies for cytokine-mediated intervention and/or therapy of radiation 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.

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 10Gy. 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-1alpha, IL-11, MIP-1alpha and SCF and no production of LIF, TNF-alpha, 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.

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.