The effect of oxidized low-density lipoprotein (ox-LDL) on radiation-induced endothelial-to-mesenchymal transition

Abstract

Purpose: Radiation-induced cardiovascular disease is a potentially severe side-effect of thoracic radiotherapy treatment. Clinically, this delayed side-effect presents as a form of accelerated atherosclerosis several years after irradiation. As general endothelial dysfunction is known to be an initiating event in radiation-induced vascular damage, we examined the effects of radiation on endothelial cells in radiation-induced atherosclerosis.

Materials and methods: The effects of radiation on human aortic endothelial cells (HAoEC) were assessed by immunoblotting and immunofluorescence assays. Radiation-induced phenotypic changes of endothelial cells (ECs) were examined using atherosclerotic tissues of irradiated apoprotein E null (ApoE^?/?) mice.

Results: Radiation induced the HAoEC to undergo phenotypic conversion to form fibroblast-like cells, called the endothelial-to-mesenchymal transition (EndMT), which leads to the upregulation of mesenchymal cell markers such as alpha-smooth muscle actin (α-SMA), fibroblast specific protein-1 (FSP-1), and vimentin, and downregulation of endothelial cell-specific markers such as CD31 and vascular endothelial (VE)-cadherin. Furthermore, compared with low-density lipoprotein (LDL), oxidized low-density lipoprotein (ox-LDL) significantly augmented radiation-induced EndMT in HAoEC. These fibrotic phenotypes of ECs were found in atherosclerotic tissues of irradiated ApoE^?/? mice with increased levels of ox-LDL.

Conclusions: Taken together, these observations suggest that ox-LDL accelerates radiation-induced EndMT and subsequently contributes to radiation-induced atherosclerosis, providing a novel target for the prevention of radiation-induced atherosclerosis.     

Role of CD4 and CD8 T-lymphocytes,B-lymphocytes and Natural Killer cells in the prediction of radiation-induced late toxicity in cervical cancer patients

Purpose:?To analyse the role of in vitro radio-induced apoptosis of lymphocyte subpopulations as predictive test for late effects in cervical cancer patients treated with radiotherapy.

Methods and materials:?Ninety-four consecutive patients and four healthy controls were included in the study. Toxicity was evaluated using the Late Effects Normal Tissue-Subjective, Objective, Management, and Analytic (LENT-SOMA) scale. Peripheral blood lymphocyte subpopulations were isolated and irradiated at 0, 1, 2 and 8 Gy, and then collected 24, 48 and 72 h after irradiation. Apoptosis was measured by flow cytometry.

Results:?Radiation-induced apoptosis increased with radiation dose and time of incubation, and data fitted to a semi-logarithmic model defined by two constants: α (percentage of spontaneous cell death) and β (percentage of cell death induced at a determined radiation dose). Higher β values in cytotoxic T-lymphocytes (CD8) and bone cells (B-lymphocytes) were observed in patients with low bowel toxicity (hazard ratio (HR)?=?0.96, p?=?0.002 for B-cells); low rectal toxicity (HR?=?0.96, p?=?0.020; HR?=?0.93, p?=?0.05 for B and CD8 subpopulations respectively); low urinary toxicity (HR?=?0.93, p?=?0.003 for B-cells) and low sexual toxicity (HR?=?0.93, p?=?0.010 for CD8-cells).

Conclusions:?Radiation-induced CD8 T-lymphocytes and, for the first time, B-lymphocytes apoptosis can predict differences in late toxicity in cervical cancer patients.     

FGF2 mediates DNA repair in epidermoid carcinoma cells exposed to ionizing radiation

Abstract

Purpose: Fibroblast growth factor 2 (FGF2) is a well-known survival factor. However, its role in DNA repair is poorly documented. The present study was designed to investigate in epidermoid carcinoma cells the potential role of FGF2 in DNA repair.

Materials and methods: The side population (SP) with cancer stem cell-like properties and the main population (MP) were isolated from human A431 squamous carcinoma cells. Radiation-induced DNA damage and repair were assessed using the alkaline comet assay. FGF2 expression was quantified by enzyme linked immunosorbent assay (ELISA).

Results: SP cells exhibited rapid repair of radiation induced DNA damage and a high constitutive level of nuclear FGF2. Blocking FGF2 signaling abrogated the rapid DNA repair. In contrast, in MP cells, a slower repair of damage was associated with low basal expression of FGF2. Moreover, the addition of exogenous FGF2 accelerated DNA repair in MP cells. When irradiated, SP cells secreted FGF2, whereas MP cells did not.

Conclusions: FGF2 was found to mediate DNA repair in epidermoid carcinoma cells. We postulate that carcinoma stem cells would be intrinsically primed to rapidly repair DNA damage by a high constitutive level of nuclear FGF2. In contrast, the main population with a low FGF2 content exhibits a lower repair rate which can be increased by exogenous FGF2.     

Impact of carbon ion irradiation on epidermal growth factor receptor signaling and glioma cell migration in comparison to conventional photon irradiation

Abstract

Purpose: Radiotherapy of malignant gliomas may be limited by an interference of radiation with the migratory potential of tumor cells. Therefore, the influence of conventional photon and modern carbon ion (¹²C) irradiation on glioblastoma cell migration and on epidermal growth factor receptor-related (EGFR) signaling was investigated in vitro.

Materials and methods: EGFR overexpressing glioblastoma cell lines U87 EGFR++ and LN229 EGFR++ were irradiated with 0, 2 or 6 Gy photons or ¹²C heavy ions. Migration was analyzed 24 h after treatment in a standardized Boyden Chamber assay. At different time points EGFR, protein kinase B (PKB/AKT) and extracellular signal-related kinases (ERK1/2) were analyzed by Western blotting.

Results: 2 Gy photon irradiation increased U87 EGFR++ migration and decreased motility of LN229 EGFR++ cells. Heavy ions decreased migration of both cell lines as a function of dose. There was a time-dependent increase of phosphorylation of EGFR, AKT and ERK1/2 in U87 EGFR++ after 2 Gy photon irradiation. After heavy ion irradiation EGFR, AKT or ERK1/2 remained unchanged.

Conclusions: Results suggest that the impact of irradiation on tumor cell migration depends on radiation type and cell line. Photons, but not heavy ions, potentially contribute to treatment failure by increasing EGFR-related tumor cell migration.     

Lymphocyte subsets and their H2AX phosphorylation in response to in vivo irradiation in rats

Abstract

Purpose: The objective of the study was to investigate differences in the radiosensitivity of rat peripheral blood lymphocyte subsets identified by expression of surface clusters of differentiation markers (CD3, CD4, CD8, CD45RA, CD161) after whole-body in vivo gamma-ray irradiation and to assess their individual histone H2AX phosphorylation as an early cell response to irradiation.

Materials and methods: The relative representations of CD45RA B-lymphocytes, CD161 natural killer cells (NK cells), CD3CD4 T-lymphocyte subset and CD3CD8 T-lymphocyte subset in the rat peripheral blood were studied 24–72 hours after irradiation in a dose range of 0–5 Gy. Their intracellular H2AX phosphorylation (γ-H2AX) after 4 Gy and 9 Gy whole-body in vivo irradiation was assessed by multicolour flow cytometry.

Results: We determined the linear dose response of radioresistant CD161 NK cells (24 h), both radiosensitive T-lymphocyte subsets (24 h) and CD45RA B-lymphocytes (72 h) after in vivo irradiation. CD45RA B-lymphocytes showed the highest radiosensitivity and we observed pronounced H2AX phosphorylation which remained expressed in these cells for over 4 h after irradiation.

Conclusion: The combination of the surface immunophenotyping together with intracellular detection of γ-H2AX offers the possibility to assess the absorbed dose of ionizing irradiation with high sensitivity post irradiation and could be successfully applied to biodosimetry.     

Ionizing radiation causes active degradation and reduces matrix synthesis in articular cartilage

Abstract

Purpose: Little is known regarding radiation effects on adult articular (joint) cartilage, though joint damage has been reported following cancer treatment or occupational exposures. The aim of this study was to determine if radiation can reduce cartilage matrix production, induce cartilage degradation, or interfere with the anabolic effects of IGF-1.

Materials and methods: Isolated chondrocytes cultured in monolayers and whole explants harvested from ankles of human donors and knees of pigs were irradiated with 2 or 10 Gy γ-rays, with or without IGF-1 stimulation. Proteoglycan synthesis and IGF-1 signaling were examined at Day 1; cartilage degradation throughout the first 96 hours.

Results: Human and pig cartilage responded similarly to radiation. Cell viability was unchanged. Basal and IGF-1 stimulated proteoglycan synthesis was reduced following exposure, particularly following 10 Gy. Both doses decreased IGF-induced Akt activation and IGF-1 receptor phosphorylation. Matrix metalloproteinases (ADAMTS5, MMP-1, and MMP-13) and proteoglycans were released into media after 2 and 10 Gy.

Conclusions: Radiation induced an active degradation of cartilage, reduced proteoglycan synthesis, and impaired IGF-1 signaling in human and pig chondrocytes. Lowered Akt activation could account for decreased matrix synthesis. Radiation may cause a functional decline of cartilage health in joints after exposure, contributing to arthropathy.     

Free Radical Formation in Crystals of Guanine Hydrochloride Dihydrate: An ESR and ENDOR Study

Summary

Radiation-induced free radical formation in single crystals of guanine hydrochloride dihydrate has been studied at temperatures between 20 and 300 K using ESR and ENDOR spectroscopy. At low temperatures three radical species are trapped. Two of these are the C8 H-addition radical R1 previously analysed by Alexander and Gordy (1967) and the O6-protonated anion radical R2. The third species (R4) remains unidentified. Upon annealing at 280 K for an extended period the protonated anion R2 transforms into a new radical R3 which exhibit a well-defined hyperfine pattern but still could not be identified unambiguously. Also radical R4 probably transforms into a new radical (R5) upon such treatment. One proton coupling due to R5 was detected. A scheme of radical reactions incorporating these five radicals is proposed. This scheme also suggests that differences in radical formation between the monohydrate and dihydrate crystals of guanine hydrochloride depends upon differences in the hydrogen bonding network.     

List of reviewers

Abstract

Purpose: Perturbations in protein folding induce endoplasmic reticulum (ER) stress, which elicits coordinated response, namely the unfolded protein response (UPR), to cope with the accumulation of misfolded proteins in ER. In this study, we characterized mechanisms underlying ionizing radiation (IR)-induced UPR signaling pathways.

Materials and methods: We analyzed alterations in UPR signaling pathways in human umbilical vein endothelial cells (HUVEC) and human coronary artery endothelial cells (HCAEC) irradiated with 15 Gy IR.

Results: IR selectively activated the eIF2α/ATF4 branch of the UPR signaling pathway, with no alterations in the IRE1 and ATF6 branches in HUVEC and HCAEC. Phosphorylation of PERK was enhanced in response to IR, and the IR-induced activation of the eIF2α/ATF4 signaling pathway was completely inhibited by PERK knockdown with siRNA. Surprisingly, chemical chaperones, which inhibit the formation of misfolded proteins and sequential protein aggregates to reduce ER stress, failed to prevent the IR-induced phosphorylation of PERK and the subsequent activation of the eIF2α/ATF4 signaling pathway.

Conclusions: PERK mediates the IR-induced selective activation of the eIF2α/ATF4 signaling pathway, and the IR-induced activation of PERK/eIF2α/ATF4 signaling in human vascular endothelial cells is independent of alterations in protein-folding homeostasis in the ER.     

Effects of IL-4 on pulmonary fibrosis and the accumulation and phenotype of macrophage subpopulations following thoracic irradiation

Purpose: Thoracic irradiation injures lung parenchyma, triggering inflammation and immune cell activation, leading to pneumonitis and fibrosis. Macrophage polarization contributes to these processes. Since IL-4 promotes pro-fibrotic macrophage activation, its role in radiation-induced lung injury was investigated.

Materials and methods: Lung macrophage subpopulations were characterized from 3–26 weeks following exposure of WT and IL-4?/? mice to 0 or 12.5 Gray single dose thoracic irradiation.

Results: Loss of IL-4 did not prevent fibrosis, but blunted macrophage accumulation within the parenchyma. At 3 weeks following exposure, cell numbers and expression of F4/80 and CD206, an alternative activation marker, decreased in alveolar macrophages but increased in infiltrating macrophages in WT mice. Loss of IL-4 impaired recovery of these markers in alveolar macrophages and blunted expansion of these populations in infiltrating macrophages. CD206+ cells were evident in fibrotic regions of WT mice only, however Arg-1+ cells increased in fibrotic regions in IL-4?/? mice only. Radiation-induced proinflammatory Ly6C expression was more apparent in alveolar and interstitial macrophages from IL-4?/? mice.

Conclusions: IL-4 loss did not prevent alternative macrophage activation and fibrosis in irradiated mice. Instead, a role is indicated for IL-4 in maintenance of macrophage populations in the lung following high single dose thoracic irradiation.     

Alpha-tocopherol succinate-mobilized progenitors improve intestinal integrity after whole body irradiation

Abstract

Purpose: The objective of this study was to elucidate the action of α-tocopherol succinate (TS)- and AMD3100-mobilized progenitors in mitigating radiation-induced injuries.

Material and methods: CD2F1 mice were exposed to a high dose of radiation and then transfused intravenously with 5 million peripheral blood mononuclear cells (PBMC) from TS- and AMD3100-injected mice after irradiation. Intestinal and splenic tissues were harvested after irradiation and cells of those tissues were analyzed for markers of apoptosis and mitosis. Bacterial translocation from gut to heart, spleen, and liver in TS-treated and irradiated mice was evaluated by bacterial culture.

Results: We observed that the infusion of PBMC from TS- and AMD3100-injected mice significantly inhibited apoptosis, increased cell proliferation in the analyzed tissues of recipient mice, and inhibited bacterial translocation to various organs compared to mice receiving cells from vehicle-mobilized cells. This study further supports our contention that the infusion of TS-mobilized progenitor-containing PBMC acts as a bridging therapy by inhibiting radiation-induced apoptosis, enhancing cell proliferation, and inhibiting bacterial translocation in irradiated mice.

Conclusions: We suggest that this novel bridging therapeutic approach that involves the infusion of TS-mobilized hematopoietic progenitors following acute radiation injury might be applicable to humans as well.     

Long-term effects of sprint interval training on expression of cardiac genes involved in energy efficiency

Purpose

The real mechanisms of intensive exercise training-induced energy efficiency have not yet been well examined. Therefore, the aim of the present study was to investigate the effects of sprint interval training (SIT) on gene expression of uncoupling proteins (UCPs) and endothelial nitric oxide synthase (eNOS).

Methods

For this purpose, 16 Albino Wistar rats (250–300 g) were randomly divided into equal groups of control and sprint training. The animals run on treadmill for 10 weeks, 5 days per week at intensity corresponding to 90–95% maximal oxygen consumption. The gene expression of UCP2, UCP3 and eNOS was analyzed by RT-PCR method in hearts. The data were analyzed by independent samples T test at P?<?0.05 level.

Results

Sprint interval training significantly decreased mRNA expression of UCP2 (t₁₄?=?4.818, P?=?0.001) and UCP3 (t₁₄?=?4.620, P?=?0.001) in cardiac muscle of rats. In contrast, mRNA expression of eNOS in cardiac muscle significantly increased following sprint interval training (t₁₄?=?7.967, P?=?0.001).

Conclusion

This study elucidates that SIT through reduction in gene expression of uncoupling proteins can improve energy efficiency. But, more studies are needed to confirm this hypothesis.

     

Meeting overview

Abstract

Purpose: The purpose of this study was to determine whether the effects of gamma rays on the regeneration of hair follicles are carried over to later hair cycles.

Materials and methods: The whole bodies of C57BL/10JHir mice in the 1st telogen phase were irradiated with ⁶⁰Co γ-rays. Mice were examined for the effects on hair follicles, including their number, morphology and pigmentation in the 3rd anagen phase. Effects of γ-rays on hair follicle stem cells were investigated by the indirect immunolabeling of keratin 15 (K15).

Results: Decreased hair follicle density and induction of curved hair follicles were observed in the dermis of irradiated skin. In addition, white hair and hypopigmented hair bulbs were found. The number of K15-positive hair follicle stem cells in the hair bulge region of irradiated skin appeared to decrease slightly but not significantly.

Conclusions: These results suggest that the effects of γ-rays are carried over to a later hair cycle to affect the number, structure and pigmentation of hair follicles in the 3rd anagen phase when stem cells and committed progenitors for keratinocytes and melanocytes are irradiated in the 1st telogen phase.     

Retention of 241Am by Some Endocrine Organs of the Rat and Its Response to DPTA-treatment

Summary

The effect of chronic irradiation on T and B cell numbers and function was studied in mice. Cobalt 60 gamma radiation at 6 R/hour reduced the numbers of anti-SRBC PFC in the spleen, with minimal levels recorded after total exposures of 1000–2000 R. Recovery was incomplete after 1000 R, reaching only 40–50 per cent of normal in four months and remaining at that level for the animal's lifetime. The long-term deficiency in PFC formation was not due to a quantitative lack of T or B cells since normal cell numbers were observed in the spleen 60–144 days after 1000 R. Adoptive transfer studies with combinations of bone marrow and thymus cells, or of splenic T and B cells, from normal and irradiated mice, revealed functional defects in both cell compartments during the first two months. Normal and near normal function of T and B cells occurred 100 days postirradiation, a time when the splenic in vivo response was still only 50 per cent of the controls. The latter observation suggests that the microenvironment of the chronically irradiated spleen alters factors regulating T and B cell interactions in response to a T-dependent antigen.     

Partially Damaged Molecules after Irradiation of Dry Invertase with Fast Electrons and Protons

Summary

Radiation-induced life-shortening in Drosophila melanogaster has been measured. Flies were irradiated at a dose-rate of 1430 rads/sec whilst breathing various mixtures of oxygen and nitrogen. Neither a detectable age dependence nor a sex difference was noted. There was no difference in sensitivity with oxygen contents of 6–21 per cent; under 100 per cent nitrogen, life-shortening was reduced. This difference in sensitivity was due to an increase in the plateau region at low doses for nitrogen. For higher doses inactivation constants for both sets of curves were similar Thus the o.e.r. was dependent on the level of damage. For the acute effects of radiation there was no protection under nitrogen. This study is relevant to the effects of radiation on post-mitotic normal tissue.     

Radiation-induced surge of macrophage foam cell formation,oxidative damage,and cytokine release is attenuated by a nanoformulation of curcumin

Purpose: We examined the potential of a dendrosomal nanoformulation of curcumin (DNC) for intervention of ionizing radiation (IR)-induced damage (particularly leading to atherosclerosis), employing an irradiated THP-1 macrophage model.

Materials and methods: Differentiated THP-1 macrophages were irradiated and treated with curcumin or DNC nanoformulation (and oxidized low density lipoprotein, ox-LDL, to promote foam cells). Chemical, biochemical, and genetics tools including viability and apoptosis, multiple ELISA, real-time PCR, Western blotting, enzyme activity, and fluorimetry assays were employed to illustrate IR damage as well as the DNC intervention potential.

Results: DNC per se at 10 μM exerted no cytotoxic effects on macrophages. However, it caused apoptosis in 2?Gy-irradiated macrophages which were treated with ox-LDL, chiefly through a caspase-dependent pathway involving caspase-3. Concurrently, 10 μM DNC prevented the IR-induced rise in lipid accumulation (72% decrease compared to IR control, p p p p p Conclusions: DNC treatment suppresses IR-induced oxidative damage, inflammation, and foam cell formation in macrophages through multiple mechanisms.     

Effect of 5-hydroxytryptamine (serotonin) receptor inhibitors on the radiation-induced bystander effect

Abstract

Purpose: To test the importance of serotonin as a signaling molecule involved in the production and response of radiation-induced bystander effects.

Materials and methods: HPV-G human keratinocyte cultures were spiked with various concentrations of Granisetron or Ketanserin and subject to either 0 Gy or 0.5 Gy X-irradiation to observe the inhibitor's effects on bystander signal production. Medium from these cultures was harvested and introduced to non- irradiated cultures of the same cell line to determine the clonogenic bystander response. Separate HPV-G cultures were set up for subsequent calcium measurements in response to irradiated cell conditioned medium (ICCM) in the presence or absence of Granisetron in an attempt to block bystander signal response.

Results: Granisetron and Ketanserin produced a dose-dependent propagation of the bystander effect in recipient cultures. Granisetron completely abolished the characteristic calcium pulse observed when non-irradiated cultures are exposed to irradiated cell medium in the presence of this drug.

Conclusions: Serotonin-dependent mechanisms appear to be involved in bystander signal production and response to radiation in this system.     

Some properties of the signals involved in unirradiated zebrafish embryos rescuing α-particle irradiated zebrafish embryos

Abstract

Purpose: The in vivo radiation-induced bystander effect (RIBE) and radiation-induced rescue effect (RIRE) induced between embryos of the zebrafish (Danio rerio) by alpha-particle irradiation were studied through the number of apoptotic signals revealed at 24 h post fertilization (hpf) through vital dye acridine orange staining.

Materials and methods: RIBE and RIRE were verified through the significant increase and decrease in apoptotic signals in the partnered bystander and irradiated embryos, respectively.

Results: The medium transfer experiment where irradiated zebrafish embryos were rescued through immersion in the medium previously conditioned by a larger number of irradiated zebrafish embryos showed (a) the involvement of a released stress signal in the induction of RIRE, and (b) RIBE and RIRE signals had the same function. With the help of 500 μM of the specific nitric oxide (NO) scavenger cPTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide), NO was confirmed as an essential signaling molecule for inducing both the RIBE and RIRE. On the other hand, the treatment with 20 μM of the carbon monoxide (CO) releasing chemical CORM-3 (tricarbonylchloro(glycinato)ruthenium (II)) suppressed the manifestations of RIBE but did not suppress RIRE.

Conclusions: In conclusion, unirradiated zebrafish embryos need NO but not NO-induced damages to rescue α-particle irradiated zebrafish embryos.