A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells.

PURPOSE: The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence. MATERIALS AND METHODS: Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence. RESULTS: Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario. CONCLUSIONS: This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

Pulp capping materials modulate the balance between inflammation and regeneration.

The interrelations between inflammation and regeneration are of particular significance within the dental pulp tissue inextensible environment. Recent data have demonstrated the pulp capacity to respond to insults by initiating an inflammatory reaction and dentin pulp regeneration. Different study models have been developed in vitro and in vivo to investigate the initial steps of pulp inflammation and regeneration. These include endothelial cell interaction with inflammatory cells, stem cell interaction with pulp fibroblasts, migration chambers to study cell recruitment and entire human tooth culture model. Using these models, the pulp has been shown to possess an inherent anti-inflammatory potential and a high regeneration capacity in all teeth and at all ages. The same models were used to investigate the effects of tricalcium silicate-based pulp capping materials, which were found to modulate the pulp anti-inflammatory potential and regeneration capacity. Among these, resin-containing materials such as TheraCal® shift the pulp response towards the inflammatory reaction while altering the regeneration process. On the opposite, resin-free materials such as Biodentine™ have an anti-inflammatory potential and induce the pulp regeneration capacity. This knowledge contradicts the new tendency of developing resin-based calcium silicate hybrid materials for direct pulp capping. Additionally, it would allow investigating the modulatory effects of newly released pulp capping materials on the balance between tissue inflammation and regeneration. It would also set the basis for developing future capping materials targeting these processes.

Light-cured Tricalcium Silicate Toxicity to the Dental Pulp.

INTRODUCTION: Numerous studies reported dentin bridge formation after pulp capping with tricalcium silicates. By contrast, pulp capping with resins leads to pulp toxicity and inflammation. Hybrid materials made up of tricalcium silicates and resins have also been developed to be used in direct pulp capping. This work was designed to study the consequences of adding resins to tricalcium silicates by investigating TheraCal (BISCO, Lançon De Provence, France) and Biodentine (Septodont, Saint Maur des Fosses, France) interactions with the dental pulp. METHODS: Media conditioned with the biomaterials were used to analyze pulp fibroblast proliferation using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test and proinflammatory cytokine interleukin 8 (IL-8) secretion using the enzyme-linked immunosorbent assay. The effects of conditioned media on dentin sialoprotein (DSP) and nestin expression by dental pulp stem cells (DPSCs) were investigated by immunofluorescence. The materials' interactions with the vital pulp were investigated using the entire tooth culture model. RESULTS: TheraCal-conditioned media significantly decreased pulp fibroblast proliferation, whereas no effect was observed with Biodentine. When DPSCs were cultured with Biodentine-conditioned media, immunofluorescence showed an increased expression of DSP and nestin. This expression was lower with TheraCal, which significantly induced proinflammatory IL-8 release both in cultured fibroblasts and entire tooth cultures. This IL-8 secretion increase was not observed with Biodentine. Entire tooth culture histology showed a higher mineralization with Biodentine, whereas significant tissue disorganization was observed with TheraCal. CONCLUSIONS: Within the limits of these preclinical results, resin-containing TheraCal cannot be recommended for direct pulp capping.

Complement Activation by Pulp Capping Materials Plays a Significant Role in Both Inflammatory and Pulp Stem Cells' Recruitment.

INTRODUCTION: The role of complement, especially through the C5a fragment, is well-known for the initiation of inflammation. Its involvement in regeneration has been shown more recently by the recruitment of mesenchymal stem cells. C5a can be produced locally by the pulp fibroblasts in response to injury or infection. This work aims to investigate the effect of different pulp capping biomaterials on complement activation and its possible influence on inflammatory and pulp stem cell recruitment. METHODS: Conditioned media were prepared from 3 pulp capping biomaterials: Biodentine (Septodont, Saint-Maur-des-Fosses, France), TheraCal (BISCO, Lançon De Provence, France), and Xeno III (Dentsply Sirona, Versaille, France). Injured pulp fibroblasts were cultured with these conditioned media to analyze C5a secretion using an enzyme-linked immunosorbent assay. Dental pulp stem cells (DPSCs) were isolated from human third molar explants by magnetic cell sorting with STRO-1 antibodies. The expression of C5a receptor on DPSCs and inflammatory (THP-1) cells was investigated by immunofluorescence. The migration of both DPSCs and THP-1 cells was studied in Boyden chambers. RESULTS: Pulp fibroblast production of C5a significantly increased when the cells were incubated with TheraCal- and Xeno III-conditioned media. The recruitment of cells involved in inflammation (THP-1 cells) was significantly reduced by Biodentine- and TheraCal-conditioned media, whereas the migration of DPSCs was reduced with TheraCal- and Xeno III-conditioned media but not with that of Biodentine. The involvement of C5a in cell recruitment is demonstrated with a C5a receptor-specific antagonist (W54011). CONCLUSIONS: After pulp injury, the pulp capping material affects complement activation and the balance between inflammation and regeneration through a differential recruitment of DPSCs or inflammatory cells.

Cardiovascular diseases related to ionizing radiation: The risk of low-dose exposure (Review).

Traditionally, non-cancer diseases are not considered as health risks following exposure to low doses of ionizing radiation. Indeed, non-cancer diseases are classified as deterministic tissue reactions, which are characterized by a threshold dose. It is judged that below an absorbed dose of 100 mGy, no clinically relevant tissue damage occurs, forming the basis for the current radiation protection system concerning non-cancer effects. Recent epidemiological findings point, however, to an excess risk of non-cancer diseases following exposure to lower doses of ionizing radiation than was previously thought. The evidence is the most sound for cardiovascular disease (CVD) and cataract. Due to limited statistical power, the dose-risk relationship is undetermined below 0.5 Gy; however, if this relationship proves to be without a threshold, it may have considerable impact on current low­dose health risk estimates. In this review, we describe the CVD risk related to low doses of ionizing radiation, the clinical manifestation and the pathology of radiation-induced CVD, as well as the importance of the endothelium models in CVD research as a way forward to complement the epidemiological data with the underlying biological and molecular mechanisms.

Characterization and angiogenic potential of xenogeneic bone grafting materials: Role of periodontal ligament cells.

Adequate revascularization is a prerequisite for successful healing of periodontal bone defects. This study characterized three different xenogeneic bone grafting materials: Gen-Os of equine and porcine origins, and anorganic Bio-Oss. We also investigated their angiogenic potential. All materials were composed of poorly crystalline calcium oxide phosphate, with Bio-Oss exhibiting a carbonated phase and larger particle size and both Gen-Os showing the presence of collagen. Both Gen-Os materials significantly enhanced vascular endothelial growth factor (VEGF) secretion by PDL cells. A significant increase in endothelial cell proliferation was observed in cultures with both Gen-Os conditioned media, but not with that of Bio-Oss. Finally, angiogenesis was stimulated by both Gen-Os conditioned media as demonstrated by an increased formation of capillary-like structures. Taken together, these findings indicate an enhanced angiogenic potential of both Gen-Os bone grafting materials when applied on PDL cells, most likely by increasing VEGF production.

Preparation and characterization of biodegradable polyhydroxybutyrate-co-hydroxyvalerate/polyethylene glycol-based microspheres.

The in vivo effectiveness of biomolecules may be limited by their rapid diffusion in the body and short half-life time. Encapsulation of these biomolecules allows protecting them against degradation and ensuring a controlled release over time. In this work, the production of polyhydroxybutyrate-co-hydroxyvalerate/polyethylene glycol-based microspheres loaded with heparin by double emulsion-solvent evaporation is investigated. Significant improvements are achieved after blending PHB-HV microspheres with PEG. First of all, an important decrease of the initial burst effect is ensured. Moreover, lower degradation of the microspheres is observed after 30days in the release medium. Finally, the release rate could be controlled using different PEG molecular weights and concentrations. A toxic effect of PHB-HV 30% PEG 1100gmol-1 microspheres is observed whereas PHB-HV and PHB-HV 30% PEG 10,000gmol-1 microspheres are not toxic. These microspheres seem to be most suited for further tissue engineering applications. The effectiveness of direct PEG blending to PHB-HV is proved, limiting the use of chemical reagents for PHB-HV/PEG copolymer synthesis and steps for chemical reagents removal from the copolymer.

Complement C3a Mobilizes Dental Pulp Stem Cells and Specifically Guides Pulp Fibroblast Recruitment.

INTRODUCTION: Complement activation is considered a major mechanism in innate immunity. Although it is mainly involved in initiating inflammation, recent data reported its involvement in other processes such as tissue regeneration. In the dental pulp, complement C5a fragment has been shown to be involved in the recruitment of dental pulp stem cells (DPSCs). This study sought to investigate the possible role of C3a, another complement fragment, in the early steps of dentin-pulp regeneration. METHODS: Expression of C3a receptor (C3aR) was investigated by immunofluorescence and reverse transcriptase polymerase chain reaction on cultured pulp fibroblasts, STRO-1-sorted DPSCs, as well as on human tooth sections in vivo. The effect of C3a on proliferation of both DPSCs and pulp fibroblasts was investigated by MTT assay. Cell migration under a C3a gradient was investigated by using microfluidic chemotaxis chambers. RESULTS: C3aR was expressed in vivo as well as in cultured pulp fibroblasts co-expressing fibroblast surface protein and in DPSCs co-expressing STRO-1. Addition of recombinant C3a induced a significant proliferation of both cell types. When subjected to a C3a gradient, DPSCs were mobilized but not specifically recruited, whereas pulp fibroblasts were specifically recruited following a C3a gradient. CONCLUSIONS: These results provide the first demonstration of C3aR expression in the dental pulp and demonstrate that C3a is involved in increasing DPSCs and fibroblast proliferation, in mobilizing DPSCs, and in specifically guiding fibroblast recruitment. This provides an additional link to the tight correlation between inflammation and tissue regeneration.

Dental Pulp Stem Cell Recruitment Signals within Injured Dental Pulp Tissue.

The recruitment of dental pulp stem cells (DPSC) is a prerequisite for the regeneration of dentin damaged by severe caries and/or mechanical injury. Understanding the complex process of DPSC recruitment will benefit future in situ tissue engineering applications based on the stimulation of endogenous DPSC for dentin pulp regeneration. The current known mobilization signals and subsequent migration of DPSC towards the lesion site, which is influenced by the pulp inflammatory state and the application of pulp capping materials, are reviewed. The research outcome of migration studies may be affected by the applied methodology, which should thus be chosen with care. Both the advantages and disadvantages of commonly used assays for investigating DPSC migration are discussed. This review highlights the fact that DPSC recruitment is dependent not only on the soluble chemotactic signals, but also on their interaction with neighboring cells and the extracellular matrix, which can be modified under pathological conditions. These are discussed to explain how these modifications lead to the stimulation of DPSC recruitment.

Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation.

Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/µm) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by γ-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-κB, may be involved in these cellular responses.

Transcriptomic profiling suggests a role for IGFBP5 in premature senescence of endothelial cells after chronic low dose rate irradiation.

PURPOSE: Ionizing radiation has been recognized to increase the risk of cardiovascular diseases (CVD). However, there is no consensus concerning the dose-risk relationship for low radiation doses and a mechanistic understanding of low dose effects is needed. MATERIAL AND METHODS: Previously, human umbilical vein endothelial cells (HUVEC) were exposed to chronic low dose rate radiation (1.4 and 4.1 mGy/h) during one, three and six weeks which resulted in premature senescence in cells exposed to 4.1 mGy/h. To gain more insight into the underlying signaling pathways, we analyzed gene expression changes in these cells using microarray technology. The obtained data were analyzed in a dual approach, combining single gene expression analysis and Gene Set Enrichment Analysis. RESULTS: An early stress response was observed after one week of exposure to 4.1 mGy/h which was replaced by a more inflammation-related expression profile after three weeks and onwards. This early stress response may trigger the radiation-induced premature senescence previously observed in HUVEC irradiated with 4.1 mGy/h. A dedicated analysis pointed to the involvement of insulin-like growth factor binding protein 5 (IGFBP5) signaling in radiation-induced premature senescence. CONCLUSION: Our findings motivate further research on the shape of the dose-response and the dose rate effect for radiation-induced vascular senescence.

Differential response to acute low dose radiation in primary and immortalized endothelial cells.

PURPOSE: The low dose radiation response of primary human umbilical vein endothelial cells (HUVEC) and its immortalized derivative, the EA.hy926 cell line, was evaluated and compared. MATERIAL AND METHODS: DNA damage and repair, cell cycle progression, apoptosis and cellular morphology in HUVEC and EA.hy926 were evaluated after exposure to low (0.05-0.5 Gy) and high doses (2 and 5 Gy) of acute X-rays. RESULTS: Subtle, but significant increases in DNA double-strand breaks (DSB) were observed in HUVEC and EA.hy926 30 min after low dose irradiation (0.05 Gy). Compared to high dose irradiation (2 Gy), relatively more DSB/Gy were formed after low dose irradiation. Also, we observed a dose-dependent increase in apoptotic cells, down to 0.5 Gy in HUVEC and 0.1 Gy in EA.hy926 cells. Furthermore, radiation induced significantly more apoptosis in EA.hy926 compared to HUVEC. CONCLUSIONS: We demonstrated for the first time that acute low doses of X-rays induce DNA damage and apoptosis in endothelial cells. Our results point to a non-linear dose-response relationship for DSB formation in endothelial cells. Furthermore, the observed difference in radiation-induced apoptosis points to a higher radiosensitivity of EA.hy926 compared to HUVEC, which should be taken into account when using these cells as models for studying the endothelium radiation response.

Short-term infusion of a fish oil-based lipid emulsion modulates fatty acid status, but not immune function or (anti)oxidant balance: a randomized cross-over study.

BACKGROUND AND AIMS: Studies suggest clinical benefits of parenteral fish oil (FO), rich in n-3 polyunsaturated fatty acids (PUFAs), over soyabean oil (SO), rich in n-6 PUFAs, in patients with pro-inflammatory conditions such as sepsis and trauma. Because the mechanisms behind these observations remain unclear, the present study explored the effects of intravenous infusion of FO and SO on fatty acid incorporation, immune functions and (anti)oxidant balance in healthy human volunteers. METHODS: Saline, a SO emulsion and a FO emulsion were administered for one hour on three consecutive days at a rate of 0·2 g/kg BW/h to eight subjects in a randomized cross-over design with a 3-week interval between treatments. Plasma phospholipid and peripheral blood mononuclear cell (PBMC) fatty acid compositions, and leucocyte counts and functions were assessed prior to the first infusion (T = 0, baseline) and 1 day (T = 4, early effects) and 8 days (T = 11, late effects) after the third infusion. RESULTS: Fish oil infusion significantly increased n-3 PUFA proportions and decreased n-6 PUFA proportions in plasma phospholipids and PBMCs. There were no differences in immune functions or (anti)oxidant balance between treatments at any time. CONCLUSIONS: The present lipid infusion protocol appears to be safe and well tolerated and provides significant incorporation of n-3 PUFAs into plasma phospholipids and PBMCs. In the absence of overt inflammation, no direct effects of FO were observed on immune function or (anti)oxidant balance. This model may be useful to evaluate effects of parenteral lipids in other settings, for example in individuals displaying an inflammatory state.