Platelets, inflammation, and purinergic receptors in chronic kidney disease.

Platelets are anucleated cells that circulate in the bloodstream. Historically, platelets were thought to perform a singular function-stop bleeding via clotting. Although platelets do play a key role in hemostasis and thrombosis, recent studies indicate that platelets also modulate inflammation, and this platelet-induced inflammation contributes to the pathophysiology of various diseases such as atherosclerosis and diabetes mellitus. Thus, in recent years, our understanding of platelet function has broadened. In this review, we revisit the classic role of platelets in hemostasis and thrombosis and describe the newly recognized function of platelets in modulating inflammation. We cover the potential use of purinergic receptor antagonists to prevent platelet-modulated inflammation, particularly in patients with chronic kidney disease, and finally, we define key questions that must be addressed to understand how platelet-modulated inflammation contributes to the pathophysiology of chronic kidney disease.

Understanding the molecular mechanism responsible for developing therapeutic radiation-induced radioresistance of rectal cancer and improving the clinical outcomes of radiotherapy - A review.

Rectal cancer accounts for the second highest cancer-related mortality, which is predominant in Western civilizations. The treatment for rectal cancers includes surgery, radiotherapy, chemotherapy, and immunotherapy. Radiotherapy, specifically external beam radiation therapy, is the most common way to treat rectal cancer because radiation not only limits cancer progression but also significantly reduces the risk of local recurrence. However, therapeutic radiation-induced radioresistance to rectal cancer cells and toxicity to normal tissues are major drawbacks. Therefore, understanding the mechanistic basis of developing radioresistance during and after radiation therapy would provide crucial insight to improve clinical outcomes of radiation therapy for rectal cancer patients. Studies by various groups have shown that radiotherapy-mediated changes in the tumor microenvironment play a crucial role in developing radioresistance. Therapeutic radiation-induced hypoxia and functional alterations in the stromal cells, specifically tumor-associated macrophage (TAM) and cancer-associated fibroblasts (CAF), play a crucial role in developing radioresistance. In addition, signaling pathways, such as - the PI3K/AKT pathway, Wnt/ß-catenin signaling, and the hippo pathway, modulate the radiation responsiveness of cancer cells. Different radiosensitizers, such as small molecules, microRNA, nanomaterials, and natural and chemical sensitizers, are being used to increase the effectiveness of radiotherapy. This review highlights the mechanism responsible for developing radioresistance of rectal cancer following radiotherapy and potential strategies to enhance the effectiveness of radiotherapy for better management of rectal cancer.

Protocol for preparation and staining of chromosomes isolated from mouse and human tissues for conventional and molecular cytogenetic analysis.

The study of chromosomes without or with molecular DNA probes provides crucial insight for understanding research findings, as well as refining diagnosis, prognosis, and therapeutics in clinical settings. Here, we present a protocol for chromosome preparation, conventional G-banding, locus-specific fluorescent in situ hybridization, and spectral karyotyping for both mouse and human samples. This protocol optimizes the preparation of chromosomes from mouse and human cells for subsequent conventional and molecular cytogenetic analysis. For complete details on the use and execution of this protocol, please refer to Binz et al.1.

Tocotrienols Provide Radioprotection to Multiple Organ Systems through Complementary Mechanisms of Antioxidant and Signaling Effects.

Tocotrienols have powerful radioprotective properties in multiple organ systems and are promising candidates for development as clinically effective radiation countermeasures. To facilitate their development as clinical radiation countermeasures, it is crucial to understand the mechanisms behind their powerful multi-organ radioprotective properties. In this context, their antioxidant effects are recognized for directly preventing oxidative damage to cellular biomolecules from ionizing radiation. However, there is a growing body of evidence indicating that the radioprotective mechanism of action for tocotrienols extends beyond their antioxidant properties. This raises a new pharmacological paradigm that tocotrienols are uniquely efficacious radioprotectors due to a synergistic combination of antioxidant and other signaling effects. In this review, we have covered the wide range of multi-organ radioprotective effects observed for tocotrienols and the mechanisms underlying it. These radioprotective effects for tocotrienols can be characterized as (1) direct cytoprotective effects, characteristic of the classic antioxidant properties, and (2) other effects that modulate a wide array of critical signaling factors involved in radiation injury.

Cytogenetic and epigenetic aberrations in peripheral lymphocytes of northwest Arkansas Marshallese.

PURPOSE: Nuclear weapons testing in the northern Marshall Islands between 1946 and 1958 resulted in ionizing radiation (IR) exposure of the thousands of Marshallese. Furthermore, numerous islands were contaminated by radioactive fallout. Significant increases in cancer and metabolic syndrome incidences have been reported among Marshallese, and potential for further increases looms due to the latency of radiation-induced health effects. The purpose of this study was to investigate the genetic and epigenetic effects of exposure to IR that could be associated with radiation-induced disease among the Northwest Arkansas (NWA) Marshallese. MATERIALS AND METHODS: We performed analysis of chromosomal aberrations and DNA methylation based on residential and exposure history of NWA Marshallese. RESULTS: Analysis of chromosomal aberrations demonstrated higher incidence of genetic rearrangements in women with self-reported history of radiation exposure (95% CI: 0.10, 1.22; p=.022). Further clustering of study participants based on their residential history demonstrated that participants who spent substantial amounts of time (≥6 months) in the northern atolls (thus, in the proximity of nuclear tests) before 1980 had more chromosomal aberrations than their peers who lived only in the southern atolls (95% CI: 0.08, -0.95; p=.021), and that this difference was driven by women. A relationship between the time spent in the northern atolls and increase in chromosomal aberrations was observed: 0.31 increase in chromosomal aberrations for every 10 years spent at northern atolls (95% CI: 0.06, 0.57; p=.020). Finally, significant inverse correlations between the chromosomal aberrations and the extent of DNA methylation of four LINE-1 elements L1PA2, L1PA16, L1PREC1, and L1P4B were identified. CONCLUSIONS: The results of this study provide first evidence of the presence of stable genetic and epigenetic rearrangements in peripheral lymphocytes of NWA Marshallese and warrant further studies to analyze the role of radiation exposure in health disparities experienced by this Pacific Island nation.

Evaluating the effects of low-dose simulated galactic cosmic rays on murine hippocampal-dependent cognitive performance.

Space exploration has advanced substantially over recent decades and plans to increase the duration of deep space missions are in preparation. One of the primary health concerns is potential damage to the central nervous system (CNS), resulting in loss of cognitive abilities and function. The majority of ground-based research on space radiation-induced health risks has been conducted using single particle simulations, which do not effectively model real-world scenarios. Thus, to improve the safety of space missions, we must expand our understanding of the effects of simulated galactic cosmic rays (GCRs) on the CNS. To assess the effects of low-dose GCR, we subjected 6-month-old male BALB/c mice to 50 cGy 5-beam simplified GCR spectrum (1H, 28Si, 4He, 16O, and 56Fe) whole-body irradiation at the NASA Space Radiation Laboratory. Animals were tested for cognitive performance with Y-maze and Morris water maze tests 3 months after irradiation. Irradiated animals had impaired short-term memory and lacked spatial memory retention on day 5 of the probe trial. Glial cell analysis by flow cytometry showed no significant changes in oligodendrocytes, astrocytes, microglia or neural precursor cells (NPC's) between the sham group and GCR group. Bone marrow cytogenetic data showed a significant increase in the frequency of chromosomal aberrations after GCR exposure. Finally, tandem mass tag proteomics identified 3,639 proteins, 113 of which were differentially expressed when comparing sham versus GCR exposure (fold change > 1.5; p < 0.05). Our data suggest exposure to low-dose GCR induces cognitive deficits by impairing short-term memory and spatial memory retention.

Radiation-Induced Intestinal Normal Tissue Toxicity: Implications for Altered Proteome Profile.

Radiation-induced toxicity to healthy/normal intestinal tissues, especially during radiotherapy, limits the radiation dose necessary to effectively eradicate tumors of the abdomen and pelvis. Although the pathogenesis of intestinal radiation toxicity is highly complex, understanding post-irradiation alterations in protein profiles can provide crucial insights that make radiotherapy safer and more efficient and allow for increasing the radiation dose during cancer treatment. Recent preclinical and clinical studies have advanced our current understanding of the molecular changes associated with radiation-induced intestinal damage by assessing changes in protein expression with mass spectrometry-based approaches and 2-dimensional difference gel electrophoresis. Studies by various groups have demonstrated that proteins that are involved in the inflammatory response, the apoptotic pathway, reactive oxygen species scavenging, and cell proliferation can be targeted to develop effective radiation countermeasures. Moreover, altered protein profiles serve as a crucial biomarkers for intestinal radiation damage. In this review, we present alterations in protein signatures following intestinal radiation damage as detected by proteomics approaches in preclinical and clinical models with the aim of providing a better understanding of how to accomplish intestinal protection against radiation damage.

Molecular Cytogenetics Reveals Mosaicism in Human Umbilical Vein Endothelial Cells.

Primary human umbilical vein endothelial cells (HUVECs) are consistently the most reliable in vitro model system for studying the inner lining of blood and lymphatic vessels or the endothelium. Primary human cells originate from freshly isolated tissues without genetic manipulation and generally show a modal number of 46 chromosomes with no structural alterations, at least during early passages. We investigated the cytogenetic integrity of HUVECs with conventional (G-banding) and molecular cytogenetic methods (spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH)). Our G-band data shows two X-chromosomes, confirming these HUVECs originate from a female donor. Notably, some cells consistently exhibit an unfamiliar banding pattern on one X chromosome toward the distal end of the long arm (Xq). Our FISH analysis confirms that approximately 50% of these HUVECs have a deletion of the Xq terminal region. SKY analysis indicates that the deleted region is apparently not integrated into any other chromosome. Finally, we demonstrated the presence of a similar Xq deletion in the daughter cell line, EA.hy926, which was generated by fusing HUVECs with A549 (a thioguanine-resistant clone of adenocarcinomic human alveolar basal epithelial cells). These findings will advance comprehension of HUVECs biology and will augment future endothelial studies.

Methods for induction and assessment of intestinal permeability in rodent models of radiation injury.

Ionizing radiation (IR) is a significant contributor to the contemporary market of energy production and an important diagnostic and treatment modality. Besides having numerous useful applications, it is also a ubiquitous environmental stressor and a potent genotoxic and epigenotoxic agent, capable of causing substantial damage to organs and tissues of living organisms. The gastrointestinal (GI) tract is highly sensitive to IR. This problem is further compounded by the fact that there is no FDA-approved medication to mitigate acute radiation-induced GI syndrome. Therefore, establishing the animal model for studying IR-induced GI-injury is crucially important to understand the harmful consequences of intestinal radiation damage. Here, we discuss two different animal models of IR-induced acute gastrointestinal syndrome and two separate methods for measuring the magnitude of intestinal radiation damage.

Ionizing Radiation Activates Mitochondrial Function in Osteoclasts and Causes Bone Loss in Young Adult Male Mice.

The damaging effects of ionizing radiation (IR) on bone mass are well-documented in mice and humans and are most likely due to increased osteoclast number and function. However, the mechanisms leading to inappropriate increases in osteoclastic bone resorption are only partially understood. Here, we show that exposure to multiple fractions of low-doses (10 fractions of 0.4 Gy total body irradiation [TBI]/week, i.e., fractionated exposure) and/or a single exposure to the same total dose of 4 Gy TBI causes a decrease in trabecular, but not cortical, bone mass in young adult male mice. This damaging effect was associated with highly activated bone resorption. Both osteoclast differentiation and maturation increased in cultures of bone marrow-derived macrophages from mice exposed to either fractionated or singular TBI. IR also increased the expression and enzymatic activity of mitochondrial deacetylase Sirtuin-3 (Sirt3)-an essential protein for osteoclast mitochondrial activity and bone resorption in the development of osteoporosis. Osteoclast progenitors lacking Sirt3 exposed to IR exhibited impaired resorptive activity. Taken together, targeting impairment of osteoclast mitochondrial activity could be a novel therapeutic strategy for IR-induced bone loss, and Sirt3 is likely a major mediator of this effect.

Synthetic Matrices for Intestinal Organoid Culture: Implications for Better Performance.

Organoids are three-dimensional (3D) self-renewing and self-organizing clusters of cells that imitate an organ's structure and function, making them an important tool in various fields ranging from regenerative medicine to drug discovery. Organoids can be developed ex vivo by isolating adult stem cells from an organ-specific tissue (e.g., intestine, brain, and lung) and allowing the stem cells to grow and differentiate in an appropriate growth media with some structural support elements. A 3D extracellular matrix (ECM) hydrogel, a network of highly hydrophilic cross-linked polymer chains, provides essential support and cues for ex vivo organoid growth. Commercially available hydrogel matrices (for example, Matrigel and collagen) are primarily derived from animal tissues. Notably, these animal-derived hydrogel matrices are not suitable for controlled modifications and pose risks of immunogen and pathogen transfer, thus diminishing their clinical application. These limitations of animal-derived hydrogel matrices can, however, be overcome using synthetic hydrogel matrices based on polymers such as polyethylene glycol, nanocellulose, alginate, hyaluronic acid, and polylactic-co-glycolic acid. This review highlights some of the current approaches and advantages of developing synthetic ECM-mimic hydrogels, focusing primarily on intestinal organoid culture.

Platelet glycoprotein Ibα provides radiation protection.

BACKGROUND AND PURPOSE: Platelet membrane glycoprotein Ibα (GPIbα), the major ligand-binding subunit of the GPIb-IX-V complex, binds to a number of ligands contributing to hemostasis, thrombosis, and inflammation. Binding to von Willebrand factor (VWF) initiates the process of hemostasis/thrombosis, while binding to the leukocyte receptor Macrophage-1 antigen (Mac-1) has been implicated in modulating the inflammatory response. Thus as GPIbα resides at the nexus of thrombosis and inflammation, we investigated the impact of GPIbα on radiation injury outcomes as this injury triggers both the thrombotic and inflammatory pathways. MATERIALS AND METHODS: We used wild-type (WT) C57BL/6J mice and a dysfunctional GPIbα mouse model, in which endogenous GPIbα is replaced with a non-functional α-subunit (hIL-4R/Ibα), to determine whether the impairment of platelet GPIbα alters radiation response. Following exposure to 8.5 Gy total body irradiation (TBI), a series of parameters including radiation lethality, platelet-neutrophil/monocyte interactions, neutrophil/monocyte activation, serum cytokine levels and intestinal injury, were compared between the strains. RESULTS: The lack of functional GPIbα resulted in higher radiation lethality, greater monocyte activation, increased levels of serum pro-inflammatory cytokines, heightened intestinal damage, and a reduction of intestinal neutrophil recovery. CONCLUSION: These data suggest that loss of platelet GPIbα enhances radiation toxicity and that GPIbα-mediated interactions may play a crucial role in limiting radiation damage. Thus, a mechanistic understanding of the biological impact of GPIbα following TBI could provide crucial insights for improving the safety of radiotherapy and minimizing the deleterious effects of accidental or occupational exposure to high-dose radiation.

Mitigation of late cardiovascular effects of oxygen ion radiation by γ-tocotrienol in a mouse model.

PURPOSE: While there is concern about degenerative tissue effects of exposure to space radiation during deep-space missions, there are no pharmacological countermeasures against these adverse effects. γ-Tocotrienol (GT3) is a natural form of vitamin E that has anti-oxidant properties, modifies cholesterol metabolism, and has anti-inflammatory and endothelial cell protective properties. The purpose of this study was to test whether GT3 could mitigate cardiovascular effects of oxygen ion (16O) irradiation in a mouse model. MATERIALS AND METHODS: Male C57BL/6 J mice were exposed to whole-body 16O (600 MeV/n) irradiation (0.26-0.33 Gy/min) at doses of 0 or 0.25 Gy at 6 months of age and were followed up to 9 months after irradiation. Animals were administered GT3 (50 mg/kg/day s.c.) or vehicle, on Monday - Friday starting on day 3 after irradiation for a total of 16 administrations. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters. Cardiac tissue remodeling and inflammatory infiltration were assessed with histology and immunoblot analysis at 2 weeks, 3 and 9 months after radiation. RESULTS: GT3 mitigated the effects of 16O radiation on cardiac function, the expression of a collagen type III peptide, and markers of mast cells, T-cells and monocytes/macrophages in the left ventricle. CONCLUSIONS: GT3 may be a potential countermeasure against late degenerative tissue effects of high-linear energy transfer radiation in the heart.

Plasma Metabolomics in a Nonhuman Primate Model of Abdominal Radiation Exposure.

The acute radiation syndrome is defined in large part by radiation injury in the hematopoietic and gastrointestinal (GI) systems. To identify new pathways involved in radiation-induced GI injury, this study assessed dose- and time-dependent changes in plasma metabolites in a nonhuman primate model of whole abdominal irradiation. Male and female adult Rhesus monkeys were exposed to 6 MV photons to the abdomen at doses ranging between 8 and 14 Gy. At time points from 1 to 60 days after irradiation, plasma samples were collected and subjected to untargeted metabolomics. With the limited sample size of females, different discovery times after irradiation between males and females were observed in metabolomics pattern. Detailed analyses are restricted to only males for the discovery power. Radiation caused an increase in fatty acid oxidation and circulating levels of corticosteroids which may be an indication of physiological stress, and amino acids, indicative of a cellular repair response. The largest changes were observed at days 9 and 10 post-irradiation, with most returning to baseline at day 30. In addition, dysregulated metabolites involved in amino acid pathways, which might indicate changes in the microbiome, were detected. In conclusion, abdominal irradiation in a nonhuman primate model caused a plasma metabolome profile indicative of GI injury. These results point to pathways that may be targeted for intervention or used as early indicators of GI radiation injury. Moreover, our results suggest that effects are sex-specific and that interventions may need to be tailored accordingly.

Differential Recovery of Small Intestinal Segments after Partial-Body Irradiation in Non-Human Primates.

In the event of a radiological attack or accident, it is more likely that the absorbed radiation dose will be heterogeneous, rather than uniformly distributed throughout the body. This type of uneven dose distribution is known as partial-body irradiation (PBI). Partial exposure of the vital organs, specifically the highly radiosensitive intestines, may cause death, if the injury is significant and the post-exposure recovery is considerably compromised. Here we investigated the recovery rate and extent of recovery from PBI-induced intestinal damage in large animals. Rhesus macaques (Macaca mulatta) were randomly divided into four groups: sham-irradiated (0 Gy), 8 Gy PBI, 11 Gy PBI and 14 Gy PBI. A single dose of ionizing radiation was delivered in the abdominal region using a uniform bilateral anteroposterior and posteroanterior technique. Irradiated animals were scheduled for euthanasia on days 10, 28 or 60 postirradiation, and sham-irradiated animals on day 60. Intestinal structural injuries were assessed via crypt depth, villus height, and mucosal surface length in the four different intestinal regions (duodenum, proximal jejunum, distal jejunum and ileum) using H&E staining. Higher radiation doses corresponded with more injury at 10 days post-PBI and a faster recovery rate. However, at 60 days post-PBI, damage was still evident in all regions of the intestine. The proximal and distal ends (duodenum and ileum, respectively) sustained less damage and recovered more fully than the jejunum.

Dietary Methionine Deficiency Enhances Genetic Instability in Murine Immune Cells.

Both cell and animal studies have shown that complete or partial deficiency of methionine inhibits tumor growth. Consequently, the potential implementation of this nutritional intervention has recently been of great interest for the treatment of cancer patients. Unfortunately, diet alteration can also affect healthy immune cells such as monocytes/macrophages and their precursor cells in bone marrow. As around half of cancer patients are treated with radiotherapy, the potential deleterious effect of dietary methionine deficiency on immune cells prior to and/or following irradiation needs to be evaluated. Therefore, we examined whether modulation of methionine content alters genetic stability in the murine RAW 264.7 monocyte/macrophage cell line in vitro by chromosomal analysis after 1-month culture in a methionine-deficient or supplemented medium. We also analyzed chromosomal aberrations in the bone marrow cells of CBA/J mice fed with methionine-deficient or supplemented diet for 2 months. While all RAW 264.7 cells revealed a complex translocation involving three chromosomes, three different clones based on the banding pattern of chromosome 9 were identified. Methionine deficiency altered the ratio of the three clones and increased chromosomal aberrations and DNA damage in RAW 264.7. Methionine deficiency also increased radiation-induced chromosomal aberration and DNA damage in RAW 264.7 cells. Furthermore, mice maintained on a methionine-deficient diet showed more chromosomal aberrations in bone marrow cells than those given methionine-adequate or supplemented diets. These findings suggest that caution is warranted for clinical implementation of methionine-deficient diet concurrent with conventional cancer therapy.

Simultaneous exposure to chronic irradiation and simulated microgravity differentially alters immune cell phenotype in mouse thymus and spleen.

Deep-space missions may alter immune cell phenotype in the primary (e.g., thymus) and secondary (e.g., spleen) lymphoid organs contributing to the progression of a variety of diseases. In deep space missions, astronauts will be exposed to chronic low doses of HZE radiation while being in microgravity. Ground-based models of long-term uninterrupted exposures to HZE radiation are not yet available. To obtain insight in the effects of concurrent exposure to microgravity and chronic irradiation (CIR), mice received a cumulative dose of chronic 0.5 Gy gamma rays over one month ± simulated microgravity (SMG). To obtain insight in a dose rate effect, additional mice were exposed to single acute irradiation (AIR) at 0.5 Gy gamma rays. We measured proportions of immune cells relative to total number of live cells in the thymus and spleen, stress level markers in plasma, and change in body weight, food consumption, and water intake. CIR affected thymic CD3+/CD335+ natural killer T (NK-T) cells, CD25+ regulatory T (Treg) cells, CD27+/CD335- natural killer (NK1) cells and CD11c+/CD11b- dendritic cells (DCs) differently in mice subjected to SMG than in mice with normal loading. No such effects of CIR on SMG as compared to normal loading were observed in cell types from the spleen. Differences between CIR and AIR groups (both under normal loading) were found in thymic Treg and DCs. Food consumption, water intake, and body weight were less after coexposure than singular or no exposure. Compared to sham, all treatment groups exhibited elevated plasma levels of the stress marker catecholamines. These data suggest that microgravity and chronic irradiation may interact with each other to alter immune cell phenotypes in an organ-specific manner and appropriate strategies are required to reduce the health risk of crewmembers.

Dietary Methionine Supplementation Exacerbates Gastrointestinal Toxicity in a Mouse Model of Abdominal Irradiation.

BACKGROUND AND PURPOSE: Identification of appropriate dietary strategies for prevention of weight and muscle loss in cancer patients is crucial for successful treatment and prolonged patient survival. High-protein oral nutritional supplements decrease mortality and improve indices of nutritional status in cancer patients; however, high-protein diets are often rich in methionine, and experimental evidence indicates that a methionine-supplemented diet (MSD) exacerbates gastrointestinal toxicity after total body irradiation. Here, we sought to investigate whether MSD can exacerbate gastrointestinal toxicity after local abdominal irradiation, an exposure regimen more relevant to clinical settings. MATERIALS AND METHODS: Male CBA/CaJ mice fed either a methionine-adequate diet or MSD (6.5 mg methionine/kg diet vs 19.5 mg/kg) received localized abdominal X-irradiation (220 kV, 13 mA) using the Small Animal Radiation Research Platform, and tissues were harvested 4, 7, and 10 days after irradiation. RESULTS: MSD exacerbated gastrointestinal toxicity after local abdominal irradiation with 12.5 Gy. This was evident as impaired nutrient absorption was paralleled by reduced body weight recovery. Mechanistically, significant shifts in the gut ecology, evident as decreased microbiome diversity, and substantially increased bacterial species that belong to the genus Bacteroides triggered proinflammatory responses. The latter were evident as increases in circulating neutrophils with corresponding decreases in lymphocytes and associated molecular alterations, exhibited as increases in mRNA levels of proinflammatory genes Icam1, Casp1, Cd14, and Myd88. Altered expression of the tight junction-related proteins Cldn2, Cldn5, and Cldn6 indicated a possible increase in intestinal permeability and bacterial translocation to the liver. CONCLUSIONS: We report that dietary supplementation with methionine exacerbates gastrointestinal syndrome in locally irradiated mice. This study demonstrates the important roles registered dieticians should play in clinical oncology and further underlines the necessity of preclinical and clinical investigations in the role of diet in the success of cancer therapy.

Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose.

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.

Methionine dietary supplementation potentiates ionizing radiation-induced gastrointestinal syndrome.

Methionine is an essential amino acid needed for a variety of processes in living organisms. Ionizing radiation depletes tissue methionine concentrations and leads to the loss of DNA methylation and decreased synthesis of glutathione. In this study, we aimed to investigate the effects of methionine dietary supplementation in CBA/CaJ mice after exposure to doses ranging from 3 to 8.5 Gy of 137Cs of total body irradiation. We report that mice fed a methionine-supplemented diet (MSD; 19.5 vs. 6.5 mg/kg in a methionine-adequate diet, MAD) developed acute radiation toxicity at doses as low as 3 Gy. Partial body irradiation performed with hindlimb shielding resulted in a 50% mortality rate in MSD-fed mice exposed to 8.5 Gy, suggesting prevalence of radiation-induced gastrointestinal syndrome in the development of acute radiation toxicity. Analysis of the intestinal microbiome demonstrated shifts in the gut ecology, observed along with the development of leaky gut syndrome and bacterial translocation into the liver. Normal gut physiology impairment was facilitated by alterations in the one-carbon metabolism pathway and was exhibited as decreases in circulating citrulline levels mirrored by decreased intestinal mucosal surface area and the number of surviving crypts. In conclusion, we demonstrate that a relevant excess of methionine dietary intake exacerbates the detrimental effects of exposure to ionizing radiation in the small intestine.NEW & NOTEWORTHY Methionine supplementation, instead of an anticipated health-promoting effect, sensitizes mice to gastrointestinal radiation syndrome. Mechanistically, excess of methionine negatively affects intestinal ecology, leading to a cascade of physiological, biochemical, and molecular alterations that impair normal gut response to a clinically relevant genotoxic stressor. These findings speak toward increasing the role of registered dietitians during cancer therapy and the necessity of a solid scientific background behind the sales of dietary supplements and claims regarding their benefits.