Nikolai V. Gorbunov

Cardioprotection by adaptation to ischaemia augments autophagy in association with BAG-1 protein.

Autophagy is an intracellular process in which a cell digests its own constituents via lysosomal degradative pathway. Though autophagy has been shown in several cardiac diseases like heart failure, hypertrophy and ischaemic cardiomyopathy, the role and the regulation of autophagy is still largely unknown. Bcl‐2‐associated athanogene (BAG‐1) is a multifunctional pro‐survival molecule that binds with Hsp70/Hsc70. In this study, myocardial adaptation to ischaemia by repeated brief episodes of ischaemia and reperfusion (I/R) prior to lethal I/R enhanced the expression of autophagosomal membrane specific protein light chain 3 (LC3)‐II, and Beclin‐1, a molecule involved in autophagy and BAG‐1. Autophagosomes structures were found in the adapted myocardium through electron microscopy. Co‐immunoprecipitation and co‐immunofluorescence analyses revealed that LC3‐II was bound with BAG‐1. Inhibition of autophagy by treating rats with Wortmannin (15 μg/kg; intraperitoneally) abolished the ischaemic adaptation‐induced induction of LC3‐II, Beclin‐1, BAG‐1 and cardioprotection. Intramyocardial injection of BAG‐1 siRNA attenuated the induction of LC3‐II, and abolished the cardioprotection achieved by adaptation. Furthermore, hypoxic adaptation in cardiac myoblast cells induced LC3‐II and BAG‐1. BAG‐1 siRNA treatment attenuated hypoxic adaptation‐induced LC3‐II and BAG‐1, and abolished improvement in cardiac cell survival and reduction of cell death. These results clearly indicate that myocardial protection elicited by adaptation is mediated at least in part via up‐regulation of autophagy in association with BAG‐1 protein.

Ciprofloxacin Modulates Cytokine/Chemokine Profile in Serum, Improves Bone Marrow Repopulation, and Limits Apoptosis and Autophagy in Ileum after Whole Body Ionizing Irradiation Combined with Skin-Wound Trauma

Radiation combined injury (CI) is a radiation injury (RI) combined with other types of injury, which generally leads to greater mortality than RI alone. A spectrum of specific, time-dependent pathophysiological changes is associated with CI. Of these changes, the massive release of pro-inflammatory cytokines, severe hematopoietic and gastrointestinal losses and bacterial sepsis are important treatment targets to improve survival. Ciprofloxacin (CIP) is known to have immunomodulatory effect besides the antimicrobial activity. The present study reports that CIP ameliorated pathophysiological changes unique to CI that later led to major mortality. B6D2F1/J mice received CI on day 0, by RI followed by wound trauma, and were treated with CIP (90 mg/kg p.o., q.d. within 2 h after CI through day 10). At day 10, CIP treatment not only significantly reduced pro-inflammatory cytokine and chemokine concentrations, including interleukin-6 (IL-6) and KC (i.e., IL-8 in human), but it also enhanced IL-3 production compared to vehicle-treated controls. Mice treated with CIP displayed a greater repopulation of bone marrow cells. CIP also limited CI-induced apoptosis and autophagy in ileal villi, systemic bacterial infection, and IgA production. CIP treatment led to LD0/10 compared to LD20/10 for vehicle-treated group after CI. Given the multiple beneficial activities of CIP shown in our experiments, CIP may prove to be a useful therapeutic drug for CI.

Up-regulation of autophagy in small intestine Paneth cells in response to total-body γ-irradiation † †

Macroautophagy (mAG) is a lysosomal mechanism of degradation of cell self‐constituents damaged due to variety of stress factors, including ionizing irradiation. Activation of mAG requires expression of mAG protein Atg8 (LC3) and conversion of its form I (LC3‐I) to form II (LC3‐II), mediated by redox‐sensitive Atg4 protease. We have demonstrated upregulation of this pathway in the innate host defense Paneth cells of the small intestine (SI) due to ionizing irradiation and correlation of this effect with induction of pro‐oxidant inducible nitric oxide synthase (iNOS). CD2F1 mice were exposed to 9.25 Gy γ‐ionizing irradiation. Small intestinal specimens were collected during 7 days after ionizing irradiation. Assessment of ionizing irradiation‐associated alterations in small intestinal crypt and villus cells and activation of the mAG pathway was conducted using microscopical and biochemical techniques. Analysis of iNOS protein and the associated formation of nitrites and lipid peroxidation products was performed using immunoblotting and biochemical analysis, and revealed increases in iNOS protein, nitrate levels and oxidative stress at day 1 following ionizing irradiation. Increase in immunoreactivity of LC3 protein in the crypt cells was observed at day 7 following ionizing irradiation. This effect predominantly occurred in the CD15‐positive Paneth cells and was associated with accumulation of LC3‐II isoform. The formation of autophagosomes in Paneth cells was confirmed by transmission electron microscopy (TEM). Up‐regulation of LC3 pathway in the irradiated SI was accompanied by a decreased protein–protein interaction between LC3 and chaperone heat shock protein 70. A high‐level of LC3‐immunoreactivity in vacuole‐shaped structures was spatially co‐localized with immunoreactivity of 3‐nitro‐tyrosine. The observed effects were diminished in iNOS knockout B6.129P2‐NOS2tm1Lau/J mice subjected to the same treatments. We postulate that the observed up‐regulation of mAG in the irradiated small intestine is at least in part mediated by the iNOS signalling mechanism. Published in 2009 by John Wiley & Sons, Ltd.

Pegylated G-CSF Inhibits Blood Cell Depletion, Increases Platelets, Blocks Splenomegaly, and Improves Survival after Whole-Body Ionizing Irradiation but Not after Irradiation Combined with Burn

Exposure to ionizing radiation alone (radiation injury, RI) or combined with traumatic tissue injury (radiation combined injury, CI) is a crucial life-threatening factor in nuclear and radiological accidents. As demonstrated in animal models, CI results in greater mortality than RI. In our laboratory, we found that B6D2F1/J female mice exposed to 60Co-γ-photon radiation followed by 15% total-body-surface-area skin burns experienced an increment of 18% higher mortality over a 30-day observation period compared to irradiation alone; that was accompanied by severe cytopenia, thrombopenia, erythropenia, and anemia. At the 30th day after injury, neutrophils, lymphocytes, and platelets still remained very low in surviving RI and CI mice. In contrast, their RBC, hemoglobin, and hematocrit were similar to basal levels. Comparing CI and RI mice, only RI induced splenomegaly. Both RI and CI resulted in bone marrow cell depletion. It was observed that only the RI mice treated with pegylated G-CSF after RI resulted in 100% survival over the 30-day period, and pegylated G-CSF mitigated RI-induced body-weight loss and depletion of WBC and platelets. Peg-G-CSF treatment sustained RBC balance, hemoglobin levels, and hematocrits and inhibited splenomegaly after RI. The results suggest that pegylated G-CSF effectively sustained animal survival by mitigating radiation-induced cytopenia, thrombopenia, erythropenia, and anemia.

Radiation Combined Injury: DNA Damage, Apoptosis, and Autophagy

Radiation combined injury is defined as an ionizing radiation exposure received in combination with other trauma or physiological insults. The range of radiation threats we face today includes everything from individual radiation exposures to mass casualties resulting from a terrorist nuclear incident, and mans of these exposure scenarios include the likelihood of additional traumatic injury. Radiation combined injury sensitizes target organs and cells and exacerbates acute radiation syndrome. Organs and cells with high sensitivity to combined injury are the skin, the hematopoietic system, the gastrointestinal tract, spermatogenic cells, and the vascular system. Among its many effects, radiation combined injury results in decreases in lymphocytes, macrophages, neutrophils, platelets, stem cells, and tissue integrity; activation of the iNOS/NF-κB/NF-IL6 and p53/Bax pathways; and increases in DNA single and double strand breaks, TLR signaling, cytokine concentrations, bacterial infection, and cytochrome c release from mitochondria to cytoplasm. These alterations lead to apoptosis and autophagy and, as a result, increased mortality. There is a pressing need to understand more about the bodys response to combined injury in order to be able to develop effective countermeasures, since few currently exist. In this review, we summarize what is known about how combined injury modifies the radiation response, with a special emphasis on DNA damage/repair, signal transduction pathways, apoptosis, and autophagy. We also describe current and prospective countermeasures relevant to the treatment and prevention of combined injury.

Ghrelin therapy improves survival after whole-body ionizing irradiation or combined with burn or wound: amelioration of leukocytopenia, thrombocytopenia, splenomegaly, and bone marrow injury.

Exposure to ionizing radiation alone (RI) or combined with traumatic tissue injury (CI) is a crucial life-threatening factor in nuclear and radiological events. In our laboratory, mice exposed to 60Co-γ-photon radiation (9.5 Gy, 0.4 Gy/min, bilateral) followed by 15% total-body-surface-area skin wounds (R-W CI) or burns (R-B CI) experienced an increment of ≥18% higher mortality over a 30-day observation period compared to RI alone. CI was accompanied by severe leukocytopenia, thrombocytopenia, erythropenia, and anemia. At the 30th day after injury, numbers of WBC and platelets still remained very low in surviving RI and CI mice. In contrast, their RBC, hemoglobin, and hematocrit were recovered towards preirradiation levels. Only RI induced splenomegaly. RI and CI resulted in bone-marrow cell depletion. In R-W CI mice, ghrelin (a hunger-stimulating peptide) therapy increased survival, mitigated body-weight loss, accelerated wound healing, and increased hematocrit. In R-B CI mice, ghrelin therapy increased survival and numbers of neutrophils, lymphocytes, and platelets and ameliorated bone-marrow cell depletion. In RI mice, this treatment increased survival, hemoglobin, and hematocrit and inhibited splenomegaly. Our novel results are the first to suggest that ghrelin therapy effectively improved survival by mitigating CI-induced leukocytopenia, thrombocytopenia, and bone-marrow injury or the RI-induced decreased hemoglobin and hematocrit.

Response of crypt paneth cells in the small intestine following total-body gamma-irradiation.

Ionizing irradiation causes damage and functional failure of irradiation-sensitive systems and tissues such as small intestine. The molecular mechanisms underlying inflammatory and adaptive responses to acute irradiation damage are poorly understood. Using a mouse model of total-body γ-irradiation, we assessed the irradiation response of crypt host-defense Paneth cells by measuring α-defensin 4 (AD4) expression and correlated the gathered data with activation of the caspase-1/IL-1β inflammatory signaling cascade. The irradiation injury was produced in CD2F1 mice exposed to 9.25 Gy γ-radiation. This dose resulted in 85–100% mortality at the 15th day post-irradiation. Small intestine tissue samples were collected at the 7th day post-irradiation. Assessment of irradiation-associated pro-inflammatory alterations in small intestine tissue and expression of AD4 in Paneth cells was conducted using confocal immunofluorescence imaging, transmission electron microscopy (TEM), light microscopy, and immunoblotting techniques. The small intestine analysis revealed an increase in the precursor form of IL-1β, the activated form of IL-1β, and the activated form of caspase-1 (p10 CASP-1) at the 7th day post-irradiation. Immunoprecipitation analysis showed increased interaction between IL-1β and p10 CASP-1 after irradiation. This effect was observed in the irradiated small intestine and CD15-positive Paneth cells using confocal imaging techniques. The pro-inflammatory alterations in Paneth cells were accompanied by increases in AD4 mRNA and its 8 kD peptide product. Paneth cell secretory activity was observed at the sites of bacterial translocation in the crypt lumens. These data suggest that Paneth cells can contribute to small intestine inflammatory remodeling during the post-irradiation period.

Adaptive Redox Response of Mesenchymal Stromal Cells to Stimulation with Lipopolysaccharide Inflammagen: Mechanisms of Remodeling of Tissue Barriers in Sepsis

Acute bacterial inflammation is accompanied by excessive release of bacterial toxins and production of reactive oxygen and nitrogen species (ROS and RNS), which ultimately results in redox stress. These factors can induce damage to components of tissue barriers, including damage to ubiquitous mesenchymal stromal cells (MSCs), and thus can exacerbate the septic multiple organ dysfunctions. The mechanisms employed by MSCs in order to survive these stress conditions are still poorly understood and require clarification. In this report, we demonstrated that in vitro treatment of MSCs with lipopolysaccharide (LPS) induced inflammatory responses, which included, but not limited to, upregulation of iNOS and release of RNS and ROS. These events triggered in MSCs a cascade of responses driving adaptive remodeling and resistance to a “self-inflicted” oxidative stress. Thus, while MSCs displayed high levels of constitutively present adaptogens, for example, HSP70 and mitochondrial Sirt3, treatment with LPS induced a number of adaptive responses that included induction and nuclear translocation of redox response elements such as NFkB, TRX1, Ref1, Nrf2, FoxO3a, HO1, and activation of autophagy and mitochondrial remodeling. We propose that the above prosurvival pathways activated in MSCs in vitro could be a part of adaptive responses employed by stromal cells under septic conditions.

Lipid Peroxidation After Ionizing Irradiation Leads to Apoptosis and Autophagy

A living cell is a dynamic biological system composed primarily of nucleic acids, carbohydrates, lipids, and proteins that structurally and functionally interact with many other molecules--organic and inorganic--to carry out normal cell metabolism. Exposure of a cell to radiation can both directly and indirectly alter molecules within the cell to affect cell viability. Radiation energy absorbed by tissues and fluids is dissipated by the radiolysis of water molecules and biomolecules [1-3]. These reactions result in redox-reactive products such as hydroxyl radical (HO*), hydrogen peroxide (H2O2), hydrated electron (e-aq), and an array of biomolecule-derived carbon-, oxygen-, sulfur-, and nitrogen-centered radicals (i.e., RC*, RO*, RS*, and RN*) that can in turn lead to the formation of organic peroxides and superoxide anion radicals ( O2*) in the presence of molecular oxygen [3, 4].

Autophagy-Mediated Defense Response of Mouse Mesenchymal Stromal Cells (MSCs) to Challenge with Escherichia coli

Abstract : Symbiotic microorganisms are spatially separated from their animal host, e.g., in the intestine and skin, in a manner enabling nutrient metabolism as well as evolutionary development of protective physiologic features in the host such as innate and adaptive immunity, immune tolerance, and function of tissue barriers . The major interface barrier between the microbiota and host tissue is constituted by epithelium reticuloendothelial tissue, and mucosa-associated lymphoid tissue (MALT) . Traumatic damage to skin and the internal epithelium in soft tissues can cause infections that account for 7% to 10% of hospitalizations in the United States (4). Moreover, wound infections and sepsis are an increasing cause of death in severely ill patients, especially those with immunosupression due to exposure to cytotoxic agents and chronic inflammation (4). It is well accepted that breakdown of the host-bacterial symbiotic homeostasis and associated infections are the major consequences of impairment of the first line of antimicrobial defense barriers such as the mucosal layers, MALT and reticuloendothelium. Under these impairment conditions of particular interest then is the role of sub-mucosal structures, such as connective tissue stroma, in the innate defense compensatory responses to infections. The mesenchymal connective tissue of different origins is a major source of multipotent mesenchymal stromal cells (i.e., colony-forming-unit fibroblasts). Recent discovery of immunomodulatory function of mesenchymal stromal cells (MSCs) suggests that they are essential constituents that control inflammatory responses.