The impact of NF-κB activation on radiation sensitivity in human embryonic kidney cells

Outer space is heavily loaded with high-energetic particles contributing to high risks for space travelers, which could lead to lethal health problems. To protect astronauts from radiation-induced immediate and late effects, countermeasures need to be developed. This requires improved knowledge on damage response pathways and possible pharmacological interaction. DNA double-strand breaks are the ultimate damages caused by radiation. This stimulates the cellular response, which aims at repairing the damaged DNA and ensuring the survival of the cell. One of such cellular responses is the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and its pathway. The role of NF-κB in cellular survival and proliferation has been well studied (Baldwin, 1996b). Recent studies also suggest a possible of role of the NF-κB activation in DNA repair (Volcic et al., 2012). Using this knowledge, the role of NF-κB in sensitizing cells by X-irradiation was studied by colony formation assay and cell cycle analysis. With the help of the reporter cell line, the NF-κB activation kinetics was clearly studied. Lastly, the role of NF-κB in X-irradiation-induced DNA damage and its repair was also investigated. The study was performed using a HEK NF-κB reporter cell line (HEK-pNFκB-DDtdtomatoC8) and HEK cells with a knockdown NF-κB subunit, RelA (HEK shRNA RelA). A pro-survival role of NF-κB in the survival of the HEK cells after X-irradiation was confirmed. The different phases of the cell cycle were analyzed after X-irradiation and it was observed that NF-κB regulates the G2/M arrest. Further, the NF-κB subunit, RelA was found to be involved in the phosphorylation of the H2AX foci, which is a known marker for DNA damage and repair (Sharma et al., 2012). This project reflects the potential of the NF-κB pathway as a target in modulating the cellular radiation response during space missions and aims at finding its role in radiosensitization of cells, which could be beneficial for medical purposes as well

MPO (Myeloperoxidase) Reduces Endothelial Glycocalyx Thickness Dependent on Its Cationic Charge

Objective The leukocyte heme-enzyme MPO (myeloperoxidase) exerts proinflammatory effects on the vascular system primarily linked to its catalytic properties. Recent studies have shown that MPO, depending on its cationic charge, mediates neutrophil recruitment and activation. Here, we further investigated MPO's extracatalytic properties and its effect on endothelial glycocalyx (EG) integrity. Approach and Results In vivo staining of murine cremaster muscle vessels with Alcian Blue 8GX provided evidence of an MPO-dependent decrease in anionic charge of the EG. MPO binding to the glycocalyx was further characterized using Chinese hamster ovary cells and its glycosaminoglycan mutantspgsA-745 (mutant Chinese hamster ovary cells lacking heparan sulfate and chondroitin sulfate glycosaminoglycan) and pgsD-677 (mutant Chinese hamster ovary cells lacking heparan sulfate glycosaminoglycan), which revealed heparan sulfate as the main mediator of MPO binding. Further, EG integrity was assessed in terms of thickness using intravital microscopy of murine cremaster muscle. A significant reduction in EG thickness was observed on infusion of catalytically active MPO, as well as mutant inactive MPO and cationic polymer polylysine. Similar effects were also observed in wild-type mice after a local inflammatory stimulus but not in MPO-knockout mice. The reduction in EG thickness was reversed after removal of vessel-bound MPO, suggesting a possible physical collapse of the EG. Last, experiments with in vivo neutrophil depletion revealed that MPO also induced neutrophil-mediated shedding of the EG core protein, Sdc1 (syndecan-1). Conclusions These findings provide evidence that MPO, via ionic interaction with heparan sulfate side chains, can cause neutrophil-dependent Sdc1 shedding and collapse of the EG structure

Myeloperoxidase aggravates pulmonary arterial hypertension by activation of vascular Rho-kinase

Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo(-/-) than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo(-/-) mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling

Rationale: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. Objective: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. Methods and Results: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin–deficient (CD11b−/−) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo−/−) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo−/− mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell–derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo−/− mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter–defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction \u3e35% undergoing elective diagnostic cardiac evaluation. Conclusions: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling

Rationale: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. Objective: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. Methods and Results: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b(-/-)) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo(-/-)) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wildtype mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo(-/-) mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo(-/-) mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. Conclusions: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia