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A myelopoiesis gene signature in circulating leucocytes, exemplified by increased myeloperoxidase (MPO) and proteinase 3 (PR3) mRNA levels, has been reported in patients with active anti-neutrophil cytoplasm antibody associated vasculitis (AAV) and to a lesser extent during remission. We hypothesized that this signature could predict disease relapse. mRNA levels of PR3, MPO, selected myelopoiesis transcription factors (CEBPA, CEBPB, SPIB, SPI1) and microRNAs (miRNAs) from patient and control peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) were analyzed and associated with clinical data. Patients in stable remission had higher mRNA levels for PR3 (PBMCs, PMNs) and MPO (PBMCs). PR3 and SPIB mRNA correlated positively in control but negatively in patient PBMCs. Statistically significant correlations existed between PR3 mRNA and several miRNAs in controls, but not in patients. PR3/MPO mRNA levels were not associated with previous or future relapses but correlated to steroid treatment. Prednisolone doses were negatively linked to SPIB and miR-155-5p, miR-339-5p (PBMCs) and to miR-221, miR-361, miR-505 (PMNs). PR3 mRNA in PBMCs correlated with time since last flare, blood leucocyte count and estimated glomerular filtration rate. Our results show that elevated leucocyte PR3 mRNA levels in AAV patients in remission do not predict relapse. The origin seems multifactorial, but to an important part explainable by prednisolone action. Gene signatures in patients with AAV undergoing steroid treatment should therefore be interpreted accordingly

Stress-vs-time signals allow the prediction of structurally catastrophic events during fracturing of immature cartilage and predetermine the biomechanical, biochemical, and structural impairment

Objective Trauma-associated cartilage fractures occur in children and adolescents with clinically significant incidence. Several studies investigated biomechanical injury by compressive forces but the injury-related stress has not been investigated extensively. In this study, we hypothesized that the biomechanical stress occurring during compressive injury predetermines the biomechanical, biochemical, and structural consequences. We specifically investigated whether the stress-vs-time signal correlated with the injurious damage and may allow prediction of cartilage matrix fracturing. Methods Superficial and deeper zones disks (SZDs, DZDs; immature bovine cartilage) were biomechanically characterized, injured (50% compression, 100%/s strain-rate), and re-characterized. Correlations of the quantified functional, biochemical and histological damage with biomechanical parameters were zonally investigated. Results Injured SZDs exhibited decreased dynamic stiffness (by 93.04 ± 1.72%), unresolvable equilibrium moduli, structural damage (2.0 ± 0.5 on a 5-point-damage-scale), and 1.78-fold increased sGAG loss. DZDs remained intact. Measured stress-vs-time-curves during injury displayed 4 distinct shapes, which correlated with histological damage (p < 0.001), loss of dynamic stiffness and sGAG (p < 0.05). Damage prediction in a blinded experiment using stress-vs-time grades was 100%-correct and sensitive to differentiate single/complex matrix disruptions. Correlations of the dissipated energy and maximum stress rise with the extent of biomechanical and biochemical damage reached significance when SZDs and DZDs were analyzed as zonal composites but not separately. Conclusions The biomechanical stress that occurs during compressive injury predetermines the biomechanical, biochemical, and structural consequences and, thus, the structural and functional damage during cartilage fracturing. A novel biomechanical method based on the interpretation of compressive yielding allows the accurate prediction of the extent of structural damage.National Institutes of Health (U.S.) (Grant R01-AR45779)Deutsche Forschungsgemeinschaft (Grant RO2511/1-1)Deutsche Forschungsgemeinschaft (Grant RO2511/2-1)Germany. Federal Ministry of Education and Research (Grant 01KQ0902B TP2

The Involvement of Lysosomes in Myocardial Aging and Disease

The myocardium is mainly composed of long-lived postmitotic cells with, if there is any at all, a very low rate of replacement through the division and differentiation of stem cells. As a consequence, cardiac myocytes gradually undergo pronounced age-related alterations which, furthermore, occur at a rate that inversely correlates with the longevity of species. Basically, these alterations represent the accumulation of structures that have been damaged by oxidation and that are useless and often harmful. These structures (so-called ‘waste’ materials), include defective mitochondria, aberrant cytosolic proteins, often in aggregated form, and lipofuscin, which is an intralysosomal undegradable polymeric substance. The accumulation of ‘waste’ reflects the insufficient capacity for autophagy of the lysosomal compartment, as well as the less than perfect functioning of proteasomes, calpains and other cellular digestive systems. Senescent mitochondria are usually enlarged, show reduced potential over their inner membrane, are deficient in ATP production, and often produce increased amounts of reactive oxygen species. The turnover of damaged cellular structures is hindered by an increased lipofuscin loading of the lysosomal compartment. This particularly restricts the autophagic turnover of enlarged, defective mitochondria, by diverting the flow of lysosomal hydrolases from autophagic vacuoles to lipofuscin-loaded lysosomes where the enzymes are lost, since lipofuscin is not degradable by lysosomal hydrolases. As a consequence, aged lipofuscin-rich cardiac myocytes become overloaded with damaged mitochondria, leading to increased oxidative stress, apoptotic cell death, and the gradual development of heart failure. Defective lysosomal function also underlies myocardial degeneration in various lysosomal storage diseases, while other forms of cardiomyopathies develop due to mitochondrial DNA mutations, resulting in an accumulation of abnormal mitochondria that are not properly eliminated by autophagy. The degradation of iron-saturated ferritin in lysosomes mediates myocardial injury in hemochromatosis, an acquired or hereditary disease associated with iron overload. Lysosomes then become sensitized to oxidative stress by the overload of low mass, redox-active iron that accumulates when iron-saturated ferritin is degraded following autophagy. Lysosomal destabilization is of importance in the induction and/or execution of programmed cell death (either classical apoptotic or autophagic), which is a common manifestation of myocardial aging and a variety of cardiac pathologies

Lysosomes in iron metabolism, ageing and apoptosis

The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH ∼4 to 5) that constantly fuse and divide. It receives a large number of hydrolases (∼50) from the trans-Golgi network, and substrates from both the cells’ outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while ‘resting’ lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place

Role of lysosomal iron in oxidative stress-induced cell death

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Lysosomal Iron, Iron Chelation, and Cell Death

Significance: Lysosomes are acidic organelles containing more than fifty hydrolases that provide for the degradation of intracellular and endocytosed materials by autophagy and heterophagy, respectively. They digest a variety of macromolecules, as well as all organelles, and their integrity is crucial. As a result of the degradation of iron-containing macromolecules (e.g., ferritin and mitochondrial components) or endocytosed erythrocytes (by macrophages), lysosomes can accumulate large amounts of iron. This iron occurs often as Fe(II) due to the acidic and reducing lysosomal environment. Fe(II) is known to catalyze Fenton reactions, yielding extremely reactive hydroxyl radicals that may jeopardize lysosomal membrane integrity during oxidative stress. This results in the release of hydrolases and redox-active iron into the cytosol with ensuing damage or cell death. Lysosomes play key roles not only in apoptosis and necrosis but also in neurodegeneration, aging, and atherosclerosis. Recent Advances: The damaging effect of intralysosomal iron can be hampered by endogenous or exogenous iron chelators that enter the lysosomal compartment by membrane permeation, endocytosis, or autophagy. Critical Issues: Cellular sensitivity to oxidative stress is enhanced by lysosomal redox-active iron or by lysosomal-targeted copper chelators binding copper (from degradation of copper-containing macromolecules) in redox-active complexes. Probably due to higher copper levels, lysosomes of malignant cells may be specifically sensitized by such chelators. Future Directions: By increasing lysosomal redox-active iron or exposing cells to lysosomal-targeted copper chelators, it should be possible to enhance the sensitivity of cancer cells to radiation-induced oxidative stress or treatment with cytostatics that induce such stress.</p

Cell sensitivity to oxidative stress is influenced by ferritin autophagy

To test the consequences of lysosomal degradation of differently iron-loaded ferritin molecules and to mimic ferritin autophagy under iron-overload and normal conditions, J774 cells were allowed to endocytose heavily iron loaded ferritin, probably with some adventitious iron (Fe-Ft), or iron-free apo-ferritin (apo-Ft). When cells subsequently were exposed to a bolus dose of hydrogen peroxide, apo-Ft prevented lysosomal membrane permeabilization (LMP), whereas Fe-Ft enhanced LMP. A 4-h pulse of Fe-Ft initially increased oxidative stress-mediated LMP that was reversed after another 3 h under standard culture conditions, suggesting that lysosomal iron is rapidly exported from lysosomes, with resulting upregulation of apo-ferritin that supposedly is autophagocytosed, thereby preventing LMP by binding intralysosomal redox-active iron. The obtained data suggest that upregulation of the stress protein ferritin is a rapid adaptive mechanism that counteracts LMP and ensuing apoptosis during oxidative stress. In addition, prolonged iron starvation was found to induce apoptotic cell death that, interestingly, was preceded by LMP, suggesting that LMP is a more general phenomenon in apoptosis than so far recognized. The findings provide new insights into aging and neurodegenerative diseases that are associated with enhanced amounts of cellular iron and show that lysosomal iron loading sensitizes to oxidative stress.Original Publication: Tino Kurz, Bertil Gustafsson and Ulf Brunk, Cell sensitivity to oxidative stress is influenced by ferritin autophagy, 2011, FREE RADICAL BIOLOGY AND MEDICINE, (50), 11, 1647-1658. http://dx.doi.org/10.1016/j.freeradbiomed.2011.03.014 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com