Davide Curzi

Melatonin Prevents Chemical-Induced Haemopoietic Cell Death

Melatonin (MEL), a methoxyindole synthesized by the pineal gland, is a powerful antioxidant in tissues as well as within cells, with a fundamental role in ameliorating homeostasis in a number of specific pathologies. It acts both as a direct radical scavenger and by stimulating production/activity of intracellular antioxidant enzymes. In this work, some chemical triggers, with different mechanisms of action, have been chosen to induce cell death in U937 hematopoietic cell line. Cells were pre-treated with 100 μM MEL and then exposed to hydrogen peroxide or staurosporine. Morphological analyses, TUNEL reaction and Orange/PI double staining have been used to recognize ultrastructural apoptotic patterns and to evaluate DNA behavior. Chemical damage and potential MEL anti-apoptotic effects were quantified by means of Tali® Image-Based Cytometer, able to monitor cell viability and apoptotic events. After trigger exposure, chromatin condensation, micronuclei formation and DNA fragmentation have been observed, all suggesting apoptotic cell death. These events underwent a statistically significant decrease in samples pre-treated with MEL. After caspase inhibition and subsequent assessment of cell viability, we demonstrated that apoptosis occurs, at least in part, through the mitochondrial pathway and that MEL interacts at this level to rescue U937 cells from death.

Antioxidants in the prevention of UVB-induced keratynocyte apoptosis.

Skin cells can respond to UVB-induced damage by counteracting it through antioxidant activation and DNA repair mechanisms or, when damage is massive by undergoing programmed cell death. Antioxidant factors, and, in particular, food compounds, have attracted much interest because of their potential use in new protective strategies for degenerative skin disorders. Melatonin, creatine and hydroxytyrosol show a variety of pharmacological and clinical benefits including anti-oxidant and anti-inflammatory activities. Here, the potential protective actions of antioxidant compounds against UVB-induced apoptosis were investigated in human keratinocytes. The cells were pre-treated with antioxidants before UVB exposure and their effect evaluated by means of ultrastructural and molecular analyses. After UVB radiation typical morphological apoptotic features and in situ DNA fragmentation after TUNEL reaction, appeared. A significant numerical decrease of apoptotic patterns could be observed when antioxidants were administrated before cell death induction. Moreover, both the intrinsic and extrinsic apoptotic pathways appeared activated after UVB radiation, and their down-regulation has been shown when antioxidants were added to cells before death induction. In conclusion, these compounds are able to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, an important role in preventing skin damage.

Impact of the Phosphatidylinositide 3-Kinase Signaling Pathway on the Cardioprotection Induced by Intermittent Hypoxia

Background Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. Methods Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 µg/kg ip) 15 min before LAD. Results We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. Conclusion We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.

Growth Hormone Plus Resistance Exercise Attenuate Structural Changes in Rat Myotendinous Junctions Resulting from Chronic Unloading

Myotendinous junctions (MTJs) are specialized sites on the muscle surface where forces generated by myofibrils are transmitted across the sarcolemma to the extracellular matrix. At the ultrastructural level, the interface between the sarcolemma and extracellular matrix is highly folded and interdigitated at these junctions. In this study, the effect of exercise and growth hormone (GH) treatments on the changes in MTJ structure that occur during muscle unloading, has been analyzed. Twenty hypophysectomized rats were assigned randomly to one of five groups: ambulatory control, hindlimb unloaded, hindlimb unloaded plus exercise (3 daily bouts of 10 climbs up a ladder with 50% body wt attached to the tail), hindlimb unloaded plus GH (2 daily injections of 1 mg/kg body wt, i.p.), and hindlimb unloaded plus exercise plus GH. MTJs of the plantaris muscle were analyzed by electron microscopy and the contact between muscle and tendon was evaluated using an IL/B ratio, where B is the base and IL is the interface length of MTJ’s digit-like processes. After 10 days of unloading, the mean IL/B ratio was significantly lower in unloaded (3.92), unloaded plus exercise (4.18), and unloaded plus GH (5.25) groups than in the ambulatory control (6.39) group. On the opposite, the mean IL/B ratio in the group treated with both exercise and GH (7.3) was similar to control. These findings indicate that the interaction between exercise and GH treatments attenuates the changes in MTJ structure that result from chronic unloading and thus can be used as a countermeasure to these adaptations.

Effects of lead pollution on Ammonia parkinsoniana (foraminifera): ultrastructural and microanalytical approaches.

The responses of Ammonia parkinsoniana (Foraminifera) exposed to different concentrations of lead (Pb) were evaluated at the cytological level. Foraminifera-bearing sediments were placed in mesocosms that were housed in aquaria each with seawater of a different lead concentration. On the basis of transmission electron microscopy and environmental scanning electron microscopy coupled with energy dispersive spectrometer analyses, it was possible to recognize numerous morphological differences between untreated (i.e., control) and treated (i.e., lead enrichment) specimens. In particular, higher concentrations of this pollutant led to numerical increase of lipid droplets characterized by a more electron-dense core, proliferation of residual bodies, a thickening of the organic lining, mitochondrial degeneration, autophagosome proliferation and the development of inorganic aggregates. All these cytological modifications might be related to the pollutant-induced stress and some of them such as the thickening of organic lining might suggest a potential mechanism of protection adopted by foraminifera.

Mercury-Pollution Induction of Intracellular Lipid Accumulation and Lysosomal Compartment Amplification in the Benthic Foraminifer Ammonia parkinsoniana

Heavy metals such as mercury (Hg) pose a significant health hazard through bioaccumulation and biomagnification. By penetrating cell membranes, heavy metal ions may lead to pathological conditions. Here we examined the responses of Ammonia parkinsoniana, a benthic foraminiferan, to different concentrations of Hg in the artificial sea water. Confocal images of untreated and treated specimens using fluorescent probes (Nile Red and Acridine Orange) provided an opportunity for visualizing the intracellular lipid accumulation and acidic compartment regulation. With increased Hg over time, we observed an increased number of lipid droplets, which may have acted as a detoxifying organelle where Hg is sequestered and biologically inactivated. Further, Hg seems to promote the proliferation of lysosomes both in terms of number and dimension that, at the highest level of Hg, resulted in cell death. We report, for the first time, the presence of Hg within the foraminiferal cell: at the basal part of pores, in the organic linings of the foramen/septa, and as cytoplasmic accumulations.

Metabolic and cytoprotective effects of in vivo peri-patellar Hyaluronic Acid injections in cultured tenocytes

Abstract The purpose of this study was to investigate tenocyte mechanobiology after sudden-detraining and to examine the hypothesis that repeated peri-patellar injections of hyaluronic acid (HA) on detrained patellar tendon (PT) may reduce and limit detrained-associated damage in tenoctyes. Twenty-four male Sprague–Dawley rats were divided into three groups: Untrained, Trained and Detrained. In the Detrained rats, the left tendon was untreated while the right tendon received repeated peri-patellar injections of either HA or saline (NaCl). Tenocyte morphology, metabolism and synthesis of C-terminal-propeptide of type I collagen, collagen-III, fibronectin, aggrecan, tenascin-c, interleukin-1β, matrix-metalloproteinase-1 and-3 were evaluated after 1, 3, 7 and 10 days of culture. Transmission-electronic-microscopy showed a significant increase in mitochondria and rough endoplasmic reticulum in cultured tenocytes from Detrained-HA with respect to those from Detrained-NaCl. Additionally, Detrained-HA cultures showed a significantly higher proliferation rate and viability, and increased synthesis of C-terminal-Propeptide of type I collagen, fibronectin, aggrecan, tenascin-c and matrix-metalloproteinase-3 with respect to Detrained-NaCl ones, whereas synthesis of matrix-metalloproteinase-1 and interleukin-1β was decreased. Our study demonstrates that discontinuing training activity in the short-term alters tenocyte synthetic and metabolic activity and that repeated peri-patellar infiltrations of HA during detraining allow the maintenance of tenocyte anabolic activity.

Morphological adaptation and protein modulation of myotendinous junction following moderate aerobic training.

Myotendinous junction is the muscle-tendon interface through which the contractile force can be transferred from myofibrils to the tendon extracellular matrix. At the ultrastructural level, aerobic training can modify the distal myotendinous junction of rat gastrocnemius, increasing the contact area between tissues. The aim of this work is to investigate the correlation between morphological changes and protein modulation of the myotendinous junction following moderate training. For this reason, talin, vinculin and type IV collagen amount and spatial distribution were investigated by immunohistochemistry and confocal microscopy. The images were then digitally analyzed by evaluating fluorescence intensity. Morphometric analysis revealed a significant increased thickening of muscle basal lamina in the trained group (53.1 ± 0.4 nm) with respect to the control group (43.9 ± 0.3 nm), and morphological observation showed the presence of an electron-dense area in the exercised muscles, close to the myotendinous junction. Protein concentrations appeared significantly increased in the trained group (talin +22.2%; vinculin +22.8% and type IV collagen +11.8%) with respect to the control group. Therefore, our findings suggest that moderate aerobic training induces/causes morphological changes at the myotendinous junction, correlated to the synthesis of structural proteins of the muscular basal lamina and of the cytoskeleton.

Effect of Different Exercise Intensities on the Myotendinous Junction Plasticity.

Myotendinous junctions (MTJs) are anatomical regions specialized in transmission of contractile strength from muscle to tendon and, for this reason, a common site where acute injuries occur during sport activities. In this work we investigated the influence of exercise intensity on MTJ plasticity, as well as on the expression of insulin-like growth factor 1 (IGF-1) and transforming growth factor beta (TGF-β) and their receptors in muscle and tendon. Three groups of rats were analyzed: control (CTRL), slow-runner (RUN-S) and fast-runner (RUN-F) trained using a treadmill. Ultrastructural and morphometric analyses of distal MTJs from extensor digitorum longus muscles have been performed. Contractile strength and hypertrophy were investigated by using in vivo tension recordings and muscle cross-sectional area (CSA) analysis, respectively. mRNA levels of PGC-1α, vinculin, IGF-1Ea and TGF-β have been quantified in muscle belly, while IGF-1Ea, TGF-β and their receptors in tendon. Morphometry revealed an increased MTJ complexity and interaction surface between tissues in trained rats according to training intensity. CSA analysis excluded hypertrophy among groups, while muscle strength was found significantly enhanced in exercised rats in comparison to controls. In muscle tissue, we highlighted an increased mRNA expression of PGC-1α and vinculin in both trained conditions and of TGF-β in RUN-F. In tendon, we mainly noted an enhancement of TGF-β mRNA expression only in RUN-F group and a raise of Betaglycan tendon receptor mRNA levels proportional to exercise intensity. In conclusion, MTJ plasticity appears to be related to exercise intensity and molecular analysis suggests a major role played by TGF-β.

Ultrastructural study of myotendinous junction plasticity: from disuse to exercise

In the musculoskeletal system, the interface between muscle and tendon, called the myotendinous junction, has the key role of transferring the contractile strength from muscular belly to its tendon. At the ultrastructural level, the myotendinous junction is characterized by tendon finger-like processes that penetrate into the muscle mass, amplifying the interaction between tissues. For many years, this anatomical region has been considered a passive interface and researchers’ attention was focused on muscle and tendon as independent structures. On the contrary, the plasticity of the myotendinous junction in response to different physiological or pathological conditions has been revealed and these changes appear at the morphological, structural and functional levels. In this work, the ultrastructural adaptations of the myotendinous junction to different physiological conditions have been described, suggesting possible causes that could control this plasticity. In particular, while muscle atrophy can reduce the contact interface between tissues, training protocols can amplify this area, allowing an improved ability to transfer increased levels of contractile strength. Moreover, given the frequency of muscle injuries at this level, the potential preventive role of exercise is discussed.