Acute reperfusion intramyocardial hemorrhage leads to regional chronic iron deposition in the heart

Intramyocardial hemorrhage commonly occurs in large reperfused myocardial infarctions. However, its long-term fate remains unexplored. We hypothesized that acute reperfusion intramyocardial hemorrhage leads to chronic iron deposition

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 pM of NO donor diethylenetriamine NONOate (DETANO) for 24 h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 uM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR Furthermore, DETANO stimulated Ala anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions

Regulation of AMPA receptor function and synaptic localization by stargazin and PSD-95

The majority of excitatory transmission in the brain is mediated by glutamatergic synapses. Rapid synaptic signaling is mediated by AMPA and kainate receptors, whereas NMDA receptors mediate slow synaptic currents. Pathophysiological activation of glutamatergic neurons can lead to excitotoxicity and neuronal death, for example in ischaemia and neurodegenerative disorders. Therefore, studying the structure and function of AMPA receptors is important for understanding general mechanisms of synaptic transmission as well as for the development of new therapies. AMPA receptors are associated with auxiliary subunits called Transmembrane AMPA Receptor Regulatory Proteins (TARPs). The first identified member of this family was stargazin. Given the structural similarity to the γ1 subunit of skeletal muscle voltage-gated Ca2+channels, stargazin is also called γ2. The stargazer mouse is a spontaneous mutant that lacks AMPA receptors in granule cells of cerebellum and suffers from ataxia. In addition to stargazin, the family includes γ3, γ4 and γ8. TARPs regulate all aspects of AMPA receptor function - from early steps of synthesis and trafficking to the cell surface, to synaptic localization and biophysical properties. TARPs interact with PSD-95, a main scaffolding protein of excitatory synapses that belongs to the Membrane-Associated Guanylate Kinases (MAGUK) family. Via this interaction AMPA receptors are localized to the synapse. PSD-95 clusters many other synaptic proteins and organizes signaling complexes in the synapse. The goal of this thesis was to investigate the role of stargazin in regulating the antagonism of AMPA receptors. I focused on the commonly used antagonists CNQX, GYKI-53655 (GYKI) and CP-465,022 (CP) and explored how stargazin changes the inhibition of AMPA receptors by these drugs. The second goal was to assess the role of PSD-95 in synaptic function. More specifically, I aimed to investigate how an increased level of PSD-95 in a neuron affects AMPA and NMDA currents, as well as the presynaptic function of a neuron. In the first part of my thesis I used the heterologous Xenopus oocyte expression system to express AMPA receptor subunits alone or with stargazin. Using the two-electrode voltage clamp, I measured the glutamate-evoked currents and obtained dose-response curves for CNQX, GYKI and CP. I found that stargazin decreases the affinity of GluR1 for CNQX, which was explained by the partial agonistic effect of CNQX in the presence of stargazin. In contrast, stargazin increases the affinity for GYKI, and has only a small effect on CP. I also tested the effect of stargazin on recently described GYKI-insensitive receptors and found that inhibition of these receptors is restored by co-expression with stargazin. My data strongly suggest that the identified residues do not constitute the full GYKI-binding site. I could also show that the ectodomain of stargazin controls the changes in antagonist sensitivity of the receptors. In the second part of my thesis I used cultured hippocampal slices and Semliki Forest virus to overexpress PSD-95:GFP in CA1 region of hippocampus. I recorded simultaneously from a cell overexpressing PSD-95 and a neighboring control cell and compared their AMPA and NMDA currents. I confirmed the finding that overexpression of PSD-95 robustly increases currents mediated by AMPA receptors. In contrast to other studies, I observed that PSD-95 increases NMDA currents, although to smaller extent. I addressed the debated role of PSD-95 in regulating the presynatic release probability and found that overexpression of PSD-95 did not change glutamate release probability. Importantly, I observed that cells overexpressing PSD-95 have a lower rectification index of synaptic AMPA receptors, strongly suggesting that PSD-95 overexpression led to an increased fraction of AMPA receptors that lack GluR2 subunit. In conclusion, the work presented in this thesis gives further insights into AMPA receptor physiology, both from the aspect of pharmacology and synaptic trafficking. The results of co-expression of stargazin with the previously described GYKI-insensitive GluR1 mutants strongly indicate that TARP interacts with the linker domains of AMPA receptors. This finding is of great importance for understanding the molecular mechanism of AMPA-TARP interaction. Furthermore, this thesis shows that PSD-95 regulates both AMPA and NMDA synaptic currents by increasing the number of synaptic receptors. In addition, my data suggest that PSD-95 enriches the number of GluR2-lacking receptors in the synapse. Given the Ca2+permeability of GluR2-lacking receptors and their implication in plasticity and excitotoxicity, this finding is important for understanding how the synaptic localization of these receptors is regulated

TRESK channel contribution to nociceptive sensory neurons excitability: modulation by nerve injury

Background: Neuronal hyperexcitability is a crucial phenomenon underlying spontaneous and evoked pain. In invertebrate nociceptors, the S-type leak K(+) channel (analogous to TREK-1 in mammals) plays a critical role of in determining neuronal excitability following nerve injury. Few data are available on the role of leak K(2P) channels after peripheral axotomy in mammals. Results: Here we describe that rat sciatic nerve axotomy induces hyperexcitability of L4-L5 DRG sensory neurons and decreases TRESK (K2P18.1) expression, a channel with a major contribution to total leak current in DRGs. While the expression of other channels from the same family did not significantly change, injury markers ATF3 and Cacna2d1 were highly upregulated. Similarly, acute sensory neuron dissociation (in vitro axotomy) produced marked hyperexcitability and similar total background currents compared with neurons injured in vivo. In addition, the sanshool derivative IBA, which blocked TRESK currents in transfected HEK293 cells and DRGs, increased intracellular calcium in 49% of DRG neurons in culture. Most IBA-responding neurons (71%) also responded to the TRPV1 agonist capsaicin, indicating that they were nociceptors. Additional evidence of a biological role of TRESK channels was provided by behavioral evidence of pain (flinching and licking), in vivo electrophysiological evidence of C-nociceptor activation following IBA injection in the rat hindpaw, and increased sensitivity to painful pressure after TRESK knockdown in vivo. Conclusions: In summary, our results clearly support an important role of TRESK channels in determining neuronal excitability in specific DRG neurons subpopulations, and show that axonal injury down-regulates TRESK channels, therefore contributing to neuronal hyperexcitability

Iron Deposition following Chronic Myocardial Infarction as a Substrate for Cardiac Electrical Anomalies: Initial Findings in a Canine Model

Purpose: Iron deposition has been shown to occur following myocardial infarction (MI). We investigated whether such focal iron deposition within chronic MI lead to electrical anomalies. Methods: Two groups of dogs (ex-vivo (n = 12) and in-vivo (n = 10)) were studied at 16 weeks post MI. Hearts of animals from ex-vivo group were explanted and sectioned into infarcted and non-infarcted segments. Impedance spectroscopy was used to derive electrical permittivity () and conductivity (). Mass spectrometry was used to classify and characterize tissue sections with (IRON+) and without (IRON-) iron. Animals from in-vivo group underwent cardiac magnetic resonance imaging (CMR) for estimation of scar volume (late-gadolinium enhancement, LGE) and iron deposition (T2*) relative to left-ventricular volume. 24-hour electrocardiogram recordings were obtained and used to examine Heart Rate (HR), QT interval (QT), QT corrected for HR (QTc) and QTc dispersion (QTcd). In a fraction of these animals (n = 5), ultra-high resolution electroanatomical mapping (EAM) was performed, co-registered with LGE and T2* CMR and were used to characterize the spatial locations of isolated late potentials (ILPs). Results: Compared to IRON- sections, IRON+ sections had higher, but no difference in. A linear relationship was found between iron content and (p1.5%)) with similar scar volumes (7.28%±1.02% (Iron (1.5%)), p = 0.51) but markedly different iron volumes (1.12%±0.64% (Iron (1.5%)), p = 0.02), QT and QTc were elevated and QTcd was decreased in the group with the higher iron volume during the day, night and 24-hour period (p<0.05). EAMs co-registered with CMR images showed a greater tendency for ILPs to emerge from scar regions with iron versus without iron. Conclusion: The electrical behavior of infarcted hearts with iron appears to be different from those without iron. Iron within infarcted zones may evolve as an arrhythmogenic substrate in the post MI period

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Interaction of rat alveolar macrophages with dental composite dust

Background: Dental composites have become the standard filling material to restore teeth, but during the placement of these restorations, high amounts of respirable composite dust (<5 mu m) including many nano-sized particles may be released in the breathing zone of the patient and dental operator. Here we tested the respirable fraction of several composite particles for their cytotoxic effect using an alveolar macrophage model system. Methods: Composite dust was generated following a clinical protocol, and the dust particles were collected under sterile circumstances. Dust was dispersed in fluid, and 5-mu m-filtered to enrich the respirable fractions. Quartz DQ12 and corundum were used as positive and negative control, respectively. Four concentrations (22.5 mu g/ml, 45 mu g/ml, 90 mu g/ml and 180 mu g/ml) were applied to NR8383 alveolar macrophages. Light and electron microscopy were used for subcellular localization of particles. Culture supernatants were tested for release of lactate dehydrogenase, glucuronidase, TNF-alpha, and H2O2. Results: Characterization of the suspended particles revealed numerous nano-sized particles but also many high volume particles, most of which could be removed by filtering. Even at the highest concentration (180 mu g/ml), cells completely cleared settled particles from the bottom of the culture vessel. Accordingly, a mixture of nano- and micron-scaled particles was observed inside cells where they were confined to phagolysosomes. The filtered particle fractions elicited largely uniform dose-dependent responses, which were elevated compared to the control only at the highest concentration, which equaled a mean cellular dose of 120 pg/cell. A low inflammatory potential was identified due to dose-dependent release of H2O2 and TNF-alpha. However, compared to the positive control, the released levels of H2O2 and TNF-alpha were still moderate, but their release profiles depended on the type of composite. Conclusions: Alveolar macrophages are able to phagocytize respirable composite dust particle inclusive nanoparticles. Since NR8383 cells tolerate a comparatively high cell burden (60 pg/cell) of each of the five materials with minimal signs of cytotoxicity or inflammation, the toxic potential of respirable composite dust seems to be low. These results are reassuring for dental personnel, but more research is needed to characterize the actual exposure and uptake especially of the pure nano fraction