Miri Assayag

Long- but not short-term heat acclimation produces an apoptosis-resistant cardiac phenotype: a lesson from heat stress and ischemic/reperfusion insults

Long-term heat acclimation (AC, 30d/34°C) is a phenotypic adaptation leading to increased thermotolerance during heat stress (HS, 2xa0h 41°C). AC also renders protection against ischemic/reperfusion (I/R, 30′ global ischemia/40′ reperfusion) insult via cross-tolerance mechanisms. In contrast to the protected AC phenotype, the onset of acclimation (34°C, AC2d) is characterized by cellular perturbations, suggesting increased susceptibility to HS and I/R insults. In this investigation, we tested the hypothesis that apoptosis resistance is part of the AC repertoire and that, at the initial phase of acclimation (AC2d), cytoprotection is impaired. TUNEL staining and caspase 3 levels in HS and I/R insulted hearts affirmed this hypothesis. To examine the role of the mitochondria in life/death decision in AC2d and 30d AC settings vs. control hearts, we studied the Bcl-2 apoptotic cascade and found increased levels of the anti-apoptotic Bcl-XL and decreased levels of the pro-apoptotic death promoter Bad in hearts from AC2d and AC animals. In these groups, cytochrome c (cyt c) was elevated in the mitochondria and remained unchanged in the cytosol. This adaptation was insufficient to negate apoptosis in AC2d rats. At this early acclimation phase (and in controls), increased caspase 8 activity confirmed activation of the extrinsic (Fas ligand) apoptosis pathway. In conclusion, the elevated Bcl-XL/Bad ratio and decreased cyt c leakage to the cytosol are insufficient to protect the heart and interactions with additional cytoprotective pathways involved in acclimation (elevated HSP70, ROS, and sarcolemmal adaptations to abolish extrinsic apoptosis pathways) are required to induce the apoptosis-resistant AC phenotype.

Mitochondrial performance in heat acclimation—a lesson from ischemia/reperfusion and calcium overload insults in the heart

Long-term heat acclimation (LTHA; 30 days, 34°C) causes phenotypic adaptations that render protection against ischemic/reperfusion insult (I/R, 30 min global ischemia and 40 min reperfusion) via heat acclimation-mediated cross-tolerance (HACT) mechanisms. Short-term acclimation (STHA, 2 days, 34 °C), in contrast, is characterized by cellular perturbations, leading to increased susceptibility to insults. Here, we tested the hypothesis that enhanced mitochondrial respiratory function is part of the acclimatory repertoire and that the 30-day regimen is required for protection via HACT. We subjected isolated hearts and mitochondria from controls (C), STHA, or LTHA rats to I/R, hypoxia/reoxygenation, or Ca2+ overload insults. Mitochondrial function was assessed by measuring O2 consumption, membrane potential (ΔΨm), mitochondrial Ca2+ ([Ca2+]m), ATP production, respiratory chain complex activities, and molecular markers of mitochondrial biogenesis. Our results, combining physiological and biochemical parameters, confirmed that mitochondria from LTHA rats subjected to insults, in contrast to C, preserve respiratory functions (e.g., upon I/R, C mitochondria fueled by glutamate-malate, demonstrated decreases of 81%, 13%, 25%, and 50% in O2/P ratio, ATP production, ΔΨm, and complex I activity, respectively, whereas the corresponding LTHA parameters remained unchanged). STHA mitochondria maintained ΔΨm but did not preserve ATP production. LTHA [Ca2+]m was significantly higher than that of C and STHA and was not affected by the hypoxia/reoxygenation protocol compared with C. Enhanced mitochondrial biogenesis markers, switched-on during STHA coincidentally with enhanced membrane integrity (ΔΨm), were insufficient to confer intact respiratory function upon insult. LTHA was required for respiratory complex I adaptation and HACT. Stabilized higher basal [Ca2+]m and attenuated Ca2+ overload are likely connected to this adaptation.

Heat acclimation memory: do the kinetics of the deacclimated transcriptome predispose to rapid reacclimation and cytoprotection?

Faster reinduction of heat acclimation (AC) after its decline indicates AC memory. Our previous results revealed involvement of epigenetic mechanisms of transcriptional regulation. We hypothesized that the decline of AC (DeAC) is a period of dormant memory during which many processes are alerted to enable rapid reacclimation (ReAC). Using a genomewide approach we studied the AC, DeAC, and ReAC transcriptomes, to uncover hallmark pathways linked to molecular memory in the cardioacclimatome. Fifty rats subjected to heat acclimation [34°C for 2d (AC2d) or 30d (AC30)], DeAC (24°C, 30 days), ReAC (34°C, 2 days), and untreated controls were used. The GeneChip Rat Gene 1.0 ST Array was employed for left ventricular (cardiac) mRNA hybridization. Three independent bioinformatic analyses showed that 1) during AC2d enrichment of DNA impair/repair-linked genes is seen, and this is the molecular on-switch of acclimation; 2) genes activated in AC30 underlie the qualitative physiological adaptations of cardiac performance; 3) particular molecular programs encompassing constitutive upregulation of p38 MAPK, Jak/Stat, and Akt pathways and targets are specifically activated during DeAC and ReAC; and 4) epigenetic markers such as linker histones (histones H1 cluster), associated with nucleosome spacing, transcriptional chromatin modifiers, poly-(ADP-ribose) polymerase-1 (PARP1) linked to chromatin compaction, and microRNAs are only altered during DeAC/ReAC. The latter are newcomers to the AC/DeAC puzzle. We suggest that these transcriptional responses maintain euchromatin and proteostasis and enable faster physiological recovery upon ReAC by rapidly reestablishing the protected acclimated cardiophenotype. We propose that the cardiac AC model can be applied to acclimation processes in general.

Cyclic nitroxide radicals attenuate inflammation and Hyper-responsiveness in a mouse model of allergic asthma.

The effects of stable cyclic nitroxide radicals have been extensively investigated both in vivo and in vitro demonstrating anti-inflammatory, radioprotective, anti-mutagenic, age-retardant, hypotensive, anti-cancer and anti-teratogenic activities. Yet, these stable radicals have not been evaluated in asthma and other airway inflammatory disorders. The present study investigated the effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (TPL) and 3-carbamoyl-proxyl (3-CP) in a mouse model of ovalbumin (OVA)-induced allergic asthma. Both 3-CP and TPL were non-toxic when administered either orally (1% w/w nitroxide-containing chow) or via intraperitoneal (IP) injection (∼300 mg/kg). Feeding the mice orally demonstrated that 3-CP was more effective than TPL in reducing inflammatory cell recruitment into the airway and in suppressing airway hyper-responsiveness (AHR) in OVA-challenged mice. To characterize the optimal time-window of intervention and mode of drug administration, 3-CP was given orally during allergen sensitization, during allergen challenge or during both sensitization and challenge stages, and via IP injection or intranasal instillation for 3 days during the challenge period. 3-CP given via all modes of delivery markedly inhibited OVA-induced airway inflammation, expression of cytokines, AHR and protein nitration of the lung tissue. Oral administration during the entire experiment was the most efficient delivery of 3-CP and was more effective than dexamethasone a potent corticosteroid used for asthma treatment. Under a similar administration regimen (IP injection before the OVA challenge), the effect of 3-CP was similar to that of dexamethasone and even greater on AHR and protein nitration. The protective effect of the nitroxides, which preferentially react with free radicals, in suppressing the increase of main asthmatic inflammatory markers substantiate the key role played by reactive oxygen and nitrogen species in the molecular mechanism of asthma. The present results demonstrate the therapeutic potential of nitroxides for the treatment of asthma.

Extra domain-A fibronectin is necessary for the development of nasal remodeling in chronic allergen-induced rhinitis

BACKGROUNDnExtra domain A-containing fibronectin (EDA-FN) is necessary for the development of allergen-induced lower airway fibrosis. The pathogenesis of fibrosis in allergic rhinitis has not been well studied.nnnOBJECTIVESnTo determine whether EDA-fibronectin is necessary for the development of nasal remodeling in a murine model of chronic allergic rhinitis and in human allergic rhinitis.nnnMETHODSnEDA(-/-) and wild-type (WT) C57Bl/6 mice were sensitized intraperitoneally and then challenged with inhaled ovalbumin (OVA) or saline for 2 and 5 weeks. Clinical signs of rhinitis and histological analysis of nasal tissue were evaluated. Immunohistological staining for EDA-FN was performed in human tissue of inferior nasal conchae from patients with allergic rhinitis and controls.nnnRESULTSnAfter 2 weeks of allergen exposure, only goblet cell hyperplasia and perivascular eosinophilia were observed. After 5 weeks, goblet cell number, thickening of the subepithelial layer, and extent and area of collagen deposition were increased in the nasal tissue of WT OVA (ovalbumin)-challenged mice as compared with saline controls (P < .0001, P < .0001, P = .018, and P = .03, respectively). Clinical signs of rhinitis were observed only in WT OVA-challenged mice. In the EDA(-/-) mice exposed to OVA, collagen deposition, collagen area, and subepithelial thickness showed no increase and were similar to saline control mice, whereas goblet cell hyperplasia was similar to WT OVA-challenged mice. EDA-FN expression was prominent in inferior conchae from patients with allergic rhinitis but was absent in control patients.nnnCONCLUSIONnEDA-containing fibronectin is necessary for the development of nasal tissue fibrotic remodeling process in both murine and human allergic rhinitis.

Pyruvate dehydrogenase has a major role in mast cell function, and its activity is regulated by mitochondrial microphthalmia transcription factor

Background We have recently observed that oxidative phosphorylation–mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. Objective We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. Methods Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. Results PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. Conclusion The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases. Graphical abstract Figure. No Caption available.

Lung Injury Repair by Transplantation of Adult Lung Cells Following Preconditioning of Recipient Mice

Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6–8 weeks post‐transplant, with donor derived structures including epithelial, endothelial, and mesenchymal cells. Epithelial cells within these donor‐derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about threefold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Our study provides proof of concept for lung regeneration by adult lung cells after preconditioning to vacate the pulmonary niche. Stem Cells Translational Medicine 2018;7:68–77

The effect of omalizumab treatment on the low affinity immunoglobulin E receptor (CD23/fc epsilon RII) in patients with severe allergic asthma

BACKGROUNDnOmalizumab is an anti-immunoglobulin E (IgE) monoclonal antibody used in the treatment of severe asthma. Its therapeutic efficacy is primarily attributed to reduction of serum-free IgE and in the expression of high-affinity IgE receptor, fc epsilon RI. However, its effect on the low-affinity IgE receptor fc epsilon RII/CD23 in vivo has not been evaluated.nnnAIMnTo determine whether CD23 plays a role in the inflammatory process in severe uncontrolled asthma and whether anti-IgE therapy modulates fc epsilon RII/CD23 expression in these patients.nnnMETHODSnWe evaluated the expression of IgE receptors fc epsilon RI, fc epsilon RII/CD23, and soluble CD23 (sCD23), and the activation state of peripheral blood monocytes (tumor necrosis factor alpha, interleukin (IL) 1-beta, transforming growth factor (TGF) beta expression) in the patients with severe asthma before and after 24 weeks of omalizumab treatment and in the healthy controls. Cytokine expression of monocytes in response to different stimulation (IL-4, IL-4 plus IgE, IL-4 plus IgE plus anti-IgE, and IL-4 plus IgE plus anti-IgE plus anti-CD23 for 72 hours) was determined by enzyme-linked immunosorbent assay.nnnRESULTSnTreatment with omalizumab (for 24 weeks) improved disease control and pulmonary function (forced expiratory volume in the first second of expiration, 64.5 versus 74%; p = 0.021). Mean ± SE expression of fc epsilon RI on monocytes was higher in the patients with asthma versus the controls (45.7 ± 12.2% versus 18.6 ± 5.8%; p = 0.04) and was reduced after omalizumab treatment (45.7 ± 12.2% versus 15.6 ± 4.4%; p = 0.027). Mean ± SE TGF-beta levels in supernatants from monocytes were reduced in the patients treated with omalizumab (211 ± 6 pg/mL versus 184 ± 9 pg/mL; p = 0.036).nnnCONCLUSIONnModulation of the low affinity IgE receptor CD23 in severe asthma is complex, and sCD23 may inversely reflect disease activity. Treatment with omalizumab was associated with reduced monocyte activation.

Modulation of allergic responses by mitochondrial STAT3 inhibitors

Recently, we have shown that mast cell mitochondrial STAT3 could serve as a new target for the regulation of the allergic response as it plays an essential role in immunologically mediated degranulation of mast cells. In the present work, we explored how two recently developed mitochondrial STAT3 inhibitors (Mitocur‐1 and Mitocur‐3) modulate the allergic response.

The association between osteopontin gene polymorphisms, osteopontin expression and sarcoidosis

Background Sarcoidosis is a systemic inflammatory disease of unknown etiology. Osteopontin (SPP1, OPN) is an extra cellular matrix glycoprotein and cytokine with a known role in granuloma formation and in autoimmune and inflammatory diseases. Objective To determine whether plasma OPN levels are elevated in patients with sarcoidosis and compare the frequency of four single nucleotide polymorphism (SNPs) variants in the OPN gene in sarcoidosis patients compared to healthy controls. Methods Demographic and clinical information, radiological studies and pulmonary function tests were evaluated in 113 patients with sarcoidosis and in 79 healthy controls. Blood samples were analyzed for SNPs of the OPN gene and for plasma OPN and CRP levels. Association between clinical features of disease and OPN levels as well as SNP frequencies was determined. Results Plasma OPN levels were higher in sarcoidosis patients than in healthy subjects, (median: 217 vs 122ng/ml, p<0.001). Area under the curve for receiver operator curves (ROC) was 0.798 (0.686–0.909 95% CI.) No differences were observed between sarcoidosis patients and controls in the frequency of any of the SNPs evaluated. Presence of lung parenchymal involvement was associated with SNP distribution at rs1126772 (p = 0.02). We found no correlation between SNPs distribution and plasma OPN levels. Conclusions Osteopontin protein levels are elevated in sarcoidosis. We found no evidence for an association between SNPs on the osteopontin gene and plasma OPN levels or the presence of sarcoidosis, however, an association between genotype and several phenotypic clinical parameters of disease was observed.