Inhalation of Silver Silicate Nanoparticles Leads to Transient and Differential Microglial Activation in the Rodent Olfactory Bulb.

Engineered silver nanoparticles (AgNPs), including silver silicate nanoparticles (Ag-SiO2 NPs), are used in a wide variety of medical and consumer applications. Inhaled AgNPs have been found to translocate to the olfactory bulb (OB) after inhalation and intranasal instillation. However, the biological effects of Ag-SiO2 NPs and their potential nose-to-brain transport have not been evaluated. The present study assessed whether inhaled Ag-SiO2 NPs can elicit microglial activation in the OB. Adult Sprague-Dawley rats inhaled aerosolized Ag-SiO2 NPs at a concentration of 1 mg/ml for 6 hours. On day 0, 1, 7, and 21 post-exposure, rats were necropsied and OB were harvested. Immunohistochemistry on OB tissues were performed with anti-ionized calcium-binding adapter molecule 1 and heme oxygenase-1 as markers of microglial activation and oxidative stress, respectively. Aerosol characterization indicated Ag-SiO2 NPs were sufficiently aerosolized with moderate agglomeration and high-efficiency deposition in the nasal cavity and olfactory epithelium. Findings suggested that acute inhalation of Ag-SiO2 NPs elicited transient and differential microglial activation in the OB without significant microglial recruitment or oxidative stress. The delayed and differential pattern of microglial activation in the OB implied that inhaled Ag-SiO2 may have translocated to the central nervous system via intra-neuronal pathways.

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis.

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood-brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1ß, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-ß. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.

A Coumarin-Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1.

Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin-porphyrin FRET break-apart probe to detect HO-1 activity, Fe-L1. We designed Fe-L1 to "break-apart" upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC-MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe-L2, we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.

P-selectin deficiency promotes liver senescence in sickle cell disease mice.

Sickle cell disease (SCD) is caused by a homozygous mutation in the ß-globin gene, which leads to erythrocyte sickling, vasoocclusion, and intense hemolysis. P-selectin inhibition has been shown to prevent vasoocclusive events in patients with SCD; however, the chronic effect of P-selectin inhibition in SCD remains to be determined. Here, we used quantitative liver intravital microscopy in our recently generated P-selectin-deficient SCD mice to show that chronic P-selectin deficiency attenuates liver ischemia but fails to prevent hepatobiliary injury. Remarkably, we find that this failure in resolution of hepatobiliary injury in P-selectin-deficient SCD mice is associated with the increase in cellular senescence and reduced epithelial cell proliferation in the liver. These findings highlight the importance of investigating the long-term effects of chronic P-selectin inhibition therapy on liver pathophysiology in patients with SCD.

Characterization and heme oxygenase-1 content of extracellular vesicles in human biofluids.

Heme oxygenase-1 (HO-1), a highly inducible stress protein that degrades heme to biliverdin, carbon monoxide, and free ferrous iron, is increased in blood and other biofluids of subjects with various systemic and neurological disorders. HO-1 does not contain an N-terminal signal peptide and the mechanism responsible for its secretion remains unknown. Extracellular vesicles (EVs) are membrane-bound inclusions that transport microRNAs, messenger RNAs, lipids, and proteins among diverse cellular and extracellular compartments. The objective of the current study was to determine whether EVs in human biofluids contain HO-1, and whether the latter may be transported in EVs from brain to periphery. Total, L1 cell adhesion molecule protein (L1CAM)-enriched (neuron-derived), and glutamate aspartate transporter 1 (GLAST)-enriched (astrocyte-derived) EVs were purified from five different human biofluids (saliva [n = 40], plasma [n = 14], serum [n = 10], urine [n = 10], and cerebrospinal fluid [n = 11]) using polymer precipitation and immuno-affinity-based capture methods. L1CAM-enriched, GLAST-enriched, and L1CAM/GLAST-depleted (LGD) EV, along with EV-depleted (EVD), fractions were validated by nanoparticle tracking analysis, enzyme-linked immunosorbent assay (ELISA), and western blot. HO-1 was assayed in all fractions using ELISA and western blot. The majority of HO-1 protein was localized to LGD, L1CAM-enriched, and GLAST-enriched EVs of all human biofluids surveyed after adjusting for age and sex, with little HO-1 protein detected in EVD fractions. HO-1 protein in human biofluids is predominantly localized to EV compartments. A substantial proportion of EV HO-1 in peripheral human biofluids is derived from the central nervous system and may contribute to the systemic manifestations of various neurological conditions.

miR-183-5p alleviates early injury after intracerebral hemorrhage by inhibiting heme oxygenase-1 expression.

Differences in microRNA (miRNA) expression after intracerebral hemorrhage (ICH) have been reported in human and animal models, and miRNAs are being investigated as a new treatment for inflammation and oxidative stress after ICH. In this study, we found that microRNA-183-5p expression was decreased in the mouse brain after ICH. To investigate the effect of miRNA-183-5p on injury and repair of brain tissue after ICH, saline, miRNA-183-5p agomir, or miRNA-183-5p antagomir were injected into the lateral ventricles of 8-week-old mice with collagenase-induced ICH. Three days after ICH, mice treated with exogenous miRNA-183-5p showed less brain edema, neurobehavioral defects, inflammation, oxidative stress, and ferrous deposition than control mice. In addition, by alternately treating mice with a heme oxygenase-1 (HO-1) inducer, a HO-1 inhibitor, a nuclear factor erythroid 2-related factor (Nrf2) activator, and Nrf2 knockout, we demonstrated an indirect, HO-1-dependent regulatory relationship between miRNA-183-5p and Nrf2. Our results indicate that miRNA-183-5p and HO-1 are promising therapeutic targets for controlling inflammation and oxidative damage after hemorrhagic stroke.

KIT ligand protects against both light-induced and genetic photoreceptor degeneration.

Photoreceptor degeneration is a major cause of blindness and a considerable health burden during aging but effective therapeutic or preventive strategies have not so far become readily available. Here, we show in mouse models that signaling through the tyrosine kinase receptor KIT protects photoreceptor cells against both light-induced and inherited retinal degeneration. Upon light damage, photoreceptor cells upregulate Kit ligand (KITL) and activate KIT signaling, which in turn induces nuclear accumulation of the transcription factor NRF2 and stimulates the expression of the antioxidant gene Hmox1. Conversely, a viable Kit mutation promotes light-induced photoreceptor damage, which is reversed by experimental expression of Hmox1. Furthermore, overexpression of KITL from a viral AAV8 vector prevents photoreceptor cell death and partially restores retinal function after light damage or in genetic models of human retinitis pigmentosa. Hence, application of KITL may provide a novel therapeutic avenue for prevention or treatment of retinal degenerative diseases.

Picroside II attenuates ischemia/reperfusion testicular injury by alleviating oxidative stress and apoptosis through reducing nitric oxide synthesis.

PURPOSE: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. METHODS: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. RESULTS: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). CONCLUSION: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.

Propofol vs desflurane on the cytokine, matrix metalloproteinase-9, and heme oxygenase-1 response during living donor liver transplantation: A pilot study.

BACKGROUND: We investigated the effects of propofol vs desflurane on ischemia and reperfusion injury (IRI)-induced inflammatory responses, especially in matrix metalloproteinase-9 (MMP-9) downregulation and heme oxygenase-1 (HO-1) upregulation, which may result in different clinical outcomes in liver transplant recipients. METHODS: Fifty liver transplant recipients were randomized to receive propofol-based total intravenous anesthesia (TIVA group, n = 25) or desflurane anesthesia (DES group, n = 25). We then measured the following: perioperative serum cytokine concentrations (interleukin 1 receptor antagonist [IL-1RA], IL-6, IL-8, and IL-10); MMP-9 and HO-1 mRNA expression levels at predefined intervals. Further, postoperative outcomes were compared between the 2 groups. RESULTS: The TIVA group showed a significant HO-1 level increase following the anhepatic phase and a significant MMP-9 reduction after reperfusion, in addition to a significant increase in IL-10 levels after the anhepatic phase and IL-1RA levels after reperfusion. Compared to DES patients, TIVA patients showed a faster return of the international normalized ratio to normal values, lower plasma alanine aminotransferase concentrations 24 hours after transplantation, and fewer patients developing acute lung injury. Moreover, compared with DES patients, TIVA patients showed a significant reduction in serum blood lactate levels. However, there were no differences in postoperative outcomes between the two groups. CONCLUSION: Propofol-based TIVA attenuated inflammatory response (elevated IL-1RA and IL-10 levels), downregulated MMP-9 response, and increased HO-1 expression with improved recovery of graft function and better microcirculation compared with desflurane anesthesia in liver transplant recipients.

Picroside II attenuates ischemia/reperfusion testicular injury by alleviating oxidative stress and apoptosis through reducing nitric oxide synthesis

Abstract Purpose: To investigate the effect of Picroside II on testicular ischemia and reperfusion (l/R) injury and the underlying mechanism. Methods: Sprague-Dawley rats were randomly divided into 4 groups: sham operated group (Sham), Sham with Picroside II treatment group (Sham+ Pic II), l/R group (l/R) and l/R with Picroside II treatment group (I/R+ Pic II). l/R model was established by rotating the left testis 720° in a clock-wise direction for 4 hours. The histopathologic and spermatogenetic evaluation was performed. The apoptosis changes and the levels of HO-1 (heme oxygenase-1), MPO (myeloperoxidase), NOX (NADPH oxidase), SOD (superoxide dismutase), XO (xanthine oxidase) and NOS (nitric oxide synthase) were measured. Results: The seminiferous tubules were damaged in l/R rats, but Picroside II alleviated the changes induced by l/R. The increased level of apoptosis was decreased by Picroside II (P=0.01, 9.05±0.35 vs. 4.85±0.25). The activities of HO-1, MPO, NOX, XO and MDA content were increased and the SOD activity was decreased in l/R (P<0.05) and could be reversed by Picroside II (P=0.03, 405.5±7.5 vs. 304±17U/mgprot; P=0.02, 0.99±0.05 vs. 0.52±0.04 mgprot; P=0.01, 260+7 vs. 189±2 mgprot; P=0.04, 10.95+0.55 vs. 8.75+0.35 U/mgprot; P=0.045, 6.8+0.7 vs. 3.75+0.35 mgprot; P=0.04, 44.5+3.5 vs. 57.5+3.5 mgprot). Western blot showed that the expression of iNOS, nNOS and eNOS were increased in l/R (P<0.05); however, they were decreased after Picroside II treatment (P<0.05). Conclusion: Picroside II attenuated testicular I/R injury in rats mainly through suppressing apoptosis and oxidative stress through reduction of nitric oxide synthesis.

Protective effect of lactobacillus plantarum on alcoholic liver injury and regulating of keap-Nrf2-ARE signaling pathway in zebrafish larvae.

This research investigated the protective effect of lactobacillus plantarum against alcohol-induced liver injury and the regulatory mechanism of Keap-Nrf2-ARE signal pathway in zebrafish. Firstly, a zebrafish alcoholic liver injury model was established using1.0mM of ethanol concentration, then two forms of lactobacillus plantarum treatment were designed to perform repair, including a lactobacillus plantarum thallus suspension (LPS) and a lactobacillus plantarum thallus breaking solution (LPBS). After 24h of alcohol injury, lactobacillus plantarum concentrations of 0, 1.0×105, 1.0×106, 1.0×107 and 1.5×107 cfu/mL were added to protect zebrafish larvae. Then with the treatment of lactobacillus plantarum after 48h, activities of alanine transaminase (ALT), aspartate transaminase (AST), superoxide dismutase (SOD) and malondialdehyde (MDA) in zebrafish tissue homogenate were respectively determined. Keap-Nrf2-ARE signal pathway related gene expression conditions were also analyzed, including nuclear factor (erythroid-derived 2)-like 2(Nrf2), Kelch like ECH associated protein 1(Keap1), catalase(CAT), hemooxygenase1(HO1) and Glutathione S-Transferase Kappa 1(gstk1). Results showed that: in comparison with the control group, the LPBS with dosage of 1.0×107 cfu/mL remarkably improved the activities of SOD, CAT, HO1and gstk1 in zebrafish larvae liver (P<0.05), resulting in significant increase of the protein expression level of Nrf2 (225.78%) and suppression of Keap1 gene expression (73.67%)(P<0.01). As confirmed by the results, lactobacillus plantarum activated the Keap-Nrf2-ARE signal pathway from the level of transcription, the up-regulation of the expression quantity of Nrf2 protected the organism from oxidative stress and maximally reduced liver injury.

In Vivo Comparative Study on Acute and Sub-acute Biological Effects Induced by Ultrafine Particles of Different Anthropogenic Sources in BALB/c Mice.

Exposure to ultrafine particles (UFPs) leads to adverse effects on health caused by an unbalanced ratio between UFPs deposition and clearance efficacy. Since air pollution toxicity is first direct to cardiorespiratory system, we compared the acute and sub-acute effects of diesel exhaust particles (DEP) and biomass burning-derived particles (BB) on bronchoalveolar Lavage Fluid (BALf), lung and heart parenchyma. Markers of cytotoxicity, oxidative stress and inflammation were analysed in male BALB/c mice submitted to single and repeated intra-tracheal instillations of 50 µg UFPs. This in-vivo study showed the activation of inflammatory response (COX-2 and MPO) after exposure to UFPs, both in respiratory and cardiovascular systems. Exposure to DEP results also in pro- and anti-oxidant (HO-1, iNOS, Cyp1b1, Hsp70) protein levels increase, although, stress persist only in cardiac tissue under repeated instillations. Statistical correlations suggest that stress marker variation was probably due to soluble components and/or mediators translocation of from first deposition site. This mechanism, appears more important after repeated instillations, since inflammation and oxidative stress endure only in heart. In summary, chemical composition of UFPs influenced the activation of different responses mediated by their components or pro-inflammatory and pro-oxidative molecules, indicating DEP as the most damaging pollutant in the comparison.

Pre-treatment with FK506 reduces hepatic ischemia-reperfusion injury in rats.

AIM: The study is aimed to investigate the protective effects and possible mechanism of tacrolimus (FK506) pre-treatment in hepatic ischemia-reperfusion injury in rats. METHODS: The rats were randomly assigned into four groups, which were S, IR, L and H group, and then all groups were subjected to 60min of 70% partial warm liver ischemia, except S group. Rats in the L and H group were pre-treated with two different doses FK506 at 60min before ischemia. The rats of the IR group received an identical volume of normal saline. All animals were sacrificed after 6h of reperfusion. Transaminases were measured by biochemistry analyzer. Elisa kit was used to detect TNF-α, IL-6 and IL-1ß levels in serum. Liver specimens were stained with hematoxylin and eosin (HE) to assess the pathologic changes. The expressions of heme oxygenase-1 (HO-1), hypoxia-inducible factor-1α (HIF-1α), nuclear factor of activated T cells (NFAT3) were measured by real-time quantitative PCR and western blotting and the Bcl-2 and the Bax protein were tested by western blotting. RESULTS: In rats pre-treated with FK506, the levels of transaminases, TNF-α and IL-1ß were reduced significantly and also liver damage was dramatically mitigated compared to those without FK506 pre-treatment. Moreover, the expression of HO-1 at the level of both transcription and translation increased clearly and the activation of the HIF-1α was found in FK506 pre-treated livers. Moreover, NFAT3 protein transportation to the nucleus was reduced and Bax protein expression was decreased, but the expression of Bcl-2 protein was markedly increased after FK506 pre-treatment. CONCLUSION: FK506 pre-treatment could lessen hepatic ischemia-reperfusion injury through up-regulating the expression of HIF-1α and HO-1, and inhibiting nuclear translocation of NFAT3 in liver tissues.

A fine-needle aspiration-based protein signature discriminates benign from malignant breast lesions.

There are increasing demands for informative cancer biomarkers, accessible via minimally invasive procedures, both for initial diagnostics and to follow-up personalized cancer therapy. Fine-needle aspiration (FNA) biopsy provides ready access to relevant tissues; however, the minute sample amounts require sensitive multiplex molecular analysis to achieve clinical utility. We have applied proximity extension assays (PEA) and NanoString (NS) technology for analyses of proteins and of RNA, respectively, in FNA samples. Using samples from patients with breast cancer (BC, n = 25) or benign lesions (n = 33), we demonstrate that these FNA-based molecular analyses (a) can offer high sensitivity and reproducibility, (b) may provide correct diagnosis in shorter time and at a lower cost than current practice, (c) correlate with results from routine analysis (i.e., benchmarking against immunohistochemistry tests for ER, PR, HER2, and Ki67), and (d) may also help identify new markers related to immunotherapy. A specific 11-protein signature, including FGF binding protein 1, decorin, and furin, distinguished all cancer patient samples from all benign lesions in our main cohort and in smaller replication cohort. Due to the minimally traumatic sampling and rich molecular information, this combined proteomics and transcriptomic methodology is promising for diagnostics and evaluation of treatment efficacy in BC.

Protective effects of hyperbaric oxygen preconditioning against LPS-induced acute lung injury in rats.

INTRODUCTION: Acute lung injury (ALI) is generally caused by oxidative damages and pulmonary overinflammations. Hyperbaric oxygen preconditioning (HBO2-PC) has been proven protective against oxidative-stress-related injuries. In this study, we investigated the effect of HBO2-PC on lipopolysaccharide (LPS)-induced ALI in rats. METHODS: Thirty-two Sprague-Dawley rats randomly assigned into Sham, HBO2-PC, ALI and HBO2-PC÷ALI groups (eight in each group) were sacrificed at 12 hours after the injection of LPS. The severity of ALI in rats was assessed in terms of histopathological changes in addition to wet/dry weight ratios. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß in serum and lung homogenates were measured by enzyme-linked immunosorbent and qRT-PCR assays. Activities by hydrogen peroxide (H2O2), malondialdehyde (MDA), myeloperoxidase (MPO) as well as superoxide dismutase (SOD) in rat lungs were tested for neutrophil infiltration. Meanwhile the oxidative stress molecular markers nuclear factor-kappa B(NF-κB) p65 and nuclear factor erythroid 2-related factor 2 (Nrf2), together with its downstream heme-oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) were also quantified. RESULTS: HBO2-PC significantly alleviated LPS-induced ALI, lowered the lung injury scores, reduced lung water content, and reduced H2O2, MDA levels as well as MPO activity, while simultaneously improving the arterial partial oxygen pressure (PaO2) and SOD activity. Furthermore, HBO2-PC inhibited the nuclear translocation of NF-κB p65 while enhancing the nuclear translocation of Nrf2, thus upregulating HO-1 and NQO1. CONCLUSIONS: Our results suggest that HBO2-PC was potentially protective for LPS-induced ALI lungs in rats, with a presumed mechanism that suppressed NF-κB while activating Nrf2. We propose that HBO2-PC should be considered a potential therapeutic strategy against ALI in rats.

Effect of combination sildenafil and gemfibrozil on cisplatin-induced nephrotoxicity; role of heme oxygenase-1.

BACKGROUND/AIM: Cisplatin-induced nephrotoxicity in large proportion of patients. The aim of this work is to clarify the effect of combination of sildenafil and gemfibrozil on cisplatin-induced nephrotoxicity either before or after cisplatin treatment and determination of nephrotoxicity predictors among the measured tissue markers. METHODS: Thirty two adult male albino rats were divided into four equal groups (G) GI control, GII received cisplatin, GIII received sildenafil and gemfibrozil before cisplatin, GIV received sildenafil and gemfibrozil after cisplatin. Creatinine and urea were measured and animals were sacrificed and kidney was taken for histopathology. The following tissue markers were measured, heme oxygenase-1 (HO-1) activity, reduced glutathione, quantitative (real-time polymerase chain reaction) RT-PCR for gene expression of tumor necrosis factor alpha (TNF-α) and endothelial nitric oxide synthase (ENOS) level. RESULTS: GII developed AKI demonstrated by significantly high urea and creatinine and severe diffuse (80-90%) tubular necrosis. TNF-α was highly and significantly elevated while the rest of tissue markers were significantly reduced in GI1 compared to other groups. GIV showed better results compared to GIII. There was a significant positive correlation between creatinine and TNF-α when combining GI and GII while there were significant negative correlation between creatinine and other tissue markers in same groups. Linear regression analysis demonstrated that HO-1 was the independent predictor of AKI demonstrated by elevated creatinine among GI and GII. CONCLUSIONS: Combination of sildenafil and gemfibrozil can be used in treatment of cisplatin-induced nephrotoxicity. HO-1 is a promising target for prevention and/or treatment of cisplatin-induced nephrotoxicity.

Simultaneous accurate quantification of HO-1, CD39, and CD73 in human calcified aortic valves using multiple enzyme digestion - filter aided sample pretreatment (MED-FASP) method and targeted proteomics.

Several proteins such as membrane-associated ectonucleotidases: ecto-5'-nucleotidase (E5NT/CD73) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), and intracellular heme oxygenase-1 (HO-1) may contribute to protection from inflammation-related diseases such as calcific aortic valve stenosis (CAS). Accurate quantification of these proteins could contribute to better understanding of the disease mechanisms and identification of biomarkers. This report presents development and validation of quantification method for E5NT/CD73, ENTPD1/CD39 and HO-1. The multiplexed targeted proteomic assay involved antibody-free, multiple-enzyme digestion, filter-assisted sample preparation (MED-FASP) strategy and a nanoflow liquid chromatography/mass spectrometry under multiple reaction monitoring mode (LC-MRM/MS). The method developed presented high sensitivity (LLOQ of 5 pg/mL for each of the analytes) and accuracy that ranged from 92.0% to 107.0%, and was successfully applied for the absolute quantification of HO-1, CD39 and CD73 proteins in homogenates of human calcified and non-calcified valves. The absolute CD39 and CD73 concentrations were lower in calcified aortic valves (as compared to non-stenotic ones) and were found to be: 1.16 ± 0.39 vs. 3.15 ± 0.37 pmol/mg protein and 1.94 ± 0.21 vs. 2.39 ± 0.39 pmol/mg protein, respectively, while the quantity of HO-1 was elevated in calcified valves (10.72 ± 1.18 vs. 4.28 ± 0.42 amol/mg protein). These results were consistent but more reproducible as compared to immunoassays. In conclusion, multiplexed quantification of HO-1, CD39 and CD73 proteins by LC-MRM/MS works well in challenging human tissues such as aortic valves. This analysis confirmed the relevance of these proteins in pathogenesis of CAS and could be extended to other biomedical investigations.

Inhibition of heme oxygenase ameliorates anemia and reduces iron overload in a ß-thalassemia mouse model.

Thalassemias are a heterogeneous group of red blood cell disorders, considered a major cause of morbidity and mortality among genetic diseases. However, there is still no universally available cure for thalassemias. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoiesis. In ß-thalassemia, ß-globin synthesis is reduced causing α-globin accumulation. Unpaired globin chains, with heme attached to them, accumulate in thalassemic erythroblasts causing oxidative stress and the premature cell death. We hypothesize that in ß-thalassemia heme oxygenase (HO) 1 could play a pathogenic role in the development of anemia and ineffective erythropoiesis. To test this hypothesis, we exploited a mouse model of ß-thalassemia intermedia, Th3/+ We observed that HO inhibition using tin protoporphyrin IX (SnPP) decreased heme-iron recycling in the liver and ameliorated anemia in the Th3/+ mice. SnPP administration led to a decrease in erythropoietin and increase in hepcidin serum levels, changes that were accompanied by an alleviation of ineffective erythropoiesis in Th3/+ mice. Additionally, the bone marrow from Th3/+ mice treated with SnPP exhibited decreased heme catabolism and diminished iron release as well as reduced apoptosis. Our results indicate that the iron released from heme because of HO activity contributes to the pathophysiology of thalassemia. Therefore, new therapies that suppress heme catabolism may be beneficial in ameliorating the anemia and ineffective erythropoiesis in thalassemias.

Bioavailable Blueberry-Derived Phenolic Acids at Physiological Concentrations Enhance Nrf2-Regulated Antioxidant Responses in Human Vascular Endothelial Cells.

SCOPE: Blueberry consumption is believed to confer a cardiovascular health advantage, but the active compounds and effects require characterization. This study aims to identify the polyphenol metabolites in plasma after blueberry juice intake and determine their bioactivity on endothelial cells. METHODS AND RESULTS: Three healthy individuals are recruited to obtain profiles of bioavailable plasma polyphenol metabolites following intake of blueberry juice. Of 33 phenolic compounds screened, 12 aglycone phenolic acids are detected and their maximum plasma concentrations and circulation time determined. Using this information, the effect of three physiologically relevant mixtures of blueberry-derived phenolic acids is investigated for their ability to induce nuclear factor erythroid 2-related factor 2 (Nrf2)-nuclear translocation and downstream gene expression in human endothelial cells. Pretreatment with the phenolic acids for 18 h results in a significant upregulation of the Nrf2-regulated antioxidant response proteins heme oxygenase 1 (HO-1) and glutamate-cysteine ligase modifier subunit (GCLM), following 6 h exposure to 2.5 µm H2 O2 . CONCLUSION: Physiologically relevant concentrations of blueberry-derived aglycone phenolic acids can induce Nrf2-regulated antioxidant response proteins in vascular endothelial cells in response to low µm concentrations of H2 O2 . Our results represent an advance over previous studies that have used single compounds or high concentrations in cell-based investigations.