Human Heme Oxygenase-1 Research Articles

Antioxidant activity of Yellow Candle bush (Cassia alata) leaves extract and Bioactive fractions through LC-QTOF-MS/MS and Molecular Docking Approach

Free radicals play a significant role in numerous cellular functions, such as cell signaling, metabolism, and defense mechanism. Cassia alata, well known as Yellow Candle bush or locally known as Ketepeng Badak, has been known to possess biological activities including antiinflamation and antidiabetic. This current study investigates the antioxidant potential of C. alata leaves crude extract and resultant fractions. DPPH, ABTS radical scavenging activity, and FRAP assay were evaluated for the determination of the antioxidant activity, while the active constituents in C. alata leaf extract and fractions were identified with LC-QTOF-MS/MS. Total phenolic, flavonoid, and DPPH radical scavenging activities were highest in the ethyl acetate fraction with IC50: 18.54±0.01µg/mL on DPPH assay. Meanwhile, butanol fraction exhibited the strongest activity in ABTS and FRAP tests with IC50 of 4.29±0.26µg/mL and 4.39±0.11µg/mL, respectively. An in silico study using a molecular docking technique was conducted to determine the free energy of binding between human heme oxygenase-1 (HO-1) with bioactive compounds contained in C. alataethanolic leaves extract. Molecular docking of the main constituents in C. alata ethyl acetate fraction showed a greater binding affinity for kaempferol-3-O-β-D-glucopyranoside (-8.95kcal/mol), followed by coclaurine (-7.94kcal/mol), quercetin (-7.66kcal/mol) and 3-Hydroxy-7-methoxy baicalein (-7.32kcal/mol). These results corroborate the potent antioxidant activity of C. alata extract and fractions and its use as possible antioxidant agents in the pharmaceutical industry.

Systematic Evaluation of Aegle marmelos-Derived Compounds: Potential Therapeutic Agents Against Inflammation and Oxidative Stress.

Aim This study aimed to evaluate the potential antioxidant and anti-inflammatory properties of Aegle marmelos active compoundsthrough a multifaceted approach. The investigation encompasses molecular docking studies, computational pharmacokinetic predictions, and in vitro assessments, with a focus on understanding their physiochemical properties, pharmacokinetics, and molecular interactions. Materials and methods This study was conducted in the Research Departmentof Biochemistry, Saveetha Medical College & Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS), Tamilnadu, India. The study employed Soxhlet and methanol extraction techniques to obtain Aegle marmelos extracts, which were then subjected to antioxidant and anti-inflammatory assays. Antioxidant activity was assessed using the H2O2 assay, while anti-inflammatory potential was determined via the egg albumin denaturation assay. Molecular docking studies were conducted with human heme oxygenase 1 (HO-1) and human zanthine oxidoreductase (XO) proteins to elucidate potential therapeutic interactions. Furthermore, computational tools like SwissADME, pkCSM, and ADMETlab 2.0 were utilized to predict physiochemical and pharmacokinetic properties, providing insights into the compoundabsorption, distribution, metabolism, and excretion profiles. This integrated approach aimed to comprehensively evaluate the therapeutic potential of Aegle marmelos-derived compounds against inflammation and oxidative stress-related disorders, paving the way for future drug development endeavors. Results In the antioxidant assay, Aegle marmelos methanolic tuber extracts showed exceptional absorption of 87.4%, surpassing the reference standard. In the anti-inflammatory assay, the extracts displayed an absorption of approximately 79%, indicating significant anti-inflammatory potential. Auraptene, imperatorin, luvangetin, and psoralen exhibited favorable pharmacokinetic properties and adherence to the Lipinski rule of 5, suggesting promising drug development potential. In molecular docking, imperatorin demonstrated the highest binding affinity to HHO-1 and XO. Conclusion The study on Aegle marmelos highlights its potential as a therapeutic agent due to its potent antioxidant and anti-inflammatory properties. Phytochemical constituents, such as auraptene, imperatorin, luvangetin, and psoralen, show promising pharmacokinetic profiles, suggesting their suitability for drug development. Molecular docking analysis reveals imperatorin as the most effective binder to key enzymes, emphasizing its therapeutic potential against inflammation and oxidative stress-related disorders.

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

Interactions of azole-based inhibitors with human heme oxygenase

Human heme oxygenase-1 (hHO-1) plays a crucial role in human physiology because of its ability to metabolize free heme. The heme degradation products, biliverdin and bilirubin, were shown to have protective antioxidant properties in cells. In the context of cancer, hHO-1 function grants cancer cells defense from standard chemotherapy treatments, leading to the development of azole-based inhibitors that target hHO-1 for potential anticancer therapy. This work reports experimental and theoretical characterization of interactions between three azole-based inhibitors and the active site of hHO-1. It was found that all three compounds have Kd values within the μM order. The electronic absorption and resonance Raman (rR) spectra indicated that they bind to the ferric heme and coordinate through a nitrogen atom. rR measurements revealed varying effects of inhibitors on the geometry of heme vinyl groups in the ferric form of hHO-1. Changes in peripheral group orientation are known to affect heme redox potential, and consequently can reflect the inhibitory properties of studied azoles. The subsequent docking studies showed that inhibitors with lower Kd values are located close to two vinyl groups, while the compound with higher Kd is situated near only one, consistent with the rR studies. Finally, the rR studies of the CO adducts showed that the inhibitors bind to the heme in a reversible manner. Altogether, the combination of ligand binding studies, UV–Vis and rR spectroscopies, as well as computational approach revealed an importance of the steric hindrance imposed by the inhibitor's side chain.

Human Heme Oxygenase-1 Promoter Activity Is Mediated by Z-DNA Formation.

In recent years, it has been shown that Z-DNA formation in DNA plays functionally significant roles in nucleic acid metabolism, such as gene expression, chromosome recombination, and epigenetic regulation. The reason for the identification of these effects is mainly due to the advancement of Z-DNA detection methods in target genome regions in living cells.The heme oxygenase-1 (HO-1) gene encodes an enzyme that degrades an essential prosthetic heme, and environmental stimuli, including oxidative stress, lead to robust induction of the HO-1 gene. Many DNA elements and transcription factors are involved in the induction of the HO-1 gene, and Z-DNA formation in the thymine-guanine (TG) repetitive sequence in the human HO-1 gene promoter region is required for maximum gene induction.Here, we describe a detailed protocol for Z-DNA detection in the human HO-1 gene promoter region based on chromatin immunoprecipitation with quantitative PCR. We also provide some control experiments to consider in routine lab procedures.

Identification of Cytoprotective Small-Molecule Inducers of Heme-Oxygenase-1.

Acute kidney injury (AKI) is a major public health concern with significant morbidity and mortality and no current treatments beyond supportive care and dialysis. Preclinical studies have suggested that heme-oxygenase-1 (HO-1), an enzyme that catalyzes the breakdown of heme, has promise as a potential therapeutic target for AKI. Clinical trials involving HO-1 products (biliverdin, carbon monoxide, and iron), however, have not progressed beyond the Phase ½ level. We identified small-molecule inducers of HO-1 that enable us to exploit the full therapeutic potential of HO-1, the combination of its products, and yet-undefined effects of the enzyme system. Through cell-based, high-throughput screens for induction of HO-1 driven by the human HO-1 promoter/enhancer, we identified two novel small molecules and broxaldine (an FDA-approved drug) for further consideration as candidate compounds exhibiting an Emax ≥70% of 5 µM hemin and EC50 <10 µM. RNA sequencing identified shared binding motifs to NRF2, a transcription factor known to regulate antioxidant genes, including HMOX1. In vitro, the cytoprotective function of the candidates was assessed against cisplatin-induced cytotoxicity and apoptosis. In vivo, delivery of a candidate compound induced HO-1 expression in the kidneys of mice. This study serves as the basis for further development of small-molecule HO-1 inducers as preventative or therapeutic interventions for a variety of pathologies, including AKI.

Dysregulation of a Heme Oxygenase-Synuclein Axis in Parkinson Disease.

α-Synuclein is a key driver of the pathogenesis of Parkinson disease (PD). Heme oxygenase-1 (HO-1), a stress protein that catalyzes the conversion of heme to biliverdin, carbon monoxide and free ferrous iron, is elevated in PD-affected neural tissues and promotes iron deposition and mitochondrial dysfunction in models of the disease, pathways also impacted by α-synuclein. Elevated expression of human HO-1 in astrocytes of GFAP.HMOX1 transgenic mice between 8.5 and 19 months of age elicits a parkinsonian phenotype characterized by nigrostriatal hypodopaminergia, locomotor incoordination and overproduction of neurotoxic native S129-phospho-α-synuclein. Two microRNAs (miRNA) known to regulate α-synuclein, miR-153 and miR-223, are significantly decreased in the basal ganglia of GFAP.HMOX1 mice. Serum concentrations of both miRNAs progressively decline in wild-type (WT) and GFAP.HMOX1 mice between 11 and 18 months of age. Moreover, circulating levels of miR-153 and miR-223 are significantly lower, and erythrocyte α-synuclein concentrations are increased, in GFAP.HMOX1 mice relative to WT values. MiR-153 and miR-223 are similarly decreased in the saliva of PD patients compared to healthy controls. Upregulation of glial HO-1 may promote parkinsonism by suppressing miR-153 and miR-223, which, in turn, enhance production of neurotoxic α-synuclein. The aim of the current review is to explore the link between HO-1, α-synuclein and PD, evaluating evidence derived from our laboratory and others. HO-1, miR-153 and miR-223 and α-synuclein may serve as potential biomarkers and targets for disease-modifying therapy in idiopathic PD.

Prometheus Unbound: The Allure of Hepatic Regeneration

The paper “HO-1 protects remnant liver against dysfunction after major hepatectomy in humans” the authors provide some of the first clinical data showing that human Heme Oxygenase (HO)-1 plays a ro...

Investigation of Cyanide Ligand as an Active Site Probe of Human Heme Oxygenase.

Human heme oxygenase (hHO-1) is a physiologically important enzyme responsible for free heme catabolism. The enzyme's high regiospecificity is controlled by the distal site hydrogen bond network that involves water molecules and the D140 amino acid residue. In this work, we probe the active site environment of the wild-type (WT) hHO-1 and its D140 mutants using resonance Raman (rR) spectroscopy. Cyanide ligands are more stable than dioxygen adducts and are an effective probe of active site environment of heme proteins. The inherently linear geometry of the Fe-C-N fragment can be altered by the steric, electrostatic, and H-bonding interactions imposed by the amino acid residues present in the heme distal site, resulting in a tilted or bent configuration. The WT hHO-1 and its D140A, D140N, and D140E mutants were studied in the presence of natural abundance CN- and its isotopic analogues (13CN-, C15N-, and 13C15N-). Deconvolution of spectral data revealed that the ν(Fe-CN) stretching and δ(Fe-CN) bending modes are present at 454 and 376 cm-1, respectively. The rR spectral patterns of the CN- adducts of WT revealed that the Fe-C-N fragment adopts a tilted conformation, with a larger bending contribution for the D140A, D140N, and D140E mutants. These studies suggest that the FeCN fragment in hHO-1 is tilted more strongly toward the porphyrin macrocycle compared to other histidine-ligated proteins, reflecting the propensity of the exogenous hHO-l ligands to position toward the α-meso-carbon, which is crucial for the HO reactivity and essential for regioselectivity.

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 μM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 μg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXβ/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the β/δ selective HemO. Moreover, at 20 μM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the β-galactosidase transcriptional reporter assay.

Heme Oxygenase-1 Deficiency and Oxidative Stress: A Review of 9 Independent Human Cases and Animal Models.

Since Yachie et al. reported the first description of human heme oxygenase (HO)-1 deficiency more than 20 years ago, few additional human cases have been reported in the literature. A detailed analysis of the first human case of HO-1 deficiency revealed that HO-1 is involved in the protection of multiple tissues and organs from oxidative stress and excessive inflammatory reactions, through the release of multiple molecules with anti-oxidative stress and anti-inflammatory functions. HO-1 production is induced in vivo within selected cell types, including renal tubular epithelium, hepatic Kupffer cells, vascular endothelium, and monocytes/macrophages, suggesting that HO-1 plays critical roles in these cells. In vivo and in vitro studies have indicated that impaired HO-1 production results in progressive monocyte dysfunction, unregulated macrophage activation and endothelial cell dysfunction, leading to catastrophic systemic inflammatory response syndrome. Data from reported human cases of HO-1 deficiency and numerous studies using animal models suggest that HO-1 plays critical roles in various clinical settings involving excessive oxidative stress and inflammation. In this regard, therapy to induce HO-1 production by pharmacological intervention represents a promising novel strategy to control inflammatory diseases.

Heteromeric complex formation between human cytochrome P450 CYP1A1 and heme oxygenase-1.

P450 and heme oxygenase-1 (HO-1) receive their necessary electrons by interaction with the NADPH-cytochrome P450 reductase (POR). As the POR concentration is limiting when compared with P450 and HO-1, they must effectively compete for POR to function. In addition to these functionally required protein-protein interactions, HO-1 forms homomeric complexes, and several P450s have been shown to form complexes with themselves and with other P450s, raising the question, 'How are the HO-1 and P450 systems organized in the endoplasmic reticulum?' Recently, CYP1A2 was shown to associate with HO-1 affecting the function of both proteins. The goal of this study was to determine if CYP1A1 formed complexes with HO-1 in a similar manner. Complex formation among POR, HO-1, and CYP1A1 was measured using bioluminescence resonance energy transfer, with results showing HO-1 and CYP1A1 form a stable complex that was further stabilized in the presence of POR. The POR•CYP1A1 complex was readily disrupted by the addition of HO-1. CYP1A1 also was able to affect the POR•HO-1 complex, although the effect was smaller. This interaction between CYP1A1 and HO-1 also affected function, where the presence of CYP1A1 inhibited HO-1-mediated bilirubin formation by increasing the KmPOR•HO-1 without affecting the Vmaxapp. In like manner, HO-1 inhibited CYP1A1-mediated 7-ethoxyresorufin dealkylation by increasing the KmPOR•CYP1A1. Based on the mathematical simulation, the results could not be explained by a model where CYP1A1 and HO-1 simply compete for POR, and are consistent with the formation of a stable CYP1A1•HO-1 complex that affected the functional characteristics of both moieties.

Geniposide Achieved an Anti-Inflammatory Effect Through the NF-κB/HIF-1α Signaling Pathway Mediated by VEGFA and HMOX1 Genes

Geniposide is a kind of iridoid glycoside compound mainly extracted from the dried and mature fruit of a Rubiaceae plant, Gardenia jasminoides J.Ellis, which has a certain anti-inflammatory effect, but mechanism of this effect remains unclear.In this study, we utilized the network pharmacology approach, molecular docking, and in vitro experiments on the pharmacodynamics and anti-inflammatory mechanism of Geniposide. We leveraged the public databases to uncover putative targets across Geniposide and inflammatory diseases and constructed the anti-inflammatory protein–protein interaction (PPI) network of Geniposide following the intersection targets between them, further molecular docking, and enrichment analysis were performed. Finally, in vitro experiments were conducted to validate the predicted target genes and pathways. A total of 59 Geniposide-related targets and 8954 inflammatory diseases related targets were screened out, and 50 intersection targets were eventually obtained. We took the key target of the top 10 for molecular docking, and vascular endothelial growth factor A (VEGFA), matrix metalloproteinase-2, recombination human heme oxygenase 1 (HMOX1), adenosine receptor A1, adenosine deaminasegenes were identified based on the free binding energy. Hypoxia-inducible factor 1 (HIF-1) was predicted as the critical path of Geniposide anti-inflammatory by the Kyoto Encyclopedia of Genes and Genomessignaling pathway enrichment analysis. The results showed that Geniposide could regulate the gene expression of VEGFA and HMOX1 and influence the protein expression levels of HIF-1α, VEGFA,HOMX1,and p-p65 in the NF-κB/HIF-1α signaling pathway. Our data revealed that the anti-inflammatory effect of Geniposide was at least through the NF-κB/HIF-1α signaling pathway mediated by VEGFA and HMOX1 genes.

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle-when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Human Heme Oxygenase-1 Induced by Interleukin-6 via JAK/STAT3 Pathways Is a Tumor Suppressor Gene in Hepatoma Cells.

Heme oxygenase-1 (HO-1) has several important roles in hepatocytes in terms of anti-inflammation, anti-apoptosis, and antioxidant properties. Interleukin-6 (IL-6) is a pleiotropic cytokine associated with liver regeneration and protection against injury. The aim of this study was to determine the potential crosstalk between HO-1 and IL-6, and to elucidate the signaling pathways involved in the induction of HO-1 by IL-6 in human hepatoma cells. Ectopic overexpression of HO-1 not only attenuated cell proliferation in vitro and in vivo, but also blocked the reactive oxygen species (ROS) induced by H2O2 and the pyocyanin in HepG2 or Hep3B cells. IL-6 expression was negatively regulated by HO-1, while IL-6 induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and HO-1 gene expression in HepG2 cells. The co-transfected HO-1 reporter vector and a protein inhibitor of the activated STAT3 (PIAS3) expression vector blocked the IL-6-induced HO-1 reporter activity. Both interferon γ and interleukin-1β treatments induced STAT1 but not STAT3 phosphorylation, which had no effects on the HO-1 expression. Treatments of AG490 and luteolin blocked the JAK/STAT3 signaling pathways which attenuated IL-6 activation on the HO-1 expression. Our results indicated that HO-1 is the antitumor gene induced by IL-6 through the IL-6/JAK/STAT3 pathways; moreover, a feedback circuit may exist between IL-6 and HO-1 in hepatoma cells.

Changes in pregnancy-related serum biomarkers early in gestation are associated with later development of preeclampsia.

BackgroundPlacental protein expression plays a crucial role during pregnancy. We hypothesized that: (1) circulating levels of pregnancy-associated, placenta-related proteins throughout gestation reflect the temporal progression of the uncomplicated, full-term pregnancy, and can effectively estimate gestational ages (GAs); and (2) preeclampsia (PE) is associated with disruptions in these protein levels early in gestation; and can identify impending PE. We also compared gestational profiles of proteins in the human and mouse, using pregnant heme oxygenase-1 (HO-1) heterozygote (Het) mice, a mouse model reflecting PE-like symptoms.MethodsSerum levels of placenta-related proteins–leptin (LEP), chorionic somatomammotropin hormone like 1 (CSHL1), elabela (ELA), activin A, soluble fms-like tyrosine kinase 1 (sFlt-1), and placental growth factor (PlGF)–were quantified by ELISA in blood serially collected throughout human pregnancies (20 normal subjects with 66 samples, and 20 subjects who developed PE with 61 samples). Multivariate analysis was performed to estimate the GA in normal pregnancy. Mean-squared errors of GA estimations were used to identify impending PE. The human protein profiles were then compared with those in the pregnant HO-1 Het mice.ResultsAn elastic net-based gestational dating model was developed (R2 = 0.76) and validated (R2 = 0.61) using serum levels of the 6 proteins measured at various GAs from women with normal uncomplicated pregnancies. In women who developed PE, the model was not (R2 = -0.17) associated with GA. Deviations from the model estimations were observed in women who developed PE (P = 0.01). The model developed with 5 proteins (ELA excluded) performed similarly from sera from normal human (R2 = 0.68) and WT mouse (R2 = 0.85) pregnancies. Disruptions of this model were observed in both human PE-associated (R2 = 0.27) and mouse HO-1 Het (R2 = 0.30) pregnancies. LEP outperformed sFlt-1 and PlGF in differentiating impending PE at early human and late mouse GAs.ConclusionsSerum placenta-related protein profiles are temporally regulated throughout normal pregnancies and significantly disrupted in women who develop PE. LEP changes earlier than the well-established biomarkers (sFlt-1 and PlGF). There may be evidence of a causative action of HO-1 deficiency in LEP upregulation in a PE-like murine model.

Glial HMOX1 expression promotes central and peripheral α-synuclein dysregulation and pathogenicity in parkinsonian mice.

α-Synuclein is a key player in the pathogenesis of Parkinson disease (PD). Expression of human heme oxygenase-1 (HO-1) in astrocytes of GFAP.HMOX1 transgenic (TG) mice between 8.5 and 19 months of age results in a parkinsonian phenotype characterized by neural oxidative stress, nigrostriatal hypodopaminergia associated with locomotor incoordination, and overproduction of α-synuclein. We identified two microRNAs (miR-), miR-153 and miR-223, that negatively regulate α-synuclein in the basal ganglia of male and female GFAP.HMOX1 mice. Serum concentrations of both miRNAs progressively declined in the wild-type (WT) and GFAP.HMOX1 mice between 11 and 19 months of age. Moreover, at each time point surveyed, circulating levels of miR-153 were significantly lower in the TG animals compared to WT controls, while α-synuclein protein concentrations were elevated in erythrocytes of the GFAP.HMOX1 mice at 19 months of age relative to WT values. Primary WT neurons co-cultured with GFAP.HMOX1 astrocytes exhibited enhanced protein oxidation, mitophagy and apoptosis, aberrant expression of genes regulating the dopaminergic phenotype, and an imbalance in gene expression profiles governing mitochondrial fission and fusion. Many, but not all, of these neuronal abnormalities were abrogated by small interfering RNA (siRNA) knockdown of α-synuclein, implicating α-synuclein as a potent, albeit partial, mediator of HO-1's neurodystrophic effects in these parkinsonian mice. Overexpression of HO-1 in stressed astroglia has previously been documented in the substantia nigra of idiopathic PD and may promote α-synuclein production and toxicity by downmodulating miR-153 and/or miR-223 both within the CNS and in peripheral tissues.

Justification of specific genetic modifications in pigs for clinical organ xenotransplantation.

Xenotransplantation research has made considerable progress in recent years, largely through the increasing availability of pigs with multiple genetic modifications. We suggest that a pig with nine genetic modifications (ie, currently available) will provide organs (initially kidneys and hearts) that would function for a clinically valuable period of time, for example, >12months, after transplantation into patients with end-stage organ failure. The national regulatory authorities, however, will likely require evidence, based on in vitro and/or in vivo experimental data, to justify the inclusion of each individual genetic modification in the pig. We provide data both from our own experience and that of others on the advantages of pigs in which (a) all three known carbohydrate xenoantigens have been deleted (triple-knockout pigs), (b) two human complement-regulatory proteins (CD46, CD55) and two human coagulation-regulatory proteins (thrombomodulin, endothelial cell protein C receptor) are expressed, (c) the anti-apoptotic and "anti-inflammatory" molecule, human hemeoxygenase-1 is expressed, and (d) human CD47 is expressed to suppress elements of the macrophage and T-cell responses. Although many alternative genetic modifications could be made to an organ-source pig, we suggest that the genetic manipulations we identify above will all contribute to the success of the initial clinical pig kidney or heart transplants, and that the beneficial contribution of each individual manipulation is supported by considerable experimental evidence.

Strong xenoprotective function by single-copy transgenes placed sequentially at a permissive locus.

Multiple xenoprotective transgenes are best grouped at a single locus to avoid segregation during breeding and simplify production of donor animals. We used transgene stacking to place a human CD55 transgene adjacent to a human heme oxygenase 1 construct at the porcine ROSA26 locus. A transgenic pig was analyzed by PCR, RT-PCR, droplet digital PCR, immunohistochemistry, immunofluorescence, and flow cytometry. Resistance to complement-mediated cell lysis and caspase 3/7 activation were determined in vitro. The ROSA26 locus was retargeted efficiently, and animals were generated by nuclear transfer. RNA and protein analyses revealed abundant expression in all organs analyzed, including pancreatic beta cells. Transgenic porcine kidney fibroblasts were almost completely protected against complement-mediated lysis and showed reduced caspase 3/7 activation. Step-by-step placement enables highly expressed single-copy xenoprotective transgenes to be grouped at porcine ROSA26.

323: Pravastatin improves pregnancy outcome and placental development in heme oxygenase-1 deficient mice

Pravastatin is currently being studied clinically for prevention of preeclampsia. Low expression of heme oxygenase-1 (HO-1) due to polymorphisms in the human HO-1 promoter region may cause impaired placental development resulting in intrauterine growth restriction (IUGR), preeclampsia, and recurrent miscarriages. We have shown that pregnant HO-1 heterozygous (HO-1+/-, Het) mice have abnormal placental development, with thinner spongiotrophoblast layers and increased Sflt-1 levels (Zhao H. et al., 2009). We have also found that statins can induce HO-1 expression. Therefore, our aim was to evaluate the effect of pravastatin on pregnancy outcome and placental development in HO-1-deficient mice. We compared untreated Het mice (control, n=5) with Het mice treated with pravastatin (10 mg/g/d; n=9) added to their drinking water 1 wk prior to timed-pregnancy breeding for a total of 3.5 wks. At E14.5, dams were sacrificed, litter sizes recorded, and livers and placentas (fetal side) harvested for total HO activity (expressed as percent of Het controls). A subset of whole placentas (control n=3, pravastatin n=4) was stained for H&E to assess placental development. At E14.5, litter sizes of the pravastatin-treated dams were significantly larger (8.0±2.5 vs 5.0±2.0 p<0.05), with increases in HO activity in livers (154%, p=0.012) and in placentas (119%, p=0.059). Histologically, placentas of pravastatin-treated mice had less apoptosis, more glycogen-laden cells, and larger vessel diameters in the spongiotrophoblast (SP) layer, reflecting improved placental development (Figs. 1 and 2). Pravastatin induces liver and placental HO activity and appears to improve pregnancy outcome and placental development in HO-1-deficient mice. We conclude that pravastatin has potential for use in the treatment of not only preeclampsia, but also of IUGR due to its effects on placental development. We speculate that among women at risk, those with low HO-1 expression may specifically benefit from pravastatin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)