Varying Time Points Research Articles

Induction of kidney endonucleases by DNase I: evidence of endonuclease network

Endonuclease‐mediated DNA fragmentation is both an immediate cause and a result of apoptosis and all other types of irreversible cell death during kidney injury. It is produced by nine enzymes: DNase I and 2 families, CAD and EndoG, which act simultaneously. However, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most active kidney endonuclease, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK‐52E cells were transfected with rat DNase I gene or its inactive mutant in pECFP expression vector, while control cells were transfected with empty vector. Cells were collected and DNA and mRNA were isolated from the cells at varying time points. mRNA expression of all nine endonucleases was studied using real‐time RT‐PCR; DNA strand breaks endonuclease genes was determined by PCR; and protein expression of the enzymes was measured by Western and quantitative immunocytochemistry. Our data showed DNase I, but not its inactive mutant, induces all other endonucleases at different time periods after transfection, DNA breaks in endonuclease genes, and protein expression of several endonucleases. This is the first evidence of an endonuclease network, in which endonucleases are orchestrated by DNA‐degrading activity of DNase I. The study was supported by NIH and VA grants to A.G.B. and S.V.S.

Provitamin A carotenoid biofortified maize as a source of vitamin A in pregnant and nursing sows (Sus scrofa domesticus)

Biofortification is an emerging technique to enhance the nutrient profile of staple crops, while being financially affordable for developing countries. Vitamin A deficiency is a problem of epidemic proportions in many countries and recent studies have emerged to study the efficacy of biofortification of maize to improve vitamin A status. Sows (Sus scrofa domesticus) were given either a biofortified provitamin A maize diet or megadose of retinyl palmitate at the beginning of pregnancy in order to determine the efficacy of maternal‐fetal transfer of vitamin A using these two routes of delivery. Sows were fed the biofortified maize diet throughout pregnancy and lactation, while control sows were fed a vitamin A‐free white maize diet, after receiving the oral vitamin A dose at insemination. Three piglets from each sow were killed at birth and 14 days, and blood was collected at varying time points. Organs collected for vitamin A analysis were liver, lung, spleen, adrenals, and kidneys and analyzed for vitamin A using HPLC methods. Organs and serums are currently being analyzed. Funded by the World Health Organization and HarvestPlus.

Neutrophil Extracellular Traps Are Generated By Reactive Oxygen Species Through Toll-Like Receptor 4

Introduction: Neutrophil extracellular traps (NETs) are complexes of DNA, granular enzymes and histone proteins released by activated neutrophils during sepsis. NETs function to bind, disarm and destroy infectious microbes extracellulary following stimulation by lipopolysaccharide. However, the role of NET formation in non-infectious stimuli, such as oxidative stress, is unknown. Therefore, we sought to determine if neutrophils activated by reactive oxygen species (ROS) release NETs. Methods: Neutrophils were isolated from femurs and tibias of rats and HeJ (TLR4 Mutant) and HeOUJ (WT) mice using a percol gradient. Cells were treated at varying time points with xanthine oxidase and its substrate hypoxanthine (XO/HX), hydrogen peroxide, phorbol-myristate-acetate (PMA, positive control) and cell culture media (negative control). Slides were fixed and stained for DNA and histones. NETs release was verified by immunofluoresence and quantified by calculating the percentage of extranuclear histones with high field microscopy analysis. Results: NETs stained positively for DNA and histone proteins, and appeared as web-like structures extending extracellularly from neutrophils (Image 1). Neutrophils treated with hydrogen peroxide or XO/HX released NETs in a time dependant manner with peak release at 12 hrs post treatment, whereas neutrophils with media treatment alone released no NETs. Furthermore TLR4 WT neutrophils co-treated with XO/HX released NETs while TLR4 Mutant neutrophils released significantly less NETs. NETs stimulated with PMA, the positive control, appeared as extracellular histones and DNA in close proximity to the neutrophil of origin while oxidative-stressed NETs extended multiple diameters further and overlapped with other cells, suggesting that NETs released due to oxidative stress may be more intense than NETs release due to PMA in murine neutrophils. Conclusions: This study demonstrates that oxidative stress can induce the release of NETs from activated neutrophils. Further, NET generation involves TLR4, a receptor found to play a critical role in both infectious and non-infectious inflammatory conditions. These results provide evidence that NETs may play a previously unrecognized role in sterile inflammation.

Molecular MRI of Acute Necrosis With a Novel DNA-Binding Gadolinium Chelate

Background— Current techniques to image cell death in the myocardium are largely nonspecific. We report the use of a novel DNA-binding gadolinium chelate (Gd-TO) to specifically detect the exposed DNA in acutely necrotic (ruptured) cells in vivo. Methods and Results— In vivo MRI was performed in 20 mice with myocardial infarction (MI). The mice were injected with Gd-TO or Gd-DTPA at varying time points after MI. MRI was performed 2 hours after probe injection, to avoid nonspecific signal from the late gadolinium enhancement effect. Cell rupture (Gd-TO uptake) was present within 2 hours of infarction but peaked 9 to 18 hours after the onset of injury. A significant increase in the longitudinal relaxation rate (R 1 ) in the infarct was seen in mice injected with Gd-TO within 48 hours of MI, but not in those injected more than 72 hours after MI (R 1 =1.24±0.08 and 0.92±0.03 s −1 , respectively, P <0.001). Gd-DTPA, unlike Gd-TO, washed completely out of acute infarcts within 2 hours of injection ( P <0.001). The binding of Gd-TO to exposed DNA in acute infarcts was confirmed with fluorescence microscopy. Conclusions— Gd-TO specifically binds to acutely necrotic cells and can be used to image the mechanism and chronicity of cell death in injured myocardium. Cell rupture in acute MI begins early but peaks many hours after the onset of injury. The ruptured cells are efficiently cleared by the immune system and are no longer present in the myocardium 72 hours after injury.

Cell of origin strongly influences genetic selection in a mouse model of T-ALL

AbstractIdentifying the normal cell from which a tumor originates is crucial to understanding the etiology of that cancer. However, retrospective identification of the cell of origin in cancer is challenging because of the accumulation of genetic and epigenetic changes in tumor cells. The biologic state of the cell of origin likely influences the genetic events that drive transformation. We directly tested this hypothesis by performing a Sleeping Beauty transposon mutagenesis screen in which common insertion sites were identified in tumors that were produced by mutagenesis of cells at varying time points throughout the T lineage. Mutation and gene expression data derived from these tumors were then compared with data obtained from a panel of 84 human T-cell acute lymphoblastic leukemia samples, including copy number alterations and gene expression profiles. This revealed that altering the cell of origin produces tumors that model distinct subtypes of human T-cell acute lymphoblastic leukemia, suggesting that even subtle changes in the cell of origin dramatically affect genetic selection in tumors. These findings have broad implications for the genetic analysis of human cancers as well as the production of mouse models of cancer.

Early identification of antigen-specific immune responses in vivo by [ 18 F]-labeled 3′-fluoro-3′-deoxy-thymidine ([ 18 F]FLT) PET imaging

Current biomarkers are unable to adequately predict vaccine-induced immune protection in humans with infectious disease or cancer. However, timely and adequate assessment of antigen-specific immune responses is critical for successful vaccine development. Therefore, we have developed a method for the direct assessment of immune responses in vivo in a clinical setting. Melanoma patients with lymph node (LN) metastases received dendritic cell (DC) vaccine therapy, injected intranodally, followed by [(18)F]-labeled 3'-fluoro-3'-deoxy-thymidine ([(18)F]FLT) PET at varying time points after vaccination. Control LNs received saline or DCs without antigen. De novo immune responses were readily visualized in treated LNs early after the prime vaccination, and these signals persisted for up to 3 wk. This selective [(18)F]FLT uptake was markedly absent in control LNs, although tracer uptake in treated LNs increased profoundly with as little as 4.5 × 10(5) DCs. Immunohistochemical staining confirmed injected DC dispersion to T-cell areas and resultant activation of CD4(+) and CD8(+) T cells. The level of LN tracer uptake significantly correlates to the level of circulating antigen-specific IgG antibodies and antigen-specific proliferation of T cells in peripheral blood. Furthermore, this correlation was not observed with [(18)F]-labeled fluoro-2-deoxy-2-D-glucose. Therefore, [(18)F]FLT PET offers a sensitive tool to study the kinetics, localization, and involvement of lymphocyte subsets in response to vaccination. This technique allows for early discrimination of responding from nonresponding patients in anti-cancer vaccination and aid physicians in individualized decisionmaking.

Dimerization of human 5-lipoxygenase

The aim of present study was to understand the mechanism of action of 2,2′-diselenobis(3-pyridinol) or DISPOL in human lung cancer (A549) cells. A549 cells were treated with 10 µM (∼IC 50 ) of DISPOL for varying time points to corelate the intracellular redox changes with its cytotoxic effect. The results indicated that DISPOL treatment led to a time dependant decrease in the basal level of reactive oxygen species (ROS). Additionally, DISPOL treatment elevated the ratio of reduced (GSH) and oxidised (GSSG) glutathione by upregulating gamma-glutamylcysteine ligase ( γ-GCL ) involved in GSH biosynthesis and inhibiting the activities of redox enzymes responsible for GSH utilization and recycling, such as glutathione-S-transferase (GST) and glutathione reductase (GR). Molecular docking analysis suggests putative interactions of DISPOL with GST and GR which could account for its inhibitory effect on these enzymes. Further, DISPOL induced reductive environment preceded G1 arrest and apoptosis as evidenced by decreased expression of cell cycle genes ( Cyclin D1 and Cyclin E1 ) and elevation of p21 and apoptotic markers (cleaved caspase 3 and cleaved PARP). The combinatorial experiments involving DISPOL and redox modulatory agents such as N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) indeed confirmed the role of reductive stress in DISPOL-induced cell death. Finally, Lipinski’s rule suggests attributes of drug likeness in DISPOL. Taken together, DISPOL exhibits a novel mechanism of reductive stress-mediated cell death in A549 cells that warrants future exploration as anticancer agent.

The influenza virus PB1-F2 protein has interferon-antagonistic activity

The aim of present study was to understand the mechanism of action of 2,2′-diselenobis(3-pyridinol) or DISPOL in human lung cancer (A549) cells. A549 cells were treated with 10 µM (∼IC 50 ) of DISPOL for varying time points to corelate the intracellular redox changes with its cytotoxic effect. The results indicated that DISPOL treatment led to a time dependant decrease in the basal level of reactive oxygen species (ROS). Additionally, DISPOL treatment elevated the ratio of reduced (GSH) and oxidised (GSSG) glutathione by upregulating gamma-glutamylcysteine ligase ( γ-GCL ) involved in GSH biosynthesis and inhibiting the activities of redox enzymes responsible for GSH utilization and recycling, such as glutathione-S-transferase (GST) and glutathione reductase (GR). Molecular docking analysis suggests putative interactions of DISPOL with GST and GR which could account for its inhibitory effect on these enzymes. Further, DISPOL induced reductive environment preceded G1 arrest and apoptosis as evidenced by decreased expression of cell cycle genes ( Cyclin D1 and Cyclin E1 ) and elevation of p21 and apoptotic markers (cleaved caspase 3 and cleaved PARP). The combinatorial experiments involving DISPOL and redox modulatory agents such as N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) indeed confirmed the role of reductive stress in DISPOL-induced cell death. Finally, Lipinski’s rule suggests attributes of drug likeness in DISPOL. Taken together, DISPOL exhibits a novel mechanism of reductive stress-mediated cell death in A549 cells that warrants future exploration as anticancer agent.

Tissue remodeling in the acute otitis media mouse model

Objectives Otitis media is an infectious, inflammatory process involving the middle ear space. Chronic inflammation is associated with fibrosis, scarring and osteogenesis within the middle ear, which may contribute to subsequent hearing loss and increase the difficulty of treatment. Methods Heat-killed Streptococcus pneumoniae was injected into the middle ears of 8–12 week old Balb/c mice. Control mice were treated with PBS middle ear injections. Middle ears were harvested at 1, 3, 5 and 7 days following injection ( n = 8 for each time point). The middle ears were processed using standard RT-PCR techniques. Up- and down-regulation of mRNA expression of various members of the Bone Morphogenetic Protein (BMP), Fibroblast Growth Factor (FGF) and Matrix Metalloproteinase (MMP) families was quantified and compared to PBS treated controls ( n = 8 for each time point). Results Significant upregulation of MMP2, MMP3 and MMP9 was observed at varying time points ( p < 0.05). Significant downregulation of BMP3, BMP4, BMP5 BMP6 and BMP8a was seen at varying time points ( p < 0.05). Significant downregulation of FGF3, FGF6, FGF10 and FGFr1 was observed at varying time points ( p < 0.05). No significant expression of BMP8b, BMP9, BMP10, FGF5, FGF8, MMP1a, MMP7 and MMP14 was detected within the middle ear. Conclusions Inflammation within the middle ear following injection of bacterial products results in changes in the regulation of several tissue remodeling cytokines and proteinases in the mouse model. Further understanding of these molecular processes may allow for the development of treatment modalities aimed at preventing middle ear tissue remodeling.

Differential Expression in Hibiscus cannabinus (Mesta) Plants After Infection with Mesta Yellow Vein Mosaic Virus as Revealed by Suppression Subtractive Hybridization

AbstractYellow vein mosaic disease induced by a whitefly transmitted monopartite begomovirus causes a devastating foliar disease of Hibiscus cannabinus (mesta) crops across India. Characterization of the causal virus at molecular level and different epidemiological factors associated with the disease have already been investigated to understand the role of driving components behind continued spread of the disease. We have investigated the global gene expression profiling to increase knowledge of transcriptional changes taken place in a compatible interaction between Mesta yellow vein mosaic virus (MeYVMV) and H. cannabinus plants by PCR‐based suppression subtractive hybridization supplemented with mirror orientation selection. Dot‐blot analysis of forward and reverse subtracted libraries with respective cDNA probes confirmed the differential regulation of 100 clones of forward subtracted library and 70 clones of reverse‐subtracted library of 220 positive colonies (proved by colony PCR and restriction release) picked for analysis (from both reactions), and these clones were sequenced. Sequence analysis and virtual Northern blot at varying time points of the infection process finally confirmed the consistent up‐regulation of 11 and down‐regulation of seven gene fragments (ESTs) in infected plant. The up‐regulated transcripts could be functionally categorized in three different groups: (i) members of signal transduction cascades, (ii) host defence‐responsive elements and (iii) factors involved in metabolism and transport. Down‐regulation of the gene encoding SGT1 protein in infected plants suggested the possible modulation by the virus to overcome host defence responses. Other down‐regulated ESTs were found to be members of photosynthetic pathways, electron transport chain and metabolism. Differential regulations of a few genes from both libraries were subsequently confirmed by Northern analysis. Our results present the first evidence of genes that might be involved in recognition and signalling routes in the mesta plant after infection with MeYVMV and facilitate the design of new crop protection strategies.

The Effect of Synovial Fluid Enzymes on the Biodegradability of Collagen and Fibrin Clots

Recently there has been a great deal of interest in the use of biomaterials to stimulate wound healing. This is largely due to their ability to centralize high concentrations of compounds known to promote wound healing at a needed location. Joints present a unique challenge to using scaffolds because of the presence of enzymes in synovial fluid which are known to degrade materials that would be stable in other parts of the body. The hypothesis of this study was that atelocollagen scaffolds would have greater resistance to enzymatic degradation than scaffolds made of gelatin, fibrin and whole blood. To test this hypothesis, collagen and fibrin-based scaffolds were placed in matrix metallopeptidase-1 (MMP-1), elastase, and plasmin solutions at physiologic concentrations, and the degradation of each scaffold was measured at varying time points. The atelocollagen scaffolds had a significantly greater resistance to degradation by MMP-1, elastase and plasmin over the fibrin based scaffolds. The results suggest that atelocollagen-based scaffolds may provide some protection against premature degradation by synovial fluid enzymes over fibrin-based matrices.

Rotavirus shedding in premature infants following first immunization

There is limited data regarding rotavirus vaccine shedding in premature infants. We describe the natural history of rotavirus shedding in premature infants in the 2-week period following first immunization with RotaTeq(®), the pentavalent rotavirus vaccine (RV5), and the risk for symptomatic transmission to household contacts (HHC). A prospective pilot study of 15 premature infants of gestational ages 26-34 weeks immunized with RV5 between 6 and 14 weeks chronological age on discharge from the NICU was conducted. Stool samples collected in the following 2 weeks and analyzed for rotavirus antigen by enzyme immunoassay (EIA), cell culture, and RT-PCR. Solicited adverse events were collected on study subjects and any symptoms of fever, vomiting and diarrhea in HHC. Rotavirus antigen shedding after immunization was detected, with positive rotavirus EIA results in 53.3% of premature infants and in 22.1% of 86 stool samples collected. Shedding rates by RT-PCR were higher with 86.7% of infants and 76.7% of samples being positive. Only 42% of EIA positive samples were positive by cell culture (8/86 total samples, 9.3%). None of 53 HHC reported symptoms of rotavirus infection during the 4 weeks following immunization of the infants. The findings of this study demonstrate that premature infants have positive stools by EIA, viral culture, and RT-PCR at varying time points during 2 weeks following first-dose immunization with RV5. RT-PCR shedding rates need to be clinically evaluated in the context of virus quantification by cell culture, which was low. No symptomatic transmission to HHC was detected in this study, supporting low transmissibility of vaccine virus shed by these infants born prematurely.

A Study of the Effectiveness of the Containment Level-4 (CL-4) Chemical Shower in Decontaminating Dover Positive-Pressure Suits

Objectives: The decontamination or physical removal of contaminants from personal protective equipment upon exit from high containment laboratories is crucial to maintain containment and safety of laboratory workers. The current methodology for exiting from high-containment level 4 (CL-4) laboratory calls for laboratory personnel to decontaminate their worn positive pressure suits via “mechanical washing” during a 5-minute chemical/water rinse cycle. This study was carried out to evaluate the effectiveness of the current shower methodology and to test the efficacy of the physical removal of any contaminants from the positive pressure suits following the application of water alone and of a milder detergent. Method: Brushed stainless steel test carriers were inoculated with Vesicular Stomatitis Virus (VSV), a surrogate for filoviruses, and a soil load based on mucin, tryptone, and bovine serum albumen (BSA) to mimic serum. The virus mixture was then dried and secured with magnets onto a pre-washed Dover positive pressure suit. The suit was subjected to varying shower cycles, and once complete, carriers were removed and further analyzed. Any remaining virus was quantified using a TCID50 assay on Vero E6 cells showing cytopathic effects after 3–5 days. Three positive control carriers with dried inoculum were left untreated by the shower cycle, and negative control carriers (no inoculum) were also included on the suit during shower testing and analyzed alongside the test carriers. Results: No residual virus was detected using the TCID50 method following the normal shower cycle (2-minute chemical wash and 3-minute water rinse). Shower cycles consisting of 5 minutes of water-only rinses were equally as effective in removing virus from the suit. Cycles consisting of only a 1-minute water rinse showed a 5.5 log decrease from an average initial 6 log viral concentration. Varying time points between 1 and 5 minutes were evaluated during the normal shower cycle as well as varying concentrations of chemical disinfectant. Conclusion: The current chemical shower protocol is effective in removing all virus from the Dover positive pressure suits worn in high-containment laboratories. However, data show that reducing the need for a high-level decontamination chemical and using any surfactant carries the same effectiveness as the current protocol used, which would serve as a more cost-effective and eco-friendly alternative to the currently implemented shower protocol.

Altered expression of tissue remodeling genes in a mouse model of acute allergic rhinitis

Osteogenesis, fibrosis, and scarring are prominent pathologic changes resulting from chronic sinonasal inflammation, and these tissue changes may increase the degree of disease symptomatology and the level of surgical difficulty. Members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families of cytokines and the matrix metalloproteinase (MMP) family of endopeptidases are known to regulate tissue remodeling in other disease processes, but their role in acute and chronic sinonasal inflammation remains undefined. A previously described mouse model of acute allergic rhinitis secondary to Aspergillus fumigatis exposure in BALB/C mice was used. Intranasal challenge was performed 1 week following intraperitoneal sensitization with A. fumigatis extract and mice were sacrificed 6 hours (n = 8) and 24 hours (n = 8) later. Additional mice were intranasally challenged 3 times per week and sacrificed at the end of 7 days (n = 8) and 21 days (n = 8). The snouts were processed for quantitative reverse-transcription polymerase chain reaction (RT-PCR) and compared to untreated controls for messenger ribonucleic acid (mRNA) expression of BMP1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10, FGF1, 2, 3, 4, 5, 6, 7, 8, 10, and MMP1a, 2, 3, 7, 8, 9, 12, and 14. Additional 21-day-old mice were prepared for sinonasal histopathology. Control mice were treated with the same protocol, with intraperitoneal phosphate-buffered saline (PBS) and intranasal PBS substituted for A. fumigatis extract. Untreated mice were used for additional comparison. Compared to both the PBS-treated and untreated control groups, statistically significant (p < 0.05) upregulation of MMP8 was observed in the 6-hour time point. Significant downregulation of MMP8 was observed at 1 week. Significant upregulation of FGF3 was observed at 1 week (p < 0.05). BMP3 and BMP5 were significantly downregulated in the 1-week group (p < 0.05). The mice exhibited histologic sinonasal changes consistent with allergic inflammation. Intranasal exposure to A. fumigatis results in altered expression of several tissue remodeling cytokines at varying time points in the acute allergic rhinitis mouse model. These changes in cytokine regulation may subsequently contribute to sinonasal osteogenesis, scarring, and fibrosis as seen in chronic rhinosinusitis.

Abstract 1708: Induction of GDF15 by cisplatin triggers apoptotic cell death in ovarian cancer cells

Abstract Ovarian cancer is the fifth most common cause of death among women. The overall five year survival is 45.6% as a result of late diagnosis when treatment is mostly ineffective. Current treatment consists of complete resection followed by a combination of a taxane and a platinum -based chemotherapy regardless of the subtype. After standard treatment, 70% of patients will develop chemoresistance, creating a major challenge in the treatment of this type of cancer. Therefore, there is great interest in understanding the mechanisms by which tumors develop resistance. From the microarray data of a high grade serous ovarian cancer cell line (A2780) treated with varying concentrations of cisplatin and at varying time points, we identified four genes (CDKN1A, FDXR, TP53I3 and GDF15) which were significantly up-regulated by cisplatin in a time and dose dependent manner. The time and dose dependent upregulation of these genes were further validated by real-time RT-PCR and Western blot analysis in A2780 and two other ovarian cancer cell lines. We further tested whether GDF15 expression correlated with cisplatin response. RNA and protein from 6 different cell lines with different IC50 to cisplatin revealed a positive correlation between GDF15 induction and sensitivity to cisplatin. To further investigate the role GDF15 plays in the chemoresponse of ovarian cancer cell, we knocked down the expression of GDF15 by shRNA. Ovarian cancer cells with GDF15 knockdown showed a decrease in PARP cleavage, suggesting a pro-apoptotic role for GDF15. Thus, our results suggest that induction of GDF15 by cisplatin is one of the mechanisms that triggers apoptotic cell death in ovarian cancer cells. On the other hand, we were not able to detect an induction of GDF15 by cisplatin in a cisplatin resistant cell line A2780cp20. Thus, the lost of GDF15 induction could be one of the mechanisms of developing chemoresistance. Future studies to decipher the mechanism by which cisplatin induces GDF15 expression have the potential of identifying new targets that could circumvent chemoresistance to cisplatin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1708. doi:10.1158/1538-7445.AM2011-1708

Protease activated receptor‐1 increases phosphorylation of endothelial nitric oxide synthase at site thr495 in human corpus cavernosum endothelial cells

Endothelial dysfunction, a common symptom of vascular diseases such as erectile dysfunction (ED), encompasses a broad array of physical and molecular defects of the endothelium that are due to low nitric oxide (NO) availability. Protease activated receptor‐1 (PAR1) induces downstream phosphorylation of endothelial nitric oxide synthase (eNOS) at site Threonine‐495 (Thr495) to inhibit NO. We hypothesize that excessive phosphorylation at this site reduces NO availability and contributes to ED, a risk factor for cardiovascular disease. Immortalized human corpus cavernosum endothelial cells (HCCECs) were lysed and PAR1 expression was examined by Western blot analysis. HCCECs were stimulated with either thrombin (10 U/mL) or TFLLR (10μM), the PAR1 activating peptide, at varying time points and eNOS‐Thr495 phosphorylation was determined by Western blot. Results were quantified by densitometry, and demonstrated that PAR1 is expressed in HCCECs. Treatment with thrombin and TFLLR induced maximal downstream phosphorylation of eNOS‐Thr495 at 10 minutes. We believe that this signal transduction pathway is amplified in males with ED, and inhibition of PAR1 will restore NO availability. Our goal is to contribute to the development of novel therapeutic agents that focus on improving NO availability in ED and other cardiovascular diseases. This project was supported, in part, by NIGMS Grant 5SC3GM086336‐02.

Irradiated Bone Marrow Environment Impairs Hematopoietic Stem Cells by Exposure to Tumor Necrosis Factor a.

AbstractAbstract 3724 Background:Hematopoietic stem cell transplantation (HSCT) represents a curative treatment for various disorders including hematopoietic malignancies. Most HSCT requires cytoreductive conditioning such as total body irradiation (TBI) and/or chemotherapy to ensure engraftment of HSCs by emptying recipients' marrow niches. Our preliminary experiments, however, revealed that TBI could induce local inflammation peaking around days 2–3 within marrow environment. Of note is that conditioning regimens widely used in the current HSCT settings are mostly compatible with HSC exposure to inflammatory storm in post-irradiation bone marrow (BM). Although certain inflammatory cytokines have been shown to affect HSC functions in in vitro, it remains unknown whether in vivo exposure to inflammatory BM environment can alter the characteristics of transplanted HSCs. We therefore sought to investigate what effects irradiated BM environment would have on transplanted donor HSCs using murine systems. Methods and Results:We first tested whether infusion of HSCs at varying timing post TBI would affect transplantation outcomes and HSC functions. To this end, fifty HSCs (CD34-negative/low, cKit+, Sca-1+, lineage marker-negative cells) obtained from Ly5.1-B6 mice were transplanted into lethally irradiated Ly5.2-B6 mice with 1 × 106 competitor Ly5.1/5.2-B6 BM cells at day 0, 3, or 5 post irradiation. No mice survived long-term in the group that received transplants 5 days post irradiation, indicating insufficient HSC engraftment and hematopoietic reconstitution. Although hematopoiesis reconstituted in long term survivors was comparable between another two groups (d0 and d3), we found significant difference in donor HSC ability when tested in a competitive repopulation assay using Ly5.1-KSL cells sorted from primary recipients: Test HSCs obtained from mice transplanted 3 days after irradiation showed poor secondary reconstitution ability, suggesting alteration of pre-engraftment HSC functions depending on transplantation protocols. We then tested whether in vivo exposure of HSCs to irradiated BM environment would have negative effects on HSC functions. Test HSCs (400 cells) from Ly5.1-B6 mice were transplanted into Ly5.2-B6 primary recipients at varying time points (day 0, 1, 2, or 3) after lethal-dose TBI. Approximately 24 h later, BM samples were subjected to a competitive repopulation assay to test secondary reconstitution ability in test HSCs that homed to irradiated BM environment. Consequently, test HSCs that were exposed for ≂f24 h to BM environment at day 2 post TBI showed marked impairment in their long-term reconstitution ability. When “BM-homed” HSCs were enumerated 24 h after infusion, we found modestly impaired HSC homing to BM irradiated 2 days prior to transplantation, compared to BM irradiated on day 0, indicating negative effects on HSCs transplanted during a peak phase of inflammatory storm. Next we examined whether inflammatory cytokines were capable of impairing HSC functions. When tested in liquid culture using purified HSCs, both IFN-g and IL-1 had little effects on colony formation. In contrast, TNF-a inhibited colony formation in a dose-dependent manner. When in vivo HSC exposure to irradiated BM environment was tested using TNF-a KO mice as primary recipients, significant improvement of HSC ability was observed, indicating a major role for this inflammatory cytokine in the HSC-inhibitory effect observed within the irradiated host tissues. We finally sought to test if blocking of TNF-a signaling in HSCs at a peak phase of inflammation could lead to better transplantation outcomes. We utilized the peptide previously shown to block TNFR signaling, and confirmed that pre-incubation of HSCs with this molecule did suppress TNF-a induced reactive oxygen species production in HSCs. Studies are ongoing to test if HSCs transplanted in a peak-phase of TBI-mediated inflammation will benefit from this shielding measure. Conclusion:We demonstrated that inflammatory response in BM environment 2–3 days after irradiation could have a negative effect on donor HSCs regarding both homing efficiency and secondary reconstitution ability. These findings provide important implications for developing the measures that enable HSCs to escape from this inhibitory effect to achieve far more improvement in clinical HSCT outcomes. Disclosures:No relevant conflicts of interest to declare.

Prostaglandin E2 Is Rapidly Produced In Response to Bone Marrow Injury and Improves Survival of Primitive Hematopoietic Cells

AbstractAbstract 407Since the hematopoietic system is exquisitely sensitive to environmental and iatrogenic injury, the bone marrow microenvironment likely provides protective mechanisms during times of injury or stress. We have previously demonstrated that prostaglandin E2 (PGE2), which can be produced by many cell types in the bone marrow, targets both the bone marrow microarchitecture and primitive hematopoietic cells when administered systemically to mice (Porter, Frisch et. al., Blood, 2009). Since PGE2 is a local mediator of injury and is known to play a protective role in other cell types, we hypothesized that it could be an important microenvironmental regulator of HSPCs during times of injury. To test this hypothesis, we injured mice with a sub-lethal dose of gamma radiation, 6.5 Gy TBI, and sacrificed mice at varying time points from 1 hour to 6 days post-radiation. Bone marrow supernatant was collected and used for quantification of local PGE2 levels by ELISA. We found that, compared to non-irradiated mice, the PGE2 levels were increased greater than two-fold by 4 hours after irradiation (p=0.0030; n=3–6 mice/group), and these levels remain elevated until at least 6 days after injury (p<0.0001 by ANOVA). These data clearly demonstrate that PGE2 production is rapidly upregulated following bone marrow injury. To determine if HSPCs could be responding to this increase in local PGE2, we sorted Lin− c-Kit+ Sca1+ (LSK) cells from murine bone marrow and assayed the expression of the four PGE2 receptors, EP1-EP4. RT-PCR analysis demonstrated that all four receptors are expressed on LSK cells, suggesting that PGE2 could be acting on these primitive hematopoietic cells during times of injury. We next tested whether supplying additional PGE2 to mice could protect hematopoietic cells after injury. Mice were subjected to 6.5 Gy TBI and were treated with 0.5 mg/kg 16,16-dimethyl-PGE2 (dmPGE2) immediately after radiation and once daily thereafter until time of sacrifice. At 24 hours after radiation injury, mice that were treated with dmPGE2 had greater than 8-fold more surviving LSK cells, a population which still retains HSC repopulating activity in competitive transplantation studies, in their bone marrow compared with vehicle treated mice (n=4/group, p=0.046). Similarly, at 72 hr post-radiation, the dmPGE2 treated mice continued to have almost 2-fold greater numbers of LSK cells remaining viable in their bone marrow compared with vehicle treated mice (n=2–3/group). These data suggest that dmPGE2 treatment after bone marrow injury may provide protection, at least in the days immediately following injury, to primitive hematopoietic cells that remain capable of regenerating the hematopoietic system. To further support this idea, we also pretreated uninjured bone marrow cells in vitro with PGE2 (1 μ M) for 90 minutes and then exposed them to the chemotherapeutic agent cytarabine (Ara-C, 10 μ M for 4 hours). Pretreatment with PGE2 results in lower levels of apoptotic LSK cells compared with vehicle pre-treated LSK cells (30.26% vs. 39.02%; n=9/group; 3 independent experiments; p=0.0012). This result correlates with our in vivo radiation injury data and suggests that PGE2 may target primitive hematopoietic cells and render them more resistant to cell death from injury.Taken together, these results suggest that PGE2, which is released in the bone marrow after radiation exposure, may be an important microenvironmental regulator of HSPC response to injury, by preventing cell death, and/or increasing their recovery. Amplification of this physiological signal by treatment with exogenous PGE2 could provide a beneficial means of protecting hematopoietic cells in clinical situations of hematopoietic system injury and bone marrow transplantation, allowing patients to tolerate bone marrow suppressive treatments or to recover more easily. Further, these results also bring forth a potential concern about the safety of blocking prostaglandin synthesis by using anti-inflammatory medications during times of bone marrow injury. Disclosures:No relevant conflicts of interest to declare.

Role of Protein Disulfide Isomerase (PDI) In the Expression of Tissue Factor (TF) Procoagulant Activity on Apoptotic Myeloblasts

AbstractAbstract 152Disseminated intravascular coagulation (DIC) is a common and potentially life-threatening complication of acute myelogenous leukemia (AML). Although various components of the procoagulant and fibrinolytic systems have been implicated in its pathogenesis, aberrant expression of TF appears to be critically involved. Despite the need for more specific hemostatic agents, the molecular pathways underlying the expression of TF procoagulant activity (PCA) in AML remain obscure. In this study, we sought to identify determinants for systemic coagulation activation in a prospective cohort of 50 patients with newly diagnosed AML and conducted a series of in-vitro experiments to further characterize TF PCA regulation on myeloblasts in light of recently provided evidence. AML patients had significantly elevated plasma D-dimer and TF-specific PCA of isolated peripheral blood mononuclear cells (PB-MNCs) compared to healthy controls (n=10) and patients with ALL (n=5) or MDS (n=7), demonstrating specific upregulation of the TF-dependent coagulation pathway. In the AML cohort, TF PCA of PB-MNCs was 30-fold higher and TF+ plasma microparticles were more frequently detectable in patients with hypofibrinogenemia and a thrombohemorrhagic syndrome (n=8) as compared to patients with normal fibrinogen levels (n=42), providing further evidence that TF over-expression by myeloblasts is indeed a determinant for DIC decompensation. Physical cell disruption resulted in a 3-fold increase in TF PCA of PB-MNCs, indicating that a significant proportion of TF was non-coagulant (i.e. cryptic) on unperturbed cells. In patients with AML, TF PCA of PB-MNCs, absolute numbers of circulating blasts and lactate dehydrogenase (LDH) serum levels, a surrogate marker for leukemic cell turnover, were significantly correlated with plasma D-dimer, suggesting that membrane alterations associated with apoptosis/necrosis played a crucial role in myeloblast TF PCA expression. Extracellular PDI, which is abundantly expressed in the endoplasmic reticulum, has recently been implicated in TF regulation via its oxidoreductase and chaperone activity. In monoblastic THP1 cells harvested at varying time points during maintenance culture (n=5), we found a significant correlation between membrane PDI expression, as assessed by flow cytometry, and cell-associated TF PCA (r=0.93). Analysis of annexin V-FITC binding revealed that PDI was predominantly expressed on apoptotic monoblasts. Treatment of THP1 cells for 24 h with 1 μ M daunorubicin (DNR), a cytotoxic drug commonly used in AML therapy, induced >80% apoptosis and increased cellular TF PCA 8 ± 3-fold (n=8) independent of de-novo protein synthesis. Consistently, apoptotic cells stained extensively positive for PDI by flow cytometry. DNR-induced TF PCA was inhibited by 70 ± 6% (n=8) when THP1 cells were coincubated with RL90, an inhibitory PDI monoclonal antibody previously shown to diminish fibrin and platelet deposition in murine models of micro- and macrovascular thrombosis. Importantly, RL90 had no effect when added to THP1 cells at the end of the 24-h culture period, indicating specific effects on the expression of cellular TF PCA rather than the coagulation reaction itself. Similarly, coincubation of DNR-treated THP1 cells with the PDI inhibitor bacitracin dose-dependently (1-5 mM) inhibited TF activation by up to 93 ± 10% (n=3), while only a 30 ± 16% inhibition was observed when bacitracin was added at the end of the culture period. Virtually identical results were obtained with the AML cell lines, HL60 and U937. Furthermore, ex-vivo treatment of isolated myeloblasts from a patient with acute promyelocytic leukemia and decompensated DIC with 1 μ M DNR for 24 h induced >90% apoptosis and increased cellular TF PCA 5-fold. Extensive PDI staining was observed on apoptotic myeloblasts, and coincubation with bacitracin (5 mM) completely inhibited DNR-induced upregulation of TF PCA while not affecting baseline TF PCA expression. Finally, treatment with DNR resulted in the leakage of both LDH and PDI into the culture supernatants of AML cell lines, and soluble PDI antigen was detectable in the plasma samples of AML patients with the highest LDH serum levels. In summary, our findings suggest that, in addition to negatively charged phospholipids, surface-expressed and/or released PDI may be required for the efficient expression of TF-specific PCA on apoptotic myeloblasts. Disclosures:No relevant conflicts of interest to declare.

Abstract 5138: The regulation of CD44 expression in malignant brain tumor cells

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain tumor that demonstrates a high growth rate and the capacity to invade surrounding brain tissue. These characteristics result in a dismal prognosis for GBM patients. Current treatment options for GBM tumors involve surgical resection followed by radiation and/or chemotherapy. Disappointingly, GBM tumors are or become resistant to radiation and chemotherapy, and the invasiveness of GBM precludes curative removal of the tumor. GBM cells infiltration has been associated with the transmembrane glycoprotein, CD44, which is the principle cell surface receptor for hyaluronic acid (HA). The molecular mechanisms that regulate CD44 in GBM are not fully understood. However, CD44 protein expression has been associated with an AP-1 transcription factor, fos related antigen 1 (Fra-1), in another aggressive cancer, mesothelioma. Therefore, we investigated the regulation of CD44 expression in human glioma cells by Fra-1. Human GBM cell lines, U-251 MG, U-1242 MG, and A-172, were exposed to either epidermal growth factor (EGF, 20 ng/ml) or hepatocyte growth factor (HGF, 50 ng/ml) at varying time points and Western blot analysis was used to determine the protein expression of CD44 and Fra-1. CD44 and Fra-1 expression levels increased with longer exposures (4 to 24 hr) to EGF and HGF. To determine whether Fra-1 takes part in the regulation of CD44 expression, we utilized transient gene knockdown using siRNA directed against fra-1. fra-1-siRNA decreased Fra-1 expression in EGF- and HGF-treated GBM cells. Following EGF stimulation and fra-1-siRNA treatments, CD44 expression also decreased. However, HGF stimulation of fra-1-siRNA treated GBM cells showed no change in CD44 expression. These data suggest that Fra-1 regulation of CD44 expression is growth factor-specific. To investigate whether CD44-mediated cell adhesion is promoted by the expression of Fra-1, we analyzed GBM cell adhesion to HA. U-251 MG and U-1242 MG cell lines demonstrated an increase adherence to HA after growth factor simulation, 4hr and 24 hr respectively. This data is comparable to the initial western blot analysis of the GBM cells after growth factor simulation. However, A-172 cells did not adhere to HA regardless of growth factor stimulation. These data suggest that CD44 binding to HA is cell line specific. Taken together, our results suggest that CD44-mediated HA binding of GBM cells can be regulated by Fra-1 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5138.