Progress 08/22/13 to 06/30/16
Outputs
Target Audience:Target Audience Target audiences are the pharmaceutical industry, farm animal veterinary care sectors, domestic animal veterinary care sectors, and farming communities Changes/Problems:Changes/Problems In 2013 and within the first year of this project, the originally proposed analysis of pancreata tissues of mesobiliverdin IXα−fed and control db/db type 2 diabetic mice was displaced by cytoprotection studies on retinal epithelium pigmented cells, angiotensin II-treated arterial smooth muscle (A7r5) cells, and microgravity skeletal C2C12 myocytes. These latter studies emerged from recent and unpredicted findings that are of direct relevance to the overall and original goals of this UAES project. The resulting findings are promising, significant for inflammation research, and offer potentially significant future therapeutic outcomes. What opportunities for training and professional development has the project provided?Opportunities This project permitted opportunities for several graduate and undergraduate students to participate in research. Graduate students Susie Robinson (M.S., completed, Biology), Charles Harding (Ph.D. candidate, Biological Engineering), Farhad Farjood (Ph.D. candidate, Biological Engineering) and graduate student Jonathan Wood (Biological Engineering) participate in several research activities including mesobiliverdin IXalpha and phycocyanobilin production, lipid peroxidation assays, and cytoprotection experiments. Seven undergraduate students also participated first-hand in research experiences. They were Garrett Hinton, Tyler Gladwin, Cody Maxfield, Gilbert Nelson, Wil Higham, Christian Peterson, Seth Drury, and Matthew Agrio. Dr. Lai Yang (Visiting Scholar) from Guangzho University, China joined the project in 2013 and investigated the supressive effects of mesobiliverdin on angiotensin II induced cardiac muscle smooth muscle inflammation. Research findings by students were presented at several meetings. They include: annual meetings of the USU USTAR Synthetic Biomanufacturing Institute in Winter 2014 and 2015, the 31st Annual Conference of the American Society for Gravitational and Space Research, Alexandria, VA, November, 2015, and the Hansen Life Sciences Graduate Symposium in 2013 and 2015. Several graduate student fellowship proposals were submitted to various agencies for supplemental funding of this project. In 2015, two grant proposals funded: a Utah NASA Space Grant Consortium Fellowship to Charles Harding and a USTAR grant on Novel Therapeutics to P.I. J. Takemoto. Several grant proposals (to the American Heart Association, NIH, and NASA) are currently pending. How have the results been disseminated to communities of interest?Dissemination Research results from this project were presented at several venues such as the USU Commercial Enterprises USTAR meetings sponsored in Winter 2014 and 2015, the 31st Annual Conference of the American Society for Gravitational and Space Research, Alexandria, VA, November, 2015, and the Hansen Life Sciences Graduate Symposium in 2013, 2014, and 2015. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work This is the final report for the period July 1, 2013 to June 30, 2016. A new grant beginning July 1, 2016 has been funded by the Utah AES (USDA NIFA program) for 3 years to continue the research on mesobiliverdin IXalpha anti-inflammatory effects initiated by the current grant. The research will focus on inflammatory diseases of the human eye (e.g. age-related macular degeneration and retinal fibrosis).
Impacts
What was accomplished under these goals?
Accomplishments 1) A mesobiliverdin IXalpha ammonium salt with improved water solubility was produced. This form of the compound was shown to protect against peroxidation of linoleic acid to linoleic hydroperoxide. This technical accomplishment solved issues with solubility and pH inconsistencies that made experimental use of the compound difficult. As a result, the generated data on the effects of mesobiliverdin IXalpha on cellular and animal inflammatory phenomena were more consistent. 2) Mesobiliverdin IXalpha was shown to suppress cell viability inhibition and lipid peroxidation of hydrogen peroxide treated CHO-1-15 cells and arterial smooth muscle A7r5 cells responding to angiotensin II. Cell viability was measured using NADH dehydrogenase-dependent tetrazolium- formazan activity and lipid peroxidation was assessed by measurement of dichlorohydrofluorescein (DCHF) fluorescence. 3) Mesobiliverdin IXalpha was shown to suppress hydrogen peroxide induced inflammatory responses in cultured human ARPE-19 cells as assessed by measurement of cell proliferation, lipid peroxidation, and heme oxygenase activities. 4) Mesobiliverdin IXalpha was shown to suppress microgravity induced cell morphological changes of C2C12 myocytes (an inflammatory response) as assessed by confocal microscopy, measurement of lipid peroxidation, and Western blot analysis of muscle proteins.
Publications
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Target Audience Target audiences are the pharmaceutical industry, farm animal veterinary care sectors, domestic animal veterinary care sectors, and farming communities Changes/Problems:Changes/Problems Changes The originally proposed analysis of pancreata tissues of mesobiliverdin IXα−fed and control db/db type 2 diabetic mice has been displaced by the above proposed studies of retinal ARPE-19 and skeletal muscle C2C12. These latter studies emerged from recent and unpredicted findings that are of direct relevance to the overall and original goals of this UAES project. The findings are promising, significant for inflammation research, and offer potentially significant future therapeutic outcomes. What opportunities for training and professional development has the project provided?Opportunities Training Activities In 2015, this project provided opportunities for research training and experiences to several undergraduate and graduate students. Graduate students Susie Robinson (M.S., completed, Biology), Charles Harding (Ph.D. candidate, Biological Engineering), Farhad Farjood (Ph.D. candidate, Biological Engineering) pursued projects on the effects of mesobiliverdin IXalpha on anti-inflammatory cytoprotection of ARPE-19 cells. Five undergraduate students (Garrett Hinton, Seth Drury, Matthew Argiro, Tyler Gladwin, and Cody Maxfield) and graduate student Jonathan Wood (Biological Engineering) participate in several research activities including mesobiliverdin IXalpha and phycocyanobilin production, lipid peroxidation assays, and ARPE-19 cytoprotection experiments. How have the results been disseminated to communities of interest?Dissemination Several posters and presentations were made at local and national meetings. These included the USU Commercial Enterprises meeting sponsored by the Utah Science, Technology, and Research (USTAR) Synthetic Biomanufacturing Institute, Logan, Utah, January 2015, the 31st Annual Conference of the American Society for Gravitational and Space Research, Alexandria, VA, November, 2015, and the Hansen Life Sciences Graduate Symposium, Wellsville, Utah, September, 2015. Several grant and graduate student fellowship proposals were submitted to various agencies for supplemental funding of this project. In 2015, two have been funded: a Utah NASA Space Grant Consortium Fellowship to Charles Harding and a USTAR grant on Novel Therapeutics to P.I. J. Takemoto. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work 1) Mesobiliverdin IXalpha activation of biliverdin reductase by retinal pigment epithelium (ARPE-19). Experiments are planned to test the ability of mesoBV to cause autophosphorylation of human biliverdin reductase - an event that occurs with substrate binding of biliverdin IXalpha to the enzyme. Biliverdin reductase autophosphorylation is the initial event in signaling pathways that lead to transcriptional induction of anti-inflammatory and inhibition of pro-inflammatory processes. The studies will be conducted using immunopreciptation methods using immunoglobulins directed against human biliverdin reductase A followed by Western blotting techniques with anti-phosphoserine/threonine monoclonal antibodies. The levels of autophosphorylation with addition of native biliverdin IXalpha, mesobiliverdin IXalpha or phycocyanobilin will be compared. Following experiments with purified human biliverdin reductase A, complementary experiments will be performed with addition of biliverdin IXalpha and mesobiliverdin IXalpha to intact ARPE-19 cells and analysis of cell extracts using immunoprecipitation methods. In both in vitro and cellular experiments, similar rates of autophosphorylation are expected with biliverdin IXalpha and mesobiliverdin IXalpha and no or lower rates are expected with phycocyanobilin. Such results will indicate the potential of mesobiliverdin IXalpha as a therapeutic for acute and chronic inflammatory conditions that benefit from physiological responses involving heme-derived metabolites. 2) Mesobiliverdin IXalpha cytoprotection of skeletal muscle C2C12 cells against oxidative stress. Experiments will be conducted on the inflammatory responses of oxidatively-stressed C2C12 skeletal muscle cells subjected to microgravity and the cytoprotection of mesobiliverdin IXalpha on these responses. Cultured C2C12 cells will be bound to glass microbeads, subjected to microgravity, and the degree of lipid peroxidation will be observed by DCHF fluorescence using confocal fluorescence microscopy. Mesobiliverdin IXalpha (5 to 100uM) will be added to the cells before, at the same time, or at various times after exposure to microgravity. Mesobiliverdin IXalpha effects on DCHF fluorescence at all concentrations will be measured. The results will determine if mesobiliverdin IXalpha cytoprotects skeletal muscle cells from oxidative-stress induced formation of reactive oxygen species as observed for ARPE-19 cells. This research with skeletal muscle cells represents a new addition to the originally proposed studies. The objectives, however, of demonstrating cytoprotection of oxidative stress by mesobiliverdin IXalpha, are identical and directly related to the originally proposed studies with CHO 1-15, A7r5, and pancreatic tissue of this UAES project. Only the targeted cells differ as they are found to be more amenable to the experimental approaches.
Impacts
What was accomplished under these goals?
Accomplishments Specific Objectives Met 1) Determined mesobiliverdin IXalpha suppression of reactive oxygen species production in oxidatively stressed cultured age-related retinal pigment epithelium-19 (ARPE-19) cells. 2) Determined that mesobiliverdin IXalpha suppression of reaction oxygen species production in ARPE-19 cells is not due solely to direct anti-oxidative suppression of lipid peroxidation. Significant Results Achieved Objective 1 The RPE of the neurosensory mammalian retina nourishes retinal visual cells and firmly attached to the underlying choroid and overlying retinal visual cells. Dysfunctions of the RPE are major factors in eye disease development, principally age-related macular degeneration, retinitis pigmentosa and diabetic retinopathy. An important contributor to the development of these diseases is oxidative stress. Earlier studies suggested that heme-derived metabolites including biliverdin IXalpha provided cytoprotection of RPE from oxidative stress. Therefore, in 2015, this project focused on the capabilities of mesobiliverdin IXalpha to suppress oxidative stress induced inflammatory responses in cultured rat ARPE-19 cells. Cultured ARPE-19 cells were stressed with exposure to H2O2 and cellular reactive oxygen species levels were assessed by measurement of dichlorohydrofluorescein (DCHF) fluorescence. Mesobiliverdin IXalpha (5 to 100uM) was added to the cells before, at the same time, or at various times after treatment with H2O2. Mesobiliverdin IXalpha inhibited DCHF fluorescence at all concentrations and did not affect growth rates or cell viabilities. Similar observations were obtained by examination of H2O2-treated ARPE-19 cells by confocal fluorescence microscopy exposed to mesobiliverdin IXα. These results indicate that as with biliverdin IXα, mesobiliverdin IXalpha cytoprotects ARPE-19 cells from cellular oxidative damage. In preliminary experiments, similar mesobiliverdin IXalpha suppressive effects were obtained with cultured rat skeletal muscle C2C12 cells that were subjected to microgravity-induced oxidative stress using a rotary cell culture apparatus (Synthecon, Inc.). Objective 2 The relative contribution of the anti-oxidative capabilities of mesobiliverdin IXalpha against lipid peroxidation to cellular cytoprotection by mesobiliverdin IXalpha was studied. Effects of mesobiliverdin IXalpha in an in vitro biochemical system were compared with those in ARPE-19 cells. In the cell-free biochemical system, 2, 2'-azobis(2,4-dimethylvaleronitrile) (AMVN) was used to initiate superoxide anion-dependent peroxidation of linoleic acid (lipid peroxidation). Mesobiliverdin IXalpha, biliverdin IXalpha, and phycocyanobilin all inhibited AMVN-initiated linoleic acid peroxidation equally well. In contrast, with H2O2-treated ARPE-19 cells, mesobiliverdin IXalpha and biliverdin IXalpha suppressed reactive oxygen species production to a greater degree than did phycocyanobilin. The results indicate that the cellular suppression effects of mesobiliverdin IXalpha and biliverdin IXalpha are not due solely to direct anti-oxidative action against lipid peroxidation). It may be speculated that mesobiliverdin IXalpha and biliverdin IXalpha promote additional anti-inflammatory systems in ARPE-19 cells that are not done so by phycocyanobilin. Among these systems, the most likely is activation of biliverdin reductase by autophosphorylation - an event known to initiate signaling pathways leading to induction of anti-inflammatory and inhibition of pro-inflammatory processes.
Publications
- Type:
Other
Status:
Other
Year Published:
2015
Citation:
Publications
Other
Takemoto, J. Y., Chen, D., Chang, C. W. T., Jonathan, W. L. (2015). Therapeutic mesobiliverdin IXalpha compositions and associated methods, Patent assigned, US9119842 B2. United States Patent Office.
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Target Audience Target audiences are the pharmaceutical industry, farm animal veterinary care sectors, domestic animal veterinary care sectors, and farming communities. Changes/Problems: Changes/Problems Changes The originally proposed analysis of pancreata tissues of mesobiliverdin IXα−fed and control db/db type 2 diabetic mice has been delayed although it continues to be in progress. The pancreatic tissues are stored at minus 20o C which affords their analyses at any time. As work for 2014 objectives 1 and 2 are completed, time and resources (limited for this project) will be diverted to the analytical pancreatic tissue work in 2015. What opportunities for training and professional development has the project provided? Opportunities Training Activities This project permitted opportunities for one undergraduate and one graduate student to participate in research experiences. Master's degree candidate Susie Robinson conducted experiments on the effects of mesobiliverdin on CHO cell growth and viability and anti-inflammatory effects against ARPE-19 retinal pigment cells. She gained valuable knowledge and research experience in animal cell culture growth and cell biological approaches and techniques. Undergraduate student Garrett Hinton conducted experiments on the effects of mesobiliverdin on lipid peroxidation initiated by superoxide anion. A visiting scholar from China, Dr. Lai Yang also participated and trained on this research from August 2013 to August 2014. Dr. Yang investigated the effects of mesobiliverdin on angiotensin II-induced oxidative stress of vascular smooth muscle cells A7r5 and is continuing the work in China in a collaborative arrangement with our group at Utah State University. How have the results been disseminated to communities of interest? Dissemination The goals and early results of this project were presented at a USU Commercial Enterprises meeting sponsored by the Utah Science, Technology, and Research (USTAR) Synthetic Biomanufacturing Institute in January 2014. The purpose of this meeting was to analyze the potential of the technology proposed for development and commercialization of human and animal therapeutics. The CHO 1-15 work was the subject for a Masters of Science degree thesis authored by Susie Robinson and published in December 2014. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work 1) Mesobiliverdin IXα effects on retinal pigment epithelium (RPE) cells. The RPE is cell layer just outside the neurosensory mammalian retina that nourishes retinal visual cells, and is firmly attached to the underlying choroid and overlying retinal visual cells. Dysfunctions of the RPE are major factors in the development of eye diseases, principally age-related macular degeneration, retinitis pigmentosa and diabetic retinopathy. An important contributor to the development and progression of these diseases is oxidative stress. Studies will focus on the capabilities of mesobiliverdin IXα to suppress oxidative stress processes in RPE cells. In particular, mesobiliverdin IXα will be tested for capabilities to provide cytoprotection against oxidative damage. This research with RPE cells represents a new addition to the originally proposed studies. It is, however, closely and directly related to the originally proposed studies with CHO 1-15, A7r5, and pancreatic tissue of this UAES project. Human ARPE-19 RPE primary cell line will be used in these studies. In November 2014, we succeeded in culturing, storing, and propagating these cells. The cells will be exposed to a series of oxidative stress initiators that include menadione, H2O2 and light + hypoxia. The major oxidation stress parameter, lipid peroxidation, will be measured using the lipophilic fluorescent dye diphenyl-1-pyrenylphosphine oxide (DPPP) which fluoresces with hydroperoxide formation. Lipid peroxidation catalyzed by superoxide anion is an early and major process leading to generalized inflammatory responses and known to occur in age-related macular degeneration (Kopitz et al., 2004; Kaemmerer et al. 2007, Yin et al. 2011) 2) Pancreata tissue analyses of mesobiliverdin IXα−fed diabetic mice. As initially proposed the following analyses are planned: a) NADPH oxidase. Immunostaining of 8-OHdG, a marker of oxidative DNA damage and gp91phox and Nox4, components of NADPH oxidase induced with oxidative stress will be examined for relative amounts in pancreatic tissues of mesobiliverdin IXα−fed and control mice. Excised pancreata fixed in 10% formaldehyde will be embedded in paraffin and sectioned (3 μm). After inactivation of endogenous peroxidase with 3% (vol/vol) H2O2 and preincubation with 1% (wt/vol) BSA, the samples will be exposed to anti-human 8-OHdG goat polyclonal antibodies, anti-human p91phox goat polyclonal antibodies, or anti-human Nox4 goat polyclonal antibodies and probed with anti-goat peroxidase-labelled IgG antibodies. Primary antibodies will be replaced with normal goat serum as negative controls. Sections will be counterstained with haematoxylin and examined under a light microscope. Peroxidase abundance will then be visualised with diaminobenzidine. b) Superoxide production. Pancreata tissues of mesobiliverdin IXα−fed and control mice will be stained with dihydroethidium by intravenous administration of this dye through the right jugular vein under anaesthesia. At 2 h after injection, the mice will be killed by transcardial perfusion with formaldehyde and the pancreata frozen immediately. Pancreata tissue will be examined for nuclear staining using Hoechst 33258 (Invitrogen) and an Olympus IX81 inverted fluorescence microscope. c) Gene expression of oxidative stress markers. Expression of Pdx1, calcyclin, synaptosomal-associated protein of 25 kDa (Snap25), Bax, activating transcription factor 3 (Atf3), Fas, glutathione peroxidase-1, gp91phox, in pancreata extracts will be determined by real-time quantitative RT-PCR. Total RNA will be extracted from frozen pancreatic islet samples of mesobiliverdin IXα−fed and control mice, converted to single-stranded cDNA using Superscript III reverse transcriptase , and subjected to PCR reactions using specific primers. β-Actin was used as internal control. The specificity of PCR amplification will be confirmed by melting curve analysis and agarose gel electrophoresis. d) Apoptosis detection. Pancreatic islet cell apoptosis will be determined by TUNEL staining in pancreatic tissue from mesobiliverdin IXα−fed and control miee. In situ detection kit (Wako) and the results will be expressed as the percentage of TUNEL-positive islet β-cells. References Kopitz, J., Holz, F.G., Kaemmerer, E., and Schutt, F. (2004). Lipids and lipid peroxidation products in the pathogenesis of age-related macular degeneration. Biochimie 86, 825-831. doi: http://dx.doi.org/10.1016/j.biochi.2004.09.029. Yin, J., Thomas, F., Lang, J.C., and Chaum, E. (2011). Modulation of oxidative stress responses in the human retinal pigment epithelium following treatment with vitamin C. J Cell Physiol 226, 2025-2032. doi: 10.1002/jcp.22532. Kaemmerer, E., Schutt, F., Krohne, T.U., Holz, F.G., and Kopitz, J. (2007). Effects of Lipid Peroxidation-Related Protein Modifications on RPE Lysosomal Functions and POS Phagocytosis. Investigative Ophthalmology & Visual Science 48, 1342-1347. doi: 10.1167/iovs.06-0549.
Impacts
What was accomplished under these goals?
Accomplishments Specific Objectives Met 1) Determine the cellular inflammatory responses suppressed by mesobiliverdin IXα in CHO 1-15 and A7r5 cells subjected to oxidative stress, 2) Measure the anti-lipid peroxidation activity of mesobiliverdin IXα with linoleic acid and oxidatively stressed growing cells. Significant Results Achieved Objective 1 Experiments were conducted on the inflammatory responses of oxidatively-stressed CHO I-15 cells and angiotensin-treated vascular smooth muscle A7r5 cells and the influences of mesobiliverdin IXα on these responses. CHO-1-15 cells. Suspension CHO 1-15 cells were stressed with exposure to H2O2 or menadione and lipid peroxidation levels were determined. Mesobiliverdin IXα was added to the cells before, at the same time, or at various times after treatment with these strong oxidants. In all cases, mesobiliverdin IXα had no effect on the growth rates or cell viabilities at concentrations up to 100 uM. At higher concentrations mesobiliverdin IXα, growth inhibition was measured. Similar results were obtained with biliverdin IXα and phycocyanobilin. A7r5 cells. Vascular smooth muscle A7r5 cells were purchased from the American Type Culture Collection (ATCC), propagated, and cultured, and exposed to angiotensin II initiated oxidative stress. The major oxidation stress parameter, lipid peroxidation, was measured using the lipophilic fluorescent dye diphenyl-1-pyrenylphosphine oxide (DPPP) which fluoresces with hydroperoxide formation. Lipid peroxidation catalyzed by superoxide anion is an early and major process leading to generalized inflammatory responses. The effects of mesobiliverdin IXα on DPPP fluorescence were then determined. The results indicate that angiotensin II caused lipid peroxidation that in turn was suppressed by mesobiliverdin IXα at concentrations between 10 and 80 ug/mL. Superoxide forming NADPH oxidase (NOX) gene expression was measured by real-time PCR of NOX1, NOX2, NOX 3, and NOX4 gene transcripts. Angiotensin II added to A7r5 caused an increase in NOX1 and NOX4 gene expression but had no measureable effects on NOX2 and NOX3 expresssion. In one initial experiment, the addition of mesobiliverdin IXα suppressed NOX4 expression, but not NOX1. These experiments are being currently pursued. Objective 2 The anti-oxidative capabilities of mesobiliverdin IXα (ammonium salt) to suppress lipid peroxidation were explored in more depth. In a cell-free biochemical system, the chemical catalyst 2, 2'-azobis(2,4-dimethylvaleronitrile) (AMVN) was used to initiate superoxide anion-dependent peroxidation of linoleic acid to its corresponding hydroperoxide. The effects of mesobiliverdin IXα, bilirubin, biliverdin IXα, and phycocyanobilin on this activity were measured. The conversion to the fatty acid hydroperoxide was measured using high-performance liquid chromatography. The results showed that mesobiliverdin IX inhibited linoleic acid lipid peroxidation in a dose-dependent manner. Similar results were achieved with biliverdin IX, bilirubin, and phycocyanoblin.
Publications
- Type:
Other
Status:
Other
Year Published:
2014
Citation:
Takemoto, J. Y., Chen, D., Chang, C.-W. T., Jonathan, W. L. (2014). Therapeutic compositions and associated methods. United States Patent Office.
- Type:
Other
Status:
Other
Year Published:
2014
Citation:
Takemoto, J. Y., Chen, D., Chang, C.-W. T., Jonathan, W. L. (2014). Therapeutic compositions and associated methods. World Intellectual Property Organization.
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Progress 08/22/13 to 09/30/13
Outputs
Target Audience: Target audiences are the pharmaceutical industry, farm animal veterinary care sectors, domestic animal veterinary care sectors, and farming communities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Training Activities. This project permits opportunities for two undergraduate students to participate in first-hand researcgh experiences. One student (G. Nelson) conducted experiments on the effects of mesobiliverdin on CHO cell growth and viability, he is gaining valuable knowledge and experince in animal cell culture growth and techniques. The second student (G. Hinton) is conducting experiments on the effects of mesobiliverdin on lipid peroxidation initiated by superoxide anion. An opportunity to have a visiting scholar from China, Dr. Lai Yang, unexpectedly arose in August 2013. Dr. Yang is a expert on angiotensin-induced inflammatory responses in vascular smooth muscle cells. She has joined the project and is investigating the effects of mesobiliverdin on this inflammatory system. This system is very similar and highly relevant to the inflammatory processes described in the original proposal. How have the results been disseminated to communities of interest? The goals and early results of this project were presented at a USU Commercial Enterprises meeting sponsored by the Utah Science, Technology, and Research (USTAR) Synthetic Biomanufacturing Institute. The purpose of this meeting was to analyze the potential of the technology proposed for development and commercialization of human and animal therapeutics. What do you plan to do during the next reporting period to accomplish the goals? Plan of Work Analysis of mesobiliverdin effects on CHO I-15 and A7r5 cells In the last 2 to 3 months, the technical difficulties related to mesobiliverdin solubility and pH toxicity of cell cultures were resolved with the production of the ammonium salt of mesobiliverdin. This “breakthrough” now opens the way for the studies aimed at examining the influences of mesobiliverdin on anti-inflammatory responses. The studies will focus on the CHO I-15 oxidative stress and angiotensin-induced inflammation of vascular smooth muscle A7r5 cells. The latter activity is a new, but highly relevant, addition to the aims of the project prompted by newly arrived visiting scholar, Lei Yang, who is a specialist on this inflammatory system. NADPH oxidase levels. A major initial event in inflammatory responses is superoxide anion production due to the activation of NADPH oxidase (Nox4). The influences of mesobiliverdin on Nox4 protein expression levels will be determined. Extracts of treated and harvested cells will be subjected to SDS-PAGE and Western blotting using anti-Nox4 (Abcam, Cambridge, MA) and anti-β-actin primary antibodies to normalize the levels. The blot membranes will be incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody or sheep anti-mouse IgG antibody, developed and imaged. NADPH oxidase enzyme activities and NADPH-dependent superoxide anion production by mesobiliverdin treated and non-treated extracts will be measured. Anti-lipid peroxidation activity of mesobiliverdin. Lipid peroxidation catalyzed by superoxide anion is an early and major process leading to generalized inflammatory responses. The anti-oxidative capabilities of mesobiliverdin (ammonium salt) to suppress this activity will be explored in depth. The chemical catalyst AMV will be used to initiate superoxide anion-dependent peroxidation of linoleic acid and other fatty acids to their corresponding hydroperoxides. The effects of mesobiliverdin, bilirubin, biliverdin, and phycocyanobilin on this activity will be measured. The conversion of fatty acids to fatty acid hydroperoxides will measured using high-performance liquid chromatography. Oxygen consumption in superoxide anion production will be measured using an oxygen detecting probe. Mice feeding experiments. These experiments will continue as originally proposed. NADPH oxidase. Immunostaining of 8-OHdG, a marker of oxidative DNA damage and gp91phox and Nox4, components of NADPH oxidase induced with oxidative stress will be examined for relative amounts in pancreatic tissues of mesobiliverdin IXα−fed and control mice. Excised pancreata fixed in 10% formaldehyde will be embedded in paraffin and sectioned (3 μm). After inactivation of endogenous peroxidase with 3% (vol/vol) H2O2 and preincubation with 1% (wt/vol) BSA, the samples will be exposed to anti-human 8-OHdG goat polyclonal antibodies, anti-human p91phox goat polyclonal antibodies, or anti-human Nox4 goat polyclonal antibodies and probed with anti-goat peroxidase-labelled IgG antibodies. Primary antibodies will be replaced with normal goat serum as negative controls. Sections will be counterstained with haematoxylin and examined under a light microscope. Peroxidase abundance will then be visualised with diaminobenzidine. Superoxide production. Pancreata tissues of mesobiliverdin IXα−fed and control mice will be stained with dihydroethidium by intravenous administration of this dye through the right jugular vein under anaesthesia. At 2 h after injection, the mice will be killed by transcardial perfusion with formaldehyde and the pancreata frozen immediately. Pancreata tissue will be examined for nuclear staining using Hoechst 33258 (Invitrogen) and an Olympus IX81 inverted fluorescence microscope. Gene expression of oxidative stress markers. Expression of Pdx1, calcyclin, synaptosomal-associated protein of 25 kDa (Snap25), Bax, activating transcription factor 3 (Atf3), Fas, glutathione peroxidase-1, gp91phox, in pancreata extracts will be determined by real-time quantitative RT-PCR. Total RNA will be extracted from frozen pancreatic islet samples of mesobiliverdin IXα−fed and control mice, converted to single-stranded cDNA using Superscript III reverse transcriptase, and subjected to PCR reactions using specific primers. β-Actin was used as internal control. The specificity of PCR amplification will be confirmed by melting curve analysis and agarose gel electrophoresis. Apoptosis detection. Pancreatic islet cell apoptosis will be determined by TUNEL staining in pancreatic tissue from mesobiliverdin IXα−fed and control mice. In situ detection kit (Wako) and the results will be expressed as the percentage of TUNEL-positive islet β-cells.
Impacts
What was accomplished under these goals?
Specific Objectives Met The specific objectives are to determine the cellular inflammatory responses suppressed by mesobiliverdin IXα in: 1) pancreatic tissues of type 2 diabetic mice fed diets containing mesobiliverdin IXα, and 2) Chinese Hamster Ovarian (CHO) cells growing in suspension under oxidative stress. Specific objective 1) PROCEDURES: Mice feeding experiments were initiated. Male db/db mice were obtained by Dr. Dale Abel, School of Medicine, University of Utah and a controlled powdered diet supplemented with mesobiliverdin IXα at two levels was formulated and produced. Feeding experiments as proposed were initiated. RESULTS: The experiments are in process and no data have been compiled yet. Specific objective 2) PROCEDURES: For specific objective 2): Procedures. CHO cell line I-15 was propagated in HyCell medium with and without antioxidant alpha-tocopherol supplementation and in suspension in a Applikon Bioreactor. At designated times hydrogen peroxide was added to induce oxidative stress, and various concentrations of mesobiliverdin were added and cell counts and viability monitored by Trypan Blue staining in a Beckman Vi cell analyzer. Controls had no bile pigment addition. At 1 to 5 h intervals, culture aliquots were removed for determination of viability. RESULTS: Addition of H2O2 to CHO I-15 cell suspensions caused a decrease in cell viability as expected of this strong oxidant. With no H2O2 added and several H2O2 concentrations, mesobiliverdin added at 25 and 50 microM in 50% methanol at acidic pH caused a further decrease in CHO cell viability. The use methanol and acidic pH was speculated to cause this cellular toxicity effect. Therefore, a procedure for making an ammonium salt of mesobiliverdin that is water-soluble was developed and used in subsequent experiments. Initial experiments showed that addition of the mesobiliverdin ammonium salt in water at pH7 to the CHO cell suspension at concentrations as high as 100 microM did not affect cell viability. The mesobiliverdin ammonium salt was observed preliminarily to protect against peroxidation of linoleic acid to linoleic hydroperoxide. Experiments to observe the effects of the mesobiliverdin ammonium salt on cell viability after addition of H2O2 are currently underway.
Publications
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