Progress 07/01/16 to 06/30/20
Outputs
(N/A)
Impacts
What was accomplished under these goals?
Cytoprotection by mesobiliverdin of human retinal pigmented epithelial cells (ARPE-19 and polarized embryonic stem cell (ESC)-derived) against oxidative stress and damage was demonstrated. It was shown that a cellular mechanism of mesobiliverdin cytoprotection against oxidative stress is to suppress cell cycle progression to cell scenescence. The suppressive mechanism was shown to occur at via cell cycle stage 1 regulatory protein kinase inhibitors p21 and p16 and by induction of heme oxygenase 1 which is a universal regulator of anti-inflammatory cell responses. These findings with human retinal pigmented epithelial cells indicate the potential of mesobiliverdin to be a therapeutic against age-related dry macular degeneration (the leading cause of blindness). To facilitate the project research, the production of pure mesobiliverdin from microalgae feedstock was accomplished and green and scalable methods for production in gram amounts were achieved. A Biotek Cytation5 Multimode Reader and Imaging System was acquired with partial support from this project. This instrument has facilitiated more recent studies on the mechanisms of mesobiliverdin cytoprotection against oxidative stress in age related macular degeneration and other animal inflammatory conditions of agricultural and medical importance. The other conditions include intestinal bowel diseases, cardiovascular diseases and osteoporosis. Recently emerging observations show a similar pattern of inhibition of hyper-inflammatory cytokine expression - most notably of IL-6 - in experimental models for all of these inflammatory disease conditions.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Takemoto, J. Y., & Chen, D. (2017, January 20). Recombinant non-animal cell for making biliverdin, US14/854,791, Issued Jan 20 2017. U.S. Patent and Trademark Office.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Zhang, W., Rhodes, J., Garg, A., Takemoto, J. Y., Qi, X., Harihar, S., Chang, C. W. T., Moon, K., & Zhou, A. (2020, September). Label-free discrimination and quantitative analysis of oxidative stress induced cytotoxicity and potential protection of antioxidants using Raman spectroscopy and machine learning algorithms. Analytical Chemistry, 1128, 221-230.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Burton, T. J. (Presenter & Author), Bryson, C. (Author Only), Takemoto, J. Y. (Author Only), Sims, R. C. (Author Only), National Conference of Undergraduate Research, "Culturing cyanobacteria in wastewater based suspension for mesobiliverdin production," Montana State University, Bozeman MT. (March 26, 2020 - March 28, 2020)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Presentations
Clegg, M. (Presenter & Author), Soboleva, T. (Author Only), Berreau, L. (Author Only), Takemoto, J. Y. (Author Only), Vargis, E. (Author Only), American Society for Gravitational and Space Research (ASGSR), "In Vitro Muscular Atrophy Model with Suspended Muscle Fibers." (October 2018)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2016
Citation:
Presentations
Takemoto, J. Y. (Author Only), Harding, C. (Author Only), Hinton, G. (Author Only), Robinson, S. (Author Only), Chen, D., Vargis, E., SBC Science and Technology Review, "Mesobiliverdin IXalpha cytoprotects retinal pigmented epithelial cells against oxidative stress," Synthetic Biomanufacturing Center, Utah State University. (November 29, 2016)
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Veterinarians Animal farmers Veterinary pharmaceutical industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for several Utah State University undergraduate students to participate in research experiences. Jordyn Brown, Justin Evans, Matt Lambert, Robert Hunt, Matt Armbrust, Christina Ngyuen, Thomas Burton and Stephen Short produce mesobiliverdin and phycocyanobilin from feedstock microalgae and biochemically analyze these metabolites that are used in the cytoprotection experiments. This project permitted opportunities for graduate students to pursue oxidative stress cell research. Matthew Clegg (M.S. Biological Engineering) and Jonathan Wood (M.S, Biological Engineering) conducted research related to mesobiliverdin and phycocyanobilin production and their cytoprotection against oxidatively stressed animal cells. This project provided opportunities for Logan High School student Ankit Garg to experience biological research and to win 2nd place in a local science fair competition. How have the results been disseminated to communities of interest?The results of this project have been disseminated through peer reviewed publications and scientific presentations. ? Publications peer reviewed in scientific journals: Takemoto, J.Y., C.W.T. Chang, D. Chen, and G. Hinton (2019) Heme-derived bilins. Isr. J. Chem. 59: 378-386 Chang, C.W. T. , J.Y. Takemoto, J. Zhan (2019) Natural bioactive compounds. Isr. J. Chem. 59:325-326. In preparation: Takemoto, JY, Spee, C., Ooyang, S. Hinton, G., Harding, C., Farjood, F., Vargis, E. and Hinton DR. 2020. Cytoprotection of oxidatively-stressed human retinal pigmented epithelial cells by mesobiliverdin IXa. Invited Talk: Takemoto, Jon. Biliverdin and Mesobiliverdin: New and (Very) Old Anti-inflammatories for Diabetes, March 29, 2019 . Diabetes Care Conference, Taichung Veterans General Hospital, Taichung, Taiwan Conference Presentations: Takemoto, Jon (Invited Lecture). Biliverdin and Mesobiliverdin: New Anti-inflammatories for Diabetes. March 31, 2019, 2019 Annual Meeting of the Diabetes Association and the Endocrinology Society of the R.O.C. Taiwan, Taipei, Taiwan. Armbrust, M., J. Y. Takemoto and R. C. Sims. (Poster) Mesobiliverdin production by conversion of cyanobacteria-derived phycocyanobilin . Intermountain Biological Engineering Conference, November 11, 2019, Utah State University Logan Utah. Burton, T. J. C. Bryson, J. Y. Takemoto and R. C. Sims. (Poster). Culturing cyanobacteria in wastewater based suspension for mesobiliverdin production. National Conference of Undergraduate Research, Montana State University, Bozeman, MT., March 26-28, 2020. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Experiments on the roles of ZO-1 protein and mitochondrial oxidation state in NaI induced oxidative stress and suppression by mesobiliverdin IXα will be completed. It is hypothesized that both cell scenescence markers will be suppressed with addition of mesobiliverdin IXα in combination with sublethal concentrations of NaI (e.g. 1mM). The results could provide conclusive evidence that mesobiliverdin IXα provides cytoprotection against oxidative stress in ARPE-19 cells by suppressing entry into NaI-induced cell scenescence. Objective 2: The effects of mesobiliverdin IXα on the enzyme biliverdin reductase A (BVRA) will be studied. BVRA catalyzes the reduction of biliverdin IXα to bilirubin. Both compounds are strong anti-oxidants, and therefore the enzyme plays a pivotal role in the antioxidant properties of biliverdin IXα and ROS suppression. Upon binding and reduction of biliverdin IXα, BVRA undergoes autophosphorylation that initiates a cell signaling cascade leading to anti-inflammatory effects including suppression of ROS production. Experiments will be conducted to determine if mesobiliverdin IXα (like biliverdin IXα) is capable of causing BVRA autophosphorylation. NaDodSO4 gel electrophoresis and Western blotting will be used to immunodetect BVRA and determine if autophosphorylation of BVRA occurs when exposed to mesobiliverdin IXα. ARPE-19 cell extracts or purified human BVRA will be heat-treated, and immunodetection of BVRA and phosphorylated BVRA will be performed using anti-BVRA polyclonal rabbit or mouse monoclonal IgG primary antibodies. It is hypothesized that mesobiliverdin IXα will cause the activation of BVRA autophosphorylation as an early step in the signaling pathway that leads to anti-inflammatory responses. Such a result will contribute to the accumulated knowledge of the cell molecular mechanisms that underlie mesobiliverdin IXα's role as a cytoprotective anti-inflammatory against responses involving oxidative stressor ROS species.
Impacts
What was accomplished under these goals?
Studies on the mechanisms of mesobiliverdin suppression of oxidative stress and stress-associated cell scenescence of retinal pigmented epithelial cells were conducted with ARPE-19 cells. Two to 3-week old serially passaged ARPE-19 subconfluent or just-confluent cells were exposed for various time intervals to the oxidant NaI (1 - 25 mM) and 1 μM to10 μM mesobiliverdin IXα, examined microscopically, and assayed for cell viability and cell scenescence -associated lysosomal leakage of beta-galactosidase (SA beta-gal) activity. At various times after exposure to NaI, cellular material was extracted and the levels of protein indicators of cell death and scenescence were determined by NaDodSO4-gel electrophoresis, Western blotting, and immunochemical detection and quantitation. A newly acquired Biotek Cytation 5 reader and imaging system was used to measure growth, cell numbers, and for conducting quantititative assays of markers indicative of oxidative stress and stress-associated cell scenescence. The treated ARPE-19 cells were analyzed for cell viability and death by MTT assay. Cells subjected to two rounds of exposure to sublethal concentrations (1 to 5 mM) of NaI for 2h on consecutive days began to show SA-beta-gal staining 24h after a second NaI treatment at the same NaI concentrations indicating the onset of cell scenescence. Controls not treated with NaI showed negligible SA-beta-gal staining. Exposure to mesobiliverdin IXα (1 or 2 μM) throughout the NaI treatments and thereafter reduced the degree of SA -beta-gal staining indicating suppression of stress-induced cell scenescence. The mesobiliverdin IXα -treated cells displayed lower degree (~ 60%) of SA-beta-gal stained cells compared to non-treated control cells (~ 90% SA beta-gal staining). In related experiments, expression of cell signaling protein markers of cell scenescence, p16 and p21 were measured in ARPE-19 cells subjected to similar regimens of 2h interval exposure to NaI at sublethal concentrations for two days as described above for the SA-beta-gal staining experiments (1 to 5mM). The effect of simultaneous exposure to mesobiliverdin IXα on the expression of these markers was determined. Exposure to sublethal levels of NaI caused increases of p16 and p21; and the simultaneous addition of mesobiliverdin IXα (1 or 2 μM) repressed p16 and p21 expression. Untreated controls showed no increases on p16 and p21. p16 and p21 are cyclin-dependent protein kinase inhibitors that impede specific steps of the cell cycle and cell division that characterize the cell scenescent state. Similar studies of cell scenescence markers, ZO-1, and mitochondrial oxidation states are currently underway. In summary, mesobiliverdin IXα at 1 to 2 μM suppressed stress-associated cell scenescence of ARPE-19 cells induced by NaI via mechanisms that involve decreased expression of cell cycle regulators p16 and p21. The results indicate that mesobiliverdin IXα cytoprotects ARPE-19 cells against oxidative damage and further suggests the potential of mesobiliverdin IXα as as a candidate therapeutic drug against age-related dry macular degeneration.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Chang, C. W. T., Takemoto, J. Y., & Zhan, J. (2019, May). Natural Bioactive Compounds. Israel Journal of Chemistry, 59, 325-326.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Takemoto, J. Y., Chang, C. W. T., Chen, D., & Hinton, G. (2019, February). Heme-derived bilins. Israel Journal of Chemistry, 59, 378-386.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Presentations
Armbrust, M. (Presenter & Author), Takemoto, J. Y. (Presenter & Author), Sims, R. C. (Presenter & Author), 1st Annual Meeting, Intermountain Biological Engineering Conference, "Mesobiliverdin production by conversion of cyanobacteria-derived phycocyanobilin," Intermountain Biological Engineering Society, Utah State University, Logan Utah. (November 11, 2019)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Presentations
Takemoto, J. Y. (Invited Lecture), Annual Meeting, Diabetes Association and Endocrinology Society of the R.O.C. Taiwan, "Biliverdin and Mesobiliverdin: New Anti-inflammatories for Diabetes," Diabetes Association and Endocrinology Society of the R.O.C. Taiwan, Taipei, Taiwan. (March 30, 2019 - March 31, 2019)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Presentations
Takemoto, J. Y. (Guest Speaker), Diabetes Care Conference, "Biliverdin and Mesobiliverdin: New and (Very) Old Anti-inflammatories for Diabetes," Taichung Veterans General Hospital, Taichung, Taiwan. (March 29, 2019)
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:veterinarians animal farmers veterinary pharmaceutical industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided opportunities for Logan High School student David Ban to experience biological research and win a American Chemical Society sponsored science fair competition in Pittsburg, Pennsylvania. This project provided opportunities for several Utah State University undergraduate students to participate in research experiences. Christian Peterson, Ty Nicholas, Jordyn Brown and Stephen Short conducted experiments and produced mesobiliverdin and its synthetic precursor phycocyanobilin used in the RPE cytoprotection experiments. This project permitted opportunities for graduate students to participate in research. Charles Harding (M.S. Biological Engineering), Farhad Farjood (Ph.D. candidate, Biological Engineering), Jonathan Wood (M.S, Biological Engineering) participated in several research activities including mesobiliverdin and phycocyanobilin production, lipid peroxidation assays, and cytoprotection experiments. How have the results been disseminated to communities of interest?Findings from this project were disseminated to the scientific community at the American Chemical Society meeting (Pittsburg section) in Pittsburgh, Pa on Feb 2, 2018. Progress reports of the project were also presented on a monthly basis throughout 2018 to the Utah State University community at meetings of the USU)/Utah Science, Technology and Research (USTAR) Synthetic Biomanufacturing Center. Participants at these meetings were composed of faculty, students, and researchers from the USU academic departments of Biology, Biological Engineering, and Chemistry and Biochemistry. What do you plan to do during the next reporting period to accomplish the goals?Using the newly acquired Biotek Cytation5 Multimode Reader and Imaging system, the mechanisms of mesobiliverdin suppression of oxidation-sensitive cell scenescence cell cycle arrest in ARPE-19 cells will be investigated. Confluent adherent ARPE-19 cells will be synchronized by a thymidine double block procedure in wells of Corning 3904 plates (4000 cells per well with 100 uL Dulbecco's Eagle's medium per well), fixed with 4% PFA. The adhered cells will be stained with DAPI or Hoechst 33342 and cell cycle progression will be monitored as described in BioTek Application Note, Held, P. (2018) Monitoring Cell Cycle Progression in Cancer Cells, Winooski, VT. The entire procedure and analyses will be repeated with various concentrations of NaI with and without 2 uM mesobiliverdin. Cell scenescence markers will be further monitored in the Biotek Cytation 5 system. The markers will include beta-galactosidase (from lysosome leakage) and expression of cell cycle regulators such as p16 and p21 and oxidative stress regulators such as HO-1, FG-2, VEGF and IL-8. These will be analyzed by immunofluorescence microscopy and fluorescence quantitation using the Biotek Cytation5 imaging system and by gel electrophoretic Western blot immunodetection and quantitation. Cell adhesion will be assessed by immunostaining of ZO-1 protein, and mitochondrial oxidation state by staining with MitoTimer reporter and quantitated using the Biotek Cytation5 system. It is hypothesized that the levels of the cell scenescence markers, ZO-1, and mitochondrial oxidation states will be suppressed with addition of mesobiliverdin in combination with 1mM NaI. This outcome will indicate that mesobiliverdin cytoprotects ARPE-19 cells against oxidative damage and is a potential candidate as a therapeutic drug against age-related dry macular degeneration.
Impacts
What was accomplished under these goals?
1) As proposed in the 2017 AD421 report for this project, experiments using ARPE-19 cells to study oxidative-induced cell scenescence in retinal pigmented epithelial (RPE) cells were conducted. ARPE-19 cells were purchased and propagated in Dulbecco's Eagle's medium. The cells were exposed to the oxidant NaI at 1 to 25 millimolar concentrations to induce cell scenescence as determined by causing cell cycle arrest at phase G0. The effects of mesobiliverdin exposure on NaI-induced cell cycle arrest of exposure to 1 and 2 micromolar mesobiliverdin were then determined. Experimental controls were exposed to mesobiliverdin alone and in the absence of both mesobiliverdin and NaI. Cell viability was determined by MTT assay. The cells were stained with propidium iodide and DNA free 3'-OH end TUNEL label to observe cell cycle arrest and apoptosis by fluorescence microscopic imaging and flow cytometry. Experiments were repeated three times. In summary, it was observed that 24h treatment of RPE-19 cells with NaI at 5 millimolar concentration induced cell cycle arrest at stage G0 and that 1 and 2 micromolar mesobiliverdin suppressed this arrest approximately 2- and 5-fold over controls, respectively. Lower concentrations of NaI did not cause arrest and NaI concentrations exceeding 10 mM were cytotoxic. The results show that the oxidizer NaI at a certain concentration (1 mM) induced ARPE-19 cells to enter cell scenescent cell cycle arrest and that mesobiliverdin prevented entry into scenescence in a dose-dependent manner. 2) A new automated cell imaging system (Biotek Cytation5 Multimode Reader and Imaging system) capable of simultaneous cell imaging and quantitation of cell cycle events was acquired and installed. The system allows further studies on the mechanisms of cell scenescence entry induced by NaI and suppression by mesobiliverdin in ARPE-19 cells.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Conference Proceedings
Takemoto, J. Y., & Ban, D. (2018). Mechanisms of Mesobiliverdin Against Oxidative Stress in Retinal Cells. American Chemical Society (Pittsburg Section). 1271
Takemoto, J. Y., & Ban, D. (2018). Mechanisms of Mesobiliverdin Against Oxidative Stress in Retinal Cells. American Chemical Society (Pittsburg Section).
|
Progress 10/01/16 to 09/30/17
Outputs
Target Audience:veterinarians animal farmers veterinary pharmaceutical industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The P.I. conducted the proposed second year research from January through July 2017 as part of a sabbatical leave with collaborators at the University of Southern California Keck School of Medicine, Pathology Department. The P.I.'s collaborator were Dr. David Hinton and other eye disease researchers who offered substantial experience and training in this field. This project provided opportunities for several Utah State University undergraduate students to participate in research experiences. Garrett Hinton, Nick Noyce, and Matt Clegg conducted experiments on cytoprotection of ARPE-19 cells by mesobiliverdin. Christian Peterson, Ty Nicholas, and Jordyn Brown conducted experiments and produced mesobiliverdin and its synthetic precursor phycocyanobilin used in the RPE cytoprotection experiments. This project permitted opportunities for graduate students to participate in research. Charles Harding (Ph.D. candidate, Biological Engineering), Farhad Farjood (Ph.D. candidate, Biological Engineering), Jonathan Wood (M.E. candidate, Biological Engineering), and Sha Ooyang (MD candidate, Univ. So. Calif Keck Medicine) participate in several research activities including mesobiliverdin and phycocyanobilin production, lipid peroxidation assays, and cytoprotection experiments. How have the results been disseminated to communities of interest?• Research results were presented at the annual meeting of the Association for Research in Vision and Opthalmology (ARVO), Baltimore, MD as a poster presentation: (Zhu DH, Takemoto JY, Parameswaran S, Hinton DR. 2017. Exploring cytoprotective molecules for polarized HESC-RPE cells. 2017 Annual Meeting of the Association for Research in Vision and Ophthalmology, Baltimore MD, May 7 -11, 2017.) • Manuscript in preparation: Takemoto, JY, Spee, C., Ooyang, S. Hinton, G., Harding, C., Farjood, F., Vargis, E. and Hinton DR. 2017. Cytoprotection of oxidatively-stressed human retinal pigmented epithelial cells by mesobiliverdin IXalpha. What do you plan to do during the next reporting period to accomplish the goals?The mechanisms of mesobiliverdin cytoprotection against oxidant stress-induced cell scenescence in RPE cells will be investigated. ARPE-19 cells as opposed to fetal eye derived H-RPE cells will be used since the latter are limited for mechanism of action studies. H-RPE are difficult to obtain, can only be passaged up to 3 or 4 times, and are not amenable to genetic alteration. These are experimental limitations that are not encountered with ARPE-19 cells. Based on a recent publication by Zhang et al. (2016. Continuous exposure to non-lethal doses of sodium iodate induces retinal pigment epithelial cell dysfunction. Scientific Reports 6, 37279), the mechanisms of mesobiliverdin suppression of oxidative-induced cell scenescence in RPE cells will be pursued with ARPE-19 cells. In brief, ARPE-19 cells propagated in Dulbecco's Eagle's medium will be exposed to the oxidant NaI at 1 to 10 millimolar concentrations in the presence or absence of 1 and 2 micromolar mesobiliverdin. Experimental controls will include exposure to mesobiliverdin alone and to no mesobiliverdin/NaI combination. Cell viability will be determined by MTT assay. The cells will be harvested stained with propidium iodide and DNA free 3'-OH end TUNEL label to examine cell cycle arrest and apoptosis by fluorescence microscopic imaging and flow cytometry. Lysosomal leakage of beta-galactosidase and expression of cell cycle regulators such as p16 and p21 and oxidative stress regulators such as HO-1, FG-2, VEGFand IL-8 will be examined by relevant procedures that include gel electrophoretic Western blot immunodetection and phase contrast and fluorescence microscopy. Cell adhesion will be assessed by immunostaining of ZO-1 protein, and mitochondrial oxidation state by staining with MitoTimer reporter and fluorescence microcopy and spectroscopy. Results from these experiments are anticipated to show that 24 h treatment of ARPE-19 cells with NaI at 5 millimolar concentration or less will induce the cell scenescence state and that mesobiliverdin will suppress this transition into cell scenescence by altering signaling pathways that regulate oxidative stress responses.
Impacts
What was accomplished under these goals?
Specific Objectives Met: 1. Observed that confluent but not subconfluent retinal pigmented epithelial cells derived from human fetal eyes (H-RPE cells) undergo cell scenescence when exposed to high levels of hydrogen peroxide. 2. Demonstrated that mesobiliverdin suppresses or reverses cell scenescence-associated events (release of lysosomal beta-galactosidase and expression of cell cycle inhibitors p16 and p21) of confluent H -RPE cells exposed to high levels of hydrogen peroxide Significant Results Achieved: Objective 1: In the previous year (year 1 of this 3-year project), it was observed that highly differentiated polarized H9-stem cell derived RPE cell monolayers propagated on Matrigel underwent cell scenescence when exposed to tert-butylhydroperoxide (t-BH) at 500 micromolar concentration. This was evident from the release of beta-galactosidase from lysosomes. Exposure to mesobiliverdin suppressed the t-BH induced release of beta-galactosidase. In 2017, H-RPE cells derived from human fetal eyes were seeded and propagated in Dulbecco's modified Eagle's medium in tissue culture 12-well plates. Non-confluent cells were shown to undergo cell death with 100 micromolar hydrogen peroxide. Simultaneous exposure to mesobiliverdin at concentrations as low as 1 micromolar prevented hydrogen peroxide-induced cell death. In contrast, confluent cells showed no cell death with hydrogen peroxide up to 500 micromolar concentration in the absence or presence of 1 or 2 micromolar mesobiliverdin. In the absence of mesobiliverdin, confluent cells exposed to a 2-hour interval exposure to 500 micromolar hydrogen peroxide for two consecutive days displayed lysosomal leakage of beta-galactosidase as revealed by staining with 5-bromo-4-chloro- 3-indolyl-β-D-galactopyranoside (Xgal). Such beta-galactosidase leakage is consistent with (but not diagnostic for) cell scenescence - a cellular state characterized by the arrest of cell division but sustained cell metabolism. Oxidative stress-induced cell scenescence of RPE is considered to be the cellular state of RPE cells that occurs during the progression of age-related macular degeneration (AMD). Therefore, the above regimen of hydrogen peroxoide exposure to confluent H-RPE may serve as an experimental model for testing potential therapeutics against AMD. Objective 2: Experiments were conducted to: 1) determine if tandem exposure of confluent H-RPE cells to high levels (e.g. 500 micromolar) of hydrogen peroxide induced a cell scenescent state as suggested from results observed in Objective 1 (above), and 2) mesobiliverdin would suppress the induction of cell scenescence. Confluent H-RPE cells were subjected to two separate 2 hour long exposures of oxidant hydrogen peroxide at 500 micromolar concentration in the presence or absence of 1 and 2 micromolar mesobiliverdin on two consecutive days as described above (in Objective 1). Experimental controls included exposures to mesobiliverdin alone and to no mesobiliverdin or hydrogen peroxide. After a 24 h recovery period in Dulbecco's modified Eagle's medium, the cells were examined for detection of cell scenescence markers, i.e. beta-galactosidase leakage and expression of cell cycle protein kinase inhibitors p16 and p21). In the absence of mesobiliverdin, hydrogen peroxide caused beta-galactosidase leakage, induction of p21, and repression of p16. However, in the presence of mesobiliverdin, these effects were reversed (beta-galactosidase leakage was reduced, p21 expression was repressed, and p16 was induced). These results indicate that the hydrogen peroxide treatment initiated cell scenescence, but with mesobiliverdin present, cell scenescence was diminished. Therefore, it is concluded that mesobiliverdin cytoprotects confluent H-RPE cells against oxidatively stressed-induced cell scenescence.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Zhu, D. H., Takemoto, J. Y., Parameswaran, S., Hinton, D. R., Annual Meeting of the Association for Research in Vision and Opthamology, "Exploring cytoprotective molecules for polarized HESC-RPE cells," ARVO, Baltimore MD. (May 7, 2017 - May 11, 2017)
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2017
Citation:
Clegg, M., Harding, C., Clegg, T., Takemoto, J. Y. (Author Only), Vargis, E., Biomedical Engineering Western regional Conference, "In vitro antioxidant stress treatment model in microgravity." (January 19, 2017 - January 20, 2017)
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Progress 07/01/16 to 09/30/16
Outputs
Target Audience:Target Audience Veterinarians Animal farmers Veterinary pharmaceutical industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Opportunities The P.I. conducted the proposed first year research while on sabbatical leave with collaborators at the University of Southern California Keck School of Medicine, Pathology Department. The P.I.'s collaborators are eye disease researchers who offer substantial experience and training in this field. This project also provides opportunities for several Utah State University undergraduate students to participate in research experiences. Garrett Hinton and Nick Noyce conducted experiments on cytoprotection of ARPE-19 cells by mesobiliverdin. Tyler Gladwin, Christian Peterson, and Ty Nicholas conducted experiments and produced mesobiliverdin and its synthetic precursor phycocyanobilin used in the RPE cytoprotection experiments. This project permitted opportunities for graduate students to participate in research. 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 and phycocyanobilin production, lipid peroxidation assays, and cytoprotection experiments. These students presented their research findings at the Synthetic Biomanufacturing Center Science and Review Meeting, November 29, 2016, Logan, Utah. How have the results been disseminated to communities of interest?Dissemination Research results were presented at the Synthetic Biomanufacturing Center (SBC) Science and Review Meeting, November 29, 2016 and in an abstract on Dec. 1, 2016 for presentation at the 2017 annual meeting of the Association for Research in Vision and Opthalmology (ARVO), Baltimore, MD, May 3-7, 2017 What do you plan to do during the next reporting period to accomplish the goals?Plan of Work 1) Mesobiliverdin cytoprotection experiments similar to those perfomed with ARPE-19 and H9-HESC RPE monolayer cells will be performed with highly differentiated, polarized fetal RPE cells. In trans-well plate cultures, these polarized fetal-derived RPE cells arrange themselves confluently in monolayers via cell junctions after long culture periods (e.g. 2 months). The monolayers functionally resemble the in situ RPE more than ARPE-19 or H9-HESC cells as revealed by high trans-epithelial resistance (TER) values characteristic of the RPE. For culturing, non-polarized/subconfluent fetal RPE cells will be plated at 5.0 × 104 cells per cm2 to reach approximately 70% confluence the following day. The cells will be plated on Matrigel-coated plates and cultured for several weeks with growth medium changes every 4 days. For oxidative stress experiments, cells will be initially treated for 24 hours with a range of H2O2 concentrations or with 0 and 600 μM t-BH. For cytoprotection experiments, the same procedures (i.e. MTT and SA-beta-galactosidase staining) described above to measure effects of mesobiliverdin treatments on oxidatively stressed ARPE-19 and non-polarized ESC-derived RPE cell monolayers will be used. We anticipate observing mesobiliverdin cytoprotection of oxidatively stressed differentiated, highly-polarized fetal RPE monolayer cells. 2) To determine the role of BVRA activation in mesobiliverdin cytoprotection of RPE cells, immunodetection experiments will be conducted with highly polarized and non-polarized fetal RPE cell monolayers and ARPE-19 cells. Cells will be exposed to mesobiliverdin at various concentrations in the presence of ATP. RPE cell lysates and purified BVRA will then be immunoprecipated with rabbit or mouse anti-phosphoserine IgG followed by anti-IgG BioBead conjugated to alkaline phosphatase or horse radish peroxidase and the precipitates washed by centrifugation. The immunoprecipates will then be washed by centrifugation, and the degree of color formation (420 nm) from hydrolysis of dinitrophenolphosphate (for alkaline phosphatase) or chemiluminescence with luminol (for horse radish peroxidase) will be measured. The immunoprecipates will also be subjected to NaDodSO4 gel electrophoresis and Western blotting using anti-human BVRA IgG conjugated to alkaline phosphatase or horse radish peroxidase to detect and assess the degree of BVRA protein autophosphorylation at serine residues. We anticipate observing mesobiliverdin-induced BVRA autophosphorylation in extracts of all three RPE cell types.
Impacts
What was accomplished under these goals?
Accomplishments Specific Objectives Met: 1. Demonstrated that addition of mesobiliverdin provides cytoprotection to ARPE-19 cells and human embryonic stem cell (HSC)-derived RPE monolayers against oxidative stress. 2. Demonstrated that human biliverdin reductase A (BVRA) is activated by mesobiliverdin Significant Results Achieved: Objective 1 A. Cytoprotection of ARPE-19 cells. ARPE-19 cells were cultured for 1 to 5 weeks after seeding and twice weekly fed with Dulbecco's modified Eagle's medium. To induce oxidative stress, H2O2 was added to cultures for 1 to 18 hours at concentrations from 0 to 100 μM. Cell viability was determined and quantitated by counting cells that exclude trypan blue and oxidative cell damage was assessed by viewing dihydrofluorescein chloride fluorescence microscopy. For cytoprotection experiments, mesobiliverdin was added to ARPE-19 cells at designated times and at concentrations between 1 and 100 microM to both H2O2 treated and non-treated cultures. Mesobiliverdin at concentrations between 5 and 100 microM showed dose-dependent cytoprotection against cellular oxidative damage (using DCF). In addition, mesobiliverdin at 10 microM concentration showed cytoprotection against lipid peroxidation when added simultaneously with 15 microM menadione and using DPPP as a fluorescent probe to detect lipid peroxidation. B. Cytoprotection of human embryonic stem cell (HESC)-derived RPE cells. H9 HESC cells were cultured to generate polarized RPE cells. Spontaneous differentiation was initiated at 7 days by changing the medium to Dulbecco's modified Eagle's medium and then every 3-4 days for 10 weeks (differentiation period). RPE cells were purified from other differentiating cell types using a double enzymatic method, plated onto Matrigel, and cultured in transwell 24-well plates as monolayers in differentiation medium with passages performed every 4 weeks. For oxidative stress experiments, the monolayer cells were treated for 24 hours with tert-butylhydroperoxide (t-BH) at 500 microM concentration. Cell viability of t-BH treated HESC-RPE cells was measured using MTT staining. Senescence-associated beta-galactosidase (SA beta-gal) staining was used to detect oxidative stress-induced cellular senescence. Mesobiliverdin (1 microM) modestly increased HESC-RPE cell viability under oxidative stress and suppressed the development of cellular senescence observed with untreated positive controls following long-term curtured growth (60% vs 90% SA beta-gal positive cells, respectively). Objective 2 The enzyme biliverdin reductase A (BVRA) catalyzes the reduction of biliverdin to bilirubin. Both compounds are strong anti-oxidants, and biliverdin - through "activation" of BVRA - also promotes anti-inflammatory processes. Therefore, BVRA plays a pivotal role in oxidative stress suppression by biliverdin. Mesobiliverdin was shown to be reduced to mesobilirubin by BVRA. Upon binding and catalytic reduction of biliverdin, BVRA (Mr ~33.5k) undergoes autophosphorylation at serines that initiates cell signaling cascades leading to anti-inflammatory effects including suppression of reactive oxygen species production. In the current project, mesobiliverdin was shown to stimulate autophosphorylation of serine residues by human BVRA dimers (Mr ~67k) and higher Mr oligomers (e.g. octomers). In summary, it was shown that mesobiliverdin: 1) has cytoprotective capabilities against cellular oxidative damage of ARPE-19 and polarized H9 HESC-derived RPE cells in monolayers, and 2) is converted to mesobilirubin by BVRA and stimulates BVRA autophosphorylation known to lead to antioxidant activity and anti-inflammatory suppression of reactive oxygen species production.
Publications
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