2016 Fiscal Year Research-status Report
Exploration of mechanisms underlying organ protective role of blockade of protease activated receptor 2 in sepsis through the modulation of VEGF angiogenic system and associated microcirculation
| Project/Area Number |
16K11394
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| Research Institution | University of Tsukuba |
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Principal Investigator |
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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | Sepsis / MODS / PAR2 blochade / VEGF / mechanism |
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| Outline of Annual Research Achievements |
Male Wistar rats at 8 weeks of age were administered with either saline solution or lipopolysaccharide (LPS) at different time points (1, 3, 6 and 10h). Additionally, we treated LPS-administered rats with PAR2 blocking peptide (PAR2BP) for 3h to assess whether PAR2 has a regulatory role on the on VEGF and ET-1 level in septic organs. Among various time points investigated, in 3h hour post LPS animals, PAR2 blockade showed significant normalizing effects of important inflammatory cytokines like circulatory TNF-alpha and IL-6. PAR2 blockade was effective in reversing LPS-induced organ injury at the earliest hours of 3h sepsis (liver, lung, kidney, heart). 3h PAR2 blockade has also significant inhibitory effects on the organ based TNF-alpha upregulation following after LPS administration. Indeed, organ based aberrant VEGF expression was also reversed with the treatment of PAR2BP for 3h in LPS-induced septic rats. Next we investigated the effects of PAR2BP on sepsis-organ based upregulation of ET-1 in LPS-administered rats and found both VEGF and ET-1 were modulated in a beneficial manner for vital organ protection in sepsis with the blockade of PAR2. Now we are investigating in details on VEGF linked ET-1 pathway reversal mechanism through PAR2BP in LPS-induced sepsis. As VEGF and ET-1 have already been reported by us and other laboratories as crucial molecules involved in the pathogenesis of organ complications in sepsis, more detailed investigations are required on the possible beneficial effects of PAR2BP in sepsis organ protection.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The current research is on the right track both from the contexts of time line and the amount of research activities scheduled to be done (100% done with target milestone achievement) with the proper management of project budget and funding. Keeping consistency with original research application and planning, in first year of this project, we already performed the generation of acute model of sepsis (1h, 3h, 6h and 10h) and then treated with PAR2BP (there were more than 10 rats per group in the experimental setting). We evaluated this sepsis model with or without PAR2BP at 3h of sepsis induction specifically from blood gas analysis, blood pressure assessment, histological analysis and molecular evaluation of target organs of multiple organ dysfunction syndrome (MODS) of sepsis (liver, heart, kidney, lung) in depth. As a molecular mechanism on the blockade of PAR2 in sepsis, the potential molecular pathway like VEGF linked ET-1 pathway has been explored in the first year of this project. It also seems to be evident that PAR2 starts to act in the organ complication of sepsis from early stage. Based on the findings of Ist year, we are going to generate chronic sepsis model in the second year of this project with preparation for PAR2 knockout mice as planned. In addition, based on the first year results and experiments, we are now preparing two manuscripts to be submitted in international high impact factor journal. There was no big trouble during the implementation of this research project last year.
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| Strategy for Future Research Activity |
In 2nd year of this project, we would like to generate chronic model of sepsis using wistar rats by LPS administration and then treat with PAR2BP. Lipopolysaccharide [LPS,15mg/kg,IP]) will be administered intraperitoneally for three weeks, Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO]. Then from blood gas analysis to molecular studies will be conducted. Detailed of VEGF and ET-1 pathway will be evaluated in heart, lung, liver and kidney. In addition, we will inject PAR2 peptide through systemic circulation in already developed sepsis model and then assess blood gas analysis, blood pressure measurement, histological assessment and molecular analysis. In addition, we will use genetically altered mice model PAR2 knockout mice and then induction of sepsis. PAR2 knockout (PAR2-KO, PAR2 +/+ or PAR2 －/－ (B6.Cg-F2rl1tm1Nwb) mice will be bought and then injected LPS to induce sepsis (10-100 μg/mouse; Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO]. Stock breeders of C57BL/6J (PAR2-WT) and B6.Cg-F2rl1tm1Mslb/J (PAR2-KO) mice will be purchased from Jackson Laboratory (Bar Harbor, ME).We will then systematically assess the organ function and other parameters.
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| Causes of Carryover |
Last year we did not hire a research assistant due to some un-avoidable factors from this grant money.
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| Expenditure Plan for Carryover Budget |
This year we will use the un-used money from last year to hire one research assistant to speed up the current research work.
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[Journal Article] Landiolol hydrochloride ameliorates acute lung injury in a rat model of early sepsis through the suppression of elevated levels of pulmonary endothelin-1.2016
Author(s)
Matsuishi Y, Jesmin S, Kawano S, Hideaki S, Shimojo N, Mowa CN, Akhtar S, Zaedi S, Khatun T, Tsunoda Y, Kiwamoto T, Hizawa N, Inoue Y, Mizutani T.
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Journal Title
Life Sci.
Volume: 166
Pages: 27-33
DOI
Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
