2017 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 blockade / VEGF / mechanism |
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| Outline of Annual Research Achievements |
Male Wistar rats at 8 weeks of age were divided into three groups according to the intravenous (IV) fluid received: saline (sham; n = 17), LPS (Escherichia coli serotype 0111:B4; n = 15) at a dose of 0.5 mg/kg/h, or a combination of PAR2 blocking peptide (PAR2BP)+ LPS (LPS at 0.5 mg/kg/h; PAR2BP at 20 ug/kg/h, 100ul PBS/hour); n = 16) by implantable pumps for 48h. The LPS dose applied here induces significant lung injury with less than 10% mortality at 72 h. The PAR2BP dose was chosen based on our pilot studies. Total lung injury scores were assessed, lung injury was significantly more severe in LPS-treated animals compared with either sham or PAR2BP+ LPS groups at 48h. As the infusion of LPS continued, the lung injury progressed in the LPS group and remained unchanged in the PAR2BP+ LPS group as demonstrated by less progression of interstitial inflammation, polymorphonuclear neutrophil infiltration, congestion, alveolar edema. 48h PAR2BP has also significant inhibitory effects on the pulmonary inflammatory cytokine upregulation following after LPS administration namely TNF-alpha. Decreased pulmonary expression of VEGF was also reversed with the treatment of PAR2BP for 48h in LPS-induced septic rats. The effects of PAR2BP on pulmonary ET-1 upregulation was also assessed in LPS-administered rats and significantly normalized with PAR2BP treatment. Last year, we reported using an acute model of sepsis generated by 3h high dose of LPS administration and the effects of PAR2BP. The current results were in the similar trend as that of last year.
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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 almost on the right track both from the contexts of time line and the amount of research activities scheduled to be done with the proper management of project budget and research facilities available. 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. This sepsis model has been assessed in depth from all perspectives 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. Based on the findings obtained in 1st year, we generated longer duration of LPS induced sepsis model focusing on the acute lung injury in 2nd year and again intensively assessed the effects of PAR2BP on sepsis-induced inflammation in lung with the elaboration of PAR2BP beneficial effects on pulmonary VEGF and ET-1 systems. Indeed, consistent organ protective effects of PAR2BP were found using different sepsis experimental setting. 1st year and 2nd year results were very similar demonstrating PAR2 as a potential therapeutic and preventive target for sepsis-induced vital organ complications. There was nothing remarkable to be mentioned as obstacle during implementation of this research project last year.
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| Strategy for Future Research Activity |
This year (2018) 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 ug/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. We will also perform in vitro studies. Target cells for in vitro studies are: vascular endothelial cells, macrophages, cardiomyocytes, pneumocytes, bronchial epithelial cells, hepatocytes, mesangial cells, renal tubular cells. In addition, we will do in depth data analysis this year, critically search the experimental trouble-shooting taken place while this project is being implemented and their future solutions, prepare for scientific publications, preparation for translational research for this current finding, consultation with well-known institutes in this research field for more extensive comprehensive future works.
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| Causes of Carryover |
Last year we could not perform PAR2 knockout mice study due to animal breeding space difficulties, this year we will use the saved money from last year to perform this mice study.
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[Journal Article] Differences in coagulofibrinolytic changes between post-cardiac arrest syndrome of cardiac causes and hypoxic insults: a pilot study.2017
Author(s)
Wada T, Gando S, Mizugaki A, Kodate A, Sadamoto Y, Murakami H, Maekawa K, Katabami K, Ono Y, Hayakawa M, Sawamura A, Jesmin S, Ieko M.
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Journal Title
Acute Med Surg.
Volume: 27;4(3)
Pages: 371-372
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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