Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2

Lee, Sung Kyun,Kim, Sang Doo,Kook, Minsoo,Lee, Ha Young,Ghim, Jaewang,Choi, Youngwoo,Zabel, Brian A.,Ryu, Sung Ho,Bae, Yoe-Sik The Rockefeller University Press 2015 The Journal of experimental medicine Vol.212 No.9

<P>Lee et al. find that phospholipase D2 deficiency increases survival and decreases organ damage during experimental sepsis in mice which could be reversed with a CXCR2 antagonist. Thus, targeting PLD2 may offer therapeutics for septic patients.</P><P>We determined the function of phospholipase D2 (PLD2) in host defense in highly lethal mouse models of sepsis using PLD2<SUP>−/−</SUP> mice and a PLD2-specific inhibitor. PLD2 deficiency not only increases survival but also decreases vital organ damage during experimental sepsis. Production of several inflammatory cytokines (TNF, IL-1β, IL-17, and IL-23) and the chemokine CXCL1, as well as cellular apoptosis in immune tissues, kidney, and liver, are markedly decreased in PLD2<SUP>−/−</SUP> mice. Bactericidal activity is significantly increased in PLD2<SUP>−/−</SUP> mice, which is mediated by increased neutrophil extracellular trap formation and citrullination of histone 3 through peptidylarginine deiminase activation. Recruitment of neutrophils to the lung is markedly increased in PLD2<SUP>−/−</SUP> mice. Furthermore, LPS-induced induction of G protein–coupled receptor kinase 2 (GRK2) and down-regulation of CXCR2 are markedly attenuated in PLD2<SUP>−/−</SUP> mice. A CXCR2-selective antagonist abolishes the protection conferred by PLD2 deficiency during experimental sepsis, suggesting that enhanced CXCR2 expression, likely driven by GRK2 down-regulation in neutrophils, promotes survival in PLD2<SUP>−/−</SUP> mice. Furthermore, adoptively transferred PLD2<SUP>−/−</SUP> neutrophils significantly protect WT recipients against sepsis-induced death compared with transferred WT neutrophils. We suggest that PLD2 in neutrophils is essential for the pathogenesis of experimental sepsis and that pharmaceutical agents that target PLD2 may prove beneficial for septic patients.</P>

Effects of Coculture With Immune Cells on the Developmental Competence of Mouse Preimplantation Embryos in Vitro and in Utero

Lee, Jaewang,Kim, Jihyun,Kim, Seok Hyun,Kang, Hee-Gyoo,Jun, Jin Hyun SAGE Publications 2015 REPRODUCTIVE SCIENCES Vol.22 No.10

<P>The aim of this study was to establish a coculture system using immune cells as well as an in vitro model for inflammatory conditioning using RAW 264.7 mouse macrophages activated by lipopolysaccharide. The direct and indirect coculture systems were applied to evaluate the influence of embryo-to-cell direct or indirect secretory molecules from the cocultured cells. Blastulation rate in vitro (94.6% vs 76.9%, <I>P</I> < .05) and implantation rate in utero (43.3% vs 17.6%, <I>P</I> < .01) were significantly increased in direct coculture with activated RAW 264.7 cells compared to control. We also found the embryotrophic effects in vitro in the indirect coculture system. Our results indicate that coculture of mouse preimplantation embryos with immune cells could improve the developmental competence in vitro and in utero. Taken together, RAW 264.7 cells secret embryotrophic molecules, and it suggests the valuable insights that immune cells could improve in vitro culture conditions of preimplantation embryos.</P>

Effects of dynamic oxygen concentrations on the development of mouse pre- and peri-implantation embryos using a double-channel gas supply incubator system

Lee, Seung-Chan,Seo, Ho-Chul,Lee, Jaewang,Jun, Jin Hyun,Choi, Kyoo Wan The Korean Society for Reproductive Medicine 2019 Clinical and Experimental Reproductive Medicine Vol.46 No.4

Objective: We aimed to evaluate the effects of different oxygen conditions (20% [high O₂], 5% [low O₂] and 5% decreased to 2% [dynamic O₂]) on mouse pre- and peri-implantation development using a novel double-channel gas supply (DCGS) incubator (CNC Biotech Inc.) to alter the oxygen concentration during in vitro culture. Methods: The high-O₂ and low-O₂ groups were cultured from the one-cell to the blastocyst stage under 20% and 5% oxygen concentrations, respectively. In the dynamic-O₂ group, mouse embryos were cultured from the one-cell to the morula stage under 5% O₂ for 3 days, followed by culture under 2% O₂ to the blastocyst stage. To evaluate peri-implantation development, the blastocysts from the three groups were individually transferred to a fibronectin-coated dish and cultured to the outgrowth stage in droplets. Results: The blastocyst formation rate was significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The total cell number was significantly higher in the dynamic-O₂ group than in the low-O₂ and high-O₂ groups. Additionally, the apoptotic index was significantly lower in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. The trophoblast outgrowth rate and spread area were significantly higher in the low-O₂ and dynamic-O₂ groups than in the high-O₂ group. Conclusion: Our results showed that a dynamic oxygen concentration (decreasing from 5% to 2%) had beneficial effects on mouse pre- and peri-implantation development. Optimized, dynamic changing of oxygen concentrations using the novel DCGS incubator could improve the developmental competence of in vitro cultured embryos in a human in vitro fertilization and embryo transfer program.

A combination of simvastatin and methylprednisolone improves the quality of vitrified-warmed ovarian tissue after auto-transplantation

Lee, Jaewang,Kim, Eun Jung,Kong, Hyun Sun,Youm, Hye Won,Lee, Jung Ryeol,Suh, Chang Suk,Kim, Seok Hyun Oxford University Press 2015 Human reproduction Vol.30 No.11

<P><B>STUDY QUESTION</B></P><P>Does the preoperative administration of simvastatin and methylprednisolone enhance mouse ovarian quality after auto-transplantation of vitrified-warmed ovarian tissue (OT)?</P><P><B>SUMMARY ANSWER</B></P><P>Treatment with combined simvastatin and methylprednisolone enhances the quality of transplanted mouse OTs.</P><P><B>WHAT IS KNOWN ALREADY</B></P><P>The prevention of ischemic injury after transplantation of OT is critical for preserving the ovarian follicles. Preoperative administration of simvastatin (a cholesterol-lowering drug) has beneficial effects on various organ transplantations. Moreover, donor treatment with simvastatin and methylprednisolone (main effects are on immune response) prevents ischemia-reperfusion injury and has a beneficial effect on allograft survival in rat cardiac allografts.</P><P><B>STUDY DESIGN, SIZE, DURATION</B></P><P>A total of 232 6-week-old B6D2F1 mice were randomly distributed into fresh control, vitrified-warmed control and experimental groups (<I>n</I> = 10–17 per group). The experimental groups were as follows: sham control, simvastatin, methylprednisolone and co-treatment groups. In the experimental groups, the mice were administered simvastatin (5 mg/kg, orally), methylprednisolone (15 mg/kg, i.v.) or a combination of simvastatin and methylprednisolone 2 h before ovariectomy, whereas the sham control mice received normal saline.</P><P><B>PARTICIPANTS/MATERIALS, SETTING, METHODS</B></P><P>Whole ovaries were removed from the mice and vitrified by two-step vitrification procedures. The vitrified ovaries were warmed 1 week later and auto-transplanted under the bilateral kidney capsules. The ovaries and blood samples were collected 2, 7 and 21 days (D) after transplantation for histological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, immunohistochemistry for CD31 and serum anti-Mullerian hormone (AMH) level estimation. Embryonic development was evaluated after IVF of oocytes obtained from the transplanted ovary.</P><P><B>MAIN RESULTS AND THE ROLE OF CHANCE</B></P><P>The group that received simvastatin and methylprednisolone showed a significantly improved intact (Grade 1) follicle ratio (D2: <I>P</I> < 0.001, D7: <I>P</I> < 0.05 and D21: <I>P</I> < 0.001), apoptotic follicle ratio (D21: <I>P</I> < 0.05), CD31-positive area (D7: <I>P</I> < 0.05 and D21: <I>P</I> < 0.05) and serum AMH level (D7: <I>P</I> < 0.001) after transplantation when compared with the sham control. However, no difference was noted in the fertilization and blastocyst formation rates, number of total and apoptotic blastomeres per blastocyst and inner cell mass/trophectoderm ratio among the four transplantation groups.</P><P><B>LIMITATIONS, REASONS FOR CAUTION</B></P><P>Although we evaluated the beneficial effects of simvastatin and methylprednisolone in the present study, we did not unravel the corresponding protective mechanisms.</P><P><B>WIDER IMPLICATIONS OF THE FINDINGS</B></P><P>Our results suggest that a combination of simvastatin and methylprednisolone has beneficial effects on the quality and functioning of transplanted OT. This combined treatment can potentially be applied clinically to humans and domestic animals subject to further studies.</P><P><B>STUDY FUNDING/COMPETING INTEREST(S)</B></P><P>This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). There are no conflicts of interest to declare.</P>

Ovarian injury during cryopreservation and transplantation in mice: a comparative study between cryoinjury and ischemic injury

Lee, Jaewang,Kong, Hyun Sun,Kim, Eun Jung,Youm, Hye Won,Lee, Jung Ryeol,Suh, Chang Suk,Kim, Seok Hyun Oxford University Press 2016 Human reproduction Vol.31 No.8

<P>What is the main cause of ovarian injury during cryopreservation and transplantation in mice: cryoinjury or ischemic injury? Post-transplantation ischemia is the main cause of ovarian injury during cryopreservation and transplantation for restoring ovarian function. During cryopreservation and the transplantation of ovaries, cryoinjury and ischemic injury inevitably occur, which has a detrimental effect on ovarian quality and reserve. A total of 80 B6D2F1 female mice were randomly allocated to 2 control and 6 experimental groups according to the presence or the absence of transplantation (n = 10/group). The control groups consisted of fresh or vitrified-warmed controls that had the whole ovary fixed without transplantation (fresh and vitri-con, respectively). The experimental groups were further divided according to the presence of vitrification (fresh or vitrified-warmed) and the transplantation period (2 [D2], 7 [D7] or 21 [D21] days). In the control groups, fresh and vitrified-warmed ovaries were immediately fixed after the collection (fresh) and the vitrification-warming process (vitrification control, vitri-con), respectively. Of those experimental groups, three were auto-transplanted with fresh whole ovary (FrOT; FrOT-D2, FrOT-D7 and FrOT-D21). For the other three groups, the ovaries were harvested and stored in liquid nitrogen for 1 week after vitrification and then warmed to auto-transplant the vitrified whole ovaries (vitrified ovary [VtOT]; VtOT-D2, VtOT-D7 and VtOT-D21). After 2, 7 or 21 days of grafting, the grafts and blood sera were collected for analysis by hematoxylin-eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, CD31 immunohistochemistry and follicle-stimulating hormone enzyme-linked immunosorbent assay. The vitrification-warming procedure decreased the proportion of intact follicles (Grade 1, G1) (vitri-con 50.3% versus fresh 64.2%) but there was a larger decrease due to ischemic injury after transplantation (FrOT-D2: 42.5%). The percentage of apoptotic follicles was significantly increased in the vitrified-warmed ovary group compared with the fresh control, but it increased more after transplantation without vitrification (fresh: 0.9%, vitri-con: 6.0% and FrOT-D2: 26.8%). The mean number of follicles per section and percentage of CD31-positive area significantly decreased after vitrification but decreased to a larger extent after transplantation (number of follicles, fresh: 30.3 +/- 3.6, vitri-con: 20.6 +/- 2.9, FrOT-D2: 17.9 +/- 2.1; CD31-positive area, fresh: 10.6 +/- 1.3%, vitri-con: 5.7 +/- 0.9% and FrOT-D2: 4.2 +/- 0.4%). Regarding the G1 follicle ratio and CD31-positive area per graft, only the FrOT groups significantly recovered with time after transplantation (G1 follicle ratio, FrOT-D2: 42.5%, FrOT-D7: 56.1% and FrOT-D21: 70.7%; CD31-positive area, FrOT-D2: 4.2 +/- 0.4%, FrOT-D7: 5.4 +/- 0.6% and FrOT-D21: 7.5 +/- 0.8%). Although there was no significant difference between the two transplantation groups at each evaluation, the serum follicle-stimulating hormone level of both groups significantly decreased over time. It is unclear how far these results can be extrapolated from mice to the human ovary. Minimizing ischemic injury should be the first priority rather than preventing cryoinjury alone, and decreasing the combination of cryoinjury and ischemic injury is necessary to improve ovarian quality after cryopreservation and transplantation. This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055). The authors have no conflict of interest to declare.</P>

Differential regulation of p53 and p21 by MKRN1 E3 ligase controls cell cycle arrest and apoptosis.

Lee, Eun-Woo,Lee, Min-Sik,Camus, Suzanne,Ghim, Jaewang,Yang, Mi-Ran,Oh, Wonkyung,Ha, Nam-Chul,Lane, David P,Song, Jaewhan Published for the European Molecular Biology Organ 2009 The EMBO journal Vol.28 No.14

<P>Makorin Ring Finger Protein 1 (MKRN1) is a transcriptional co-regulator and an E3 ligase. Here, we show that MKRN1 simultaneously functions as a differentially negative regulator of p53 and p21. In normal conditions, MKRN1 could destabilize both p53 and p21 through ubiquitination and proteasome-dependent degradation. As a result, depletion of MKRN1 induced growth arrest through activation of p53 and p21. Interestingly, MKRN1 used earlier unknown sites, K291 and K292, for p53 ubiquitination and subsequent degradation. Under severe stress conditions, however, MKRN1 primarily induced the efficient degradation of p21. This regulatory process contributed to the acceleration of DNA damage-induced apoptosis by eliminating p21. MKRN1 depletion diminished adriamycin or ultraviolet-induced cell death, whereas ectopic expression of MKRN1 facilitated apoptosis. Furthermore, MKRN1 stable cell lines that constantly produced low levels of p53 and p21 exhibited stabilization of p53, but not p21, with increased cell death on DNA damage. Our results indicate that MKRN1 exhibits dual functions of keeping cells alive by suppressing p53 under normal conditions and stimulating cell death by repressing p21 under stress conditions.</P>

Phospholipase D2 drives mortality in sepsis by inhibiting neutrophil extracellular trap formation and down-regulating CXCR2

Lee, Sung Kyun,Kim, Sang Doo,Kook, Minsoo,Lee, Ha Young,Ghim, Jaewang,Choi, Youngwoo,Zabel, Brian A.,Ryu, Sung Ho,Bae, Yoe-Sik The Rockefeller University Press 2015 The Journal of cell biology Vol.210 No.5

Combined Simvastatin and Methylprednisolone improve the quality of vitrified-warmed ovarian tissue after transplantation

Jaewang Lee,Eun Jung Kim,Hyun Sun Kong,Hye Won Youm,Jung Ryeol Lee,Byung Chul Jee,Chang Suk Suh,Seok Hyun Kim 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

Objective : To investigate the effects of Simvastatin and Methylprednisolone on ovarian tissue cryopreservation and transplantation using mouse models. Methods : The mice were randomly distributed into 1 control and 3 experimental groups. The B6D2F1 mice were given oral Simvastatin (5 mg/kg), intravenous Methylprednisolone (15 mg/kg), or a combination of both at 2 hours before ovariectomy. Same volume of normal saline was given perorally in the control group at 2 hours before ovariectomy. The ovarian tissues were vitrified accrording to our protocols. The vitrified ovaries were warmed 1 week later and auto-transplanted under bilateral kidney capsules. The ovaries and blood sera were collected at 2, 7 or 21 days after transplantation. Histological analysis, TUNEL assay, immuno-histochemistry for CD31, serum AMH level and embryonic development after in vitro fertilization were assessed for evaluation. Results : With regard to the total grade 1 follicle rate, both Simvastatin or Methylprednisolone treated groups were significantly increased at 2, 7 or 21 days after transplantation (except Simvastatin treated group at 7 days). A combination of Simvastatin and Methylprednisolone group was significantly improved in terms of the total G1 follicle rate, apoptotic follicle rate, CD31 positive area and serum AMH after ovarian tissue transplantation. However, there were no statistically difference with respect to the oocyte maturation rate, blastulation rate, and the other embryonic development parameters after in vitro fertilization procedure among the four groups. Conclusion : Our results suggest that combined donor Simvastatin and Methylprednisolone have beneficial effects on the quality and function of transplanted ovarian tissues.

Endothelial Deletion of Phospholipase D2 Reduces Hypoxic Response and Pathological Angiogenesis

Ghim, Jaewang,Moon, Jin-Sook,Lee, Chang Sup,Lee, Junyeop,Song, Parkyong,Lee, Areum,Jang, Jin-Hyeok,Kim, Dayea,Yoon, Jong Hyuk,Koh, Young Jun,Chelakkot, Chaithanya,Kang, Byung Jun,Kim, Jung-Min,Kim, Ky American Heart Association, Inc. 2014 Arteriosclerosis, thrombosis, and vascular biology Vol.34 No.8

<P><B>Objective—</B></P><P>Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1α and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo.</P><P><B>Approach and Results—</B></P><P><I>Pld2</I> knockout ECs exhibited decreased hypoxia-induced cellular responses in survival, migration, and thus vessel sprouting. Analysis of hypoxia-induced gene expression revealed that PLD2 deficiency disrupted the upregulation of hypoxia-inducible factor-1α target genes, including <I>VEGF</I>, <I>PFKFB3</I>, <I>HMOX-1</I>, and <I>NTRK2</I>. Consistent with this, PLD2 contributed to hypoxia-induced hypoxia-inducible factor-1α expression at the translational level. The roles of PLD2 in hypoxia-induced in vivo pathological angiogenesis were assessed using oxygen-induced retinopathy and tumor implantation models in endothelial-specific <I>Pld2</I> knockout mice. <I>Pld2</I> endothelial-specific knockout retinae showed decreased neovascular tuft formation, despite a larger avascular region. Tumor growth and tumor blood vessel formation were also reduced in <I>Pld2</I> endothelial-specific knockout mice.</P><P><B>Conclusions—</B></P><P>Our findings demonstrate a novel role for endothelial PLD2 in the survival and migration of ECs under hypoxia via the expression of hypoxia-inducible factor-1α and in pathological retinal angiogenesis and tumor angiogenesis in vivo.</P>

차량용 전자제어기 소프트웨어플랫폼 개발

이재왕(Jaewang Lee),연제명(Jaemyoung Youn),원동훈(Donghoon Won) 한국자동차공학회 2011 한국자동차공학회 부문종합 학술대회 Vol.2011 No.5

A useful software platform (SWP) and a development environment for Electronic Control Unit (ECU) of the Electric Vehicle (EV) and Hybrid Electric Vehicle (HEV) are designed. The designed SWP has the layered architecture for increasing reusability. The development environment is designed to help the application engineers who design the algorithm by model-based development (MBD) method or who calibrate the parameters. The application software (ASW) modules generated from the TargetLink model to control the vehicle and the developed SWP modules are integrated and verified on vehicle. Because the SWP has layered structure, it is possible to reuse in another ECU except the dependent part of CPU. This software Platform is also applied to HMC’s ECUs for the EV and HEV project.