범발성혈관내응고증환자의 혈중 Tissue-Type Plasminogen Activator, Type 1 Plasminogen Activator Inhibitor 및 von Willebrand Factor변동에 관한 연구

정철원,안진석,박선양,김병국,김노경 大韓血液學會 1994 大韓血液學會誌 Vol.29 No.2

Plasminogen Activator Inhibitor-1 Promotes Synaptogenesis and Protects Against Aβ_1-42-Induced Neurotoxicity in Primary Cultured Hippocampal Neurons

Cho, Harim,Joo, Yuyoung,Kim, Seonghan,Woo, Ran-Sook,Lee, Sang Hyung,Kim, Hye-Sun Informa Healthcare 2012 International journal of neuroscience Vol.123 No.1

<P>Plasminogen activator inhibitor-1 (PAI-1) is a soluble factor that is released from astrocytes, the most abundant type of glial cell in the brain. PAI-1 was initially identified as inhibiting two types of plasminogen activators, that is, tissue-type plasminogen and urokinase activators that are known to lead to the proteolytic degradation of the extracellular matrix. Recently, PAI-1 was reported to mediate the neuroprotective activity of transforming growth factor-β1 against N-methyl-D-aspartate receptor-mediated excitotoxicity and to be involved in angiogenesis following ischemic stroke, independently of the effects via the inhibition of tissue-type plasminogen and urokinase-type plasminogen activators. In this study, we examined whether PAI-1 influences synaptogenesis and neurotoxicity induced by amyloid beta peptide<SUB>1-42</SUB> (Aß<SUB>1-42</SUB>) in rat primary hippocampal neurons. Using immunostaining, treatment with PAI-1 for 24 h was found to significantly upregulate synaptophysin, postsynaptic density-95, and the polysialylated form of neural cell adhesion molecule, compared to treatment with vehicle alone. In addition, PAI-1 has neuroprotective effects against Aβ<SUB>1-42</SUB>-induced cytotoxicity in rat primary cultured hippocampal neurons. Taken together, our results suggest that PAI-1 has therapeutic potential in Alzheimer's disease by promoting synaptogenesis and by demonstrating neuroprotective effects against Aβ<SUB>1-42</SUB>-oligomer-induced neurotoxicity in rat primary cultured hippocampal neurons.</P>

소아 신증후군 환자에서 Plasminogen Activator Inhibitor Type 1 유전자 다형성

김영민,홍현기,김성도,조병수,Kim Young-Min,Hong Hyun-Kee,Kim Sung-Do,Cho Byoung-Soo 대한소아신장학회 2004 Childhood kidney diseases Vol.8 No.1

목 적 : 소아 신증후군 환자에서 과응고성 경향을 가지고 있으며 Plasminogen activator inhibitor-1(PAI-1)은 강력하게 섬유소의 용해를 감소시키는 당단백으로 최근 몇몇 연구에 의하면 신증후군에서 증가된 PAI-1과 사구체내의 섬유소원이나 섬유소와 관련된 항체의 침착이 연관되어 있음을 시사하고 있다. 저자는 PAI-1 유전자 다형성 중에서 A-844 G 대립유전자 다형성의 빈도와 유전형을 소아의 신증후군에서 임상경과와의 연관성을 비교 평가하였다. 방 법 : 2001년 10월부터 2003년 1월까지 경희대학교 부속병원 동서신장병연구소에 방문한 146명의 신증후군 환아와 249명의 대조군을 대상으로 하였고 신증훈군 환아는 infrequent relapser(IR)와 frequent relapser(FR)로 다시 나누었다. 이들에 대해 PAI-1 promoter gene의 A-844 G에 대한 중합효소 연쇄반응-제한효소절편길이 다형현상(PCR-RFLP)을 이용하여 유전자형을 A/A, A/G, G/G로 분류하여 비교 분석하였다. 통계는 Graph Pad Prism 통계분석 소프트웨어 version 2.0을 사용하였고 95% 신뢰구간과 P value는 0.05보다 작은 것을 의미 있게 보았다. 결 과 : PAI-1 promoter gene의 A- 844G 다형성의 분포는 대조군애서 G/G 81(32.5%), A/A 42(16.9%), G/A 126(50.6%)이였고 신증후군 그룹 중 IR 그룹은 G/G 29(34.1%), A/A 15(17.7%), G/A 41(48.2%)이였으며 FR 그룹에서는 G/G 17(27.9%), A/A 18(29.5%), G/A 26(42.6%)이었다. 단지 PAI-1 gene의 A-844G의 다형성중 A/A 유전자형이 신증후군 환아 중 FR군에서만 대조군에 비하여 유의하게 증가하였다(16.9% vs 29.5%, OR=2.06, P=0.0251). 반면 A/G 유전자형(OR=0.73, P=0.2639)이나 G/G 유전자형(OR=0.80, P=0.4828)은 통계학적인 의의가 없었고 IR군에서 대조군에 비하여 A/A(OR=1.06, P=0.8690), A/G(OR=0.91, P=0.7063), G/G(OR=1.07, P=0.7880)으로 모두 통계학적으로 유의하지 않았다. 결 론 : A-844G AA 유전자형을 가진 신증후군 환자와 FR와 통계적인 상관관계가 있음을 알수 있었다. 이는 향후 PAI-1 유전자와 과응고성과 관련된 생화학적 검사와의 연관성에 대한 추가적인 조사와 특히, 신증후군의 과응고성과 신증후군 재발과의 연관성이 있는지에 관한 연구가 더 필요할 것으로 사료된다. Purpose : Hypercoagulability is present in patients with nephrotic syndrome. Plasminogen activator inhibitor type 1(PAI-1) is a major inhibitor of plasminogen activators. PAI-1 inactivates both tissue plasminogen activator(tPA) and urokinase plasminogen activator(uPA) by rapid formation of inactive 1:1 stoichiometric complexes. Recently some studies showed that the enhanced PAI-1 expression may be involved in the intraglomerular fibrinogen/fibrinrelated antigen deposition seen in nephrotic syndrome. Methods : PAI-1 gene promoter -844(G/A) polymorphism was evaluated in 146 children with minimal change nephrotic syndrome(MCNS) and 230 control subjects. The patients with MCNS were subdivided into 85 infrequent-relapser(IR) group and 61 frequent relapser(FR) group. PCR of PAI-1 gene promoter region including -844(G/A) and RFLP using the restriction enzyme Xhol were performed for each DNA samples extracted from the groups. Results : The distribution of PAI-1 genotype in the control group was G/G 81(32.5%), A/A 42(16.9%), and G/A 126(50.6%). The distribution of PAI-1 genotypes in the IR group of MCNS was G/G 29(34.1%), A/A 15(17.7%), and G/A 41(48.2%). The distribution of PAI-1 genotype in the FR group of MCNS was G/G 17(27.9%), A/A 18(29.5%), and G/A 26(42.6%). There was a significantly increased frequency of A/A genotype(P=0.0251) in the FR group of MCNS. Conclusion : Our results indicate that the PAI-1 gene promoter A/A genotype may be associated with the FR in MCNS.

쥐의 골세포에서 PGE² 합성과 plasminogen activator 활성 조절에 의한 IL-ß의 골 흡수유도와 TGF-ß에 의한 골 흡수 억제 기전에 관한 연구

김영훈(Young-Hun Kim),이영준(Young-Jun Lee),정규림(Kyu-Rhim Chung),박영국(Young-Guk Park) 대한치과교정학회 2000 대한치과교정학회지 Vol.30 No.6

골세포는 골대사에 영향을 미치는 다양한 성장인자와 싸이토카인을 생성하여 골 기질로 유리시킨다. 이연구는 쥐의 장골 세포 배양 모델에서 recombinant human IL-1β가 PGE₂ 합성과 plasminogen activator의 활성 조적을 통한 골흡수 유도 기전의 일단을 구명하고, 이와 동시에 TGF-β에 의한 골흡수 억제 기전을 해명하는데 그 목적이 있다. 쥐의 장골 세포를 배양하여 통법의 골모세포 phenotype을 발현하는 세포를 분리하고 세포 배양능, alkaline phosphatase assay, PG assay, 골흡수능 측정들을 시행하여 다음의 결과를 얻었다. 1. IL-1β는 쥐의 골모세포 증식, PGE₂ 생성 및 palsmongen activator의 활성을 촉진하였다. 2. IL-1β는 쥐의 골모세포에서의 alkaline phosphatase활성을 감소시켰다. 3. rhIL-1β는 골 흡수를 촉진시켰다. 4. TGF-ILβ는 쥐의 장골 세포에서 골의 흡수를 억제하였으며, Vitamin D₃에 의하여 유도된 골 흡수를 억제하였다. 이상의 연구 결과는 IL-1β에 의한 골 파괴의 병인과 관련하여 골 세포 대사의 병리학적 조절에 있어서의 IL-1β의 역할을 지지하며, 이와 동시에 골 흡수 억제에 있어서의 TGF-β의 역할을 확인시켜주는 것으로 생각된다. Bone cells produce multiple growth factors and cytokines that have effects on bone metabolism and can be incorporated into the bone matrix. The present study was designed to extend these observations by examining the interactions between transforming growth factor-Il (TGF-β) or interleukin-1β (rhIL-1β) and bone cells in a rat long bone culture model. IL-1β regulates several activities of the osteoblast cells derived from rat long bone explants in vitro. IL-1β stimulated cellular proliferation as well as the synthesis of prostaglandin E₂and plasminogen .activator activity in the cultured cells in a dose-dependent manner. TGF-β is present in the bone matrix and potentially released during bone resorption. TGF-β reduced basal bone resorption and inhibited vitamin D₃ [1,25(OH)₂D₃]-induced bone resorption in rat long bone cells. These results support the role.of IL-1β in the pathological modulation of bone cell metabolism, with regard to implication in the pathogenesis of osteoporosis by IL-1β, and that TGF-β positively inhibits the bone resorption.

Interleukin-1β induces bone resorption by regulation of prostaglandin E₂ synthesis and plasminogen activator activity, and TGF-β inhibits bone resorption of rat bone cells

Kim, Young-Hun,Lee, Young-Jun,Chung, Kyu-Rhim,Park, Young-Guk 대한치과교정학회 2000 대한치과교정학회지 Vol.30 No.6

골세포는 골대사에 영향을 미치는 다양한 성장인자와 싸이토카인을 생성하여 골 기질로 유리시킨다. 이 연구는 쥐의 장골 세포 배양 모델에서 recombinant human IL-1β가 PGE2 합성과 plasminogen activator의 활성 조절을 통한 골흡수 유도 기전의 일단을 구명하고, 이와 동시에 TGF-β에 의한 골흡수 억제 기전을 해명하는데 그 목적이 있다. 쥐의 장골 세포를 배양하여 통법의 골모세포 phenotype을 발현하는 세포를 분리하고 세포 배양능, alkaline phosphatase assay, PG assay, 골흡수능 측정들을 시행하여 다음의 결과를 얻었다. 1.IL-1β는 쥐의 골모세포의 증식, PGE2 생성 및 palsmonogen activator의 활성을 촉진하였다. 2.IL-1β는 쥐의 골모세포에서의 alkaline phosphatase 활성을 감소시켰다. 3.rhIL-1β는 골 흡수를 촉진시켰다. 4.TGF-β는 쥐의 장골 세포에서 골의 흡수를 억제하였으며, Vitamin D3에 의하여 유도된 골 흡수를 억제하였다. 이상의 연구 결과는 IL-1β에 의한 골 파괴의 병인과 관련하여 골 세포 대사의 병리학적 조절에 있어서의 IL-1β의 역할을 지지하며, 이와 동시에 골 흡수 억제에 있어서의 TGF-β의 역할을 확인시켜주는 것으로 생각된다. Bone cells produce multiple growth factors and cytokines that hale effects on bone metabolism and can be incorporated into the bone matrix. The present study was designed to extend these observations by examining the interactions between transforming growth factor-β (TGF-β) or interleukin-1β (rhIL-1β) and bone cells in a rat long bone culture model. IL-1β regulates several activities of the osteoblast cells derived from rat long bone explants in vitro. IL-1β stimulated cellular proliferation as well as the synthesis of prostaglandin E2 and plasminogen activator activity in the cultured cells in a dose-dependent manner. TGF-β is present in the bone matrix and potentially released during bone resorption. TGF-β reduced basal bone resorption and inhibited vitamin D3 [1,25(OH)2D3]-Induced bone resorption in rat long bone cells. These results support the role of IL-1β in the pathological modulation of bone cell metabolism, with regard to implication in the pathogenesis of osteoporosis by IL-1β, and that TGF-β positively inhibits the bone resorption.

Purification and Biochemical Characteristics of Fibrinolytic Enzyme from Streptomyces corcohrussi JK-20

You Jung Kim(김유정),Jeong Uck Park(박정욱),Min Jeong Seo(서민정),Min Jeong Kim(김민정),Hye Hyeon Lee(이혜현),Se Hun Jin(진세훈),Byoung Won Kang(강병원),Yung Hyun Choi(최영현),Yong Kee Jeong(정영기) 한국생명과학회 2010 생명과학회지 Vol.20 No.6

토양에서 생육하는 Streptomyces corcohrussi의 혈전용해효소가 DEAE-Sephadex A-50 그리고 Sephadex G-50 젤 여과를 이용한 크로마토그라피 방법에 의해 순수분리 되었다. SDS-PAGE 분석결과, 분리된 효소는 단일 단백질이고, 그 분자량은 약 34 kDa 이라는 것을 알 수 있었다. 순수분리된 효소의 혈전용해활성은 plasminogen-rich fibrin plate 에서 0.8 U/ml 이었으나, plasminogen-free fibrin plate 에서의 그 효소활성은 0.36 U/ml 이하이었다. 이러한 결과로, 순수 분리된 효소가 plasminogen activator 로 작용한다는 것을 알 수 있었다. 단백질 저해제인 ε-ACA, t-AMCHA 와 mercuric chloride 의 존재시에 그 혈전용해활성은 24% 이하이었는데, 이러한 결과는 이들 plasmin 저해제 그리고(혹은) fibrinogen을 fibrin으로 전환시키는 과정과 관련된 fibrinogen 저해제에 의해 이 효소가 조절될 수 있음을 나타낼 수 있다. 한편으로, 중금속 이온인 Zn<SUP>2+</SUP>은 그 활성을 58% 감소시켰다. 순수 분리된 효소의 최적 온도는 약 50℃ 이었고, 그 효소활성의 92% 이상은 pH 5.0과 8.0 사이에서 유지되었다. 그러므로, 이러한 결과들은 하나의 강력한 혈전용해효소를 제공해서, S. corcohrussi 유래 새로운 혈전용해제의 개발에 기여하도록 한다. A fibrinolytic enzyme of Streptomyces corcohrussi from soil sediment was purified by chromatography using DEAE-Sephadex A-50 and Sephadex G-50. The analysis of SDS-polyacrylamide gel suggested that the purified enzyme is a homogeneous protein and the molecular mass is approximately 34 kDa. The purified enzyme showed activity of 0.8 U/ml in a plasminogen-rich fibrin plate, while its activity in a plasminogen-free fibrin plate was only 0.36 U/ml. These results suggested that the purified enzyme acts as a plasminogen activator. The fibrinolytic activity of the enzyme under the supplementation of protease inhibitors, ε-ACA, t-AMCHA and mercuric chloride in the enzyme reaction was less than 24%, indicating that it could be modulated by the plasmin and/or fibrinogen inhibitors involved in the fibrinogen-to-fibrin converting process. As time passed, Zn<SUP>2+</SUP>, a heavy metal ion, inhibited the activity to 34.1%. The optimum temperature of the purified enzyme was approximately 50℃ and over 92% of the enzyme activity was maintained between pH 5.0 and 8.0. Therefore, our results provide a potential fibrinolytic enzyme as a noble thrombolytic agent from S. corcohrussi.

Effects of Cumulus Cells and Follicular Fluid on Plasminogen Activator Activity during In Vitro Maturation of Porcine Oocytes

Ann Ji-Young,Sa Soo-Jin,Cao Yang,Lee Sang-Young,Cheon Hee-Tae,Yang Boo-Keun,Park Choon-Keun The Korean Society of Animal Reproduction 2006 Reproductive & developmental biology Vol.30 No.2

The present study was conducted to investigate the effects of cumulus cells and porcine follicular fluid (pFF) on plasminogen activator (PA) activity and oocytes maturation in vitro in the pig. The cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) were incubated in NCSU-23 medium with or without 10% pFF for 0, 24, or 48 hr. In the presence of cumulus cells, the proportions of oocytes matured to metaphase-II stage were significantly (P<0.05) higher in medium with pFF than without pFF (69.8 vs. 37.7%, respectively). When COCs and DOs were cultured in the presence of pFF, tissue-type PA (tPA), urokinase-type PA (uPA), and tPA-PA inhibitor (tPA-PAI) were observed in COCs, and PA activities were higher at 48 hr than 24 hr. When COCs and DOs were cultured in the absence of pFF, tPA and tPA-PAI were observed in COCs, and PA activities were increased as duration of culture increased. No PA activities were detected in DOs regardless of pFF supplementation. When porcine oocytes were cultured in the presence of pFF for 24 and 48 hrs, the activities of tPA-PAI, tPA, and uPA were observed in both COCs and DOs. In medium of absence of pFF, PA activities were observed in oocytes with cumulus cells only. On the other hand, three plasminogen-dependent lytic bands (tPA-PAI, tPA, and uPA) were observed in pFF cultures. Particularly uPA activity was higher than the other kinds of PA activity. When oocytes and cumulus cells were separated from porcine COCs at 0 hr of culture, tPA-PAI, tPA, and uPA were detected in cumulus cells at 48 hr of culture, but no PA activities were in DOs. The presence of pFF and cumulus cells in maturation medium stimulated not only nuclear and cytoplasmic maturation in porcine COCs, but also PA production by cumulus cells and COCs. It is possible that PAs produced by cumulus cells migrated through the gap junction between oocyte and cumulus cells. These results suggest that porcine oocytes have no ability to produce PA themselves.

멸치젓갈유래의 혈전용해호소에 대한 특성

양웅석,임학섭,정경태,김영희,허만규,최병태,최영현,정영기,Yang Woong-Suk,Lim Hak-Seob,Chung Kyung Tae,Kim Young-Hee,Huh Man Kyu,Choi Byung Tae,Choi Yung Hyun,Jeong Yong Kee 한국생명과학회 2005 생명과학회지 Vol.15 No.3

멸치 액젖을 이용하여 ammonium sulfate침전, ion exchange, gel filtration, ethanol침전등의 과정을 거쳐 혈전분해 효소(myulchikinase, MK)를 분리 및 정제하였다. 이 정제된 효소를 SDS-PACE gel 전기영동한 결과 분자량은 약 28 kDa 이었다. MK의 활성에 대한 특성을 조사한 결과 NaCl $30\%$까지 활성이 $80\%$이상 유지되는 것으로 보아 내염성 효소로 판단된다. 온도에 대한 효소활성을 조사한 결과, MK의 온도에 대한 안정성은 $40^{\circ}C$까지는 안정하였으나 $50^{\circ}C$이상의 온도에서는 급격하게 활성 떨어졌고, 최적온도는 $40^{\circ}C$였다 MK의 활성은 $pH6\~9$범위에서 매우 안정하였고, 최적 pH는 8이였다. 또한 2가 금속 양이온에 대한 효과는 $Hg^{2+} (1mM)$에 의하여 완전히 활성저해를 보였고, $Zn^{2+}(1mM)$에 의하여 약 $50^{\circ}C$의 저해되는 것을 알았다. Fibrinogen-rich plate와 Fibrinogen-free plate에서 활성을 측정 한 결과, Fibrinogen-rich plate에서는 fibrin 분해능이 있었지만 Fiberinogen-free Plate에서는 분해활성이 없는 것으로 보아 본 효소는 plasminogen activator type의 혈전용해효소로 사료된다. In the previous study, we isolated a myulchikinase (MK), which has fibrinolytic activity and cytotoxicity to the tumor cell line, from myl- chi-jeot-gal. In this study, the effect of NaCl concentration, metallic ions, pH, temperature, and plasminogen on the activity of MK was analysed. The MK activity was maintained at least $80\%$ activity up to $30\%$ NaCl, which indicates that the enzyme may be halotolerant. The optimal pH and temperature were 8 and $40^{\circ}C$, respectively. The fibrinolytic activity of MK was completely inhibited with 0.5 mM $Hg^{2+}$ and inhibited to $50^{\circ}C$ with 1 mM $Cu^{2+}\;and\;Zn^{2+}$. The MK showed strong activity in plasminogen- rich fibrin plate but not in plasminogen-free fibrin plate. The result indicates that the MK may be a plasminogen activator type fibrinolytic enzyme.

Plasminogen Activator Inhibitor-1 Antisense Oligodeoxynucleotides Abrogate Mesangial Fibronectin Accumulation

Ha, Hun Joo,Park, Je Hyun,Seo, Ji Yeon 梨花女子大學校 藥學硏究所 2011 藥學硏究論文集 Vol.- No.21

Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-β1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 µM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-β1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-β1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-β1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.

Inhibition of Plasminogen Activator Inhibitor-1 Expression in Smoke-Exposed Alveolar Type 2 Epithelial Cells Attenuates Epithelial-Mesenchymal Transition

( Jeong Sup Song ),( Chun Mi Kang ) 대한결핵 및 호흡기학회 2011 Tuberculosis and Respiratory Diseases Vol.70 No.6

Background: Smoking is a risk factor for idiopathic pulmonary fibrosis (IPF), but the mechanism of the association remains obscure. There is evidence demonstrating that plasminogen activator inhibitor-1 (PAI-1) is involved in the progression of pulmonary fibrosis. This study was to determine whether the administration of small interfering RNA (siRNA) targeting PAI-1 or PAI-1 inhibitor to the cigarette smoking extract (CSE)-exposed rat alveolar type II epithelial cells (ATII cells) limits the epithelial-mesenchymal transition (EMT). Methods: ATII cells were isolated from lung of SD-rat using percoll gradient method and cultured with 5% CSE. The EMT was determined from the ATII cells by measuring the real-time RT PCR and western blotting after the PAI-1 siRNA transfection to the cells and after administration of tiplaxtinin, an inhibitor of PAI-1. The effect of PAI-1 inhibitor was also evaluated in the bleomycin-induced rats. Results: PAI-1 was overexpressed in the smoking exposed ATII cells and was directly associated with EMT. The EMT from the ATII cells was suppressed by PAI-1 siRNA transfection or administration of tiplaxtinin. Signaling pathways for EMT by smoking extract were through the phosphorylation of SMAD2 and ERK1/2, and finally Snail expression. Tiplaxtinin also suppressed the pulmonary fibrosis and PAI-1 expression in the bleomycin-induced rats. Conclusion: Our data shows that CSE induces rat ATII cells to undergo EMT by PAI-1 via SMAD2-ERK1/2-Snail activation. This suppression of EMT by PAI-1 siRNA transfection or PAI-1 inhibitor in primary type II alveolar epithelial cells might be involved in the attenuation of bleomycin-induced pulmonary fibrosis in rats.