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dc.contributor.advisor박성희-
dc.contributor.author최주연-
dc.creator최주연-
dc.date.accessioned2016-08-26T04:08:22Z-
dc.date.available2016-08-26T04:08:22Z-
dc.date.issued2016-
dc.identifier.otherOAK-000000120816-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/214948-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000120816-
dc.description.abstractFabry disease is an X-linked recessive metabolic disorder that is caused by deficiency of lysosomal α-galactosidase A, resulting in accumulation of globotriaosylceramide (Gb3) in almost all cells and tissues. Among various cardiovascular complications of Fabry disease, thoracic ascending aortic aneurysm is relatively a common finding, which is characterized by abnormal extracellular matrix composition. It has long been accepted that Gb3 accumulation is in charge of the development of various complications of Fabry disease. However the pathophysiology of these complications could not be explained by excess accumulation of Gb3 alone, and recently lyso-Gb3 which is increased in urine and plasma of Fabry patients is suggested as a new biomarker in Fabry disease. Therefore, the aims of this study were to investigate whether lyso-Gb3 can have a possible role in the ascending aortic aneurysm development of Fabry patients, and to consider what the underlying mechanisms are. First, the role of fibroblasts in the pathogenesis of thoracic ascending aortic aneurysm in Fabry disease was examined: 1) Growth, differentiation into myofibroblasts and collagen synthesis were compromised in fibroblasts from Fabry patients. 2) Accumulation of lyso-Gb3 in fibroblasts induced these compromised responses. 3) Lyso-Gb3 reduced KCa3.1 channel expression and activity in fibroblasts. 4) These impairments by lyso-Gb3 were rescued by activating KCa3.1 channel or increasing intracellular Ca2+ concentration. Next, detailed mechanisms of reduced KCa3.1 channel expression by lyso-Gb3 were investigated, especially focused on the role of kinases which is related to the regulation of KCa3.1 channel activity: 1) cAMP were increased by lyso-Gb3 and inhibited ERK phosphorylation through PKA pathway, leading to the inhibition of KCa3.1 channel synthesis. 2) KCa3.1 channel surface expression was reduced by decreasing intracellular levels of PI(3)P which is modulated by the lyso-Gb3-induced reduction of PI3KC2β. Consequently, the increased cAMP by lyso-Gb3 inhibited both KCa3.1 channel synthesis and KCa3.1 channel surface expression. Furthermore, the fibroblast proliferation and differentiation into myofibroblasts were compromised in KCa3.1 dysfunctional fibroblasts by lyso-Gb3, resulted in the inhibited collagen synthesis in fibroblasts. Therefore, this study suggest that the modulation of KCa3.1 channel through these investigated mechanisms could provide a means to attenuate and prevent development of thoracic ascending aortic aneurysm in Fabry disease.;파브리병이란 X염색체 이상에 따른 유전질환이며, 원인은 알파-갈락토시다아제A(α-galactosidase A) 효소의 결핍에 의해 나타난다. 이는 세포와 조직에 글로보트리아오실세라마이드 (globotriaosylceramide, Gb3) 가 축적되어 심혈관 질환을 포함하여 다양한 질환을 야기하지만, 이러한 합병증의 발병원인을 단지 Gb3의 축적만으로는 충분히 설명하기 어렵다. 최근 연구에서, 파브리병 환자의 혈장에서 글로보트리아오실스핑고신 (globotriaosylsphingosine, lyso-Gb3)가 크게 증가 되었다는 보고되어, lyso-Gb3가 파브리병의 새로운 바이오표지자로서 제안 되고 있다. 상행대동맥류는 파브리병의 합병증으로 나타나는 심혈관 질환 중에서 비교적 흔하게 발병되는 질환이다. 이는 세포외기질층 (Extracellular matrix) 구성의 이상으로 인해 발병되는데, 파브리병의 합병증으로 발병하는 상행대동맥류가 어떤 기전에 의한 것인지는 아직 밝혀진 것이 없다. 따라서 먼저, 섬유모세포(fibroblasts)가 파브리병에 의해 이차적으로 발병하는 상행대동맥류 발병기전에서 어떤 역할을 하는지 밝히고자 하였다. 1) 파브리환자에서 섬유모세포의 성장, 근섬유 세포 (myofibroblasts)로 분화, 콜라겐 합성에 모두 이상이 있음을 알 수 있었다. 2) 이와 같은 반응은 lyso-Gb3의 축적에 의한 것임을 밝혔다. 3) 또한 이러한 결과는 KCa3.1 이온통로의 이상으로 인해 칼슘이온이 세포 밖에서 안으로 들어오는 과정이 억제되어 나타나는 것임을 확인 하였다. 4) 그러므로 KCa3.1 이온통로를 활성화시킴으로써 이상 반응들은 회복 될 수 있었다. 이어서, lyso-Gb3에 의해 KCa3.1 이온통로 발현이 감소하는 상세한 기전을 KCa3.1 이온통로의 활성화를 조절하는데 관련된 것으로 알려진 인산화효소(kinases)에 초점을 맞추어 연구하였다. 1) 사이클릭 아데노신일인산(cyclic adenosinemonophosphate; cAMP)은 lyso-Gb3에 의해 농도가 증가되고, 증가된 cAMP는 A-인산화효소(Protein kinase A; PKA)를 활성화시켜 세포외신호조절인산화효소 (Extracellular signal-regulated kinase; ERK)인산화를 억제시켰다. 이러한 과정을 통해 KCa3.1 이온통로의 합성은 감소되었다. 2) 뿐만 아니라, lyso-Gb3에의해 억제된 포스포이노시타이드-3-인산화 효소 (The class 2 beta phosphoinositide-3 kinase; PI3KC2β)는 세포 내 포스파티딜이노시톨-3-인산(Phosphatidylinositol 3-phosphate; PI(3)P)의 농도를 감소시켜 KCa3.1 이온통로가 세포막에 발현되는 것을 억제시켰다. 결과적으로, lyso-Gb3에 의해 KCa3.1 이온통로의 이상이 나타남으로써 섬유모세포에서의 성장이 방해되고, 근섬유세포로의 분화가 잘 안되어 콜라게 합성을 감소시킨다는 것을 밝혔다. 이러한 KCa3.1 이온통로의 이상 기전은 lyso-Gb3에 의해 증가된 세포 내 cAMP가 KCa3.1 이온통로의 합성과 세포막에 KCa3.1 이온통로가 발현되는 것을 방해하는 것이다. 따라서, 본 연구는 KCa3.1 이온통로의 이상이 나타나는 기전을 규명하고, 이러한 기전을 바탕으로 KCa3.1 이온통로를 조절함으로서, 파브리병에서 이차적으로 나타나는 상행대동맥류의 치료표적을 제시하였다.-
dc.description.tableofcontentsI. Introduction 1 1.1 Fabry disease 2 1.1.1 General information 2 1.1.2 Lyso-Gb3 as a biomarker in Fabry disease 6 1.1.3 Ascending thoracic aortic aneurysm in Fabry disease 9 1.2 Fibroblasts and myofibroblasts 12 1.2.1 The importance of adventitial layer 12 1.2.2 Role of adventitia in arterial remodeling 14 1.2.3 Differentiation of adventitial fibroblasts into myofibroblasts 17 1.3 KCa3.1 channel as a therapeutic target 20 1.3.1 Intermediate conductance Ca2+ activated K+ channels 20 1.3.2 The physiological roles of KCa3.1 channels 22 1.3.3 Modulation of KCa3.1 channel expression and activity 24 1.4 cAMP signaling pathway 29 1.4.1 cAMP 29 1.4.2 Activation or inhibition of cell proliferation by cAMP: ERK-dependent mechanisms 31 1.5 Specific aims 34 1.5.1 The role of fibroblasts in pathogenesis of ascending aortic aneurysm secondary to Fabry disease 34 1.5.2 The role of KCa3.1 dysfunction by lyso-Gb3 in ascending aortic aneurysm development secondary to Fabry disease 35 II. Materials and Methods 37 2.1 Cells 38 2.1.1 Cell culture 38 2.2 Molecular biological methods 39 2.2.1 Resazurin reduction cell viability assay 39 2.2.2 RNA analysis 39 2.2.3 Western blotting 42 2.2.4 Collagen assay 45 2.2.5 cAMP assay 46 2.2.6 Biotinylation of cell-surface membrane proteins 47 2.3 Electrophysiological recordings 48 2.3.1 Patch-clamp technique 48 2.4 Chemicals and reagents 49 2.5 Data analysis 51 III. Results 52 3.1 The compromised collagen production of Fabry patients fibroblasts and its mechanism 53 3.1.1 Growth, differentiation into myofibroblasts and collagen synthesis are compromised in fibroblasts from Fabry disease 53 3.1.2 Compromised fibroblast proliferation and differentiation into myofibroblasts are related to the lyso-Gb3 accumulation 57 3.1.3 Compromised collagen synthesis in fibroblasts is related to the lyso-Gb3 accumulation 60 3.2 The effect of KCa3.1 channel dysfunction in Fabry patients fibroblasts or lyso-Gb3 accumulated fibroblasts 63 3.2.1 The expression and activity of KCa3.1 channel is inhibited in fibroblasts from Fabry patients 63 3.2.2 Lyso-Gb3 induces KCa3.1 channel dysfunction in fibroblasts 66 3.2.3 KCa3.1 channel dysfunction by lyso-Gb3 reduces differentiation into myofibroblasts and collagen expression 68 3.2.4 The compromised biological responses by lyso-Gb3 are rescued by increasing intracellular Ca2+ concentration 71 3.3 The mechanisms of lyso-Gb3-induced KCa3.1 dysfunction in fibroblasts 73 3.3.1 Lyso-Gb3 leads to the elevation of cAMP 73 3.3.2 Lyso-Gb3 inhibits ERK cascade by PKA pathway, thereby inducing KCa3.1 channel dysfunction 75 IV. Discussion 80 4.1 General discussions 81 4.1.1 Downregulation of KCa3.1 channel by lyso-Gb3 contributes ascending aortic aneurysm development in Fabry disease 81 4.1.2 Lyso-Gb3 inhibits synthesis and surface expression of KCa3.1 channel 85 4.2 Limitations and suggestions 88 References 95 Abstract (in Korean) 109-
dc.formatapplication/pdf-
dc.format.extent3380227 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleDownregulation of KCa3.1 channel by lyso-globotriaosylceramide results in the inhibition of collagen synthesis in fibroblasts-
dc.typeMaster's Thesis-
dc.title.translated섬유모세포에서 lyso-Gb3에 의한 KCa3.1 이온 통로 이상이 가져오는 콜라겐 합성의 방해-
dc.format.pagexvii, 110 p.-
dc.contributor.examiner서석효-
dc.contributor.examiner정성철-
dc.contributor.examiner박성희-
dc.identifier.thesisdegreeMaster-
dc.identifier.major대학원 의과학과-
dc.date.awarded2016. 2-
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