본 연구실은 동맥 수준 (artery)의 새로운 혈관을 만들고자 1) 혈관신생 유전자의 복합적인 투여 기술 개발과, 2)최근 혈관신생에 관여한다고 알려진 혈관내피전구세포 (endothelial progenitor cell; EPC)의 특성연구를 통하여 ″혈관신생 치료법 (therapeutic angiogenesis)″이 아닌 ″동맥신생 치료법 (therapeutic arteriogenesis)″을 개발하고자 하였다. 지난 2년간의 연구를 통하여 대표적으로 1) 혈관신생에 관여하는 전사인자인 Egr-1을 이용하여 복합적인
본 연구실은 동맥 수준 (artery)의 새로운 혈관을 만들고자 1) 혈관신생 유전자의 복합적인 투여 기술 개발과, 2)최근 혈관신생에 관여한다고 알려진 혈관내피전구세포 (endothelial progenitor cell; EPC)의 특성연구를 통하여 ″혈관신생 치료법 (therapeutic angiogenesis)″이 아닌 ″동맥신생 치료법 (therapeutic arteriogenesis)″을 개발하고자 하였다. 지난 2년간의 연구를 통하여 대표적으로 1) 혈관신생에 관여하는 전사인자인 Egr-1을 이용하여 복합적인 혈관신생유전자들을 과발현시켜 혈관신생을 유도함으로써 하지 허혈을 감소시킬 수 있음을 증명하였고 2) 다양한 혈관신생유전자들의 혈관신생능 (angiogenic potential)을 테스트할 수 있는 간편하고 효율적인 angiogenesis assay법을 개발하였으며 3) 허혈에 의하여 유전자 발현이 turn-on/off 되는 hypoxia-inducible vector를 제조하였고 4) EPC의 배양법과 기능 분석법을 확립하여 만성신부전 환자에서 EPC의 수 감소와 혈관형성능이 저하되었음을 보고하였고 5) 동맥경화 유발 물질인 C-reactive protein (CRP)에 의하여 EPC의 혈관형성능 및 동맥형성능이 감소함을 증명하였다. 1단계 연구기간동안 총 9 편의 논문이 완성되었고, 출판기준으로 전체 SCI IF는 18.1 이다. 그밖에 1건의 산업화를 위한 임상시험 실적, 17 건의 국내외 학회발표, 다양한 공공기능 수행 실적 등이 있다
Abstract▼
Development of combined angiogenic gene therapy for ischemic cardiovascular diseases Cardiovascular diseases are the leading cause of death and the number of patients suffering from ischemic diseases are growing rapidly. The narrowing and/ or occlusion of blood vessel following atherosclerosis pr
Development of combined angiogenic gene therapy for ischemic cardiovascular diseases Cardiovascular diseases are the leading cause of death and the number of patients suffering from ischemic diseases are growing rapidly. The narrowing and/ or occlusion of blood vessel following atherosclerosis prevents free flow of blood and necessarily entails ischemic injury to the organ such as heart, brain and limb. The clinical symptoms of ischemia ranges from stable/unstable anginas to acute ischemic syndrome such as acute myocardial infarction where the initial mortality reaches upto 50 % if left untreated. The clinical outcome of ischemic disease is altered by the collaterals which alleviate the severity of ischemia by bypassing blood flow around the clogged region. Early growth response factor-l (Egr-l) gene therapy Here, we sought to develop a novel angiogenic strategy. where formation of collaterals is stimulated by combined administration of angiogenic growth factors. To facilitate such combination effects, a novel transcription factor, Egr-l, that can induce expression of multiple angiogenic genes such as bFGF, PDGF-A, PDGF-B, IGF-Ⅱ and TGF-β1, was used together with its constitutively active form, Egr-l*. In vitro, ex vivo, and in vivo analyses of Ad-Egr-1* confirmed its angiogenic potential as evidenced by increased proliferation/ migration of endothelial cells, promiscuous sprouting from the explanted skeletal muscle, and enhanced tissue perfusion in a murine model of hindlimb ischemia. With its important role in vascular recovery after occlusion, Egr-l * represents a potential target for therapeutic revascularization. Development of a potent hypoxia-inducible vector A potent chimeric enhancer vector that allows hyper-elevated expression of target genes under hypoxic condition was created. The new chimeric enhancer approach combines different cis-acting elements, such as the early growth response factor-l (Egr-l)-binding site (EBS), the metal-response element (MRE) from metallothionein-Ⅰ, and the hypoxia-response element (HRE) from phosphoglycerate kinase 1, to achieve highly inducible expression from the minimal SV40 promoter. One of the optimized combinations (EBS-MRE-3HRE) displayed a hypoxic induction ratio of 69, conferring robust and tightly regulated gene expression. The expression induced from this novel chimeric vector was significantly higher than the expression from the HCMV promoter in most of the cells tested. High-level expression was also induced by various hypoxia mimetics, including deferoxamine, which is widely used clinically. With its hyper-induction capacity and versatile means of modulation, our chimeric enhancer vector should be an invaluable tool for the gene-based treatment of cancer and ischemic diseases. Development of a gene-based angiogenesis assay A novel angiogenesis assay that offers simple and reproducible means of quantifying angiogenic activity of certain genes was developed. The new method utilizes explant culture of skeletal muscle that had been transfected with the plasmid harboring gene of interest coupled with the optimized procedure of electroporation. The new assay could successfully predict in vivo behavior of connective tissue growth factor in muscle including its interaction with VEGF. Considering the fact that most angiogenesis assays are limited in their clinical relevance and often lack quantification, our method would be very useful in evaluating putative pro-angiogenic and/ or anti-angiogenic genes. Endothelial progenitor cell (EPC) dysfunction in chronic renal failure (CRF) Increased risk of cardiovascular disease in patients with CRF has been explained by accelerated atherosclerosis and impaired angiogenesis, in which EPC may play key roles. We hypothesized that altered EPC biology may contribute to the pathophysiology of CRF. Indeed, CRF patients showed markedly decreased numbers of EPC (44.6%) and colonies (75.3%) when compared to the controls (p<0.001). These findings were corroborated by 30.5% decrease in EPC migratory function in response to vascular endothelial growth factor (VEGF) (p=0.040) and 48.8% decrease in EPC incorporation into human umbilical vein endothelial cells (HUVEC) (p<0.001). In addition, Framingham's risk factor score of both CRF (r=-0.461, p=0.010) and normal group (r=-0.367, p=0.0l6) significantly correlated with the numbers of EPC. The circulating EPC level was significantly lower in CRF patients than in normal group under the same burden of risk factors (p<0.001). A significant correlation was also observed between dialysis dose (Kt/V) and EPC incorporation into HUVEC (r=0.427, p=0.004). These results indicate that EPC biology, which is critical for neovascularization and the maintenance of vascular function, is altered in CRF.
목차 Contents
표지 ...1
제출문 ...2
보고서 초록 ...3
요약문 ...4
SUMMARY ...8
CONTENTS ...11
목차 ...12
제 1 장 연구개발과제의 개요...13
1-1. 연구개발의 경제.사회.기술적 중요성 ...13
1-2. 연구개발의 범위 ...17
제 2 장 국내외 기술개발 현황...18
2-1. 국외의 연구 개발 실적 ...18
제 3 장 연구개발수행 내용 및 결과...20
3-1. Angriogenesis/arteriogenesis 관련 유전자들의 기능과 상호작용 연구 ...20
3-2. 혈관신생 치료법 연구를 위한 혈과 신생 assay법 개발 ...21
3-3. 허혈유도 유전자 발현에 대한 연구 및 hypoxia-inducible vector의 개발 ...23
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