[보고서]지방줄기세포 및 혈관생성인자를 이용한 음경혈관신생유도 및 이를 이용한 발기부전의 국소 치료법 개발

서준규

지방줄기세포 및 혈관생성인자를 이용한 음경혈관신생유도 및 이를 이용한 발기부전의 국소 치료법 개발
Development of curative treatment modality in erectile dysfunction through induction of cavernous neoangiogenesis using local delivery of adipose-tissue derived stem cells and angiogenic factors 원문보기

보고서 정보
주관연구기관	인하대학교 산학협력단
연구책임자	서준규
참여연구자	김우진 , 류지간 , 송재숙 , 최민지 , 신선화
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2010-11
과제시작연도	2008
주관부처	보건복지가족부
사업 관리 기관	한국보건산업진흥원 Korea Health Industry Development Institute
등록번호	TRKO201100006494
과제고유번호	1355059473
사업명	(보건의료기술연구개발)보건의료기술연구개발
DB 구축일자	2013-04-18
키워드	발기 부전.활관생성.지방줄기세포.혈관생성인자.국소치료.erectile dysfunction.angiogenesis.adipose fissue-derived stem cells.angiogenic factors.local therapy.

초록 ▼

Ⅰ. 연구개발의 목적 및 필요성
인체와 가장 유사한 다양한 질환-특이 발기부전 동물모델에서 국소적 혈관생성기술, 즉
- 지방줄기세포에서 고효율로 혈관조직으로 분화할 수 있는 순수세포 분리기술 확립
- 지방조직유래 혈관신생 줄기세포치료
- 지방조직유래 혈관신생 줄기세포와 혈관생성인자의 병용치료
- 고효율의 비바이러스 유전자전달체 개발
- 비바이러스 유전자전달체를 이용한 혈관생성인자의 유전자치료를 통해서 음경신생혈관생성을 유도하고 이를 토대로 발기부전의 근본적인 치료법을 개발함.

Ⅱ. 연구개발의 내용 및 범위
1. 지방줄기세포에서 고효율로 혈관조직으로 분화할 수 있는 순수세포 분리기술 확립
- GFP + 생쥐와 dsRed + 생쥐의 지방줄기세포를 분리하고, FACS와 MACS를 사용하여 지방줄기세포에서 고효율로 혈관생성으로 분화할 수 있는 순수세포 분리기술을 확립함.
2. 지방조직유래 혈관신생 줄기세포치료
- 다양한 질환-특이 발기부전 동물모델에서 신선하게 분리된 지방조직유래 혈관신생 줄기세포(adipose tissue-derived stromal vascular fraction, AD-SVF)의 혈관생성 및 발기력 개선효과를 평가함.
3. 지방조직유래 혈관신생 줄기세포와 혈관생성인자의 병용치료
- 다양한 질환-특이 발기부전 동물모델에서 신선하게 분리된 지방조직유래 혈관신생 줄기세포(adipose tissue-derived stromal vascular fraction. AD-SVF)와 혈관생성인자의 병용치료의 혈관생성 및 발기력 개선효과를 평가함.
4. 고효율의 비바이러스 유전자전달체 개발
- 다양한 혈관성 불기부전 동물모델의 음경에서 유전자전달에 가장 효과적인 유전자전달체 (RTP801 promoter, Epo enhancer, Epo enhancer + ODD domain etc.)를 개발함.
5. 혈관생성인자의 유전자/단백질치료
- 다양한 질환-특이 발기부전 동물모델에서 혈관생성인자 (Angiopoietin-1, variant form of Angiopoietin-1, VEGF-A, VEGF-E, etc.)의 유전자/단백질치료의 혈관생성 및 발기력 개선효과를 평가함.

Ⅲ. 연구개발결과
1. 지방줄기세포에서 고효율로 혈관조직으로 분화할 수 있는 순수세포 분리기술 확립
- 신선하게 분리된 AD-SVF는 혈관내피세포들의 역동적인 재조합을 통해서 새로운 혈관을 생성하고, 하지허혈 동물모델에서도 성공적으로 혈관생성을 유도함
2. 지방조직유래 혈관신생 줄기세포치료
- 신선하게 분리된 AD-SVF가 당뇨성 발기부전 동물모델에서 음경혈관내피세포 재생을 통해서 발기력을 부분적으로 획복시킴.
3. 지방조직유래 혈관신생 줄기세포와 혈관생성인자의 병용치료
- 신선하게 분리된 AD-SVF를 VEGF-A와 angiopoietin-1으로 전처치한 후 투여한 결과, 당뇨성 발기부전 동물모델에서 음경혈관내피세포 재생을 통해서 발기력을 완전하게 회복시킴.
4. 고효율의 비바이러스 유전자전달체 개발
- 고지질혈증 발기부전 모델의 음경에서 Epo enhancer (pEpo-SV-luc)가 유전자 전달효과를 현저하게 증강시킴.
5. 혈관생성인자의 유전자/단백질치료
- Angiopoietin-1 합성 유전자 또는 단백질이 당뇨성 발기부전 동물모델에서 음경혈관내피세포 재생을 통해서 발기력을 부분적으로 회복시킴.

Ⅳ. 연구개발결과의 활용계획
1. 기술적 측면
- 발기부전의 치료에 있어서, 질환 및 그 병인에 근거한 새로운 세포/유전자/단백질치료법의 임상적용
- 기존의 치료법에 실패한 많은 수의 발기부전 환자에게 근본적인 치료의 기회를 제공
- 발기부전뿐만 아니라 혈관질환을 포함한 여러 관련 질환의 치료에도 상당한 파급효과
ㆍ기능적/치료적 혈관생성유도 : 허혈성 심혈관/뇌혈관질환, 당뇨성 혈관합병증, 말초혈관질환 (버거씨병 등)
ㆍ비바이러스 유전자전달체 개발 : 심혈관계, 근골격계 등. 여러 분야의 유전자치료에 적용
2. 경제/산업적 측면
- 음경혈관 재생기술의 기술수요 및 시장전망 : 전체 발기부전시장의 50% 이상을 점유할 것으로 기대
ㆍ예상 매출규모: 국매. 500-1000억원/년: 전 세계, 2-4조원/년
- 고부가가치 치료제개발 산업 및 연구로 재편 (치료 패러다임의 변화)
- 세포/유전자/단백질치료 및 관련 바이오산업의 활성화

Abstract ▼

Ⅰ. Purpose
To deve lop curative treatment modalities for erectile dys function (ED) using the techniques controlling local angiogenesis in various disease-specific animal models.
- Establishment of technique to isolate pure cells from adipose-derived stromal vascular fraction (AD-SVF) that can be differentiated into the vascular stru ctures with high efficiency .
- Investi gation of the efficacy of freshly iso lated AD-SVF in animal models for ED.
- Investigation of the efficacy of combined treatm ent with freshly isolated AD-SVF and angiogenic factors in animal models for ED.
- Development of nonviral gene delivery systems.
- Investigation of the efficacy of angiogenic factor gene therapy by using nonviral gene delivery systems in animal models for ED.

Ⅱ . Contents
1. Establishment of technique to isolate pure cells from AD-SVF that can be differentiated into the vascular stru ctures with high efficiency.
2. Investigation of the efficacy of freshly isolated AD-SVF in animal models for ED.
3. Investigation of the efficacy of combined tr eatm ent with freshly isolated AD-SVF and angiogenic factors in animal models for ED.
4. Development of nonviral gene delivery systems.
5. Investigation of the efficacy of angiogenic factor gene or protein therapy in animal models for ED.

Ⅲ . Results
1. Establishment of technique to isolate pure cells from AD-SVF th at can be differentiated into the vascular stru ctures with high efficiency.
- Freshly isolated AD-SVF efficien tly induced new vessel form ation through the dynamic reassembly of blood endothelial cells and achieved therapeutic neovascularization in hind limb ischemia model.
2. Investigation of the efficacy of freshly isolated AD-SVF in animal models for ED.
- Intracavernous delivery of fr eshly isolated AD-SVF partially restored erectile function by enhancing cavern ous endothelial regeneration in an animal mode l of streptozotocin-induced diabetic ED.
3. Investigation of the efficacy of combined tr eatm ent with freshly isolated AD-SVF and angiogenic factors in animal models for ED.
- Priming of freshly isolated AD-SVF with VEGF-A and angiopoietin-1 completely restored erectile function by enhancing cavern ous endothelial regeneration in an animal model of streptozotocin-induced diabetic ED.
4. Development of nonviral gene delivery systems.
- Epo enhancer (pEpo-SV-luc) significantly induced reporter gene expression in the penis of high-cholesterol diet-induced ED model.
5. Investigation of the efficacy of angiogenic factor gene or protein therapy in animal models for ED.
- Intracavern ous delivery of synthetic angiogenic-1 gene or protein partially restored erectile function by enhancing cavernous regeneration in an animal model of streptozotocin-induced diabetic ED.

Ⅳ. App lications
1. Technical aspects
- Application of new field of therapy. disease-specific and pathology-oriented curative cell / gene/ protein therapy. in ED.
- Introduction of curative treatment modality for majority of patients witheD who did not respond to conventional therapies.
- Positive influence on other medical fields related with vascular disease. or nonviral gene therapy.
ㆍFunctional or therapeutic angiogenesis: ischemic cardiovascular or cerebrovascular disease. diabetic vascular complications. peripheral vascu lar disease (Burger' s disease)
ㆍNonviral gene delivery systems: gene therapy for other field of disease including cardiovascular or musculoskeletal diseases.
- Potential demand of te chnique for controlling cavern ous angiogeneis: expected to possess more than half of the total ED market.
ㆍ Expected market value: domestic. 50-1 00 billion won /year: international. 2000-4000 billion won /year.
- Paradigm shift to develop high value- added product.
- Revitalize the bioindustry related with the development of cell / gene/protein therapeutics.

목차 Contents

Ⅰ. 총괄현황 ...1
1. 겉표지 ...2
2. 제출문 ...4
3. 보고서 요약서 ...5
4. 요약문 (한글) ...6
5. 요약문 (영문) ...8
6. 연구 성과 실적 및 향후 계획 ...10
7. 참여연구원 현황표 ...17
Ⅱ. 총괄연구과제 연구결과 ...18
1. 연구개발과제의 배경 및 필요성 ...19
2. 국내외 기술개발 현황 ...25
3. 연구개발과제의 추진체계 ...26
4. 연구개발수행 내용 및 결과 ...28
5. 목표달성도 및 관련분야 기여도 ...49
6. 향후 연구성과 추진 계획 ...53
7. 연구개발결과의 파급효과 ...54
8. 연구개발결과의 활용계획 ...56
9. 연구개발과정에서 수집한 해외과학기술정보 ...57
10. 참고문헌 ...57
Ⅳ. 제1 세부 연구과제 연구결과 ...58
제 1 세부 연구과제 연구결과 ...58
1. 연구개발과제의 목표 ...59
2. 연구개발과제의 추진체계 ...61
3. 연구개발수행 내용 및 결과 ...63
4. 참고문헌 ...83
Ⅴ. 제2 세부 연구과제 연구결과 ...85
제 2 세부 연구과제 연구결과 ...85
1. 연구개발과제의 목표 ...86
2. 연구개발과제의 추진체계 ...88
3. 연구개발수행 내용 및 결과 ...89
4. 참고문헌 ...107
Ⅳ. 첨부서류 ...108
1. 자체평가의견서 ...110
2. 실적 증빙자료 ...114
3. 기타 첨부서류 ...116

표/그림 (68)

표 발기부전은 전신혈관질환과 동일한 위험인자 및 병리기전을 가지며, 음경동맥의 내구경이 심 혈관 (관상동맥), 뇌혈관 (내경동맥), 하지혈관 (대퇴동맥)에 비해서 작기 때문에 가장 초기에 증상이 발현됨
표 The structures of the pSV-Luc, pRTP801-Luc, pEpo-SV-Luc, pEpo-SV-Luc-ODD, and pEpo-SV-Luc-sODD plasmids. E indicates the erythropoietin (Epo) enhancer. ODD = oxygendependent degradation; sODD = short oxygen-dependent degradation.
표 HIF-1 expression in the corpus cavernosum α tissue of control and hypercholesterolemic mice. (A) Western blot analysis showing the protein expression of HIF-1α. (B) Immunohistochemical staining of cavernous tissue performed with antibody to HIF-1α. The results were similar from four independent samples. HIF-1α = hypoxia-inducible factor-1α.
표 Luciferase expressions by reporter plasmids in the corpus cavernosum tissue of control and hypercholesterolemic mice. Luciferase activity in age-matched control (A) or hypercholesterolemic mice (B) 3 days after intracavernous injection of pSV-Luc, pRTP801-Luc, pEpo-SV-Luc, pEpo-SV-Luc-ODD, pEpo-SV-Luc-sODD (100 μg/40 μL, respectively), or saline (40 μ L). Each bar depicts the mean values (± standard deviation) from N = 5 animals per group.
표 Luciferase expression area in hypercholesterolemic mouse corpus cavernosum tissues. Immunofluorescent double staining of cavernous tissue performed with antibodies to luciferase and PECAM-1, or with antibodies to luciferase and smooth muscle -α actin. The results were similar from four independent samples. PECAM-1 = platelet/endothelial cell adhesion molecule-1.
표 AD-SVF transfer restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve.
$transfer increases cavernous endothelial content. (A) Anti-CD31 and smooth muscle α -actin staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 2 wk after receiving intracavernous injection of PBS (20μl) or AD-SVF (1×105cells/20 μl). Scale bar=200 μm. (B,C) Quantitative analysis of endothelial cell and smooth muscle cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean values (± SE) from n=6 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs untreated and PBS-treated diabetic groups. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; C = control; N = no treatment; P = phosphate-buffered saline; DM = diabetes mellitus.$ 표 transfer increases cavernous endothelial content. (A) Anti-CD31 and smooth muscle α -actin staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 2 wk after receiving intracavernous injection of PBS (20μl) or AD-SVF (1×105cells/20 μl). Scale bar=200 μm. (B,C) Quantitative analysis of endothelial cell and smooth muscle cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean values (± SE) from n=6 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs untreated and PBS-treated diabetic groups. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; C = control; N = no treatment; P = phosphate-buffered saline; DM = diabetes mellitus.
$AD-SVF transfer increases VEGF-A expression in the corpus cavernosum tissue. Anti-CD31 and VEGF-A staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 1 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1×105 cells/20 μl). Scale bar=100μ m. STZ = streptozotocin; AD-SVF = adipose tissuederived stromal vascular fraction; GFP, green fluorescent protein; VEGF-A, vascular endothelial growth factor-A.$ 표 AD-SVF transfer increases VEGF-A expression in the corpus cavernosum tissue. Anti-CD31 and VEGF-A staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 1 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1×105 cells/20 μl). Scale bar=100μ m. STZ = streptozotocin; AD-SVF = adipose tissuederived stromal vascular fraction; GFP, green fluorescent protein; VEGF-A, vascular endothelial growth factor-A.
$AD-SVF transfer recruits CD34(+)CD31(-) progenitor cell population in the corpus cavernosum tissue. Anti-CD31 (green) and CD34 staining (red) of cavernous tissue from age-matched control or diabetic mice 1, 3, and 7 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar=200 μm. Results were similar from three independent experiments. On merged images, red color represents CD34(+)CD31(-) progenitor cell population. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction.$ 표 AD-SVF transfer recruits CD34(+)CD31(-) progenitor cell population in the corpus cavernosum tissue. Anti-CD31 (green) and CD34 staining (red) of cavernous tissue from age-matched control or diabetic mice 1, 3, and 7 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar=200 μm. Results were similar from three independent experiments. On merged images, red color represents CD34(+)CD31(-) progenitor cell population. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction.
표 Engraftment and differentiation of donor AD-SVF into the host cavernous endothelial cells. Anti-CD31 staining of cavernous tissue from diabetic mice 3 d after receiving intracavernous injection of AD-SVF (1 × 105 cells/20 μl). Arrows indicate the GFP(+) cells that expressed CD31.Scale bar=100 μm. GFP = green fluorescent protein.
표 Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 completely restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve.
$Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 additively increases cavernous endothelial content. Anti-CD31 staining of cavernous tissue from age-matched control, or diabetic mice stimulated at 2 wk after intracavernous injection of PBS (20 μl), AD-SVF (1×105 cells), AD-SVF + VEGF-A, AD-SVF + COMP-Ang1, or AD-SVF + VEGF-A + COMP-Ang1. DM = diabetes mellitus; SVF = adistromal vascular fraction.$ 표 Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 additively increases cavernous endothelial content. Anti-CD31 staining of cavernous tissue from age-matched control, or diabetic mice stimulated at 2 wk after intracavernous injection of PBS (20 μl), AD-SVF (1×105 cells), AD-SVF + VEGF-A, AD-SVF + COMP-Ang1, or AD-SVF + VEGF-A + COMP-Ang1. DM = diabetes mellitus; SVF = adistromal vascular fraction.
표 Adenoviral COMP-Ang1 gene or COMP-Ang1 protein transfer restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve.
표 COMP-Ang1 protein transfer increases cavernous endothelial content through enhanced endothelial cell proliferation.
표 COMP-Ang1 protein transfer decreases cavernous superoxide anion production.
표 COMP-Ang1 protein transfer decreases apoptosis in cavernous endothelial cells and smooth muscle cells.
표 COMP-Ang1 protein transfer restores endothelial cell-to-cell junction proteins and decreases cavernous endothelial permeability.
표 Schematic representation of a proposed model for how COMP-Ang1 protein restores erectile function in diabetic mice. EC, endothelial cell; O2·-,superoxide anion; iNOS, inducible nitric oxide synthase; NO·, nitric oxide; ONOO-, peroxynitrite; eNOS, endothelial nitric oxide synthase; ZO-1, zonula occludens-1; VE-cadherin; vascular endothelial-cadherin.
표 지방줄기세포가 혈관조직을 생체 내에서 형성함.
표 혈관성장인자에 2. 의해 지방줄기세포유래 혈관의 기능성이 향상됨.
표 혈관성장억제인자에 3. 의해 지방줄기세포유래 혈관신생이 억제됨.
표 대식세포에 4. 의해 지방줄기세포유래 혈관신생이 조절됨.
표 지방줄기세포가 만드는 혈관은 지방조직 내의 5. 혈관내피세포가 분리-재조합되는 과정을 통해 형성됨.
표 지방줄기세포유래의 혈관에 6. 의해 생쥐하지허혈모델의 허혈성 괴사가 방지됨.
표 The structures of the pSV-Luc, pRTP801-Luc, pEpo-SV-Luc, pEpo-SV-Luc-ODD, and pEpo-SV-Luc-sODD plasmids. E indicates the erythropoietin (Epo) enhancer. ODD = oxygendependent degradation; sODD = short oxygen-dependent degradation.
표 HIF-1α expression in the corpus cavernosum tissue of control and hypercholesterolemic mice. (A) Western blot analysis showing the protein expression of HIF-1α. (B) Immunohistochemical staining of cavernous tissue performed with antibody to HIF-1α. The results were similar from four independent samples. HIF-1α = hypoxia-inducible factor-1α.
표 Luciferase expressions by reporter plasmids in the corpus cavernosum tissue of control and hypercholesterolemic mice. Luciferase activity in age-matched control (A) or hypercholesterolemic mice (B) 3 days after intracavernous injection of pSV-Luc, pRTP801-Luc, pEpo-SV-Luc, pEpo-SV-Luc-ODD, pEpo-SV-Luc-sODD (100 g/40 L, respectively), μ μ or saline (40 μ L). Each bar depicts the mean values (± standard deviation) from N = 5 animals per group.
표 Luciferase expressions by reporter plasmids in the corpus cavernosum tissue of hypercholesterolemic mice. Luciferase activity 3 days after intracavernous injection of pSV-Luc, pRTP801-Luc, pEpo-SV-Luc (100 μg/40 μL, respectively). Each bar depicts the mean values (± standard deviation) from N = 5 animals per group.
표 Time-course expression of luciferase protein by pSV-Luc or pEpo-SV-luc in hypercholesterolemic mouse corpus cavernosum tissues. Luciferase activity in hypercholesterolemic mice 3, 7, 14, and 28 days after intracavernous injection of pSV-Luc or pEpo-SV-Luc (100 g/40 L,respectively). Each bar depicts μ μ the mean values (± standarddeviation) from N = 5 animals per group and per each time point. *P < 0.01 compared with the pSV-Luc group.
표 Luciferase expression area in hypercholesterolemic mouse corpus cavernosum tissues. Immunofluorescent double staining of cavernous tissue performed with antibodies to luciferase and PECAM-1, or with antibodies to luciferase and smooth muscle α-actin. The results were similar from four independent samples. PECAM-1 = platelet/endothelial cell adhesion molecule-1.
표 Physiologic and metabolic parameters ; 2 weeks after treatment
표 AD-SVF transfer restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve.
$AD-SVF transfer increases cavernous endothelial content. (A) Anti-CD31 and smooth muscle α -actin staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 2 wk after receiving intracavernous injection of PBS (20μl) or AD-SVF (1×105 cells/20 μl). Scale bar=200 μm. (B,C) Quantitative analysis of endothelial cell and smooth muscle cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean values (± SE) from n=6 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs untreated and PBS-treated diabetic groups. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; C = control; N = no treatment; P = phosphate-buffered saline; DM = diabetes mellitus.$ 표 AD-SVF transfer increases cavernous endothelial content. (A) Anti-CD31 and smooth muscle α -actin staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 2 wk after receiving intracavernous injection of PBS (20μl) or AD-SVF (1×105 cells/20 μl). Scale bar=200 μm. (B,C) Quantitative analysis of endothelial cell and smooth muscle cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean values (± SE) from n=6 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs untreated and PBS-treated diabetic groups. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; C = control; N = no treatment; P = phosphate-buffered saline; DM = diabetes mellitus.
$Time-course survival of AD-SVF and expression of CD31 in the corpus cavernosum tissue. (A) Anti-CD31 staining of cavernous tissue from age-matched control or diabetic mice 1, 3, and 7d and 2wk after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1×105 cells/20 μl). Nuclei were labeled with the DNA dye DAPI. Scale bar = 200 μm. (B) Number of GFP-positive AD-SVF in the corpus cavernosum tissue. Each bar depicts the mean values (± SE) from n=4 animals per group. (C) Quantitative analysis of endothelial cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean values (± SE) from n=4 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs PBS-treated group. †p < 0.05 vs control group. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; GFP = green fluorescent protein; DAPI = DM = diabetes mellitus; C = control; P = phosphate-buffered saline.$ 표 Time-course survival of AD-SVF and expression of CD31 in the corpus cavernosum tissue. (A) Anti-CD31 staining of cavernous tissue from age-matched control or diabetic mice 1, 3, and 7d and 2wk after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1×105 cells/20 μl). Nuclei were labeled with the DNA dye DAPI. Scale bar = 200 μm. (B) Number of GFP-positive AD-SVF in the corpus cavernosum tissue. Each bar depicts the mean values (± SE) from n=4 animals per group. (C) Quantitative analysis of endothelial cell content in cavernous tissue was performed by using an image analyzer. Each bar depicts the mean values (± SE) from n=4 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs PBS-treated group. †p < 0.05 vs control group. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; GFP = green fluorescent protein; DAPI = DM = diabetes mellitus; C = control; P = phosphate-buffered saline.
$AD-SVF transfer increases the number of dividing endothelial cells in the corpus cavernosum tissue. Immunohistochemical staining of cavernous tissue using antibodies to CD31 (red) and phosphohistone H3 (PH3; green) in age-matched control or diabetic mice 1, 3, and 7 d and 2 wk after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar = 100 μm. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; GFP = green fluorescent protein.$ 표 AD-SVF transfer increases the number of dividing endothelial cells in the corpus cavernosum tissue. Immunohistochemical staining of cavernous tissue using antibodies to CD31 (red) and phosphohistone H3 (PH3; green) in age-matched control or diabetic mice 1, 3, and 7 d and 2 wk after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar = 100 μm. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; GFP = green fluorescent protein.
$AD-SVF transfer induces cavernous eNOS phosphorylation and increases cGMP concentration. (A) Anti-CD31 (red) and phospho-eNOS staining (Ser1177; green) of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 2 wk after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar=200μm. (B) Each bar depicts the mean values (± SE) from n=6 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs untreated and PBS-treated diabetic groups. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; C = control; N = no treatment; P = phosphate-buffered saline; DM = diabetes mellitus.$ 표 AD-SVF transfer induces cavernous eNOS phosphorylation and increases cGMP concentration. (A) Anti-CD31 (red) and phospho-eNOS staining (Ser1177; green) of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 2 wk after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar=200μm. (B) Each bar depicts the mean values (± SE) from n=6 animals per group. *p < 0.01 vs control and AD-SVF-treated diabetic groups, #p < 0.01 vs untreated and PBS-treated diabetic groups. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction; C = control; N = no treatment; P = phosphate-buffered saline; DM = diabetes mellitus.
$AD-SVF transfer increases VEGF-A expression in the corpus cavernosum tissue. Anti-CD31 and VEGF-A staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 1 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1×105 cells/20 μl). Scale bar=100μ m. STZ = streptozotocin; AD-SVF = adipose tissuederived stromal vascular fraction; GFP, green fluorescent protein; VEGF-A, vascular endothelial growth factor-A.$ 표 AD-SVF transfer increases VEGF-A expression in the corpus cavernosum tissue. Anti-CD31 and VEGF-A staining of cavernous tissue from age-matched control, untreated diabetic mice, or diabetic mice 1 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1×105 cells/20 μl). Scale bar=100μ m. STZ = streptozotocin; AD-SVF = adipose tissuederived stromal vascular fraction; GFP, green fluorescent protein; VEGF-A, vascular endothelial growth factor-A.
$AD-SVF transfer recruits CD34(+)CD31(-) progenitor cell population in the corpus cavernosum tissue. Anti-CD31 (green) and CD34 staining (red) of cavernous tissue from age-matched control or diabetic mice 1, 3, and 7 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar=200 μm. Results were similar from three independent experiments. On merged images, red color represents CD34(+)CD31(-) progenitor cell population. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction.$ 표 AD-SVF transfer recruits CD34(+)CD31(-) progenitor cell population in the corpus cavernosum tissue. Anti-CD31 (green) and CD34 staining (red) of cavernous tissue from age-matched control or diabetic mice 1, 3, and 7 d after receiving intracavernous injection of PBS (20 μl) or AD-SVF (1 × 105 cells/20 μl). Scale bar=200 μm. Results were similar from three independent experiments. On merged images, red color represents CD34(+)CD31(-) progenitor cell population. STZ = streptozotocin; AD-SVF = adipose tissue-derived stromal vascular fraction.
표 Engraftment and differentiationof donor AD-SVF into the host cavernous
$Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 protein completely restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve. Representative ICP responses for age-matched control, or diabetic mice stimulated at 2 wk after intracavernous injection of PBS (20 μ l), AD-SVF (1×105 cells), AD-SVF + VEGF-A, AD-SVF + COMP-Ang1, or AD-SVF + VEGF-A + COMP-Ang1. The stimulus interval is indicated by a solid bar. Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group. Each bar depicts the mean values (± SE) from n=4 or 5 animals per group. *p < 0.01 vs PBS-treated diabetic groups. STZ=streptozotocin; AD-SVF=adipose tissue-derived stromal vascular fraction; C, control; P, PBS; S, SVF; SV, SVF + VEGF-A; SA, SVF + COMP-Ang1; SVA, SVF + VEGF-A + COMP-Ang1.$ 표 Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 protein completely restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve. Representative ICP responses for age-matched control, or diabetic mice stimulated at 2 wk after intracavernous injection of PBS (20 μ l), AD-SVF (1×105 cells), AD-SVF + VEGF-A, AD-SVF + COMP-Ang1, or AD-SVF + VEGF-A + COMP-Ang1. The stimulus interval is indicated by a solid bar. Ratios of mean maximal ICP and total ICP (area under the curve) to mean systolic blood pressure (MSBP) were calculated for each group. Each bar depicts the mean values (± SE) from n=4 or 5 animals per group. *p < 0.01 vs PBS-treated diabetic groups. STZ=streptozotocin; AD-SVF=adipose tissue-derived stromal vascular fraction; C, control; P, PBS; S, SVF; SV, SVF + VEGF-A; SA, SVF + COMP-Ang1; SVA, SVF + VEGF-A + COMP-Ang1.
표 Physiologic and metabolic parameters : 2 weeks after treatment
$Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 protein additively increases cavernous endothelial content. Anti-CD31 staining of cavernous tissue from age-matched control, or diabetic mice stimulated at 2 wk after intracavernous injection of PBS (20 μl), AD-SVF (1×105 cells), AD-SVF + VEGF-A, AD-SVF + COMP-Ang1, or AD-SVF + VEGF-A + COMP-Ang1. DM = diabetes mellitus; SVF = adistromal vascular fraction.$ 표 Pretreatment of AD-SVF with combined VEGF-A and COMP-Ang1 protein additively increases cavernous endothelial content. Anti-CD31 staining of cavernous tissue from age-matched control, or diabetic mice stimulated at 2 wk after intracavernous injection of PBS (20 μl), AD-SVF (1×105 cells), AD-SVF + VEGF-A, AD-SVF + COMP-Ang1, or AD-SVF + VEGF-A + COMP-Ang1. DM = diabetes mellitus; SVF = adistromal vascular fraction.
표 In vivo COMP-Ang1 or β-gal expression in diabetic corpus cavernosum tissue.
표 Adenoviral COMP-Ang1 gene or COMP-Ang1 protein transfer restores intracavernous pressure (ICP) elicited by electrical stimulation of the cavernous nerve.
표 COMP-Ang1 protein transfer increases cavernous endothelial content through enhanced endothelial cell proliferation.
표 COMP-Ang1 protein transfer induces cavernous eNOS phosphorylation and increases cGMP concentration.
표 COMP-Ang1 protein transfer decreases cavernous superoxide anion production.
표 COMP-Ang1 protein transfer decreases cavernous nitrotyrosine production. A:
표 COMP-Ang1 protein transfer decreases p47phox and iNOS expression in the corpus cavernosum tissue.
표 COMP-Ang1 protein transfer decreases apoptosis in cavernous endothelial cells and smooth muscle cells.
표 COMP-Ang1 protein transfer restores endothelial cell-to-cell junction proteins and decreases cavernous endothelial permeability.
표 Schematic representation of a proposed model for how COMP-Ang1 protein restores erectile function in diabetic mice. EC, endothelial cell; O2·-,superoxide anion; iNOS, inducible nitric oxide synthase; NO·, nitric oxide; ONOO-, peroxynitrite; eNOS, endothelial nitric oxide synthase; ZO-1, zonula occludens-1; VE-cadherin; vascular endothelial-cadherin.
표 COMP-Ang1 protein-induced cavernous angiogenesis and recovery of erectile function is NOS-dependent.
표 지방줄기세포가 혈관조직을 생체 내에서 형성함.
표 지방줄기세포가 혈관조직을 생체 내에서 형성하고 수용 (recipient) 동물 혈관과 연결됨.
표 구형배양 된 지방줄기세포는 혈관신생능력이 떨어짐.
표 혈관성장인자에 의해 지방줄기세포유래 혈관의 기능성이 향상됨.
표 혈관성장인자에 의한 지방줄기세포유래 혈관의 sprouting 현상의 촉진됨.
표 혈관성장억제인자에 의해 지방줄기세포유래 혈관신생이 억제됨.
표 대식세포에 의해 지방줄기세포유래 혈관신생이 조절됨.
표 지방조직내에 발달된 혈관 구조와 지방줄기세포에서의 혈관내피세포의 비율.
표 지방줄기세포가 만들어내는 혈관은 지방조직내의 혈관내피세포유래임.
표 지방줄기세포가 만드는 혈관은 지방조직 내의 혈관내피세포가 분리-재조합되는 과정을 통해 형성됨.
표 지방줄기세포유래의 혈관에 의해 생쥐하지허혈모델의 허혈성 괴사가 방지됨.
표 생쥐하지허혈모델에서 지방줄기세포는 기능성을 가지는 혈관을 만듦.
표 지방줄기세포는 혈관성장인자의 도움을 받아 피하지방 이식물에서 일어나는 지방의 체내 흡 수를 방지함.
표 지방줄기세포와 피하지방이 동시이식된 이식물에서 지방줄기세포, 피하지방, 수용동물의 혈 관이 같이 연결되는 모습이 관찰됨.
표 지방줄기세포의 타가이식 시에 비정상적인 (allograft) 혈관구조와 비정상적인 혈관내피세포 결 합이 관찰됨.

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