서론 재료및방법 연구동물 풍선도관에의한혈관손상 782 Korean Circulation J 1998;285:

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1 Original Articles Korean Circulation J 1998;285: 토끼의장골동맥에서, 과확장 - 손상후일어나는세포증식과사망 의경시적변화양상과이에미치는고콜레스테롤혈증의영향 이무용 2 최영진 1,3 채인호 1,3 김효수 1,3 손대원 1,3 오병희 1,3 이명묵 1,3 박영배 1,3 최윤식 1,3 이영우 1,3 Effect of Hypercholesterolemia on Cell Proliferation and Apoptosis in Rabbit Iliac Artery after Balloon-injury Moo-Yong Rhee, MD 2, Hyo-Soo Kim, MD 1,3, In-Ho Chae, MD 1,3, Young-Jin Choi, MD 1,3, Dae-Won Sohn, MD 1,3, Byung-Hee Oh, MD 1,3, Myoung-Mook Lee, MD 1,3, Young-Bae Park, MD 1,3, Yun-Shik Choi, MD 1,3 and Young-Woo Lee, MD 1,3 1 Heart Research Institute, Seoul National University, 2 Department of Internal Medicine, Dankook University College of Medicine, Cheonan, 3 Seoul National University College of Medicine, Seoul, Korea ABSTRACT Objective and MethodsTo evaluate the effect of hypercholesterolemia on apoptosis APOP & proliferation PROL after vascular injury, we examined iliac arteries on 1, 3, 7 & 14 days after balloon injury N5 at each time in rabbits with hypercholesterolemia HC and normocholesterolemia NC. ResultsIn media immediately after injury, APOP occurred massively & then decreased TUNEL index at D1, % at D14. HC did not affect early massive APOP but significantly p0.01 increased APOP, % at D14. Massive early APOP in media was followed by active PROL PCNA index at D7, % at D14). HC sustained the high activity of PROL upto D % at D14p0.01. In intima where cells were scanty initially, PROL activity reached peak at D7 and then decreased % at D7, % at D14. HC significantly p0.05 enhanced PROL at D % at D14). In intima PROL was accompanied by low-grade APOP % at D7, % at D14. HC significantly p0.05 enhanced this low-grade APOP at D %. These effects of HC on APOP & PROL result in a significantly increased area of intima in HC, in NC & media in HC, in NCp0.01. Fundamental difference between HC & NC was infiltration of macrophage in HC, which was colocalized with APOP & PROL activities. ConclusionsBalloon injury induces early massive APOP followed by PROL in media, whereas in intima, it induces active PROL followed by low-grade APOP. Hypercholesterolemia does not affect early massive APOP, but enhances PROL & low-grade APOP at late phase, which results in intimal & medial hyperplasia. Korean Circulation J 1998;285: KEY WORDSApoptosis proliferation Hypercholesterolemia Macrophage Vascular injury. 781

2 서론 재료및방법 연구동물 풍선도관에의한혈관손상 782 Korean Circulation J 1998;285:

3 조직채취및처리 TdT mediated In-Situ Nick End Labeling (TUNEL) 면역조직화학적염색 분석 783

4 결과 혈관손상후내막과중막의증식, 그리고이에미치는고콜레스테롤혈증의영향 Fig. 1. Cross sectional areas of intimal and media mm 2 of iliac artery in hypercholesterolemic HC and normocholesterolemic NC rabbits 1, 3, 7, and 14 days after overstretchinjury. Intimal area of hypercholesterolemic rabbits at day 7 and 14, and medial area at day 14 were larger than that of normocho-lesterolemic rabbits. Results are expressed as meanstandard deviations.*p0.01, hypercholesterolemic vs. normocholesterolemic rabbits. 784 Korean Circulation J 1998;285:

Fig. 2. Microphotographs showing hematoxylin and eosin staining of iliac arteries of normocholesterolemic and hypercholesterolemic rabbits on 14 days after overstretch-injury.

Intimal and medial hyperplasia are more prominent in hypercholesterolemic than in normocholestrolemic rabbit. The intima (i), media (m), and internal elastic lamina (arrow) are indicated.

Microphotographs showing proliferating macrophage and smooth muscle cells around macrophage infiltration at day 14 after injury in hypercholesterolemic rabbit, which was doubled-stained with antibody

Upper panel Proliferating macrophages shown as brown nucleus and red cytoplasm, were frequently observed around internal elastic lamina.

5 Fig. 2. Microphotographs showing hematoxylin and eosin staining of iliac arteries of normocholesterolemic and hypercholesterolemic rabbits on 14 days after overstretch-injury. Upper panel The intima and media of normocholesterolemic rabbit are composed of compact cells. Lower panel The intima and media of hypercholesterolemic rabbit are composed of foam cells. Intimal and medial hyperplasia are more prominent in hypercholesterolemic than in normocholestrolemic rabbit. The intima (i), media (m), and internal elastic lamina (arrow) are indicated. Magnification, 200. Fig. 8. Microphotographs showing proliferating macrophage and smooth muscle cells around macrophage infiltration at day 14 after injury in hypercholesterolemic rabbit, which was doubled-stained with antibody for PCNA (dark brown nucleus arrow heads) and RAM-11 antibody (red cytoplasm). Upper panel Proliferating macrophages shown as brown nucleus and red cytoplasm, were frequently observed around internal elastic lamina. Lower panel Proliferating smooth muscle cells shown as brown nucleus and blue cytoplasm, were also found in intima and media. IEL (arrow), Magnification, 400. Fig. 7. Microphotographs showing serial sections of iliac artery stained with α-actin and macrophage in hypercholes-terolemic rabbits at day 14 after injury. Upper panel Immunostaining with HHF-35 for alpha actin (dark brown color). Lower panel Immunostaining with RAM-11 anti-body for rabbit monocyte/macrophage (dark brown color). Macrophages infiltrated in the outer intima and inner media adjacent to the internal elastic lamina. In some area, macrophage infiltrated whole layers of the intima and media. Magnification, 100. Fig. 9. Microphotographs showing macrophage and smooth muscle cells to undergo apoptosis at day 14 after injury in hypercholesterolemic rabbit, which was doubled-stained with TUNEL (dark brown nucleus arrow head) and RAM-11 antibody (red cytoplasm). Upper panel Macrophages in intima underwent apoptosis, which was shown as apoptotic brown nucleus with red cytoplasm. Lower panel Smooth muscle cell in intima underwent apoptosis, which was shown as apoptotic brown nucleus with blue cytoplasm. Magnification,

혈관 손상후 복구과정에서의 세포증식의 변화와 이에 로서 3 7일에 절정에 도달하였다가 14일째에는 감소 미치는 콜레스테롤의 영향 하였다. 한편, 고콜레스테롤혈증 군에서 3일째 5.

3 and 4), 손상 후 14일째까지 지속적으 1.78%, 7일째 5.99±3.30%, 14일째 3.91±2.77%으 로 세포증식이 증가하는 양상이었다. 세포 증식의 활 Fig. 3. Microphotographs showing PCNA immunostaining of iliac arteries.

A, B and C normocholesterolemic rabbits at day 3, day 7, and day 14 after injury.

The proliferative activity is higher in hypercholesterolemic than in normocholesterolemic rabbits especially on 14 days after

4. PCNA index (%) of intima and media of hypercholesterolemic (HC) and normocholesterolemic (NC) rabbits 1, 3, 7, and 14 days after

There was no difference in PCNA index between the two groups until day 7.

6 혈관 손상후 복구과정에서의 세포증식의 변화와 이에 로서 3 7일에 절정에 도달하였다가 14일째에는 감소 미치는 콜레스테롤의 영향 하였다. 한편, 고콜레스테롤혈증 군에서 3일째 5.85± 중막의 PCNA index는 3일째부터 나타나기 시작하 2.75%, 7일째 7.46±2.74%, 14일째 8.88±2.57%으 였는데, 정상콜레스테롤혈증 군에서는 3일째 5.40± 로서(Figs. 3 and 4), 손상 후 14일째까지 지속적으 1.78%, 7일째 5.99±3.30%, 14일째 3.91±2.77%으 로 세포증식이 증가하는 양상이었다. 세포 증식의 활 Fig. 3. Microphotographs showing PCNA immunostaining of iliac arteries. PCNA positive nuclei are indicated by arrow heads (dark brown color). A, B and C normocholesterolemic rabbits at day 3, day 7, and day 14 after injury. D, E, and F hypercholesterolemic rabbits at day 3, day 7, and day 14 after injury. The proliferative activity is higher in hypercholesterolemic than in normocholesterolemic rabbits especially on 14 days after overstretchinjury. The intima (i), media (m), and internal elastic lamina (arrow) are indicated. Magnification, 400. A D B E C F Fig. 4. PCNA index (%) of intima and media of hypercholesterolemic (HC) and normocholesterolemic (NC) rabbits 1, 3, 7, and 14 days after injury. Proliferative activity was found from day 3 aftr injury. There was no difference in PCNA index between the two groups until day 7. But at day 14, proliferative activity of intima and media of hypercholesterolemic rabbits was higher than that of normocholesterolemic rabbits. Proliferative activity tended to decrease after day 7 in normocholesterolemic rabbits, but it was sustained in hypercholesterolemic rabbits. Results are expressed as meanstandard deviations. *p<0.05, hypercholesterolemic vs. normocholesterolemic rabbits. 786 Korean Circulation J 1998;28(5):

A, B, C and D Normocholesterolemic rabbits at day 1, day 3, day 7, and

E, F, G and H Hypercholesterolemic rabbits at day 1, day 3, day 7, and

normocholesterolemic (NC) rabbits 1, 3, 7, and 14 days after injury.

7 A E B F C G D H Fig. 5. Microphotographs showing T-dT mediated In-Situ nick end labeling (TUNEL) of sections from iliac arteries. TUNEL positive cells are indicated by arrow heads (dark brown color). A, B, C and D Normocholesterolemic rabbits at day 1, day 3, day 7, and day 14. E, F, G and H Hypercholesterolemic rabbits at day 1, day 3, day 7, and day 14. Apoptosis of medial smooth muscle cells was observed massively on 1 and 3 days after injury and then decreased. Apoptotic activity was higher in hypercholesterolemic than in normocholesterolemic rabbit on 14 days. The intima (i), media (m), and internal elastic lamina (arrow) are indicated. Magnification, 400. Fig. 6. TUNEL index (%) of intima and media of hypercholesterolemic (HC) and normocholesterolemic (NC) rabbits 1, 3, 7, and 14 days after injury. Apoptosis of medial smooth muscle cells was observed massively on 1 and 3 days after injury and then decreased. Apoptotic activities both in intima and media, were higher in hypercholesterolemic than in normocholesterolemic rabbit on 14 days. Results are expressed as meanstandard deviations. *p<0.05, hypercholesterolemic vs. normocholesterolemic rabbits. 787

8 혈관손상후복구과정에서의세포사망의변화와이에미치는콜레스테롤의영향 고콜레스테롤혈증의작용기전 : 대식세포의침윤, 세포증식과고사 788 고찰 Korean Circulation J 1998;285:

9 실험동물과혈관-손상방법의선택 혈관손상후세포증식의변화와그기전 789

10 790 혈관손상후세포고사의변화와그기전 Korean Circulation J 1998;285:

11 서론 : 요약 791

12 재료및방법 : 결과 : 792 결론 : 중심단어 REFERENCES 1) Nobuyoshi M, Kimura T, Nosaka H, Mioka S, Ueno K, Yokoi H, et al. Restenosis after successful percutaneous transluminal coronary angioplastyserial angiographic follow-up of 229 patients. J Am Coll Cardiol ) Serruys PW, Luijiten HE, Beatt KJ, Geuskens R, defeyter PJ. Incidence of restenosis after successful coronary angioplasty a time-related phenomenon a quantitative angiographic study in 342 consecutive patients at 1, 2, 3, and 4 months. Circulation ) Popma JJ, Califf RM, Topol EJ. Clinical trials of restenosis after coronary angioplasty. Circulation ) Essed CE, van den Brand M, Becker AE. Transluminal coronary angioplasty and early restenosisfibrocellular occlusion after wall laceration. Br Heart J ) Austin GE, Ratliff NB, Hollmann J, Tabei S, Phillips DF. Intimal proliferation of smooth muscle cells as an explanation for recurrent coronary artery stenosis after percutaneous transluminal coronary angioplasty. J Am Coll Cardiol ) Steele PM, Chesebro JH, Stanson AW, Holmes DR Jr, Dewanjee MK, Badimon L, et al. Balloon angioplasty Natural history of the pathophysiological response to injury in a pig model. Cir Res ) Franklin SM, Faxon DP. Pharmacologic prevention of restenosis after coronary angioplasty review of the randomized clinical trials. Coronary Artery Dis ) O Brien ER, Schwartz SM. Update on the biology and clinical study of restenosis. Trend Cardiovas Med ) Kerr JFR. Shrinkage necrosis a distinct mod of cellular death. J Pathol ) Kerr JFR, Winterford CM, Harmon BV. Apoptosis its significance in cancer and cancer therapy. Cancer 1994 Korean Circulation J 1998;285:

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388 The Korean Journal of Hepatology : Vol. 6. No COMMENT 1. (dysplastic nodule) (adenomatous hyperplasia, AH), (macroregenerative nodule, MR

388 The Korean Journal of Hepatology : Vol. 6. No COMMENT 1. (dysplastic nodule) (adenomatous hyperplasia, AH), (macroregenerative nodule, MR 6 3 2000 ; 387-392 (3) Dysplastic Nodule Young Nyun Park, M.D., Chanil Park, M.D. Department of Pathology, Yonsei University College of Medicine BRIEF HISTORY 56. AST/ALT 72/73 IU/L, total bilirubin 0.7