| 研究生: |
黃智勇 Huang, Chih-Yung |
|---|---|
| 論文名稱: |
白血球在低密度脂蛋白處理後之內皮細胞表面黏著程度的變化 Adhesiveness of leukocytes on low-density lipoprotein pretreated endothelial cells |
| 指導教授: |
任卓穎
Jen, Chauying J. |
| 學位類別: |
碩士 Master |
| 系所名稱: |
醫學院 - 生理學研究所 Department of Physiology |
| 論文出版年: | 2004 |
| 畢業學年度: | 92 |
| 語文別: | 中文 |
| 論文頁數: | 122 |
| 中文關鍵詞: | 白血球 、低密度脂蛋白 、氧化低密度脂蛋白 、流體室 |
| 外文關鍵詞: | oxidized low density lipoprotein, leukocyte, low density lipoprotein, chamber |
| 相關次數: | 點閱:128 下載:1 |
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血管中白血球的黏著在許多心血管疾病的發展過程扮演一個相當重要的角色。已有許多文獻指出,在動脈硬化潰瘍部位上的內皮細胞,會異常表現各類的黏著分子。而當這些黏著分子被抑制住時,可以看到白血球及脂肪的堆積在動脈硬化潰瘍部位有減少的結果出現。此外,氧化的低密度脂蛋白(ox-LDL)會刺激內皮細胞上各類的黏著分子表現量增加。因此,ox-LDL可能藉著增加內皮細胞黏著分子表現使內皮細胞功能失調,進而促進白血球的黏著及穿越內皮細胞進入血管內層堆積的反應,最後使動脈硬化潰瘍發展加劇。這篇論文主要的目的,是想觀察及量化白血球在LDL處理後之內皮細胞黏著程度的變化。為了達成此一目的,我們運用一個扇形流體室,其特性為管腔中的流體剪力呈線性變化。我們將人類臍靜脈內皮細胞培養在玻片上,再組裝於流體室中,之後讓白血球沉降及黏著,以測試白血球的黏著力。由正常男子的週邊血液分離出白血球,注入流體室中,經一段時間沉降後,通入一固定流率之緩衝液,在不同流體剪力的沖刷下,觀察白血球黏著之情形。結果顯示,受到ox-LDL前處理後的內皮細胞,在測試的各個剪力強度下的白血球黏著總數及攤平之白血球黏著數目皆大幅上升。反之,若以自然態的低密度脂蛋白前處理內皮細胞,則不影響白血球的黏著。如果在白血球注入流體室前,先處理以VCAM-1抗體,則會使得白血球粘著總數及攤平之白血球數目下降,尤其是後者降幅更大。此外,以ICAM-1抗體及或thrombomodulin lectin domain處理白血球也看到相同的結果。我們的結論是,當內皮細胞受到ox-LDL(尤其是氧化八小時的ox-LDL)的前處理後,白血球與內皮細胞之間的黏著強度會增強;這可能是因為處理後的內皮細胞表現了較多的 VCAM-1、ICAM-1及P-selectin之故。本研究之結果,不僅確認黏著分子在白血球黏著於內皮細胞上所扮演的重要角色,對LDL調控此種重要的細胞間交互作用也有更深一層的瞭解。
Intravascular adhesion of leukocytes plays an important role in the pathogenesis of various vascular diseases. Previous studies have indicated that the endothelium overlying atherosclerotic lesions express various adhesion molecules. Blocking of these adhesion molecules reduced leukocytes and lipid accumulation in the lesion area. Various forms of low-density lipoprotein (LDL), especially oxidized LDL (ox-LDL), have been reported to increase the expression of adhesion molecules in endothelial cells. Therefore, ox-LDL may contribute to the development of arterial lesions by altering the function of endothelium, promoting the adhesion of leukocytes and their migration from vascular lumen to intima. This study was undertaken to obtain the quantitative information regarding the adhesiveness between leukocytes and lipoprotein-treated endothelial cells. To address this subject, we used a tapered flow chamber that has a linear variation of shear stress across it flow channel. Cultured human umbilical vein endothelial cells (HUVECs) on a glass slide served as the testing surface of the chamber to allow sedimentation and adhesion of leukocytes. Peripheral leukoocytes collected from normal male subjects were loaded into this chamber and their adhesiveness to HUVECs was accessed by flushing them with a buffer under specific local shear stresses. Our results showed that the numbers of leukocytes and spreaded leukocytes which adhered to oxLDL-pretreated HUVECs increased significantly at all shear stress levels tested. In constrast, native LDL-pretreatment was ineffective. When VCAM-1 antibody was added to a leukocyte suspension before filling into the chamber, the number of adherent leukocytes on ox-LDL-pretreated HUVECs decreased significantly. This was mainly caused by the absence of spreaded leukocytes.Similar results were obtained by the addition of ICAM-1 antibody or thrombomodulin lectin domain. We concluded that the adhesiveness between leukocytes and endothelial cells was enhanced by ox-LDL-pretreated HUVECs. Moreover, the expression of VCAM-1, ICAM-1 antibody and P-selectin on HUVECs may play a role in the enhanced adhesiveness between leukocytes and endothelial cells. This study should provide valuable information regarding not only the adhesion between leukocytes and endothelial cells, but also the roles of LDL in modulating this important cell-cell interaction.
1.Allen, S., Khan, S., Al-Mohanna, F., Batten, P., and Yacoub, M. (1998). Native low density lipoprotein-induced calcium transients trigger VCAM-1 and E-selectin expression in cultured human vascular endothelial cells. J Clin Invest 101, 1064-1075.
2.Auge, N., Pieraggi, M. T., Thiers, J. C., Negre-Salvayre, A., and Salvayre, R. (1995). Proliferative and cytotoxic effects of mildly oxidized low-density lipoproteins on vascular smooth-muscle cells. Biochem J 309 ( Pt 3), 1015-1020.
3.Berliner, J. A., Territo, M. C., Sevanian, A., Ramin, S., Kim, J. A., Bamshad, B., Esterson, M., and Fogelman, A. M. (1990). Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest 85, 1260-1266.
4.Blann, A. D., Nadar, S. K., and Lip, G. Y. (2003). The adhesion molecule P-selectin and cardiovascular disease. Eur Heart J 24, 2166-2179.
5.Brandley, B. K. (1992). Sticking around. Adhesion: its role in inflammatory disease edited by John M. Harlan and David Y. Liu, W. H. Freeman, 1992. pound 27.95 (xii + 202 pages) ISBN 0 7167 7010 5. Trends Cell Biol 2, 248-249.
6.Breslow, J. L. (1996). Mouse models of atherosclerosis. Science 272, 685-688.
7.Burnett, M. S., Gaydos, C. A., Madico, G. E., Glad, S. M., Paigen, B., Quinn, T. C., and Epstein, S. E. (2001). Atherosclerosis in apoE knockout mice infected with multiple pathogens. J Infect Dis 183, 226-231.
8.Carlos, T. M., and Harlan, J. M. (1994). Leukocyte-endothelial adhesion molecules. Blood 84, 2068-2101.
9.Chow, S. E., Lee, R. S., Shih, S. H., and Chen, J. K. (1998). Oxidized LDL promotes vascular endothelial cell pinocytosis via a prooxidation mechanism. Faseb J 12, 823-830.
10.Choy, J. C., Granville, D. J., Hunt, D. W., and McManus, B. M. (2001). Endothelial cell apoptosis: biochemical characteristics and potential implications for atherosclerosis. J Mol Cell Cardiol 33, 1673-1690.
11.Collins, R. G., Velji, R., Guevara, N. V., Hicks, M. J., Chan, L., and Beaudet, A. L. (2000). P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. J Exp Med 191, 189-194.
12.Cox, D. A., and Cohen, M. L. (1996). Effects of oxidized low-density lipoprotein on vascular contraction and relaxation: clinical and pharmacological implications in atherosclerosis. Pharmacol Rev 48, 3-19.
13.Davies, M. J., Gordon, J. L., Gearing, A. J., Pigott, R., Woolf, N., Katz, D., and Kyriakopoulos, A. (1993). The expression of the adhesion molecules ICAM-1, VCAM-1, PECAM, and E-selectin in human atherosclerosis. J Pathol 171, 223-229.
14.Dong, Z. M., Chapman, S. M., Brown, A. A., Frenette, P. S., Hynes, R. O., and Wagner, D. D. (1998). The combined role of P- and E-selectins in atherosclerosis. J Clin Invest 102, 145-152.
15.Drake, T. A., Hannani, K., Fei, H. H., Lavi, S., and Berliner, J. A. (1991). Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells. Am J Pathol 138, 601-607.
16.Esterbauer, H., Dieber-Rotheneder, M., Striegl, G., and Waeg, G. (1991). Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr 53, 314S-321S.
17.Esterbauer, H., Jurgens, G., Quehenberger, O., and Koller, E. (1987). Autoxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res 28, 495-509.
18.Esterbauer, H., Puhl, H., Waeg, G., Krebs, A., Tatzber, F., and Rabl, H. (1992). Vitamin E and atherosclerosis: an overview. J Nutr Sci Vitaminol (Tokyo) Spec No, 177-182.
19.Faggiotto, A., Ross, R., and Harker, L. (1984). Studies of hypercholesterolemia in the nonhuman primate. I. Changes that lead to fatty streak formation. Arteriosclerosis 4, 323-340.
20.Ghaisas, N. K., Shahi, C. N., Foley, B., Goggins, M., Crean, P., Kelly, A., Kelleher, D., and Walsh, M. (1997). Elevated levels of circulating soluble adhesion molecules in peripheral blood of patients with unstable angina. Am J Cardiol 80, 617-619.
21.Gimbrone, M. A., Jr. (1999). Vascular endothelium, hemodynamic forces, and atherogenesis. Am J Pathol 155, 1-5.
22.Glagov, S., Zarins, C., Giddens, D. P., and Ku, D. N. (1988). Hemodynamics and atherosclerosis. Insights and perspectives gained from studies of human arteries. Arch Pathol Lab Med 112, 1018-1031.
23.Glass, C. K., and Witztum, J. L. (2001). Atherosclerosis. the road ahead. Cell 104, 503-516.
24.Gutteridge, J. M., and Halliwell, B. (1990). The measurement and mechanism of lipid peroxidation in biological systems. Trends Biochem Sci 15, 129-135.
25.Heinecke, J. W. (1998). Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 141, 1-15.
26.Huo, Y., and Ley, K. (2001). Adhesion molecules and atherogenesis. Acta Physiol Scand 173, 35-43.
27.Itabe, H. (2003). Oxidized low-density lipoproteins: what is understood and what remains to be clarified. Biol Pharm Bull 26, 1-9.
28.Itabe, H., Mori, M., Fujimoto, Y., Higashi, Y., and Takano, T. (2003). Minimally modified LDL is an oxidized LDL enriched with oxidized phosphatidylcholines. J Biochem (Tokyo) 134, 459-465.
29.Itabe, H., Yamamoto, H., Imanaka, T., Shimamura, K., Uchiyama, H., Kimura, J., Sanaka, T., Hata, Y., and Takano, T. (1996). Sensitive detection of oxidatively modified low density lipoprotein using a monoclonal antibody. J Lipid Res 37, 45-53.
30.Jaffe, E. A. (1985). Physiologic functions of normal endothelial cells. Ann N Y Acad Sci 454, 279-291.
31.Jeng, J. R., Chang, C. H., Shieh, S. M., and Chiu, H. C. (1993). Oxidized low-density lipoprotein enhances monocyte-endothelial cell binding against shear-stress-induced detachment. Biochim Biophys Acta 1178, 221-227.
32.Johnson-Tidey, R. R., McGregor, J. L., Taylor, P. R., and Poston, R. N.
(1994). Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1. Am J Pathol 144, 952-961.
33.Jordan, J., Beneke, R., Hutler, M., Veith, A., Haller, H., and Luft, F. C. (1997). Moderate exercise leads to decreased expression of beta1 and beta2 integrins on leucocytes. Eur J Appl Physiol Occup Physiol 76, 192-194.
34.Kaikita, K., Ogawa, H., Yasue, H., Sakamoto, T., Miyao, Y., Suefuji, H., Soejima, H., Tayama, S., Hayasaki, K., Honda, T., and Kamijikkoku, S. (1997). Increased plasma soluble intercellular adhesion molecule-1 levels in patients with acute myocardial infarction. Jpn Circ J 61, 741-748.
35.Kansas, G. S. (1996). Selectins and their ligands: current concepts and controversies. Blood 88, 3259-3287.
36.Khan, B. V., Parthasarathy, S. S., Alexander, R. W., and Medford, R. M. (1995). Modified low density lipoprotein and its constituents augment cytokine-activated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest 95, 1262-1270.
37.Komoriya, A., Green, L. J., Mervic, M., Yamada, S. S., Yamada, K. M., and Humphries, M. J. (1991). The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine. J Biol Chem 266, 15075-15079.
38.Kritharides, L., Jessup, W., Gifford, J., and Dean, R. T. (1993). A method for defining the stages of low-density lipoprotein oxidation by the separation of cholesterol- and cholesteryl ester-oxidation products using HPLC. Anal Biochem 213, 79-89.
39.Lang, D., Kredan, M. B., Moat, S. J., Hussain, S. A., Powell, C. A., Bellamy, M. F., Powers, H. J., and Lewis, M. J. (2000). Homocysteine-induced inhibition of endothelium-dependent relaxation in rabbit aorta: role for superoxide anions. Arterioscler Thromb Vasc Biol 20, 422-427.
40.Lee, R. T., Schoen, F. J., Loree, H. M., Lark, M. W., and Libby, P. (1996). Circumferential stress and matrix metalloproteinase 1 in human coronary atherosclerosis. Implications for plaque rupture. Arterioscler Thromb Vasc Biol 16, 1070-1073.
41.Libby, P., Aikawa, M., Kinlay, S., Selwyn, A., and Ganz, P. (2000). Lipid lowering improves endothelial functions. Int J Cardiol 74 Suppl 1, S3-S10.
42.Lin, J. H., Zhu, Y., Liao, H. L., Kobari, Y., Groszek, L., and Stemerman, M. B. (1996). Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Atherosclerosis 127, 185-194.
43.Mertens, A., and Holvoet, P. (2001). Oxidized LDL and HDL: antagonists in atherothrombosis. Faseb J 15, 2073-2084.
44.Muller, K., Hardwick, S. J., Marchant, C. E., Law, N. S., Waeg, G., Esterbauer, H., Carpentar, K. L., and Mitchinson, M. J. (1996). Cytotoxic and chemotactic potencies of several aldehydic components of oxidised low density lipoprotein for human monocyte-macrophages. FEBS Lett 388, 165-168.
45.Muller, W. A. (2002). Leukocyte-endothelial cell interactions in the inflammatory response. Lab Invest 82, 521-533.
46.Nakashima, Y., Raines, E. W., Plump, A. S., Breslow, J. L., and Ross, R. (1998). Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vasc Biol 18, 842-851.
47.Navab, M., Fogelman, A. M., Berliner, J. A., Territo, M. C., Demer, L. L., Frank, J. S., Watson, A. D., Edwards, P. A., and Lusis, A. J. (1995). Pathogenesis of atherosclerosis. Am J Cardiol 76, 18C-23C.
48.Navab, M., Imes, S. S., Hama, S. Y., Hough, G. P., Ross, L. A., Bork, R. W., Valente, A. J., Berliner, J. A., Drinkwater, D. C., Laks, H., and et al. (1991). Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest 88, 2039-2046.
49.O'Brien, K. D., McDonald, T. O., Chait, A., Allen, M. D., and Alpers, C. E. (1996). Neovascular expression of E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 in human atherosclerosis and their relation to intimal leukocyte content. Circulation 93, 672-682.
50.Parthasarathy, S., Santanam, N., and Auge, N. (1998). Oxidized low-density lipoprotein, a two-faced Janus in coronary artery disease? Biochem Pharmacol 56, 279-284.
51.Pearson, J. D. (2000). Normal endothelial cell function. Lupus 9, 183-188.
52.Poston, R. N., Haskard, D. O., Coucher, J. R., Gall, N. P., and Johnson-Tidey, R. R. (1992). Expression of intercellular adhesion molecule-1 in atherosclerotic plaques. Am J Pathol 140, 665-673.
53.Pritchard, K. A., Jr., Tota, R. R., Lin, J. H., Danishefsky, K. J., Kurilla, B. A., Holland, J. A., and Stemerman, M. B. (1991). Native low density lipoprotein. Endothelial cell recruitment of mononuclear cells. Arterioscler Thromb 11, 1175-1181.
54.Ramos, C. L., Huo, Y., Jung, U., Ghosh, S., Manka, D. R., Sarembock, I. J., and Ley, K. (1999). Direct demonstration of P-selectin- and VCAM-1-dependent mononuclear cell rolling in early atherosclerotic lesions of apolipoprotein E-deficient mice. Circ Res 84, 1237-1244.
55.Ross, R. (1999). Atherosclerosis--an inflammatory disease. N Engl J Med 340, 115-126.
56.Saku, K., Zhang, B., Ohta, T., Shirai, K., Tsuchiya, Y., and Arakawa, K. (1999). Levels of soluble cell adhesion molecules in patients with angiographically defined coronary atherosclerosis. Jpn Circ J 63, 19-24.
57.Salvayre, R., Auge, N., Benoist, H., and Negre-Salvayre, A. (2002). Oxidized low-density lipoprotein-induced apoptosis. Biochim Biophys Acta 1585, 213-221.
58.Shih, P. T., Brennan, M. L., Vora, D. K., Territo, M. C., Strahl, D., Elices, M. J., Lusis, A. J., and Berliner, J. A. (1999). Blocking very late antigen-4 integrin decreases leukocyte entry and fatty streak formation in mice fed an atherogenic diet. Circ Res 84, 345-351.
59.Smithies, O., and Maeda, N. (1995). Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension. Proc Natl Acad Sci U S A 92, 5266-5272.
60.Sonoki, K., Yoshinari, M., Iwase, M., Iino, K., Ichikawa, K., Ohdo, S., Higuchi, S., and Iida, M. (2002). Glycoxidized low-density lipoprotein enhances monocyte chemoattractant protein-1 mRNA expression in human umbilical vein endothelial cells: relation to lysophosphatidylcholine contents and inhibition by nitric oxide donor. Metabolism 51, 1135-1142.
61.Springer, T. A. (1994). Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76, 301-314.
62.Steinberg, D. (1997). Low density lipoprotein oxidation and its pathobiological significance. J Biol Chem 272, 20963-20966.
63.Steinberg, D., and Witztum, J. L. (2002). Is the oxidative modification hypothesis relevant to human atherosclerosis? Do the antioxidant trials conducted to date refute the hypothesis? Circulation 105, 2107-2111.
64.Steinbrecher, U. P. (1987). Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem 262, 3603-3608.
65.Takei, A., Huang, Y., and Lopes-Virella, M. F. (2001). Expression of adhesion molecules by human endothelial cells exposed to oxidized low density lipoprotein. Influences of degree of oxidation and location of oxidized LDL. Atherosclerosis 154, 79-86.
66.Uittenbogaard, A., Shaul, P. W., Yuhanna, I. S., Blair, A., and Smart, E. J. (2000). High density lipoprotein prevents oxidized low density lipoprotein-induced inhibition of endothelial nitric-oxide synthase localization and activation in caveolae. J Biol Chem 275, 11278-11283.
67.Usami, S., Chen, H. H., Zhao, Y., Chien, S., and Skalak, R. (1993). Design and construction of a linear shear stress flow chamber. Ann Biomed Eng 21, 77-83.
68.van Eeden, S. F., Granton, J., Hards, J. M., Moore, B., and Hogg, J. C. (1999). Expression of the cell adhesion molecules on leukocytes that demarginate during acute maximal exercise. J Appl Physiol 86, 970-976.
69.Van Gaal, L. F., Vertommen, J., and De Leeuw, I. H. (1998). The in vitro oxidizability of lipoprotein particles in obese and non-obese subjects. Atherosclerosis 137 Suppl, S39-44.
70.Vora, D. K., Fang, Z. T., Liva, S. M., Tyner, T. R., Parhami, F., Watson, A. D., Drake, T. A., Territo, M. C., and Berliner, J. A. (1997). Induction of P-selectin by oxidized lipoproteins. Separate effects on synthesis and surface expression. Circ Res 80, 810-818.
71.Witztum, J. L., and Steinberg, D. (1991). Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 88, 1785-1792.
72.Yamaguchi, Y., Kunitomo, M., and Haginaka, J. (2002). Assay methods of modified lipoproteins in plasma. J Chromatogr B Analyt Technol Biomed Life Sci 781, 313-330.
73.Zimmerman, G. A., Prescott, S. M., and McIntyre, T. M. (1992). Endothelial cell interactions with granulocytes: tethering and signaling molecules. Immunol Today 13, 93-100.