Identification of Differentially Expressed Gene(DEG) in Atherosclerotic Lesion by Annealing Control Primer (ACP)-Based Genefishing™ PCR
Atherosclerosis was identified as a chronic inflammatory process resulting from interactions between plasma lipoproteins, cellular components (monocyte, macrophages, T lymphocytes, endothelial cells and smooth muscle cells) and the extracellular matrix of the arterial wall. Several types of genes were known to express during formation of atherosclerosis. This study is carried out to identify unknown differentially expressed gene (DEG) in atherogenesis. Rabbit’s aorta tissues were stained by H&E for histomorphology. GeneFishing™ PCR analysis was performed from total RNA extracted from the aorta tissues. The DNA fragment from DEG was cloned, sequenced and validated by Real-time PCR. Histomorphology showed intimal thickening in the aorta. DEG detected from ACP-41 was identified as cathepsin B gene and showed upregulation at week-8 and week-12 of atherogenesis. Therefore, ACP-based GeneFishing™ PCR facilitated identification of cathepsin B gene which was differentially expressed during development of atherosclerosis.
[1] Newby, A. C., George, S.J., Ismail, Y., Johnson, J.L., Sala-Newby, G.B. and Thomas, A.C. "Vulnerable atherosclerotic plaque metalloproteinases
and foam cell phenotypes". Thromb Haemost, vol. 101(6): pp. 1006-11,2009.
[2] Newby, A.C. "Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degradingmatrix and non-matrix substrates". Cardiovasc Res, vol. 69(3): pp. 614-24, 2006.
[3] Matsumoto, S., Kobayashi, T., Katoh, M., Saito, S., Ikeda, Y., Kobori,
M., Masuho, Y. and Watanabe, T. "Expression and localization of
matrix metalloproteinase-12 in the aorta of cholesterol-fed rabbits:
relationship to lesion development". Am J Pathol, vol. 153(1): pp. 109-
19, 1998.
[4] Watanabe, N. and Ikeda, U., Matrix metalloproteinases and
atherosclerosis. Curr Atheroscler Rep, 6(2): p. 112-20, 2004.
[5] Newby, A.C. and Zaltsman, A.B. Fibrous cap formation or destruction-
-the critical importance of vascular smooth muscle cell proliferation,
migration and matrix formation. Cardiovasc Res, 41(2): p. 345-60, 1999.
[6] Blake, G.J. and Ridker, P.M. Inflammatory bio-markers and
cardiovascular risk prediction. J Intern Med, 252(4): p. 283-94, 2002.
[7] Beaudeux, J. L., Giral, P., Bruckert, E., Foglietti, M. J., & Chapman, M.
J. Matrix metalloproteinases, inflammation and atherosclerosis:
therapeutic perspectives. Clin Chem Lab Med, 42(2): p. 121-31, 2004.
[8] Koga, T.,Kwan, P., Zubik, L., Ameho, C., Smith, D.and Meydani, M.
Vitamin E supplementation suppresses macrophage accumulation and
endothelial cell expression of adhesion molecules in the aorta of
hypercholesterolemic rabbits. Atherosclerosis, 176(2): p. 265-72, 2004.
[9] Bobryshev, Y.V. Monocyte recruitment and foam cell formation in
atherosclerosis. Micron, 37(3): p. 208-22, 2006.
[10] Aikawa, M. and Libby, P. Lipid lowering therapy in atherosclerosis.
Semin Vasc Med, 4(4): p. 357-66, 2004.
[11] Aikawa, M. and Libby, P. Lipid lowering reduces proteolytic and
prothrombotic potential in rabbit atheroma. Ann N Y Acad Sci, 902: p.
140-52, 2000.
[12] Kang, J. G., Patino, W. D., Matoba, S., & Hwang, P. M. Genomic
analysis of circulating cells: a window into atherosclerosis. Trends in
cardiovascular medicine, 16(5): p. 163-8, 2006.
[13] Satterthwaite G, Francis SE, Suvarna K, Blakemore S, Ward C, Wallace
D, Braddock M, and Crossman D. Differential gene expression in
coronary arteries from patients presenting with ischemic heart disease:
Further evidence for the inflammatory basis of atherosclerosis. American
heart journal, 150(3): p. 488-499, 2005.
[14] Sinnaeve, P. R., Donahue, M. P., Grass, P., Seo, D., Vonderscher, J.,
Chibout, S. D., Kraus, W.E., Michael Sketch, M.J., Nelson, C.,
Ginsburg, G.S., Goldschmidt-Clermont, P.J., Granger, C.B. Gene
expression patterns in peripheral blood correlate with the extent of
coronary artery disease. PLoS One, 4(9): p. e7037, 2009.
[15] Kim, Y. J., Kwak, C. I., Gu, Y. Y., Hwang, I. T., & Chun, J. Y.
Annealing control primer system for identification of differentially
expressed genes on agarose gels. Biotechniques, 36(3): p. 424-6, 2004.
[16] Ryu, H. H., Jung, S., Sun, H. S., Jung, T. Y., Jin, S. G., Jin, Y. H., Kim,
I.Y., Jeong, Y.I1 and Kang, S.S. Screening for motility-associated genes
in malignant astrocytoma cell lines. J Neurooncol, 82(2): p. 125-31,
2007.
[17] Hwang, I.-T.K., Y-J; Kim, S-H; Kwak, CIl. and a.J.-Y.C. Young-Yun
Gu, Annealing control primer system for improving specificity of PCR
amplification. BioTechniques, 35(6): p. 1180-1184, 2003.
[18] Kim, Y. S., Hwan, J. D., Bae, S., Bae, D. H., & Shick, W. A.
Identification of differentially expressed genes using an annealing
control primer system in stage III serous ovarian carcinoma. BMC
Cancer, 10: p. 576, 2010.
[19] Lutgens, S. P., Cleutjens, K. B., Daemen, M. J., & Heeneman, S.
Cathepsin cysteine proteases in cardiovascular disease. Faseb J, 21(12):
p. 3029-41, 2007.
[20] Ma, Y., Malbon, C.C., Williams, D.L. and Thorngate, F.E. Altered gene
expression in early atherosclerosis is blocked by low level
apolipoprotein E. PLoS One, 3(6): p. e2503, 2008.
[1] Newby, A. C., George, S.J., Ismail, Y., Johnson, J.L., Sala-Newby, G.B. and Thomas, A.C. "Vulnerable atherosclerotic plaque metalloproteinases
and foam cell phenotypes". Thromb Haemost, vol. 101(6): pp. 1006-11,2009.
[2] Newby, A.C. "Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degradingmatrix and non-matrix substrates". Cardiovasc Res, vol. 69(3): pp. 614-24, 2006.
[3] Matsumoto, S., Kobayashi, T., Katoh, M., Saito, S., Ikeda, Y., Kobori,
M., Masuho, Y. and Watanabe, T. "Expression and localization of
matrix metalloproteinase-12 in the aorta of cholesterol-fed rabbits:
relationship to lesion development". Am J Pathol, vol. 153(1): pp. 109-
19, 1998.
[4] Watanabe, N. and Ikeda, U., Matrix metalloproteinases and
atherosclerosis. Curr Atheroscler Rep, 6(2): p. 112-20, 2004.
[5] Newby, A.C. and Zaltsman, A.B. Fibrous cap formation or destruction-
-the critical importance of vascular smooth muscle cell proliferation,
migration and matrix formation. Cardiovasc Res, 41(2): p. 345-60, 1999.
[6] Blake, G.J. and Ridker, P.M. Inflammatory bio-markers and
cardiovascular risk prediction. J Intern Med, 252(4): p. 283-94, 2002.
[7] Beaudeux, J. L., Giral, P., Bruckert, E., Foglietti, M. J., & Chapman, M.
J. Matrix metalloproteinases, inflammation and atherosclerosis:
therapeutic perspectives. Clin Chem Lab Med, 42(2): p. 121-31, 2004.
[8] Koga, T.,Kwan, P., Zubik, L., Ameho, C., Smith, D.and Meydani, M.
Vitamin E supplementation suppresses macrophage accumulation and
endothelial cell expression of adhesion molecules in the aorta of
hypercholesterolemic rabbits. Atherosclerosis, 176(2): p. 265-72, 2004.
[9] Bobryshev, Y.V. Monocyte recruitment and foam cell formation in
atherosclerosis. Micron, 37(3): p. 208-22, 2006.
[10] Aikawa, M. and Libby, P. Lipid lowering therapy in atherosclerosis.
Semin Vasc Med, 4(4): p. 357-66, 2004.
[11] Aikawa, M. and Libby, P. Lipid lowering reduces proteolytic and
prothrombotic potential in rabbit atheroma. Ann N Y Acad Sci, 902: p.
140-52, 2000.
[12] Kang, J. G., Patino, W. D., Matoba, S., & Hwang, P. M. Genomic
analysis of circulating cells: a window into atherosclerosis. Trends in
cardiovascular medicine, 16(5): p. 163-8, 2006.
[13] Satterthwaite G, Francis SE, Suvarna K, Blakemore S, Ward C, Wallace
D, Braddock M, and Crossman D. Differential gene expression in
coronary arteries from patients presenting with ischemic heart disease:
Further evidence for the inflammatory basis of atherosclerosis. American
heart journal, 150(3): p. 488-499, 2005.
[14] Sinnaeve, P. R., Donahue, M. P., Grass, P., Seo, D., Vonderscher, J.,
Chibout, S. D., Kraus, W.E., Michael Sketch, M.J., Nelson, C.,
Ginsburg, G.S., Goldschmidt-Clermont, P.J., Granger, C.B. Gene
expression patterns in peripheral blood correlate with the extent of
coronary artery disease. PLoS One, 4(9): p. e7037, 2009.
[15] Kim, Y. J., Kwak, C. I., Gu, Y. Y., Hwang, I. T., & Chun, J. Y.
Annealing control primer system for identification of differentially
expressed genes on agarose gels. Biotechniques, 36(3): p. 424-6, 2004.
[16] Ryu, H. H., Jung, S., Sun, H. S., Jung, T. Y., Jin, S. G., Jin, Y. H., Kim,
I.Y., Jeong, Y.I1 and Kang, S.S. Screening for motility-associated genes
in malignant astrocytoma cell lines. J Neurooncol, 82(2): p. 125-31,
2007.
[17] Hwang, I.-T.K., Y-J; Kim, S-H; Kwak, CIl. and a.J.-Y.C. Young-Yun
Gu, Annealing control primer system for improving specificity of PCR
amplification. BioTechniques, 35(6): p. 1180-1184, 2003.
[18] Kim, Y. S., Hwan, J. D., Bae, S., Bae, D. H., & Shick, W. A.
Identification of differentially expressed genes using an annealing
control primer system in stage III serous ovarian carcinoma. BMC
Cancer, 10: p. 576, 2010.
[19] Lutgens, S. P., Cleutjens, K. B., Daemen, M. J., & Heeneman, S.
Cathepsin cysteine proteases in cardiovascular disease. Faseb J, 21(12):
p. 3029-41, 2007.
[20] Ma, Y., Malbon, C.C., Williams, D.L. and Thorngate, F.E. Altered gene
expression in early atherosclerosis is blocked by low level
apolipoprotein E. PLoS One, 3(6): p. e2503, 2008.
@article{"International Journal of Medical, Medicine and Health Sciences:61209", author = "M. Maimunah and G. A. Froemming and H. Nawawi and M. I. Nafeeza and O. Effat and M. Y. Rosmadi and M. S. Mohamed Saifulaman", title = "Identification of Differentially Expressed Gene(DEG) in Atherosclerotic Lesion by Annealing Control Primer (ACP)-Based Genefishing™ PCR", abstract = "Atherosclerosis was identified as a chronic inflammatory process resulting from interactions between plasma lipoproteins, cellular components (monocyte, macrophages, T lymphocytes, endothelial cells and smooth muscle cells) and the extracellular matrix of the arterial wall. Several types of genes were known to express during formation of atherosclerosis. This study is carried out to identify unknown differentially expressed gene (DEG) in atherogenesis. Rabbit’s aorta tissues were stained by H&E for histomorphology. GeneFishing™ PCR analysis was performed from total RNA extracted from the aorta tissues. The DNA fragment from DEG was cloned, sequenced and validated by Real-time PCR. Histomorphology showed intimal thickening in the aorta. DEG detected from ACP-41 was identified as cathepsin B gene and showed upregulation at week-8 and week-12 of atherogenesis. Therefore, ACP-based GeneFishing™ PCR facilitated identification of cathepsin B gene which was differentially expressed during development of atherosclerosis.
", keywords = "Atherosclerosis, GeneFishing™ PCR, cathepsin B gene.", volume = "6", number = "7", pages = "325-6", }
