Thrombospondin-1 첫 번째 type I repeat 펩티드의 신혈관형성 억제작용
Inhibition of Angiogenesis by the First Type I Repeat Peptides of Thrombospondin-1
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초록
신혈관형성 (angiogenesis)은 척추동물의 발생과정 또는 질병 시에 수반되는 중요한 생물학적 현상이다. 혈관형성 억제물질의 하나로 알려진 Thrombospondin-1 (TSP-1)은 CSVTCG 아미노산 서열을 포함하는 두 번째와 세 번째type I repeat 부분이 혈관내피세포의 이동을 억제함으로써 혈관형성을 차단하는 것으로 알려져 있으나 CSVTCG 서열과 다른 서열을 갖는 첫 번째 type I repeat 부분의 역할은 잘 알려져 있지 않았다. 따라서 본 연구에서는 아미노산 서열의 차이를 나타내는 사람과 소 TSP-1의 첫 번째 type I repeat 부분 (TSR-1)이 신혈관형성 억제능력에 있어서 어떠한차이를 나타내는지 규명하였다. 이를 위해 TSR-1 펩티드 또는 PBS를 섞은 Matrigel에 bFGF를 첨가하여 생쥐복부피하에 주사함으로써 신혈관형성을 유도하였고 일주일 후에 헤모글로빈 양, 혈관의 개수, Matrigel의 색수치 등을 비교하여 신혈관형성 억제능력을 비교하였다. 또한 계란의 융모막 시험법을 통해서도 혈관형성을 억제하는지 확인하였다. 시험관내분석을 위해서는 TSR-1 펩티드를 배양 중인 HUVEC 세포에 처리하여 세포의 증식, 이동 및 맥관형성능력 등을 비교하였다. TSR-1 펩티드의 세포사멸효과는 유세포분석법으로 비교하였다. 실험 결과 소와 사람의 TSR-1 펩티드는 in vivo Matrigel assay와 CAM assay에서 신혈관형성을 억제하였으며, 소의TSR-1 펩티드가 사람의 TSR-1 또는 대조군 펩티드 (CSVTCG)에 비해 강한 억제효과를 나타내었다. 그리고 소와 사람 TSR-1 펩티드 및 CSVTCG 펩티드는 모두 HUVEC 세포의 증식은 억제하지 못했지만 세포이동 및 세포사멸을 조절함으로써 신혈관형성을 억제하였다. 이상의 실험 결과를 통해 사람과 소의 TSR-1 펩티드는 아미노산 서열은 다르지만 CSVTCG 서열이 아니더라도 유사한 펩티드로서 신혈관형성을 억제하는 능력을 나타낸다는 사실을 확인할 수 있었다.
Abstract
Angiogenesis is the fundamental biological phenomenon in the development of vertebrates and various pathophysiological process such as cancer, inflammation and wound healing. Thrombospondin-1 is a well-known anti-angiogenic molecule which is distributed in the extracellular matrix of various tissues. The second and third type I repeats of human TSP-1 have inhibitory effects on endothelial cell migration and induce angiogenesis inhibition. However the role of the first type I repeat was not elucidated. In addition, the first type I repeat of bovine TSP-1 has CSVTCG amino acid sequence which is known to have anti-angiogenic activity. In the present study, we compared the inhibition of angiogenesis to investigate the role of the first type I repeat of the human and bovine TSP-1. Matrigel was mixed with or without TSR-1 peptides and then injected into C57BL/6J mice. We compared angiogenesis inhibition activity by hemoglobin assay, microvessel density and optical density value after 7 days. Furthermore, inhibition of angiogenesis was confirmed on CAM assay by TSR-1 peptides. For in vitro angiogenesis assay, TSR-1 peptides were treated on the proliferation, migration, and tube formation assay of HUVEC. Apoptosis effect of TSR-1 peptides was confirmed by apoptosis assay kit and flow cytometry. Bovine and human TSR-1 peptides blocked neovascularization in in vivo Matrigel plug assay and CAM assay at 10 μM. Bovine TSR-1 peptides have shown stronger angiogenesis inhibition in bFGF-induced angiogenesis than human TSR-1 and CSVTCG peptides. However, all of TSR-1 peptides inhibit migration and tube formation of HUVEC in in vitro. Furthermore, these peptides also induced apoptosis of HUVEC. These results suggest that TSR-1 peptides of bovine and human TSP-1 have angiogenesis inhibition activity.
참고문헌 (46)
- Folkman J, "Clinical applications of research on angiogenesis", New Engl J Med, 333, pp.1757~1763 (1995)
- Folkman J, "Angiogenesis and apoptosis", Semin Cancer Biol, 13, pp.159~167 (2003)
- Bouck N., "How tumors become angiogenic", Adv Cancer Res, 69, pp.135~174 (1996)
- Felmeden DC., "Angiogenesis : basic pathophysiology and implications for disease", Eur Heart J, 24, pp.586~603 (2003)
- Folkman J, "What is the role of angiogenesis in metastasis from cutaneous melanoma?", Eur J Cancer Clin Oncol, 23, pp.361~363 (1987)
- Koch AE., "Stimulation of neovascularization by human rheumatoid synovial tissue macrophages", Arthritis Rheum, 29, pp.471~479 (1986)
- Kraling BM., "A simplified method for growth of human microvascular endothelial cells results in deceased senescence and continued responsiveness to cytokines and growth factors", In Vitro Cell Dev Biol Anim, 34, pp.308~315 (1998)
- Leikens S., "Angiogenesis : regulators and clinical applications", Biochem Pharmacol, 61, pp.253~270 (2001)
- Cornelius LA. ., "Human dermal microvascular endothelial cells produce matrix metalloproteinases in response to angiogenic factors and migration", J Invest Dermatol, 105, pp.170~176 (1995)
- Johnson MD., "Inhibition of angiogenesis by tissue inhibitor of metalloproteinase", J Cell Physiol, 160, pp.194~202 (1994)
- Taraboletti G., "Platelet thrombospondin modulates endothelial cell adhesion, motility and growth : a potential angiogenesis regulatory factor", J Cell Biol, 111, pp.765~772 (1990)
- Kanda S., "Role of Thrombosponidn-1-derived peptide, 4N1K, in FGF-2-induced angiogenesis", Exp Cell Res, 252, pp.262~272 (1999)
- Good DJ., "A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin", Proc Natl Acad Sci USA, 87, pp.6624~6628 (1990)
- Morandi V, "Roleof thrombospondin in the adhesion of human endothelial cells in primary culture", In Vitro Cell Dev Biol Anim, 29A, pp.585~591 (1993)
- Dreyfus M., "The build-up of the thrombospondin extracellular matrix. An apparent dependence on synthesis and on preformed fibrillar matrix", Eur J Cell Biol, 47, pp.275~282 (1988)
- Lawler J, "The functions of thrombospondin-1 and-2", Curr Opin Cell Biol, 12, pp.634~640 (2000)
- Wang TN., "The effect of thrombospondin on oral squamous carcinoma cell invasion of collagen", Am J Surg, 170, pp.502~505 (1995)
- Bein K., "Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinas activity", J Biol Chem, 275, pp.32167~32173 (2000)
- Iruela-Arispe ML., "Inhibition of angiogenesis by thrombospondin-1 is mediated by 2 independent regions within the type 1repeats", Circulation, 100, pp.1423~1431 (1999)
- Chen H., "Metabolism of thrombospondin-2 binding and degradation by 3T3 cells and glycosaminoglycan-variant chinese hamster ovary cells", J Biol Chem, 271, pp.15993~15999 (1996)
- Petzelbauer P., "Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture", J Immunol, 151, pp.5062~5072 (1993)
- Greenwalt DE., "Membrane glycoprotein CD36 : a review of its roles in adherence, signal transduction, and transfusion medicine", Blood, 80, pp.1105~1115 (1992)
- Swerlick RA., "Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro", J Immunol, 148, pp.78~83 (1992)
- Frazier WA, "Thrombospondin : a modular adhesive glycoprotein of platelets and nucleated cells", J Cell Biol, 105, pp.625~632 (1987)
- Rigotti A., "The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids", J Biol Chem, 270, pp.16221~16224 (1995)
- Nicholson AC., "Oxidized LDL binds to CD36 on human monocyte-derived macrophages and transfected cell lines. Evidence implicating the lipid moiety of the lipoprotein as the binding site", Arterioscler Thromb Vasc Biol, 15, pp.269~275 (1995)
- Nozaki S., "Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects", J Clin Invest, 96, pp.1859~1865 (1995)
- Endemann G., "CD36 is a receptor for oxidized low density lipoprotein", J Biol Chem, 268, pp.11811~11816 (1993)
- Stern M., "Human monocyte-derived macrophage phagocytosis of senescent eosinophils undergoing apoptosis. Mediation by αvβ3", CD36, Am J Pathol, pp.1996, 149[DOI]
- Ren Y., "CD36 gene transfer confers capacity for phagocytosis of cells undergoing apoptosis", J Exp Med, 181, pp.1857~1862 (1995)
- Silverstein RL., "Sense and antisense cDNA transfection of CD36 (Glycoprotein IV) in melanoma cells. Role of CD36 as a thrombospondin receptor", J Biol Chem, 267, pp.16607~16612 (1992)
- Bagavandoss P., "Specific inhibition of endothelial cell proliferation by thrombospondin", Biochem Biophys Res Commun, 170, pp.867~872 (1990)
- de Fraipont F., "Thrombospondins and tumor angiogenesis", Trends Mol Med, 7, pp.401~407 (2001)
- Guo N., "Thrombospondin 1 and type I repeat peptides of thrombospondin 1specifically induce apoptosis of endothelial cells", Cancer Res, 57, pp.1735~1742 (1997)
- Tolsma SS., "Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity", J Cell Biol, 122, pp.497~511 (1993)
- Jaffe EA., "Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria", J Clin Invest, 52, pp.2745~2756 (1973)
- Passaniti A., "Methods in laboratory investigation; A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor", Lab Invest, 67, pp.519~528 (1992)
- Akhrat N., "The sponge", Matrigel angiogenesis assay, 2002, pp.5, 75~80
- Bandyopadhyay A., "Extracellular domain of TGF-βtype III receptor inhibits angiogenesis and tumor growth in human cancer cells", Oncogene, 21, pp.3541~3551 (2002)
- Weinstat-Saslow D., "Transfection of thrombospondin 1complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis", Cancer Res, 54, pp.6504~6511 (1994)
- Streit M., "Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas", Am J Pathol, 155, pp.441~452 (1999)
- Auerbach R., "Angiogenesis assays : A critical overview", Clin Chem, 49, pp.32~40 (2003)
- Tolsma SS., "Lumen formation and other angiogenic activities of cultured capillary endothelial cells are inhibited by thrombospondin-1", Microvasc Res, 54, pp.13~26 (1997)
- Friedlander M., "Definition of two angiogenic pathways by distinct αν integrins", Science, 270, pp.1500~1502 (1995)
- Dawson DW., "CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells", J Cell Biol, 138, pp.707~717 (1997)
- Hua Li, "Caffeine-induced endothelial cell death and the inhibition of angiogenesis", Anatomy and Cell Biology, 46(1), pp.57~67 (2013), 대한해부학회