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KMID : 0388420020120010043
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Konkuk Journal of Medical Sciences
2002 Volume.12 No. 1 p.43 ~ p.55
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Inhibitory Effect of an N-terminal Recombinant Fragment of Human Thrombospondin-2 on Migration of Human Dermal Microvascular Endothelial Cells
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Noh Yun-Hee
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Abstract
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Thrombospondin-2(TSP-2) has been known to modulate tumor growth, wound healing, and inflammation through inhibition of angiogenesis based on the findings from TSP-2 knock-out mice and overexpression studies. However, the molecular mechanisms of human TSP-2(hTSP-2) for inhibition of angiogenesis or the feasibility to develope as a new therapeutic angiogenesis inhibitor for curing cancer have not been intensively studied because so far the whole molecule of hTSP-2 is not available due to difficulties in extraction of useful amount of hTSP-2 from tissues and limitations on production of full length recombinant hTSP-2 in mammalian or insect cells. Therefore, an. N-terminal 80 kDa fragment of hTSP-2 encompassing N-terminal globular region through typeI repeats(hTSP-2/NTF) was expressed in human embryonal kidney 293 EBNA cells to investigate whether this fragment has antiangiogenic effect on primary cultured human endothelial cells(ECs). The recombinant vector was constructed as follows: an 1.67 kbp of cDNA of hTSP-2(nt 213 to 1883) was amplified by PCR from human placenta cDNA library and ligated into Kpn I and Bgl II sites of the modified pCEP 4 expression vector. The recombinant protein was purified from the serum free conditioned medium of transfected 293 EBNA cells using ammonium sulfate precipitation and gelatin- and heparin-sepharose columns. Yield was 2.1 mg/1 of conditioned medium and the purified protein was confirmed by N terminal peptide sequencing. To see the fragment has anti-angiogenic activity its. ulIro, migration assay was performed on human dermal microvascular ECs(H Y, ECs) with the recombinant protein, demonstrating inhibition of migration by 30 4,o at 1 mg/ml of hTSP-2/NTF as compared to control group. This effect was not neutralized by anti-CD 36 antibody, suggesting a different receptor other than CD 36, to which TSP 1 binds and inhibits EC migration, may be invoked in the inhibitory mechanism of h T SP-2/NTF on the migration of HDMECs. Father studv to see in t;i:)o antiangiogenic activity of hTSP 2/NTF is on -going to evaluate the validity of this fragment as a new antiangIogenic agent.
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KEYWORD
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Thronuhospondin-2, angiogencsis, migration, endothelial cells
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