Angiogenic factors such as bFGF have been incorporated in extracellular matrices to stimulate angiogenesis for tissue regeneration. However, the biological activity of protein-type growth factors did not last long because of their poor stability. Our previous studies have demonstrated that ginsenoside Rg1 (Rg1) and Re (Re) extracted from Panax ginseng are effective angiogenic factors.
This study was designed to further investigate the stabilities of Rg1 and Re, using bFGF, a commonly used angiogenic factor, as a control. In the study, bFGF, Rg1, and Re were stored separately at different temperatures, pH values, and solvents for distinct durations. Subsequently, the remaining activity of each test agent on HUVEC proliferation, migration, and tube formation were studied in vitro. Additionally, angiogenesis in a Matrigel plug incorporated with bFGF, Rg1 or Re implanted subcutaneously in a C57/BL6 mouse model was investigated.
The in vitro results showed that the activity of bFGF on proliferation, migration, and tube formation of HUVECs was significantly affected by its storage temperature, pH and solvent, while that of Rg1 or Re remained the same. Additionally, the in vivo results demonstrated that bFGF induced angiogenesis in a storage-condition-dependent manner, but neither Rg1 nor Re was significantly affected. The aforementioned results suggested that the stabilities of Rg1 and Re are significantly greater than that of bFGF.

1. Andersson MM, Breccia JD, Hatti-Kaul R. Stabilizing effect of chemical additives against oxidation of lactate dehydrogenase. Biotechnol Appl Biochem 2000;32:145–53.
2. Vemuri S, Beylin I, Sluzky V, Stratton P, Eberlein G, Wang YJ. The stability of bFGF against thermal denaturation. Pharm Pharmacol 1994;46:481-6.
3. Yoo HS, Shin JW, Cho JH, Son CG, Lee YW, Park SY, Cho CK. Effects of cordyceps militaris extract on angiogenesis and tumor growth. Acta Pharmacol Sin 2004;25:657-665.
4. Griffith LG, Naughton G. Tissue engineering: Current challenges and expanding opportunities. Science 2002;295:1009-14.
5. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249-57.
6. Colton CK. Implantable biohybrid artificial organs. Cell Transplant 1995;4:415-36.
7. Smith MK, Peters MC, Richardson TP, Garbern JC, Mooney DJ. Locally enhanced angiogenesis promotes transplanted cell survival. Tissue Eng 2004;10:63-71.
8. Shea LD, Smiley E, Bonadio J, Mooney DJ. DNA delivery from polymer matrices for tissue engineering. Nat Biotechnol 1999;17:551-4.
9. Lee KY, Halberstadt CR, Holder WD, Mooney DJ. Breast reconstruction, In: Lanza RP, Langer R, Vacanti J, editors. Principles of tissue engineering. New York: Academic, 2000. p. 409-423.
10. Tabata, Y. Tissue regeneration based on growth factor release. Tissue Eng 2003;9: S5-S15.
11. Moon EJ, Lee YM, Kim KW. Anti-angiogenic activity of conjugated linoleic acid on basic fibroblast growth factor-induced angiogenesis. Oncology Reports 2003;10:617-21.
12. Mizuno K, Yamamura K, Yano K, Osada T, Saeki S, Takimoto N, Sakurai T, Nimura Y. Effect of chitosan film containing basic fibroblast growth factor on wound healing in genetically diabetic mice. J Biomed Mater Res 2003; 64A:177-81.
13. Chan RYK, Chen WF, Dong A, Guo D, Wong MS. Estrogen-like activity of ginsenoside-Rg1 derived from panax notoginseng. J Clin Endocrinol Metab 2002;87:3691-5.
14. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology multiple constituents and multiple actions. Biochem Pharmacol 1999;58:1685-93.
15. Morisaki N, Watanabe S, Tezuka M, Zenibayashi M, Shiina R, Koyama N, Kanzaki T, Saito Y. Mechanism of angiogenic effects of saponin from Ginseng Radix rubra in human umbilical vein endothelial cells. Br J Pharmacol 1995;115:1188-93.
16. Lee YJ, Chung E, Lee KY, Lee YH, Huh B, Lee SK. Ginsenoside-Rg1, one of the major active molecules from Panax ginseng, is a functional ligand of glucocorticoid receptor. Mol Cell Endocrinol 1997;133:135-40.
17. Morales DE, McGowan KA, Grant DS, Maheshwari S, Bhartiya D, Cid MC, Kleinman HK, Schnaper HW. Estrogen promotes angiogenic activity in human umbilical vein endothelial cells in vitro and in a murine model. Circulation 1995;91:755-63.
18. Losordo DW, Isner JM. Estrogen and angiogenesis. Arterioscler Thromb Vasc Biol 2001;21:6-12.
19. Scott GI, Colligan PB, Ren BH, Ren J. Ginsenosides Rb1 and Re decrease cardiac contraction in adult rat ventricular myocytes: role of nitric oxide. Br J Pharmacol 2001;134:1159-65.
20. Jin ZQ, Liu CM. Effect of ginsenoside Re on the electrophysiological activity of the heart. Planta Med 1994;60:192-3.
21. Jin ZQ. The action of ginsenoside Re on inotropy and chronotropy of isolated atria prepared from guinea pigs. Planta Med 1996;62:314-6.
22. Babaei S, Stewart DJ. Overexpression of endothelial NO synthase induces angiogenesis in a co-culture model. Cardiovas Res 2002;55:190-200.
23. Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA, Pauly RR, Grant DS, Martin GR. 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 1992;67(4):519-28.
24. Hotchkiss KA, Ashton AW, Mahmood R, Russell RG, Sparano JA, Schwartz EL. Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): Association with impaired repositioning of the microtubule organizing center. Mol Cancer Ther 2002;1:1191-200.
25. Terai Y, Abe M, Miyamoto K, Koike M, Yamasaki M, Ueda M, Ueki M, Sato Y. Vascular smooth muscle cell growth-promoting factor/F-Spondin inhibits angiogenesis via the blockade of integrin □vβ3 on vascular endothelial cells. J Cell Physiol 2001;188:394-402.
26. Sakamoto H, Mashima T, Kizaki A, Dan S, Hashimoto Y, Naito M, Tsuruo T. Glyoxalase I is involved in resistance of human leukemia cells to antitumor agent-induced apoptosis. Blood 2000;95:3214-8.
27. Facchiano F, Lentini A, Fogliano V, Mancarella S, Rossi C, Facchiano A, Capogrossi MC. Sugar-induced modification of fibroblast growth factor 2 reduces its angiogenic activity in vivo. Am J Pathol 2002;161:531-41.
28. Malinda KM, Ponce L, Kleinman HK, Shackelton LM, Millis AJT. Gp38k, a protein synthesized by vascular smooth muscle cells, stimulates directional migration of human umbilical vein endothelial cells. Exp Cell Res 1999;250:168-73.
29. Bryant SJ, Anseth KS. Controlling the spatial distribution of ECM components in degradable PEG hydrogels for tissue engineering cartilage. J Biomed Mater Res 2003;64A:70-9.
30. Bader A, Schiling T, Teebken OE. Tissue engineering of heart valves-human endothelial cell seeding of detergent acellularized porcine valves. Euro J Cardio Thoracic Sur 1998;14:279-84.
31. Tabata Y, Miyao M, Yamamoto M, Ikada Y. Vascularization into a porous sponge by sustained release of basic fibroblast growth factor. J Biomater Sci Polymer Edn 1999;10:957-68.
32. Gospodarowicz D, Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol 1986;128(3):475-84.
33. Akhtar N, Dickerson EB, Auerbach R. The sponge/Matrigel angiogenesis assay. Angiogenesis 2002;5:75-80.
34. Min JK, Han KY, Kim EC, Kim YM, Lee SW, Kim OH, Kim KW, Gho YS, Kwon YG. Capsaicin inhibits in vitro and in vivo angiogenesis. Cancer Res 2004;64(2):644-51.
35. Goerges AL,Nugent MA. Regulation of vascular endothelial growth factor binding and activity by extracellular pH. J Biol Chem 2003;278(21):19518-25.
36. Privalov PL, Khechinashvili N. A thermodynamic approach to the problem of globular protein structure: acalorimetric study. J Biol 1974;86:665-84.
37. Miyamoto E, Odashima S, Kitagawa I, Tsuji A. Stability kinetics of ginsenosides in aqueous solution. J Pharm Sci 1984;73(3):409-10.
38. Gospodarowicz D. Purification of brain and pituitary FGF. In Methods in Enzymology. D. Barnes and D. Sirbasku, Eds. Acad Press, New York, 1986.
39. Gospodarowicz D. Purification of a fibroblast growth factor from bovine pituitary. J Biol Chem 1975;250;2515-20.
40. Zheng YJ, Ornstein. A molecular dynamics and quantum mechanics analysis of the effect of DMSO on enzyme structure and dynamics: subtilisin. J Am Chem Soc 1996;118:4175-4180.
41. Popovich DG, Kitts DD. Structure-function relationship exists for ginsenosides in reducing proliferation and inducing apoptosis in the human leukemia (THP-1) cell line. Arch Biochem Biophys 2002;406(1):1-8.
42. Grant DS, Lelkes PI, Fukuda K, Kleinman HK. Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro. In Vitro Cell Devel Biol 1991;27A:327-36.
43. Kibbey MC, Royce LS, Dym MS, Baum BJ, Kleinman HK. Glandular morphogenesis of a human submandibular cell line by basement membrane components in vitro. Exp Cell Res 1992;198:343-57.
44. Seely KA, Aggeler J. Modulation of milk protein synthesis through alteration of the cytoskeleton in mouse mammary epithelial cells cultured on a reconstituted basement membrane. J Cell Physiol 1991;146:117-30.
45. Taub M, Wang Y, Szcesney TM, Kleinman HK, Martin GR. Transforming growth factor alpha is required for kidney tubulogenesis in Matrigel cultures in serum-free medium. Proc Natl Acad Sci U S A 1990;87:4002-6.
46. Perets A, Baruch Y, Weisbuch F, Shoshany G, Neufeld G, Cohen S. Enhancing the vascularization of three-dimensional porous alginate scaffolds by incorporating controlled release basic fibroblast growth factor microspheres. J Biomed Mater Res 2003;65A:489-97.
47. Westal FC, Rubin R, Gospodarowicz D. Brain derived fibroblast growth factor: A study of its inactivation. Life Sci 1983;33:2425-9.